Diseases of Immunity

General Features of the Immune System

The immune system is a defensive system whose primary functions are to protect against infectious organisms, such as bacteria, viruses, fungi, and parasites, and the development of cancer. The complexity by which these functions occur is evidenced not only by the cell types, recognition molecules, and soluble factors involved and interactions with other systems (e.g., endocrine, nervous) but also by the ability to recognize virtually any foreign antigen. Immunologic responses result in pathologic processes, primarily inflammatory responses, as a result of either normal immune responses to foreign antigens (e.g., microbial pathogens) or from aberrations of the immune system as in the case of hypersensitivity reactions and autoimmune diseases. Finally, the importance of a normal functional immune system cannot be more evident than in instances in which it is deficient as the result of a genetic defect or as the result of an acquired immunodeficiency disease.

Immunity is the result of nonspecific (innate) and specific (adaptive) responses that together provide effective protection. The immune system’s recognition and response functional capabilities are key components of both innate and adaptive immune responses. The recognition capabilities are highly specific and allow immune responses to develop against a diverse group of foreign (nonself) antigens and prevent the development of immune responses to self-antigens. Innate and adaptive immune responses feature effector mechanisms for eliminating or neutralizing the antigen, whereas adaptive immunity has the additional feature of memory. The emphasis of this chapter is on diseases that are the result of inadequate or inappropriate immune responses. Before one can understand the pathogenic mechanisms of these diseases, one must first have an understanding of the basic elements of the immune system. The chapter begins with an overview of our current understanding of innate and adaptive immunity, cells of the immune system, cytokines, and major histocompatibility complex (MHC) molecules. This overview facilitates the discussion of disorders of the immune system that includes hypersensitivity reactions, autoimmunity, and immunodeficiency. This chapter concludes with a discussion of amyloidosis, a diverse group of conditions characterized by the deposition of a pathologic extracellular protein. One of the conditions is associated with the deposition of immunoglobulin components. Although the focus of this text is on the pathologic basis of veterinary diseases, with an emphasis on domestic species, in this chapter we use the vast knowledge base regarding human and rodent immunology (applicable to most mammalian species studied to date) as our basis and interject major known relevant species differences as appropriate.

Innate Immunity (Nonspecific Immunity)

As stated previously, the function of the immune system is to protect against infectious pathogens and the development of cancer. There are two categories of immune responses that are based in part on their specificity for the antigen: (1) innate immunity and (2) adaptive immunity (Fig. 5-1). Innate immune responses are considered the first-line defense mechanisms, are not specific to the antigen, and lack memory. These defense mechanisms are the result of anatomic (e.g., skin, mucosal epithelia, cilia) and physiologic (e.g., stomach pH, body temperature) properties, and phagocytic and inflammatory responses. Major components of innate immunity are intact epithelial barriers, phagocytic cells, natural killer (NK) cells, and a number of plasma proteins, the most important of which are the proteins of the complement system. Phagocytic cells are recruited to sites of infection during an inflammatory response where they have a number of functions, two of which are to ingest and destroy pathogenic organisms and neutralize toxins. Neutrophils, monocytes, and tissue macrophages are the major cells involved in phagocytosis. These cells recognize components of microbial pathogens through the expression of several membrane receptors, including receptors for mannose residues and N-formyl-methionine containing peptides and a family of pattern recognition receptors (PRRs) that, when activated by microbial components, signal the activation of transcription factors that facilitate the microbicidal mechanisms of the phagocytic cell and are discussed later. NK cells are the cytotoxic cells of innate immunity and are also discussed later. The complement system, discussed in Chapter 3, is a complex cascade of proteins that has a number of biologic functions, including the formation of the membrane attack complex that efficiently lyses plasma membranes of microbial pathogens. The complement system can be activated by either the innate immune system (alternative and mannose/lectin pathways) or the adaptive immune system (classical pathway). Other important plasma proteins of the innate immune system include mannose-binding protein and C-reactive protein; two of the functions of these proteins are to facilitate phagocytosis through opsonization of pathogens and complement activation. Inflammatory responses comprise vascular, permeability, and cellular phases that act in response to damage to vascularized tissue. The features of the inflammatory response are also presented in Chapter 3.

Fig. 5-1 Innate (nonspecific) and adaptive (specific) immunity are depicted in relation to the time course of an infection.
NK, Natural killer. (From Kumar V, Abbas AK, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005, Saunders.)

Recognition Molecules of Innate Immunity

The recognition molecules of innate immunity provide an opportunity to recognize pathogens by cells other than lymphocytes. These germline-encoded molecules function to sense molecular structures shared by microbes and endogenous molecules associated with inflammation. The recognition molecules associated with microbes are generally referred to as pathogen-associated molecular patterns (PAMPs). The function of the innate immune system extends beyond microbial pathogen recognition to include endogenous molecules associated with cell damage and inflammation which are generally referred to as danger or damage associated molecular patterns (DAMPs). The activation of the innate immune system through these invariant PRRs not only precedes lymphocyte activation but also is required to initiate the adaptive immune responses. PRRs molecular patterns are associated with microbial pathogens and classified as secreted, transmembrane, and cytosolic forms. Collectins and pentraxin are examples of secreted PRRs that largely function to bind to microbial surfaces and activate the complement system. Collectins, having properties of collagen and lectins, include mannose binding lectins and pulmonary surfactants A and D. Pentraxin is comprised of five identical subunits to form a pentamer and include C-reactive protein, an activator of the classical pathway of complement. Toll-like receptors (TLRs) and C-type lectins are examples of the transmembrane PRRs, and they have limited cellular distribution, including macrophages, NK cells, and dendritic cells. TLRs are expressed on the plasma membrane or in endosomal/lysosomal organelles. The cytosolic PRRs are more widely distributed, including all nucleated cells, and include retinoic acid–inducible gene (RIG)-1–like receptors (RLRs) and nucleotide-binding domain (NOD) and leucine-rich repeat–containing receptors (NLRs). These recognition molecules provide the host with the ability to sense “danger” either through PAMPs in the case of microbial infections or through DAMPs in the presence of cellular damage or stress. This allows the innate immune system to detect and initiate immune responses in response to infectious and noninfectious causes. NLR molecules as immune sensors are not unique to mammals because they have been found in plants and urchins. The molecules are designated based on the structure of the molecule NLR with a suffix of P (pyrin domain) or C (caspase activation and recruitment domains [CARD]), referring to the N-terminal moiety followed by a number (e.g., NLRP1, NLRP2, or NLRP3). The function of these pattern recognition molecules is also extended beyond the initiation of adaptive immunity to also include regulation of cell death (apoptosis). As is often the case in immunology, our understanding of immune responses is greatly enhanced through the identification of genetic immunologic disorders. Deficiencies in components of the NLRs have been described in humans and are most frequently associated with inflammatory disorders (e.g., Crohn’s disease and inflammatory bowel disease).

Analogous to the adaptive immune response, which has developed the ability to defend the host against a diverse array of microbial pathogens (humoral versus cell-mediated responses), the innate immune system is now seen as also having an ability to distinguish specific types of pathogens. Intracellular (e.g., viruses) and extracellular (extracellular bacteria) pathogens require different types of immune responses to control them. The innate immune system has developed cell-intrinsic and cell-extrinsic recognition mechanisms dependent on if it is mediated by an infected cell or an uninfected cell. Cell-extrinsic recognition mechanisms are a way in which an uninfected cell can participate in the immune response and are mediated through transmembrane receptors (e.g., TLRs) on specialized cells of the innate immune system such as macrophages and dendritic cells. Cell-intrinsic recognition mechanisms are essential for recognizing intracellular pathogens and involve type I interferon (IFN) gene transcription signaling by members of the RLRs. Three members of the RLR family are RIG-1, melanoma differentiation-associated gene-5 (MDA-5), and laboratory of genetics and physiology gene 2 (LPG-2). In viral sensing, the RLRs are highly discriminating with regards to the cytoplasmic localization and sensing of specific RNAs. They specifically detect RNA molecular patterns not normally present in the cytoplasm. Abnormal RNA patterns include chemical modifications, secondary or tertiary RNA conformations, specific RNA sequences, or double-stranded RNA. While much of what is currently known about RLRs is centered around the sensing of RNA viruses, there is preliminary evidence that similar intracellular mechanisms may exist for sensing DNA viruses and some intracellular bacteria.

Members of the NLR are central regulators of immunity and inflammation largely through activation of transcription factors like nuclear factor (NF) kappa B, interferon regulatory factor (IRF), or nuclear factor of activated T lymphocytes (NFAT). Some members of the NLR family form multiprotein complexes with the cysteine protease pro-caspase-1 and the adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD) referred to as inflammasomes (also see Fig. 3-12). The inflammasome is a multiprotein complex that activates caspase-1. PAMPs and DAMPs are sensed through activation of the inflammasome complex, resulting in activation of caspase-1, which elicits effector functions through proteolytic cleavage of cytosolic proinflammatory cytokines (e.g., pro-interleukin [IL]-1β and pro-IL-18), which are then secreted in their active form. The inflammasome model is analogous to the model for the activation of the apoptotic caspases by CD95/Fas death–inducing signaling complex (DISC) and the Apaf-1 apoptosome. IL-1β functions in localized and systemic responses to infections and injury, to cause fever, activation of lymphocytes, and the extravasation of leukocytes at sites of injury or infection. IL-18 functions to induce IFN-γ by activated T lymphocytes and NK cells during a T_H1 response and to induce secondary inflammatory cytokines, chemokines, cell adhesion molecules, and nitric oxide (NO) synthesis. Other members of the NLR family are involved in inflammasome-independent (noninflammasome) innate immune responses and signal through different multicomponent signalosomes such as nodosomes, transcriptosomes, mito-signalosomes. Noninflammasome-mediated innate immune responses occur through NF kappa B activation, mitogen-activated protein kinase (MAPK) activation, cytokine and chemokine production, antimicrobial reactive oxygen species production, type I interferon (IFN-α and IFN-β) production, and ribonuclease L activity.

Toll-Like Receptors

TLRs are the mammalian homologue of the Toll receptor originally identified in Drosophila. It has not only an embryologic function but also an immunologic function. In mammals, TLRs are membrane molecules that function in cellular activation by a wide range of microbial pathogens. TLRs are classified as PRRs because they recognize PAMPs and signal to the host the presence of an infection. Pathogen associated molecules include lipopolysaccharide (LPS) from Gram-negative bacteria, peptidoglycan from Gram-positive bacteria, double-stranded RNA from viruses, or α-glucans from fungi (Table 5-1). In general, TLRs 1, 2, 4, and 6 recognize unique bacterial products that are found on the cell surface, and TLRs 3, 7, 8, and 9 are involved in viral detection and nucleic acid recognition within endosomes. The specificity of TLRs for microbial products depends on interactions between TLRs and non-TLR adapter molecules. All TLRs contain an extracellular domain characterized by a leucine-rich repeat motif flanked by a cysteine-rich motif (Fig. 5-2). They also contain a conserved intracellular signaling domain, Toll/IL-1 receptor (TIR), that is identical to the cytoplasmic domain of the IL-1 and IL-18 receptors. Fig. 5-2 illustrates how TLRs function in the recognition of LPS. In the blood or extracellular fluid, the binding of LPS to LPS-binding protein (LBP) facilitates the binding of LPS to CD14, a plasma protein and glycophosphatidylinositol-linked membrane protein present on most cells. The binding of LPS to CD14 results in the dissociation of LBP and the association of the LPS-CD14 complex with TLR4. An accessory protein, MD2, complexes with the LPS-CD14-TLR4 molecule and results in LPS-induced cell signaling.

TABLE 5-1

Toll-Like Receptors (TLRs) and TLR Ligands and Their Microbial Source

CpG, Cytosine and guanine linked oligonucleotide; GPI, glycosyl phosphatidyl inositol; HSP60, heat shock protein 60; LPS, lipopolysaccharide.

Modified from Kumar V, Abbas AK, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005, Saunders.

Fig. 5-2 Signaling pathway for Toll-like receptor 4 (TLR4) in response to bacterial lipopolysaccharide (LPS).
The binding of LPS to TLR4 results in activation of a signal transduction pathway, leading to gene transcription and the elicitation of an inflammatory response. AP-1, Activating protein 1; NF-κB, nuclear factor-kappa B; TIR, Toll/interleukin-1 receptor. (From Kumar V, Abbas AK, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005, Saunders.)

Briefly, TLR signaling through the binding of PAMP to a TLR leads to the activation of TIR, which forms a complex with the cytoplasmic adapter protein MyD88, an IL-1 receptor-associated kinase (IRAK), and tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6). Activated TRAF then activates the MAPK cascade leading to the activation of NF kappa B, a transcription factor. MyD88 is a universal signaling molecule for NF kappa B activation, and MyD88-deficient mice are incapable of activation by TLR, IL-1, and IL-18. Recent information suggests that there are also signaling mechanisms unique to individual TLRs.

TLRs and their pathogen-associated ligands are important recognition molecules for the innate immune system and trigger a number of antimicrobial and inflammatory responses. Up to 15 different TLR genes have been identified. The importance of these receptors in immunity is further supported by the observation of polymorphisms in the genes encoding them.

Although the individual TLRs exhibit ligand specificity, they differ in their cellular expression patterns and the signal pathways they activate, similar to that described for cytokines, which exhibit pleiotropy, redundancy, synergy, and antagonism. There are constitutively and inducibly expressed TLRs in different tissues. TLRs regulate cell recruitment to sites of infection through the upregulation of the expression of adhesion molecules, chemokines, and chemokine receptors during an inflammatory response. TLRs activate leukocytes (primarily neutrophils and NK cells of the innate immune system) and epithelial, endothelial, and hematopoietic cells. TLRs are also hypothesized to be essential for linking the innate immune response to the adaptive immune responses. Central to this hypothesis is the TLR-dependent dendritic cell-mediated control of T lymphocyte activation. Dendritic cells are important antigen-presenting cells for T lymphocyte activation. Dendritic cells uptake microbial antigens in the peripheral tissues and migrate to regional lymph nodes where they present peptide fragments, in the context of MHC molecules, to naïve T lymphocytes. In addition to the expression of the peptide-MHC signal, dendritic cells are also required to provide a second, co-stimulatory signal through the expression of B7, the ligand for the CD28 molecule on naïve T lymphocytes. The activation and maturation pathway related to the co-stimulatory signal occurs through TLR recognition of PAMPs.

There are species differences in the ligand specificity of TLRs and in the cellular responses elicited. Although sequences for canine, feline, and chicken TLR4 have been identified, no functional data have been published. With regard to domestic animals, a significant body of literature exists on TLRs of cattle.

Finally, TLRs have been implicated in “innate autoimmunity,” with several reports of TLRs recognizing fibrinogen, heat-shock proteins, or DNA. There are also reports of TLR binding of DNA as a factor in directing antibody production by autoreactive B lymphocytes contributing to the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus (SLE). Further studies are necessary to more fully understand these observations and the underlying immunopathogenesis.

Adaptive Immunity (Specific Immunity)

Adaptive immunity in general consists of cell-mediated immunity, mediated by T lymphocytes against intracellular pathogens, and humoral immunity, mediated by B lymphocytes against extracellular pathogens and toxins (Fig. 5-3). The adaptive immune response is the second-line defense mechanism and is characterized by antigen specificity, diversity, memory, and self-/nonself-recognition. Antigen specificity and self-/nonself-recognition are the result of distinct membrane molecules. Mature B lymphocytes are activated by a specific antigen-binding molecule on its membrane. The antigen receptor is membrane-bound immunoglobulin. Mature T lymphocytes express a specific antigen-binding molecule, the T lymphocyte receptor (TCR), on their membrane. Unlike membrane-bound immunoglobulin on the B lymphocyte, which can recognize antigen alone, TCRs can recognize only antigens that are associated with cell membrane proteins called MHC molecules. Self-/nonself-recognition is the result of MHC molecules. There are two major classes of MHC molecules. Class I molecules are present on all nucleated cells, and class II molecules are present primarily on antigen-presenting cells. T lymphocytes and B lymphocytes are the major cells of adaptive immunity.

Fig. 5-3 Overview of humoral and cell-mediated arms of adaptive immunity.
CTL, Cytotoxic T lymphocyte. (Adapted from Goldsby RA, Kindt TJ, Osborne BA: Kuby immunology, ed 4, New York, 2000, WH Freeman.)

Cells and Tissues of the Immune System

T Lymphocytes

T lymphocytes are small nongranular cells that constitute 50% to 70% of the peripheral blood mononuclear cells. They originate in the bone marrow and migrate to the thymus (thus the “T” designation), where they undergo differentiation, selection, and maturation processes before exiting to the periphery as effector lymphocytes. In secondary lymphoid tissues, they are located primarily in the paracortical regions of lymph nodes and the periarteriolar sheaths (PALS) of the spleen. These specific anatomic sites elaborate chemoattractant cytokines (chemokines), for which the T lymphocytes express receptors. The definitive marker for T lymphocytes is the TCR, the polymorphic antigen-binding molecule. The antigen specificity of individual lymphocytes is attributed to their respective TCR, which is genetically determined. TCRs are classified as either αβ-TCR or γδ-TCR based on the composition of their disulfide-linked heterodimers. The individual polypeptide chains of the heterodimers contain variable (antigen-binding) and constant regions. In mammals, most peripheral blood T lymphocytes express αβ-TCR; however, in ruminants and swine, these lymphocytes make up only 10% to 50%, and 10% of peripheral blood T lymphocytes, respectively. Both TCRs are associated with CD3, and together they form the TCR-CD3 complex. There are significant activation and functional differences between αβ-TCR– and γδ-TCR–expressing lymphocytes. Unlike membrane-bound immunoglobulin on B lymphocytes that can recognize soluble antigen, the αβ-TCR can only recognize antigen after it has been processed into peptide fragments and associated with MHC molecules (the MHC is discussed later). In most instances, the antigen is associated with the MHC on the surface of an antigen-presenting cell, a virally infected cell, a neoplastic cell, or a cell of a foreign tissue graft. Individual αβ-TCRs are covalently linked to a cluster of five polypeptide chains, three comprising the CD3 molecule and two comprising the β-chain. The CD3 molecule and the β-chain are invariant, and although they do not bind antigen, they do function in the signal transduction after antigen binding by the TCR. Each T lymphocyte expresses a unique TCR with regard to structure and antigen specificity. The genes that encode α-, β-, γ-, and δ-chains of the TCR can undergo somatic rearrangements during their development in the thymus, resulting in tremendous diversity for antigen recognition. Not only are these rearrangements important for diversity, but also they can be used to molecularly phenotype proliferating populations of T lymphocytes as a diagnostic tool for the identification of clonal populations (neoplastic) and polyclonal populations (nonneoplastic) (see Chapter 6).

In most species, a minority of T lymphocytes express γδ-TCR. The γδ-TCR lymphocytes develop in the thymus and migrate to the epithelium of the skin and intestine, mammary gland, and reproductive organs. Although these cells can be found within regional lymph nodes and the lamina propria, in these organs they primarily reside as intraepithelial lymphocytes (IELS). As stated, in some species, notably ruminants, γδ-TCR lymphocytes are the predominant circulating population of T lymphocytes. In contrast to the αβ-TCR lymphocytes, γδ-TCR lymphocytes can recognize native antigen in the absence of MHC binding and they do not rely exclusively on the δ-chain as a signal transducer. Most γδ-TCR lymphocytes use the γ-chain for signal transduction after activation. The diversity of antigens recognized by γδ-TCR is limited in most species except ruminants and swine, indicating their importance in these species. Some have suggested that they may provide early cell-mediated immune responses in neonates. The precise function of γδ-TCR lymphocytes remains unknown. Another small subset of T lymphocytes, called NK-T lymphocytes, expresses molecules found on NK cells in addition to a limited diversity of TCRs. NK-T lymphocytes primarily recognize glycolipids that are associated with an MHC-like molecule, CD1. The function of NK-T lymphocytes remains unknown.

Although all T lymphocytes express the TCR-CD3 complex, they are further classified according to accessory CD4 and CD8 molecules. These nonpolymorphic accessory molecules include CD4, CD8, CD2, integrins, and CD28. CD4 and CD8 functionally subdivide T lymphocytes into CD8⁺ cytotoxic T lymphocytes (CTL [T_C]) and CD4⁺ helper T lymphocytes (T_H). During antigen presentation, CD4⁺ lymphocytes only recognize antigen bound to MHC class II molecules (Fig. 5-4), whereas CD8⁺ lymphocytes only recognize antigen bound to MHC class I molecules. This coreceptor requirement is commonly referred to as MHC class I and MHC class II restriction, the basis for positive selection in the thymus. Although there have been reports in some species of CD4 lymphocytes that are functionally cytotoxic and CD8 lymphocytes that are functionally “helper”-like, these appear to be anomalies and for the purposes of this text are excluded. In most species, peripheral blood T lymphocytes express either CD4 or CD8. Except for ruminants and swine, lymphocytes negative for both CD4 and CD8—“double negative” lymphocytes—are rare in the peripheral blood. So-called “double positive” lymphocytes, positive for both CD4 and CD8, are rare, except in swine, where they can approach 25% of the T lymphocytes in the peripheral circulation. T lymphocytes require two signals for activation. Signal 1 is provided by the TCR and the MHC-antigen complex and the CD4 or CD8 MHC complex. Signal 2 is provided by another accessory molecule expressed by T lymphocytes, the CD28 molecule. The ligands for CD28 are B7-1 (CD80) and B7-2 (CD81) expressed on activated dendritic cells, B lymphocytes, and macrophages (see Fig. 5-4). An inability to deliver the second signal results in an unresponsive T lymphocyte that either undergoes apoptosis or remains anergic. These molecules provide an important co-stimulatory signal for T lymphocyte activation, and are discussed in more detail later in the chapter regarding anergy and the development of tolerance with regard to autoimmunity. When T lymphocytes are activated by antigen and receive the appropriate co-stimulatory signals, they clonally expand as a result of their secretion of IL-2. This clonally expanded population of T lymphocytes, of the same antigen specificity, differentiates into populations of effector lymphocytes and memory lymphocytes.

Fig. 5-4 The T lymphocyte receptor complex (T lymphocyte receptor [TCR]).
A, TCR-α and TCR-β chains complexed with CD3 γ-, δ-, and ε-chains and the invariant, ζ-chains. B, Illustrating how the TCR recognizes antigen in the context of major histocompatibility complex on the antigen-presenting cell (top) and how the ζ-chains and CD3 γ-, δ-, and ζ-chains deliver one of the two required signals for activation of the T lymphocyte. The second required signal is delivered by the co-stimulator molecules CD28 on the T lymphocyte and B₇ on the antigen-presenting cell. MHC, Major histocompatibility complex. (A from Kumar V, Abbas AK, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005, Saunders. B from Kumar V, Abbas AK, Fausto N, et al: Robbins & Cotran pathologic basis of disease, ed 8, Philadelphia, 2009, Saunders.)

T_H lymphocytes can be classified based on their functional capacity and ability to elicit primarily an antibody response or a cell-mediated immune response. After activation of T_H lymphocytes, by recognition of antigen bound to MHC class II molecules on the surface of an antigen-presenting cell, there is clonal expansion of T_H lymphocytes of the same antigen specificity. These clonally expanded lymphocytes are important in directing the immune response as either primarily an antibody response or a cellular response. The type of response is dictated by a restricted cytokine profile that primarily activates B lymphocytes in the case of an antibody response or activates CTL (T_c) and macrophages in a cellular response. The restricted cytokine profile for T_H lymphocytes allows for their classification as either T_H1 or T_H2 lymphocytes (Web Table 5-1). T_H1 lymphocytes synthesize and secrete IL-2 and interferon-γ (IFN-γ), stimulating CTL (T_c) and macrophages, and induce a cell-mediated immune response. T_H2 lymphocytes synthesize and secrete IL-4, IL-5, IL-6, and IL-13 that stimulate B lymphocytes to develop into antibody-secreting plasma cells and inhibit macrophage functions, and induce an antibody response. The type of immune response (antibody versus cell-mediated) can have a profound influence on the outcome of a disease. In the instance of an intracellular protozoal infection, a T_H2 type response results in rapid proliferation of the organism and death of the host, whereas a T_H1 type response results in elimination of the organism and survival of the host. Similarly, a T_H2 response to an allergen results in the elaboration of immunoglobulin (Ig) E, through IL-4 production, stimulation of eosinophils, through IL-5 production, and the development of an allergic reaction. The exact regulation of the T_H1 versus the T_H2 lymphocyte response is unknown, but studies suggest that IL-12 produced by activated macrophages stimulates the T_H1 response, whereas IL-4 inhibits the T_H1 response, allowing the T_H2 response to dominate. T_H lymphocytes predominately drive the immune response to microbial pathogens by activating macrophages or B lymphocytes. Another functionally distinct subpopulation of CD4⁺ T lymphocytes is the regulatory T lymphocyte (T reg). T reg lymphocytes function to suppress the response of self-reactive CD4 lymphocytes that have escaped the negative selection process in the thymus. They are distinguished from other CD4 T lymphocytes by the expresson of CD25 on the cell surface. Like T_H1 and T_H2 lymphocytes, T reg lymphocyte differentiation is driven by cytokine environments; however, where T_H1 and T_H2 lymphocyte activation occurs through transcripional activators T-bet and GATA-3, respectively, T reg lymphocytes are activated through the transcriptional repressor FoxP3 (Fig. 5-5). This subpopulation of T reg lymphocytes are often referred to as Fox3P lymphocytes and produce the immunosuppressive and antiinflammatory cytokines IL-4, IL-10 and TGF-β. FoxP3 lymphocytes are an intense area of investigation for their role as suppressor lymphocytes of immunity and inflammation. T reg lymphocytes have been shown to have a role in the prevention of organ-specific autoimmune diseases and in modulation of immune responses to microbial pathogens to prevent overwhelming inflammatory reactions. Finally, a subpopulation of CD4 lymphocytes characterized by the ability to produce IL-17 is designated as T_H17 lymphocytes. T_H17 lymphocyte differentiation is driven by TGF-β, IL-6, IL-1, and IL-23. T_H17 lymphocytes, through production of chemokines IL-17 and IL-22, induce the recruitment of monocytes and neutrophils to sites of inflammation. Again, one must recognize that this is an oversimplification of a complex regulatory mechanism and that as additional knowledge is gained about T_H1, T_H2, T reg, and T_H17 lymphocyte responses, we will be able to understand pathogenic mechanisms of diseases, which will lead to the development of more specific therapeutic targets.

Fig. 5-5 Differentiation and expression of CD4 T lymphocytes.
Cytokines in the extracellular tissue fluids direct the differentiation of CD4 T lymphocytes to specific secretory types, such that they secrete specific cytokines that have specific functions on other lymphocytes in the environment. (Adapted from Parham P: The immune system, ed 3, 2009, Garland Science.)

WEB TABLE 5-1

Basic Cytokine and Functional Profiles of T_H1 and T_{_H}2 Lymphocytes

Cytokine	T_H1	T_H2
IL-2	+
IFN-γ	++
TNF-β	++
GM-CSF	++	+
IL-3	++	++
IL-4		++
IL-5		++
IL-13		++
Function
Antibody*	+	++
IgE		++
Eosinophils, mast cells		++
Macrophages	++
Type IV hypersensitivity	++
Cytotoxic T lymphocytes	++

GM-CSF, Granulocyte-macrophage colony-stimulating factor; IFN-γ, interferon-γ; IgE, immunoglobulin E; IL, interleukin; TNF-β, tumor necrosis factor-β.

^*Opsonizing and complement fixing antibodies.

B Lymphocytes

B lymphocytes constitute 5% to 20% of the peripheral blood mononuclear cells. B lymphocyte development occurs in two phases, an antigen-independent phase in the primary lymphoid tissues, followed by an antigen-dependent phase in secondary lymphoid tissues. B lymphocytes can be found in primary lymphoid tissues, such as the bone marrow and ileal Peyer’s patches (a primary lymphoid tissue in some species because it is the site of B lymphocyte development, rather than the bone marrow), and in secondary lymphoid tissues, such as the spleen, lymph nodes, tonsils, and Peyer’s patches. Within secondary lymphoid tissues, B lymphocytes are aggregated in the form of distinct lymphoid follicles, which on activation expand to form prominent pale regions called germinal centers (Fig. 5-6). This anatomic localization, similar to T lymphocytes in the PALS and paracortex, is the result of elaboration of chemokines for which the B lymphocyte has receptors. The antigen receptor of the B lymphocyte is the membrane bound immunoglobulin. After the antigen-independent phase of development, B lymphocytes express IgM and IgD on their surface that signifies a mature B lymphocyte. In the antigen-dependent phase, antigen-activated mature B lymphocytes differentiate into IgM-secreting plasma cells or switch to another antibody isotype. Immunoglobulins can be generated against an almost unlimited number of antigenic determinants through the rearrangement of genes encoding the light chain and heavy chain components. As in the case of the TCR, an evaluation of the rearranged genes of a B lymphocyte can be used to molecularly phenotype B lymphocyte neoplasms (see Chapter 6).

Fig. 5-6 Histology of a hyperplastic lymph node.
A, Outer cortex, containing numerous secondary lymphoid follicles with characteristic pale centers, and inner medulla are easily identifiable. B, Localization of B lymphocytes (labeled with a B lymphocyte marker conjugated with a green fluorochrome) and T lymphocytes (labeled with a T lymphocyte marker conjugated with a red fluorochrome). Immunofluorescence photomicrograph. C, Higher magnification of a secondary lymphoid follicle illustrating the central pale, germinal center, which contains primarily proliferating B lymphocytes, CD4⁺ lymphocytes and dendritic cells, surrounded by densely packed small B lymphocytes. H&E stain. (A, B, and C from Kumar V, Abbas AK, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005, Saunders.)

Like the T lymphocyte, the B lymphocyte also has accessory molecules that function to form the antigen receptor complex (Fig. 5-7). These nonpolymorphic molecules are nonpolymorphic heterodimers composed of Ig-α (CD79a) and Ig-β (CD79b) that do not bind antigen but do interact with the transmembrane portion of surface immunoglobulin involved in cell activation. B lymphocytes, unlike T lymphocytes, can recognize soluble antigens. Additional nonpolymorphic molecules that are important to B lymphocyte functions are CD21 and CD40. The CD21 molecule is the complement receptor-2 molecule whose ligands are C3b and C3d. B lymphocyte responses to protein antigens are dependent on cytokines produced by activated T lymphocytes (CD4⁺). The CD40 molecule interacts with CD40 ligand on the surface of T_H lymphocytes and functions to allow B lymphocyte development into antibody-secreting plasma cells. A failure to express CD40 ligand has been associated with an inability to isotype switch, resulting in a hyper-IgM syndrome. B lymphocytes activated by antigen develop into antibody secreting plasma cells and memory lymphocytes of the same antigenic specificity.

Fig. 5-7 The B lymphocyte antigen receptor complex.
Membrane IgM (or IgD, not shown) and the signaling molecules Igα and Igβ. CD21, also known as complement receptor-2, binds complement components and activates B lymphocytes. Ig, Immunoglobulin. (Courtesy Dr. Alex McPherson, University of California, Irvine.)

Mononuclear Phagocytic System (Monocyte-Macrophage System)

The preferred term today for the functionally and phenotypically diverse population of mononuclear phagocytic cells is the mononuclear phagocytic system (MPS). It is also referred to as the monocyte-macrophage system. These cells have a broad range of functions contributing to immunity, inflammation, and tissue remodeling and repair. A brief history on the recognition of the defensive mechanism of phagocytosis not only facilitates our current understanding of how these cells play a crucial role in innate and adaptive immunity but also explains some of the terminology that often leads to confusion for students. Elie Metchnikoff (1845-1916), a comparative developmental zoologist, is credited with recognizing and establishing the process of phagocytosis, an important defensive mechanism for organisms. He recognized the association between the systemic clearance of microorganisms and the presence of the microorganisms in the spleen and liver. Subsequent studies evaluating the systemic clearance of dyes suggested that Kupffer cells and endothelial cells lining the sinusoids of the liver were responsible because both cell types internalized the dyes. Because the investigators considered macrophages (Kupffer cells) and endothelial cells to have a common biologic function, phagocytosis, they proposed that they be encompassed as a system, the reticuloendothelial system. We now understand that the mechanism of uptake by endothelial cells is not by phagocytosis and as a result the term reticuloendothelial system is no longer used. The term mononuclear phagocytic system was developed to differentiate lymphoid cells (mononuclear T and B lymphocytes), granulocytes (polymorphonuclear leukocytes), and endothelial cells from the lineage-committed precursors in the bone marrow, blood monocytes and tissue macrophages, and dendritic cells that currently comprise the MPS. It is very likely that as we learn more about the precursors and differentiated cells that constitute the MPS, there will be proposals for new, more specific classification schemes.

In general, a circulating MPS cell in the blood is designated as a monocyte, whereas the tissue-based cell is designated as a macrophage. The blood monocyte-to-tissue macrophage is well recognized, although the mechanisms that allow for differentiation of the circulating monocyte pool into the tissue-based pool are still largely unknown. The monocyte is a bone marrow–derived cell of the myeloid lineage that is the precursor cell to the terminally differentiated macrophage that has limited recirculation and replication capacity. The myeloid dendritic cell represents a specific type of mononuclear cell present in nonlymphoid tissues with unique migratory properties and is discussed separately. As opposed to granulocytic myeloid cells, macrophages are long-lived (days to months) and can exist as quiescent “resident” cells widely distributed throughout the body. It is becoming increasingly clear that there is heterogeneity in the circulating monocyte pool that corresponds with the ultimate tissue localization of resident macrophages. The phenotypic characterization of the cells the MPS contains is often used in an attempt to identify specific populations of MPS (Fig. 5-8). Morphologically, monocytes are variably sized with an irregular shape, oval or kidney-shaped nucleus, prominent cytoplasmic vesicles, and a high cytoplasm to nucleus ratio. These features are not unique to monocytes, and as a result, they are difficult to distinguish from circulating dendritic cells, activated lymphocytes, and NK cells based on morphology or by light scatter (flow cytometry). This section covers basic concepts attributable to monocytes, tissue macrophages, and myeloid dendritic cells and refers to specific organ systems. See additional chapters in the section on pathology of organ systems for details on organ-specific cells of the MPS. Certain organ systems have specific names for their resident macrophages, whereas other organ systems only refer to them as macrophages (Table 5-2).

TABLE 5-2

Nomenclature and Location of Nonlymphoid Monocyte-Macrophage Cell Types

Organ/Tissue	Name	Location
Lung	Alveolar macrophages Pulmonary intravascular macrophages	Alveolar spaces Capillaries of the lung
Connective tissues	Histiocytes	Interstitium
Kidney	Mesangial cells	Glomerular tuft
Brain	Microglial cells	Neuroparenchyma and perivascular areas
Bone	Osteoclasts	Bone marrow
Blood	Monocytes	Circulation
Liver	Kupffer cells	Hepatic sinusoids

Fig. 5-8 Differentiation of mononuclear phagocytes based on antigen markers. (Adapted from Paul WE: Fundamental immunology, ed 6, Philadelphia, 2008, Lippincott Williams & Wilkins.)

Growth and differentiation of monocytes is regulated by specific growth factors, such as IL-3, colony-stimulating-factor-1 (CSF-1), granulocyte-macrophage CSF (GM-CSF), IL-4, and IL-13, and inhibitors such as type I interferons and transforming growth factor-β (TGF-β). CSF-1 is the most important because it controls the proliferation, differentiation, and survival of the monocyte. Monocytes represent approximately 4% to 10% of blood leukocytes and are identified in mammals, birds, amphibians, and fish. They are largely viewed as accessory cells that importantly link inflammation and innate immune responses to adaptive immunity. Hematopoietic stem cells (HSC) give rise to the common myeloid progenitor (CMP), the precursor cell to the granulocyte/macrophage progenitor (GMP) and macrophage/dendritic cell progenitor (MDP). The MDP is the common progenitor cell for monocytes, macrophages, and conventional dendritic cells. Monocytes express the CSF-1 receptor (CD115) and the chemokine receptor CX3CR1, are differentiated from polymorphonuclear cells (PMNs), NK cells, and lymphoid cells, and do not express CD3, CD19, or CD15. Monocyte heterogeneity based on surface marker expression and function has identified subsets that are an area of intense investigation and are best characterized in humans and rodents. Subpopulations of blood monocytes can be phenotyped based on the expression of surface markers CD14 and CD 16 (FcγR-III). Two additional subpopulations of blood monocytic cells are the myeloid blood dendritic cells, which are negative for CD14 and CD16. In the mouse, the main subset of CD115⁺ monocytes are characterized as large cells expressing Ly6C, the chemokine receptor CCR2 and the adhesion molecule L-selectin (CD62L), and CX3CR1 (Fig. 5-9). These are referred to as inflammatory monocytes or inflammatory-monocyte-derived macrophages that are preferentially recruited to inflamed tissues and lymph nodes where they produce high levels of TNF-α and IL-1. The emigration of Ly6C+ monocytes from the bone marrow to the periphery depends on the chemokine receptor CCR2 and its ligands CCL7 and CCL2. Ly6C negative monocytes have less of an influence on inflammatory reactions and appear to function more importantly as tissue resident cells and as cells involved in healing and regeneration associated with vascular injury. In those species characterized to date, there appear to be two major functional subpopulations of monocytes, one that is recruited and differentiated into macrophages at the site of inflammation and expresses higher levels of MHC class II and adhesion molecules and one that is responsible for repopulating resident tissue macrophages. Both populations can give rise to dendritic cells (see Fig. 5-8). Classification schemes are forever evolving, and there are definite species differences that may explain species differences in rates of infection and types of clinical disease associated with specific microbial agents. Similar phenotypic and functional heterogeneity exists with tissue-based macrophages.

Fig. 5-9 Monocyte differentiation into dendritic cells and tissue macrophages. (Adapted from Serbina NV, Jia T, Hohl HM, et al: Ann Rev Immunol 26:421-452, 2008.)

The MPS of hematopoietic and lymphoid organs is diverse with functionally and phenotypically distinct parallel subpopulations in humans, rodents, and pigs. In primary lymphoid organs, mature macrophages are involved in the production, differentiation, and destruction of all lineages of hematopoietic cells in the bone marrow, and in positive and negative selection processes in the thymus. In secondary lymphoid organs, macrophages are uniquely positioned to enhance their phagocytic properties to endogenous and exogenous material and to influence other cell types through their products. The resident macrophages in the spleen differ in microscopic localization, phenotype, and function. The reason for this complexity is largely attributable the spleen’s role as a hematopoietic organ and as a secondary lymphoid organ. Distinct subpopulations of macrophages are present in the red pulp, white pulp, and marginal zone with some recognized species differences (see Chapter 13). The red pulp macrophage functions in the phagocytosis of hematogenous pathogens and senescent erythrocytes. Erythrophagocytosis primarily occurs in the spleen and liver and allows for the removal of senescent erythrocytes and recycling of iron, often evident as cytoplasmic accumulations of pigments. The white pulp macrophages are also actively phagocytic, and a morphologically distinct cell, the “tingible body macrophage,” can often be easily identified histologically and represents macrophages involved in the uptake and removal of apoptotic T and B lymphocytes. The marginal zone of the spleen provides a complex environment for the interface for the red and white pulp, specifically an important transit area for cells leaving circulation and entering the white pulp, and for B lymphocyte differentiation. The development of granulomatous inflammatory reactions in the spleen as a response to hematogenous microorganisms often begins in the marginal zone. The two subpopulations of macrophages present in the marginal zone are the metallophilic macrophage and the marginal zone macrophage. Marginal zone macrophages express high levels of PRRs and scavenger receptors that facilitate the clearance of pathogens from the circulation. The function of metallophilic macrophages is unknown. Tissue macrophages are derived from a combination of precursors in the blood (monocytes) and from local proliferation of precursors that varies for individual organ systems.

Much has been written about the relationship between monocytes, macrophages, and dendritic cells. The inclusion of dendritic cells as a component of the MPS is in part attributable to the fact that they are derived from a common myeloid precursor, influenced by similar growth factors (e.g., CSF-1), express common surface markers, and have no unique properties as antigen-presenting cells that allow them to be differentiated from macrophages. Like macrophages, dendritic cells in specific organ systems may have a specific name or designated only as dendritic cells preceded by the organ or in some instances a specific anatomic location within an organ. Dendritic cell subsets are also an intense area of investigation with regards to phenotypic and functional heterogeneity. The so-called conventional dendritic cells are present as immature cells in the interstitial tissues of all organs except the brain.

Macrophages

Mononuclear phagocytic cells include circulating monocytes and tissue-based macrophages. In the spleen, macrophages are located in the marginal zone, white pulp, and red pulp, where they function primarily as phagocytic cells. In the lymph node, macrophages are located in the subcapsular sinus, which is analogous to the marginal zone of the spleen, and the medulla. These physical locations, the subcapsular sinus of lymph nodes and marginal zone of the spleen, facilitate their exposure to potential antigens. Nonlymphoid tissue-based macrophages have different functions and are named according to the tissue in which they reside (see Table 5-2). One primary function of these cells is phagocytosis, as discussed in Chapter 3. Macrophages express Fc receptors (FcR) for antibody and can phagocytose antigens opsonized by antibody or complement components. Another primary function is their involvement in the immune response as antigen-presenting cells. In this instance, they phagocytose antigen and process it into peptide fragments, which are then presented to T lymphocytes, and the induction of cell-mediated immune responses. Although all nucleated cells express MHC class I molecules and could be considered antigen-presenting cells, only three cell types normally express MHC class II molecules and are regarded as the major antigen-presenting cells. They are the macrophage, dendritic cell, and B lymphocyte. Whereas B lymphocytes and dendritic cells constitutively express MHC class II molecules, macrophages express MHC class molecules on activation.

Macrophages also have an important role in the generation of a cell-mediated immune response and are essential to type IV hypersensitivity reactions. Activated T_H1 lymphocytes synthesize IFN-γ, a potent activator of macrophages. Under the influence of IFN-γ, macrophages have increased phagocytic activity and are more efficient at killing.

Dendritic Cells

Dendritic cells comprise a distinct population of cells that are characterized by elongate cell processes. Most dendritic cells are antigen-presenting cells, which process antigens and present fragments to T lymphocytes. They are more efficient than macrophages and B lymphocytes at antigen presentation. Antigen-presenting dendritic cells are nonphagocytic, bone marrow–derived cells. They are the most important antigen-presenting cell for initiating primary immune responses to protein antigens (Fig. 5-10). Antigen-presenting dendritic cells express a number of molecules, such as TLRs and mannose receptors, that make them efficient at capturing and responding to antigens. They also express high concentrations of MHC class II molecules and B7 co-stimulatory molecules. By expressing chemokine receptors similar to T lymphocytes, they have the ability to localize in T lymphocyte regions of lymphoid tissue. By colocalizing to these areas, they are uniquely positioned to present antigens to recirculating T lymphocytes. Antigen-presenting dendritic cells function to capture antigen and then migrate to T lymphocyte areas of secondary lymphoid organs, where they present fragments of the antigen on their surface and increase their expression of co-stimulatory molecules that activate T lymphocytes. Specifically, migrating dendritic cells, derived from Langerhans’ cells that have captured antigen, enter the lymph node through efferent lymphatics and localize in lymphoid organs, where they present antigenic peptides to T lymphocytes that facilitate B lymphocyte activation and the production of antibody-secreting plasma cells. In addition to their function as antigen-presenting cells, they are also important in the process of negative selection in the thymus and in the maintenance of peripheral tolerance. The four types of antigen-presenting dendritic cells and their locations are listed in Table 5-3. Circulating dendritic cells, also known as veiled cells, make up less than 1% of peripheral blood mononuclear cells. The second type of dendritic cell, the follicular dendritic cell, is primarily located in lymphoid follicles. These cells are not derived from the bone marrow, do not express MHC class II molecules, and do not function as an antigen-presenting cell. Follicular dendritic cells have FcR and receptors for C3b. They store antigen-antibody and antigen-C3b complexes and are thought to be involved in the development and maintenance of memory B lymphocytes.

TABLE 5-3

Antigen-Presenting Dendritic Cells and Their Primary Location

Dendritic Cells	Location
Langerhans’ cells	Skin, mucous membranes, iris, ciliary body
Interstitial dendritic cells	Most major organs
Interdigitating dendritic cells	T lymphocyte area of secondary lymphoid tissue and thymic medulla
Circulating dendritic cells	Peripheral blood

Fig. 5-10 Dendritic cell functions.
Specialized dendritic cells in the epidermis (Langerhans’ dendritic cells) capture antigen via phagocytosis or endocytosis and migrate to regional lymph nodes, where they present peptide fragments of the antigen to naïve T lymphocytes. (From Kumar V, Abbas AK, Fausto N, et al: Robbins & Cotran pathologic basis of disease, ed 8, Philadelphia, 2009, Saunders.)

Natural Killer Cells

NK cells are nonspecific cytotoxic cells that are important in early responses to tumor cells and viral infections. NK cells are bone marrow–derived, large granular lymphocytes that make up 5% to 15% of the peripheral blood mononuclear cells. Their size, slightly larger than that of a small lymphocyte, and the presence of abundant granular cytoplasm distinguish them from T lymphocytes (Fig. 5-11). They are commonly referred to as large granular lymphocytes. The cytoplasm of NK cells and CTL (T_c) is characterized by cytotoxic granules that contain perforin and granzymes, two potent pathways mediating lysis of the target cell. NK cells and T lymphocytes express numerous similar surface molecules and kill virus infected cells and tumor cells by similar mechanisms. Two membrane molecules, CD16 and CD56, are commonly used to identify NK cells. NK cells express FcγR (CD16) and the β-subunit of the IL-2 receptor (CD2). They do not express antigen-specific TCR or CD3 molecules. In contrast to cytotoxic lymphocytes, NK cells are not MHC restricted, are constitutively cytolytic, and do not develop memory cells. Because NK cells are activated early in an immune response and do not require a previous sensitization phase to develop memory cells after activation, they are the cytotoxic cell of innate immunity, the counterpart to the adaptive immune response’s CTL (T_c).

Fig. 5-11 Activated natural killer cell with numerous cytoplasmic granules that are characteristic of these large granular lymphocytes. (Courtesy Dr. Noelle Williams, Department of Pathology, University of Texas Southwestern Medical School, Dallas.)

Although NK cells do not express any antigen-specific molecules, they are very efficient at recognizing and killing altered or virally infected cells. NK cell activity is regulated through activating and inhibitory receptor molecules expressed on their cell surface (Fig. 5-12). These NK cell receptor molecules fall into two distinct categories: the immunoglobulin-like NK receptors and the C-type lectin-like NK receptors. Ligands for these receptors are cell surface molecules whose expression has been altered as a result of infection or damage. Ligands for activating receptors that stimulate NK cell activity commonly include viral and stress-induced proteins. Ligands for inhibitory receptors that block NK cell activity most commonly involve class I MHC molecules. A decreased expression of class I MHC molecules makes cells susceptible to NK cell–mediated lysis. A decrease in MHC class I expression often occurs in virus-infected cells and in neoplastic cells, making them susceptible to attack by NK cells. Normal cells are protected from NK cell killing because all nucleated cells express class I MHC molecules. This is an oversimplification of the “opposing-signals” model of NK cell regulation of how cytotoxic activity is limited to altered self-cells. Recent studies on the molecular mechanisms of NK cell regulation indicate that the absence of an inhibitory stimulus by itself is insufficient for triggering NK cell killing. NK cells also require triggering of activating receptors. Several activating receptors have been identified. One is the NKG2D receptor, a C-type lectin-like molecule that recognizes a number of stress-induced proteins. These stress-induced proteins are normally only constitutively expressed in the intestinal epithelium or as a result of cellular distress caused by infection or neoplastic transformation. There are a number of additional activating receptors, some of which recognized viral proteins, that are structurally similar to class I MHC molecules.

Fig. 5-12 Regulation of natural killer (NK) cell activity through activating and inhibitory receptors.
MHC, Major histocompatibility complex. (From Kumar V, Abbas AK, Fausto N, et al: Robbins & Cotran pathologic basis of disease, ed 8, Philadelphia, 2009, Saunders.)

Because NK cells express FcγR (CD16), they can also function in antibody-dependent cellular cytotoxicity (ADCC). In the case of NK cells, ADCC allows for antibody-bound targets to be identified and targeted for NK cell–induced lysis.

NK cells also facilitate the early response to viral infections not only by responding to cytokines produced early in a viral infection, but also by producing cytokines that help direct the immune response. NK cells are activated by IFN-α and IFN-β, released by virus-infected cells, and by IL-12, released by macrophages. After activation, NK cells have the ability to produce IFN-γ, a major cytokine directing the development of T_H1-type immune response early in the infection. IL-2 and IL-15 stimulate NK cell proliferation, and IL-12 enhances NK cell killing.

Cytokines: Messenger Molecules of the Immune System

General Properties of Cytokines

Cytokines make up a vast group of low molecular weight soluble glycoprotein proteins that are produced by immune and nonimmune cells, are largely produced locally, and act locally to direct the immune response. The expression of cytokine receptors and their respective ligands is highly regulated and contributes to the complexity of the systemic organization of the immune response. Cytokines are involved in every aspect of leukocyte biology and the immune response and are essential to leukocyte development, recirculation, differentiation, and activation and in maintaining self-tolerance. Cytokines can influence the cytokine-producing cell itself (autocrine), other cells present locally (paracrine), or distant cells (endocrine) (see Fig. 12-1). Cytokines use common signal transducing pathways, converting an extracellular signal through a cell surface receptor to activate or inhibit the target cell.

The nomenclature of cytokines has evolved from a system that originally named them according to their cellular source (e.g., lymphokine from lymphocytes and monokines from monocytes) to one that named them according to not only the cellular source but also the target cell (e.g., interleukin, a cytokine produced by a leukocyte that influences another leukocyte) or to the primary function (e.g., chemokine, a cytokine that affects chemotaxis). As cytokines were characterized, it became apparent that they have pleiotropic, redundant, synergistic, and antagonistic features that do not allow for such a simplistic classification scheme. More recently, cytokines and their receptors have been classified based on their molecular structure and common signaling pathways. Many cytokines share a similar, α-helix structure that is also shared by their respective receptors and are classified as type I cytokines and receptors. As is discussed later, the classification of cytokines and cytokine receptors according to their structural similarities allowed for the identification of the cause of the profound cytokine defects associated with X-linked severe combined immunodeficiency disease in humans and dogs. Unfortunately, type I cytokines also have now been found to include other regulatory proteins such as growth hormone, prolactin, erythropoietin, thrombopoietin, and leptin. Type II cytokines include type I interferons (IFN-α and IFN-β), type II interferon (IFN-γ), and the IL-10 family of cytokines.

The breadth and depth of knowledge regarding the function, regulation, and control of cytokines is overwhelming; however, an overview of the major cytokines and their primary functions is important for understanding the pathogenic mechanism of many disease processes. Some of the major cytokines and their primary biologic activities are presented here and in Chapter 3 as they pertain to acute and chronic inflammatory responses.

Cytokines that broadly influence innate and adaptive immune responses include IL-1, interferons (type 1), IL-6, and TNF-α. These cytokines are produced and influence a wide array of cell types. Cytokines that are involved in hematopoiesis and lymphocyte development include IL-2, IL-3, IL-4, IL-5, IL-12, IL-15, TGF-β, and GM-CSF to mention a few. Chemokines are a large group of cytokines that influence leukocyte development, trafficking, and function. They are organized into subfamilies, with distinct functions, based on the position of cysteine residues. The C-X-C subfamily of chemokines is primarily produced by activated macrophages and tissue cells (e.g., endothelium), and the C-C subfamily is largely produced by activated T lymphocytes. Chemokines are responsible for the anatomic localization (“homing”) of lymphocytes within lymphoid and nonlymphoid tissue. Chemokines and the other proinflammatory cytokines are more thoroughly covered in Chapter 3. The most important functional group of cytokines related to the pathogenesis of a number of diseases of immunity are those involved in the regulation of T_H lymphocytes. As discussed previously, T_H lymphocytes are classified based on their functional capacity and ability to elicit primarily an antibody response or a cell-mediated immune response rather than on their expression of specific cell markers (Fig. 5-13). T_H1 lymphocytes are activated by IL-12 and IL-18 and produce primarily IL-2, IFN-γ, and TNF-β to direct a cell-mediated immune response. T_H2 lymphocytes are activated by IL-4 and produce primarily IL-3, IL-4, IL-5, IL-6, IL-10, and IL-13 to direct a humoral immune response. As discussed later in the chapter, the type of response (T_H1 versus T_H2 type) may determine if a diseased state will occur. IL-15 regulates the growth and activity of NK cells. As indicated in Fig. 5-13, some cytokines, such as IL-10 and TGF-β, downregulate immune responses. In summary, cytokines produced by the T_H1 or T_H2 subset of lymphocytes not only promote the activation and functional capacities of the subset that produces them (autocrine effect) but also inhibit the development and activity of the other subset. This is known as cross-regulation and has important implications with regard to protective immune responses and adverse immune responses, as is discussed later.

Fig. 5-13 Cross-regulation of immunity.
Cross-regulation of T_H1 and T_H2 lymphocytes in part determines if an immunity is primarily a cell-mediated response or a humoral response. T_H1 lymphocytes, activated primarily by IL-12 and IL-18, promote cell-mediated immunity (CMI) by activating macrophages and cytotoxic T lymphocytes. T_H2 lymphocytes, activated primarily by IL-4, promote humoral immunity by producing cytokines that activate B lymphocytes to develop into antibody-secreting plasma cells. T_H2 lymphocytes also produce cytokines that activate mast cells and eosinophils in the pathogenesis of allergic diseases. The cross-regulation of T_H1 and T_H2 lymphocytes provides an inverse relationship between cell-mediated and humoral immunity. IFN-γ, Interferon-γ; Ig, immunoglobulin; IL, interleukin; NK, natural killer; TNF-β, tumor necrosis factor-β. (Adapted from Goldsby RA, Kindt TJ, Osborne BA: Kuby immunology, ed 4, New York, 2000, WH Freeman.)

Finally, a number of cytokine inhibitors were identified, and one of the more studied ones includes a factor called IL-1 receptor antagonist, which is produced by macrophages, hepatocytes, and keratinocytes. This factor inhibits the local and systemic effects of IL-1 by blocking the IL-1 receptor. Another group of inhibitors are the soluble cytokine receptors, which are the enzymatic product of cleavage of the extracellular domain of cytokine receptors that bind their respective cytokines, preventing interaction with the membrane-bound receptor form. The best characterized soluble cytokine receptor is the soluble IL-2 receptor. A number of pathogenic organisms have adapted this as an evasion strategy by producing cytokine-binding proteins or mimics to influence the development of the immune response. Although soluble receptors have been identified in a number of human diseases, their exact role remains to be determined.

Structure and Function of Histocompatibility Antigens

The MHC represents a complex of genes that encode specialized molecules involved in intercellular recognition and the distinguishing of self from nonself. These cell surface molecules have immunologic and nonimmunologic functions. The histocompatibility designation originated from the identification of these molecules in determining the compatibility of transplanted tissues. The MHC is an essential component in humoral and cell-mediated immunity. Most T lymphocytes only recognize fragments of antigen when they are bound to MHC molecules, and this requirement is the basis for MHC restriction. The repertoire of MHC molecules is genetically controlled and determines an individual’s ability or inability to respond to specific antigens. MHC molecules are present throughout vertebrates and are maintained in gene clusters, each of which encodes different MHC products, which are linked. The primary function of cell surface MHC molecules is to bind peptide fragments of foreign proteins for presentation to antigen-specific T lymphocytes. There are three major classes of genes, and these encode for MHC molecules that are grouped according to their structure, tissue distribution, and function. Class I and class II genes encode cell surface molecules. Class III genes encode components of the complement system, enzymes 21-hydroxylase A and B, cytochrome p450, TNF-α and TNF-β, and heat shock protein 70.

MHC class I molecules are present on all nucleated cells (and platelets in some species). Their major function is the presentation of peptide fragments of antigens to CTL (T_c) (CD8⁺). This requirement results in CD8⁺ lymphocytes being MHC class I restricted. MHC class I molecules are further subdivided into highly polymorphic loci, referred to as Ia, and relatively nonpolymorphic loci, referred to as Ib, Ic, and Id. Each class I molecule is composed of a heterodimer consisting of a polymorphic α-chain that is linked to a nonpolymorphic β₂-microglobulin. The extracellular region of the α-chain consists of three domains (α₁, α₂, and α₃). The α₁ and α₂ domains form a groove where the peptide fragments bind the MHC molecule (Web Fig. 5-1). Although MHC class I molecules differ in their ability to bind peptide fragments, they are not as restrictive as antibodies and TCRs. The intracellular processing of antigen into peptide fragments, the association of those fragments with MHC class I molecules, and their transport to the cell surface is a complex process.

Web Fig. 5-1 Schematic diagram and crystal structures of class I and II major histocompatibility complex (MHC) molecules. (Courtesy Dr. P. Bjorkman, California Institute of Technology, Pasadena.)

Antigen uptake by antigen-presenting cells is by phagocytosis or endocytosis. Antigen processing is the degradation of an antigen into peptide fragments, which are complexed with MHC molecules. Antigen presentation is the transport of the peptide-MHC complex to the membrane, where they are displayed. Antigens can arise intracellularly (endogenous) and extracellularly (exogenous), and the immune system most effectively eliminates these antigens through the elaboration of CTL (T_c) or secretion of antibody. The immune system uses two different processing pathways for antigen processing and antigen presentation. Intracellular antigens are processed in a cytosolic pathway and presented in association with MHC class I molecules (Fig. 5-14). Extracellular antigens are processed in an endocytic pathway and presented in association with MHC class II molecules. Endogenous antigens are degraded into small peptide fragments within the cytoplasm by the proteasome complex. Peptide fragments are transported to the endoplasmic reticulum (ER) by an adenosine triphosphate–binding peptide transporter (TAP). Within the ER, the newly synthesized MHC class I α-chain and associated β₂-microglobulin bind the antigenic peptide and form a complex that is transported from the ER to the Golgi and then to the plasma membrane for presentation to CD8⁺ CTL (T_c). The antigen recognition molecule of the cytotoxic T lymphocyte (T_C) recognizes the MHC-peptide complex by means of its CD8 molecule, which functions as a co-receptor, binding to the nonpolymorphic α₃ domain of the MHC class I heavy chain. Because CTL (T_c) only recognize peptides when they are presented as a complex with MHC class I molecules, they are referred to as being MHC class I restricted. MHC restriction is the result of positive selection of lymphocytes during T lymphocyte development in the thymus. Portions of some antigens, those portions not processed for presentation, and in some cases, entire antigens are completely degraded by exopeptidases of the antigen-presenting cell into amino acids and do not initiate an immune response. Endogenous antigens are most frequently encountered during viral infections and thus CTL (T_c) are an important defense mechanism for eliminating virally infected cells.

Fig. 5-14 Antigen processing and presentation by an antigen-presenting cell, and antigen recognition by T lymphocytes.
A class I major histocompatibility complex (MHC)–restricted CD8⁺ lymphocyte is depicted. ER, Endoplasmic reticulum; TCR, T lymphocyte receptor. (From Kumar V, Abbas AK, Fausto N: Robbins & Cotran pathologic basis of disease, ed 7, Philadelphia, 2005, Saunders.)

Class II molecules have a variable tissue distribution depending on the species of domestic animal, but in general they are present on antigen-presenting cells (B lymphocytes, dendritic cells, and macrophages) and can be induced on T lymphocytes, keratinocytes, and endothelial cells by IFN-γ. There are significant species differences with regard to the constitutive expression of MHC molecules. The major function of MHC class II molecules is the induction of T_H lymphocytes. Class II MHC molecules are heterodimers consisting of an α-chain and a β-chain. The antigen-binding site of the class II molecule, unlike that of the class I molecule, is formed by portions of both the α₁ and β₁ domain. Additionally, as with the class I MHC molecules, polymorphism of the class II MHC molecules is associated with determining an individual’s response to antigens of infectious organisms (see next section). Peptides that bind to class II MHC molecules are in general derived from exogenous antigens, which have been internalized and processed within endosomes and lysosomes of antigen-presenting cells. Antigen-presenting cells can internalize antigen by phagocytosis or endocytosis (receptor mediated or pinocytosis). Macrophages are the only cell type capable of both because other antigen-presenting cells are poorly phagocytic. Extracellular antigens are processed into peptide fragments in an endocytic pathway and presented in association with MHC class II molecules on the cell membrane. During the synthesis of a class II molecule within the ER, it associates with another protein called the invariant chain, which prevents the molecule from binding endogenously derived peptides. The complex is then transported from the ER to the Golgi complex and into an endocytic compartment (vesicles) that contains the antigenic peptide fragments derived from exogenous antigens. Proteolytic cleavage of the invariant chain allows for the association of the peptide fragment with the class II MHC molecule. The peptide-class II MHC complex is then transported to the cell surface for presentation to CD4⁺ T_H lymphocytes. The CD4 molecule acts as a coreceptor for induction of T_H lymphocyte activation. Because T_H lymphocytes only recognize peptides when they are presented as a complex with class II MHC molecules, they are referred to as class II MHC restricted. It is worth mentioning that of the two scientists, Peter Doherty and Rolf Zinkernagel, who received the Nobel Prize in Medicine in 1996 for discovering MHC restriction, Dr. Doherty is an Australian veterinarian.

MHC molecules are important in regulating T lymphocyte development in the thymus and in the peripheral lymphoid tissues determining specific responses to different forms of antigens. During the development and maturation process in the thymus, only T lymphocytes capable of recognizing self-MHC molecules are selected (positive selection) for export to the peripheral lymphoid tissues. These developmental processes influence an individual’s T lymphocyte repertoire, which is the functional population that influences immunity. An individual’s ability to mount an effective immune response is determined in part by his or her ability to recognize endogenous and exogenous antigens and their MHC haplotype. Thus, in one case, the association of an antigenic peptide fragment with a specific MHC molecule may result in a protective antibody response with elimination of an infectious agent, whereas in another case, the association of an antigenic peptide fragment with a specific MHC molecule may result in an inappropriate immune response to an innocuous antigen, producing an allergic reaction.

The organization (chromosomal location) and characterization (e.g., number of loci) of the MHC of each species of domestic animals appears to be fairly conserved and present in higher vertebrates and mammals. Most mammalian species studied have class I, II, and III genes, with differences between species being the arrangement and number of genetic loci comprising the MHC. In general, class I genes are more closely related within a species than between species, and the avian MHC is smaller and less complex, with many genes found in the mammalian MHC being absent. Although it is beyond the scope of this chapter to discuss the details regarding the differences between domestic species, the preceding overview is relevant to most domestic species.

Major Histocompatibility Complex and Disease Association

The MHC influences transplant acceptance or rejection, immune responsiveness, and the pathogenesis of a number of diseases. The MHC represents a complex of genes that encode specialized molecules involved in antigen presentation and thus regulate immune responses. The ability of the immune system to respond to an antigen is determined in part by the binding of peptide fragments to MHC molecules, which are then presented on the surface of antigen-presenting cells. There is a growing body of information associating certain MHC alleles with increased or decreased susceptibility to certain diseases (Web Table 5-2). Because the MHC genes of most domestic species are not well characterized, it is difficult to distinguish if the observed effects are due to the MHC itself or to other tightly linked genes. These conclusions are generally the result of an observation that some MHC alleles occur at a higher frequency among individuals affected with the disease, as compared with the general population. The association of an increased risk with certain MHC alleles is never the sole basis for determining if an individual will develop the disease, as often other hereditary and environmental factors also play an important role. The diseases most often associated with certain MHC alleles have a pathogenesis that incorporates a significant immunologic component. The types of diseases identified are diverse; however, they frequently include autoimmune, infectious, and allergic diseases. Additionally, MHC diversity may increase or decrease the susceptibility to infectious diseases. There is evidence that MHC polymorphisms may significantly impact disease resistance and is best illustrated in species in which there is a loss of MHC diversity attributed to a limited breeding pool of animals. In the case of the cheetah and Florida panther, the current breeding stocks of both species are derived from a limited genetic pool, thus resulting in reduced MHC diversity. In both species, there is an increased susceptibility to infectious agents that is not seen in other species of big cats.

WEB TABLE 5-2

Major Histocompatibility Complex (MHC) Polymorphisms Related to Resistance or Susceptibility to Diseases

Although there are a number of hypotheses to account for the role of MHC molecules in disease susceptibility, the actual mechanisms remain elusive. The most often hypothesized mechanism is attributed to the role of MHC alleles in determining responsiveness or nonresponsiveness to a particular pathogen. On the one hand, through antigen presentation and activation of cytotoxic or T lymphocytes, it determines whether a protective immune response is generated to a particular pathogen. On the other hand, certain MHC alleles may encode molecules that are used by infectious agents, as in the case of receptors for viruses or bacterial toxins, and facilitate their infectivity or pathogenicity.