immune system

 155

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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