Immune system and cancer

 215

THE CLINICAL CONTEXT

The annual incidence of cancer in the United States is estimated at 439.2 per 100,000 per-

sons, with an annual rate of deaths due to can- cer of 163.5 per 100,000 persons. As a cause of death in the United States, cancer ranks second, just behind heart disease. 1 Data from 2013 to 2015 indicate that about 38% of men and women in the United States will be diagnosed with cancer sometime in their lifetime. 2 Worldwide estimates of cancer cases are 18.1 million new cancer cases and 9.6 million cancer deaths annually. 3

Over the past 25 years, death rates have dropped in the United States for cancers of the lung and bronchus, prostate, colon and rectum, and stomach, while liver cancer death rates have increased. At least 42% of cancer cases in the United States may be preventable with lifestyle changes such as smoking cessation, weight loss, physical activity, alcohol use reduction or avoid- ance, improved nutrition, use of sunblock, and avoidance of tanning devices. Vaccination or anti- biotic use can reduce incidence of cancer-causing infections such as those due to hepatitis B and C viruses, human papillomavirus, and Helicobacter pylori . Clinicians should educate their patients about these cancer-reduction strategies as well as promoting evidence-based screening tests to reduce cancer morbidity and mortality. 4

OVERVIEW OF CANCER PATHOPHYSIOLOGY

In the United States, cancer remains a leading cause of death, with nearly one in four deaths resulting from this disease. 5 Unique to cancer cells is the acquisition of

traits that impart a proliferative capacity that bypasses many of the inherent safety features designed to pre- vent abnormal growth. This uninhibited cell division, even in a single tissue or organ, harbors the potential to cause demise of the entire body. In line with this, the ability of cancer cells to spread to additional organs and form new tumors remains the most clinically rele- vant aspect of the disease. As discussed in this chapter, a diverse array of molecular alterations leads to key changes in cellular function, survival, and proliferation.

Although cancer remains heterogeneous in its development, experimental molecular evidence, ani- mal model characterization, and analysis from clini- cal studies have elucidated key features common to cancer cells. Most cases of cancer arise sporadically from the accumulation of changes in DNA that may be infl uenced by environmental interactions. This feature is highlighted by the fact that overall cancer incidence increases with age. Characterization of heritable forms of cancer in individuals with a strong family history has further increased our understanding of the genetic basis of cancer. Importantly, our knowledge in this area has been supplemented by genomic-based approaches that continue to delineate the complex and interrelated changes underlying cancer initiation and progression. Taken together, these studies have propelled the devel- opment of novel therapies aimed at disrupting the sig- naling mechanisms responsible for promoting various aspects of cancer cell function.

As uncontrolled cellular growth is the central fl aw in a cancer cell, we begin this chapter with a brief over- view of the cell cycle to highlight important aspects of growth initiation and control. We then expand our discussion to encompass tumor terminology and phe- notypic changes that drive the aggressive qualities of a cancer cell and distinguish it from its normal coun- terparts. Finally, we examine specifi c examples of mutations that initiate and promote the progression of cancer from a clinical perspective, and discuss geno- typing in cancer diagnosis and treatment.

NEOPLASIA Kolbrun (Kolla) Kristjansdottir , Thomas M. Bodenstine , and Sandhya Noronha

7

Copyright Springer Publishing Company. All Rights Reserved. From: Advanced Physiology and Pathophysiology DOI: 10.1891/9780826177087.0007

216 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

THE CELL CYCLE

The steps of cell division, collectively referred to as the cell cycle, encompass a complex system of interacting molecules. In multicellular organisms, coordinated cell division gives rise to tissues and organs during embryo- genesis that are subsequently maintained by a balance between cell growth and cell death. Rates of cell divi- sion vary widely among mammalian cells. For example, mature cardiac cells and neurons exhibit low rates of division, whereas cells lining the gastrointestinal tract and blood cell precursors of the bone marrow divide rapidly. The steps of the cell cycle must proceed in a careful, regulated manner to ensure proper production of new cells. Consequently, the cell cycle exhibits dis- tinct phases, each with its own molecular signatures and specific functions to accomplish the generation of viable new cells. To achieve this feat, cells not only manage their internal machinery, but integrate cues from the extracellular environment, including the influ- ence of growth factors, availability of nutrients, and physical interactions with neighboring cells. During this process, tight regulation of cellular proliferation must exist to avoid pathological consequence. As dis- cussed later in this chapter, an inability to control cell division remains the fundamental defect in cancer.

Although researchers have learned much about the regulation of cell division from experimental models such as the fruit fly, nematode, and amphibian, our discussion focuses on the division of human somatic cells, which account for the majority of cells present within the body. (Stem and reproductive cells exhibit specialized forms

Cell prepares for division

G1

S

M

G2

G1

S

M

DNA replicated

Cell grows, RNA/ protein synthesis, organelle duplication begins

Repair DNA errors, continued growth Interphase: S G2G1

G2

Cell divides

Cytokinesis, cellular contents divided

Mitosis, DNA separated, two nuclei form

Mitotic phase: M

(a)

(b)

FIGURE 7.1 Two major phases of the cell cycle. (a) During interphase, the cell prepares for division. In G

1 , the cell grows, organelles begin duplication, and RNA and protein synthesis are

increased to complete replication of DNA in S phase. Integrity of DNA is assessed and repaired if necessary during G

2 . (b) The mitotic phase (M phase) details the process by which replicated DNA

is separated, forming two nuclei. During cytokinesis, the contents of the cell are separated as two new cells form.

of division.) In its simplest categorization, the cell cycle is divided into two broad stages (Figure 7.1). Interphase describes the period in which the cell grows, replicates its DNA, and activates factors necessary for cell divi- sion. M phase, or the mitotic phase, entails separation of chromosomes and cytoplasm. Thus, cells prepare for division in interphase and carry out this division in M phase. Although the molecular complexities of the cell cycle and its regulation are extensive, brief descriptions of its basic components are provided here.

INTERPHASE Cells spend the majority of their time in interphase. It is during this period that they evaluate whether conditions are appropriate for cell division, irreversibly commit to the process, and complete the necessary preparations required for successful duplication. Interphase is char- acterized by three subphases referred to as G

1 (first gap

phase), S (synthesis), and G 2 (second gap phase), as

illustrated in Figure 7.1a. G

1 is the most variable portion of interphase in regard

to duration, and the amount of time cells spend in this period depends on cell type. Cells with high rates of divi- sion spend less time in G

1 than cells with less frequent

division. Throughout G 1 , high RNA and protein synthesis

rates support cell growth. Organelles such as mitochon- dria and lysosomes begin their own process of biogen- esis in preparation for providing each new cell with the necessary repertoire of organelles that will support cell function. It is also during this time that cells make a commitment to completing the remaining steps of cell division.

Chapter 7 • Neoplasia 217

S phase is so named because the primary function involves DNA synthesis within the nucleus. During this phase, DNA from all 46 chromosomes is replicated, with each new copy remaining linked to the original by cohesive proteins. These connected chromosomes are referred to as sister chromatids . Production of histone proteins increases during S phase, and DNA becomes tightly coiled around these proteins. This creates a DNA–histone complex known as chromatin , which helps organize, condense, and package DNA in later stages of the cell cycle.

Following DNA duplication, the cell enters G 2 , in

which integrity of the DNA is checked for errors and corrected if necessary by DNA repair pathways. This ensures that new cells inherit DNA that is free from mistakes that would compromise its ability to carry out the vital tasks of the cell, tissue, or organ. Following completion of G

2 , the cell has reached a critical size,

doubled its internal contents, replicated and checked its DNA, and is now ready to divide.

M PHASE (MITOTIC PHASE) Upon completing interphase, the cell must separate its DNA and cellular contents to properly form two new cells in the intricate process known as M phase (Figure 7.2).

This phase can be described in two stages: (a) Mitosis, with its own set of subphases, encompasses the process of breaking down the nuclear membrane and dividing the now duplicated chromosomes, while (b) cytokinesis entails equal splitting of the cell membrane and all of the components contained within it ( Figure 7.1b ).

Mitosis involves fi ve distinct subphases: prophase, prometaphase, metaphase, anaphase, and telophase. These processes are shown in Figure 7.2a and described in Box 7.1 . Interpreting the steps of mito- sis allows us to understand the process by which cells perform the critical task of dividing their genomic content.

Cytokinesis completes the fi nal phase of the cell cycle by separating the cellular contents and fi nal- izing the formation of the new cells, each with their own nuclei. By this time, a ring composed of actin and myosin has formed and been positioned toward the center of the cell. Known as the cleavage furrow, the ring begins to separate the cytoplasm as it contracts, ultimately sealing off the plasma membrane on each side ( Figure 7.2b ). At the end of cytokinesis, the cell cycle is now complete, resulting in the production of two daughter cells, each with its own set of DNA and cytoplasm to support function and survival.

1. Prophase 3. Metaphase 4. Anaphase 5. Telophase 2. Prometaphase

M it

o si

s C

yt o

ki ne

si s

“Cleavage furrow” Ring contracts

Centromere regions

Attachment of kinetochores to centromeres

Microtubules attach to kinetochores

Sister chromatids separate Microtubules shorten

(a)

(b)

FIGURE 7.2 Details of M phase. (a) The process of mitosis involves condensation of DNA into chromosomes during prophase while centromeres move toward opposite ends of the nucleus. Kinetochore complexes attach to centromere regions of chromosomes in prophase, while the nuclear membrane begins to break down, allowing binding of microtubules to kinetochores. Chromosomes are lined up during metaphase and separated by shortening microtubules in anaphase. Nuclear membranes form around newly separated chromosomes, which begin to decondense during telophase. (b) During cytokinesis, an actin and myosin ring aligned at the center of the dividing cell begins to contract. This serves to separate the cellular components and seal off the membranes of each new cell.

218 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

EXIT AND REENTRY OF THE CELL CYCLE While replicating cells continue to move through addi- tional rounds of the cell cycle, a cell may leave this pro- gression and enter a state known as G

0 . In G

0 , cells remain

both viable and functional, but do not actively prolifer- ate. A cell may permanently leave the cycle and enter G

0

once it has matured and performs a specific function. This is true for cells such as cardiac myocytes and neu- rons. These cells are said to be terminally differentiated, indicating that they carry out their function but no longer divide. This is a primary reason that ischemic damage of heart and brain often results in long-term and potentially permanent consequences. In addition to differentiation, most cells have a finite number of division cycles that they can complete as the ends of their chromosomes (telomeres) become progressively shorter with each divi- sion, a process known as replicative senescence. Once a critical length is reached, DNA cannot be appropriately

replicated and the cells enter a permanent state of G 0 .

Collectively, terminally differentiated and senescent cells are thus thought to be in a state of irreversible G

0 .

Compared to heart and brain, liver cells maintain a much higher capability of compensatory growth, and portions of liver can regrow following injury. These liver cells have the capacity to exit G

0 and reenter the

cell cycle. Cells possessing this ability are said to be quiescent, in that they can enter G

0 but later resume the

cell cycle if necessary by returning to G 1 . In an addi-

tional example, memory T cells of the immune system follow patterns of quiescence. During exposure to a pathogen, activated T cells increase in number as part of the immune response. Following resolution of the infection, a portion of these cells remains in the body in a quiescent state of G0

. Should the body again encoun- ter the same pathogen, these cells will rapidly reenter the cell cycle and proliferate.

BOX 7.1 Subphases of Mitosis

PROPHASE

• During the initial step of mitosis, prophase, the chromatin condenses into chromosomes, a process that involves regulated compaction of DNA while the nuclear membrane remains intact.

• A protein complex known as the kinetochore assembles on each sister chromatid at specialized regions known as centromeres. These structures will be important for separation of the sister chromatids by microtubules, dynamic protein structures that constitute part of the cytoskeleton.

• In each cell, microtubules are organized in a structure known as the centrosome. Centrosomes duplicate during cell division and move toward opposing sides of the nucleus during prophase, awaiting dissolution of the nucleus.

PROMETAPHASE

• In prometaphase, the nuclear membrane breaks down and exposes the sister chromatids to the microtubules of the centrosome.

• Microtubules from opposite centrosomes bind to the bound kinetochore protein complexes of each sister chromatid, forming a tight connection and creating tension on the proteins holding them together.

METAPHASE

• During metaphase, the microtubules align sister chromatids toward the center of the cell. This creates an appearance sometimes referred to as the metaphase plate or equatorial plane due to the alignment of the sister chromatids along the center. The next phase of mitosis will not occur until all kinetochores are attached to microtubules and properly aligned.

ANAPHASE

• With sister chromatids aligned at the center of the cell and microtubules attached to kinetochores, the proteins linking the chromatids are released and bound microtubules begin to shorten during anaphase. As this occurs, the chromatids are effectively separated and pulled to opposite ends of the cell by the shortening microtubules.

TELOPHASE

• Following this separation, in telophase, kineto- chores and their attached microtubules disas- semble while two nuclear membranes begin to form around the now separated and decondensing chromosomes. At this stage, the replicated DNA has been split and moved to opposite ends, but the cell must still effectively divide the cytoplasm.

Chapter 7 • Neoplasia 219

CONTROL OF THE CELL CYCLE While the cell cycle itself has been well characterized experimentally and its phases documented in detail through microscopy, the underpinnings of these func- tional events lie at the molecular level in an enor- mously complex network of signaling interactions. Any discussion of these mechanisms must include a basic understanding of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs). Although these molecules are not the only contributors to control of the cell cycle, our understanding of the regulation of cell division stems largely from what has been discov- ered about them.

CDKs are a family of kinase proteins that, when active, phosphorylate a specifi c set of protein sub- strates. These phosphorylation events set in motion various stages and transition points in the cell cycle by activating or inhibiting a multitude of proteins at key times. The activity of CDKs is regulated by their own phosphorylation signatures, and as the name implies, depends on the binding of proteins known as cyclins, which increase CDK function. As such, active CDKs exist as a heterodimer with cyclins and have both a cyclin-binding domain and a kinase domain that increases in activity following cyclin binding. Various CDKs have roles in the cell cycle (e.g., CDK1, CDK2, CDK4, CDK6), and different combinations of CDKs and their cyclins are important for regulation (e.g., cyclin D/ CDK4, cyclin E/CDK2, cyclin B/CDK1). A key feature of these interactions is that while cellular levels of CDKs remain relatively constant, the levels of cyclins rise and fall as the cell cycle progresses ( Figure 7.3 ). Thus, cell cycle regulation is controlled in part by fl uctuations in the amount and type of cyclins present. This allows

the process to occur in a sequential order, and activity of some CDK/cyclin complexes will upregulate levels of the cyclin required for the next phase. For exam- ple, cyclin D/CDK6, which guides the cell through G

1 ,

upregulates the levels of cyclin E, which is important for the transition from G

1 to S phase through its associ-

ation with CDK2. Of equal importance to cell cycle progression is

the ability to halt the cycle when necessary. If a cell experiences alterations to its DNA through damage or mutations, the cell must be prevented from proceeding through cellular division so as not to produce daughter cells with the same genetic alterations. Additionally, cells must ensure that the necessary nutrients and building blocks are present before proceeding through the steps of division. One regulatory protein of cell division, the retinoblastoma protein (pRb), inhibits the cell cycle from progressing through G

1 by binding and

inhibiting the activity of necessary transcription fac- tors. As levels of cyclin D rise in response to growth factor—and nutrient-induced signaling—the cyclin D–activated CDKs phosphorylate pRb, leading to a change in its structure. This structural shift causes the release of the bound transcription factors and allows the cell to continue through G

1 .

CKIs represent additional modes of cell cycle con- trol and include the in hibitors of k inase 4 (INK4) family (p15, p16, p18, p19) and C DK- i nteracting p rotein/ k inase i nhibitory p rotein (CIP/KIP) inhibitors (p21, p27, p57). These molecules have potent inhibitory activities on numerous cyclin/CDK complexes and interactions. Of these inhibitors, p21 is of particular note in that it is capable of disrupting numerous cyclin/CDK combina- tions and, as a result, possesses the ability to halt the cell cycle at multiple stages. 6 , 7

G1 Phase G2 PhaseS Phase

C on

ce nt

ra tio

n

Mitosis

Cyclin E Cyclin A Cyclin B

Cyclin D

FIGURE 7.3 Levels of cyclins vary through the cell cycle. While cellular levels of cyclin-dependent kinases stay relatively constant throughout the cell cycle, levels of their activating proteins, the cyclins, fl uctuate with the cell cycle phases. Along with additional activating mechanisms, these fl uctuations determine the timing of transitions between phases, controlling the rate of cell division.

220 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Equally important as the inhibitors are the molecules that control inhibitor expression and activity. The TP53 gene encodes the protein p53, which is critical to halt- ing the cell cycle when DNA is damaged or the cell has suffered injury—two scenarios in which progression through the cell cycle would be detrimental. Several proteins and complexes survey the genome for damage and, when present, initiate signaling pathways that lead to activation of p53. The p53 protein enters the nucleus and functions as a transcription factor, inducing the expression of numerous genes capable of halting the cell cycle, activating DNA repair pathways, or inducing cell death if defects cannot be reversed. In the case of its inhibitory cell cycle effects, p53 mediates this in part, by increasing the expression of p21. The link between p53 and the suppression of neoplastic growth was fi rst proposed following experiments utilizing colorectal carcinoma cells in which loss of p53 function was con- sistently observed. 8 This fi nding was later supported by additional evidence in other types of cancer, solidifying the idea that loss of the p53 brakes of the cell opened the door to unregulated cell proliferation. 9 , 10

CHECKPOINTS Multiple checkpoints are present within the cell cycle machinery to ensure the cell undergoes division appro- priately. Progression through a G

1 checkpoint, also

known as the restriction point, ensures the presence of necessary growth conditions and commits the cell to the remainder of the division process. Up to this point, the cell is infl uenced by the presence of external growth factors, which increase the activation of signaling path- ways that promote cell division by increasing cyclin D, as previously discussed. Thus, this checkpoint ensures that nutrients and growth factors are present and the extracellular environment is favorable for cell division. Once the cell has reached necessary levels of this acti- vation, it moves through this checkpoint and proceeds through the remainder of the cell cycle without the need for extracellular factors. Additional checkpoints exist within the cell; for example, a DNA damage checkpoint ensures that DNA has been correctly replicated and is free of alterations prior to mitosis, while an M-phase checkpoint assesses that all kinetochores are bound to microtubules before chromosome separation begins. Failure to pass any of these checkpoints results in ces- sation of the cell cycle until the issues are corrected. Each of these checkpoints is regulated by an elaborate system of coordinated molecular interactions.

Collectively, cell division is characterized by numer- ous phases and an abundance of coordinated intracel- lular activity. This setup ensures that these complex functions occur in a regulated manner. Nonetheless, con- trol mechanisms are often circumvented in cancer cells, leading to unregulated growth that threatens the body.

Thought Questions

1. What is the fundamental feature of all cancer cells?

2. How does regulation of the cell cycle relate to cancer development?

PROPERTIES OF NEOPLASMS

TUMOR TERMINOLOGY A tumor is defi ned as “[a]n abnormal mass of tissue that results when cells divide more than they should or do not die when they should.” 11 Similarly, a neo- plasm refers to a new and uncontrolled proliferation of cells that can be benign (noncancerous) or malig- nant (cancerous). It is important to note that not all malignant cellular proliferations form a tumor. Hematological malignancies are the most common examples of those that do not form discrete tumors. For example, acute lymphoblastic leukemia (ALL) is a malignant proliferation of lymphoblasts in the bone marrow and the peripheral blood without overt for- mation of a tumor. Histopathological examination of a tissue biopsy sample, or observation of a blood smear, together with knowledge of the salient clinical features (derived from the history, physical examina- tion, and imaging results), helps to make a diagnosis of a malignancy.

A benign tumor is typically slow growing and on macroscopic examination appears to be well circum- scribed. On microscopic examination, the tumor cells do not infi ltrate into the adjacent tissue. The term dys- plasia is used to describe a change or an alteration in a cell. Dysplastic cells, when viewed on a stained tissue section, show variation in the size and shape of their nuclei (nuclear pleomorphism), and the nuclei may appear darkly stained (hyperchromatic). An increased number of mitoses may be seen in tissue sections along with mitotic fi gures that are atypical. For example, infection of human ectocervical epithelial cells with the human papillomavirus (HPV) can result in dysplasia. Dysplasia may be low grade or high grade. In low-grade dysplasia, the cells in the lower third of the ectocervical epithelium are altered, and in high-grade dysplasia, the altered cells extend into the middle and upper thirds of the cervical epithelium ( Figure 7.4) .

The term carcinoma in situ has been used to describe aggregates of abnormal cells that have not extended beyond the basement membrane. For exam- ple, ductal carcinoma in situ of the breast refers to a condition in which neoplastic cells fi ll some of the

Chapter 7 • Neoplasia 221

ducts in breast tissue. The neoplastic cells are con- tained within the ducts and do not breach the basement membrane, which is a specialized matrix to which the cells are attached. An invasive cancer occurs when the neoplastic cells break through this basement mem- brane and extend into the underlying stroma.

A malignant tumor is composed of cells that are able to infi ltrate into the adjacent stroma or connective tis- sue. Macroscopic examination of the tumor reveals the tumor margins to be poorly circumscribed. Microscopic examination reveals infi ltration of the tumor cells into the surrounding connective tissue, which may allow the malignant cells to extend into lymphatics or blood vessels and to disseminate to distant organs where the tumor cells can form new tumors—a process known as metastasis . Malignant tumors (cancers) are named based on the tissue from which they develop. A carci- noma, for instance, is a malignant tumor derived from cells of epithelial origin; examples are squamous cell carcinoma, adenocarcinoma, and small cell carcinoma.

• A squamous cell carcinoma is a malignant tumor arising from squamous epithelial cells. For example, a squamous cell carcinoma of the skin is derived from squamous cells in the epidermis of the skin.

• Adenocarcinomas are malignant tumors arising from glandular epithelial cells. For example, an ade- nocarcinoma of the lung is derived from glandular epithelial cells lining the tracheobronchial tree in the lungs.

• A small cell carcinoma arises from neuroendo- crine cells that occur normally in various tissues in the body. For example, a small cell carcinoma of the lung arises from neuroendocrine cells in lung tissue.

Sarcomas are malignant tumors arising from mes- enchymal tissue (e.g., blood vessels, cartilage, bone,

muscle) and are rare in comparison to carcinomas. The prefi x of a sarcoma designates its origin; for example, an osteosarcoma arises from bone tissue, whereas a liposarcoma indicates a sarcoma arising from adipose tissue. Additional examples are chondrosarcoma (car- tilage), leiomyosarcoma (smooth muscle), rhabdomyo- sarcoma (skeletal muscle), and angiosarcoma (blood vessels).

In hematopoietic neoplasms, there is an abnor- mal proliferation of cells in the bone marrow, periph- eral blood, or other hematopoietic tissues such as the lymph nodes, spleen, or liver. A lymphoma refers to a malignant tumor that results from a monoclonal pro- liferation of lymphoid cells such as B lymphocytes or T lymphocytes. Leukemia occurs secondary to a monoclonal proliferation of early hematopoietic cells (blast cells) in the bone marrow, accompanied by an arrest in the normal maturation of the cells.

Other malignant tumors likely to be encountered in clinical practice include melanoma, brain tumors, and teratoma. Melanoma arises from melanocytes such as those seen within the skin. Tumors of the brain may arise from a multitude of cells in the ner- vous system, including neurons, glial cells, choroid plexus, and meninges. Brain tumors commonly seen in clinical practice include glial tumors, meningioma, and secondary tumors that have metastasized to the brain from other sites in the body. A teratoma is a tumor that arises from germ cells and is composed of tissues derived from one or more of the embryo- logical germ cell layers. For example, a mature cystic teratoma, also known as a dermoid cyst, may occur in organs such as the testes or ovaries, and may be com- posed of an aggregate of mature tissues representing various organs in the body; for example, skin and hair (ectoderm), lungs and intestines (endoderm), and bone and cartilage (mesoderm).

(a) (b)

FIGURE 7.4 Micrographs of normal cervical epithelium (a) and level II cervical intraepithelial neoplasia (b). Abnormal cells (b) have larger and darker nuclei and are found in the lower third of the epithelial layer but do not breach the basal lamina.

222 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Thought Questions

3. What features help to diff erentiate a benign tumor from a malignant tumor?

4. What tissues give rise to a carcinoma, a sarcoma, and a lymphoma?

CHARACTERISTICS OF A CANCER CELL The development of a malignant neoplasm, or cancer, is a stepwise process that includes a series of genetic changes in a normally functioning cell that gradually transform the cell into a cancer cell. These genetic changes result in the inability of the body to restrain cell division and can lead to the spread of the cancer cells around the body. Collectively, the characteristics that a cell acquires when it becomes cancerous are referred to as the hallmarks of cancer . 12 , 13 Some of these char- acteristics include uncontrolled proliferative signal- ing, evading growth suppressors, genomic instability, enabling cell immortality, resisting cell death, hijacking or generating blood supply sources for nourishment, and acquiring invasive and metastatic abilities.

Proto-Oncogenes, Oncogenes, and Tumor Suppressor Genes As a general principle, the genetic changes that promote the stepwise progression from normal cell function to malignancy alter the amount, activity, or regulation of two types of genes.

First, some genes possess the ability to promote cellular proliferation and survival, but are subject to careful regulation to limit these functions to appro- priate circumstances. Genes with these characteris- tics are known as proto-oncogenes, and the majority of these proto-oncogenes are involved in regulation of the cell cycle and responses to growth factor acti- vation. Proto-oncogene mutations may lead to loss of proper regulation and result in disease due to overac- tivity of their inherent properties. This conversion from proto-oncogene to oncogene leads to sustained activ- ity of their encoded proteins, resulting in unchecked growth that may progress to neoplasia. Such mutations are referred to as gain-of-function mutations because the resulting protein has an increase in activity.

Second, many protective genes encode proteins that inhibit uncontrolled cell division and conduct surveil- lance of DNA. Signals of DNA damage or deranged cell function result in DNA repair (if possible) or initiation of apoptosis if the cell is beyond repair. These genes can collectively be referred to as tumor suppressor genes . As described next, the loss of normal function of a single tumor suppressor gene allele is not suffi cient to induce loss of normal protein function; both copies of

the gene must be mutated in order for decreased tumor suppressor activity and promotion of cancer. These mutations are referred to as loss-of-function mutations because absence of the normal protein function is inte- gral to cancer promotion ( Figure 7.5 ).

Uncontrolled Proliferative Signaling An oncogene may be formed as a result of mutations within the proto-oncogene or as a consequence of larger alterations such as chromosomal translocations that disrupt normal controls on oncogene expression. One of the most commonly observed oncogenes is mutated Ras , found in about 20% of all human cancers. Ras is an important intracellular signaling protein that, when properly stimulated, transmits signals that activate cell growth for a brief time before being turned off. Mutated Ras results in continuous stimulation that fuels tumor growth. Another proto-oncogene codes for the mem- brane epidermal growth factor receptor (EGFR) a mem- ber of the ErbB family of growth-promoting tyrosine kinases. Increased levels of EGFR or mutated forms of EGFR are frequently present in cancers, resulting in unregulated cell division due to the continual activation of its signaling properties. Inhibitors that bind to EGFR and decrease cellular growth are used in the treatment of some breast, pancreatic, and colon cancers.

Evading Growth Suppressors Cells have many mechanisms to regulate cell prolifera- tion in response to activation of oncogenes or cell stress, including halting cell division, inducing DNA repair, and initiating cell death. The tumor suppressor genes controlling these processes include many cell cycle inhibitors and regulators. When a tumor suppressor is inactivated or turned off , the likelihood of cancer devel- opment is increased. As such, the actions of a normally functioning tumor suppressor will inhibit proliferation, but upon loss of its function unregulated growth can occur. Over half of all human cancers have mutations in the TP53 tumor suppressor gene, illustrating its vital importance in maintaining genome integrity and elimi- nating irreversibly altered cells. When the p53 protein is not functional, DNA damage goes unrepaired and muta- tions accumulate in cells, leading to acquisition of other cancer cell characteristics. RB1 represents an additional tumor suppressor gene and its product, pRb, has the capacity to stop the cell cycle in G

1 . The consequence of

losing this function is underscored by the common fi nd- ings of RB1 mutations in retinoblastoma, osteosarcoma, and carcinomas of breast, lung, and colon.

Genomic Instability Defects in DNA repair pathways are not directly onco- genic; however, because the DNA cannot be effectively repaired, DNA mutations accumulate. The genome becomes unstable, signifi cantly increasing the risk of mutations occurring in processes that regulate cell

Chapter 7 • Neoplasia 223

growth, such as tumor suppressors, proto-oncogenes, and cell death pathways. For example, patients with the disease xeroderma pigmentosum have germ- line mutations in nucleotide excision repair pathways, decreasing their ability to correct DNA damage caused by ultraviolet (UV) light. As a result, these patients are extremely susceptible to UV-induced burns and skin damage. Without protection from sun or other UV sources, about half of these patients will develop their fi rst skin cancer by age 10 years. 14

Enabling Replicative Immortality The human body must maintain equilibrium between the replacement of old cells and generation of new ones. The cell cycle continuously produces new cells, but each cell has a limited number of cell divisions before it undergoes a process called cellular aging, or senescence . Senescent cells function but are no longer able to enter the cell cycle and divide. A hallmark of cancer cells is the ability to bypass senescence and continue cell divi- sion. This occurs, in part, because of increased levels of telomerase , an enzyme that maintains chromosome length, making cancer cells in essence immortal. This property is supported by the fact that many cancer cell lines derived from cancer patients can survive indefi – nitely in the laboratory if maintained under appropriate growth conditions. Telomerase inhibitors are promising

anticancer agents because telomerase is expressed in the majority of cancer cells and is not present, or is pres- ent in very low amounts, in normal cells.

Resisting Cell Death Cell death can occur through two primary routes. Necrosis represents an uncontrolled form of cell death that often occurs in response to an acute cellular injury. The plasma membrane is ruptured and intracellular con- tents spill into the surrounding tissues, promoting infl am- mation and tissue damage. Apoptosis is a regulated and programmed form of cell death. Under normal condi- tions, cells that sustain extensive or unrepairable damage to their DNA undergo apoptosis due to intracellular sur- veillance mechanisms. Abnormal or pathogen-infected cells will also be targeted for apoptosis or outright lysis by immune cells. Thus, apoptotic mechanisms serve a protective function in the body and represent a challenge to the development of cancer. Cancer cells, however, have found ways to evade apoptosis by upregulating anti- apoptotic factors such as inhibitors of apoptosis (IAPs) or decreasing production of proapoptotic factors such as Fas. This enables cancer cells to resist induction of apoptosis. Avoiding this built-in mechanism of cell death allows a cell with mutated DNA to continue to grow and divide, gaining mutations that make the cell more tumor- igenic and ultimately malignant.

(a)

(b)

Normal cell

Normal cell

Overactive mutation (gain of function)

Single mutation event creates oncogene

Activating mutation enables oncogene to promote cell transformation

Cells en route to cancerUnderactive mutation (loss of function)

Mutation event inactivates

tumor suppressor

gene No effect of mutation in

one gene copy

Second mutation event

inactivates second gene

Two inactivating mutants functionally eliminate the tumor suppressor gene,

promoting cell transformation

FIGURE 7.5 Two major groups of genetic mutations favor progression of cells to malignancy. (a) A proto-oncogene within a normal cell is mutated, becoming an oncogene. The oncogene product stimulates excessive and unregulated cell division. (b) A tumor suppressor gene is inactivated by a mutation. The remaining normal allele sustains cell protection from proliferation; however, if the second allele develops an inactivating mutation, tumor suppressor function and this pathway of protection from malignancy are lost.

224 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Promotion of Angiogenesis Blood vessels provide oxygen and nutrients to tissues and are crucial for cell function and survival. Tumors that are not in close proximity to blood vessels are lim- ited in growth to several millimeters diameter before cells in the hypoxic core become quiescent or die. In order for tumors to continue to grow, they must develop an angiogenic ability to generate new blood vessels or expand the existing vascular tree. The resulting vascu- lature feeds growing tumors with the newly developed blood supply, allowing tumors to enlarge and the cancer cells to invade adjacent tissues, promoting metastasis. To accomplish this, many cancer cells have increased local levels of proangiogenic factors such as vascular endothelial growth factor (VEGF). Molecules such as these normally induce blood vessel formation during development or in response to vascular injury but are repurposed by cancer cells to provide themselves with a blood supply. Scientists have developed angiogene- sis inhibitors with the goal of starving the cancer of its needed blood supply. Bevacizumab is an example of a VEGF inhibitor used in the treatment of cancers that have metastasized or recurred, including glioblastoma multiforme, renal cell cancer, and ovarian cancer.

Invasive and Metastatic Ability Cancer cells have the ability to spread from the origi- nal tumor site to distant areas of the body, through the process of metastasis. In order to metastasize, cells must separate from the original tumor; invade the sur- rounding tissues; enter and survive in the circulation, lymphatics, or peritoneal space; and settle in a distant target organ where they adapt, survive, and proliferate. To do this, metastatic cancer cells typically develop alterations in their shape and in their attachment to other cells and to the extracellular matrix. The epi- thelial–mesenchymal transition (EMT) is an import- ant process that allows transformed epithelial cells to invade tissues, resist apoptosis, and spread. Increased expression of EMT transcriptional regulators results in a loss of adherence, an associated conversion from an epithelial to a fibroblastic, or spindle-like morphol- ogy, expression of matrix-degrading enzymes, and increased cell motility. E-cadherin, a key adhesion mol- ecule, is lost in many cancer cells, allowing tumor cells to detach from surrounding cells, and increasing the risk of invasion and metastasis. The cancer hallmarks and examples of proteins and processes altered are summarized in Table 7.1.

RECENTLY IDENTIFIED CANCER CELL CHARACTERISTICS The concept of cancer hallmarks was further elabo- rated in 2011, with the inclusion of evasion of immune destruction and altered metabolism as additional emerging hallmarks.13

Evasion of Immune Destruction Classical immunology proposes that cytotoxic T lym- phocytes, among other effector cells, are capable of killing not only virus-infected self-cells, but also self- cells that have undergone cancer-causing mutations and unregulated proliferation. Interestingly, having cancer promotes a state of increased generalized inflammation, with elevated cytokine levels both within a tumor and systemically that may promote cancer cell proliferation. In addition, lymphocytes are found within tumor tissue, but they are not always cytotoxic lymphocytes. Rather, regulatory T cells (Tregs) may be present in tumors and downregulate the ability of the immune system to clear the tumor cells. Cancer cells may develop characteristics that activate immune sys- tem inhibitory signaling, particularly through the acti- vation of cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which downregulates immune respon- siveness. Similarly, cancer cells produce a ligand that activates programmed cell death 1 (PD-1) receptors found on T cells, B cells, and natural killer cells, and suppresses immune activity, hindering clearance of the cancer cells.15

Immune-based cancer treatments continue to evolve.16 Chimeric antigen receptor–T cell (CAR-T cell) is a method of altering T-cell receptors to attack antigens associated with a tumor. Monoclonal anti- bodies are also successfully used, and several types have been developed that specifically target proteins unique to a given cancer type. Monoclonal antibodies can opsonize tumor cells, marking them for phago- cytic destruction. At the same time, destruction of the cells increases the reactions of antigen-presenting cells that can drive T-helper– and cytotoxic T-lymphocyte– mediated tumor destruction. Finally, some tumors have

TABLE 7.1 Cancer Characteristics and Exemplar Causative Factors

Cancer Cell Characteristics or Hallmarks

Associated Protein or Key Process

Uncontrolled proliferative signaling

Ras, EGFR

Evading growth suppressors p53, pRb

Genomic instability DNA repair

Enabling replicative immortality Telomerase

Restricting cell death Apoptosis

Promotion of angiogenesis VEGF

Invasive and metastatic ability EMT, E-cadherin

EGFR, epidermal growth factor receptor; EMT, epithelial– mesenchymal transition; VEGF, vascular endothelial growth factor.

Chapter 7 • Neoplasia 225

signals that inhibit lymphocyte proliferation by acti- vating checkpoints that block their entry into the cell cycle needed for clonal proliferation. Checkpoint inhib- itor drugs block those cell membrane signals, allow- ing the lymphocytes to respond with proliferation and immune-mediated eradication of tumor cells.

Cancer Cell Metabolic Alterations As previously noted, a growing tumor will have vary- ing degrees of oxygen supply, depending on prox- imity to existing or newly formed blood vessels. The collection of cells will consist of tumor cells in vary- ing states of genetic alteration, each having somewhat different characteristics and energy requirements, plus

surrounding tissue cells, called the stroma. Cell types making up the tumor include the cancer stem cells that propagate their own growth, immune cells (lym- phocytes, macrophages, dendritic cells) stimulated by infl ammatory signals of the tumor, fi broblasts, and cells undergoing the EMT. The microenvironment of the tumor depends on interactions between these cell types and determines the type of metabolism needed by cells in different stages of tumor progression. One model proposes that hypoxia-stressed cells in the center of a tumor adopt the properties of increased survival, treatment resistance, and immune evasion, ultimately becoming more likely to develop mutations favoring invasion and metastasis 17 ( Figure 7.6 ).

Blood vessels

N or

m ox

ia H

yp ox

ia

Tumor-derived factors • Cytokines/ Chemokines • Metabolites

Stressor tumor conditions • Hypoxia • Acidosis

Invasion and metastasis

Tumor cell adaptations • Aggressive phenotype • Growth advantage • Increased cell survival (autophagy) • Acquisition of EMT and CSC phenotype • Acquired treatment resistance • Immune escape and tolerance • Tumor progression

CTL-sensitive cell

CTL-resistant cancer cell

Cancer stem cell

CTL Macrophage Dendritic cell

Mesenchymal cell (EMT)

FIGURE 7.6 Events in tumor progression. A tumor is made up of CSCs, cancer cells, stroma cells (of the original tissue), lymphocytes, macrophages, dendritic cells, and cells in some phase of the EMT. The tumor region closest to a blood vessel has normal oxygen supply and has cells that may maintain normal sensitivity to killing by CTLs. The tumor region at a distance from the blood vessel has a hypoxic and acidotic environment that induces cell stress. This may promote acquisition of cancer cell immune resistance and development of immune tolerance, as well as favoring mutations that enable invasion and metastasis. CTL, cytotoxic T lymphocyte; CSC, cancer stem cell; EMT, epithelial–mesenchymal transition.

226 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Early studies of tumor cell metabolism indicated a high rate of glucose uptake due to increased levels of glucose transporter 1 (GLUT1). This alteration is clin- ically useful, as the accelerated glucose uptake forms the basis of PET scanning for fl uorodeoxyglucose uptake to localize tumors and metastases. Rapidly dividing and aggressive tumor cells often rely on gly- colysis for energy production. The glycolytic pathway does not produce as much adenosine triphosphate (ATP) as the usual metabolic pathways of glycolysis, Krebs cycle, and oxidative phosphorylation, but it is rapid and produces lactate, which can be used to syn- thesize new cell components to support further cell division. 18

In summary, accumulation of sporadic mutations can result in the acquisition of cancer cell characteris- tics. A mutated cell acquires more mutations over time, resulting in a heterogeneous tumor composed of cells with a range of cancer cell characteristics. Genomic instability accelerates mutation rates, and some of those mutations further promote the rate of cellu- lar growth and division. A benign neoplasm can, over time, become malignant, highlighting the importance of early identifi cation and treatment. Even established malignant tumors continue to accumulate mutations, adapting to their surroundings and acquiring more can- cer characteristics. Each hallmark of cancer presents a pathway of targeted therapy development to improve clinical outcomes.

Thought Questions

5. What is an oncogene?

6. How do cancer cells obtain suffi cient oxygen and nutrients in a large tumor?

7. Why does genomic instability increase cancer risk?

CLINICAL ASPECTS OF NEOPLASIA

PATHOPHYSIOLOGY OF CANCER MANIFESTATIONS AND TREATMENT SEQUELAE Cancer and cancer treatments are associated with a number of pathophysiological alterations at the sys- temic level, in addition to localized manifestations resulting from solid tumors. Chief among these are infl ammation, with elevated cytokine production that may cause fevers and suppress appetite while also promoting clotting. Hypercoagulability also commonly accompanies cancer, particularly in early stages. Some patients originally diagnosed with deep vein thrombosis

and pulmonary embolism are subsequently found to have cancer. In addition, fatigue that is disproportional to effort is common, particularly in more advanced cancer. Poor appetite and wasting can also occur in advanced cancer, as well as resulting from chemother- apy-induced nausea and vomiting. Endocrine-related syndromes can result from tumors that are ectopic sources of hormones and hormone-like substances. An example is parathyroid hormone–related protein, which can cause hypercalcemia and bone loss, and tumor-pro- duced vasopressin, which can cause the syndrome of inappropriate antidiuretic hormone secretion (SIADH).

Tumor lysis syndrome is an acute generalized reaction to massive cell death caused by cancer treat- ment. Although tumor lysis syndrome can be a sponta- neous event, it is generally precipitated after initiating a round of treatment that results in robust and rapid killing of malignant cells. The cells then release their contents, causing hyperuricemia, hyperkalemia, hyper- phosphatemia, and hypocalcemia. The ensuing elec- trolyte imbalance can cause instability of excitable tissues, resulting in cardiac arrhythmias and neuro- logical seizures. Acute renal failure is also a potential outcome. Risk stratifi cation to reduce tumor lysis syn- drome is aided by estimating tumor volume, cell lysis potential of the treatment, and patient factors such as fl uid and electrolyte status and renal function. Careful monitoring is required to manage the onset of this com- plication with fl uid supplementation and measures that reduce uric acid, phosphate, and potassium. 19

Other cancer- and therapy-associated complica- tions and symptoms include pain, anemia, neutropenia, thrombocytopenia, nausea and vomiting, stomatitis, fatigue, radiation-induced tissue injury, sleep distur- bance, and dysphoria. Providers in oncology centers continue to refi ne evidence-based strategies to care for patients with cancer with the aim of optimizing qual- ity of life and function. This is a critical step with the striking evolution of novel, highly effective gene- and immune-based therapies that have a high risk of unpre- dictable adverse reactions.

BIOLOGICAL ASPECTS OF GENE MUTATIONS AND CANCER RISK FACTORS Cancers may occur as a result of hereditary or sporadic gene mutations in somatic or germline cells. Germline mutations are mutations that occur in the DNA of germ cells such as ova and spermatozoa. Inheritance of a ger- mline mutation results in every cell in the body having the mutation. Somatic mutations are mutations that occur in the cells spontaneously or as a result of muta- gen exposures. For example, skin cells that have been repeatedly exposed to UV rays may develop a somatic mutation. A somatic mutation is inherited by the prog- eny of the cell with the mutation but does not occur in all cells in the body and cannot be inherited by offspring.

Chapter 7 • Neoplasia 227

Sporadic gene mutations occur more commonly than inherited gene mutations. It has been estimated that only about 5% to 10% of cancers are inherited. 20

Typically, sporadic gene mutations accumulate in the tissues over time prior to the development of a tumor. For example, benign adenomatous polyps of the colon accumulate gene mutations over time before developing into a malignant tumor. Screening pro- cedures such as colonoscopy are extremely import- ant to detect and remove benign polyps of the colon before they become malignant. Some risk factors thought to play a role in the development of cancer include age, race and ethnicity, smoking, alcohol con- sumption, excessive exposure to UV light, exposure to environmental toxins, lack of exercise, and obesity. For example,

• Smoking has been identifi ed as the leading cause of lung cancer and is linked to many other cancers.

• Excessive exposure to UV light has been linked to the development of skin cancer.

• Age is a risk factor for the development of prostate cancer, which typically occurs in older men, and men of African descent appear to have a higher risk of prostate cancer.

• A diet low in fruits and vegetables and rich in red meats is a risk factor for the development of colon cancer.

• Excessive consumption of alcohol is a risk factor for liver and other cancers.

• Exposure to asbestos has been linked to the devel- opment of mesothelioma, a tumor arising from the pleura of the lung.

• Obesity and a lack of physical activity have also been implicated as increasing development of many cancers.

Hereditary gene mutations also play a role in the development of neoplasia, and typically occur in patients at an earlier age than neoplasia due to spo- radic mutations. For example, the inheritance of germline mutations in DNA mismatch repair genes such as hMSH2 , hMLH1 , hMSH6 , and hPMS2 has been implicated in the development of Lynch syn- drome, also known as hereditary nonpolyposis col- orectal cancer (HNPCC). Mismatch repair genes are genes that play a role in ensuring the accurate pair- ing of DNA base pairs. Microsatellites are short DNA sequences that may be altered due to mutations in the mismatch repair genes, which may then result in a genomic instability called microsatellite instability. Patients with mismatch repair gene mutations are at an increased risk for the development of tumors, especially colorectal and endometrial cancers, as well as cancers in organs such as the duodenum, kid- neys, liver, stomach, and ovaries. 21

Mutations in tumor suppressor genes may be inher- ited. Affected individuals are heterozygous, being born with two alleles, of which one has normal func- tion and the other lacks that function. In Li-Fraumeni syndrome, a germline mutation of the tumor suppres- sor gene TP53 is inherited. Patients with Li-Fraumeni syndrome are at an increased risk for the devel- opment of tumors at an early age. Tumors include sarcoma of the bone and soft tissue, breast cancer, brain tumors, and tumors of the adrenal cortex. 22 The breast cancer genes 1 and 2 ( BRCA1 and BRCA2 ) are tumor suppressor genes that are often germline muta- tions, and inheritance of a copy of the mutated gene signifi cantly increases the risk for the development of early breast and ovarian cancer. The inheritance of mutated BRCA1 and BRCA2 has also been associated with the development of cancers in other organs such as the fallopian tube, peritoneum, prostate, and the male breast. 23

Chromosomal alterations due to deletions or trans- locations may also result in oncogene activation and the development of neoplasia. For example, a follicu- lar lymphoma may develop following the overexpres- sion of the oncoprotein (resulting protein produced from an oncogene) BCL-2 in B lymphocytes. In the vast majority of patients, BCL-2 overexpression occurs secondary to a t(14;18) chromosomal translo- cation. The overexpression of BCL-2 prevents normal cell death or apoptosis of B lymphocytes, resulting in the development of a B-cell lymphoma. Acute promy- elocytic leukemia (APL; or acute myeloid leukemia– M3) is a condition in which a t(15;17)(q24.1;q21.2) chromosomal translocation leads to the production of an oncoprotein that prevents the normal matu- ration of promyelocytes to neutrophils in the bone marrow.

The chromosomal translocation t(9;22), a bal- anced translocation between the breakpoint cluster region gene BCR on chromosome 22 and the ABL gene on chromosome 9, results in the generation of the Philadelphia chromosome, fi rst identifi ed at two research laboratories in Philadelphia. The transloca- tion is visible upon karyotyping and labeling, and the chromosomal rearrangement forms an abnormal BCR- ABL fusion gene ( Figure 7.7 ). This abnormal gene pro- duces oncoproteins that have proliferation-promoting tyrosine kinase activity that can no longer be switched off . Uncontrolled proliferation of granulocytic cells ensues and is associated with the development of a chronic myeloid leukemia (CML).

VIRAL CAUSES OF CANCER Adding to the potential mechanisms of cancer ini- tiation, several viruses have been implicated in the development of human cancers. The Epstein–Barr

228 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

virus is a herpesvirus that can infect B lymphocytes. The virus has been associated with a t(8;14) chromo- somal translocation and the development of a B-cell lymphoma called Burkitt lymphoma. The hepatitis B virus is a DNA virus that has been associated with the development of hepatocellular carcinoma. The mechanism by which hepatitis B viruses may induce the development of a tumor is complex but is likely related to changes that occur secondary to the inte- gration of the hepatitis B viral DNA into the genome of patients with chronic hepatitis B. Hepatitis C is caused by an RNA virus that also increases risk of hepatocellular carcinoma. 24

HPV is a small, sexually transmitted DNA virus. There are more than 200 genotypes of HPV. Subtypes 6 and 11 are associated with the development of lesions such as cutaneous and anogenital warts in adults and laryngeal papillomas in children, and HPV subtypes 16 and 18 have been implicated in the devel- opment of head and neck and anogenital cancers. The mechanism by which HPV induces neoplasia has been studied extensively. The virus produces six early (E) proteins and two late (L) proteins. The E6 and E7 proteins are involved in degradation of the pRb tumor suppressor, which allows viral repli- cation in the cell infected with HPV. The E6 protein

Normal chromosome 9

Changed chromosome 9

Normal chromosome 22

Changed chromosome 22

(Philadelphia chromosome)

ABL

BCR

Chromosomes break

BCR-ABL

FIGURE 7.7 A chromosomal translocation activates an oncogene. Chromosomal translocation between chromosomes 9 and 22 produces a mutant Philadelphia chromosome that positions a BCR (breakpoint cluster region) gene next to the ABL proto-oncogene. The protein encoded by the fusion gene BCR-ABL is a continually activated protein tyrosine kinase that stimulates cells to continually divide, bypassing normal controls of the cell cycle. Presence of this gene is common in leukemias, particularly in chronic myelogenous leukemia.

also interacts with the p53 protein, resulting in the degradation of p53 and a decrease in apoptosis or cell death. In addition to p53 and pRb, many other cellular proteins are also targeted, which leads to an uncontrolled proliferation of cells and an increased risk for neoplasia. 25 Vaccines are available against viruses such as hepatitis B and HPV. Immunization with these vaccines is important to decrease the risk of infection with the viruses and, therefore, decrease the risk of developing a cancer. Newer therapies for hepatitis C have played a signifi cant role in clearing the virus from the body and reducing the risk of can- cer development.

Thought Questions

8. What is the molecular basis for the development of the Lynch syndrome and the Li-Fraumeni syndrome?

9. What is the Philadelphia chromosome?

10. What viruses are associated with the development of neoplasia?

Chapter 7 • Neoplasia 229

GENOTYPING IN CANCER DIAGNOSIS AND TREATMENT

Approaches to the treatment of cancer depend on the type, location, and progression of the tumor at the time of diagnosis. Included in these options are tradi- tional strategies such as surgery, radiation, and che- motherapy. Surgery is commonly used to excise solid tumors and has the greatest success when tumors have not spread and remain accessible. A subset of cancers responds to radiation treatment, and the tech- nology and accuracy of this approach has dramatically improved over the past several decades.

Given that cancer cells often have fast growth rates, chemotherapy can be used to nonspecifi cally target any rapidly growing cells by damaging DNA or inhibiting its replication. Because chemotherapy is distributed sys- temically, it can be used to treat advanced cancers that have spread from their primary site of origin. However, because normal cells are also affected by the chemother- apeutic agents, side effects of chemotherapy are frequent and often severe. The discovery of common cancer cell characteristics, the mechanism behind cancer develop- ment, and technological advancements have resulted in the identifi cation of numerous targeted cancer drugs that have revolutionized diagnosis and treatment.

Cancers can now be genotyped, identifying cancer-associated genetic abnormalities using a wide variety of techniques, including whole genome sequencing, targeted polymerase chain reaction (PCR), and immunological methods. These genetic abnormal- ities can affect patient outcomes and can be used for diagnosis, prognosis, and, in some cases, to determine therapeutic approach. For example, the detection of the BCR-ABL fusion gene, described earlier, confi rms the diagnosis of CML. Treatment with a specifi c tyro- sine kinase inhibitor such as imatinib will switch off

the growth-promoting activity of the BCR-ABL fusion protein and has resulted in a dramatic improvement in the prognosis for patients with CML. Patients with APL can be diagnosed by the translocation causing the disease, and then treated with all-trans retinoic acid, which induces the promyelocytes to differentiate to neutrophils. The addition of all-trans retinoic acid to the treatment regimen of patients with APL has sig- nifi cantly reduced the mortality and morbidity rates of patients with this disease.

Amplifi cation of the HER2 gene occurs in about 30% of breast cancers and causes the cancer cells to grow and divide rapidly. 26 Patients with breast cancer are routinely tested for HER2 amplifi cation to deter- mine whether they are candidates for treatment with drugs such as trastuzumab or lapatinib that can turn off HER2 activity. Interestingly, diverse cancers can have the same genetic abnormalities. For example, some metastatic stomach or gastroesophageal junction cancers also have an amplifi cation of the HER2 gene and can thus be treated with the same HER2 -targeting drugs as those used in breast cancer. Thus, genetic mutations shared by tumors, irrespective of their tissue of origin, make it possible to treat vastly different can- cers with the same targeted drugs. These newer drug therapies are sometimes combined with radiation ther- apy and chemotherapy.

Thought Questions

11. What is the benefi t of testing for genetic abnormalities in cancers?

12. Why is it sometimes possible to treat diff erent types of cancer with the same targeted drug?

 230

PEDIATRIC CONSIDERATIONS Terri Kyle

BOX 7.2 Signs and Symptoms of Cancer in Children

Continued, unexplained weight loss

Headaches, often with early-morning vomiting

Increased swelling or persistent pain in bones, joints, back, or legs

Lump or mass, especially in the abdomen, neck, chest, pelvis, or armpits

Development of excessive bruising, bleeding, or rash

Constant infections

A whitish color behind the pupil

Nausea that persists or vomiting without nausea

Constant tiredness or noticeable paleness

Eye or vision changes that occur suddenly and persist

Recurrent or persistent fevers of unknown origin

OVERVIEW OF PEDIATRIC CANCER

Cancer in children differs from that in adults in that it is not often of epithelial origin, as it is in adults, and can- not be explained by environmental exposure. The most commonly occurring broad categories of cancer in chil- dren are of hematopoietic origin, followed by nervous system tumors and those of embryonic origin.26 Just as the origin of cancer in children differs from that in adults, the presenting signs and symptoms may differ27,28 (Box 7.2).

LEUKEMIAS

Leukemia is classified as either lymphocytic or myelogenous; each type may occur as either an acute or chronic form of malignancy. The leukemias account for at least a third of all childhood cancer, and ALL accounts for 75% of cases of childhood leukemia.29 Chronic leukemias occur less frequently in children. A primary malignancy of the bone marrow, leukemia, results in the normal bone marrow components being supplanted by abnormal white blood cells. The abnor- mal cells demonstrate a growth advantage over nor- mal cells. Rampant overgrowth of the abnormal cells causes displacement of other blood cells, which can result in pancytopenia leading to anemia and bleed- ing. The exact cause of leukemia remains unknown,

although numerous chromosomal and genetic abnor- malities have been identified in leukemic cells.30

ACUTE LYMPHOBLASTIC LEUKEMIA Risk factors for the development of acute lympho- blastic leukemia (ALL) include numerous genetic conditions, with Down syndrome being the most fre- quent. Ionizing radiation exposure is a known environ- mental risk factor.30 The presentation of ALL is usually nonspecific and may include intermittent low-grade fever, anorexia, malaise, fatigue, and irritability. Lower extremity bone pain may also occur. ALL may metas- tasize to the central nervous system, resulting in signs of increased intracranial pressure such as headache, vomiting, or vision changes. Genetic studies indicate that aneuploidy (having more or less than two copies of each chromosome), particularly trisomy of chromo- somes 4, 10, and 17, predicts likelihood of treatment success. On the other hand, children born with trisomy 21 (Down syndrome) are ten to 20 times more likely to develop ALL.31

ACUTE MYELOGENOUS LEUKEMIA Risk factors for the development of acute myelogenous leukemia (AML) are similar to those for ALL. Signs and symptoms are also similar, although in AML subcutane- ous hemolytic purpuric nodules (often termed blueberry muffin lesions) may occur.30

Source: From Feist P. Signs of childhood cancer. Pediatric Oncology Resource Center. http://www. ped-onc.org/diseases/SOCC. html#anchor75392.

Chapter 7 • Neoplasia 231

BONE TUMORS

In children and adolescents, bone tumors result in localized pain, which may be worse at night or with activity; tender soft tissue mass; and limp or movement limitations. 29 , 32 Osteosarcoma is an aggressive bone tumor affecting the long bones near the metaphyseal plate. It accounts for less than 2% of childhood cancer and is most frequently diagnosed in teenagers. 28 Ewing sarcoma is a small, round cell undifferentiated tumor that is believed to be of neural crest origin. Children with a small nonmetastatic Ewing sarcoma have a good prognosis, but if metastasis is present at diagnosis, the long-term survival rate is much poorer. 32

NERVOUS SYSTEM TUMORS

The second most frequently occurring type of cancer in children and adolescents is malignant brain and spi- nal cord tumors. 27 , 29 Exposure to ionizing radiation or certain inherited disorders may be risk factors for brain tumor development. Presenting symptoms of nervous system tumors are most often consistent with signs and symptoms of increased intracranial pressure resulting simply from tumor presence or blockage of cerebrospi- nal fl ow, or both.

There are more than 100 histological catego- ries of brain tumors. In children, medulloblastoma (primitive neuroectodermal tumor) and pilocytic astrocytoma are the most common, although several other central nervous system tumors may also occur. Medulloblastoma is an embryonic cerebellar tumor, diagnosed most often by the ages of 5 to 7 years, which can spread via cerebrospinal fl uid and can cause fourth ventricle obstruction. Cerebellar dysfunction is often present with this tumor. Astrocytoma also occurs most often in the cerebellar area. Histologically, in the com- pact area of the tumor, Rosenthal fi bers (condensed glial fi lament masses) are present. 33

NEUROBLASTOMA Neuroblastoma occurs only in children, usually younger than 10 years of age, with an average age at diagnosis

of 18 months. The tumor arises from primordial neural crest cells (neuroblasts) of the sympathetic nervous system. The tumors can develop in the adrenal medulla and sympathetic ganglia, and commonly have metas- tasized by the time of diagnosis. Neuroblastoma cells have gene and chromosomal alterations, in most cases involving the MYCN and ALK genes. Approximately half of all children who develop neuroblastoma before age 12 months will experience complete spontaneous regres- sion, whereas children diagnosed later are more likely to require treatment. The most disabling complications of neuroblastoma are spinal cord compression in up to 10% of patients, and a rare condition termed opsoclonus myoclonus syndrome . 34

RETINOBLASTOMA Retinoblastoma is a rare cancer that occurs only in children. It develops either as a hereditary disease, due to an abnormality of the RB1 gene, or sporadically (70% of cases). 29 , 35 Located on chromosome 13q14, the RB1 gene is responsible for encoding pRb, which is a tumor suppressor protein. In the heritable form, the RB1 gene mutation is inherited through germinal cells, with a second mutation occurring in somatic retinal cells. The noninherited type of retinoblastoma occurs as a result of two mutations in the somatic retinal cells. The tumor arises from the inner surface of the retina and then spreads into the retina, resulting in leukocoria—a white appearance to the red refl ex, commonly called cat-eye refl ex—which is most often fi rst identifi ed by the child’s parents. 35

WILMS TUMOR Wilms tumor usually presents in young children as a unilateral, painless abdominal mass that is most often initially observed by parents. 29 , 36 An embryonal malig- nancy of the kidney, it is thought to be due to a genetic predisposition to nephrogenic rests (fragments of embryonic tissue retained in the developed kidney). Rests that persist are thought to develop into Wilms tumor after undergoing further genetic mutation. Genes for Wilms tumor continue to be identifi ed. In addition to abdominal mass presence, some children may exhibit hematuria or hypertension. 36

 232

GERONTOLOGICAL CONSIDERATIONS Rita M. Jakubowski and Janet H. Van Cleave

Between 2015 and 2050, the segment of the U.S. popu- lation aged 65 years and older is projected to undergo rapid growth from nearly 48 million people to 88 million.37 As older adults carry a disproportionate share of the cancer burden in the United States, this increase has implications for cancer care. Adults aged 65 years and older currently make up 15% of the U.S. population, yet account for 53% of cancer diagnoses.37,38 As a result, the incidence of cancer in the United States has been projected to increase by approximately 45% between 2010 and 2030.39

To deliver appropriate care for older adults with can- cer, clinicians must have a fundamental understanding of the association between aging and cancer. This sec- tion describes the physiological processes of aging that may promote cancer development and implications for practice.

PHYSIOLOGICAL PROCESSES OF AGING THAT MAY PROMOTE CANCER DEVELOPMENT

Four processes of aging that may promote cancer devel- opment are (a) a favorable environment for cancer, (b) an accumulation of cellular mutations, (c) a decline in immune function, and (d) alterations in hematopoi- etic stem cells.

FAVORABLE ENVIRONMENT FOR CANCER Research supports the observation that cancer increases with aging,39–41 and in fact, its incidence increases exponentially beginning at approximately the midpoint of the life span.42 The mechanisms under- lying this association have not been fully determined. A number of explanations for increased cancer inci- dence with increased age are summarized in Figure 7.8 and include the following:

• Longer time of exposure to environmental and endogenous sources of DNA mutations, leading to accumulating mutations

• Decline in DNA repair mechanisms • Inevitable telomere shortening that may destabilize

DNA structure • Decreased immune surveillance • Increase in senescent cell number and progression

to the senescence-associated secretory phenotype that promotes chronic inflammation43

ACCUMULATION OF CELLULAR MUTATIONS As outlined earlier in this chapter, cancer originates from the mutation of DNA sequences in cells that reroute pathways regulating tissue homeostasis, cell survival, or cell death.44 These mutations may result in the acti- vation of oncogenes or the loss of tumor-suppressing proteins. Often multiple mutations must occur over many years before the cell actually becomes a cancer stem cell, thus explaining the increased incidence of cancer with aging. As we age, our cells are more likely to accumulate mutations and to develop one that trig- gers the development of cancer.45 When such mutations disrupt genes that regulate cell division and growth, the cells begin to grow uncontrollably. A few cells quickly multiply and then increase rates of cell division, even- tually becoming a tumor. These abnormal cells acquire phenotypes that increase their ability to proliferate, migrate, and colonize at abnormal sites within the body, to survive hostile tissue environments, and to escape immune system surveillance.

DECLINE IN IMMUNE FUNCTION (IMMUNE SENESCENCE) The immune system is a major defense mechanism against the development of cancer, monitoring tissue homeostasis to protect against invading pathogens and eliminate damaged cells.44 It performs these functions by:

• Eliminating or suppressing viral infections to pro- tect the host from virus-induced tumors

• Eliminating pathogens and promptly resolving inflammation to prevent an environment conducive to the development and growth of tumors

• Identifying and eliminating tumor cells by the recog- nition of specific antigens45

The thymus is the major site of T-cell development and maturation. A gradual decline in thymic output of T cells has been proposed as another aspect of the aging process that can aid the development of cancer through a decline in immune function (immunosenescence).46 The decline in functional immunity is not only more permissive to tumor formation but may also promote it by contributing to chronic low-level inflammation. Age-related declines in T-cell numbers and responsive- ness (described in Chapter 6 , The Immune System and Leukocyte Function) contribute to reduced immune surveillance. They help to explain the decreased

Chapter 7 • Neoplasia 233

ability of the elderly to resist infections to which they were not previously exposed, or to respond to the appearance of tumor antigens, as well as to respond adequately to reinfection or to retain memory for anti- gens expressed by relapsing tumors.

ALTERATIONS IN HEMATOPOIETIC STEM CELLS The unique ability of stem cells to proliferate, differen- tiate, and self-renew allows them to play a major role in homeostasis, replacing cells that are weakened or destroyed by aging. 47 This activity occurs through pre- cise coordination of signaling processes throughout the body. Aging brings a cascade of changes in homeo- stasis, including a decline in organ function that affects the hematopoietic system, primarily stem cells. To replace blood cells that are constantly being lost due to splenic destruction or tissue utilization, hemato- poietic stem cells continuously regenerate circulating cells of the blood and immune system throughout life. Hematopoietic reserves, however, are depleted during

aging, and their ability to renew deteriorates. The abil- ity of stem cells to differentiate into different cell types is also altered, with maintenance of myeloid cell pro- duction better than lymphoid cell production.

With aging, there is a decline in stem cell function- ing but not numbers. 48 Because of their long life span and ability to replicate, stem cells are subject to dam- age from both intracellular and extracellular sources. Intracellular sources include the oxidative chemical reactions occurring within the cell. Stem cells undergo repetitive DNA replications during their lifetime, and such repetitive replications can cause random errors. As a result, genetic damage occurs and may accumu- late. Instability of stem cells exists to a greater degree in bone marrow of older adults, which suggests a progressive decrease in DNA repair process. 49 Such a decrease or defect in this process may contribute to the increased incidence of leukemia and myelodysplastic syndrome (MDS) in older individuals.

Cancers of the hematopoietic system (leukemias) are thought to originate within the normal stem cell

Telomere Shortening DNA Repair

Immune Surveillance

Chromosomal Stability Host Resistance

Premalignancy

Time

Age

Malignancy

Microenvironment Imbalance

Apoptosis Resistance

Invasion Metastasis

Angiogenesis Initiation

Free Radicals

Oncogene Activation/Mutation

Tumor-Suppressor Gene Loss

Apoptosis Gene Loss

Carcinogens

Viruses Promotion

FIGURE 7.8 Increased cancer incidence with aging can be related to a number of changes across the life span. There are potentially multiple sporadic DNA-damaging exposures (free radicals, carcinogens, viruses), leading to altered expression of oncogenes, tumor suppressor genes, apoptosis genes; initiation of altered phenotype with increased cell proliferation; accumulation of additional gene alterations; promotion of the transition to malignancy with gradual acquisition of invasive and metastatic capacity; and tumor expansion later supported by angiogenesis—all of which are occurring against the natural backdrop of declining host resistance. When factors favoring malignancy outweigh factors inhibiting malignancy, cancer develops. Source: From Halter JB, et al. Hazzard’s Geriatric Medicine and Gerontology. 7th ed. McGraw- Hill Education. www.accessmedicine.com. All rights reserved.

234 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

through the acquisition of mutations, genetic alter- ations, and chromosomal translocations.50 Over time, these changes transform cells from normal to malig- nant. As previously discussed, the altered cells are able to escape apoptosis and have an endless ability to rep- licate. AML has an increased incidence among older adults and is an example of a cancer that is thought to originate from an accumulation of genetic mutations occurring over the lifetime of an individual.

AGING AND CANCER: PRACTICE IMPLICATIONS

Screening and treatment recommendations for cancer in older adults take into consideration the functional status of the individual, indications of frailty, and per- sonal preference. Frequency of mammography, cervi- cal cancer screening, and colonoscopy may be reduced after the age of 75, depending on an individual’s risk fac- tors and desire for testing. Similarly, frail older adults with reduced functional reserve may be at higher risk for delayed recovery or severe adverse effects after cancer surgeries and conventional chemotherapies. Altered liver and renal drug clearance must be assessed prior to chemotherapy initiation. For these reasons, careful evaluation and consultation with the patient and family are imperative in designing treatment strate- gies for older adults with a new cancer diagnosis.

Remarkable scientific advances over the past 30 years have generated new therapies that are chang- ing the landscape of cancer treatment. First, the grow- ing number of people with AML or MDS, in conjunction with improvements in transplantation science, has increased the use of allogeneic hematopoietic cell trans- plantation as a treatment in older adults. Allogeneic hematopoietic cell transplantation is a process whereby stem cells from either a related or an unrelated donor are infused into a patient with the goal of replacing the patient’s immune and hematopoietic systems. Data from 103 transplant centers between 2000 and 2013 show that the percentage of allogeneic hematopoietic

cell transplantations in adults aged 70 years and older increased from 0.1% of transplants in 2000 to 3.85% in 2013. Nearly 40% of this population was alive 2 years after transplantation.51 These findings suggest that, in the context of a growing older adult population, the use of hematopoietic cell transplantation in older adults will continue to increase. Furthermore, advances in hematopoietic stem cell transplantation have allowed the development of reduced-intensity conditioning as a preparative regimen prior to transplantation for some populations of patients. These regimens use less che- motherapy or radiation therapy, or both, than standard myeloablative conditioning regimens, thereby decreas- ing the potential for organ toxicity. This option has afforded older patients the opportunity for potentially curative therapy.

Second, myriad novel, targeted biological therapies to kill cancer cells are either being used in clinical prac- tice or are under development. One example of a novel targeted therapy is ipilimumab, an immune checkpoint inhibitor, which has demonstrated improvement in survival rates in patients who have undergone surgical resection for stage III melanoma. This therapy blocks CTLA-4 to augment antitumor immune responses.52 However, while proven to be beneficial, such drugs can induce varying degrees of adverse reactions ranging from rashes to pneumonitis.53

One of the major issues in the care of older adults with cancer is the ability of older adults to tolerate these novel therapies. Some evidence supports the con- tention that older adults tolerate these novel therapies as well as their younger counterparts.54 However, older adults are underrepresented in cancer clinical trials; thus, the data on the toxicities of targeted therapies experienced by this special population are limited.55 Because of this limited knowledge regarding the tox- icities of novel therapies in older adults, it is important that practitioners who care for older adults with cancer understand the physiological basis of these novel tar- geted therapies to enable early detection and interven- tion of treatment toxicities.

Chapter 7 • Neoplasia 235

Beth Boyer

Patient Complaint: “ About 2 months ago the skin on my chest felt itchy for several days. It started gradually, but after several days seemed persistent and wouldn’t go away. I performed a self-exam and noticed a new lump in the upper outer corner of my right breast that hadn’t been there before. I’m very concerned because of my family history of breast cancer.”

History of Present Illness/Review of Systems: Your patient is a 55-year-old postmenopausal African American woman who fi rst noticed itching of both breasts that began in the inframammary ridge area but extended to both breasts. The itching was not relieved with use of oral antihistamine and hot and cold packs. She does not have a visible rash or hives, and has noticed no change to the skin. She performed a breast self-examination and noted a lump in the superior right breast. She has not been on any hormone replacement and experienced menopause at age 50.

The review of systems is negative for fever and infections, vision or hearing changes, headaches, and memory loss. The patient reports no abdominal pain and no nausea, vomiting, diarrhea, or constipation. There are no enlarged lymph nodes and no new problems with bleeding or bruising, no back pain or bone pain, and no peripheral neuropathy. She continues to experience mild itching over her right breast without any rash or hives; however, she noted that this is much better than when she fi rst noticed the itching sensation. She reports no itching or skin changes anywhere else. She has no chest pain, palpitations, or dyspnea, and remainder of her review of systems is negative.

Past Medical/Social/Family History: The patient’s past medical history is notable for hypertension and hyperlipidemia. Obstetrical history includes gravida 2, para 2, and pregnancy number 1 at age 29 years; she breastfed both her son and her daughter. Her surgical history includes left knee surgery in 2008 and left axilla lipoma excision in 1992. Social history includes exercise one to two times per week (running) and alcohol intake of one glass of wine daily. She is a former smoker of one pack per month for 10 years; she quit 17 years ago. Oncological family history is negative for ovarian cancer but positive for breast cancer in

her sister at age 44, her maternal aunt at age 61, and her maternal grandmother in her 60s. There is no other contributory family history.

Physical Examination: Findings are as follows: temperature of 98.4°F, blood pressure of 110/76 mm Hg, heart rate of 84 beats/min, respirations of 14 breaths/min. Body mass index (BMI) is 30 kg/m 2 . In general, the patient appears well and in no apparent distress. Her heart rate is regular, lungs are clear, and abdomen is soft. Breast examination is performed with the patient sitting and supine. Examination of the left breast demonstrates no evidence of dominant mass, skin change, nipple discharge, or changes within the nipple–areolar complex on the left side. Examination of the right breast reveals a 2- to 3-cm dominant fi rm mass at the 12 o’clock position, with no overlying skin change, nipple discharge, or change the nipple–areolar complex. There is no supraclavicular or infraclavicular axillary adenopathy on either side.

Laboratory and Diagnostic Findings: Both the CBC with differential and the chemistry panel are normal. Bilateral digital diagnostic mammogram and static ultrasound images are obtained, as follows:

• Mammogram indicates that breast tissue is heterogeneously dense. In the left breast, there are no signifi cant fi ndings or changes. In the right breast at 12 o’clock, there is a suspicious mass, correlating with an area of palpable concern as indicated by a metallic marker. Spot compression views show a few punctate calcifi cations associated with the mass. There are no other suspicious fi ndings in the right breast; specifi cally, there is no mammographic abnormality in an additional area of palpable concern in the inferior right breast as indicated by a metallic marker.

• Static ultrasound images labeled right breast 12:30 , 5 to 6 cm from the nipple, demonstrate a suspicious solid mass with calipers demar- cating measurements of 12 × 18 × 14 mm. This correlates with both the palpable area in the superior breast and mammographic fi nding. There is no sonographic abnormality in the other area of palpable concern in the right breast 4 to 5 o’clock position.

CASE STUDY 7.1: A Patient With Breast Cancer

Beth Boyer

CASE STUDY 7.1:

(continued)

236 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Ben Cocchiaro

PRINCIPLES OF ASSESSMENT History and Physical Examination • Constitutional symptoms : weight loss, night

sweats, anorexia, fever, and fatigue • Organ-speci� c signs and symptoms :

❍ Lung: chronic cough, hemoptysis, chest pain, dyspnea, hoarseness

❍ Colon: change in bowel habits, hematochezia, change in stool caliber, bowel obstruction

❍ Pancreas: jaundice, abdominal pain, nausea, dark urine, hepatomegaly

❍ Breast: breast mass, nipple discharge (especially unilateral)

❍ Prostate: urinary retention, nodular, lumpy prostate on examination

❍ Skin: lesions with high potential for malignancy often feature asymmetry, uneven borders, multiple colors, diameter greater than ¼″, and change over time

• Vaccination history : The HPV and hepatitis B vaccines prevent cancers of the cervix and liver, respectively

Screening Tests • Age- and risk-appropriate cancer screenings : As

many neoplasms have long asymptomatic periods in which they are more easily treatable, screening programs have been developed for many of the most common cancers. These include cancers of the breast, colon, cervix, and lung. See recommendations from the USPSTF for more information.

Diagnostic Tools • Imaging studies : The diagnostic evaluation of

isolated masses frequently begins with ultrasonography. In contrast, patients with suspected neoplasia of unknown origin often undergo CT scanning of the chest and abdomen. PET imaging is a useful tool for identifying metastases as it highlights areas with high metabolic activity.

• Biopsy : Once a suspicious mass has been identifi ed, tissue must be obtained from the mass and sometimes from nearby lymph nodes in order to guide treatment. Genotyping and special histological stains for tumor cell surface proteins can identify the tissue of origin of a mass as well as guide therapy.

• TNM staging : To help in prognosis and treatment of most cancers, a standardized staging classifi cation has been developed based on characteristics of the primary tumor (T), lymph node (N) biopsies, and the identifi cation of metastases (M).

Laboratory Evaluation • Complete blood count and peripheral smear: useful

in the diagnosis of hematological malignancies • Speci� c tumor markers: Several dozen biomarkers

have been associated with various cancers, but lack of sensitivity and specifi city limits their utility to measuring therapeutic response or recurrence in previously diagnosed patients. PSA is a notable example in that while it is still used for prostate cancer screening, providers are encouraged to

BRIDGE TO CLINICAL PRACTICE

• Pathology ultrasound-guided needle core biopsy: Right breast, 12:30, 5 to 6 cm from nipple: Invasive ductal carcinoma, Nottingham grade 3 of 3; 1 cm in greatest dimension. Lymphovascular invasion is identifi ed.

• Immunohistochemical stains performed at an outside institution demonstrate that the neoplastic cells are immunoreactive for ERs and PRs (75% to 100% strong). The

HER2 (sometimes referred to as HER2/neu) immunohistochemical stain performed at the outside institution is negative (1+).

CASE STUDY 7.1 QUESTIONS • What consequences does this patient’s case

have for the daughter of the patient? Is there a rationale for genetic testing in this case?

• Research tamoxifen and describe the rationale for treating the patient with tamoxifen.

CBC, complete blood count; ERs, estrogen receptors; HER2, human epidermal growth factor receptor 2; PRs, progesterone receptors.

(continued)

Chapter 7 • Neoplasia 237

have in-depth discussions with patients regarding the risks of false-positive test results.

MAJOR THERAPEUTIC MODALITIES AND DRUG CLASSES Surgery: • Surgical removal of affected and adjacent tissue

❍ Surgery may be augmented by immediate analysis (frozen section) of the primary lesion and surrounding tissue to evaluate completion of removal of malignancy.

❍ PET scanning can also identify patterns of lymph node drainage to target lymph nodes to biopsy for signs of metastasis.

• Chemotherapeutic or radioactive (brachyth- erapy) beads are sometimes implanted within cancerous tissue.

Radiotherapy: • Taking advantage of cancer cells’ limited ability

to repair DNA damage compared with healthy cells, ionizing radiation is frequently used in cancer treatment to target tumors.

Chemotherapy drug classes: • Nucleic acid synthesis (DNA or RNA) inhibitors • Protein synthesis inhibitors • Microtubule inhibitors • Enzyme inhibitors (intracellular signaling cascades) • Immune checkpoint inhibitors (promote endoge-

nous immune attack) • Hormone receptor antagonists • Tumor-targeting T lymphocytes (CAR-T cells) .

CAR-T cells, chimeric antigen receptor–T cells; HPV, human papillomavirus; PSA, prostate-specifi c antigen; USPSTF, United States Preventive Services Task Force.

KEY POINTS

• The cell cycle is a series of events in the life of a cell in which cell components are grown and DNA is completely replicated, followed by mitosis: division into two daughter cells, each containing the identical genetic information as the parent cell.

• Cells spend varying amounts of time in G 1 (or

in the dormant state of G 0 ) before cell signals,

particularly levels of cyclin proteins, initiate entry into the cell cycle. After a number of divi- sions, cells may enter replicative senescence after which they will not reenter the cell cycle.

• Control of the cell cycle is dependent on tissue needs, but also on signals internal to the cell indicating that suffi cient supplies are available for DNA duplication and, later, that DNA repli- cation has produced a normal result. Cell cycle checkpoints assure cell readiness and healthy status prior to cycle progression.

• The protein p53 is a critical signal for blocking cell cycle progression when DNA damage has occurred.

• Cancer is a state in which cell proliferation proceeds uninhibited by the usual control mechanisms, producing tumors made up of progressively more abnormal cells.

• Malignant tumors are named by their tissue of origin and share the properties of invasion

(cell growth beyond usual tissue boundaries) and metastasis (ability of tumor cells to break off from the original site and travel through blood or lymph to distant organs).

• Cancer is a genetic disorder in which a single cell with a few gene mutations produces generations of cells that have progressive mutations favoring the development of uncontrolled cell division and, ultimately, invasion and metastasis.

• Proto-oncogenes may be activated into onco- genes (a gain-of-function mutation), stimulat- ing unregulated cell proliferation.

• Tumor suppressor genes may be turned off (loss-of-function mutation), reducing protec- tive reactions such as DNA repair and cell cycle arrest when DNA damage has occurred.

• The hallmarks of cancer include uncontrolled proliferative signaling, evasion of growth suppressors, genomic instability, telomerase activity, apoptosis evasion, angiogenesis pro- motion, epithelial–mesenchymal transition that contributes to invasion and metastasis, evasion of immune destruction, and metabolic alterations that promote cell survival.

• Cancer may be localized to a specifi c organ or may affect bone marrow blood cell precursors, producing leukemias and lymphomas. Whether localized or generalized, cancer can produce systemic changes and symptoms, including infl ammation, hypercoagulability, changes in

238 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

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Chapter 7 • Neoplasia 239

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THE CLINICAL CONTEXT

The immune system is exceedingly complex in its constituent cells, molecules, and signaling

pathways. Each major component of the immune system is critical for survival; immune activity protects against infections that would quickly be lethal without immune defenses and eliminates cells in the stages of cancerous transformation.

The most common and major disorders of the immune system are related to immune activity that exceeds physiological needs. Hypersensitivity in the form of allergies occurs in 10% to 20% of the population. The prevalence of allergies increased in the developed world from the 1960s through the early 2000s, after which it began to plateau. Allergies were less common in the developing world but are now increasing in prevalence. Autoimmune disorders affect 2% to 5% of peo- ple, with a similar pattern of greater prevalence in developed countries but a trend of increasing numbers of cases in the developing world. 1

Although less common than immune hyperac- tivity, disorders in which immune activity is below normal leave an individual susceptible to dangerous infections. In some individuals, immune activity is compromised to an extent that those affected are at risk for major illness or even death. Primary immu- nodefi ciency disorders, which generally present in childhood as a result of genetic mutations, are rare but very severe. In 2012, the U.S. prevalence of primary immunodefi ciency disorders was 126.8 per 100,000. 2 The most common immunodefi ciency disorder occurs secondary to HIV infection. In the United States, an estimated 1.1 million people were

living with HIV infection at the end of 2015, about 0.3% of the population. 3 Globally, in 2017, it was esti- mated that 36.9 million people were living with HIV, almost 0.5% of the population. 4

ROLE OF THE IMMUNE SYSTEM

The human body is warm, moist, and full of nutrients, with a stable pH—all optimal living conditions for many microorganisms. There are at least as many bacterial cells as human cells in the human body, as well as count- less viruses, protozoa, and fungi. Collectively, these organisms are known as the human microbiota , and for the most part they consist of commensal organisms that coexist with humans without causing overt disease— even providing physiological benefi ts such as protec- tion against pathogens and aiding digestion and nutrient absorption. However, the human body constantly faces attack by disease-causing microorganisms known as pathogens. Pathogens express virulence factors that allow them to outcompete their commensal counter- parts and, in doing so, cause damage and disease.

To defend ourselves against pathogenic microbial attack, and to maintain homeostasis with commensal organisms, humans have evolved a complex and highly orchestrated system of cells, tissues, and organs that are collectively referred to as the immune system . Cytokines are circulating protein molecules produced and secreted by immune cells to promote proliferation and activation of other immune cells. Among the cytokines, there are dozens of interleukins (ILs), so-named because they were originally identifi ed as secreted products of leuko- cytes (white blood cells). Cells of the immune system are equipped with cell-surface receptors and secreted

THE IMMUNE SYSTEM AND LEUKOCYTE FUNCTION

Jo Kirman and Raff aela Ghittoni

6

Copyright Springer Publishing Company. All Rights Reserved. From: Advanced Physiology and Pathophysiology DOI: 10.1891/9780826177087.0006

156 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

molecules that act as environmental sensors to detect and respond to foreign or non-self molecules and to molecules that are commonly associated with patho- gens. The potent immune response systems are held in check by many inhibitory signals and mechanisms, which prevent undue hyperresponsiveness.

In addition to external threats, the immune system is capable of responding to abnormal self molecules, such as mutated self-proteins expressed by tumor cells, or organs received from a donor in the case of transplan- tation. Therefore, the immune system is a multifaceted defense force that serves to protect the human body from infection, toxins, cancer, and any tissue that is detected as an invader or non-self. The cells of the immune sys- tem circulate in the bloodstream to the body tissues and via the lymphatic system back to the bloodstream, con- ducting constant surveillance and attacking invaders.

Like other body systems, in some individuals, the immune system fails to function normally. This can occur from inherited or spontaneous mutations in crit- ical genes encoding molecules of the immune system, or it can be acquired from infectious or environmental exposures. Failure of the immune system to function appropriately can result in aberrant immune responses to harmless molecules in the environments (such as food or pollen, causing allergy or hypersensitivity) or to self-molecules (leading to autoimmune disease). Alternatively, the impaired immune system might fail to respond to pathogens or fail to maintain homeosta- sis with harmless microbes or commensal organisms, which can lead to severe disease (immunodeficiency).

This chapter summarizes the basic concepts of immunology, which is the biomedical science that stud- ies all aspects of the immune system: its cells, mole- cules, and functions in health and disease. Unlike the other organ systems covered in this text, tools to study the intricacies of immune cell function took many years to develop, and many aspects of immune function are still being elucidated. The complexity and rapid pace of discovery in immunology are exciting and clinically promising, even as they present challenges in staying current in this fast-moving field.

INTRODUCTION TO HOST DEFENSES

Similar to an army that defends its host country by establishing multiple specialized branches, each with highly trained soldiers and equipment, the immune system has three different layers of defense, each with its own specialized cells and molecules. These layers of defense are (a) the physical and chemical barriers, (b) the innate immune system, and (c) the adaptive immune system (Figure 6.1). General mechanisms of each of these layers are described in this section,

followed by more detailed descriptions of innate and adaptive immune cells and functions.

OVERVIEW OF PHYSICAL AND CHEMICAL BARRIERS The first layer of immune defense encountered by a pathogen comprises the physical and chemical barriers of the skin and mucosal surfaces (Figure 6.2).

• Skin covers the outside of the human body, with an outer layer of dead cells and underlying layers of densely packed living cells (the epidermis and dermis) that provide a strong physical barrier against penetra- tion by pathogenic microorganisms. Regular shedding of the outer, dead cells of the epidermal layer further contributes to the physical removal of microbes.

• Skin also produces chemical defenses against inva- sion and colonization through the secretion of sebum (a low-pH, oily, waxy substance) and sweat (high in salt, with antimicrobial enzymes) by sebaceous glands and sweat glands in the dermal layer. Antimicrobial peptides secreted by the skin, such as cathelicidins, can be directly antimicrobial and also can activate production of other effector immune molecules. The acidic, salty environment, as well as the production of antimicrobial peptides and enzymes, function together to limit or prevent microbial growth on the skin.

Physical and chemical barriers

PATHOGENS Viruses, bacteria, fungi, protozoa, helminths

Innate immunity

Adaptive immunity

FIGURE 6.1 Levels of protection against microorganisms. There are three levels of protection against pathogens: physical and chemical barriers include surface protections intrinsic to skin and mucous membranes, innate immunity that is nonspecific and results in acute inflammation while also initiating the next step of protection, and adaptive immunity that is specific and long-lasting.

Chapter 6 • The Immune System and Leukocyte Function 157

• Mucosal surfaces line the cavities of the body that are exposed to air and ingested substances. In contrast to the skin, the outer layer of the mucosa is alive and contains specialized cells that pro- duce mucus, a thick, viscous fl uid that coats the outer layer of cells and functions to trap and expel microbes.

• Cells that line the respiratory tract have short hair- like projections on their surface called cilia . In

healthy individuals, the cilia beat in tandem to expel particles trapped in the mucus up and out of the upper and lower respiratory tract. Failure of the mucociliary transport system can lead to lung dis- ease, such as chronic obstructive pulmonary dis- ease, pneumonia, and increased risk of infection. Cigarette smoking impairs the structure and func- tion of cilia, contributing to the development of smoking-induced respiratory disease.

Hair shaft

Sebaceous gland

Blood vessels

Hair follicle

Mucus layer

Goblet cell

Basal cell

Basement membraneFibroblast Lamina propria

Cilia

Columnar epithelial cell

Nucleus

Blood vessel

Subcutaneous (hypodermis) adipose tissue

(a) Skin

(b) Mucosal membrane (respiratory mucosa)

Opening of sweat duct

Epidermis

Dermis

Sweat duct

Sweat gland

FIGURE 6.2 Physical barriers of skin and mucosa. These regions physically interact with substances and microorganisms in the environment and commensals.

158 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

• The antimicrobial enzyme lysozyme is abundant in mucus and tears, acting as a chemical barrier for mucosal surfaces. Lysozyme functions by disrupting the cell walls of certain bacteria, causing the bacte- rial cells to rupture and die.

The gastrointestinal tract has its own extensive immune system, summarized briefly here and described in more detail in Chapter 13, Gastrointestinal Tract.

The initial defenses of the immune system effectively parry a vast array of microbial assaults. Nonetheless, at times pathogenic microbes are able to bypass or avoid these physical and chemical barriers, taking advantage of damage or abrasions to enter through the physical barriers or by producing substances that allow them to resist chemical defenses.

OVERVIEW OF THE INNATE IMMUNE RESPONSE The second layer of immune defense is the innate arm of the immune system. The innate immune system comprises fast-acting cells and molecules that recog- nize common features of pathogens, rather than spe- cific types of pathogens.

• This broad response is orchestrated by circulat- ing white blood cells and tissue-resident scavenger cells, which act quickly to recruit additional immune cells to sites of infection and trauma using chemi- cal gradients and signals from molecules known as cytokines and chemokines. The innate immune cells become activated to ingest and destroy any invading microorganisms.

• Cells of the innate immune response are generated continuously from multipotent bone marrow stem cells and can rapidly proliferate in response to appro- priate signals of infection and pathogen invasion.

• Molecules of innate immunity include proteins of the complement cascade, a system that directly attacks and lyses bacteria, while promoting other aspects of innate and adaptive immunity. The timing of the cell and molecular innate responses is crucial to con- tain the replication of pathogens and to prevent the spread of the disease in the host.

Innate immunity is generally very efficient, and is not specific to a particular pathogen. Importantly, the cells of the innate arm of the immune response are able to interact and communicate with cells of the third layer of immune defense—the adaptive immune response (Figure 6.3).

OVERVIEW OF THE ADAPTIVE IMMUNE RESPONSE Although slower to respond than the innate arm of the immune system, the adaptive immune response is highly specific for a particular pathogen and qualita- tively and quantitatively improves during the course of the response. Upon resolution of the immune response,

a small number of these highly specialized cells are retained in the body and can persist for decades. Some of these cells, such as T-helper lymphocytes, are modu- latory and function to promote the activity of other cells. Other adaptive immune cells, such as B lymphocytes and cytotoxic T lymphocytes (CTLs), are effector cells that are directly involved in targeting and destroying antigens and foreign cells. Upon any future encounters, these memory cells are able to efficiently promote pathogen neutralization or elimination so quickly that there may not be clinical signs of infection. This remarkable feature of the adaptive response is known as immunological memory. Immunological memory by adaptive immune cells is the mechanism upon which vaccination is based.

• B and T lymphocytes mediate adaptive immune responses. Both B and T cells recognize highly spe- cific parts of pathogens, called antigens. B cells recognize free antigen located in extracellular com- partments of the body, whereas T cells can recognize both intracellular and extracellular peptide antigen but require other cells to present the peptides to them on cell-surface molecules called the major his- tocompatibility complex (MHC). A given B or T cell always only recognizes one type of antigen; there- fore, each B or T cell has a single specificity.

• When activated, B cells synthesize and secrete highly specific Y-shaped molecules called antibodies that can inactivate, neutralize, or label pathogens or tox- ins. B cells express the same antibody on their cell surface, which forms the antigen-recognition compo- nent of the B-cell receptor (BCR). Signaling a naive or antigen-inexperienced B cell through the BCR enables that B cell to become activated and produce

Antibodies

Macrophage

NK cell or ILC

DC CD8 T cell

CD4 T cell

B cell

Neutrophil

Granulocyte

AdaptiveInnate

Chemokines and cytokines

Antigen presentation

Co-stimulation

FIGURE 6.3 Cellular and molecular communication between innate and adaptive immune cells. Both arms of the immune system have signals that modulate the responses of the other. DC, dendritic cell; ILC, innate lymphoid cell; NK, natural killer (cell).

Chapter 6 • The Immune System and Leukocyte Function 159

antibodies. Activated B-cell clones start to divide, leading to clonal expansion . This expansion can result in many, many thousands of B cells, which all recognize and produce antibody for the same antigen. The antibody-mediated immune response is some- times referred to as the humoral immune response .

• T cells recognize antigen through highly specifi c receptors, known as T-cell receptors (TCRs), expressed on their cell surface. Although many cop- ies of the TCR are present on the surface of an indi- vidual T cell, the TCRs of that cell are all of the same specifi city. However, owing to the genetic variation introduced through somatic gene recombination events in the thymus, there are millions of different T cells present in the body, each with their own TCR specifi city and the capacity to proliferate upon rec- ognition of their specifi c antigen.

• There are two types of T cells: the CD4 T cell , which is sometimes referred to as a T-helper cell ( Th cell ), and the CD8 T cell , sometimes referred to as a CTL. CD4 T cells primarily function to produce cyto- kines, which can activate innate cells and support B-cell and CD8 T-cell responses. CD8 T cells func- tion to lyse cells infected by intracellular bacteria,

virus-infected cells, or tumor cells. CD refers to cluster of differentiation, and signifi es specifi c mem- brane proteins found on the surface of lymphocytes and many immune cells.

Although the innate and adaptive immune responses have distinct characteristics (summarized in Table 6.1 ), their functions overlap and are interconnected. The innate and adaptive immune cells cooperate through a mutual exchange of signals and mediators to provide effi cient protection from pathogens, toxins, and can- cer (see Figure 6.3 ). These processes are described in more detail in the next sections.

Thought Questions

1. What is the “big picture” of the role of the immune system in maintaining homeostasis?

2. What are the general principles involved in protection provided by the innate and adaptive immune systems?

TABLE 6.1 Characteristics of Innate and Adaptive Immunity

Feature Innate Adaptive

Pathogen recognition Broad—pathogen recognition receptors for: • PAMPs

❍ Pathogen surface markers: lipopolysaccharide, fl agellin, di- and tri-acyl-lipopeptides, peptidoglycan, zymosan, mannose

❍ Intracellular pathogen markers—dsRNA, ssRNA, unmethylated CpG, DNA

• DAMPs—tissue injury signals (sterile infl ammation)

Highly specifi c—epitopes of microorganisms, foreign proteins, modifi ed self-proteins, modifi ed self-cells

Initiation time Fast; minutes to hours Slow; days to weeks

Memory Absent or broad enhancement “ trained immunity ”

Specifi c enhanced (faster and better quality) responses to subsequent exposure

Diversity of response Low Extremely high; increases during the course of the response

Major molecules and mechanisms Physical and chemical barriers; antimicrobial molecules; phagocytosis; complement; cytokines; chemokines

Antigen-specifi c receptors: BCR and TCR, antibodies, cytokines, cytolysis

Major cell types Phagocytes (macrophages, dendritic cells); granulocytes (neutrophils, eosinophils, mast cells, basophils); innate lymphoid cells (NK cells and ILCs)

B and T lymphocytes

BCR, B-cell receptor; unmethylated CpG, dinucleotide cytosine-guanine sequences common to microbes; DAMP, danger-associated molecular pattern; dsDNA, double-stranded DNA; ILC, innate lymphoid cell; NK, natural killer; PAMP, pathogen-associated molecular pattern; ssRNA, single-stranded RNA; TCR, T-cell receptor.

160 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

FUNCTIONAL ANATOMY OF THE IMMUNE SYSTEM

The specialized organs and tissues of the immune system are termed lymphoid organs and lymphoid tissues. They are separated into primary and sec- ondary lymphoid organs and tissues based on their function (Figure 6.4). Primary lymphoid organs and tissues are the site of immune cell development and include the thymus, where T cells develop, and the bone marrow, where B cells develop. Both T and B cells, as well as cells of the innate immune system, develop from multipotent precursor stem cells that reside in the bone marrow. The secondary lymphoid organs and tissues are the sites of adaptive immune cell activation and include the lymph nodes and lym- phatic vessels, in which cells respond to damage and infection of the tissues, and the spleen, where immune responses to blood-borne pathogens are ini- tiated. The lymphatic network provides T and B cells

Thoracic duct

Spleen

Intestinal lymph nodes

Peyer’s patches in intestinal wall

Tonsils

Cervical lymph node

Entrance of thoracic duct into subclavian vein

Axillary lymph node

Right lymphatic duct

Inguinal lymph nodes

Thymus

Bone marrow

FIGURE 6.4 Anatomy of the immune system. Cells of the immune system circulate from the bone marrow to primary and secondary lymphoid tissues, moving through blood and lymph vessels, and sometimes stationary in primary and secondary lymphoid tissues.

with the ability to recirculate through the tissues, lymph, and blood, allowing them to constantly patrol and survey the body for signs of infection.

HEMATOPOIESIS In humans, hematopoiesis occurs in the bone mar- row, where multipotent precursor stem cells, known as hematopoietic stem cells (HSCs), are supported to survive, proliferate, and differentiate by mesenchymal stem cells. HSCs are self-renewing, meaning that they can divide to make more precursor cells. The HSCs can develop into the major cellular constituents of blood: red blood cells, platelets, and white blood cells of the innate and adaptive immune system (Table 6.2).

The three blood lineages that develop from HSCs are the erythroid lineage, which develops into red blood cells (also referred to as erythrocytes) and platelet-pro- ducing megakaryocytes, and the myeloid and lymphoid lineages, which both can develop into white blood cells (also referred to as leukocytes). The myeloid lineage

Chapter 6 • The Immune System and Leukocyte Function 161

originates from a specialized progenitor cell derived from the HSC, known as the common myeloid progen- itor, which can differentiate into granulocytes (neutro- phils, eosinophils, basophils, and mast cells) as well as mononuclear cells, including monocytes, macrophages, and certain dendritic cells. The lymphoid lineage devel- ops from the common lymphoid progenitor and gives rise to T and B lymphocytes, natural killer (NK) cells, innate lymphoid cells (ILCs), and some types of den- dritic cell ( Figure 6.5 ).

CELLS AND TISSUES OF THE IMMUNE SYSTEM B Cells Develop in the Bone Marrow To recognize the vast array of different carbohy- drates and proteins expressed by different pathogens with a high degree of specifi city, an equally vast range of B cells, each with its own unique antigen specifi c- ity, must exist. As previously noted, B cells recognize antigen through their BCR. The BCR has the same structure and specifi city as the antibody it goes on to secrete during an immune response. Each B cell expresses ~10 5 BCRs on its cell surface, all with the same antigen specifi city. Therefore, billions of differ- ent B cells exist in the body, each able to respond to a unique specifi c antigen.

To encode such a vast range of different BCRs, the human genome, which encodes only ~20,000 proteins, would need to be orders of magnitude larger. To cir- cumvent this, the genes that encode the antigen-binding (variable) region of the BCR are expressed in segments ( Box 6.1 and Figure 6.6 ). Dozens of different options for each gene segment combine together in a kind of mix-and-match fashion to create the great BCR diver- sity required with very few genes. This process, called

somatic gene rearrangement , occurs in B-cell precur- sors in the bone marrow. Because the BCR has two chains, heavy and light, and each rearranges its genes encoding the variable region independently, this pro- cess increases the possibility of generating different unique antigen-binding sites on the receptor.

Gene rearrangement does not depend on antigen, so an antigen-specifi c B cell can develop well before exposure to a given pathogen. As the gene rearrange- ment process occurs, the new BCRs are tested. During development, B cells with nonproductive BCRs or self- reactive BCRs are deleted or made nonresponsive so that the B cells that survive are functional and unlikely to lead to autoimmune disease. Each day bil- lions of new B cells enter the circulation from bone marrow. Naive B cells survive for only a few weeks if they do not encounter antigen. Therefore, the process of developing new B cells continues in the bone mar- row throughout life.

T Cells Develop in the Thymus The thymus is a primary lymphoid organ critical for T-cell development early in life. At birth, the human thymus is fully developed; however, after 1 year of age, the thymus begins to involute (shrink) and its function reduces. Naive (antigen-inexperienced) T cells gener- ated by the thymus are long-lived or self-renewing in the periphery, meaning that loss of thymic function with age does not impair T-cell driven immunity over a lifetime.

Progenitor cells from the bone marrow enter the thymus, becoming thymocytes —cells that are com- mitted to the T-cell lineage. In a manner similar to the way B-cell precursors in the bone marrow rearrange the antibody variable genes, thymocytes in the thymus undergo a process of somatic gene rearrangement that leads to the development of an antigen-specifi c TCR.

TABLE 6.2 Concentration and Frequency of Cells in Human Blood

Cell Type Cells/mm 3 Total Leukocytes (%)

Red blood cells 5.0 × 10 6

Platelets 2.5 × 10 5

Leukocytes 7.3 × 10 3

Neutrophil 3.7–5.1 × 10 3 50–70

Lymphocyte 1.5–3.0 × 10 3 20–40

Monocyte 1–4.4 × 10 2 1–6

Eosinophil 1–2.2 × 10 2 1–3

Basophil <1.3 × 10 2 <1

Source: From Owen JA, Punt J, Stranford SA. Kuby Immunology. 7th ed. W.H. Freeman Company; 2013, Table 2-1.

162 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

The TCR is made up of two polypeptide chains: most commonly, the TCR α  chain and the TCR β  chain (Box 6.2 and Figure 6.7). Each chain has a variable region and a constant region. The variable domain includes the region that recognizes peptide antigens that are bound to MHC molecules. Similar to the gene segments that encode the antigen-binding regions of BCRs, the TCR variable region is also encoded by gene segments, and for each segment of the TCR there are multiple distinct alternatives encoded in the germline genome. In a developing T cell, one option for each gene segment is selected to recombine with the other selected segments to create a unique TCR. In this way,

enormous diversity in the TCRs expressed in the human body is achieved with a limited number of genes.

Each developing T cell will express multiple TCRs of a single specificity on its cell surface. Therefore, each T cell will recognize only one type of antigenic peptide. Most T cells will express an α β  TCR, which is made up of the α  and β  chains. However, a small number of T cells express a different type of receptor made up of γ  and δ  chains; these cells are referred to as γ δ  T cells. The γ δ  TCR is encoded by a different set of gene segments, and this dif- ferent type of TCR leads to different cellular functions.

Developing T cells commit to becoming a CD4 T cell or a CD8 T cell in the thymus, by expressing

After division, some cells remain stem cells (self-renewing)

Multipotent hematopoietic stem cell (hemocytoblast)

The remaining cell goes down one of two paths depending on the chemical signals received

Common lymphoid progenitor

Lymphoid stem cell

Common myeloid progenitor

Myeloid stem cell

Megakaryoblast Proerythroblast Myeloblast Monoblast

Reticulocyte

Erythrocyte Basophil

Mast cell Macrophage Dendritic cell

Neutrophil Eosinophil Monocyte

T lymphocyte

Lymphoblast

Natural killer cell (large granular

lymphocyte) or innate lymphoid cell (ILC)

B lymphocyte

Megakaryocyte

Platelets

FIGURE 6.5 Hematopoiesis. Generation of red and white blood cells occurs in the bone marrow. A multipotent stem cell can follow a myeloid or lymphoid path before further differentiating to final forms of the cells shown.

Chapter 6 • The Immune System and Leukocyte Function 163

either the CD4 or the CD8 co-receptor along with the TCR on their cell surface. Although these co-receptors do not directly bind antigen, they do bind to the MHC molecules that present the antigen to the TCR. Because CD4 T cells recognize only MHC class II and CD8

T cells recognize only MHC class I, the type of peptide antigens recognized by CD4 T cells and CD8 T cells is different. Once mature, CD4 and CD8 T cells go on to play distinct roles in the immune response, described later in this chapter.

BOX 6.1 B-Cell Receptor Gene Rearrangement

• B-cell receptors and antibodies are composed of two heavy (H) chains and two light (L) chains forming the shape of the letter Y ( Figure 6.6 ). The stem is the Fc (constant fragment) region, consisting only of heavy chain components and having consistent amino acid composition determined by an antibody’s class. The two antigen-binding regions (Fab) are made up of the terminal ends of heavy and light chains.

• The genes coding for these regions have multiple diverse segments (shown as different blocks within the gene’s diversity and variable regions)

that are shuffled during B-cell development. Unused portions of the genes are discarded, and the remaining portions are rejoined in a relatively random fashion, producing the rearranged DNA . Because of this gene rearrangement, proteins translated from the fi nal light and heavy chain genes are extraordinarily diverse.

• Each B cell recognizes its specifi c antigen out of millions of possible antigens, and the millions of B cells constitute a repertoire able to respond to most if not all possible antigens encountered throughout a lifetime.

C domain

V domain

V domain

Variable

VariableConstant

Constant

H

H

L

L

C domain

Diversity

Joining

Joining

Light chain

Fab region (antigen-binding)

Fc region (mediates biological activity)

Disul�de bond

Heavy chain

Antigen- binding

site

Germline DNA of a lymphoid progenitor cell

Rearranged DNA in a circulating B cell

Protein: Antibody (secreted antibody) or B-cell receptor (cell membrane bound)

FIGURE 6.6 B-cell receptor gene arrangement and antibody structure.

164 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

The gene rearrangement process enables the devel- opment of highly diverse TCRs to create a broad and rich repertoire of antigen-specific T cells throughout the body. This is important so that T cells are able to recog- nize the great diversity of antigens expressed by patho- gens to which the human body is routinely exposed. A cost of the gene rearrangement process is that some of the TCRs either will not be functional or will recog- nize and respond to self-antigen. Because recognition of self-antigen can lead to autoimmunity, the newly

expressed TCRs are tested in the thymus to ensure they are functional (a process termed positive selection), and they are tested for self-reactivity and deleted if they are activated by self-antigen (a process termed negative selection). Negative selection prevents the release of lymphocytes that could potentially damage body tissues and trigger an autoimmune response. Alternatively, some developing self-reactive T lymphocytes survive but become unresponsive (anergic) or differentiate into suppressive cells known as regulatory T cells (Tregs).

BOX 6.2 T-Cell Receptor Gene Rearrangement

• The process of gene rearrangement with variable and constant regions is similar between B cells and T cells. Most T-cell receptors (TCRs) are of the α β  type, where the gene coding for α  chains has several possible variable (V) and joining (J) regions and a constant (C) region, and the gene coding for the β  chains has V and J regions as well as diversity (D) regions (Figure 6.7).

• As with B cells, rearrangement of coding sequences for both α  and β  chains confers tremendous diversity of antigen-recognition sites of TCRs, and a broad repertoire of recognition sites for antigens encountered throughout the life span.

Germline alpha-chain DNA of a lymphoid progenitor cell

69

V�n V�2 V�1 J� C�

�

�

V�n V�1 D�1 J�1 C�1 D�2 J�2 C�2

Rearranged DNA in a circulating T cell

Protein: T Cell Receptor (cell membrane bound)

Rearranged DNA in a circulating T cell

Germline beta-chain DNA of a lymphoid progenitor cell

91

FIGURE 6.7 T-cell receptor gene rearrangement.

Chapter 6 • The Immune System and Leukocyte Function 165

B Cells and T Cells Circulate Through the Lymphatic System Lymphatic vessels drain interstitial fl uid and allow movement of white blood cells from the tissues. After entering these vessels, the fl uid is known as lymph. The fl uid moves through the network of lymph nodes that are located at the junction of lymphatic vessels, eventually returning to the blood via the thoracic duct (see Figure 6.4 ). Lymph fl ow is powered by muscle contractions during body movement, and backfl ow is prevented by one-way valves in the lymphatic vessels. Lack of movement or damage to the lymphatic vessels or valves can lead to accumulation of lymph in the tis- sues, known as edema. This type of swelling in the arms and hands is a common complication following radiation therapy or cancer surgery and is due to dam- age to the local lymphatic network.

The lymph node has a highly structured architecture that aids immune surveillance ( Figure 6.8 ). Cells and fl uid from the tissues enter the lymph node via afferent lymphatic vessels. Then, responding to chemical gra- dients, newly arriving T and B cells move into separate parts of the lymph node. Lymphoid follicles are predom- inantly populated with B cells, and as B cells respond and proliferate in response to antigen they form circular structures known as germinal centers within the B-cell follicle. The germinal center is highly dynamic, increas- ing in size as the immune response progresses and decreasing in size as the response resolves. This can lead to enlarged lymph nodes, which are common during infection, particularly under the chin (submandibular) or in the neck (cervical), armpits (axillary), and groin (fem- oral and inguinal lymph nodes). Discrete T-cell areas are

adjacent to the lymphoid follicles, allowing T and B cells to interact. Cells leave the lymph node to return to the blood via the efferent lymphatic vessel .

The Spleen The spleen is composed of red pulp, where damaged or senescent red blood cells are removed, and scattered nodules of white pulp, in which T and B cells respond to blood-borne antigen ( Figure 6.9 ). Like the lymph node, the white pulp of the spleen is organized into clear T- and B-cell areas, with T cells mainly located near the arteriole in the periarteriolar lymphoid sheath and B cells within the lymphoid follicle, adjacent to the T cells. A unique feature of the spleen is the presence of the marginal zone, which surrounds the white pulp and is bordered by the perifollicular zone. Resident within the marginal zone are specialized macrophages as well as innate-like B cells, known as marginal zone B cells, which are clonally distinct from conventional adaptive B cells. In humans, marginal zone B cells can circulate throughout the body, and they are associated with pro- tection from bacterial and viral infections, including HIV.

Following injury or rupture, the spleen may be removed to stem internal bleeding. Individuals whose spleen is removed, as well as those with the rare con- dition of congenital asplenia, are predisposed to the development of several bacterial infections. This sus- ceptibility highlights the critical function of the spleen in controlling blood-borne pathogens.

Gut-Associated Lymphoid Tissue The mucosal immune system, separate from the immune system surveying the blood, protects the surfaces where

Efferent lymphatic vessel (out to blood)

Lymphoid follicle (mostly B cells)

Afferent lymphatic vessel (in

from tissues)

Paracortical area (mostly T cells)

Germinal center

FIGURE 6.8 The lymph node. Lymph nodes are located along lymphatic vessel pathways from the tissues to the thoracic and right lymphatic ducts that empty into the systemic venous circulation. Within lymphoid follicles, T cells and B cells that are specifi c to the same antigen can be drawn together and provide co-stimulation that promotes development of mature lymphocytes, antibody production, and ultimately memory cells.

166 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

pathogens often invade: body orifices and surfaces that come in contact with the environment (eyes, nose, mouth, throat, gut, lungs, vagina, uterus). Here we high- light the highly developed gut-associated lymphoid tissue (GALT). GALT comprises three secondary lymphoid tis- sue types: the Peyer patches, the isolated lymphoid folli- cles, and the mesenteric lymph nodes. The GALT is the largest lymphoid organ in the human body, functioning to support tolerance to highly diverse commensal gut microorganisms, while maintaining the ability to respond to food-borne pathogens. As with the spleen and periph- eral lymph nodes, GALT tissues have a highly organized structure with distinct T- and B-cell zones. Outside the secondary lymphoid tissue, effector lymphocytes are scattered throughout the gut epithelium and lamina propria. In addition to their presence in the blood and lymphoid tissues, noted previously, cells of the immune system can be found throughout the body. Innate and adaptive immune cells are strategically positioned within the peripheral tissues, fat, and organs, as well as in skin and mucosal tissue, poised to combat invading patho- gens, neutralize toxins, and kill cancerous cells.

Thought Questions

3. What are the major tissues and organs of the immune system, and what is the role of each?

4. How does gene rearrangement endow B and T cells with the ability to recognize a wide variety of potential antigens?

Afferent splenic artery

Collecting vein

Venous sinus

Cords

Central arteriole

(a)

Follicle T-cell zone

Marginal zone

Perifollicular zone

Central arteriole

Central arteriole

(b)

Follicle

T-cell zone

Marginal zone

FIGURE 6.9 The spleen has both hematological and immunological functions. (a) The splenic artery enters the organ and branches into central arterioles. White-pulp areas represent lymphoid zones, containing a T-cell zone, B-cell follicles, and arterioles. The red pulp consists of cords transitioning into venous sinuses that drain to the splenic vein. (b) The white pulp.

INNATE IMMUNITY

Innate immunity immediately leaps into action when protective barriers such as skin and mucous membranes are breached. There are many different cells of the innate immune system, each with its own specialized function. A major function of the innate immune response is to vacuum up debris and pathogens in a process known as phagocytosis. The term phagocyte comes from the Greek words phagos meaning eat and cyte meaning cell ; therefore, in phagocytosis the cell ingests solid particles, such as microorganisms or cellular debris. Neutrophils, macrophages, and dendritic cells are all highly phago- cytic cells that help engulf and then destroy pathogens.

Various molecules—including those derived from arachidonic acid, cytokines, reactive nitrogen and oxygen species, proteases, and enzymes of the com- plement system—contribute to escalate the innate immune response and destroy invading pathogens to restore healthy tissue structure and function.

ACUTE INFLAMMATION Acute infl ammation develops as a consequence of innate immune activation. Mast cells, macrophages, and related sentinel cells detect tissue damage and invasion, rapidly initiating the infl ammatory cascade. Pathogen- specifi c signals activate tissue-resident innate cells and trigger components of the complement system, leading to the production of chemotactic signals that stimu- late the movement of leukocytes (mainly neutrophils and monocytes) from the circulation into the tissue at the site of injury or infection. Activated mast cells and

Chapter 6 • The Immune System and Leukocyte Function 167

macrophages at the site of injury or infection release chemical mediators, such as histamine, that increase blood vessel permeability and cause local blood vessels to dilate, increasing blood fl ow to the area. Endothelial cells lining the blood vessels upregulate membrane pro- teins that bind to leukocytes to assist their movement from blood to tissue. Once in the tissue, neutrophils and monocytes differentiate into a highly active state, with increased ability to phagocytose pathogens and generate additional infl ammatory mediators. Activated phagocytes kill microbes and contribute to escalating vasodilation, leukocyte recruitment, and infl ammation. Box 6.3 and Figure 6.10 visually summarize these steps in the acute infl ammatory response.

INNATE IMMUNE CELLS Innate immunity depends on circulating leukocytes and on sentinel cells within tissues that conduct ongo- ing surveillance for chemical signals of tissue damage and pathogen invasion. White blood cells include the granulocytes (containing cytoplasmic granules) and agranulocytes (see Table 6.2 ). Neutrophils, eosino- phils, and basophils are the granulocytes, in descend- ing order of abundance in the blood. Mast cells are similar to basophils in containing histamine granules, but they reside in tissues, rather than in the circula- tion. Agranulocytes have smaller granules, not visi- ble by light microscope. Monocytes and lymphocytes are the circulating agranulocytes. Monocytes con- tinuously leave the circulation and enter the tissues, where they enlarge and differentiate, becoming mac- rophages. Lymphocytes circulate between the blood, lymph vessels, and lymph nodes. The principal leuko- cytes that mediate acute infl ammation are neutrophils and monocytes/macrophages.

Neutrophils Neutrophils, also known as polymorphonuclear leuko- cytes (PMNs) for the multilobed shape of their nucleus, are highly motile and phagocytic and become activated quickly in response to tissue injury and infection. 5 They are by far the most abundant white blood cells circulating in the blood, representing 50% to 70% of all leukocytes.

A complete blood count with differential white cell count ( CBC with differential ) is the laboratory evalua- tion of absolute and relative numbers of leukocytes (see Table 6.2 ). In cases of acute infection, it is common to observe leukocytosis, an increase in white cell count, generally associated with an increase in neutrophil count. The neutrophil production rate can increase up to ten times in response to infection and the associated increase in colony-stimulating factors secreted by macrophages.

In severe acute infection, the blood smear composi- tion can reveal signifi cantly increased numbers and pro- portions of immature neutrophils. The relative increase in immature cells detected in the blood sample is the result of rapid movement of mature neutrophils into tissues,

and increased bone marrow production and release of immature neutrophils. The immature cells, called band or stab cells, are easily recognizable by the characteristic shape of their nucleus. Compared with mature neutro- phils, in which chromatin appears in three to fi ve clumps, the nucleus of immature neutrophils is less segmented, appearing as a single band across the cell.

The neutrophil life span averages 24 hours. After entering the circulation from the bone marrow, neu- trophils circulate in the vascular system for up to 12 hours before being recruited into tissues to respond to injury or invasion. Once in the tissue, the neutrophils conduct their job of phagocytosis of invaders before dying. Macrophages remove the cellular debris of dead neutrophils and other remnants of the infl ammatory response from tissues.

Neutrophils are highly phagocytic cells, able to ingest and kill thousands of bacteria before dying. They have two mechanisms of bactericidal activity: expo- sure of bacteria to toxic oxygen compounds known as reactive oxygen species (ROS), and degradation of bacteria by protease and other enzymes. Specialized neutrophil organelles and enzymes generate ROS as oxygen-derived free radicals via a burst of oxidative chemical activity. Together with ROS, phagocyte lyso- somes contain many proteases that are able to break down bacterial proteins. These chemicals are very effi cient in eliminating pathogens but are also highly toxic, potentially causing secondary tissue damage to the host. Consequently, the oxidative burst is limited to states of strong neutrophil activation resulting from the presence of bacteria or other alert signals. Moreover, the body has endogenous processes protecting against the ROS and the proteases released from neutrophils and macrophages. One example of these protective compounds is the enzyme α 

1 -antitrypsin, which coun-

teracts the action of particular proteases. An additional defense mechanism restricted to neu-

trophils has been recently identifi ed. When faced with overwhelming numbers of bacteria, neutrophils can combine and organize themselves into a neutrophil extracellular trap (NET). Activated neutrophils in the process of dying release their DNA into the extracellular space ( Figure 6.11 ). At this point, chromatin from many neutrophils starts to aggregate, generating a sticky trap for bacteria. Antimicrobial enzymes and ROS contained in neutrophil granules are also released in the extracel- lular compartment, targeting the entrapped bacteria for destruction. NETs are implicated in the pathophysiology of cystic fi brosis complications, preeclampsia, and some autoimmune diseases. 6

Basophils, Mast Cells, and Eosinophils Among the granulocytes, three other cell types are present in the blood or tissue: basophils, mast cells, and eosinophils. Basophils and mast cells are related nonphagocytic granulocytes. Although rare, basophils

168 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

BOX 6.3 Steps in the Acute Inflammatory Response

TISSUE DAMAGE ACTIVATES RESIDENT CELLS (FIGURE 6.10a)

• Trauma damages tissue and allows pathogen invasion, signaled by danger- and pathogen- associated molecular patterns.

• Mast cells and macrophages release histamine and cytokines, respectively.

• Vasodilation and increased vascular permeability begin, increasing blood flow and delivery of cells and proteins to the injured region.

WHITE BLOOD CELLS MOVE TO THE INJURED REGION BY CHEMOTAXIS (FIGURE 6.10b)

• Cytokines, chemokines, and complement protein fragments promote chemotaxis of neutrophils and monocytes to the injured region.

• Antibodies and complement proteins coat bacteria to aid phagocytosis.

• Increased capillary filtration promotes local swelling and edema formation.

• Neutrophils phagocytose bacteria, killing them via an oxidative burst that generates reactive oxygen species and via fusion with lysosomes containing degradative enzymes, particularly proteases.

INFLAMMATION CESSATION AND REPAIR INITIATION (FIGURE 6.10c)

• Macrophages engulf apoptotic neutrophils, cleaning up the injury debris, while releasing antiinflammatory mediators and cytokines to recruit fibroblasts and cells capable of rebuilding the tissue.

RESOLUTION AND RECOVERY (FIGURE 6.10d)

• Macrophage signals stimulate fibroblasts and endothelial cells to support regrowth of tissue and its vascular supply, restoring tissue integrity.

Vasoactive amines

cytokines

Cellular debris

Microbial pathogens

Mast cell

(a) Detection of danger

Tissue stromal cell

PRR

clot Platelet/fibrin

Inflammatory

Macrophage

Complement

Antibody Plasma exudate

Monocyte

Neutrophil

(b) Leukocyte recruitment and elimination of stimuli

Phagocytosis of opsonized pathogens

and cellular debris

Leukocyte recruitment

Macrophage

mediators Anti-inflammatory

(c) Resolution

Phagocytosis of apoptotic neutrophils

Macrophage

Angiogenesis

and fibroblasts

(d) Wound healing

Reepithelialization

Fibroblast

Collagen deposition

Macrophage

Growth factors for endothelial cells

FIGURE 6.10 Steps of acute inflammation: (a) detection of danger; (b) leukocyte recruitment and elimination of stimuli; (c) resolution; (d) wound healing. PRR, pattern recognition receptor.

Source: From Raynes JG. Inflammation: Acute. Wiley Online Library: eLS (Encyclopedia of Life Sciences). https://doi. org/10.1002/9780470015902.a0000943.pub3

Chapter 6 • The Immune System and Leukocyte Function 169

Histone modi�cations

allow chromatin unwinding

LPS TLR-4

Microbial entrapment Microbial killing

Antimicrobial peptides

Histones

Granule proteases

Extracellular trap formation

Proinflammatory effects (e.g., contact system activation)

Neutrophil activation

In�ammatory mediators

Microbial pathogens

ROS generation

“ETosis” cell death pathway

FIGURE 6.11 Neutrophil extracellular traps. (a) When neutrophils encounter large numbers of pathogens, they can respond by extruding their DNA and granules as a sticky net that traps and rapidly kills many bacteria at one time. (b) Electron micrograph of bacteria (orange) caught in neutrophil extracellular trap (yellow). Although effective at microbe removal, because this is an extracellular reaction, it has the potential to create secondary tissue injury. ET, extracellular trap; LPS, lipopolysaccharide; ROS, reactive oxygen species; TLR, toll-like receptor. Source: (b) From Max Planck Institute for Infection Biology.

(a)

(b)

170 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

circulate in the bloodstream, while mast cells reside in tissues, particularly in the connective tissue of skin, on mucosal surfaces, and adjacent to blood vessels. Basophils and mast cells contain granules of pro inflammatory chemicals such as histamine. Immediately after tissue injury or during the early phase of an allergic response, these cells degranulate, releasing histamine, a potent mediator of vasodilation. In an allergic response, prompt antihistamine adminis- tration can counteract the action of basophils and mast cells and reduce the systemic allergic reaction.

Eosinophils, like neutrophils, are phagocytic gran- ulocytes. Their cytoplasm is filled with granules con- taining toxic enzymes that are released by exocytosis onto specific targets. Eosinophil granules are capable of killing large targets, such as parasites (helminths), which are too big for phagocytosis. However, eosino- phils have a critical role in allergy and hypersensitivity reactions because they tend to participate in the late phase of allergic responses. In asthma, eosinophils in the lung contribute to airway hyperresponsiveness, mucus production, tissue damage, and airway remodel- ing, as described in Chapter 11, Lungs.

Monocytes and Macrophages Monocytes, macrophages, and dendritic cells repre- sent other groups of highly phagocytic cells among the agranulocytes. Monocytes generated in the bone mar- row enter the bloodstream where they circulate as an immature form of a macrophage. Monocytes migrate into tissues before differentiating into macrophages. In the tissue, monocytes increase their size and phago- cytic capacity. There are two types of macrophages. The first type operates beneath epithelial tissue and in several organs, patrolling as sentinels ready to ingest any microorganism able to get through the tissue. The second type circulates between lymph and blood- stream, conducting surveillance for any foreign mate- rial. Some cells of the macrophage lineage reside in specific tissue locations to exert their function and are identified by unique names (Table 6.3). For example,

macrophages found in the liver are known as Kupffer cells. In the central nervous system, microglia display macrophage functions. Synovial macrophages share similar activity in the joints, and alveolar macrophages are found in the lung airways. Macrophages are also located throughout the primary and secondary lym- phoid tissues, where they exhibit a range of different phenotypes and functions, depending on their specific location and local signaling mediators.

When infection or injury occurs, sentinel macro- phages detect cell damage and migrate to that area by chemotaxis, the movement of a cell toward or away from a chemical stimulus (see Figure 6.10). Phagocytes use pseudopods to move toward microorganisms or damaged cells to arrive promptly at the site of infection. Pseudopods are temporary cytoplasmic extensions, reaching out from the cell to allow movement. The term pseudopod is derived from the Greek words pseudes and podos, meaning false feet. Chemotactic substances that attract monocytes and macrophages include micro- bial products, components of damaged cells, chemi- cals released by other white blood cells, and peptides derived from the complement system. Upon activation, macrophages begin to secrete cytokines, particularly IL-1 and tumor necrosis factor (TNF), that contribute to chemotaxis, activate other leukocytes, and signal bone marrow to produce more leukocytes.

Dendritic Cells The dendritic cell is the most critical cell type for com- munication between the innate and adaptive arms of the immune response. Derived from either myeloid or lym- phoid precursors, dendritic cells are a diverse subset of immune cells located throughout the body, including the skin (Langerhans cells and dermal dendritic cells), pri- mary lymphoid tissues (e.g., follicular dendritic cells in the thymus), secondary lymphoid tissues (e.g., splenic or lymph node dendritic cells), and blood (Figure 6.12). Immature dendritic cells are highly phagocytic.

Upon maturation, which occurs following rec- ognition of broad features of pathogens or damage through receptors on their surface, dendritic cells lose their phagocytic abilities and instead focus on a process termed antigen presentation. Maturation triggers dendritic cells in the tissues to move to sec- ondary lymphoid tissues, where they activate cells of the adaptive immune response through antigen pre- sentation and co-stimulation.

Opsonization and Phagocytosis Neutrophils, macrophages, and dendritic cells are able to conduct phagocytosis of pathogens as one of their functions within immune protection. The first step in the process of phagocytosis is adherence of the plasma membrane of phagocytes to glycoproteins on the surface of the microorganism (Figure 6.13). To promote this first step, antibodies or complement proteins from the host coat the microbe surface, a

TABLE 6.3 Specialized Cells of the Macrophage Lineage

Location Cell Type

Lung Alveolar macrophage

Bone Osteoclast

Connective tissue Histiocyte

Liver Kupffer cell

Brain Microglia

Intestine Intestinal macrophage

Joint Synovial macrophage

Chapter 6 • The Immune System and Leukocyte Function 171

process termed opsonization. These proteins can then bind to receptors on the phagocyte cell, facilitat- ing adherence to the cell. The proteins that coat the microbe and thereby enhance phagocytosis are called opsonins .

The next steps include the formation of a pseu- dopod, which extends out from the cell, surround- ing the particle, and engulfi ng it. The cell membrane fuses together, forming the phagosome, a circular membrane-bound body fi lled with extracellular fl uid and the engulfed particles that is situated in the cytoplasm of the phagocytic cell. The phagosome then merges with lysosomes , small fl uid-fi lled vesi- cles that contain digestive enzymes, ROS, and other

antimicrobial substances. The resulting structure is called a phagolysosome . Digestion of most bacteria is complete within 10 to 30 minutes. After phago- cytosis, the remaining debris is eliminated from the cell by exocytosis , in which the phagolysosome fuses with the plasma membrane and expels its contents.

INNATE LYMPHOID CELLS A group of innate cells that derive from the lymphoid lineage were only recently identifi ed. Like CD4 T cells, their main function is cytokine production; however, unlike T cells, these cells do not express a genetically recombined antigen receptor. Known as ILCs, these

Epidermis

Dermis

Skin

Clec9A CD141

CD1a CD14

LC

Migratory DC

Migratory DC

Resident DC

Resident DC

pDC

Lymph nodes

Blood

Clec9A CD141

CD1a CD14 LC

Clec9A CD141

CD1c

Clec9A CD141

CD1c pDC

FIGURE 6.12 DCs are professional antigen-presenting cells (they express MHC class II proteins and can activate T cells) and are found in locations throughout the body. Resident DCs tend to remain in lymph nodes, while migratory DCs are able to circulate between tissues, blood, and lymph nodes. DCs, including LCs of the skin, are perfectly positioned to encounter antigens arriving by the cutaneous route. After engulfi ng the antigen or microbe, the DCs move to lymph nodes where they can interact with resident cells and lymphocytes newly arrived from the blood to activate the next steps of the immune response. DC, dendritic cell; LC, Langerhans cell; MHC, major histocompatibility complex; pDC, plasmacytoid dendritic cell; CD, cluster of differentiation; Clec, C-type lectin receptor.

172 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

cells do not express the lineage markers of other immune cells but instead respond to their local envi- ronment by recognizing cytokines, chemokines, and other chemical signals through receptors on their cell surface. In this way, they function to augment the non- specific reactions of innate immunity, providing broad protection against a variety of pathogens.

ILCs can be categorized according to their func- tions and cytokine expression (Figure 6.14). These include ILC1s, a group that includes cytotoxic NK cells; ILC2s, which are found in fat, liver, lung, and skin, are thought to be important for fighting helminth infection, and may mediate asthma and wound repair; and ILC3s, which are found in the gut and lung and may be important for maintaining homeostasis in mucosal tissues. The ILC3 group also includes lymphoid tissue inducer cells, which are critical for establishing sec- ondary lymphoid structures during development.7

NK cells were identified decades before the other ILCs, and are therefore much better characterized. NKs can recognize and directly kill cells that appear abnormal, such as virus-infected or tumor cells. Armed by a series of surface receptors and cyto- plasmic granules containing toxic substances, NK cells scan for the presence of unusual ligands on cell

plasma membranes. Ligand and receptor interactions can modulate NK cell effector properties. For exam- ple, the absence of normal MHC class I expression on the surface of a target cell, which can occur in virus-infected cells or certain tumors, can induce the activation program in NK cells.

Once activated, NK cells orientate and release their granules in the extracellular space toward the target cells. Granules contain mainly two substances: perfo- rin and granzyme B. Perforin creates a channel in the plasma membrane, altering the membrane integrity and inducing cytolysis of the target cell. Moreover, membrane pores allow the entrance of the second com- pound, granzyme B, a protease able to engage the apop- tosis pathway leading to cell destruction.

ACTIVATION OF THE INNATE INFLAMMATORY RESPONSE Recognition of Pathogens and Tissue Damage Unlike T cells and B cells, which recognize pathogens with great specificity, innate immune cells respond to pathogen invasion by recognizing molecules that are common to entire classes of pathogens. They are also able to sense tissue damage by recognizing molecules

Microbes are coated with antibodies and complement

Microbes

Phagocytic cell

Lysosome containing enzymes

Phagosome

1

1

Pseudopodia formation2

2

Engulfment3

3

Phagosome/lysosome fusion4

4

Killing and digestion5

5

Elimination (exocytosis)6

6

FIGURE 6.13 Phagocytosis. The steps in phagocytosis depend on the ability of professional phagocytes like macrophages to deform their plasma membrane to move toward a microbe by pseudopod formation, attach to the microbe (a process facilitated by opsonization), ingest the microbe, fuse the phagosome with an intracellular lysosome, and kill off the microbe.

Chapter 6 • The Immune System and Leukocyte Function 173

produced by damaged or stressed tissue. The innate cells sense these molecules through cell surface and cytoplasmic protein receptors called pattern recogni- tion receptors (PRRs).

Among the PRRs are several types of toll-like recep- tors (TLRs) that recognize common bacterial cell wall components such as lipopolysaccharide from gram-negative bacteria, viral or bacterial nucleic acids, and fungal carbohydrates ( Figure 6.15 ). The generic term for such ligands is pathogen-associated molecu- lar patterns (PAMPs). PRRs are found within and on the surface of macrophages and dendritic cells as well as epithelial cells. Local engagement of these recep- tors promptly stimulates phagocytosis and triggers the

secretion of other signaling agents, including chemo- kines and cytokines that mediate acute infl ammation and enhance antigen processing and presentation.

The cells of innate immunity can also recognize and become activated by signals of tissue damage, cellular stress, and cell death. These triggers can occur in the presence or absence of infection, and are referred to collectively as danger-associated molecular patterns (DAMPs). When tissue damage occurs without patho- gen invasion and infection (think of a bad ankle sprain that occurs without a break in the skin, or a myocardial infarction), the innate immune response is referred to as sterile infl ammation , and the DAMPs are the orig- inating signals. DAMPs are also recognized by PRRs,

Type 1

Intracellular microbes Transformed (tumor) cells

IL-12 IL-15 IL-18

NK ILC1 ILC2 ILC3

Granzymes Perforin

IFN-γ

Macrophage activation

Oxygen radicals Cytotoxicity

Result of activation

Effector cytokines

Tissue signals

Macrophage activation Oxygen radicals

Vasodilation Mucus production Extracellular matrix

production/tissue repair

Antimicrobial peptides Phagocytosis

Tissue preservation

IFN-g

TNF-a

IL-4 IL-5 IL-13 Areg

IL-17 IL-22

LT-a1b2 GM-CSF

Allergens Large parasites

Tissue injury

IL-25 IL-33 TSLP

Extracellular microbes: Bacteria

Fungi

IL-1b

IL-23

Type 2 Type 3

FIGURE 6.14 Innate lymphoid cells. The role of NK cells in innate immunity has long been recognized, particularly their ability to recognize and kill virus-infected cells. Recently, additional lymphoid cell types were found to contribute to innate immunity, reinforcing the work of the myeloid cells, which are the workhorses of acute infl ammation. Areg, amphiregulin; GM-CSF, granulocyte/macrophage colony-stimulating factor; IFN-γ , interferon gamma; IL, interleukin; ILC, innate lymphoid cell; LT-α 

1 β 

2 , lymphotoxins alpha-1, beta-2; NK,

natural killer; TNF-α , tumor necrosis factor alpha; TSLP, thymic stromal lymphopoietin.

174 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

including TLRs and an intracellular multiprotein com- plex called the inflammasome.

The inflammasome is able to recognize PAMPs as well as DAMPs. The inflammasome is an essential medi- ator of innate immunity and inflammation by activating caspase-1 and the cytokines IL-1 and IL-18. Secretion of these cytokines can lead to activation and cytokine secretion by other innate cells, including ILCs and NK cells, as well as cell death of infected cells.

SEQUENCE OF INFLAMMATION AND INFLAMMATORY MEDIATORS Initiation of the innate immune response triggers inflammation. The inflammatory response can be local and confined, or systemic, involving the entire body (e.g., fever, leukocytosis). The cardinal signs of inflam- mation are redness in the area, swelling, pain, and heat—and in some cases, loss of function.

Acute inflammation occurs in order to destroy any injurious agent causing the tissue damage, while simul- taneously preventing spread of the infectious agent,

limiting its effects on the body, and later leading to tissue repair (see Figure 6.10). During the first stages of inflammation, PAMPs and DAMPs activate endo- thelial cells, ILCs, and tissue-resident sentinel macro- phages to release early phase, local proinflammatory chemicals. Histamines, prostaglandins, kinins, and leu- kotrienes cause local vasodilation, increase capillary permeability, and stimulate pain fibers. Macrophages release cytokines such as IL-1 and TNF locally and to the circulation, stimulating the bone marrow to pro- duce more leukocytes and the liver to secrete another class of proinflammatory substances, known as acute phase proteins. These acute phase proteins include C-reactive protein (CRP) and serum amyloid A protein. Concentrations of both proteins increase in the blood by several hundredfold during inflammation or tissue damage, and therefore can be used diagnostically as an indicator of infection or inflammation. Another import- ant acute phase protein is mannan binding lectin (MBL), which can bind to mannose sugars, found in abundance on some microbial pathogens, particularly yeasts.

GPI anchor Zymosan

LTA

Diacyl lipopeptides

TLR6

Cytosol

TLR2 TLR1 TLR2 TLR4 TLR5

TLR3

CpG DNA

Endosome

TLR7

TLR8

TLR9

Triacyl lipopeptides LPS Flagellin

dsRNA

ssRNA

Parasite

Virus

Bacteria

Bacteria

Yeast

FIGURE 6.15 TLRs are a subset of pattern recognition receptors that mediate pathogen recognition in the innate immune system. There are many types and combinations of TLRs, as shown here. Many TLRs recognize components of bacterial, yeast, or parasite cell membranes; other TLRs are found on endosomes and recognize components of invading bacteria and viruses. This is a nonselective way of distinguishing between self and non-self that characterizes innate immunity. CpG, pathogen DNA sequence of unmethylated CG dinucleotides recognized by TLR9; dsRNA, double-stranded RNA; GPI, glycosylphosphatidylinositol of protozoal membranes; LPS, lipopoly- saccharide; LTA, lipoteichoic acid of gram-positive bacteria cell wall; ssRNA, single-stranded RNA; TLR, toll-like receptor. Source: From Zaru R, European Bioinformatics Institute EMBL-EBI, UK. Pattern recognition recep – tors (PRRs): toll-like receptors. © British Society for Immunology. https://www.immunology .org/public-information/bitesized-immunology/receptors-and-molecules/pattern-recognition -receptors-prrs.

Chapter 6 • The Immune System and Leukocyte Function 175

These proinfl ammatory chemical mediators induce systemic responses, including fever, anorexia, hypo- tension, increased heart rate, and the release of the stress hormone cortisol. Histamine and other proin- fl ammatory molecules cause local vasodilation and increased vascular permeability, facilitating migration of cells and molecules out of the bloodstream and into the infl amed area. This increased blood fl ow and local swelling results in redness, heat, and pain associated with infl ammation. At the same time, if there is damage to blood vessels, other components of the blood lead to activation of the coagulation system , which drives the formation of blood clots. The blood clots act to seal off the area, preventing the infection from spreading.

Chemotactic infl ammatory mediators attract circu- lating neutrophils and monocytes. In response, these leukocytes adhere to blood vessel walls through a pro- cess called margination ( Figure 6.16 ). IL-1 and TNF

released by macrophages cause the endothelial cells to upregulate cell membrane proteins called selectins. Selectins have affi nity for selectin ligand proteins on the surface of neutrophils. Neutrophils become attracted and loosely attach to the vessel wall, rolling along the surface of the blood vessel. Neutrophils upregulate a membrane protein called integrin, which can bind to integrin receptors also upregulated on the endothelial cells. This interaction provides a tight connection, so the neutrophil stops rolling and becomes fi rmly attached to the side of the blood vessel. The adhered neutrophils squeeze through the epithelial cells in a process called diapedesis to reach the site of infl ammation.

After leaving the vessel, neutrophils and mono- cytes, which mature into macrophages or infl am- matory dendritic cells, rapidly engulf microbes by phagocytosis and destroy them through intracellu- lar killing mechanisms, such as ROS production and

Random contact

Rolling

Adherence

Chemotaxis and diapedesis

PECAM-1

PECAM-1 ICAM-1 VCAM-1

Selectin ligand

Blood

Tissue

Selectins

Chemoattractant secretion

sLexIntegrin

CD11/ CD18

Endothelial cell

FIGURE 6.16 Margination and diapedesis of neutrophils. Neutrophils are the most numerous and active cells responding to a penetrating injury with bacterial invasion. Chemotactic signals generated in injured tissue upregulate selectins on vascular endothelial cells, promoting binding of selectin ligands on neutrophils. The neutrophils begin to roll along the blood vessel wall, responding to the chemotaxic compounds by upregulating additional adhesion molecules that allow fi rm sticking to endothelial cell adhesion molecules ICAM–1 and VCAM-1. Finally, neutrophils activate their membrane adhesion molecule, PECAM-1, and begin to move out of the blood into the tissue by squeezing between endothelial cells (diapedesis). ICAM-1, intercellular adhesion molecule 1; PECAM-1, platelet-endothelial cell adhesion molecule 1; sLex, sialyl Lewis X motif; VCAM-1, vascular cell adhesion molecule 1.

176 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

release of lysosomal enzymes. Antigen-presenting cells (APCs), such as dendritic cells, traffic to the local draining lymph nodes, where they initiate the adap- tive immune response through antigen presentation. Other leukocytes begin to arrive at the inflammatory site and start producing their mediators, including arachidonic acid metabolites (prostaglandins) and cytokines, which amplify the inflammatory response in a cascade effect. After engulfing large numbers of microorganisms and damaged tissue, the phagocytes die. In some cases, dying phagocytes form pus; in other cases, significant numbers of phagocytes (par- ticularly neutrophils) die and release their content of killing mediators, including ROS and proteases. This can cause a second wave of tissue injury, as seen in reperfusion injury after myocardial infarction. The principal cell types and mediators of inflammation are summarized in Table 6.4.

Resolving an Inflammatory Response The final stage of inflammation is tissue repair. After an inflammatory event or tissue injury, innate and adap- tive immune cells as well as nonhematopoietic cells, including fibroblasts, epithelial cells, and endothelial cells, work together to resolve inflammation and initi- ate and regulate the wound-healing response. Ideally,

this leads to restoration of normal tissue architecture; however, aberrant wound-healing responses can yield fibrotic and scarred tissue that interferes with normal function or leads to development of chronic wounds. Macrophages can be subdivided into two types, one of which is acutely activated and proinflammatory, secret- ing proinflammatory cytokines such as IL-1 and TNF-α ; the other type is antiinflammatory, secretes IL-10 and transforming growth factor-β (TGF-β ), and participates in wound healing.

As inflammation resolves and progresses to the stage of wound healing, activity of antiinflammatory macrophages increases, resulting in increased levels of platelet-derived growth factor (PDGF), vascular endo- thelial growth factor alpha (VEGF-α ), and TGF-β 1. The cytokine IL-13, produced by other types of innate cells and certain T cells, also can promote TGF-β  production. Together, these growth factors promote cell proliferation and development of blood vessels. Macrophages also produce chemokines to recruit fibro- blasts and myofibroblasts to mediate wound closure, and additionally regulate the balance of matrix metallo- proteinases (enzymes that control extracellular matrix turnover and fibrosis). Wounds characterized by per- sistence of proinflammatory macrophages producing IL-1 and TNF are associated with impaired or delayed

TABLE 6.4 Inflammatory Mediators

Class of Mediators Principal Sources Major Functions

Vasoactive amines (histamine, serotonin) Mast cells, basophils, platelets Histamine: vasodilation, increased capillary permeability Serotonin: vasoconstriction

Kinins (bradykinin, kallikrein) Liver Vasodilation, increased capillary permeability, pain, coagulation

Complement Liver Microbe opsonization, chemotaxis, bacterial killing via membrane attack complex

Arachidonic acid metabolites (prostaglandins, leukotrienes, lipoxins)

Leukocytes, endothelial cells Proinflammatory—vasodilation, increased capillary permeability, pain Anti-inflammatory (lipoxins)

Reactive oxygen species (superoxide, hydrogen peroxide, hypochlorous acid)

Neutrophils and macrophages Bacterial killing, release into tissues perpetuates injury

Lysosomal degradative enzymes (proteases, lysozyme)

Neutrophils and macrophages Bacterial killing, release into tissues perpetuates injury

Acute phase proteins (C-reactive protein, serum amyloid A)

Liver Markers of inflammatory response, inhibit pathogen growth, proinflammatory

Cytokines and chemokines Leukocytes Proinflammatory: IL-1, IL-6, TNF, IL-18, others Antiinflammatory: IL-10, TGF-β  Chemokines: CCL and CXL families— chemotaxis and co-stimulators

CCL, CC-motif chemokine ligand; CXL, CX-motif chemokine ligand; IL, interleukins; TGF-β , transforming growth factor-beta; TNF, tumor necrosis factor .

Chapter 6 • The Immune System and Leukocyte Function 177

tissue repair after injury, infection, or sterile infl amma- tion, leading to chronic infl ammation.

THE COMPLEMENT PATHWAY The complement pathway is a series of proteins that are produced by the liver, and can be found through- out the body in extracellular fl uids, lymph, and blood ( Box 6.4 ). Many of the complement proteins are pro- teolytic enzymes that exist in an inactive state, similar to proteins of the coagulation cascade (see Chapter 8, Blood and Clotting). When activated by signals of pathogen invasion, complement proteins initiate a cas- cade of activation whereby one enzyme cleaves and activates the next in the series.

There are three initiating pathways for the comple- ment cascade. In the classical pathway, either anti- body (produced by B cells or plasma cells) or the acute phase protein CRP binds to a pathogen. The antibody- or CRP-bound bacterium then interacts with the C1 protein complex triggering its activation and releas- ing an active protease. The active protease cleaves C2 and C4, generating C2a and C4b ( Figure 6.17 ). C2a and C4b combine to form C4bC2a , also known as a C3 convertase . This is the point in the cascade at which C3 is cleaved to produce C3a and C3b.

The main pathway triggering the complement cas- cade early in infection is the alternative pathway. This pathway is initiated as a result of the ability of C3 and protein B to bind to the surface of the invading microbe, leading to cleavage of C3 and protein B, with formation of an active and potent C3 convertase known as C3bBb . Thus, activation on the bacterial surface of a small amount of C3 and factor B to C3bBb can trigger the activation of greater numbers of C3 molecules.

The most recently discovered pathway of initia- tion of the complement cascade is the lectin pathway.

Mannan-binding lectin (MBL) is an acute phase protein produced by the liver during an infl ammatory response. MBLs circulating in plasma bind to mannose sugars on the surface of the pathogen. This binding triggers the MBL polyprotein to become enzymatically active and able to cleave C4 and C2 to form the C4bC2a C3 con- vertase as seen in the classical pathway. It is here that the pathway converges with the alternative and classical pathways, and complement effector functions are elic- ited by the C3 cleavage products: C3a and C3b. The com- plement cascade interacts with both innate and adaptive immunity, with three main outcomes ( Figure 6.18 ):

1. Cleaving larger precursor proteins C3 and C5 gener- ates smaller proteins, C3a and C5a, that are potent infl ammatory signals. They are also known as ana- phylatoxins because they promote mast cell degran- ulation with histamine release. C5a is particularly effective at promoting leukocyte chemotaxis.

2. C3b is an opsonin that binds to bacterial cell walls to promote effi cient phagocytosis by neutrophils and macrophages. C3b also promotes B-cell antibody production.

3. The fi nal steps of the complement cascade on bacte- rial surfaces involve sequential activation of comple- ment proteins 5, 6, 7, and 8, leading up to insertion of several copies of C9 into bacterial membranes, forming the membrane attack complex (MAC). MACs form cylinder-shaped large pores in the cell wall or membrane of the invading pathogen, which allows water to move into the pathogen, causing swelling and resulting in cell death by cytolysis. Gram-negative bacteria have two membrane layers separated by only a thin peptidoglycan layer and are more susceptible to cytolysis than are gram-positive bacteria.

BOX 6.4 Overview of the Complement Pathway

• Complement proteins are designated with an uppercase C and are numbered from 1 to 9, based on order of discovery rather than the order in which they act. Complement proteins that are activated by cleavage produce two subunits that are indicated with lowercase letters a or b .

• There are three different ways in which the complement system can be activated: the classical, alternative, or lectin pathways. All three pathways directly or indirectly lead to death of the invading pathogen (see Figure 6.17 ).

• Although they are initiated in different ways, all three pathways converge at the cleavage of C3 into C3a and C3b (see Figure 6.18 ). From this point, the complement pathways lead to the outcomes of (a) recruiting inflammatory cells to the site of infection; (b) labeling or opsonization of pathogens for enhanced uptake and destruction by phagocytic cells; or (c) formation of the membrane attack complex (MAC) , which forms pores in pathogen membranes.

178 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

The complement cascade is the target of regulatory proteins that suppress activation in the absence of pathogen invasion. In addition, activated complement proteins are quickly degraded to prevent excess tissue damage. Mutations in complement genes may predis- pose to greater incidence of infection. Notably, defi- ciencies in C5 to C9 result in exquisite susceptibility to Neisseria spp., some of which cause life-threatening invasive meningococcal meningitis.

INNATE RESPONSES TO VIRAL INFECTION Viruses are exclusively intracellular pathogens. After gaining access to the body via respiratory, gastroin- testinal, and other routes, viruses use the host cell machinery to conduct replication, transcription,

and translation of viral DNA and RNA. During viral infection, viral nucleic acid can be detected in the cytoplasm by cytosolic PRRs. Binding to intracellu- lar PRRs—RIG-I-like receptors (RLRs), nucleotide- binding domain-leucine-rich repeat-containing molecules (NLRs), or cytosolic DNA sensors such as cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS)—leads to the production of type I interferons (IFNs) and other proinflammatory cytokines. Type I IFNs are a group of structurally related cytokines that induce antiviral responses within infected cells and alert neighboring cells to upregulate molecules that antagonize virus replication. Produced early during viral infection, type I IFNs are also essential for activating NK cell effector functions.

Classical pathway

Ag-Ab

C4b2a

C3 convertase

C3a

C5

C3b

C3

Microbes C3b

C3Bb

CFB

C3 convertase

C5 convertase (C3b2Bb)

C5b

Cell lysis

Anaphylatoxins

MAC

C9

C8

C7

C6

C5b-9

C5a

C4

MASP-1

C1

C4, C2

MASP-2

Lectin pathway Alternative pathway

MBL

FIGURE 6.17 Pathways of complement activation. In the classical pathway, bacteria are coated by antibodies that attract and activate the complement cascade. In the alternative pathway, complement binds to the bacterial wall itself, initiating its own cascade. The lectin pathway depends on MBL (a circulating protein synthesized by the liver) to attach to the bacteria before activating the complement cascade. Ag-Ab, antigen-antibody; CFB, complement factor B; MAC, membrane attack complex; MASP, mannan-binding lectin-associated serine protease; MBL, mannan-binding lectin.

Chapter 6 • The Immune System and Leukocyte Function 179

Upon binding their cell-surface receptors, type I IFNs stimulate expression of more than 300 genes (known as IFN-stimulated genes), which leads to inhi- bition of viral replication, assembly, and release, as well as promoting adaptive immune responses.

CHRONIC INFLAMMATION Cancer, autoimmunity, allergy, obesity and meta- bolic dysfunction, infection, genetic diseases, aberrant responses to the microfl ora, and aging can all lead to chronic infl ammation. While acute infl ammation is short lived, chronic infl ammation occurs when an infl am- matory response fails to resolve, leading to sustained

infl ammation that lasts months or even years. Chronic infl ammation heightens the risk of developing many harmful diseases, including cardiovascular disease, chronic kidney disease, cancer, and even depression.

Infl ammatory responses typically resolve when neu- trophil recruitment to the tissue stops, and those neu- trophils present in the tissue die and are engulfed by phagocytic macrophages. The local macrophages then adopt a wound-healing, antiinfl ammatory phenotype. Other cells, including regulatory or IL-13–producing T cells, also contribute to the resolution of infl ammation.

Multiple mechanisms can lead to failure of the antiin- fl ammatory response, resulting in chronic infl ammation.

C3

C3a

C3b

C5a

C5b

C3a

C3b

Microbe

MicrobesC3a

C3a

C3b

C5

C5a

C5a

C5b

C5

C6

C7

C8

C9

Splits into activated

C3a and C3b

(c)

(b)

(a)

Channel

C3b protein

C3a receptor

C5a receptor

Mast cell

Histamine

Phagocyte

Phagocytes

The complement proteins are assembled into a membrane

attack complex. This pore pierces the bacterial cell wall and allows

water entry that causes the pathogen to burst.

C3b is able to serve as an opsonin, promoting phagocytosis

of pathogens.

Capillary permeability increases, promoting phagocyte chemotaxis.

1

1

2

2

21

3

FIGURE 6.18 Mechanisms of complement effector functions diverge after C3 splits (step 1). (a) C3a and C5a are small fragments (anaphylatoxins) that promote histamine release and phagocyte chemotaxis (step 2). (b) C3b fragments opsonize (coat) microbes (step 2). (c) C3b initiates the full complement cascade (steps 2 and 3), resulting in MAC formation and pathogen lysis.

180 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Genetic susceptibility can contribute to chronic inflam- mation; for example, certain variants of the IL-1b gene are associated with lower or higher serum concentra- tions of proinflammatory cytokines. Noncoding micro- RNAs (miRNAs) that can regulate inflammation decrease with aging and may contribute to the inflam- matory phenotype observed in older people. Other causes of chronic inflammation include production of proinflammatory mediators by adipocytes in obesity; increased gut permeability leading to translocation of gut organisms into the bloodstream; alterations in the gut microflora leading to proinflammatory responses; the inflammatory tumor microenvironment in cancer; type 2 diabetes, which leads to a heightened risk for car- diovascular disease, chronic inflammation, and infec- tions; and chronic infectious processes such as lung granuloma formation in tuberculosis, cirrhosis of the

liver in hepatitis B, or recurrent severe lower respira- tory tract infections that lead to fibrosis and permanent structural airway damage in cystic fibrosis and bronchi- ectasis. In some individuals, chronic inflammation per- sists after an infection or injury has resolved. In other cases, it occurs when there have been no obvious initi- ating factors.

Early modulation of chronic inflammation is import- ant to prevent health decline. Broad-acting pharmaceu- ticals already in clinical use, such as aspirin, metformin, and rapamycin, are known to have antiinflammatory activity and can improve the overall health and life span in animal models. Other tissue- and disease-spe- cific biologically targeted therapeutics are available, such as the TNF inhibitors used in rheumatoid arthritis (Box 6.5 and Figure 6.19) and inflammatory bowel disease, and many more are in development.

BOX 6.5 Rheumatoid Arthritis Exemplifies Chronic Inflammation

Rheumatoid arthritis (RA) is an autoimmune disease found in 0.5% to 1% of persons globally. RA is two to three times more common in women than in men, and generally develops at a younger age in women. Clinical hallmarks include joint swelling, pain, and stiffness; fevers; anemia; increased erythrocyte sed- imentation rate; and increased acute phase protein

levels, as well as antibodies to citrulline-modified proteins (anti citrullinated protein antibodies, or ACPAs) and the autoantibody rheumatoid factor. Although the precipitating event has not been iden- tified, genetic risk contributes to RA development, with 15% concordance in monozygotic twins and 5% concordance in dizygotic twins.

Healthy joint

Rheumatoid joint

Pannus Synovial membrane

Cartilage Synovial fluid contains activated immune cells

Cartilage erosion Cartilage loss

Bone erosion

(continued)

(a)

Chapter 6 • The Immune System and Leukocyte Function 181

The focus of tissue damage in RA is the synovial joints, with the accumulation of Th1 and Th17 cells, macrophages, B cells, fi bro- blasts, and synovial cells ( Figure 6.19b ). The joints in individuals affected by RA are charac- terized by chronic, nonresolving infl ammation. The persistence of T cells chronically activates macrophages to secrete IL-1, TNF, and IL-6,

promoting osteoclast activity and bone resorp- tion, and causing progressive bone deformity, joint degeneration, swelling, fl uid accumula- tion, and pain (Figure 6.19a). Targeted therapy with disease-modifying antirheumatic drugs (DMARDs) usually involves monoclonal anti- bodies that inactivate one of these cytokines or their receptors. 8

BOX 6.5 Rheumatoid Arthritis Exemplifi es Chronic Infl ammation (continued)

Th1 cell

Activation

Macrophage

IL-17

TNF-a

Synovial fibroblast

TNF-a

IL-17 IL-17

IL-6

Osteoblast

Osteoclast

Precursor

RANKL

RANK

Bone resorption

(b) Osteoclast

IL-6

IL-1

Th17 cell

+ +

+–

+

+ +

FIGURE 6.19 (a) The joint damage from chronic infl ammation in RA includes loss of cartilage cushioning the joint, loss of bone contributing to deformity, excess synovial fl uid containing infl ammatory cells and mediators, and thickening of the synovial membrane with pannus formation. (b) Cells and mediators of the chronic infl ammatory process in RA. Although synovial fl uid normally has very few cells, RA is associated with infi ltration of infl ammatory cells, including macrophages, dendritic cells, B cells, and T-helper cells (Th1 and Th17). Fibroblasts and osteoclasts can also accumulate in the joint. Infl ammatory fl uid and cells cause proliferation of the synovial membrane, which enlarges, forming a pannus. The chronic infl ammatory cells produce proinfl ammatory cytokines, particularly TNF, IL-6, and IL-17. These cytokines promote activation of synovial fi broblasts, which proliferate and also begin to secrete IL-6. The cytokine milieu alters the activity of nearby osteoblasts and osteoclasts with subsequent degradation of bone at the margins of the joint and adjacent cartilage, creating the joint deformities associated with RA. IL, interleukin; RA, rheumatoid arthritis; RANK, receptor activator of nuclear factor kappa B; RANKL, RANK ligand; TNF, tumor necrosis factor .

182 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

TRAINED INNATE IMMUNITY The ability to react more effectively and efficiently against specific pathogens is a mechanism restricted to the adaptive immune system. The innate immune system, however, possesses a type of immunological memory, known as trained immunity, which lacks specificity but still offers enhanced protection against pathogenic organisms.

Evidence from epidemiological and preclinical studies has hinted at this mechanism for some time, but the science of trained immunity of the innate sys- tem has only recently gained attention. It has been known for many years that vaccines can have benefi- cial off-target effects. For example, even as far back as its first use in the 1920s, the tuberculosis vaccine, bacille Calmette-Guérin (BCG), was suggested to pre- vent death in neonates that extended far beyond its ability to prevent tuberculosis. This was subsequently confirmed by epidemiological and preclinical studies, and other vaccines have been shown to act in a sim- ilar way.

It was only in the last decade that the mechanism for this curious feature of the innate immune system was elucidated. It was discovered that monocytes could undergo epigenetic and metabolic reprogramming after exposure to certain organisms or molecules from pathogens. This reprogramming results in a heightened functional state that enables the monocyte to respond more effectively against a subsequent infectious insult, whether it be by the same pathogen or another, unre- lated pathogen.

Trained immunity may play an important role in chronic inflammation. Trained monocytes have been proposed to drive atherosclerotic lesion development, leading to cardiovascular disease, and they may also play a role in the development and maintenance of autoimmune and autoinflammatory diseases. This area of research is still very much in its infancy, but offers great potential for providing new therapeutic targets.

TRANSITION FROM INNATE TO ADAPTIVE IMMUNITY: ANTIGEN PRESENTATION AND THE MAJOR HISTOCOMPATIBILITY COMPLEX In certain phagocytic cells, some of the proteins from the pathogen are broken down further in the phagoly- sosome into small peptides. These peptides (typically 13 to 25 amino acids long) associate with a transmem- brane protein called MHC class II or human leukocyte antigen (HLA)-DM, -DO, -DR, -DQ, or -DP. MHC mole- cules present the peptide antigen in a cleft of their tri- dimensional protein structure (Figure 6.20). Antigen processing and presentation on MHC II is a critical process that involves dendritic cells, B cells, and

macrophages—collectively referred to as APCs—but not neutrophils. Unlike B cells, which can recognize free antigen, T cells can only recognize antigen that is displayed to them on MHC by APCs. Antigen presenta- tion is thus a critical step linking innate immunity with adaptive immunity.

There are two main classes of MHC proteins: MHC I and MHC II. MHC II is expressed only by APCs; how- ever, MHC I is expressed by nearly all nucleated cells. While MHC II presents peptides from phagocytized material of extracellular organisms, the peptides pre- sented on MHC I are processed in a different way and are predominantly sourced from intracellular patho- gens such as viruses or intracellular bacteria. Because viruses can infect many different cell types, it is important that T cells can be alerted to which cells are infected through antigen presentation on MHC I. CD4 T cells recognize antigen on MHC II; CD8 T cells recog- nize antigen on MHC I.

Smaller peptides (typically eight to ten amino acids long) are presented on MHC I. These cytosolic peptides are produced from within the cell using the immunoproteasome, which produces peptides able to bind to MHC I. These peptides are transported to the endoplasmic reticulum by a special transporter associated with antigen processing (TAP). The pep- tides are loaded onto MHC I in the endoplasmic retic- ulum, then the MHC I–peptide complex is exported to the cell surface, ready for presentation to CD8 T cells (Figure 6.21).

Each individual expresses a genetically distinct combination of MHC molecules of both classes. There are six MHC I isotypes: HLA-A, HLA-B, HLA- C, HLA-E, HLA-F, and HLA-G. There are multiple dif- ferent alleles of each isotype, with HLA-A, HLA-B, and HLA-C being the most polymorphic. For MHC II, HLA-DP, HLA-DQ, and HLA-DR are also highly polymorphic.

Besides their role in antigen presentation, highly polymorphic MHC I molecules, which are expressed by almost every cell in the body, play an important role in rejection of transplanted tissue. The antigenic differences between host MHC and donor MHC can lead to an immune response initiated against the graft, leading to transplant rejection. Conversely, HSCs used in bone marrow transplantation can develop into T cells that recognize the host MHC as a foreign anti- gen, leading to a potentially serious condition known as graft-versus-host disease. Tissue typing prior to transplantation evaluates the degree of similarity of donor and recipient HLA proteins—the closer these proteins are, the less likely that transplant rejection will occur. Therefore, correct matching of HLA of the donor and recipient is necessary for successful tissue transplantation.

Chapter 6 • The Immune System and Leukocyte Function 183

Thought Questions

5. What is the role of the endothelium in acute infl ammation?

6. What is the role of neutrophils in acute infl ammation?

7. What is the role of complement in acute infl ammation?

8. How do macrophages contribute to tissue surveillance, acute infl ammation, and chronic infl ammation?

9. How do dendritic cells aid the transition from innate to adaptive immunity?

Protease

Exogenous protein

Early endosome

MIIC

Golgi

Ii

HLA-DM

+

APC

α β

MHC class II ER

TCR MHC class II

Peptide

CLIP

CD4+ T cells

FIGURE 6.20 Presentation of exogenous, extracellular antigens on MHC II proteins is conducted by professional antigen-presenting cells. MHC II presentation is a multistep process, initiated by synthesis of MHC II. The antigen-binding site of MHC II is protected by an invariant peptide (Ii). MHC II can move to the membrane or to an intracellular compartment, the MIIC. Foreign proteins are brought into the APC by endocytosis, forming the early endosome, where they are broken down by proteases into small fragments. Fusion of the endosome with the MIIC allows cleavage of the invariant protein and replacement of the resulting CLIP with the antigen fragment. The fi nal complex of antigen fragment and MHC II protein are displayed on the membrane of the APC for presentation to CD4+ T cells. APC, antigen-presenting cell; CLIP, class II-associated Ii peptide; ER, endoplasmic reticulum; HLA, human leukocyte antigen; MHC, major histocompatibility complex; MIIC, MHC class II compartment; TCR, T-cell receptor.

ADAPTIVE IMMUNITY

The adaptive immune response contrasts with the broad innate immune response by being highly spe- cifi c. Its precision is such that adaptive immune cells can recognize a particular strain of a pathogen while failing to recognize a different strain of the same pathogen. An example of the problems posed by such specifi city relates to the large number of different viral strains that cause infl uenza. Immunizations to reduce infection with and transmission of the infl u- enza virus attempt to cover viral strains that are predicted to be in circulation over the coming year, because it is not practical to immunize against all possible strains. If the predictions are incorrect, the

184 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

flu vaccine may be relatively ineffective in any given year. Fortunately, other viruses that are targeted by vaccines, such as measles, chickenpox, and hepatitis A, have stable structures and routine immunizations are very effective at preventing infections.

A remarkable feature of the adaptive immune response is its ability to specifically recognize and respond more quickly and more effectively to a sec- ond exposure with the same pathogen. This is the underlying principle of vaccination. B and T cells are the main cellular mediators of adaptive immu- nity; however, they are heterogeneous groups of cells and vary greatly in the types of responses they produce (Figure 6.22). The cells, mediators, and mechanisms of adaptive immunity are detailed in this section.

B CELLS ARE RESPONSIBLE FOR HUMORAL IMMUNITY The main effector mechanism of the B-cell response is the secretion of antibodies, a specialized class of circu- lating glycoproteins that are a soluble version of the BCR, as described earlier (Figure 6.22a). Circulating antibod- ies are effector proteins that bind antigen on pathogens or toxins with high specificity. This binding provides pro- tection through three general mechanisms, as follows:

1. Antibody binding may neutralize target proteins. For example, a patient with tetanus can be treated with antitoxin—antibodies that bind to tetanus toxin mol- ecules throughout the circulation and tissues and prevent their effects on the body.

2. Coating a microbe with antibodies is a form of opsonization that facilitates phagocytosis and destruction by neutrophils and macrophages.

3. Antibodies can also lead to bacterial killing by trig- gering activation of the classical complement path- way (discussed earlier; Figure 6.23).

Antibody Structure As noted earlier, all antibodies generally share common molecular and structural characteristics, as illustrated in Figure 6.24. They are made up of four protein chains: two heavy chains and two light chains. These are held together by disulfide bonds, forming a Y-shaped mole- cule. Each arm of the immunoglobulin consists of a light chain held to the upper portion of the heavy chain. Then the two heavy chains, or stalk, are also held together by disulfide bonds. Functionally, antibodies have two regions: one end (with two arms) to bind antigen and one end that is constant across their class and is able to bind to receptors and recognition sites. When cleaved by an enzyme, papain, the antibody generates two frag- ments. The portion with variable amino acid sequence based on B-cell gene rearrangement is within the frag- ment antigen-binding (Fab) region. Although the Fab regions are extremely variable and differ between immunoglobulins of different B-cell clones, the remain- ing constant fragment, or Fc, region is the same in all immunoglobulin molecules of a given class.

The Fc region of an antibody interacts with different immune effector cells, such as neutrophils, mast cells, NK cells, and macrophages, by binding to immunoglob- ulin class-specific Fc receptors expressed on the cell membrane. The specificity of the Fc receptors for the different immunoglobulin isotypes and the type of cells expressing the Fc receptor determines the different effector outcomes.

Antibody Classes The five immunoglobulin classes or antibody iso- types are IgM, IgD, IgG, IgA, IgE; their functions are

TAP

Peptides Proteasome

Proteins in the cytosol or nucleus

Peptide

β2m

TCR

CD8+ T cells

ER

MHC class I

ERAD

Golgi apparatus

FIGURE 6.21 Intracellular antigenic peptides are presented on MHC class I molecules to CD8 T cells following antigen degradation by the proteasome. The peptides generated are translocated via TAP into the lumen of the ER, then loaded onto MHC I molecules. Peptide–MHC I complexes are transported to the plasma membrane via the Golgi apparatus for presentation to CD8 T cells. β 

2 m, β 

2 -microglobulin; ER, endoplasmic reticulum; ERAD,

ER-associated protein degradation; MHC, major histocom- patibility complex; TAP, transporter associated with antigen processing; TCR, T-cell receptor.

Chapter 6 • The Immune System and Leukocyte Function 185

summarized in Table 6.5 . Each heavy chain gene is given a Greek letter that corresponds to the class of antibody it encodes. For example, the IgM heavy chain is encoded by µ, IgD is encoded by δ , and so on. Monomeric IgM and IgD are the fi rst immunoglobulin types expressed on the surface of a mature naive B cell. These cell-surface antibodies function as the BCR.

IgM is the fi rst secreted antibody of the immune response and is secreted as a pentamer. IgM can be expressed by some B cells even in the absence of prior immunization and antigen exposure, contribut- ing to natural humoral immunity. Although the anti- gen-binding sites of IgM are, in general, low affi nity, their multivalent structure helps them to create very effective antigen–antibody complexes. Compared with the effector functions of other immunoglobulin

classes, IgM has the highest ability to activate com- plement to help destroy the invading pathogen. Secretion of IgD is rarely observed; however, IgD is used as a marker for B-cell maturation because its surface expression changes according to different B-cell maturation stages. The functions of IgD are still largely unknown, despite its being a highly con- served immunoglobulin across many species. There are indications that IgD on B-cell membranes pro- motes responses to foreign proteins while reducing responses to self-antigens. Secreted IgD, although present in relatively low concentrations, may contrib- ute to mucosal immunity.

Three quarters of circulating immunoglobulin is monomeric IgG, the most abundant immunoglobu- lin isotype. IgG is good at activating the complement

T-cell receptor

Killing of infected cells

T

Foreign proteins or infectious agents

Cell-mediated response (T lymphocytes)

Antigen Antigen

T-cell receptor

Antigen- selected T cells

Cytokine secretion

Humoral response (B lymphocytes)

B cell

(a) (b) (c)

B-cell receptor

Antigen

Antibody

Antigen elimination

B

Antigen-selected antibody-secreting B cell

B T

T T

+ AA+ AA+

FIGURE 6.22 Overview of adaptive immunity. Adaptive immunity is divided into humoral responses mediated by B cells and antibody secretion (a), and cell-mediated responses (b and c). Cell-mediated immunity is carried out by cytotoxic T lymphocytes (c) that can kill virus-infected or abnormal (cancerous) self-cells. Both humoral and cell-mediated immunity require stimulatory factors produced by related antigen-specifi c T-helper lymphocytes (b).

186 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

B-cell activation by antigen and helper T cells

Cytokines

Antibody secretion by plasma cells

1. Neutralization

Antibody prevents bacterial adherence

Antibody promotes phagocytosis

Antibody activates complement, which enhances opsonization

and lyses some bacteria

2. Opsonization 3. Complement activation

Complement

B cell Helper T cell

Expansion of B-cell clone and differentiation to plasma cells

FIGURE 6.23 The effector actions of antibodies. Antibodies provide protection from toxins and pathogens by three major mechanisms: (1) In neutralization, antitoxin antibodies bind toxin molecules into antigen–antibody complexes that can be removed by macrophages. (2) In opsonization, antibodies bind to pathogens as opsonins, facilitating macrophage phagocytosis. (3) Antibodies binding to pathogens initiate complement via the classical pathway of complement activation, leading to MAC formation and cytolysis. MAC, membrane attack complex.

Chapter 6 • The Immune System and Leukocyte Function 187

S S

S S S S

S S

IgM

IgG IgA

Antigen- binding site

Antigen

Antigen within antigen-binding

site

Fab

Fc

Light (L) chain

Disul�de bonds

Carbohydrate

CH3CH3

CH2 CH2

CH1 CH1

CL CL

VLVL

VH VH

Hinge region

Heavy (H) chain

FIGURE 6.24 Antibody structure. Note that IgA is generally secreted as a dimer, while IgM is usually a pentamer. C

H , constant domain heavy chain; C

L , constant domain light chain; Fab, fragment, antigen-binding;

Fc, fragment, crystallizable (or constant); IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, Immunoglobulin M; V

H , variable domain heavy chain; V

L , variable domain light chain.

188 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

cascade and eliminating pathogens, promoting opsonization and MAC complex formation on microbe surfaces. With a half-life of approximately 3 weeks, IgG is a long-lasting immunoglobulin. Fc receptors specific for IgG antibodies are highly expressed on macro- phages and neutrophils, making this isotype the most efficient at promoting opsonization and phagocytosis.

Notably, IgG is the only isotype able to cross the pla- centa, providing protection to the fetus during pregnancy and the neonate in the weeks after birth. While greatly beneficial in most cases, the ability of IgG to cross the placenta can cause fetal loss or death of the newborn if the mother produces IgG antibodies against the blood type (rhesus factor) of the fetus. This can occur if the mother and fetus have incompatible blood types. This condition is now easily detectable and treatable.

IgA is secreted as either a monomer or dimer. Monomeric IgA represents about 15% of the total immunoglobulin circulating in the bloodstream. One of its exclusive characteristics is the ability to cross epi- thelial membranes and to be expressed abundantly in body secretions that protect mucosal tissues and body openings. Mucus in the airways and in the urogenital and gastrointestinal tracts, as well as tears, sweat, and saliva, contain dimeric IgA. In these locations, it is stra- tegically positioned to bind and inactivate invading pathogens, preventing their entrance into the body. IgA is also abundant in breast milk and provides protection for neonates and infants whose immune systems are still developing.

In the healthy individual, IgE is the least abundant immunoglobulin class, making up less than 1% of total immunoglobulin in the bloodstream. IgE binds to mast cells and basophils interacting with its specific Fc recep- tor that is present on these innate cells. Binding to the Fc receptors triggers the release of preformed granules

containing histamines and inflammatory molecules. IgE is particularly useful for eliminating parasites such as helminth worms. Despite its extremely low blood concentration, IgE is very potent and can cause tissue damage; it is associated with hypersensitivity type 1 dis- eases, also known as atopic diseases or allergies.

Steps in B-Cell Activation and Maturation B cells are activated when the BCRs on their cell surface bind to free antigen. In some cases, when the antigen is repetitive in nature (e.g., flagellin on flagellated bacteria), the BCRs become cross-linked or aggregate together on the cell surface, and the B cell receives a strong enough signal to be activated without any T-cell help. In most cases, though, B cells require help from T cells in order to be fully activated (Figures 6.22b and 6.25). B cells can internalize their BCR and process the antigen for presentation to CD4 T cells on the MHC II molecules. CD4 T cells that recognize the antigen can then support B-cell activation through the upregulation of cell-surface co-stimulatory receptors or through cytokine produc- tion. The activated B-cell clone, while proliferating, starts simultaneously to increase the production of IgM, and differentiates into effector cells (antibody-secreting plasma cells). This process constitutes the first phase of the adaptive humoral immune response.

Follicular helper T (T fh

) cells are specialized CD4 T cells that are located within B-cell follicles in second- ary lymphoid tissues. T

fh cells are critical for the for-

mation and maintenance of germinal centers, the site of intense B-cell proliferation, because they support B-cell survival and differentiation. Following the first stage of activation, and with the support of T

fh cells, B

cells form the germinal center. There, they begin to pro- liferate and some cells differentiate into plasma cells. Plasma cells have prominent secretory organelles and

TABLE 6.5 Characteristics of the Major Antibody Classes

Feature IgM IgD IgG IgA IgE

Heavy chains μ δ  γ  α  ε

Number of units 5 1 1 1 or 2 1

% of total immunoglobulin (Ig) 10 <1 75 15 <1

Complement activation ++++ − ++ − −

Crosses placenta − − + − −

Binds to macrophages and neutrophils

− − + − −

Binds to mast cells and basophils

− − − − +

Crosses epithelial membranes − − − + −

Chapter 6 • The Immune System and Leukocyte Function 189

serve as antibody factories , secreting large amounts of specifi c antibody that circulates through the blood, lymph, and extracellular fl uids.

The daughter B cells and plasma cells usually bear the same BCR on their surface and produce the same type of secreted antibody as the parent B cell. However, as the germinal center reaction progresses, the B cells undergo a process called isotype class-switching . During isotype class-switching, the B cells, which ini- tially produce pentameric IgM, are able to class switch and swap the gene segment used to encode the con- stant region of the heavy chain. This process is gov- erned by the local cytokine environment induced by the T

fh cells. Thus, in the fi rst days of a B-cell immune

response, membrane-bound and secreted IgM antibod- ies are produced by the B cell; however, at later stages different isotypes of antibody are expressed, with IgG being the most common.

Another fascinating feature of B cells, touched on earlier, is their propensity to undergo point mutations in antigen-binding regions of their heavy and light chain variable domains during the germinal center reaction. This process is called somatic hypermuta- tion and can affect the strength of binding between the antibody and antigen. The tighter the bond, the more effective the antibody. Some mutations, of course, are deleterious or have no effect; however, this is a small price to pay for the chance to produce a better antibody. B cells that produce a more tightly binding antibody undergo a process known as affi n- ity maturation . In this process, B cells that produce higher affi nity (or more tightly binding) antibody preferentially survive and outcompete their lesser counterparts. Therefore, as the germinal center pro- gresses, the affi nity of the antibody for the antigen improves. The B cells and their daughter cells and plasma cells are now able to produce high-affi nity

antibody of the most appropriate class to fi ght the infection or toxin.

B-Cell Memory A further remarkable feature of B cells is their ability to remember ( Figure 6.26 ). During the B-cell response, high-affi nity, class-switched B cells are able to differen- tiate into long-lived memory B cells, which can survive for many years. These memory cells, now present in high numbers, are at the ready to produce high levels of high-affi nity, class-switched antibody upon reexposure to the same pathogen. The impressive ability of memory B cells to recall over long periods of time was illustrated by the resistance of the elderly population in the remote Faroe Islands of the North Atlantic to the measles virus. An epidemic of measles had occurred in the islands 65 years earlier, with no further measles cases until an out- break many decades later. 9 That immunity to the virus had persisted for 65 years is nothing short of amazing!

B cells can also differentiate into long-lived plasma cells able to secrete antibody for months after infec- tion. These cells exist in the bone marrow, and their survival is supported by bone marrow stromal cells. Long-lived plasma cells help prevent reinfection with the same organism, particularly if it remains circulating in the population during an epidemic.

Antibody Production Compared with the immediacy of the innate immune response, the B-cell response process takes at least 3 to 4 days before being detectable (latent or lag phase; see Figure 6.26 ). IgM levels peak in the serum between 5 and 10 days after infection and then decrease, becom- ing undetectable in about 20 days, although the kinetics of the antibody response may differ according to the type of pathogen or toxin, exposure route, dose of anti- gen, and intensity of the initial innate response.

TCR MHC II

Cytokines

Plasma cell

CD4+

T cells

B

FIGURE 6.25 CD4 T-cell help for B-cell activation. B cells can interact with the T fh

cells specifi c for the same antigen by way of antigen presentation. Like macrophages and dendritic cells, B cells express MHC class II receptors for antigen presentation to T

fh cells and other helper T cells. Within

lymphoid follicles, B and T fh

cells programmed to respond to the same antigen encounter each other and bind through their complementary TCRs and MHC protein. The T

fh cell then secretes

cytokines that expands the clone of B cells and promotes maturation to plasma cells that act as prolifi c antibody factories. MHC, major histocompatibility complex; TCRs, T-cell receptors; T

fh cell, follicular helper T cell.

190 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

During the first exposure to a pathogen, IgG pro- duction is delayed compared with IgM, and peak levels of IgG are lower than peak levels of IgM. On the other hand, IgG antibodies are long lasting, and during the late stages of the response long-lived plasma cells are able to maintain an active serum level of IgG, which provides protection for weeks and months after the infection has resolved.

Clinical Applications of Antibodies Antibodies or immunoglobulins constitute about 20% of human plasma proteins. Concentrations of immu- noglobulins (titers) specific for antigens on pathogens

such as rubella, hepatitis B, and measles viruses can be measured in the blood. Analysis of antibodies for a pathogen-specific antigen can reveal whether an indi- vidual has been previously exposed to or vaccinated against that pathogen, and can determine whether the infection is recent (IgM-dominated) or had occurred in the past (IgG-dominated).

As previously mentioned, neutralizing antibodies are often administered as a treatment against specific toxins. Purified preparations of antibodies are used in the ED to neutralize foreign toxins. Snake antivenom is a preparation of purified antibodies specific against snake toxins. Prompt injection of antivenom helps to

Primary response

Log phase

A nt

ib od

y co

nc en

tr at

io n

in s

er um

(l og

s ca

le ) Total

antibody

Plateau phase

Decline phase

Secondary response Total antibody

IgG

IgG

IgM

IgMLatent period

First exposure to antigen

B cells

IgM

IgG

Plasma cells

Second exposure to antigen

10 days5 days15 days10 days

IgM IgG Plasma cells

IgG

Memory cells

IgG

IgG

IgGIgM

IgG

Memory cells

FIGURE 6.26 Primary and secondary (memory) B-cell responses. First presentation of antigen, either due to infection or vaccination, produces a delayed and relatively weak response, with IgM produced first, transitioning to IgG. This stage results in B cells diverging to become either plasma cells or memory cells. Upon a second exposure (or booster shot), antibodies are rapidly produced, the total amount of antibody released increases, and there is very little IgM response. The clone of memory cells expands further. Any further exposures to the antigen will elicit a robust response, providing protection that can last a lifetime. IgM, immunoglobulin M; IgG, immunoglobulin G. Source: From Willey J, Sherwood L, Woolverton CJ. Prescott’s Microbiology. 10th ed. McGraw-Hill; 2016, Figure 34.16.

Chapter 6 • The Immune System and Leukocyte Function 191

neutralize snake toxin and allows its clearance from the body before it causes long-term effects and irre- versible tissue damage.

Large amounts of pure antibody can be produced for clinical use. Fusing a B-cell clone to an immortal tumor cell creates a B-cell hybridoma. Each B-cell hybrid- oma can divide seemingly endlessly in culture while producing antibodies that bind to the same antigenic epitope, meaning that all of the antibodies produced have exactly the same specifi city. The antibody can be designed to specifi cally target certain molecules, recep- tors, or cytokines that modify the response in vivo to reduce or eliminate disease. Over the past 10 years, the use of monoclonal antibodies as therapeutic agents has escalated dramatically. Monoclonal antibodies belong to a class of drug known as biologics that have pro- pelled us into an exciting new era of highly targeted drug development. The earliest cytokine-directed antibodies for autoimmune diseases targeted IL-1 and TNF; the targets have now expanded to include IL-17A, IL-22, IL-23, and many others. In addition, monoclonal antibodies are used to treat conditions as varied as cancer, hypercholesterolemia, postpercutaneous cor- onary intervention, and recurrent Clostridioides diffi – cile diarrhea. For a brief list of monoclonal antibodies approved by the Food and Drug Administration (FDA), their targets, and indications, see Table 6.6 .

Thought Questions

10. How do B cells recognize their antigen and the T fh cells that facilitate their activation?

11. What are the general structural characteristics of antibodies, and what are the features and characteristics of the diff erent antibody types?

T-CELL FUNCTIONS The T-cell response, sometimes referred to as the cell-mediated immune response , involves activation of T cells through the highly specifi c TCRs, which recog- nize antigen presented to them in the context of MHC, as previously described. T cells respond to activation by proliferating and producing effector molecules, which include cytokines and cytotoxic molecules. Cytokines are able to act on other cells, including B cells and many cells of innate immunity, sending signals through cytokine receptors that trigger effector mechanisms of immune protection.

Although the functions of CD4 and CD8 T cells over- lap, broadly speaking, CD4 T cells function to produce cytokine and are sometimes termed T-helper ( Th ) cells whereas CD8 T cells have cytolytic functions in addi- tion to their cytokine-producing abilities. Therefore,

CD8 T cells are often referred to as CTLs. CTLs play a critical role in combating intracellular bacterial and viral infections, as well as cancer cells. Many infec- tions lead to CD4 and CD8 T-cell responses, as well as B-cell responses. The balancing of the different types of response depends on the context of exposure to the antigen and the type of antigen. Cooperation between CD4 and CD8 T cells, B cells, and cells of the innate immune system is responsible for the incredible effec- tiveness of the adaptive immune system. Humans depend on this system to neutralize and kill pathogens before they cause disease and to limit the duration of a disease once it is established. For this reason, many infectious diseases are acute and limited in duration, and for some diseases, just one infection with the pathogen may protect against having the same disease again.

Major Histocompatibility Complex Restriction As T cells develop and mature in the thymus, they become committed to expressing either the CD4 co- receptor or the CD8 co-receptor. These receptors dic- tate the type of antigen that the T cell will recognize. As noted earlier, CD4 T cells only recognize antigen pre- sented by MHC II found on specialized APCs; CD8 T cells only recognize antigen presented by MHC I found on most nucleated cells. This is referred to as MHC restriction . Because T cells respond only to antigen that is presented on an MHC molecule, the ability of MHC proteins to associate with different antigen epi- topes is critical for determining whether an adaptive immune response will develop against a specifi c anti- gen. To optimize and enhance antigen presentation to T cells, the HLA gene locus for MHC class I and MHC class II each encodes for three different co-dominantly expressed proteins. Both sets of inherited alleles are normally expressed. Each MHC gene is highly polymor- phic, so thousands of different gene variants (alleles) exist in the human population. This varability in MHC genes and their resulting proteins contributes to phe- nomena such as transplant rejection. Improving diver- sity in antigen presentation is an evolutionary strategy to ensure development of effi cient adaptive immune responses. However, this diversity may also create dif- ferent vulnerabilities such that people may have vary- ing levels of susceptibility to a given pathogen or to developing autoimmune diseases.

CD4 T-Cell Responses CD4 T cells have a key role of coordinating the acti- vation of both arms of the adaptive immune response as well as the effector function of innate cells; most CD4 T-helper cells work to promote and sustain other immune cells to expand, differentiate, or elicit their effector functions (Figure 6.22b). Conversely, some CD4 T cells—the Tregs—do exactly the oppo- site and prevent other immune cells from expanding,

192 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

differentiating, or exerting their effector functions. CD4 T cells recognize antigen in the context of MHC class II, which is expressed only on professional APCs.

Antigen on MHC class II molecules mostly origi- nates from extracellular sources. Antigen displayed on MHC class II molecules is detected by the TCR at an immunological synapse along with T-helper cell CD3 and CD4 proteins. An additional set of membrane proteins provide co-stimulation signals, the APC CD80 or CD86 proteins bind to T-helper cell CD28. Activation of both the TCR complex and the co-stimulation complex are required for full T-helper cell activation (Figure 6.27). Once this dual activation occurs, T-helper cells upregu- late expression of genes that encode cytokines and

molecules that can stimulate and support other T cells, B cells, and innate cells. Simultaneously, acti- vated CD4 T cells start to replicate and can generate antigen- specific memory T cells as well. CD4 T cells differentiate into different types of helper cells that are classified based on their cytokine secretion pro- file and their expression of certain transcription fac- tors that are associated with their effector function.

T-Helper Subsets The context of antigen presentation to naive CD4 T cells is an important factor for determining the type of T-helper response that will develop. Important factors affecting the context of presentation include (a) type of antigen, (b) amount of antigen, (c) type

TABLE 6.6 Monoclonal Antibodies Approved for Clinical Use

Antibody Target Indication

Abciximab Platelet glycoproteins Percutaneous coronary intervention

Adalimumab TNF Rheumatoid arthritis

Alirocumab PCSK9 Hypercholesterolemia

Belimumab B-lymphocyte stimulator Systemic lupus erythematosus

Bezlotoxumab Clostridioides difficile toxin B Recurrent C. difficile diarrhea

Certolizumab TNF Crohn disease

Daclizumab IL-2R Multiple sclerosis

Denosumab RANKL Postmenopausal osteoporosis

Golimumab TNF Rheumatoid arthritis, ankylosing spondylitis

Infliximab TNF Crohn disease, other autoimmune diseases

Ipilimumab CTLA-4 Metastatic melanoma

Natalizumab α 4 integrin Multiple sclerosis

Nivolumab PD-1, immune checkpoint Metastatic melanoma

Omalizumab IgE Asthma

Pembrolizumab PD-1 Metastatic melanoma

Rituximab CD20 B-cell non-Hodgkin lymphoma

Secukinumab IL-17A Plaque psoriasis

Tocilizumab IL-6R Autoimmune arthritis (several)

Trastuzumab HER2 Metastatic breast cancer

Ustekinumab IL-12 IL-23

Plaque psoriasis

CTLA-4, cytotoxic T lymphocyte–associated protein 4; IgE, immunoglobulin E; HER2, human epidermal growth factor receptor 2; IL, interleu- kin; PCSK9, proprotein convertase subtilisin/kexin type 9; PD-1, programmed cell death protein 1; RANKL, receptor activator of nuclear factor kappa-B ligand; TNF, tumor necrosis factor.

Chapter 6 • The Immune System and Leukocyte Function 193

of APC, and (d) local cytokine environment at the time of activation. APCs produce different cytokines depending on the PRR signals they receive and the different receptors they express, so early innate events can lead to different local cytokine environ- ments, which dictate the type of adaptive T-helper response that develops.

The cytokines produced by APCs can reprogram the patterns of gene expression within the T-helper cell, enabling the cell to produce the most effective immune response for a given pathogen. Naive T-helper cells have been characterized as developing into one of fi ve broad T-helper subsets currently described: Th1, Th2, Th17, T

fh , and Tregs ( Figure 6.28 ).

• Th1 cells are characterized by their expression of the transcription factor Tbet, and their secretion of the cytokine interferon gamma (IFN-γ ). Th1 cells promote mechanisms of innate cells, such as phagocytosis and antigen presentation, and on the adaptive side, support development of CTLs against intracellular pathogens.

• Th2 cells express the transcription factor GATA3 and secrete the cytokines IL-4, IL-5, IL-9, and IL-13. This subset supports B-cell responses and drives isotype class-switching to IgE, which is important for degranulation of mast cells and basophils to fi ght helminth infections. On the one hand, the Th2 response has an important role in tissue repair following injury; on the other hand,

it can contribute to the acute allergic responses, progressive fi brosis, and chronic infl ammation that occur in asthma, atopic dermatitis, and other allergic disorders.

• T fh

cells, discussed earlier in this chapter, are found in secondary lymphoid tissues, where they support maturation and proliferation of B cells.

• IL-17–producing Th17 cells express the transcrip- tion factor retinoic-acid receptor-related orphan nuclear receptor gamma (RORγ t) and are import- ant for immune responses against extracellular bacteria as well as fungal infections. Like Th2 cells, Th17 cells have a dichotomous nature. Th17 cells can drive infl ammatory disorders, such as the auto- immune disease multiple sclerosis; yet Th17 cells are also critical for maintaining homeostasis and barrier protection in the gut. It may be that Th17 cells differ from one another depending on how they are induced; however, this area requires more study.

• Lastly, Tregs function to damp down immune responses, and are essential for preventing auto- immune responses, providing tolerance to the gut microbiota and quenching inflammation once a pathogen has been eliminated. There are two types of Tregs. The fi rst type develops in the thymus; the second type develops when a naive T cell is stim- ulated in a particular cytokine environment that encourages the expression of FoxP3, the transcrip- tion factor associated with Treg function. Tregs exert their effect through immune-suppressive cytokines, such as IL-10, or through cell-to-cell con- tact and expression of inhibitory molecules, such as cytotoxic T lymphocyte–associated protein 4 (CTLA-4). Blocking inhibitory receptors such as CTLA-4 has revolutionized cancer therapy and led to the development of a new class of medicines known as checkpoint inhibitors.

CD8 T-Cell Responses Activated through their TCR complex following rec- ognition of antigen on MHC I in conjunction with co-stimulatory signals, CD8 T cells divide and differ- entiate into effector CTLs. These effector cells are able to identify virus-infected cells or cancer cells through their expression of antigen on MHC I and eliminate them directly by inducing cell lysis or burst ( Figures 6.22c and 6.29 ). Cytolysis protects nearby cells from virus infection and limits tumor cell prolif- eration. Cytotoxic T cells also produce cytokines such as IFN-γ  that can lead to macrophage activation to destroy engulfed pathogens.

In the case of infected or cancerous cells, pep- tides within the cytosol (from the virus) or abnormal self-proteins (found within tumor cells) are presented on top of MHC class I. These complexes are recognized

MHC Peptide

Co-stimulation (2nd key for activation)

Two “keys” required

APC

TCR

CD28

CD80/86

Cell membrane

Immunological synapse

Cell membrane

T cell

CD3CD4

FIGURE 6.27 Antigen presentation to activate T cells. In addition to the TCR–MHC interaction (the first key for T cell activation), effective antigen presentation requires additional co-stimulatory signals and changes in gene expression mediated by the transcription factor CREB in the T cell nucleus. APC, antigen-presenting cell; CREB, cyclic adenosine mono- phosphate response element-binding protein MHC, major histocompatibility complex; TCR, T cell receptor.

194 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

by CTLs, which in turn release proteolytic granules to kill the infected or cancerous cell, activating apoptosis. Alternatively, expression of Fas ligand on the mem- brane of the cytotoxic T cell can initiate apoptosis in the target cell by engaging the apoptotic receptor, Fas.

Unconventional T Cells: γ δ  T Cells, Mucosal-Associated Invariant T, and Invariant Natural Killer T Cells The T cells discussed previously are known as conven- tional T cells and express a TCR known as the α β  TCR. However, a minor subset of T cells that also develop in thymus are termed γ δ  T cells; these cells express a TCR known as the γ δ  TCR. They make up only 1% to 5% of circulating T cells but are much more common in epithelial tissues, such as the skin and intestine. γ δ  T cells are important for tissue homeostasis, thermogen- esis in adipose tissue, and epithelial repair. They also

contribute to immune responses against infection and cancer.

Mucosal-associated invariant T (MAIT) cells, as their name suggests, are found in mucosal sites, as well as the skin, liver, and blood. They express an α β  TCR but have an invariant α  chain, meaning that they recognize a reduced set of small molecules. These are predomi- nantly vitamin B metabolites from bacteria and fungi, and are not presented on MHC, but rather a related molecule called MR1. MAIT cells can also be activated independently of MR1, through cytokines such as IL-12 and IL-18. MAIT cells are important for controlling bac- terial and fungal infections at mucosal sites through secretion of cytokines. They also produce cytolytic granules like conventional CD8 T cells. Owing to their reduced antigen specificity and ability to be activated without antigen, these cells straddle the innate and adaptive arms of the immune response.

IL- 12

, IL -1

8

IL -4

IL -6

, I L-

21

IL-6 + TG F-b

TGF-b/retinoic acid

Th1

IFN-γ activates macrophages to kill intracellular

bacteria

IL-4 activates macrophages to expel parasitic

worms

IL-17 attracts neutrophils

IL-22 induces antimicrobial

peptide production

Tregs inhibit immune

responses via cell surface

molecules of cytokines

such as IL-10

Tfh cells make IL-21 and IL-4 and

express CD40L and ICOS that

help B cells make high-af�nity

class-switched antibody

Th2

Tfh

Th17

Treg

FIGURE 6.28 T-helper (Th) cell subsets and functions. ICOS, inducible T-cell co-stimulator; IFN, interferon; IL, interleukin; T

fh cell, follicular helper T cell;

TGF-β , transforming growth factor beta; Treg, regulatory T cell.

Chapter 6 • The Immune System and Leukocyte Function 195

Key:

Cytotoxic T lymphocyte (CTL)

Activation of caspase enzymes for apoptosis

Virus-infected cell

(a) (b) Virus-infected cell

Activation of caspase enzymes for apoptosis

Granule with perforins and granzymes

Cytotoxic T lymphocyte (CTL)

MHC I with bound peptide

Perforin

Granzyme TCR

CD8

Fas

FasL

FIGURE 6.29 Mechanisms of cytolysis by CD8 T cells. (a) Upon recognizing a virus-infected self-cell, the T cell releases perforin, which penetrates the membrane of the infected cell, and granzyme, an enzyme that enters the cell and initiates apoptosis. (b) In addition, the T cell FasL engages the target cell Fas protein, initiating apoptosis of the infected cell. FasL, Fas ligand; MHC, major histocompatibility complex; TCR, T-cell receptor.

A further unconventional, innate-like T-cell subset comprises the invariant NK T (iNKT) cells. These cells have a semi-invariant TCR that recognizes self and microbial lipid-based antigen presented by MHC I–like CD1d molecules. iNKT cells rapidly produce cytokines after stimulation through their TCR and also have cyto- toxic activity. iNKT cells have roles in autoimmunity, infectious diseases, and cancer.

T-Cell Memory As an adaptive immune response resolves, most responding T cells that have clonally expanded in response to the antigen die. However, a small pool of T cells remains, and these cells survive long term to act as memory T cells that can respond more quickly and more effectively upon subsequent exposures to the same antigen.

Because of the higher numbers of antigen-specifi c T cells that exist in the memory T-cell pool compared with the naive T-cell pool, when antigen is reencoun- tered, the T cells can expand to higher numbers than in the fi rst, naive T-cell response. Moreover, epigenetic changes that have occurred during the fi rst exposure to antigen endow the remaining memory T cells with the ability to produce effector molecules more quickly than naive T cells. The T cells retain their functional polar- ization that was established during the initial antigen exposure. For example, if a CD4 T cell had been polar- ized to a Th1 phenotype and developed into a memory cell, when it reencounters its cognate antigen, it would

continue to express the transcription factor Tbet and produce IFN-γ .

Memory T cells are very heterogeneous. Both CD4 and CD8 T cells form distinct subsets of memory T cells that can be identifi ed based on their anatomical loca- tion, recirculation patterns, and the types of cell-surface receptors that they express. Resident memory T cells establish in the tissue in which the immune response fi rst occurred; for example, the skin or the lung. These cells do not recirculate but are strategically located to respond to a second infection, which is most likely to occur in a similar location. Effector memory T cells cir- culate among the tissues, blood, and lymphatics, and central memory T cells, which have a more resting phe- notype, are typically located in the bone marrow and secondary lymphoid tissues.

As humans age, the ratio of naive T cells to mem- ory T cells changes, with naive T cells predominat- ing at fi rst, during the years in which thymic output is at its greatest. Memory T cells accumulate follow- ing infectious exposures, exposure to the commensal microbiota, and vaccination during childhood and ado- lescence. By adulthood, memory T cells outnumber naive T cells. Interestingly, cytomegalovirus (CMV), which infects the majority of the human population but is usually asymptomatic in immune-competent individuals, leads to a very strong T-cell response. In the elderly, the memory CD8 T-cell pool is often domi- nated by CMV-specifi c CD8 T cells in a process known as memory infl ation.

196 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

PATHOGEN–HOST IMMUNE EVASION MECHANISMS Despite their amazing and intricate immune system, humans still succumb to infectious disease. As the immune system has evolved, so too have pathogens. Viruses, bacteria, fungi, protozoa, and helminths have all devised ways to escape the immune assault that they encounter in the human body.

Many viruses evade immune responses by con- tinually changing their antigens; they do so by introducing mutations in the genes that encode the immunogenic proteins. High antigenic variation can lead to evasion of the adaptive immune response, enabling the virus to infect its host unchecked. Influenza is an example of such a virus, with differ- ent variants predominating each year, each encoding antigenically distinct hemagglutinin and neuramini- dase viral glycoproteins.

Even though viruses have very small genomes, most pathogenic viruses include genes that encode immune-subverting molecules. Some viruses encode proteins that bind to their nucleic acid, precluding its sensing by host PRRs. Other viruses encode mole- cules, such as proteases, that degrade components of the inflammasome, preventing its activation. Yet oth- ers encode molecules that interfere with antigen pre- sentation, cytokine production, or cytokine signaling. These virally encoded molecules can cleave host pro- teins or act as cytokines or as decoy cytokine receptors that bind host cytokines, preventing normal immune activation.

Similar to viruses, bacteria also encode proteins to evade host immune responses. Group B streptococ- cus, which causes severe disease in the very young and very old, blocks the type I IFN response. Group A streptococcus (Streptococcus pyogenes), which causes a range of diseases including pharyngitis, scarlet fever, and skin infections, expresses a range of different proteins that inactivate or inhibit com- ponents of the complement cascade. Another major cause of human skin infections and more severe sys- temic disease, Staphylococcus aureus, can inhibit complement. S. aureus also possesses mechanisms enabling it to modulate and evade neutrophil bacteri- cidal actions, such as producing leukocidins that form pores in the neutrophil membranes and cause them to lyse. Some bacteria, including S. aureus, escape phagocytosis by secreting extracellular material that forms a capsule or slime layer. Mycobacterium tuberculosis, the cause of the potentially deadly lung disease tuberculosis, also interferes with phagocy- tosis by preventing formation of the phagolysosome, enabling its survival intracellularly.

More complex organisms, such as the Plasmodium species that cause malaria, possess multiple immune evasion strategies. Different stages of the

Plasmodium life cycle expose the immune system to different antigens, and the organisms are located in different tissues at each stage of the life cycle. This means an adaptive immune response against one stage of the life cycle may not affect the next stage. Notably, during the blood stage Plasmodium organ- isms live intracellularly within erythrocytes, one of the few cell types that does not express MHC I on its surface. This enables the organisms to evade the CD8 T-cell response.

Many other evasion mechanisms exist among pathogens. The complexity and intricacy of the immune system can be appreciated and under- stood, given the ongoing battle that exists between the human host and the invading microorganisms that constantly develop new mechanisms to escape immune attack.

VACCINATION Vaccination is the medical procedure by which immu- nological memory is induced in an individual without requiring an active infection. This prevents the indi- vidual from developing severe disease when he or she is subsequently exposed to a given pathogen. If sufficient individuals in a community are immunized against a pathogen, it causes herd immunity and prevents disease transmission within the population (Figure 6.30).

Vaccines generally contain noninfectious mate- rial from a pathogen that still includes the pathogen- specific antigens necessary to stimulate the adap- tive immune response. This material can be in many forms, such as proteins, live weakened (attenuated) viruses or bacteria, inactivated viruses, virus-like particles, and inactivated toxins (toxoids). Live vac- cines typically elicit a more robust immune response and greater protection than other types of vaccines. Live vaccines are able to replicate in the immunized individual and therefore amplify the antigen available. They also contain PAMPs that can elicit strong innate responses.

Nonlive vaccines require the use of adjuvants, which potentiate the immune response by activating PRRs to drive an innate immune response alongside the adap- tive response. Moreover, nonlive vaccines often require more boosts than live vaccines. Boosting occurs when an individual is revaccinated to create a larger antigen- specific memory B- and T-cell pool. Most vaccines require at least one boost to induce a protective mem- ory immune response.

Vaccination has led to major strides in human health. The devastating lethal disease caused by infec- tion with smallpox virus was eradicated through vac- cination. Poliovirus, which causes a potentially deadly paralytic disease in some infected individuals, has been eradicated in many countries through vaccination.

Chapter 6 • The Immune System and Leukocyte Function 197

In countries with strong immunization programs, dis- eases that were once commonplace, such as measles, diphtheria, mumps, rubella, and tetanus, are now very infrequent.

Vaccines are under development and in clini- cal trials for the global big three infectious disease

killers: tuberculosis, HIV, and malaria. Although the BCG vaccine already exists for tuberculosis and is used to protect neonates and young children in countries where the disease is endemic, it does not reliably protect adults against the disease. As new infectious diseases emerge, or old threats reemerge,

No one is immunized

Contagious disease spreads through the population

Some of the population gets immunized

Contagious disease spreads through some of the population

Most of the population gets immunized

Spread of contagious disease is contained

Not immunized but still healthy

Immunized and healthy

Not immunized, sick, and contagious

Key:

FIGURE 6.30 Herd immunity. In general, the greater the percentage of the population that receives recommended vaccinations, the broader the protection for all.

198 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

developing new and better vaccines remains a global health priority.

Although vaccines are subject to rigorous clini- cal testing for safety, very rarely they can have seri- ous adverse effects. Some people may have a serious allergic response to a vaccine component, so careful monitoring immediately after vaccination is necessary. Immune-compromised individuals are cautioned to avoid vaccination with live vaccines, as there is a risk they can develop disease. Maintaining high levels of vac- cination in healthy individuals is, therefore, very import- ant to protect vulnerable individuals in the community through herd immunity. Among the very common mild adverse effects after vaccination are short-lived pain at the vaccination site, fever, and malaise.

Despite the great improvements in human health that vaccination has achieved, there is a small but very vocal resistance against it. This antivaccination movement gained attention in the past few decades through a study published in a reputable medical jour- nal that falsely linked the early childhood vaccine for measles–mumps–rubella (MMR) with autism. Even though some of the data in the study were fabricated, the study design was faulty, and the publication was eventually retracted, the hype that the original publica- tion generated led to some intense public mistrust of vaccination. Evidence for a link between the MMR vac- cine and autism has not been supported by large pop- ulation studies.10 Furthermore, studies of safety of all vaccines include continuous and ongoing monitoring of adverse events to ensure that the benefits of vaccine use far outweigh any risks.11

Despite these and other sources supporting vaccine safety and the greater danger of vaccine avoidance to the individual and population health, false information about the dangers of vaccination is widely available on the Internet, and social media enables the fast spread of this type of misinformation. It is therefore vital that healthcare professionals appreciate the real fears that parents may have around vaccination, and take the time to explain the importance of vaccination for their own children and why vaccination is critical for preventing disease transmission to vulnerable members of society.

HYPERSENSITIVITY In a large and increasing proportion of individuals in developed countries, the immune system mistakenly gen- erates an immune response to a harmless molecule from the environment, such as food or pollen. Reexposure to that molecule can lead to a strong inflammatory response that causes varying degrees of discomfort and, in very severe cases, death. This type of reaction is known as hypersensitivity, atopy, or allergy. There are four types of hypersensitivity reaction (Figure 6.31).

Antigen–antibody complex

(d)

Mast cell

Mediators

Fc receptor

Antibody

Complement In�ammatory cell

Allergen

IgE

Macrophage

Cytokines

CD8+

T cell

CD4+

T cell

Blood vessel wall Neutrophils

(a)

(b)

(c)

FIGURE 6.31 Types of hypersensitivity. (a) Immediate hyper- sensitivity (type 1). Mast cell degranulation releases histamine, prostaglandins, and cytokines. Cytokines recruit eosinophils and other granulocytes; leukotrienes and eosinophil mediators perpetuate tissue damage. (b) Antibody-mediated (type 2). Specific antibodies alter function of receptors and other membrane proteins. Antibodies bound to tissues attract and activate complement and granulocyte attack mechanisms. (c) Immune complex–mediated (type 3). Antigen–antibody complexes deposit in subendothelial tissues, particularly in the kidneys and joints. Complement activation worsens tissue damage. (d) T cell–mediated (Type 4). Helper T cells and their cytokines provoke local cell recruitment and inflammation. Cytotoxic T cells kill altered self-cells. Fc, constant fragment; IgE, immunoglobulin E.

Chapter 6 • The Immune System and Leukocyte Function 199

Type 1: Immediate Hypersensitivity Type 1 reactions are known as immediate hypersensitiv- ity ( Figures 6.31a and 6.32 ). This response is mediated by the production of IgE that recognizes an environ- mental antigen (in this case, referred to as an allergen ). Once synthesized and secreted by B cells, IgE circulates throughout the body and binds to high- affi nity IgE recep- tors on the membranes of mast cells and basophils. Upon later exposures, the allergen binds to IgE, causing mast cell and basophil degranulation with immediate release of histamine and development of allergic infl ammation. The conditions that develop from type 1 reactions include hives, eczema, allergic rhinoconjunctivitis (hayfever, allergic rhinitis), allergic asthma, food allergy, and anaphylaxis. Although the initial allergic reaction occurs immediately, late-stage reactions occur several hours after allergen exposure as newly synthesized effector molecules take effect. Sustained allergic infl ammation can occur, for example, in chronic asthma and eczema.

The pathogenesis of type 1 hypersensitivity is not completely understood. It is thought to involve an inter- action between genetic predisposition and environ- mental exposures. There is a strong familial risk, with identical twins having 50% concordance for allergic dis- orders. 1 The major step leading to the development of allergies occurs when the body reacts to an allergen with activation of Th2 cells and a subset of T

fh cells that direct

a stimulated B cell to switch from IgM or IgG production to IgE production. The cytokines released ( Figure 6.32 ) direct this class- switching within the lymphoid follicle tissue, after which the B cell produces IgE specifi c to the allergen. Allergen exposures may come via the skin, causing atopic dermatitis (eczema); through the airways, producing asthma; or through the gastrointestinal tract, producing food allergies. As these disorders commonly present in childhood, additional information is provided in the later section on Pediatric Considerations.

Type 2: Antibody-Mediated Hypersensitivity Type 2 hypersensitivity reactions can occur when a host cell surface molecule becomes modifi ed by exposure to a chemically reactive molecule, leading the immune system to perceive the host molecule as non-self ( Figure 6.31b ). B cells produce IgM or IgG against the modifi ed host molecule, and IgG binding to host tissue leads to infl ammation and cell death mediated by com- plement and cell-mediated cytotoxicity. An example of this is hypersensitivity caused by the antibiotic penicillin. In other situations, type 2 hypersensitivity can be a mech- anism in autoimmune disease (see later discussion).

Type 3: Immune Complex–Mediated Hypersensitivity In type 3 hypersensitivity responses, IgM or IgG is produced against soluble foreign protein, leading to immune complex formation. Immune complexes are complexes of antigen, usually with IgG, that circulate in

the blood and can also form deposits in tissues and acti- vate the complement cascade, leading to infl ammation within that tissue and tissue damage. Examples include poststreptococcal glomerulonephritis, in which immune complexes deposit in the kidneys, and rheuma- toid arthritis, in which immune complexes to endoge- nous antigens are deposited in the joints ( Figure 6.31c ).

Type 4 (Delayed): T-Cell–Mediated Hypersensitivity Type 4 hypersensitivity is known as delayed-type hyper- sensitivity because it occurs several days after exposure to the antigen ( Figure 6.31d). This response is mediated by CD4 Th1 cells or CD8 T cells and most commonly manifests as a blistering skin rash. The delayed-type hypersensitivity response can be elicited by metals in jewelry such as nickel. Nickel ions that enter the skin can be chelated by histidine residues in host proteins, which are processed and presented to CD4 T cells as non-self. Other examples include the reaction that occurs to the oils on the leaves of poison ivy, and the reaction to pro- teins from tuberculosis that have been applied to the skin of an infected individual (formerly used as a diag- nostic test for tuberculosis [the Mantoux test]).

AUTOIMMUNITY Autoimmune diseases occur as an aberrant and destructive immune response against self. These responses can occur when immunological tolerance mechanisms break down. In primary lymphoid tissues (bone marrow and thymus), only B and T cells that do not respond to self-proteins, and therefore are tolerant to self, are able to survive and mature. This is known as central immunological tolerance.

Secondary mechanisms of inducing tolerance also exist, because primary immunological tolerance is not perfect, and some self-reactive cells do escape nega- tive selection in the primary lymphoid tissues and go on to mature. These peripheral mechanisms of tolerance include inducing unresponsiveness (anergy) or death in self-reactive cells when they encounter antigen without any additional activating signals, and inducing self-reac- tive cells to become Tregs or to be suppressed by Tregs.

When both central and peripheral tolerance fail, auto- immunity can occur. The diseases caused by autoimmu- nity are wide and varied in terms of the tissues and organs affected and the severity of disease. Some autoimmune disorders target one particular cell and tissue type (e.g., pancreatic β  cells in type 1 diabetes mellitus, acetylcho- line receptors in myasthenia gravis), while others have evidence of a variety of targets (e.g., rheumatoid arthritis [see Box 6.5, earlier], systemic lupus erythematosus, Sjögren syndrome). In many cases, assessment of anti- body titers is part of the diagnosis, indicating inappropri- ate activity of both B cells and T cells. Despite the rarity of individual autoimmune diseases, considered together, they are a major cause of disease in developed countries.

200 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

FcεRI

Vasoactive amines, prostaglandins

and leukotrienes

Immediate hypersensitivity

reaction (minutes after repeat exposure to

allergen)

Late phase reaction (6–24

hours after repeat exposure

to allergen)

Cytokines

Mast cell

IgE-secreting plasma cell

IgE

Th2/Tfh cell

B cell

Allergen

Mediators

First exposure to allergen

Allergen stimulates helper T cells. Th2 cell cytokines

promote B cell class-switching. IgE production begins.

IgE-producing B-cell clones differentiate to plasma cells.

IgE production increases.

IgE binds to mast cell Fc receptors, creating sensitized

mast cells around mucosal tissues and blood vessels.

Subsequent allergen exposure results in binding to mast

cell–bound IgE

Allergen binding to IgE triggers mast cell degranulation and

release of histamine. Mast cells synthesize prostaglandins and leukotrienes from arachidonic

acid. Mast cell cytokine secretion promotes chemotaxis

of granulocytes, particularly eosinophils.

FIGURE 6.32 Pathogenesis of type 1 hypersensitivity. Note that a critical step in the production of allergies is class-switching, which stimulates B cells to change from IgM or IgG to IgE production. Because mast cell membranes have IgE receptors, the IgE molecules can bind onto mast cells and sensitize them to degranulate (release granules of histamine) upon the next antigen exposure. Fc, constant fragment; FcεRI, high-affinity IgE receptor; IgE, immunoglobulin E; IgG, immun- oglobulin G; IgM, immunoglobulin M; T

fh cell, follicular helper T cell; Th2, T-helper type 2 cell.

Chapter 6 • The Immune System and Leukocyte Function 201

Factors that contribute to autoimmunity risk include female sex, older age, environmental exposures (ciga- rette smoking, obesity), and genetic factors (e.g., HLA genes, loss of function mutations in transcription fac- tors required for negative selection in the thymus or for Treg development). Notably, primary immunode- fi ciency is a strong risk factor for developing autoim- munity, with over a quarter of patients with primary immunodefi ciency reporting one or more autoimmune or infl ammatory symptoms. One further relatively new risk for developing autoimmunity is through the use of checkpoint inhibitors in cancer immunotherapy.

Although many of the risks associated with the devel- opment of autoimmunity are known, aside from the few loss of function genetic mutations that have been iden- tifi ed, the specifi c mechanisms involved in this process are largely unknown and are an area of intense research. The earliest stages of autoimmunity evade clinical detec- tion until proposed environmental triggers, acting on someone with a genetic susceptibility, result in the pro- duction of autoantibodies at detectable levels. As the condition progresses, tissues come under direct attack by antibodies and complement, as well as by immune complex deposition in tissues resulting in disease signs and symptoms. Ongoing stimulation of T and B cells increases cytokine levels, perpetuating the immune response, and producing tissue destruction and fi brosis ( Figure 6.33 ).

Increasingly, biologic agents, such as monoclonal antibodies, are being used to provide a more targeted approach for treating autoimmune disease than the older, broadly immunosuppressive drugs, thus reducing the risk of infection. It is important to acknowledge that all cur- rent therapies are disease modifying rather than curative.

IMMUNODEFICIENCY There are two main categories of immunodefi ciency: (a) primary, which are inherited or genetic immune defi ciencies, and (b) secondary, which are acquired during life as a result of infection, other diseases, med- ical intervention, or environmental factors such as stress and poor nutrition.

Primary immune defi ciencies affect over 10 million people worldwide. More than 150 different types, affecting both innate and adaptive arms of the immune response, have been defi ned. Primary immune defi cien- cies typically present early in life, as soon as protection against infection mediated by maternal antibody in the newborn has waned. 12

Primary immune defi ciencies can result from loss of function mutations in immune cell receptors, cytokines, chemokines, transcription factors, or complement com- ponents. These can lead to failure of a particular cyto- kine pathway or the complement cascade, or to broader

defi ciencies involving loss of whole immune cell subsets (e.g., severe combined immunodefi ciency [SCID]). Many primary immune defi ciencies are X-linked; thus, males are fully affected whereas females are chimeric and retain normal function in some cells.

Defects in specifi c pathways lead to susceptibil- ity to certain pathogens but not others. For example, individuals with defi ciencies in the MAC proteins of the complement cascade are exquisitely susceptible to infection with Neisseria species, but are able to defend against many other bacterial infections and have nor- mal responses against viral infection.

Loss of function mutations in the enzymes or cyto- kine receptors that orchestrate BCR and TCR gene rearrangement and B- and T-cell development, respec- tively, can lead to SCID as B and T cells fail to develop in the primary lymphoid organs. Affected individuals are devoid of conventional B and T cells and therefore are highly susceptible to a broad range of infections.

For severe primary immunodeficiency, the best outcomes occur with hematopoietic stem cell trans- plantation. For specifi c immune defi ciencies, regular intravenous immunoglobulin, careful monitoring, pro- phylactic antimicrobial use, and increased hygiene can help prevent severe infection.

Genetic susceptibility

Environmental triggers

Early autoreactivity

Autoantibodies detected

Antibody-tissue binding immune complex

deposition

Cell in�ltration, in�ammation

Tissue destruction, �brotic changes

FIGURE 6.33 General mechanisms in autoimmunity. The mechanisms underlying predisposition to autoimmune disease are not known, but family history is usually positive for members with one or more types of autoimmune disease. Against this background, it is thought that environmental exposures or prior infections can trigger autoreactivity. Autoantibodies are often present before any overt disease symptoms. Over time, the autoimmune response increases, with tissue damage resulting from immune and complement activation.

202 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

IMMUNE RESPONSES ACROSS THE LIFE SPAN

The immune system is signifi cantly weaker in infancy and older adulthood than during the rest of the life span ( Figure 6.34 ). During prenatal development, the fetus is protected by maternal antibodies that cross the placenta and confer passive immunity. The fetus can tolerate exposure to maternal antigens due to the higher activity of Tregs and Th2 cells, combined with low activity of Th1 and B cells. Similarly, the preg- nant woman is tolerant of fetal antigens, due in part to increases in Treg and Th2 activity with lower Th1 activity. At birth, a neonate immediately begins to experience colonization with commensal bacteria: from the vagina during a vaginal delivery, from the skin in a cesarean delivery, and via skin, gastrointestinal, and respiratory routes thereafter. In infancy, maternal passive immunity continues in the form of IgA that is secreted into milk during lactation.

Throughout early childhood development, natu- ral and vaccine exposures increase the immunolog- ical repertoire, helped by progressive maturation of lymphocyte function and lymphoid tissue devel- opment and function. Exposure to pathogens trains helper and effector cells that reach full functioning between the ages of 10 and 20 years. In older adults, Th2 responses are preserved, while activity of Th1 and B cells declines. In older adults, infections such as infl uenza have higher morbidity, mortality, and risk of developing secondary bacterial pneumonia. 13 , 14

Specifi c concerns in pediatric and gerontological populations follow.

The most prevalent cause of secondary immunode- fi ciency is HIV infection. HIV infects CD4 T cells and some macrophages, and if untreated, leads to signifi cant loss of immune cells throughout the body. This results in broad immune suppression, known as AIDS, in which infected individuals become susceptible to opportu- nistic infection with organisms that typically do not cause disease in immune-competent individuals. Until recently, HIV/AIDS was the leading cause of death by a single infectious agent. Robust public health measures to reduce transmission through sexual contact and needle sharing, together with the development and implemen- tation of antiretroviral therapy (ART) that can eliminate the risk of transmission, have contributed to the impres- sive global reduction in deaths due to HIV/AIDS.

Thought Questions

12. What are the similarities and diff erences in development, antigen recognition, and function between CD4- and CD8-bearing T cells?

13. What are major diff erences in cytokine secretion and function between the T-cell subtypes: Th1, Th2, Th17, T fh , and Tregs?

14. How would a tissue biopsy sample from a region of allergic infl ammation compare with one from a region of acute injury–induced (nonallergic) infl ammation, in terms of cell types and mediators?

S tr

en gt

h of

t he

re sp

on se

Years

1 10 20 30 40 50 60 70 80 90

Pregnancy

Maternal antibodies

Th1

Th2 B and innate

Treg

FIGURE 6.34 Immune responses across the life span. Th, T-helper cell; Treg, regulatory T cell.

• The Immune System And Leukocyte Function 203

PEDIATRIC CONSIDERATIONS Jane Tobias

As noted earlier, most or all of a child’s exposure to bacteria starts at birth, beginning the development of immune responsiveness. These exposures can vary with mode of birth (vaginal versus cesarean delivery), antibiotic exposure of the mother or infant, and adop- tion of breastfeeding. Bacterial colonization of the gut is thought to have primary importance in shaping immune responses throughout the childhood 15 ( Figures 6.35 and 6.36 ). Tolerance of commensal bacteria requires a balance of immunosuppressive activity during early development, with greater activity of Treg and Th2 cells and cytokines IL-10, IL-6, and IL-23. Adaptive immune responses are weak in infants, and immuno- globulin class-switching and affi nity maturation do not occur. Thus, infants are susceptible to infections, and vaccines must be boosted to achieve protection. 15

Later in childhood, environmental exposures to greater numbers of people and animals (siblings and extended family, pets, daycare) and locations (urban versus rural) continue to contribute challenges that mature the immune system (see Figure 6.35 ). This leads to greater activity of the innate immune system and greater lymphocyte diversity. The hygiene hypoth- esis posits that increased prevalence of type 1 hyper- sensitivity disorders in the developed world is due to decreased diversity of microbial exposures in early life. Trends in allergy prevalence are consistent with this hypothesis, with allergies increasing and then plateau- ing in resource-rich countries over the past 40 years, while allergies are now increasing in developing coun- tries that are acquiring more resources for clean air, water, and vaccinations. 16

Gestation

Th1/Th2 balance

Second trimester

Tolerance Immunological responsiveness

Third trimester

First trimester

Birth Infancy

Immune stem cell migration and expansion

Treg-cell upregulation and maternal antibodies transmission

Colonization of immune cells to effect site

Increased Treg levels and limited T-cell and antibody production

Increase in effector and memory T/B cells

Maturation of the innate and adaptive immunity

Balanced presence of effector cells/Tregs

Maternal immune system skewed tolerance

Maternal passive immunity

Bacterial colonization of the gut

Th1

Th2

FIGURE 6.35 Stages of immune development from gestation through infancy. Before birth, the fetal immune system is suppressed and does not respond to maternal antigens. Passive immunity is derived from the maternal antibodies that cross the placenta (before birth) and maternal IgA in breast milk (after birth). After birth, normal fl ora begins to colonize the gut and skin. The gradual increase in microbial diversity occurs against a background of increasing immune competence of the infant such that immune protection increases dramatically in the fi rst year of life. Th, T-helper cell; Treg, regulatory T cell.

204 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Increase in microbial diversity

Vaginal vs. cesarean birth, term vs. preterm birth, genetics of the host

Birth Diet Weaning

Breast fed vs. formula fed, antibiotics

Adult

310

Early life events associated with differences in intestinal microbial composition

Age (year)

Cessation of breast- feeding, transition to

solid food

FIGURE 6.36 Postnatal development of diversity of commensal bacteria. The trajectory of colonization with normal flora and infant microbial diversity is influenced by mode of birth (vaginal versus cesarean delivery), breastfeeding versus bottle-feeding, antibiotic exposure, and introduction of solid foods, as well as environmental exposures. Microbial diversity influences immune development and may influence later risk of autoimmune and hypersensitivity disorders.

TYPE 1 HYPERSENSITIVITY IN CHILDREN AND ADOLESCENTS

The global prevalence of allergy-related disorders in adolescents is reported to be greater than 40% (for any disorder), with allergic rhinoconjunctivitis (12.9%) and atopic dermatitis (8.1%) having the highest prevalence. There is a high rate of comorbidity for allergic rhinitis, atopic dermatitis, and asthma.17 Allergies often begin in childhood, with atopic dermatitis a common disor- der in early childhood, and they may wane in intensity through adulthood.18 Family history generally is posi- tive for siblings or parents with a history of allergy, although a specific gene has not yet been identified for all allergies.

MEDIATORS IN TYPE 1 HYPERSENSITIVITY The central pathways of type 1 hypersensitivity have been identified (see Figure 6.31 and earlier dis- cussion). Allergen exposure and release of IL-4 and IL-13 stimulate the Th2 phenotype, promoting B-cell class-switching from IgG production to IgE production. IgE production is critical to allergic responses as it is a step that sensitizes mast cells. Upon repeat exposure

to allergen, the mast cell coated with IgE degranulates, immediately releasing histamine, proteases, and cyto- kines, and begins to synthesize additional mediators.

Histamine rapidly produces vasodilation and increased vascular permeability, in addition to produc- ing the sensation of itch (in atopic dermatitis) or muco- sal irritation (in allergic rhinitis and asthma). Proteases produce local tissue damage and perpetuate inflam- mation. Cytokines induce chemotaxis in granulocytes (particularly eosinophils), and continue to perpetuate the immune response.

Of the arachidonic acid metabolites formed during an allergic response, prostaglandin D2 and leukotriene C4 have end-organ effects that contribute to the state of allergic inflammation. Prostaglandin D2 promotes vasodilation, facilitating granulocyte chemotaxis. Leukotrienes promote irritation and inflammation. In asthma, leukotrienes are implicated in bronchocon- striction and epithelial damage. Hours after an initial provocation, the late phase of an allergic response may result from the release of major basic protein, a toxic eosinophil mediator1,19 (Figure 6.37). Allergies are similar to disorders of chronic inflammation in that the affected tissue has persistent changes with the presence of long-lived immune cells: T cells, B cells,

Chapter 6 • The Immune System and Leukocyte Function 205

and macrophages. Differences between these chronic states include the predominance of IL-4 and IL-13 in allergic infl ammation, along with systemic titers of IgE, and local persistence of eosinophils in an aller- gic focus. Both states may produce local scar tissue and fi brotic changes that lead to permanent structural alterations.

ATOPIC DERMATITIS

The natural history of atopic dermatitis is given here as an example of the pathogenesis of allergic disorders. An early insult that disrupts the epithelial layer initi- ates the cascade of events shown in Figure 6.38 . The potential role of epithelial damage is highlighted by the fact that some individuals with atopic dermatitis and other immunological skin disorders have mutations in the fi laggrin gene that encodes the epidermal cell pro- tein fi laggrin. Abnormal function of this protein is one pathway to epidermal disruption leading to antigen entry. Langerhans cells are APCs of the skin and are the

fi rst responders to antigens that come through the dam- aged skin barrier. They interact with dermal dendritic cells and migrate to the draining lymph node, where activation of Th2 cells occurs. Th2 cells secrete IL-4, promoting B-cell class-switching to IgE production for the invading antigen. IgE binds to mast cells, which are now primed to degranulate upon further encounters with antigen, releasing histamine, which causes vaso- dilation, redness, and intense itching. 20 A vicious cycle ensues, with additional skin disruption, antigen inva- sion, and immune cell proliferation that ramps up the infl ammatory response.

Th2 cytokines, IL-4, IL-13, and IL-31, are particu- larly involved in perpetuation of the infl ammatory response, but several categories of T cells contribute. As with other atopic disorders, eotaxin is secreted and recruits eosinophils to the region. Eosinophil secre- tion of major basic protein contributes to tissue injury and infl ammation. A child or adolescent with atopic dermatitis is more prone to begin to react to other skin-associated antigens, resulting in allergic contact dermatitis. These children will have a positive skin

Antigen (allergen)

Antigen (allergen)

Mast cell activation

Epithelium basement membrane

B cell

TCR

IL-4

MHCII IgE production IgE

FcεRI IgE

Histamine, PAF, PGs, LTs

Degranulation

Cytokine secretion

AA, arachidonic acid products

Prostaglandin D2, leukotriene C4

IL-4, IL-5, IL6

Ca+2

Th2 cell

MC

APC

TNF-a, IL-1b , IL-13. Allergic in�ammation

FIGURE 6.37 Cells and mediators of type 1 hypersensitivity. Th2 cytokines IL-4 and IL-13 contribute to B-cell class-switching. Primed MCs reinforce the cytokine milieu with secretion of IL-4, IL-5, and IL-6. Allergen exposure causes mast cell degranulation and production of infl ammatory PGs, LTs, and PAF, while also recruiting eosinophils and neutrophils to the allergic infl ammatory site. Hours after the acute episode, tissue damage can result from eosinophils releasing major basic protein, a toxic protein. AA, arachidonic acid; APC, antigen-presenting cell; FcεRI, high-affi nity IgE receptor; IgE, immunoglobulin E; IL, interleukin; LTs, leukotrienes; MCs, mast cells; MHC II, major histocompatibility complex class II; PAF, platelet-activating factor; PGs, prostaglandins; TCR, T-cell receptor; Th2, T-helper 2; TNF-α , tumor necrosis factor alpha.

206 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

prick response to allergy testing, as well as positive tests for circulating IgE.20

FOOD ALLERGIES

In the pediatric patient, almost any food can produce an immune response triggering a reaction. Reactions to foods can occur at any age; however, food intolerance usually manifests in early childhood. Although gastro- intestinal exposure to most foreign proteins is usually tolerance inducing, up to 6% of children experience food allergic reactions in the first 3 years of life, includ- ing approximately 2.5% with cow’s milk allergy, 1.5% with egg allergy, and 1% with peanut allergy. Peanut allergy prevalence has tripled over the past decade. Most children outgrow milk and egg allergies, but approximately 80% to 90% of children with peanut, nut, or seafood allergy retain their allergy for life.21

Food intolerances result from a variety of mecha- nisms, whereas the pathophysiology of food allergy is predominantly IgE and cell mediated.22 Similar to other atopic disorders, exposure to the particular food in a susceptible pediatric patient results in the formation of specific IgE antibodies that bind to receptors on

the mast cells, basophils, macrophages, and dendritic cells. Air-borne exposure to vegetable antigens may also result in allergies to the ingested food. It is through the release of histamine and other mediators that the pediatric patient demonstrates both local and systemic reactions.

Symptoms manifested during IgE-mediated food reactions are not restricted to the gastrointestinal tract. Skin reactions include urticaria, angioedema, and flushing. Respiratory symptoms include nasal con- gestion, rhinorrhea, nasal pruritus, sneezing, laryngeal edema, dyspnea, and wheezing. Gastrointestinal symp- toms include oral pruritus, nausea, abdominal pain, vomiting, and diarrhea. Severe reactions may proceed to shock and anaphylaxis. Food reactions are the sin- gle most common cause of anaphylaxis seen in hospital EDs in the United States.

Until recently, families were encouraged to avoid the exposure of infants to allergenic foods. This manage- ment actually tended to increase rates of food allergy. Current recommended practice is exclusive breastfeed- ing for 4 to 6 months, followed by gradual introduction of all foods. Slow introduction should include common food allergens including cow’s milk, other dairy prod- ucts, eggs, wheat, peanut, and soy.

Antigen

LC

iDEC Eotaxin

Lymph node

Nerve T cell

IL-4

T cell

T cell

B cell

Th2

Th2

Th1Th22Th1/

IgE class- switching

TSLP

IL-4 IL-13 IL-31

IL-22TNF histamine

Blood vessel

EOS

EOS Mast cell Mast cell

IFNγIL-17 IL-22

MBP TNFα

TSLPR

OX40L

OX40

KCs

Disturbed barrier ( TEWL)

dDC

dDC

DCdDC

LC

LC

FIGURE 6.38 Cells and mediators in atopic dermatitis. Atopic dermatitis is another form of type 1 hypersensitivity. The cell types involved begin their interaction in the lymph node, but long-term alterations in cytokine levels predispose to ongoing lesion production in affected skin. DC, dendritic cell; dDC, dermal DC; EOS, eosinophil; iDEC, inflammatory dendritic epidermal cell; IFN-γ , interferon gamma; IgE, immunoglobulin E; IL, interleukin; KCs, keratinocytes; LC, Langerhans cell; MBP, (eosinophil) major basic protein; OX40, stimulatory receptor on Th2 cell; OX40L, OX40 ligand; TEWL, transepidermal water loss; TNF, tumor necrosis factor; TSLP, thymic stromal lymphopoietin; TSLPR, TSLP receptor.

Chapter 6 • The Immune System and Leukocyte Function 207

GERONTOLOGICAL CONSIDERATIONS Nancy Tkacs

TABLE 6.7 Immune Changes With Aging

Type of Tissue Changes

Primary lymphoid tissue: bone marrow ↓ lymphoid progenitors ↓ progenitor self-renewal ↑ DNA damage

Primary lymphoid tissue: thymus ↓ thymus tissue mass (involution) ↓ T-cell seeding from bone marrow ↓ thymus cellular output ↑ adipose deposition in thymus

Secondary lymphoid tissue: spleen and lymph nodes ↓ naïve T-cell numbers ↓ follicular cell developmental network ↓ dendritic cell antigen presentation ↑ infl ammatory cytokines ↑ TGF-β  ↑ pool of memory T cells

Tissues and organ systems ↓ specifi c immune protection ↓ proliferation of naïve and memory T cells ↓ eff ector molecule production and secretion ↑ nonspecifi c infl ammation

TGF-β , transforming growth factor beta.

Immune system activity decreases with age, with adaptive immunity impaired more than innate immu- nity ( Table 6.7 ). This phenomenon has been referred to as immunosenescence . 23 Age-related immune system alterations lead to an increased number of infections, as well as greater infection-associated morbidity and mor- tality rates; diminished wound healing; decreased effec- tiveness of adaptive immunity, including responses to vaccines; and decreased immune surveillance, contrib- uting to increased cancer incidence. At the same time, there are two opposing changes in innate immunity: (a) Decreased function of neutrophils, monocytes/macro- phages, dendritic cells, and NK cells increases vulnerabil- ity to infections, while (b) there is a general increase in infl ammatory mediators, particularly IL-1β  and IL-6, and TNF-α . The process of immunosenescence explains, in part, why older adults often have multiple comorbid con- ditions and conditions with an infl ammatory component.

IMMUNE CELLULAR SENESCENCE AND DISEASE

Throughout the life, the adaptive immune system builds up an armament against pathogens through T-cell–directed cellular and humoral activity. Both T cells and B cells acquire memory of pathogens fol- lowing exposures to their antigens and are able to

launch fast and effective responses following subse- quent exposures. These cells and related cell recep- tors of the adaptive immune system are maintained into the sixth decade, and then gradually decline with age. Multipotent stem cells in the bone marrow gener- ally reduce replication rates, potentially due to short- ening of telomeres. The bone marrow’s lymphocyte production rate specifi cally decreases, with a shift to greater numbers of cells progressing to the myeloid lineage. Specifi c to T cells, throughout adulthood, the thymus gland undergoes involution, leading to a decline of naive T cells. Thymic involution acceler- ates after age 70. With slow but progressive losses in naive and memory T and B cells, susceptibility to pathogens increases. 13 , 14

The progressive weakening of adaptive immunity with aging is manifested in a number of ways:

• Vaccinations are less effective and may need addi- tional boosters, adjuvants, or higher doses.

• Latent infections may reemerge—the most common of these is the reactivation of varicella zoster virus, producing shingles.

• Cancer occurrence increases owing to inadequate immune surveillance.

• An additional consequence is that the recently devel- oped immune-based cancer therapies may not be as effective in older adults as in younger adults.

208 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Stimuli Effectors Increasing levels of:

PAMPs bacterial

(environmental, altered

microbiota), viral (CMV, in�uenza)

DAMPs dying + senescent

cells, cancer, degenerative

disorders, adiposity, accumulated cell damage: DNA,

proteins, mitochondria

Multiple cells change activity

via: PRR NF-κB

in�ammasome, epigenetic

modi�cations, miRNAs

IL-1b IL-6 IL-8

TNF-a

IFN-g

CRP

FIGURE 6.39 Inflammaging. A number of age-related exposures and changes contribute to increased levels of chronic inflammatory cytokines and other mediators in some older adults. This inflammatory milieu may then accelerate age-related dysfunction. CMV, cytomegalovirus; CRP, C-reactive protein; DAMP, danger-associated molecular pattern; IFN-γ , interferon gamma; IL, interleukin; miRNA, microRNA; NF-kB, nuclear factor kappa B; PAMP, pathogen-associated molecular pattern; PRR, pattern recognition receptor; TNF, tumor necrosis factor.

INFLAMMAGING

The concept of inflammaging is based on obser- vations that the levels of inflammatory mediators generally increase with aging. Accelerated stimula- tion by pathogens (PAMPs of bacteria and viruses) and altered self-proteins and cells (DAMPs released from damaged and dying cells) are detected by PRRs, stimulating the pathways of innate inflam- mation, including cytokine release. At the cellu- lar level, cell senescence is a state reached as cells stop replicating because of injury or aging and telo- mere shortening. These cells can transition to the senescence-associated secretory phenotype (SASP) in which they release damage signals and proin- flammatory mediators. Proinflammatory conditions such as atherosclerosis, insulin resistance and type

2 diabetes mellitus, increased adiposity, cancer, and alterations in gut microbiota all contribute to this heightened inflammatory state in a vicious cycle that also worsens those conditions (Figure 6.39).24–28

With the increased inflammatory state, the dam- aging effects of innate immune activation are per- petuated, including oxidative stress; nucleic acid and protein damage; and cell, tissue, and organ dys- function. Heightened inflammation may contribute to frailty, loss of function, and earlier death. Rates of age-related progression into this state are very vari- able and are influenced by genetics, environment, diet, activity level, stress, social determinants of health, and numerous psychosocial factors. The biological path- ways and mechanisms connecting aging to the height- ened inflammatory state and its associated effects on morbidity and mortality remain to be elucidated.

Chapter 6 • The Immune System and Leukocyte Function 209

Linda W. Good

Chief Complaint: “I’m here for my sinuses. I know what I have, and I always need a Z-pak to get over this,” the patient states as he enters the examination room. This healthy-appearing 27-year-old Caucasian man then describes 10 days of alternately stuffy and runny nose, postnasal drip, sneezing, and “that deep itch that I can’t get to scratch.” He explains, “This happens every year at this time and I take Sudafed, which keeps me from sleeping and leaves me tired and less able to work, so now I’ve started Afrin instead, because I can’t breathe without it.”

History of Present Illness/Review of Systems: You elicit the following additional facts: The patient has had no fevers and no pain or pressure in the ears or face, but has had mild sore throat that is worse in the mornings. Upon closer questioning, you suspect that the patient’s inability to breathe is related to nasal obstruction and not the lungs. He denies wheezing, shortness of breath, coughing, or sputum production, but admits to tearing and burning of his eyes. Review of systems is negative for any cardiovascular, gastrointestinal, musculoskeletal, or neurosensory concerns. His sleep has been somewhat disrupted, and appetite and energy a little low, which he attributes to the decongestant medication.

Past Medical/Family History: The patient’s past medical history is signifi cant for childhood asthma

and eczema. He had otitis media frequently growing up and his mother told him he was on repetitive courses of antibiotics as a child. Both parents were smokers, but he never smoked. He grew up with cats and thought that they might have caused his asthma, so he has only had dogs as pets since he left home. He works as an inspector for the water company, which requires going into basements of homes.

Physical Examination: Findings are as follows: temperature of 97°F, blood pressure of 116/70 mm Hg, heart rate of 60 beats/min, and respirations of 12 breaths/min. BMI is 22 kg/m 2 . You note good landmarks and normal color of the tympanic membranes; no palpable facial tenderness or cervical adenopathy, but mildly erythematous oropharynx with cobblestoning of the posterior pharyngeal wall. Nasal turbinates are swollen bilaterally, with clear rhinorrhea visible anteriorly. Lungs are clear and abdominal examination is benign. Routine CBC and CMP results are normal.

CASE STUDY 6.1 QUESTIONS • What factors in the patient’s history indicate

sources of upper airway irritation? • What mediator released during a type 1 hypers-

ensitivity reaction can contribute to mucosal edema and swelling, nasal stuffi ness, and itchy nose and eyes?

CASE STUDY 6.1: A Patient With Allergic Rhinitis CASE STUDY 6.1:

BMI, body mass index; CBC, complete blood count; CMP, comprehensive metabolic panel.

210 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

Ben Cocchiaro

PRINCIPLES OF ASSESSMENT

History Evaluate: • Constitutional symptoms: fevers, pain (joint pain

and other), weight loss, anorexia, fatigue • Potential exposures in suspected allergies: pets,

work environment, associations with food and drink

• System-speci� c symptoms related to: ❍ Allergic rhinitis: ENT—stuffy nose, runny

nose, sneezing, itchy eyes, postnasal drip, and cough

❍ Asthma—chest tightness, dyspnea, cough, wheezing, worsening with exposures

❍ Atopic dermatitis—intense itching, infl am- matory papules

❍ Rheumatoid arthritis—joint pain, swelling, and stiffness, diffi culties with activities of daily living, constitutional symptoms

• Suggestive � ndings in immune disorders: patients may have more than one autoimmune or hyper- sensitivity disorder; family history is common; note history of exacerbations and remissions

• History of drug allergies must focus on speci� c reac- tions: nonspecifi c rash, nausea, and vomiting (less likely to be true allergy); hives, diffi culty breathing (more likely to be true allergy)

• Recurrent infections may be a sign of immuno- defi ciencies

Physical Examination Findings may be specifi c to organ system: • ENT: boggy nasal mucosa, red and watery eyes,

erythema and cobblestoning of pharynx • Asthma: wheezing, use of accessory muscles

of respiration, cyanosis if airflow severely limited

• Arthritis: swollen, deformed joints with redness and warmth

• Atopic dermatitis: redness, popular rash, scaly, thickened skin

Diagnostic Tools • Radiography for joint-related disorders • MRI—can be used in multiple sclerosis • Tissue biopsy

Laboratory Evaluation • Complete blood count with differential • C-reactive protein (acute phase protein): non-

specifi c indicator of infl ammation • ESR: nonspecifi c indicator of infl ammation • Immunoassays of IgG, IgM (autoantibodies, vaccine

titers): specifi c antibodies are diagnostic for cer- tain autoimmune disorders ❍ Systemic lupus erythematosus—ANA ❍ Rheumatoid arthritis—rheumatoid factor and

anticyclic citrullinated peptide antibodies ❍ Type 1 diabetes—antiislet antibodies, anti-

glutamic acid decarboxylase (GAD) antibodies • Allergy skin testing

MAJOR DRUG CLASSES • Glucocorticoid steroids : generally immuno-

suppressive, may be directly applied to target tissues (inhaled corticosteroids for asthma, topi- cal corticosteroids for atopic dermatitis, intraar- ticular injection for joint disorders). Localized or short-term systemic use is preferable to avoid adverse effects of long-term administration

• Antihistamines : often used to reduce allergic responses

• Nonsteroidal antiin� ammatory drugs : inhibit pro- duction of arachidonic acid metabolites prosta- glandins and leukotrienes. Adverse effects can occur, particularly in older adults.

• Disease-modifying antirheumatic drugs (DMARDs)

• Synthetic immunosuppressants : methotrexate, aza- thioprine, chloroquine, cyclophosphamide, cyc- losporine, lefl unomide, sulfasalazine, tofacitinib

• Biologics (monoclonal antibodies): adalimumab, etanercept, infl iximab, rituximab, golimumab (see also Table 6.6 )

BRIDGE TO CLINICAL PRACTICE BRIDGE TO CLINICAL PRACTICE

ANA, antinuclear antibodies; ENT, eyes/nose/throat; ESR, erythrocyte sedimentation rate; GAD, glutamic acid decarboxylase; IgG, immunoglobulin G; IgM, immunoglobulinM.

Chapter 6 • The Immune System and Leukocyte Function 211

KEY POINTS

• The immune system comprises body mecha- nisms that protect from microbial invaders and cancerous cells at the levels of physical barri- ers (skin and mucous membranes); a rapidly activated, nonselective, innate immune sys- tem; and a slowly developing but highly spe- cifi c adaptive immune system.

• The innate immune response recognizes gen- eral molecular patterns of pathogens and rap- idly recruits many phagocytic cells to engulf and destroy the pathogens.

• The adaptive immune response recognizes spe- cifi c patterns of pathogens called antigens and stimulates limited numbers of B and T lympho- cytes responsive to those antigens to prolifer- ate and generate immune defenses.

• The primary lymphoid organs are the bone marrow, site of lymphocyte production and B-cell development, and the thymus gland, where T cells develop.

• Lymph nodes are secondary lymphoid organs and are the sites of communication between antigen-presenting cells, T cells, and B cells during the developing immune response to antigen.

• B lymphocytes express B-cell receptors with antigen-recognizing characteristics similar to the antibodies they will eventually secrete. Gene rearrangement is the method by which a small number of B-cell receptor genes can code for a huge number of potential antibodies.

• T lymphocytes express T-cell receptors from genes that have undergone gene rearrange- ment, creating a great diversity of antigen- recognition possibilities. T cells, however, can only recognize antigens presented on mem- brane proteins termed major histocompatibil- ity class (MHC) proteins.

• The lymph nodes, spleen, and gut-associated lymphoid tissues are the major sites of antigen presentation and interactions of T cells and B cells.

• The innate immune response is initiated when tissue injury and invasion stimulates sentinel cells such as macrophages and mast cells.

• Acute infl ammation ensues, with release of chemotactic factors that rapidly recruit addi- tional neutrophils and monocytes into the region of injury.

• Leukocyte chemotaxis is added by the release of histamine and prostaglandins that pro- duce vasodilation and increased capillary permeability.

• Neutrophils and macrophages phagocytose invading organisms and release cytokines that stimulate further chemotaxis, as well as pro- moting bone marrow leukocyte proliferation.

• Complement proteins are synthesized by the liver and circulate as pro-proteins that need cleavage for activation (similar to the clot- ting cascade). Complement is activated by bacterial exposure, antibody binding, and mannose-binding lectin. The cascade gener- ates proteins that directly opsonize and kill bacteria, and promote chemotaxis.

• NK cells are innate lymphoid cells in that they have lymphoid origins but function like cells of the innate immune system. They are partic- ularly active at killing virus-infected and can- cerous cells. The roles of recently identifi ed innate lymphoid cells are still being defi ned.

• Chronic infl ammation is a pathological state in which a localized immune response does not proceed to normal wound healing. Instead, long-lived macrophages, T cells, and B cells perpetuate a response to one or more tissue antigens, creating pain, swelling, and impaired function.

• Adaptive immunity allows for specifi c target- ing of one antigen or invading organism and neutralizing it by a combination of antibodies that mark circulating extracellular antigens for destruction and cell-mediated immunity: lysis of self-cells infected with intracellular pathogens.

• B cells recognize circulating antigens through their B-cell receptors, migrate to lymph nodes and tissues, and are stimulated to maturation and activation by T-helper cells selective for the same antigen.

• Once mature, B cells proliferate and secrete antibodies, beginning with IgM and maturing to IgG secretion. Continued presence of the anti- gen stimulates affi nity maturation of B cells to produce antibodies with increasing affi nity for antigen.

• B cells ultimately differentiate into (a) plasma cells—large cells with greatly enhanced capac- ity for antibody secretion, and (b) memory cells—long-lived cells that can rapidly activate in response to a later encounter with their anti- gen. Memory B cells can provide protection for many years to decades.

• Antibodies can bind to and neutralize toxins, opsonize bacteria to promote phagocytosis, and bind to bacteria to activate complement- mediated killing. When a pathogen is encoun- tered for the second time, the rapid production

212 Advanced Physiology and Pathophysiology: Essentials for Clinical Practice

of antibodies clears the pathogen, often before any illness is experienced.

• IgG is the most abundant antibody, whereas IgA predominates at mucosal surfaces. IgE is present at very low levels but is elevated in individuals with allergies.

• T cells can be subdivided based on their mem- brane proteins. The marker CD4 is associated with T-helper cells, whereas the marker CD8 is associated with cytotoxic T cells.

• CD4 T cells recognize antigen presented by specialized APCs on the APC cell MHC II mol- ecules. Once activated, many CD4 cells stim- ulate B-cell responses and other responses of adaptive immunity. These cells are further subdivided into Th1, Th2, Th17, T

fh , and Tregs,

each with a unique spectrum of activity. • All nucleated cells express MHC I proteins on

their membranes. Intracellular protein turn- over is linked to the presentation of protein fragments on MHC I. When a virus-infected or cancerous cell begins to display abnormal pro- tein antigens on MHC I, CD8 cells bind via their T-cell receptors.

• CD8 cells release perforin and granzyme enzymes to lyse infected/abnormal cells, and can also trigger apoptosis via the Fas pathway.

• Additional lymphocyte classes provide protec- tion of epithelia, secrete cytokines, and have NK cell attributes.

• Memory T cells of both the CD4 and CD8 types are produced throughout the life span. They may reside in the tissue where their antigen was first encountered, or circulate through the blood, tissues, and lymph tissues.

• Through vaccination, people are exposed to antigenic determinants or whole (weakened) injections of major infectious pathogens, pro- voking a primary immune response and mem- ory cell formation. A booster dose leads to a secondary immune response and long-lasting protection. Empirical evidence determines the need for additional booster shots at certain intervals.

• Hypersensitivity reactions are excessive immune responses to normally innocuous stimuli. The most common form, type 1 hyper- sensitivity, is also known as atopy or allergy. In allergy, individuals respond to allergen expo- sure with Th2-promoted class-switching of B cells to produce IgE instead of IgG. Mast cells become sensitized through IgE binding to mast cell membrane receptors for the IgE constant

region. Subsequent exposures lead to mast cell degranulation and generation of an allergic inflammatory response in the exposed tissue.

• Hypersensitivity can also overlap with autoimmunity when antibodies are pro- duced to self-proteins. This is termed type 2 hypersensitivity.

• Autoimmunity is the pathological immune response to one’s own cells and tissues. The genetics of autoimmunity involves, in part, certain classes of HLA genes that code for the MHC proteins. Vulnerable individu- als often have more than one autoimmune disorder.

• Immunodeficiency disorders are less common than hypersensitivity and autoimmunity. The most common immunodeficiency is secondary, occurring in patients with HIV infection who have loss of CD4 cells to viral attack.

• Fetal development includes development of the main features of the immune system, but the cells and tissues are in a latent stage until birth. At birth, neonates have circulating IgG that crossed the placenta from the mother’s blood. In the early postnatal period, vulnerabil- ity to infections is high, as this passive protec- tion wanes.

• Colonization with commensal bacteria begins at birth, and the spectrum of bacteria var- ies with method of delivery (vaginal versus cesarean). In the first several months of life, the infant is exposed to many environmental microorganisms, and often has many immuni- zations. Thus, within the first year of life, the immune system has gained experience and strength in responding to pathogens.

• Early in life, treatment with antibiotics or fail- ure to experience certain exposures may lead to later life immune vulnerabilities, particu- larly allergies.

• In older adults, the thymus gland involutes, with decreased immune function and accumu- lation of fatty, nonfunctional tissue. Few naive T cells are produced, and the diversity of the T-cell repertoire decreases. Infections induce greater morbidity and mortality, and vaccines are less effective.

• The cells of innate immunity are less active in older adults, but other cells can develop an inflammatory phenotype. Cytokine levels tend to be higher in older adults, and contribute to age-related decline in function, in the phenom- enon of inflammaging.

Chapter 6 • The Immune System and Leukocyte Function 213

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