Diseases of Immunity

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.

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.

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).

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.

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).

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.

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.

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).

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.

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.

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.

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.

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.

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.

Type I Hypersensitivity (Immediate Hypersensitivity)

Type I hypersensitivity reactions are most commonly the result of an IgE-mediated immune response directed against environmental antigens (i.e., allergens) and parasite antigens. Harmful IgE-mediated responses to innocuous environmental antigens resulting in allergic reactions are termed hypersensitivity, whereas similar IgE-mediated protective responses to parasite antigens are considered immunity. This distinction emphasizes the fact that these are not unique immunologic reactions but rather misdirected or inappropriate “normal” immune responses. Type I hypersensitivity occurs in a previously sensitized host and is initially manifested as acute inflammatory process that occurs within minutes (“immediate hypersensitivity”) of exposure to the specific antigen. In many instances, the reaction progresses from an early acute inflammatory response to a late-phase response and/or chronic inflammatory lesion that persists (Figs. 5-15 and 5-16). The basic pathogenesis involves a sensitization phase and an effector phase. The sensitization phase occurs during the initial exposure to an antigen when the host develops an antigen-specific IgE response, which results in sensitization of the host by the binding of the antigen-specific IgE to Fcε receptors on the surface of mast cells (Fig. 5-17). The host is now sensitized, and either through a second exposure or prolonged initial exposure to the IgE-specific antigen, there is cross-linking of two or more IgE molecules on the surface of the mast cell. This results in its activation and release of preformed and newly synthesized mediators, resulting in the effector phase. The effector phase can be limited to an acute inflammatory reaction (occurring within minutes), resulting primarily from the release of mast cell mediators, or can progress to a late-phase reaction (over a period of hours), or to a chronic reaction (persisting for days to years). The acute reaction is characterized by responses associated with release of preformed vasoactive amines from the mast cell and includes increased vascular permeability, smooth muscle contraction, and influx of inflammatory cells. The late-phase and chronic reactions, often associated with repeated or prolonged antigen exposures, are largely the result of a more intense inflammatory cell infiltration (primarily eosinophils, neutrophils, macrophages, and T lymphocytes) and tissue damage. Because the mast cell is central to the pathogenesis of a type I hypersensitivity reaction, their biologic features and primary functions are reviewed.

Mast cells are a heterogeneous population of bone marrow–derived cells that reside in vascularized tissue. Mast cells are easily identified by their abundant metachromatic cytoplasmic granules. Metachromasia is defined as the staining of a tissue component so that the color (absorption spectrum) of the tissue-dye complex differs from the color of the original dye and of the other stained tissue. In other words, the metachromatic substance is a different color from those of the dye and the other stained tissue. For example, toluidine blue is a metachromatic dye, and it stains most tissues blue, but mast cell granules are purple. Other commonly used metachromatic dyes include methylene blue and thionine. Wright’s and Giemsa stains are dye mixtures that include a metachromatic dye. Mast cells can be divided into mucosal and connective tissue subpopulations, not only based on their location but also on their phenotypic, morphologic, histochemical, and functional characteristics. This suggests that individual subpopulations of mast cells may have specific functions in normal and pathologic responses that are a result of their activation. The tyrosine kinase receptor, c-kit, expressed on mast cells, their precursors, and its ligand—stem cell factor (SCF)—is essential to mast cell development and function. Alterations in c-kit have been used to molecularly identify poorly differentiated mast cell tumors.

Mast cell activation can occur through a number of immunologic and nonimmunologic mechanisms. In addition to the activation of mast cells through cross-linking of membrane-bound IgE by antigen, other substances and stimuli can also activate mast cells. Mast cells can be activated by Fcε receptor–independent mechanisms, including cytokines (IL-8), complement products (the anaphylatoxins C3a and C5a), drugs (nonsteroidal antiinflammatory drugs, codeine, and morphine), and physical stimuli (heat, cold, and trauma). Non–IgE-mediated activation of mast cells is referred to as an anaphylactoid reaction, whereas the IgE-mediated activation is referred to as type I hypersensitivity. There are species and tissue differences in how type I reactions are manifested, and these are attributable to the types and proportions of mediators produced by the mast cell. Mast cells are a heterogeneous population of cells with regard to their structure and function. Although they are generally divided into mucosal-based and connective tissue–based populations, in either case they are primarily found adjacent to blood vessels and nerves where their mediators have their greatest influence. Mediators released by mast cells are broadly classified as preformed (primary) or newly synthesized (secondary), and as presented in Web Table 5-3 and Fig. 5-17, they influence local tissues and other cell types. Primary mediators are stored in cytoplasmic granules and include the vasoactive amines histamine, serotonin, and adenosine; chemotactic factors for eosinophils and neutrophils; enzymes, including neutral proteases and acid hydrolases; and proteoglycans, such as heparin and the chondroitin sulfates. Newly synthesized mediators consist largely of the lipid mediator products of cyclooxygenase and lipoxygenase metabolism of arachidonic acid (see Chapter 3), a number of cytokines and platelet-activating factor (PAF). The major products of arachidonic metabolism are the prostaglandins and leukotrienes, of which prostaglandin D₂ and leukotrienes C₄, D₄, and E₄ are most important. The major cytokines released from mast cells during a type I reaction include IL-4, IL-5, IL-6, and TNF-α. IL-4 and IL-5 contribute to B lymphocyte activation and IgE synthesis. IL-5 is chemotactic for eosinophils. IL-6 and TNF-α are involved in the pathogenesis of shock during a systemic type I (anaphylactic) reaction. The biochemical events involved in IgE-mediated activation and mediator release by mast cells are similar to those described for leukocyte activation in Chapter 3. The primary actions of preformed and newly synthesized mediators are attributable to cellular infiltration, vasoactive responses, and smooth muscle contraction. PAF, which was first identified as an initiator of platelet aggregation and degranulation, functions not only in the acute phase by increasing vasodilation and vascular permeability but is also important early in the late phase by recruiting and activating inflammatory cells. Finally, it is of note that recent studies have identified TLR pathways that mediate interactions between dendritic cells, T lymphocytes, and mast cells, thus modulating type I responses.

A type I reaction begins as an acute inflammatory reaction mediated largely by the vasoactive amines released by degranulation of mast cells. It is during this early stage that mast cells also release large quantities of chemotactic factors and cytokines. These mediators recruit and activate the inflammatory cells that will not only sustain the inflammatory response in the absence of antigen but also cause tissue damage. The immediate response is characterized by increased blood flow, increased vascular permeability (edema), and smooth muscle spasm. As the reaction progresses, additional leukocytes are recruited, and they release biologically active substances that cause cell damage. Of these leukocytes, eosinophils are particularly important.

Eosinophils are recruited to the sites of type I hypersensitivity reactions by chemokines, such as eotaxin, and their survival is influenced by IL-3, IL-5, and GM-CSF, which are largely derived from T_H2 lymphocytes. Eosinophils recruited during the early response play an active role in the late-phase response by releasing components of their granules, synthesizing lipid mediators, and producing cytokines. The basic proteins released by eosinophils are toxic to parasites and host tissue. In particular, eosinophil major basic protein is toxic not only to parasites but also to tumor cells and normal cells. These proteins contribute to the epithelial cell damage associated with chronic type I reactions. Lipid mediators synthesized by activated eosinophils include PAF, leukotrienes, and lipoxins. Cytokines produced and released by eosinophils include growth factors, chemokines, cytokines involved in inflammation and repair, and regulatory cytokines. Macrophages and lymphocytes also participate in the late-phase response to varying degrees.

Epithelial cells further contribute to the inflammation by becoming activated and producing factors that recruit and activate additional inflammatory cells. It is this complex series of cell activation, recruitment, and mediator release that amplifies the immune response and sustains the inflammatory reaction long after the antigen has gone.

The factors that determine whether a host will develop a type I hypersensitivity reaction are complex. The genetic makeup of the host and the dose and route of antigen exposure are most important. These factors influence whether the individual will have a T_H1 or T_H2 response. The development of an IgE-secreting B lymphocyte from an immature (naïve) B lymphocyte depends on activated CD4⁺ lymphocytes of the T_H2 type. The cytokines that define a T_H2-lymphocyte response have important roles in regulating the cells involved in a type I hypersensitivity reaction. IL-3, IL-4, and IL-10 influence mast cell production; IL-4 is involved in isotype switching to IgE; and IL-3 and IL-5 influence eosinophil maturation and activation. IL-13 promotes the production of IgE. The major cytokine that defines a T_H1 response, IFN-γ, inhibits the T_H2 response. Thus an animal that develops predominantly a T_H2 response to a particular antigen would be more likely to develop a type I hypersensitivity reaction as compared with one that develops predominantly a T_H1 response. The CD4⁺ T lymphocyte plays a central role in the pathogenesis of a type I hypersensitivity. In humans, additional genetic influences can be linked to the human leukocyte antigen (HLA)–linked immune response genes. These genes appear to control allergen-specific IgE responses. As mentioned previously, the association of specific class I MHC molecules with an increased susceptibility to atopy in the dog has been proposed. As with the mast cell and the eosinophil, a role for the CD4⁺ T lymphocyte in the late-phase response has also been described. Studies suggest that the continued production of T_H2 cytokines contributes to the chronic inflammation associated with some chronic type I hypersensitivity reactions.

In summary, type I hypersensitivity is a complex disease process that occurs in sensitized hosts, which can result in three types of responses: (1) an acute inflammatory response, (2) a late-phase response, and (3) a chronic inflammatory response. In sensitized hosts, the cross-linking of IgE on the surface of mast cells results in the immediate release of mediators that influence local tissue and recruit additional inflammatory cells. The acute response is dependent on resident mast cells, whereas the late-phase and chronic responses are dependent on recruited cells, especially the eosinophil. Central to the pathogenesis of a type I hypersensitivity reaction are the T_H2 lymphocytes and the cytokines they produce, which influence IgE production and the recruitment and activation of leukocytes.

Systemic and localized type I hypersensitivity reactions occur in animals. The pathogenesis of many infectious and noninfectious diseases involves the production of IgE and the development of a type I hypersensitivity reaction. Type I hypersensitivity is an allergic reaction that occurs within minutes of exposure to an antigen to which the host has been previously sensitized. Allergy has become synonymous with type I hypersensitivity. By definition, type I hypersensitivity reactions are mediated by IgE. Systemic type I hypersensitivity reactions are called anaphylaxis. Atopy is the genetic predisposition to develop localized type I hypersensitivity reactions to innocuous antigens. Atopy is often limited to an organ or tissue such as in allergic dermatitis and rhinitis, food allergies, and asthma. Non–IgE-mediated allergic-like reactions are referred to as anaphylactoid reactions.

Type II Hypersensitivity (Cytotoxic Hypersensitivity)

In the original Gell and Coombs classification, the type II hypersensitivity reaction was designated as antibody-mediated cytotoxic hypersensitivity. This type of hypersensitivity most often occurs as the result of the development of antibodies directed against antigens on the surface of a cell or in a tissue, with the result that the cell or tissue is destroyed. Antigens may be either endogenous (normal cellular or tissue protein) or exogenous (e.g., a drug or microbial protein adsorbed to the cell). In some instances, the antigen may be a cell surface receptor and the antibody may activate or block the activation of the cell rather than cause cytotoxicity. The pathogenesis of many immune-mediated and autoimmune diseases is centered on the development of antireceptor or antisurface antigen antibodies and a type II hypersensitivity reaction. The largest group of “cytotoxic” hypersensitivity reactions involves the hematologic diseases, with antibodies directed against antigens present on the surface of red blood cells and platelets. Type II hypersensitivity reactions are mediated by antibodies directed against antigens on the surface of tissue or cells so that the tissue or cell is destroyed or the function of the cell is altered. Type II hypersensitivity reactions most frequently involve IgM and IgG and occur within hours after exposure in a sensitized host.

There are three basic antibody-mediated mechanisms that result in type II hypersensitivity (Fig. 5-18). Complement-dependent reactions occur as a result of the complement activating capability of IgG and IgM. Complement activation can mediate cytotoxicity by either the formation of the membrane attack complex, resulting in cell lysis, or the fixation of C3b fragments (opsonization) to the surface facilitating phagocytosis (see Chapter 3). Antibody-dependent reactions can similarly opsonize cells, facilitating phagocytosis, or result in cell lysis by antibody-dependent cellular cytotoxicity. Opsonization of cells by antibody makes them susceptible to destruction by macrophages, neutrophils, NK cells, and eosinophils, all of which bear FcR. This is commonly referred to as antibody-dependent cellular cytotoxicity (ADCC). Finally, antibodies directed against surface receptors may result in altered cell or tissue function. The antireceptor antibodies can function as agonists, stimulating cell function, or as antagonists, blocking receptor function.

Diseases with a type II hypersensitivity pathogenesis are presented in Table 5-5. The physical and biochemical properties of red blood cells, platelets, and leukocytes make them susceptible to cytotoxic reactions. Two properties of red blood cells make them uniquely susceptible to being involved in type II reactions. First, their surface contains a complex array of blood group antigens that can become targets of antibody responses as is commonly the case in transfusion reactions or immune-mediated hemolytic disease of the newborn. Second, the biochemical properties of red blood cells make them prone to adsorb substances such as drugs or antigenic components of infectious agents or tumors. In these instances, the red blood cell may be either directly targeted because the substance alters a surface protein to an extent that it is now recognized as foreign, or indirectly targeted if there is an antibody response to the substance itself. Finally, in autoimmune forms of hemolytic anemia, agranulocytosis, and thrombocytopenia, there is a breakdown of tolerance and the subsequent development of antibodies to normal cells, and as a result they are destroyed.

The majority of cytotoxic type II diseases result in a decrease or loss of a population of cells (e.g., anemia, thrombocytopenia). Noncytotoxic type II diseases are initially characterized by activation or inhibition of cell or tissue function followed by inflammation, which may cause inflammatory damage to the targeted organ. In a type II reaction, the pathogenesis commonly begins with cell surface antigens eliciting an antibody response, whereby the antibodies bind to the cell and the cell is either lysed or complement components attract phagocytic cells that damage tissues by releasing proteolytic enzymes.

Type III Hypersensitivity (Immune Complex Hypersensitivity)

Type III hypersensitivity is designated as immune complex hypersensitivity. This reaction occurs through the formation of antigen-antibody complexes that activate complement and result in tissue damage (Fig. 5-19). The cell or tissue injury is similar to a type II hypersensitivity reaction, although the underlying pathogenesis is different. With a type III reaction, the cell or tissue is being destroyed not because the antibody is being directed against that cell or tissue, but rather because immune complexes either become “stuck” to that cell or are deposited in that tissue. Think of it as an “innocent bystander” reaction: The targeted tissue is not a direct target of the immune response. The pathogenesis begins with the formation of immune complexes that become lodged or are formed in or deposited in tissue and are capable of activating the complement system. Products of complement activation such as anaphylatoxins and chemotactic factors result in neutrophil infiltration and activation. On activation, neutrophils release their enzymes and these result in tissue damage. Like type II hypersensitivity reactions, type III hypersensitivity reactions most frequently involve IgM and IgG and occur within hours after exposure in a sensitized host.

Antigen-antibody complexes form as a part of a normal immune response and usually facilitate the clearance of antigen by the phagocytic system without resulting in a type III hypersensitivity reaction. Although a number of factors determine whether a type III reaction will occur, the most important is the relationship of the antibody response to the quantity of antigen. When antibody is in great excess of antigen, the antigen-antibody complexes formed are large and insoluble, and easily removed by the phagocytic system. When antigen is in great excess of the quantity of antibody, the antigen-antibody complexes formed are too small to be capable of becoming lodged in tissues or of activating the complement system. However, when antigen is in slight excess of antibody, these small soluble complexes can become lodged in tissue and activate the complement system. When this type of small soluble antigen-antibody complex is formed in the circulation, their accumulation in tissue is essentially the result of anatomic and physiologic processes and has no immunologic basis. Finally, it has also been suggested that in some instances immune complex hypersensitivity may be the result of the normal phagocytic system being overwhelmed. Immune complex deposition can be localized to a tissue or generalized if the complexes are formed in circulation. Blood vessels, synovial membranes, glomeruli, and the choroid plexus are particularly vulnerable to deposition of immune complexes. The concentration and size of the complexes determine the sites of deposition.

Type III reactions can develop from antibody responses to endogenous or exogenous antigens and immune complexes can be deposited in a number of tissues (Table 5-6). Although a number of diseases of domestic species involve a type III hypersensitivity pathogenesis, a majority of diseases are the result of persistent infections, autoimmune disease, or inhalation of foreign antigen. Organisms that result in persistent infections are often characterized by a weak antibody response and the development of immune complex formation. A number of autoimmune and immune-mediated diseases result in the development of antibody responses to self-antigens or antigens complexed to self-proteins, and these are capable of generating complement-activating immune complexes. Immune complexes formed against commonly inhaled environmental antigens can lead to the development of an allergic alveolitis. Type III hypersensitivity reactions are mediated by the formation of antigen-antibody complexes, which results in complement activation leading to an influx of neutrophils and subsequent cell or tissue destruction. Antigen-antibody complexes may be formed in the circulation and lodge in tissue or may be formed in the tissue directly. The cell or tissue injury is largely determined by physiologic or anatomic properties rather than an immunologic basis. The pathogenesis of a number of diseases of domestic animals have a type III hypersensitivity basis.

Type IV Hypersensitivity (Delayed-Type Hypersensitivity)

Type IV hypersensitivity is also known as cell-mediated hypersensitivity because it is the result of the interaction of T lymphocytes and the specific antigen to which they have been sensitized. The resulting immune response is mediated either by direct cytotoxicity by CD8⁺ T lymphocytes or by the release of soluble cytokines from CD4⁺ lymphocytes, which act through mediator cells (primarily macrophages) to produce chronic inflammatory reactions (Fig. 5-22). Because these responses are dependent on sensitized T lymphocytes and require 24 to 48 hours to develop, they are also referred to as delayed-type hypersensitivity (DTH). Unlike type I, II, and III hypersensitivity reactions, type IV hypersensitivity is not dependent on an antibody. We first discuss the response mediated primarily by activated CD4⁺ lymphocytes. The prototypical DTH reaction is the localized tuberculin response. After an intradermal exposure of tuberculin, a purified protein derivative (PPD) of the tubercle bacillus, a previously sensitized host will develop a localized type IV reaction at the site of inoculation at 24 to 72 hours. The intradermal antigens are taken up and processed by dendritic Langerhans’ cells, which present antigenic peptides to antigen-specific CD4⁺ lymphocytes that are activated to produce and secrete cytokines that attract and activate other inflammatory cells. Grossly the site appears as a swollen, firm nodule. Microscopically the nodule is composed of interstitial edema and a mononuclear infiltrate that is primarily centered around blood vessels. Early (<12 hours), the infiltrate is primarily neutrophilic, which is replaced largely by macrophages and lymphocytes (>12 hours). The DTH response is generally minimal and short lived because the concentration of PPD injected is small and rapidly degraded. A similar DTH reaction can be used to test for previous exposures to a number of intracellular organisms.

In addition to the tuberculin response, type IV hypersensitivity is the underlying pathogenesis for allergic contact hypersensitivity and granulomatous inflammatory responses. As mentioned with the other hypersensitivity reactions, the components of a type IV hypersensitivity reaction can be considered beneficial (protective immunity) when they occur as an appropriate response to intracellular organisms, or they can be considered harmful (hypersensitivity), for example, when they occur as an inappropriate response to exogenous chemicals or substances that are complexed with proteins, as in the case of allergic contact hypersensitivity.

In the tuberculin reaction, the quantity of antigen limits the extent of the inflammatory response, and resolution of the inflammation generally occurs in 5 to 7 days. This is in contrast to chronic infections with persistent intracellular organisms or poorly degradable intracellular antigens (Table 5-7) that develop into a specific type of chronic inflammatory response called granulomatous inflammation. DTH reactions frequently occur in response to intracellular organisms and cause extensive tissue damage. These diseases are characterized by granulomatous inflammation. In this type of response, the host is unable to destroy or eliminate the organism, resulting in antigen persistence. Compared with the tuberculin reaction, the type of inflammatory infiltrate is different. As discussed in Chapter 3, granulomatous inflammation designates that the inflammatory infiltrate has specific attributes, notably the presence of morphologically transformed macrophages into epithelial-like cells commonly called epithelioid macrophages (Figs. 5-23 and 5-24). Concurrently, there may be many multinucleated giant cells that represent fused macrophages. A number of fusion-related monocyte-macrophage surface proteins have been identified and include receptors for mannose and β₁ integrin, Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1), and the chemoattractant chemokine ligand 2. Lymphocytes can also represent a significant component of the inflammatory infiltrate. Generally, CD4⁺ lymphocytes are interspersed with the macrophages, and CD8⁺ lymphocytes are localized to the periphery. As these lesions progress, they may become organized into nodules commonly called granulomas (see Fig. 5-23, A). Depending on the inciting antigen, there may also be varying proportions of necrosis (often as a necrotic center), calcification of the necrotic tissue, and peripheral fibrous encapsulation. These features are largely the result of lytic enzymes released from activated macrophages. Nonimmunologic granulomas can occur in cases of foreign-body type granulomas, which typically have fewer lymphocytes. In either case, the body is trying to limit the spread or wall-off the inciting antigen.

The type IV hypersensitivity reaction is immunologically specific and like all the hypersensitivity reactions involves a sensitization phase and an effector phase. The sensitization phase occurs with the initial exposure to the antigen and results in the development of antigen-specific memory T lymphocytes. These CD4⁺ lymphocytes recognize peptides presented in the context of class II molecules on the surface of antigen-presenting cells. In this context, the naïve CD4⁺ T lymphocytes develop into functional T_H1 lymphocytes. These activated T_H1 lymphocytes are sometimes designated as T_DTH lymphocytes. Once the host is sensitized, a prolonged exposure or repeat exposure to the antigen results in the development of an effector phase. The effector phase can occur as a cytotoxic response mediated by CD8⁺ lymphocytes or more commonly as a T_H1 response through the elaboration of cytokines by CD4⁺ lymphocytes (see Fig. 5-24). T_H1 cytokines (most importantly, IL-2, IL-3, IFN-γ, and TNF-β) and chemokines (IL-8, macrophage chemotactic and activating factor, and macrophage-inhibition factor) enhance the function of cytokine-producing T lymphocytes (autocrine and paracrine fashion) and attract and activate macrophages. IL-2 induces the proliferation and long-term survival of T lymphocytes. IL-3 supports the growth and differentiation of T_H1 lymphocytes and NK cells. IFN-γ, the key mediator of type IV hypersensitivity, activates macrophages not only to enhance their phagocytic and killing mechanisms but also to enhance their ability to present antigen by inducing increased expression of class II MHC molecules. Activated macrophages and dendritic cells produce IL-12, which also facilitates the development of T_H1 lymphocytes. Activated macrophages also produce IL-1 and TNF-α, both of which act locally to increase the expression of adhesion molecules on endothelial cells, which further facilitates the extravasation of additional inflammatory cells. The production of cytokines and chemokines by the CD4⁺ T_H1 lymphocytes influences macrophage function and mediates the production of cytokines that influence CD4⁺ lymphocytes, resulting in a response that potentially goes from a beneficial protective response (immunity) to a harmful response that results in tissue damage (hypersensitivity).

The beneficial protective response of T lymphocyte–mediated hypersensitivity is not limited to intracellular organisms. It also can be a primary component of transplant rejection and immunity to cancer. There are other harmful T lymphocyte–mediated responses that result in disease. One example is allergic contact hypersensitivity. In allergic contact hypersensitivity, the antigen is often too small to elicit an immune response by itself. These antigens must be complexed with other, larger proteins to become antigenic and are specifically referred to as haptens or generally called contact antigens (Box 5-1). Allergic contact hypersensitivity also depends on processing and presentation of the antigen by dendritic Langerhans’ cells to CD4⁺ lymphocytes in regional lymph nodes. In the case of allergic contact dermatitis, the keratinocyte may also participate by producing a number of cytokines that activate Langerhans’ cells, mast cells, and other inflammatory cells. In the sensitization phase, the protein-hapten complex is taken up and processed by Langerhans’ cells that migrate to regional lymph nodes. In the paracortex region of the lymph node (T lymphocyte area), they present antigenic components to CD4⁺ lymphocytes. The host develops a population of memory lymphocytes and is now sensitized to the antigen. In a sensitized host, continuous exposure to the antigen, or more commonly, repeat exposure to the antigen, results in an effector phase response seen as epidermal vesicle formation with dermal and epidermal infiltrates of mononuclear inflammatory cells. The result is tissue damage that is disproportionate to any beneficial effects of the immune response.

Finally, as mentioned earlier, another form of DTH can occur that is mediated by direct cytotoxicity by CD8⁺ T lymphocytes. This response is most commonly associated with viral infections. CD8⁺ T lymphocytes, bearing viral antigen-specific TCRs, kill antigen-expressing target cells. These cells are commonly referred to as CTL (T_c). The expression of viral proteins on the surface of an infected cell in association of class I MHC molecules serves as the recognition signal for the TCR-CD3 membrane complex. Following recognition of antigen by the CTL (T_c), there is upregulation of adhesion molecules on the CTL (T_c) and the target cell, resulting in a CTL (T_c)–target cell conjugate. This stimulates an activating signal pathway that results in death of the target cell by apoptosis. The two principal mechanisms of CTL (T_c)-mediated apoptosis are (1) the directional delivery of cytotoxic proteins and (2) the interaction of membrane-bound Fas ligand on the CTL (T_c), with the Fas receptor on the target cell. Both depend on the activation of caspases. Perforins and granzymes are preformed cytotoxic proteins contained in the cytoplasmic granules of CTL (T_c). Perforin, released between the conjugated CTL (T_c) and the target cell, is polymerized in the presence of Ca²⁺ and forms pores in the plasma membrane of the target cell, not only causing lysis but also permitting the delivery of granzymes. Granzymes activate caspases, normally present in an inactive proenzyme form, that ultimately result in apoptotic death of the cell. The cross-linking of Fas by its ligand, membrane-bound Fas ligand, results in the activation of the extrinsic (death-receptor–initiated) pathway of apoptosis, which is covered in greater detail in Chapter 1.

Immunologic Tolerance

When exposed to an antigen, the immune system can be responsive and develop an immune response or it can be nonresponsive and develop a state of tolerance. In either case, responsive or nonresponsive, the reaction is immunologically specific and has to be carefully regulated, as a response to a self-antigen or a nonresponse to a pathogen could be equally detrimental. Immunologic tolerance is an active physiologic process and is not simply the lack of an immune response. Immunologic tolerance is defined as a failure of the immune system to respond to a specific antigen after previous exposure to that antigen. It is an absence of a functional response rather than a lack of any response at all. The development of autoimmunity can be simply described as an escape from the mechanism by which self-tolerance is maintained.

Tolerance is maintained by a number of mechanisms including deletion, anergy, and suppression. Deletion, or self-tolerance, is the process of clonally eliminating self-reactive lymphocytes. For T lymphocytes, self-tolerance occurs as a developmental process of immature lymphocytes in the thymus, termed central tolerance, and as a component of mature effector cells on exposure to antigens in the peripheral tissues, known as peripheral tolerance (Fig. 5-26). Although immature B lymphocytes undergo selection processes in the bone marrow, independent of antigen, these are not referred to as central tolerance. Not all self-reactive B and T lymphocytes are deleted during their development. However, there are mechanisms of anergy and suppression that prevent the activation of self-reactive T or B lymphocytes outside the thymus and bone marrow.

Failure of Peripheral Tolerance

The mechanisms of self-tolerance, as previously discussed, serve as a basis for presenting how a failure to maintain those mechanisms can contribute to the pathogenesis of autoimmunity.

Genetic Factors in Autoimmunity

The majority of autoimmune diseases in humans have a strong genetic predisposition. The most well-studied genetic component centers around the MHC molecules. As previously discussed, MHC molecules are important in the development of lymphocytes and in the regulation of peripheral effector lymphocytes. Just as autoreactive lymphocytes have been identified in normal individuals without autoimmune disease, the presence of certain MHC molecules themselves is not sufficient to result in autoimmune disease. These observations would suggest that the expression of an autoimmune phenotype is not likely to be the result of a single-gene defect. Other genes that regulate proteins involved in other aspects of the immune response, or that are involved in the inflammatory or healing response, may also be involved. Additionally, experimental variations in the expression and activity of transcription factors can influence the expression of certain autoimmune diseases.

Several strains of mice with specific genetic mutations of factors involved in the maintenance of central and peripheral tolerance that results in autoimmune disease have been identified. Mice with defects of Fas or FasL have disruption of the activation-induced cell death signal in lymphocytes, resulting in autoimmune disease. Mice lacking the transcriptional factor AIRE, which is responsible for thymic expression of self-antigens, and mice defective in the expression of CTLA-4, the inhibitory receptor involved in T lymphocyte anergy, also develop autoimmunity. An important regulatory cytokine—IL-2, the major growth factor for lymphocytes—is also required for the development and function of regulatory T lymphocytes. Mice lacking either IL-2 or the IL-2 receptor develop autoimmune disease characterized by inflammatory bowel disease, anti-DNA antibodies, and autoimmune hemolytic anemia. The proposed mechanism of autoimmunity in these mice is thought to involve T lymphocytes and be a result of a failure of suppression by regulatory T lymphocytes and a failure of activation-induced cell death, two mechanisms of peripheral tolerance. These mouse models of autoimmune disease have facilitated the identification of pathogenic mechanisms of autoimmunity. Although all of these mechanisms have now been identified in human autoimmune diseases, it is likely only a matter of time before they are identified in other species.

Of the domestic species, there have been a number of autoimmune diseases in dogs documented to have a familial tendency, and the mechanism is in part attributed to certain MHC alleles. These recognized associations of specific autoimmune diseases and MHC molecules have been limited to a few specific breeds. It should also be noted that other diseases of immunity, for example, immunodeficiency and atopy, also have a higher incidence in some breeds.

Microbial Agents in Autoimmunity

The recognition that certain infections may result in the development of an autoimmune disease is the result of two observations. First, experimentally, one can induce autoimmunity in specific strains of mice by infection with certain strains of virus. Second, many spontaneous autoimmune diseases occur after viral infections, although attempts to isolate and identify viral agents in patients with autoimmune diseases have been equivocal. Again, these observations would suggest that these diseases have a complex pathogenesis with a genetic and an environmental component.

The role of infections as environmental factors in the pathogenesis of autoimmune diseases may be explained by understanding how infectious agents may cause a breakdown of anergy to self-molecules (loss of self-tolerance). As discussed earlier, not all self-reacting B and T lymphocytes are eliminated during the differentiation and development process. These potentially self-reacting lymphocytes are regulated in the periphery by clonal anergy. Therefore a loss of this regulation could explain how autoimmune diseases develop. Plausible mechanisms of why infectious agents cause aberrations of peripheral clonal anergy are twofold (Fig. 5-29). One mechanism may be the result of nonspecific disruption of the regulatory cells, which results in the induction of co-stimulatory molecules on antigen-presenting cells that are expressing self-molecules. This mechanism is not specific to the antigens of the infectious agent and is likely the result of the overall inflammatory response to the pathogen. Additionally, during an inflammatory response, some cells are induced by the inflammatory cytokine IFN-γ to increase their expression of MHC molecules. This can result in the expression of MHC molecules by cells that normally do not express them. Although the expression of MHC molecules in the absence of co-stimulatory molecules will not activate T lymphocytes, it does increase the potential to do so if co-stimulatory molecules are inappropriately expressed. The second mechanism is specific to the antigens of the infectious agent and is the result of cross-reactivity of T lymphocytes with an infectious agent’s antigen and a self-antigen. Many infectious agents express antigens that have similar peptide sequences to those of normal peptides as a part of their immune evasion mechanism. Therefore the potential exists for any immune response to an infectious agent to cross-react with a normal peptide, resulting in an immune response directed against self-cells or self-tissues. This mechanism is called molecular mimicry. The result is the activation of T lymphocytes that recognize the infectious agent peptide–MHC complex. These T lymphocytes can potentially attack self-peptide–MHC complexes that are cross-reactive.

Once an autoimmune disease is initiated, the clinical course is generally progressive and characterized by cyclical periods of exacerbation and remission. As with any immune-mediated disease, antigen persistence is required to maintain the functional immune response. In autoimmune diseases, antigen persistence is thought in part to occur through epitope spreading. Epitope spreading is the process by which the immune response spreads from one epitope of an antigenic molecule to another, non–cross-reacting, epitope of the same antigenic molecule, or from one epitope of different peptides that are a part of a large complex. The epitopes involved are frequently ones that the immune response has not developed tolerance against because they are not normally presented by MHC molecules in sufficient concentrations. These so-called cryptic epitopes are normally not expressed at sufficient concentrations or are “hidden” during differentiation and development of lymphocytes. However, during an infection or inflammatory response, there may be tissue or cell damage that results in the release or expression of cryptic or hidden epitopes of self-antigens, and these become targets for the immune response. Because these epitopes were hidden, the immune system has not developed tolerance to them. Epitope spreading is thought to maintain the initiated immune response by means of the continued recruitment of autoreactive T lymphocytes specific for normal “cryptic” self-peptides.

With this basic understanding of self-tolerance and the possible molecular mechanisms involved in the pathogenesis of autoimmunity, we can present some of the more common autoimmune diseases of domestic species. Autoimmune diseases can be organ specific or systemic. With many of the organ-specific diseases, an immunologically mediated pathogenesis is suspected because of the finding of a lymphocytic inflammatory reaction within the affected tissue. Rarely, autoantibodies may be identified in the circulation. The organ-specific autoimmune diseases are discussed in the appropriate chapters covering individual organ systems. This chapter focuses on some of the systemic autoimmune diseases of domestic species, beginning with the multisystemic disease SLE.

Etiology and Pathogenesis

The cause of SLE remains undetermined, although the presence of autoantibodies directed against a number of tissues and cellular components suggests that the underlying immunologic abnormality is a failure to maintain self-tolerance. Antibodies against nuclear and cytoplasmic components, which are neither organ specific nor species specific, and those directed against cell surface antigens, particularly red blood cell antigens, are central to the pathogenesis of the disease. Detection of autoantibodies also facilitates the diagnosis and monitoring of human patients with SLE. The autoantibodies and self-antigens form immune complexes, which can be deposited in glomeruli (glomerulonephritis), blood vessels (vasculitis), skin (dermatitis), and joints (arthritis), resulting in the major clinical signs associated with the disease.

ANAs are found in a high percentage of patients with SLE. In humans, antinuclear antibodies are grouped into four categories: (1) antibodies against DNA, (2) antibodies against histones, (3) antibodies to nonhistone proteins bound to RNA, and (4) antibodies against nucleolar antigens. The most common method of measuring antinuclear antibodies is indirect immunofluorescence. The pattern of immunofluorescence is used to help identify the type of autoantibody present. Other methods can be used to more specifically identify the target of ANA. In humans, the majority of ANAs are directed against nucleic acids in native double-stranded DNA, in contrast to the dog, in which the majority of ANAs are directed against nuclear proteins such as histones and extractable nuclear antigens (ENAs). ANAs are also found in normal patients and in patients with other diseases; however, their frequency is much lower. In the dog, the incidence of ANA in normal dogs and dogs with other canine diseases is 16% and 20%, respectively, compared with 97% to 100% in dogs with SLE. The indirect immunofluorescence test for ANA is sensitive but not specific because of the relatively high incidence in normal dogs and those with non-SLE diseases. Two anti-ENA antibodies appear to be specific for canine SLE. These are the anti-Sm and the anti-T1 antibodies.

Patients with SLE frequently have autoantibodies in an array of tissue and cells. Many patients have rheumatoid factors and thus have a positive Coombs’ test for anti-IgG antibodies. Antibodies directed against cellular antigens on red blood cells, platelets, and lymphocytes are frequently noted. In these instances, they may lead to clinical signs of hemolytic anemia (anti-RBC antibodies), thrombocytopenia (antiplatelet antibodies), and immune system abnormalities (antilymphocyte antibodies). Other autoantibodies to components of muscle (myositis) and skin (dermatitis) are also frequently detected. While there can be an extensive number of autoantibodies identified in patients with SLE, the major clinical signs are attributed to the deposition of immune complexes in the joint, skin, and kidney and the elaboration of a type III hypersensitivity reaction. The tissues most frequently involved are in the joint, skin, and kidney.

Canine lupus primarily affects middle-aged dogs and in some studies has been reported to occur more frequently in males than females. Breeds overrepresented are the Shetland sheepdog, German shepherd, Old English sheepdog, Afghan hound, beagle, Irish setter, and poodle. Affected dogs usually present with a spectrum of clinical signs and the disease has a progressive clinical course. Common clinical findings include fever, nonerosive polyarthritis, glomerulonephritis, mucocutaneous lesions, lymph node and splenic enlargement, and hematologic abnormalities (e.g., anemia, thrombocytopenia, and leukopenia). ANAs are the most common immunologic finding. In some reports, up to 100% of affected animals have been positive for ANAs. Dogs can also have antierythrocyte antibodies (Coombs’ positive), anti-IgG antibodies (rheumatoid factor positive), circulating immune complexes and deposits of these in the skin. Only the direct Coombs’ test is valid in the dog. In the dog, as in other species, the immunologic abnormalities involve both humoral and cellular immunity. The abnormalities in humoral immunity are largely attributed to the already discussed presence of autoantibodies and are centered on the activation of self-reactive B lymphocytes. The abnormalities in cellular immunity include a lymphopenia that is characterized by a decrease in the percentage and absolute number of CD8⁺ lymphocytes and a concurrent increase in the percentage and a decrease in the absolute number of CD4⁺ lymphocytes. This translates into a high CD4⁺:CD8⁺ ratio (as high as 6 in dogs with SLE versus <2 in normal dogs).

Lesions of Systemic Lupus Erythematosus

A wide spectrum of morphologic lesions is associated with canine SLE. The most common finding, polyarthritis, is characterized as a nonerosive lesion that commonly affects the intervertebral, carpal, tarsal, and temporomandibular joints. The acute arthritis is characterized by exudation of neutrophils and fibrin into the synovial membrane and concurrent perivascular cuffing by mononuclear cells. The primary differential diagnosis for the arthritis is rheumatoid arthritis, which is an erosive lesion. The renal lesion, the result of immune complex deposition, involves the glomerulus, blood vessels, and the basement membranes of renal tubules (Web Fig. 5-3). The resulting glomerulonephritis is variable in appearance and ranges from slight mesangial alterations to diffuse proliferative lesions. The renal lesions have a common pathogenic mechanism that is a result of the deposition of immune complexes and the activation of complement (type III hypersensitivity). Glomerular lesions are often indicated by a persistent proteinuria (>0.5 g/dL).

Skin lesions are highly variable and nonspecific in canine SLE. The face, ears, and digital extremities are frequently involved and characterized by erythema, ulceration, and exfoliative dermatitis. The distribution of the lesions suggests that photosensitization may play a role. Histologically, the epidermis is characterized by basal cell vacuolation and necrosis, and the dermis is variably edematous with a superficial infiltration of mononuclear inflammatory cells at the dermal-epidermal junction (interface dermatitis). Additionally, there may be a panniculitis, composed primarily of lymphocytes and plasma cells, and vasculitis, with fibrinoid necrosis of the vessel wall. By indirect immunofluorescence, there are deposits of immunoglobulin and complement components at the dermal-epidermal junction. Other dermatologic variants of lupus are covered in Chapter 17. The definitive diagnosis of SLE is based on accepted criteria rather than pathognomonic findings. Using 11 established criteria for the dog, modified from the American Rheumatism Association criteria for humans, a definitive diagnosis requires the presence of four or more criteria. A “probable” diagnosis is based on the presence of three criteria or the presence of polyarthritis with the identification of ANAs. Other diagnostic schemes, using “major” and “minor” signs along with a positive ANA or SLE prep test, are also used to make definitive and probable diagnoses.

In the cat, SLE is less well recognized and manifests with fever, glomerulonephritis, dermatitis, and hemolytic anemia. The ANA test in cats is less reliable because many normal cats can have positive tests results. Horses with SLE similarly present with generalized skin disease and may also have glomerulonephritis, arthritis, and hemolytic anemia.

Genetic Factors

SLE in humans is characterized as a disease with a complex genetic component with MHC and multiple non-MHC genes involved. Extensive genetic studies in domestic animals are lacking, yet it is reasonable to suggest that the disease in other species is also characterized by involvement of multiple genes. The association of SLE with certain MHC alleles in human beings indicates that the MHC genes that regulate the production of specific antibodies are involved—specifically, MHC alleles that are linked to the production of anti–double-stranded DNA, anti-Sm, and antiphospholipid antibodies. Other than the observation of breed predilections for the dog and cat and the report of an association with the canine MHC allele DLA-A7, there are no definitive genetic studies similar to those reported in humans. Interestingly a low percentage of humans with SLE also have inherited deficiencies of complement components such as C2, C4, or C1q. Because complement components are important in the removal of circulating immune complexes by the monocyte-macrophage system, such a deficiency may contribute to the deposition of circulating complexes into tissue rather than to their removal. There is an increase in lupus-like autoimmunity in mice lacking certain complement components. Finally a well-described animal model of SLE is the NZB ∞ NZW mouse strain, in which a number of genetic loci have been identified as being associated with the development of the disease.

Environmental Factors

In addition to the genetic factors, SLE in humans has also been associated with a number of environmental factors. Specifically, drugs such as hydralazine, procainamide, and d-penicillamine can induce an SLE-like disease. In domestic animals, exposure to specific drugs and viral infections are suspected. Exposure to ultraviolet (UV) light is known to exacerbate the disease in the dog. These patients present with dermatologic manifestations localized to areas exposed to sunlight (e.g., face and dorsal regions) or areas lacking adequate hair coat (e.g., axillary region). A similar association has been noted in humans with SLE. The influence of UV radiation may be attributed to tissue damage and inflammation that result in activation of keratinocytes and the elaboration of IL-1, or the modification of DNA through the induction of apoptosis that renders the DNA immunogenic. The influence of sex hormones on the occurrence and manifestations of SLE in humans has not been documented in domestic species.

Immunologic Factors

As discussed previously, SLE is characterized by a number of immunologic abnormalities and is clinically noted to have manifestations attributable to specific immune components. It is therefore reasonable to suggest that the pathogenesis of SLE involves aberrations of humoral and/or cell-mediated immunity. Previously, as a result of the documentation of autoantibodies in patients with SLE, it was hypothesized that the pathogenesis centered on an intrinsic B lymphocyte defect. Additionally, polyclonal B lymphocyte activation is a common immunologic abnormality seen in patients with SLE and in animals that are models for the disease. Recent studies, however, indicate that the autoantibodies associated with the development of clinical SLE are not the result of polyclonal B lymphocyte activation but rather the result of antigen-specific T_H lymphocyte–dependent B lymphocyte responses. This observation is consistent with our overall understanding of autoimmunity, and the current hypothesis is that these diseases are more likely to be the result of an immunologic dysregulation centered on T_H lymphocytes. The currently proposed model for the pathogenesis of SLE is presented in Fig. 5-30. This model is an oversimplification of a complex disease with genetic and nongenetic factors that contribute to the development of a complex multisystemic disease with numerous clinical presentations, a progressive disease course, and an absence of specific cause.

Although the underlying cause of autoantibody production in SLE remains unknown, the elaboration of antibody-peptide complexes is central to the mechanism of tissue damage. The majority of lesions in SLE are the result of immune complex disease (type III hypersensitivity). Antibodies directed against cell surface antigens also lead to the destruction of leukocytes, red blood cells, and platelets through direct cell lysis and enhanced removal by opsonization and phagocytosis. ANAs bind to cell-free nuclei to produce characteristic hematoxylin bodies or lupus erythematosus (LE) bodies. These bodies are frequently found in the skin, kidney, lung, lymph node, spleen, and heart of patients with SLE. These ANAs can also lead to the formation of LE cells, which are typically present in the bone marrow and are a phagocytic cell (macrophage or neutrophil) that has engulfed an opsonized nucleus. This phenomenon is also used as an in vitro diagnostic test to demonstrate the presence of ANA (LE test or LE prep).

In summary, SLE represents the prototypical multiorgan autoimmune disease with a highly variable clinical presentation, a complex cause, and pathogenesis involving multiple genetic and environmental factors and a multitude of immunologic abnormalities. The current pathogenesis suggests that these factors contribute to the activation of T and B lymphocytes, resulting in the production of autoantibodies directed against a number of self-constituents, namely molecules within the nucleus, in the cytoplasm, or on the cell surface.

Etiology and Pathogenesis

The keratoconjunctivitis sicca (dry eyes) and xerostomia (dry mouth) result from the lymphocytic infiltration and fibrosis of lacrimal and salivary glands (lymphoplasmacytic sialoadenitis). In humans the infiltrate is primarily composed of activated CD4⁺ lymphocytes and fewer B lymphocytes and plasma cells. Affected dogs are frequently hypergammaglobulinemic and less frequently have identifiable ANAs and rheumatoid factors. Many human patients have ANAs, rheumatoid factors, and nonorgan specific autoantibodies. Two autoantibodies specific to Sjögren syndrome in humans are directed against ribonucleoproteins, SS-A (Ro) and SS-B (La), which are considered serologic markers of the disease. Although autoantibodies can be identified, there is no direct evidence that they are the primary cause of tissue injury in any species evaluated to date. With the identification of autoantibodies and the presence of T lymphocytes within affected tissues, it is likely that the disease is the result of immunologic dysregulation centered on helper T lymphocytes. Sjögren syndrome in humans also is weakly correlated with certain MHC alleles, suggesting that as in SLE, the presence of certain MHC alleles may predispose the development of the disease.

As with many autoimmune diseases, viruses are suspected as potential causal agents. In most species in which viruses have been implicated, the evidence is largely circumstantial and Koch’s postulates have rarely been satisfied. The mechanisms by which infectious agents can induce autoimmunity were discussed earlier.

Clinical Signs and Lesions

Dogs with Sjögren-like syndrome have an adult onset of conjunctivitis and keratitis. Other findings include gingivitis and stomatitis. A case reported in the cat was characterized by dry eyes and enlarged salivary glands. The keratoconjunctivitis frequently leads to blepharospasm and conjunctival hyperemia. The xerostomia leads to dysphagia. Involvement of tissues other than salivary and lacrimal glands, as is reported in about one-third of human cases, has not been documented in the dog and cat. Human patients have lymph node involvement that is characterized as a pleomorphic infiltrate with increased mitoses and are at a fortyfold increased risk for developing lymphoid malignancies. Microscopically the salivary and lacrimal glands are infiltrated predominately by lymphocytes (Fig. 5-31). In the cat, immunohistochemical analysis of lesions in these glands indicated that the predominant cell type was positive for CD79 (B lymphocyte marker) with fewer, scattered CD3⁺ lymphocytes (T lymphocyte marker) and plasma cells. A mild interstitial fibrosis was also noted.

Dermatomyositis

Dermatomyositis is an inflammatory disease of the skin, muscles, and vasculature affecting primarily young dogs. The cause and pathogenesis are unknown. The disease has a higher incidence in the collie and Shetland sheepdog breeds, and in these breeds, it is often referred to as canine familial dermatomyositis, an inheritable inflammatory disease of the skin and muscle. The disease has also been diagnosed in a number of other breeds. A sex predilection has not been reported. The clinical and pathologic findings suggest that an immune-mediated or an autoimmune mechanism is involved in the pathogenesis.

Clinical Signs and Immunologic Abnormalities

The classic presentation is a febrile (104° F to 107° F) young dog with anorexia, hunched stance, cervical pain, and an unwillingness to move the head and neck. The disease has a cyclic course, with two to seven periods of signs that resolve. They have a moderate to notable leukocytosis, with neutrophilia, nonregenerative anemia, and hypoalbuminemia. Cerebral spinal fluid analysis indicates a neutrophilic pleocytosis with mild to moderate increases in microprotein. On serum protein electrophoresis, they have a high α₂-globulin fraction. ANA tests, LE preparations, and rheumatoid factor tests are generally negative. Immunologic abnormalities include an increase in serum IgA concentration, an increase in the percentage of peripheral B lymphocytes, and a decrease in the percentage of total peripheral T lymphocytes, a marked suppression of the blastogenic response to mitogenic stimulation, an inability to generate immunoglobulin-secreting plasma cells after polyclonal activation, and evidence of monocyte-macrophage activation. There are increased concentrations of IL-6 in the serum of acutely affected dogs, and these return to base-line concentrations during periods of remission or after corticosteroid therapy.

Lesions

Severe necrotizing vasculitis and perivasculitis with thrombosis of small- to medium-sized blood vessels in the leptomeninges of the cervical spinal cord, cranial mediastinum, and heart are commonly seen (Fig. 5-32). In severe cases, a more widespread distribution of vascular lesions occurs and commonly involves the thyroid gland, small intestine, testes, diaphragm, esophagus, and urinary bladder. Most patients experience multiple episodes, but some may have only one to two episodes before becoming normal, and clinical signs appear to resolve in all but the most severely affected patients by 12 to 18 months of age. Some dogs that experience repeated acute episodes develop splenic, hepatic, and renal amyloidosis. The pathogenesis of amyloidosis is attributed to serum amyloidal A production and the development of reactive systemic amyloidosis secondary to the vascular inflammation.

Primary Immunodeficiencies

Most primary immunodeficiency diseases are the result of a genetic defect (inherited or congenital) and affect specific (i.e., humoral and cell-mediated arms of the adaptive immune response) or nonspecific immunity (i.e., components of innate immune responses, such as complement, phagocytosis, NK cells, and so forth). Specific defects in adaptive immune responses can be divided into those affecting T lymphocytes or B lymphocytes or both (Fig. 5-33). As already discussed, interactions between B and T lymphocytes are necessary for the development of many immune responses. In some instances, the clinical distinction between a primary B lymphocyte defect and a T lymphocyte defect may not be obvious with respect to humoral immunity. For example, T lymphocyte defects almost always result in impaired antibody synthesis and as such are indistinguishable from B lymphocyte defects or combined B lymphocyte and T lymphocyte defects. In general, most primary deficiencies manifest themselves early in life and affected patients clinically have a failure to thrive and a susceptibility to recurrent infections. In many instances, the type of infection suggests to some extent the likely component of the immune system that is defective (Table 5-8). It is beyond the scope of this chapter to present all of the forms of human and rodent immunodeficiency, and thus only a few are referenced for clarity to facilitate understanding diseases that have been characterized in domestic species.

Primary Immunodeficiencies of Specific Immunity

Primary Immunodeficiencies of Nonspecific Immunity

Chemical Nature of Amyloid

Amyloid contains approximately 95% fibrillar proteins and 5% P component and other glycoproteins. Of the 15 biochemically distinct forms of amyloid recognized in humans, 3 are most common: (1) amyloid light chain (AL), derived from immunoglobulin light chains of plasma cells; (2) amyloid associated (AA), derived from the acute phase protein serum amyloid A (SAA); and (3) Aβ amyloid, derived from amyloid precursor protein (APP). These forms of amyloid are also recognized in domestic species.

The AL form of amyloid can contain complete immunoglobulin light chains, the NH₂-terminus portion of immunoglobulin light chains, or both. Of the two immunoglobulin light chains, λ and κ, most AL forms consist of λ-light chains or their fragments. Immunoglobulin-secreting cells, B lymphocytes, and plasma cells are associated with deposition of AL amyloid.

The AA form of amyloid consists of a proteolytic fragment of a larger acute phase protein SAA, which is found in the plasma and synthesized in the liver and released during systemic inflammatory reactions.

The Aβ form of amyloid contains a proteolytic fragment of the APP and is associated with the cerebral amyloid angiopathy of Alzheimer’s disease in humans and with some forms of neurodegeneration in the canine brain.

Of the large number of other biochemically distinct proteins that have been associated with the formation of amyloid in a number of human clinical diseases, only a few are relevant to domestic species. One of these is islet amyloidosis, affecting the feline pancreas. Islet amyloid is derived from islet amyloid polypeptide (IAPP), which is a hormone synthesized by the β cells of the pancreatic islets. All forms of amyloid contain other minor components, including serum amyloid P protein, proteoglycans, and glycosaminoglycans. Serum amyloid P may function to stabilize the fibrils and decrease their susceptibility to proteolysis.

Classification of Amyloidosis

In addition to the classification based on the primary biochemical constituent as discussed previously, it is also useful from a pathologist’s perspective to categorize these conditions according to their clinical disease and pathogenesis (Table 5-9). Amyloid can be systemic (generalized), with deposits in many organ systems, or it can be localized, with deposits limited to a single organ. The systemic or generalized category can be further subclassified into primary amyloidosis (AL), when associated with immunocyte dyscrasia, or secondary amyloidosis (SAA), when associated with a chronic inflammatory or destructive tissue process. Hereditary or familial amyloidosis is in a separate heterogeneous category with several distinctive patterns of organ involvement that are primarily recognized in humans.

Pathogenesis of Amyloidosis

Amyloid deposition is the result of abnormal folding of pathogenic proteins, deposited extracellularly as fibrils organized into β-pleated sheets. Although there are many diverse proteins associated with the formation of amyloid, they are all characterized by misfolded proteins leading to the formation of fibrils that are unstable and self-associated. Equally diverse are the number of conditions that can be associated with the formation of amyloid, and each of these conditions is characterized by excessive production of amyloidogenic proteins that are prone to misfolding. The two general categories of amyloidogenic proteins are (1) those that are normal proteins that have an inherent tendency to misfold and form fibrils when produced in excess quantities and (2) those that are abnormal protein products as a result of genetic mutations that are structurally unstable and form fibrils that self-associate. Misfolded proteins are normally degraded intracellularly within proteasomes or extracellularly by macrophages. In amyloidosis, these degradative processes are inadequate and may be responsible for the accumulation of misfolded proteins extracellularly. A simplified pathogenic scheme for the major forms of amyloidosis is presented in Fig. 5-37. As discussed previously, the overproduction of a precursor protein, although necessary, by itself is not sufficient to result in the formation of amyloid. In secondary reactive systemic amyloidosis, a component of a systemic inflammatory reaction results in the activation of macrophages that elaborate endogenous pyrogens IL-1 and IL-6, which stimulate hepatocytes to synthesize and secrete SAA.

During an inflammatory reaction, the quantity of SAA in the serum may increase several hundred times normal concentrations; however, not all systemic inflammatory reactions result in the formation of AA amyloid. Why is it that only some inflammatory reactions lead to amyloidosis? Two theories, one based on a defective degradation system for SAA and one based on abnormal SAA protein that is resistant to degradation, have been proposed. SAA is normally degraded to soluble protein products by enzymatic components of the monocyte-macrophage system. An enzymatic defect in this system may result in incomplete degradation and the production of insoluble AA molecules that form fibrils. Alternatively, a mutation of an SAA gene may result in the synthesis of an abnormal SAA protein that is resistant to degradation and prone to producing insoluble AA molecules that form fibrils. Finally, in the AL form of amyloidosis, plasma cells synthesize excessive quantities of immunoglobulin light chains that are resistant to complete degradation and susceptible to forming insoluble fibrils. Although there have been numerous attempts to identify specific sequence motifs unique to AA and AL forms of amyloid, to date none has been identified. Another research interest has focused on the existence of the diversity of SAA isoforms and their relationship to the formation of AA amyloid.

Morphology of Amyloidosis

Although there are no consistent or characteristic patterns for the distribution of amyloid deposition in any of the categories discussed, a few generalizations can be made. Reactive systemic amyloidosis, secondary to chronic inflammatory conditions, is often the most severe of the systemic forms. Liver, kidney, spleen, lymph nodes, and adrenal glands are most commonly affected. Animals with renal amyloidosis frequently die from renal failure.

Grossly affected organs are often enlarged, moderately firm, and abnormally discolored. In some instances, the application of an iodine solution (e.g., Lugol’s iodine) to an affected tissue may stain the deposits brown, which develops into a blue-violet color when exposed to dilute sulfuric acid solution.

Microscopically the diagnosis of amyloid is based on its extracellular location and staining characteristics. As discussed previously, Congo red is the most commonly used stain for the identification of amyloid. Congo red stains amyloid orange to red under light microscopy, which is seen under polarized light as an apple-green birefringent material. All forms of amyloid stain with Congo red, as the reaction is based on the presence of fibril organized into β-pleated sheets. Immunohistochemistry can also be used not only to identify amyloid deposits but also to identify the specific constituents composing the deposits (e.g., anti–β-light chain antibodies). By electron microscopy, amyloid fibrils are characterized as nonbranching, 7.5- to 10-nm diameter tubules. Descriptions of individual organ system involvement are covered in their respective chapters.