Cytokines and Chemokines

Cytokines are secreted from cells and function in communicating with surrounding cells (Table 8-2). They are soluble proteins or glycoproteins that affect the growth, differentiation, function, and activation functions of other cells. These soluble hormonelike molecules were initially discovered in association with lymphocytes and termed lymphokines, or monocytes and termed monokines. As it was discovered that many other cells produced the same substances, these terms, and others such as secretory regulins and peptide regulatory factors, were no longer considered appropriate. Cytokines are transiently produced and exert their biologic activities via specific cell-surface receptors of target cells, which may be expressed only after activation of the cell. Each mediator usually has multiple overlapping activities. Numerous cytokines have been described, and typically, they may perform several different functions, depending on the tissue they interact with and the other cytokines that may be present. In different environments, the same cytokine may even have opposite effects.

TABLE 8-2 Immunologic Properties of Cytokines

Cytokines may affect the same cell in a permissive, inhibitor, additive, or suppressive manner. Cytokines are involved in virtually every facet of immunity and inflammation, including antigen presentation, bone marrow differentiation, cellular recruitment and activation, adhesion molecule expression, and acute-phase reactions (see Table 8-2). The particular cytokines produced in response to an immunologic insult will determine whether an immune response develops and whether the response will be humoral, cell-mediated, or allergic. Certain cell types, particularly T-lymphocytes, may secrete different patterns of cytokines, and this has been used to subclassify these cells and their associated different functions. A large group of cytokines have been identified that have as their sole or major purpose the direction of the movement of cells involved in inflammation and the immune response. These have been termed chemokines (Table 8-3).

TABLE 8-3 Chemokines and Their Actions

Adhesion Molecules

Glycoproteins critical for cell-to-cell and cell-to-matrix adhesion, contact, and communication, adhesion molecules play an integral role in cutaneous inflammation and immunology (see Chapter 1) (Table 8-4). The integrin family includes membrane glycoproteins with α and ß subunits, such as vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells, which binds T-lymphocytes and monocytes via vascular leukocyte adherin-4 (VLA-4), and fibronectin and laminin, which bind keratinocytes and mast cells. The immunoglobulin gene superfamily contains intercellular adhesion molecule-1 (ICAM-1) found on keratinocytes, Langerhans cells, and endothelial cells, which bind leukocytes via leukocyte function-associated antigen-1 (LFA-1) or CD11a/CD18. The selectin family includes lectin adhesion molecule-1 (LECAM-1 or L-selectin) on lymphocytes, which binds endothelial leukocyte adhesion molecule-1 (ELAM-1 or E-selectin), and Gmp-140 (P-selectin) as a “homing” mechanism. The cadherin family is important in desmosome function (see Chapter 1).

TABLE 8-4 Cell Adhesion Molecules

Major Histocompatibility Complex

The major histocompatibility complex (MHC) is a cluster of genes that encodes a range of molecules of fundamental importantance to the immune system. MHC class I loci encode the classical histocompatibility antigens, which are expressed by all nucleated cells of the body and are target antigens in the rejection of incompatible tissue grafts. Products of the MHC class III loci include a number of factors of the complement pathways, two cytokines (tumor necrosis factor-α [TNF-α] and TNF-ß), and two heat-shock proteins. The MHC class II loci encode a series of transmembrane molecules with restricted expression by cells of the immune system, particularly the antigen-presenting cells (APCs): Langerhans cells, dermal dendritic cells, macrophages, and activated T-lymphocytes. Granulocytes do not express MHC class II, which can be upregulated on keratinocytes and endothelial cells by cytokines (e.g., interferon-γ [IFN-γ]).

SALT

The immune system and its inflammatory component are complex models of biologic activity and interaction. There is a tendency to dissect the immune response into its individual components and to discuss them as autonomous functional units. Immune responses are interwoven and interdependent, however, and manipulation of one component influences others. The skin itself is an integral and active component of the immune system. In fact, the skin is a functioning lymphoid organ; as such, it has been referred to as skin-associated lymphoid tissue (SALT). Even prior to the development of the SALT concept, it was suggested that the skin functions as a primary immunologic organ. The term skin immune system (SIS) has also been proposed to describe the components of the skin, excluding the regional lymph nodes, that constitute SALT. In the following sections, the specifics of the SIS are briefly reviewed, so that the reader is familiar with the gains in knowledge and future avenues for work that will lead to further discoveries.

Keratinocytes

Keratinocytes do much more than produce keratin, surface lipids, and intercellular substances (see Chapter 1). They are intimately associated with Langerhans cells and play a major role in the SIS. Keratinocytes produce a wide variety of cytokines that have important roles in mediating cutaneous immune responses, inflammation, wound healing, and the growth and development of certain neoplasms. Keratinocytes also produce eicosanoids, prostaglandin (PG) E2, and neuropeptides such as propiomelanocortin and α MSH. Though some of these are proinflammatory, some such as PGE2 and the neuropeptides also have anti-inflammatory effects. Keratinocytes, especially when perturbed by exposure to IFN-γ, express MHC II antigens. This expression is required for cells to be APCs for T-lymphocyte responses. Keratinocytes are capable of phagocytosis. Keratinocytes may also be stimulated to produce the leukocyte adhesion molecule, ICAM-1. They are the primary epidermal source for cytokines. Probably the most immunologically important is interleukin-1 (IL-1). Keratinocytes store IL-1, which is readily released following damage to the cells. In fact, release of IL-1 from keratinocytes is essentially a primary event in skin disease. Other cytokines derived from keratinocytes include IL-3, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-16 and IL-18, TNF-α, and a variety of growth factors and granulocyte-monocyte-macrophage stimulating and activating factors. Depending on what cytokines are produced, keratinocytes may affect the type of immune response. Keratinocytes produce both IL-12 and IL-10, which may skew which type of T-lymphocytes are activated or downregulate inflammation, depending on what stage T-lymphocytes are exposed to them. Keratinocytes may also play a role in tissue repair by production of multiple growth factors. Therefore, it becomes apparent that keratinocytes are important in stimulating and controlling inflammation and repair of tissue.

Langerhans Cells

Langerhans cells are interdigitating dendritic cells, which appear as suprabasilar clear cells on skin sections stained routinely with hematoxylin and eosin (H&E) (see Chapter 1). They are members of a family of highly specialized APCs termed dendritic cells. They are localized at the interface between organism and environment and are important sentinels of the immune system. Langerhans cells are the major APCs of the epidermis. They are bone marrow-derived monocyte/macrophage-type cells. The Langerhans cell is characteristically identified in the epidermis by the electron-microscopic presence of Birbeck granules (see Chapter 1). Cutaneous dendritic APCs include both epidermal Langerhans cells and dermal dendritic cells, both of which express abundant CD1 molecules. A unique feature of CD1 antigen presentation is the ability to present nonpeptide antigens to T-lymphocytes. The epidermal Langerhans cells do not express CD90 (Thy 1), while the dermal dendritic cells do. Langerhans cells express MHC class II antigens and receptors for C3b, Fc-IgG, and Fc-IgE. The main function of Langerhans cells is antigen-specific T-lymphocyte activation. Antigenic peptides derived from endogenous protein synthesis (e.g., viral antigens, transplantation antigens, tumor-associated antigens) are generally presented in the context of MHC class I molecules, which are expressed on the surface of essentially all nucleated cells, and recognized by CD8+ antigen-specific cytotoxic T-lymphocytes. Exogenous antigens (not synthesized within APCs [e.g., extracellular bacteria, bacterial toxins, dermatophytes, vaccines, pollens, dust mites]) are presented via CD4+ helper T-lymphocytes that recognize antigenic peptides bound to MHC class II antigen selectively expressed by professional APCs (e.g., macrophages, dendritic cells, B-lymphocytes). Langerhans cells bind epidermal antigens and then present the antigens along with costimulatory molecules, the so-called “second signal” to the lymphoid tissues (regional lymph node), where helper T-lymphocytes in particular are activated. MHC II molecules are produced in the endoplasmic reticulum where they then migrate to the Golgi and endo-lysosomal compartments. The processed protein results in antigen peptides that are incorporated into the MHC II molecules at various points in this migration from endoplasmic reticulum to the cell surface. At the surface, the antigenic peptide-MHC II complex is presented to the T-cell receptor (TCR) on the surface of the T-lymphocyte, resulting in antigen-specific T-lymphocyte activation. Effective T-lymphocyte activation requires costimulators and cytokines that promote clonal expansion of the antigen-specific T-lymphocyte. The costimulator or second signal is often supplied by the expression of the B7 family of cell-surface molecules. These molecules may be expressed following Langerhans cells exposure to lipopolysaccharide, TNF-α, IL-1, and other signals. Langerhans cells produce cytokines such as IL-1 and some lipid mediators that direct the T-lymphocyte response.

Langerhans cells express high levels of E-cadherin (dermal dendritic cells do not) that is important in selective adhesion to keratinocytes. Epidermal Langerhans cells are, thus, highly specialized cells expressing molecules that allow them to home to skin and localize in the epidermis. Tissue injury, microbial infection, and other perturbations of epidermal homeostasis provide a “danger” signal leading to local production of proinflammatory cytokines that, in turn, induce mobilization and migration of Langerhans cells to lymphoid tissue.

Lymphocytes

Lymphocytes are all derived from a common stem cell in the bone marrow and may be divided into three main types: B (bursa- or bone marrow-derived) cells, T (thymus-dependent) cells, and natural killer (NK) cells.

B-lymphocytes are characterized by possessing unique surface Igs, Fc receptors, CD21, CD79, and C3b receptors. B-lymphocytes mature into plasma cells following recognition of its specific antigen and activation, which produce the Igs IgG, IgM, IgA, and IgE, and they are responsible for antibody immunity. The growth and development of B-lymphocytes occurs in two phases. The first is antigen-independent and yields B-lymphocytes that express IgM and IgD. These initial antibodies constitute the majority of the primary antibody response to antigens, but are low affinity. The second phase or memory response is a response to a specific antigen that, with T-lymphocytes, induces differentiation into IgA-, IgG-, and IgE-secreting or memory B-lymphocytes. The second phase requires T-lymphocyte activation and is partly controlled by cytokines released by T-lymphocytes, with IL-1, IL-2, IL-4, IL-5, and IL-10 all being shown to play roles in growth or differentiation. IL-4 and IL-13 are particularly important for B-lymphocytes to switch into IgE-producing plasma cells, and this effect is enhanced by IL-5, IL-6, and TNF-α. In humans, B-lymphocytes are rarely found in normal skin and, even in dermatologic disease, are much less common than T-lymphocytes. Humoral immunity is described as providing primary defense against invading bacteria and neutralization activity against circulating viruses.

NK cells are large granular lymphocytes that do not express antigen-specific receptors, but do have receptors that recognize the self MHC I molecule. When they encounter nucleated cells with self MHC I molecules, these receptors inhibit killing (killer inhibitory receptors or KIR). Many viral or tumor cells fail to express self MHC I and will be killed. NK cells also have receptors for Fc receptor and may mediate antibody-dependent cytotoxicity.

T-lymphocytes are formed in the thymus, express CD3 when mature, and are divided into two major types: helper and suppressor/cytotoxic T-lymphocytes. The two major types are differentiated by their activity and their TCR, which is the part of the T-lymphocyte that recognizes an antigen. Classically, T-lymphocytes are considered responsible for cell-mediated immunity, activation of memory B-lymphocytes, and stimulation of NK cells. T-lymphocytes play a central role in directing and modifying the immune response. Functions of T-lymphocytes include: (1) helping B-lymphocytes make antibody and directing what type of antibody is made (helper T-lymphocytes), (2) suppressing B-lymphocyte antibody production (suppressor T-lymphocytes), (3) directly damaging “target” cells (cytotoxic T-lymphocytes), (4) mediating delayed hypersensitivity reactions, (5) suppressing delayed hypersensitivity reactions mediated by other T-lymphocytes, (6) regulating macrophage function, (7) modulating the inflammatory response with chemokines and cytokines, (8) inducing graft rejection, and (9) producing graft-versus-host reactions. T-lymphocyte cytokines may amplify or dampen phagocytic activity, collagen production, vascular permeability, and coagulation phenomena. T-lymphocytes can kill microorganisms and other cells, or they can recruit effector cells to perform this function. T-lymphocyte function is known to be suppressed by numerous infections, cancers, and drugs.

As T-lymphocytes mature in the thymus, they develop surface receptor molecules. The receptor molecules expressed on T-lymphocytes are critical in determining the future function of the cell. CD3 represents the TCR complex and marks all mature T-lymphocytes. MHC II-expressing T-helper (Th) cells express CD4, and MHC I-restricted suppressor/cytotoxic T-cells express CD8. Subpopulations of helper and suppressor/cytotoxic T-lymphocytes exist. These subpopulations have different cytokine production profiles. The most studied are the Th1 and Th2 subpopulations of CD4+ T-lymphocytes. Th2 cells produce IL-4, IL-5, and IL-10, classically favoring allergic reactions. Th1 cells produce IL-2, IFN-γ, and TNF-α, classically endowing cell-mediated immunity.

Tissue Macrophages

The end-stage of the mononuclear phagocyte system (MPS) is the tissue macrophage. These cells are bone marrow-derived and pass through the blood circulation as monocytes that, in general, are CD14+. These cells have a wide variety of activities and morphologic appearances, especially in inflammation. Tissue macrophages, epithelioid cells, and multinucleated histiocytic giant cells are all MPS cells found in a variety of inflammatory diseases. They serve the critical function of the afferent arm of the immune system in processing and presenting antigens, especially for T-lymphocyte activation, yet are also important in the efferent arm of the immune response. The MPS also plays major roles in wound healing, granulopoiesis, erythropoiesis, and antimicrobial defense (especially against intracellular pathogens). Monocytes and macrophages may secrete numerous enzymes, cytokines, inflammatory mediators, histamine-releasing factors, and inhibitors when stimulated. Some mediators upgrade inflammation, whereas others inhibit inflammatory activity to prevent too much tissue destruction from inflammation.

Mast Cells

Mast cells are derived from hematopoietic stem cells in the bone marrow and migrate as immature unrecognizable cells in the blood and then localize in connective or mucosal tissues (see Chapter 1). Once present in tissue, they proliferate and differentiate into mature recognizable mast cells. The regulation of mast cell proliferation and differentiation is the subject of much research. Cytokines from fibroblasts, stem cell growth factor, T-lymphocytes, and IL-3 are particularly important.

Mast cells combine characteristics of both innate and acquired immune responses: they can (1) bind certain bacteria and phagocytose/kill them, (2) elaborate and secrete several biologically active products, and (3) serve as an APC and promote clonal expansion of CD4+ helper T-lymphocytes. The importance of mast cells in immediate hypersensitivity diseases is well-documented. However, their role in other skin diseases, such as contact dermatitis and bullous pemphigoid, and in the process of fibrosis has only recently been recognized. Mast cells have diverse effects and interactions with other cells and structures of the skin (Fig. 8-1). Mast cells serve as repositories for or synthesizers of numerous inflammatory mediator substances. The mediators present vary by species studied and according to the type of the mast cells. Some mediators are universally present, such as histamine, leukotrienes, eosinophil chemotactic factor of anaphylaxis (ECF-A), and proteolytic enzymes. There are two main categories of mediators. Preformed mediators are produced and stored in mast cell granules, which are modified lysosomes that develop from the Golgi apparatus (Table 8-5). Mast cells also produce mediators that are newly synthesized at the time of activation and degranulation (Table 8-6). Mast cells have the potential to synthesize many cytokines (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, granulocyte-macrophage colony-stimulating factor, TNF-α, IFN-γ) and many chemokines (e.g., macrophage inflammatory protein, MIP1α). Mast cells, therefore, may play many roles in the mediation of immune and inflammatory responses. Classically they are known for the recruitment of eosinophils and neutrophils, Igs, and complement from the circulation and the regulation of the immunologic response (see Tables 8-5 and 8-6). In addition, mast cells can: (1) produce IL-12 to drive Th1 responses; (2) produce IL-4, which is essential for the conversion of Th0 to Th2 cells; (3) produce IL-5 and IL-10 to drive Th2 responses; and (4) activate B-lymphocytes without surface contact.

Figure 8-1 Schematic representation of the documented interactions of human mast cells with other cells and structures in the skin.

(From Goldstein Sm, Wintroub BV: The cellular and molecular biology of the human mast cell. In Fitzpatrick TB et al, editors: Dermatology in general medicine V, New York, 1993, McGraw-Hill, p 365.)

TABLE 8-5 Preformed Mediators in Mast Cells

TABLE 8-6 Pharmacologic Activities of Newly Generated Mast Cell Mediators

Mast cell degranulation may be initiated by a variety of substances, including allergens cross-linking two surface IgE (or possibly IgG [T]) molecules, complement components C3a and C5a, eosinophil major basic protein, some hormones (estrogen, gastrin, somatostatin), substance P, and a group of cytokines referred to as histamine-releasing factors. Other exogenous compounds known to cause mast cell degranulation include anti-IgE, compound 48/80, opiates, concanavalin A, and calcium ionophores. The different types of mast cells may be affected differently, depending on the compound that causes degranulation.

Interestingly, histamine has two types of effects on hypersensitivity reactions, proinflammatory and anti-inflammatory. The proinflammatory effects of histamine are mediated through histamine 1 (H₁) receptors and resultant decreases in intracellular cyclic adenosine monophosphate (cAMP). Also, H₁ receptors mediate pruritus, and the trauma associated with pruritus may lead to further tissue and keratinocyte damage. The anti-inflammatory effects of histamine (inhibition of the release of inflammatory mediator substances from mast cells, neutrophils, lymphocytes, and monocyte-macrophages) are mediated through H₂ receptors and resultant increases in cAMP.

Endothelial Cells

The vascular endothelium is a very active cell type that is important in inflammation, immune responses, and tissue repair (see Chapter 1). In response to various cytokines, endothelial cells express adhesion molecules (integrins, selectins, and Ig supergene family [ICAMs]) on their surfaces. The selectins (E-selectin and P-selectin) are expressed on endothelial cells following certain inflammatory stimuli and act to slow down and cause rolling of leukocytes along the vascular endothelium. The leukocyte activation results in integrin expression and binding to Ig supergene family molecules such as ICAM-1 and VCAM-1 on endothelial cells, resulting in adhesion. Transendothelial migration occurs following adhesion. Then, in response to chemokines, the migrating lymphocytes, monocytes, and granulocytes will move toward the site of inflammation. Without this ability to home, the circulating effector cells could not respond to an immunologic or inflammatory event. In addition, activated endothelial cells can synthesize and secrete numerous substances such as cytokines (including IL-1, IL-6, and IL-8), fibronectin, collagen IV, proteoglycans, blood clotting factors, growth factors, and granulocyte-macrophage colony-stimulating factor. Defects in endothelial cell adhesion molecule expression may result in disorders that mimic immunodeficiencies owing to defective migration of lymphocytes, monocytes, or granulocytes.

Granulocytes

Neutrophils have, as their major roles, the function of phagocytosis and subsequent destruction and elimination of phagocytized material. In a sense, they are the scavengers of immunologically identified debris. They are considered most important in containing infection. Owing, however, to their numerous chemoattractants (Table 8-7) and intracellular products (Table 8-8) that may be released at sites of inflammation, neutrophils are omnipresent participants in most immune and virtually all inflammatory reactions.

TABLE 8-7 Chemoattractants for Neutrophils

TABLE 8-8 Neutrophil Products

Eosinophils, effector cells in hypersensitivity reactions, also participate in the downgrading of inflammation and defense of the host against extracellular parasites. They are also phagocytic (immune complexes, mast cell granules, aggregated Igs, and certain bacteria and fungi). Eosinophils have a tremendous ability to communicate with surrounding cells by the expression of surface receptors and cytokine secretion. Over 60 receptors for a variety of adhesion molecules, Ig Fc receptors, cytokines and lipid mediators have been found on the eosinophil membrane (Table 8-9). Eosinophil chemotaxis has been the subject of much research. Many molecules are considered good candidates for influencing eosinophil chemotaxis in vivo. These include platelet-activating factor (PAF), leukotrienes (LTB4, LTD4), dihydroxyeicosatetraenoic acid, and the C-C subfamily of chemokines. The C-C chemokines considered most potent and selective for eosinophil chemotaxis are eotaxin, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4.

TABLE 8-9 Secretory Products of Eosinophils

Major basic protein (also found in basophils), eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase are potent toxic mediators and all have been shown to be effective at killing a variety of parasites. Eosinophil proteases may contribute to host tissue damage and wound healing as the collagenase degrades type I and III collagen and gelatinase degrades type XVII collagen. Additionally, eosinophil degranulation results in the production of membrane-derived mediators such as LTs and PAF.

Basophils, effector cells in some hypersensitivity reactions, may also play a role in downgrading delayed-type hypersensitivity reactions. Basophils are somewhat similar to mast cells, in that they have high-affinity receptors for IgE and contain high levels of histamine, but they also are the only leukocytes to share features once thought specific for eosinophils. Basophils contain major basic protein and the Charcot-Leyden crystals (lysophospholipase). Basophils express over 30 surface receptors and have preformed granule-stored mediators and newly synthesized mediators following degranulation or activation. Basophils are important in host defense, which has been most conclusively shown in the rejection of ticks. In skin diseases, basophils are particularly important in cutaneous basophil hypersensitivity (a T-lymphocyte-controlled reaction important in host responses to various ectoparasites) and in late-phase, immediate hypersensitivity reactions, an important mechanism in the pathogenesis of allergic skin diseases.

Humoral Components

The humoral components as described in the SIS include Igs, complement components, fibrinolysins, cytokines, eicosanoids, neuropeptides, and antimicrobial peptides. The changes observed in inflammation are mediated, however, by numerous substances derived from the plasma, from cells of the damaged tissue, and from infiltrating monocytes, macrophages, lymphocytes, and granulocytes. The interactions among cells, neurons, expression of cell receptors, cytokines, and other soluble mediators determine the inflammatory response. Some mediators augment inflammation, and others suppress it. Some mediators antagonize or destroy other mediators, and others amplify or generate other mediators. All mediators and cells normally act together in a harmonious fashion to maintain homeostasis and to protect the host against infectious agents and other noxious substances. Many mediators may be preformed and stored with the effector cells; other mediators are produced only in response to damage or appropriate receptor activation.

Complement is a group of plasma and cell membrane proteins that induce and influence immunologic and inflammatory events. The critical step in the generation of biologic activities from the complement proteins is the cleavage of C3. There are two pathways for the cleavage of C3. The classic pathway requires the presence of Ig and immune complexes. The alternative (properdin) pathway does not require Ig and may be directly activated by bacteria, viruses, and some abnormal cells. There is also a pathway to amplify C3 cleavage and an effector sequence. The final effect of this sequence is the production of a membrane attack complex, which causes cell lysis. As the effector sequence progresses, a variety of complement components that have other effects are formed. These other components play a role in neutralization of viruses, solubilization of immune complexes, and interaction with receptors on other cells. Many inflammatory cells have receptors for degradation products of C3 and C5. These activated receptors are important in phagocytosis, immune regulation, and mast cell and basophil degranulation.

Lipid mediators (PAF and eicosanoids) are newly synthesized unstored molecules derived from cell membranes. Cell membranes contain phospholipids, one of which is phosphatidylcholine, the parent molecule of PAF. Arachidonic acid is stored in cells as an ether in phospholipids and is the dominant fatty acid attached to the glycerol portion of PAF. Though arachidonic acid is also present in other phospholipids, this is a major source in inflammatory cells. Following specific receptor stimulation and after cellular injury, phospholipases cause the degradation of phospholipids. Phospholipase A₂ enzymes break down phosphatidylcholine into a molecule of free arachidonic acid and one of PAF. Phospholipase C activity will also result in free arachidonic acid, but it is the predominance of phospholipase A that contributes the most to the generation of inflammatory mediators. Glucocorticoids inhibit the action of phospholipases at pharmacologically achieved levels. This is thought to be a major anti-inflammatory mechanism of glucocorticoid therapy.

PAF is not an eicosanoid, but a phospholipid that also acts as an inflammatory mediator. It is produced from a variety of cells, although neutrophils and eosinophils produce the largest amounts. PAF primes cells to have augmented responses to other stimuli and has its greatest effects on eosinophils and monocytes. It is an extremely potent eosinophil chemoattractant and stimulates their degranulation and release of LTs. A chemoattractant for mononuclear cells, PAF stimulates their release of IL-1, IL-4, and TNF-α.

The metabolites of the oxidation of arachidonic acid are termed eicosanoids and are potent biologic mediators of a variety of physiologic or pathologic responses. Though arachidonic acid and the oxidative enzymes to degrade it are found in all cells, only mast cells, leukocytes, endothelial cells, epithelial cells, and platelets have enough to play a major role in allergic diseases. Free arachidonic acid is oxidized by one of two enzyme classes: cyclooxygenase (COX) and lipoxygenase (LOX).

The eicosanoids include two main types of molecules, the prostanoids and LTs. Prostanoids (prostaglandins [PGs] and thromboxanes) are derived by the metabolism of arachidonic acid by COX. Two forms of COX are known: COX-1 in the endoplasmic reticulum, and COX-2 in the nuclear envelope, and they serve different functions and are preferentially important in different tissues. Aspirin and the nonsteroidal anti-inflammatory drugs primarily function by blocking COX. COX metabolism results in PGH₂ formation, which is subsequently metabolized by specific terminal enzymes that result in the formation of PGE₂, PGF₂, PGD₂, PGI₂, or thromboxane A₂. Different cell types have variations in which enzyme system is present and, therefore, which metabolites are produced.

Leukotrienes (LTA, LTB, LTC, LTD and LTE) and their precursors—hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs)—are derived by the metabolism of arachidonic acid by the three enzymes of lipoxygenation, 5-, 12-, and 15-lipoxygenase. Different tissues express variable levels of the cytosolic LOXs. The 5-lipoxygenase pathway predominates in neutrophils, monocytes, macrophages, and mast cells, whereas the 15-lipoxygenase pathway predominates in eosinophils and in endothelial and epithelial cells. The 12-lipoxygenase pathway predominates in platelets. The 5-lipoxygenase pathway results in the production of LTA₄ that, depending on the enzymes present in the cells, is converted to LTB₄, or LTC₄, and LTD₄ or LTE₄ are produced from LTC₄. Typically, eicosanoids have autocrine and paracrine functions that are important locally for host defense, and then they are inactivated or degraded. Abnormalities in production or control mechanisms may occur, however, leading to local or systemic tissue damage and disease. The actions of eicosanoids are quite diverse and variable according to the species, tissue, cellular source, the presence of stereospecific receptors, and the generation of secondary mediators. The effects of arachidonic acid formation and some of the activities that eicosanoids may have in skin disease are summarized in Table 8-10.

TABLE 8-10 Effects of Eicosanoids in Skin Disease

Treatment Plans

Most horses with chronic allergies, particularly those with atopic dermatitis, require a combination of therapeutic agents for optimal control of symptoms.* When possible, the optimum treatment of all allergies is avoidance of the offending allergen(s). If this is not possible, most treatments are directed at blocking the effects of the allergic reaction. Prevention may also occur with allergen-specific immunotherapy (ASIT) (hyposensitization, desensitization) (see discussion on Atopic Dermatitis) and, possibly, with some immunomodulatory drugs. When allergen avoidance and/or ASIT are ineffective, partially effective, or when clients decline these approaches, other medical options will be required. Often the optimum control will require different therapeutic protocols over the life of the animal. Therefore, a treatment plan will be required that takes the different problems, goals of the clients, and types of therapeutic agents into consideration. In general, these are aimed at treating the by-products of the allergic reaction that has already occurred. The majority of therapeutic protocols are used to help alleviate pruritus, the major symptom of most allergic diseases, to treat specific problems or secondary infections, to avoid or decrease exposure to offending allergens, and to decrease inflammation. These therapeutic protocols may be specific for a problem or etiology or nonspecific. The most commonly recommended therapeutic protocols are listed in Table 8-11. These treatment options vary in their ease of administration, risks, efficacy, expense, and monitoring required. In many cases, combinations of treatments may be used, and an overall plan to control those aspects of the problem considered most bothersome by the client is the most effective way to manage these cases long term.

TABLE 8-11 Therapeutic Regimens

Avoidance

Avoidance requires the identification of allergens contributing to the allergy. The offending allergens may be determined by historoclinical findings, provocation testing, and “allergy testing.” Avoidance frequently is effective for allergies such as insect-bite hypersensitivity and food allergy, but may be ineffective or impractical in atopic dermatitis. However, even if the offending allergens cannot be completely avoided, it is helpful to eliminate exposure as much as possible. Some methods of avoidance for atopic dermatitis will be covered in that section.

Topical Therapy

Topical therapy is often incorporated into treatment plans and is discussed in more detail in Chapter 3.⁷ The major disadvantages are the time and effort needed for administration. Expense may also be an important factor. Total body bathing and/or rinses are required for regional or generalized pruritus and often reduce pruritus by rehydrating the stratum corneum and by removing surface debris, microbial by-products, and allergens that may contribute to the pruritic load. Hydrocortisone (1%) shampoo is not significantly absorbed and may help treat allergic reactions. Treatment with ointments, creams, lotions, and sprays may be possible for localized areas. Topical glucocorticoids are most effective, but generally limited to localized allergic reactions. Cold water, hypoallergenic and moisturizing shampoos and creme rinses, colloidal oatmeal, and shampoos and creme rinses containing pramoxine (local anesthetic) can reduce pruritus for up to 72 h (see Chapter 3). Topical therapy unfortunately is often overlooked and not presented as an option to clients. Appropriately administered topical therapy can reduce the need for systemic treatment in many patients.

Systemic Therapy

Several systemic agents can be used in the medical management of allergic horses. The clinician needs to be familiar with which of these agents must be withdrawn prior to shows and competitions. Medicines capable of improving racing performance of horses are classified by the Association of Racing Commissioners International (ARCI) based on their performance-enhancing potential (www.arci.com). Antihistamines are in Classes 3 and 4, and glucocorticoids are in Class 3.

Fatty Acids

Fatty acids and their role in normal skin and coat are discussed in Chapter 3. As these agents are relatively benign, diets or supplements containing appropriate amounts and ratios could be used in most allergic horses.

The proposed mechanism of action of omega-3 and omega-6 fatty acids, besides the inhibition of arachidonic acid metabolism, relates to metabolic by-products of fatty acid metabolism. Supplements used for pruritus usually contain one or both of γ-linolenic acid (GLA) and eicosapentaenoic acid (EPA). GLA is found in relatively high concentrations in evening primrose, borage, and black currant oils. It is elongated to dihomo(D)GLA, which directly competes with arachidonic acid as a substrate for COX and 15-lipoxygenase. The result of DGLA metabolism is the formation of PGE1 and 15-hydroxy-8,11,13-eicosapentaenoic acid, both of which are believed to have anti-inflammatory effects.

EPA, which is usually supplied by using cold water marine fish oils, also competes as a substrate for COX and 5- and 15-lipoxygenase. The metabolism of EPA by the LOX enzymes results in the formation of LTB5 and 15-hydroxyeicosapentaenoic acid. These two products are believed to inhibit LTB4, which is a potent proinflammatory mediator. Fig. 8-2 demonstrates the interactions of GLA, EPA, and arachidonic acid. Flaxseed contains α-linolenic acid, which is metabolized to EPA.

Figure 8-2 I, N-6 fatty acid metabolism with production of anti-inflammatory eicosanoids. II, Arachidonic acid cascade with production of proinflammatory eicosanoids. III, N-3 fatty acid metabolism with production of anti-inflammatory eicosanoids. 13-Hode, 13- hydroxyoctadecadienoic acid; PG, prostaglandin; E, elongase; Δ-6-D, Δ-6-desaturase; LA, linoleic acid; GLA, γ-linolenic acid; EPO, evening primrose oil; DGLA dihomo-γ-linolenic acid; AA, arachidonic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; DES, desaturase; PLA2, phospholipase A2; CO, cyclooxygenase; O, lipoxygenase; HETE, hydroxyeicostetrasenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; HEPE, hydroxyeicosapentaenoic acid; 15-HetrE, 15-hydroxy-8,11,13-eicosatriaenoic acid; LT, leukotriene; ⋆, arachidonic acid-derived eicosanoids identified in inflammatory skin disease; ?, inhibitory or anti-inflammatory eicosanoid (number of slash lines indicates degree of inhibition.

(From White P: Essential fatty acids: use in management of canine atopy, Comp Cont Educ 15:451, 1993.)

Little information has been published on the usefulness of these fatty acids in horses with inflammatory dermatoses. The oral administration of linseed or flaxseed oil (50-58% α-linolenic, 13-18% linoleic) inhibited inflammation, the production of eicosanoids, and the production of TNF in experimental studies in horses.^7,11,12 Although linseed oil and flaxseed oil are both produced from flaxseed, the extraction processes are different. Linseed oil is extracted under conditions of high temperatures and petroleum extraction. Flaxseed oil is cold pressed with no solvent used. Linseed oil may cause depression, anorexia, and mild colic in horses, whereas flaxseed oil does not.⁷ In two double-blinded, placebo-controlled clinical studies, no significant reduction in pruritus occurred in allergic horses treated orally (PO) with linseed oil or a commercial product (evening primrose oil and cold water marine fish oil).⁷ These studies are difficult to interpret because: (1) it was not clear how many horses had insect-bite hypersensitivity, atopic dermatitis, or both; and (2) the horses’ base diets were not analyzed. In addition, as horses may have low levels of Δ-6-desaturase and Δ-5-desaturase activity,¹² supplementation with specific omega-3 (e.g., EPA) and omega-6 (e.g., GLA) fatty acids may be more effective. A milled flaxseed supplement reduced reactivity to intradermal injections of Culicoides antigen in horses with insect-bite hypersensitivity.¹⁹

Many clinicians feel that omega-6/omega-3 fatty acid supplementation is beneficial in some allergic horses, especially those with atopic dermatitis.* Although fish oils are occasionally unpalatable to some horses, a commercial fatty acid supplement (DVM Derm Caps 100s), given PO at 1 capsule/50-100 kg every 12 h appears to be well-tolerated and effective in some horses. Platinum Performance Equine Wellness and Performance Formula (omega-3 fatty acids, tract minerals, vitamins, antioxidants) and Platinum Vet Skin and Allergy Formula (algal omega-3 fatty acids, quercetin, purified calf thymus) are popular commercial products, but no scientific studies substantiate their benefits in equine skin disease. These fatty acid supplements may also have synergistic effects when administered in conjunction with glucocorticoids and/or antihistamines.^7,23,46 Fatty acid supplements should be given for 3-12 weeks before a judgment is made as to their benefits.

Nonsteroidal Anti-inflammatory Agents

Nonsteroidal anti-inflammatory agents are classically used to decrease pain and inflammation, but have shown little benefit for pruritus or atopic disease. A number of nonsteroidal drugs have been used to treat equine pruritus. Little or no work has been done to document the effect of these, including phenylbutazone, diethylcarbamazine, flunixin, ketoprofen, orgotein (metalloprotein, nonsteroidal anti-inflammatory), phenothiazine tranquilizers, barbiturates, and levamisole.⁷ Experimental studies have indicated that 5-lipoxygenase inhibitors, a leukotriene synthesis inhibitor, and PAF receptor antagonists inhibit various aspects of the equine inflammatory response,⁷ but therapeutic trials with such agents have not been reported. Montelukast (a leukotriene receptor antagonist) administered PO at 0.11 mg/kg/day was of no benefit in horses with chronic obstructive pulmonary disease (COPD).¹³

Pentoxifylline has a variety of immunomodulatory effects, and the benefits may result from different mechanisms, depending on the disease being treated (see Chapter 9).^14,15,21 Pentoxifylline may be useful—especially for reducing required glucocorticoid doses and frequencies—in some allergic horses.^7,23

Methylsulfonylmethane (MSM EQ, Vetri-Science) is an organosulfur compound and a metabolite of dimethyl sulfoxide. It is recommended by the manufacturer to “support proper joint function and connective tissue health” in horses. Anecdotal reports indicate that MSM can be used in combination with other antipruritic agents in allergic horses.^6,23 The powder is sprinkled on the food at 10-12 gm/500 kg every 12 h, and then every 24 h. The product is well-tolerated, but its benefits in equine dermatology are unproven.

Antihistamines

Histamine is a potent chemical mediator that has variable actions, depending on what receptors and tissues are stimulated. The effects of histamine can be blocked in three ways: by physiologic antagonists, such as epinephrine; by agents that reduce histamine formation or release from mast cells and basophils; and by histamine receptor antagonism. Antihistamines work by the latter two mechanisms. In equine dermatology, the primary indication for antihistamine therapy is the treatment of inflammation and pruritus mediated by stimulation of H₁ receptors, usually associated with allergic reactions.

First-generation (classic or traditional) antihistamines are H₁ blockers. Because antihistamines are metabolized by the liver, they should be used with caution in patients with hepatic disease. In addition, their anticholinergic properties contraindicate their use in patients with glaucoma, gastrointestinal atony, and urinary retentive states. Some antihistamines are teratogenic in various laboratory animals. No information on teratogenicity is available for horses; however, this issue should be considered before treating pregnant mares. Finally, the efficacy of antihistamines is notoriously unpredictable and individualized in a given patient. Part of this variation may be dose-related, as antiallergic effects are concentration dependent, and some dose ranges will be antiallergic while in others may enhance mediator release. Thus, the clinician may try several antihistamines and doses before finding the one that is beneficial for a given patient.

H₁ receptors are primarily responsible for pruritus, increased vascular permeability, release of inflammatory mediators, and recruitment of inflammatory cells. In addition to their histamine-blocking action, some of these antihistamines have sedative, antinausea, anticholinergic, antiserotoninergic, and local anesthetic effects. They will block mediator release if present prior to allergen challenge and if at the appropriate concentration. Most of the second-generation, or nonsedating, antihistamines block mediator release. Some, such as cetirizine, also block the allergen-induced late-phase cutaneous reaction, decrease the influx of eosinophils, and downregulate TNF-α-induced hyperactivation of nuclear factor kappa beta (NF-κβ).^17,18 Second-generation antihistamines (less likely to cross blood-brain barrier) typically have less side effects than first-generation antihistamines (cross blood-brain barrier).

It is important to remember that antihistamines function best as preventive antipruritic agents. They will not rapidly reduce severe ongoing pruritus and inflammation. Thus, a short course of glucocorticoids may have to be given to control the pruritus. Then the ability of an antihistamine to prevent recurrence of the pruritus or inflammation can be assessed.

There is little published information on the use of antihistamines in horses. Most clinicians consider hydroxyzine (1-2 mg/kg every 8-12 h PO) to be the antihistamine of choice in the horse (Table 8-12).* It is not known what percentage of horses respond to any given antihistamine. However, as the effects of different antihistamines vary from one horse to another, it is important to try several different ones before concluding that the pruritus or inflammation is not responsive to these agents. Other antihistamines suggested for use in allergic horses include chlorpheniramine (0.25-0.5 mg/kg every 12 h PO) and diphenhydramine (1-2 mg/kg every 8-12 h PO). Tripelenamine and pyrilamine are not usually effective.^7,10,23 Clemastine and fexofenadine have very poor oral bioavailability in horses and are unlikely to be clinically useful.^7,16,22 Cetirizine (0.2-0.4 mg/kg every 12 h PO) has good bioavailability in horses and could be clinically useful,^17,18 but no clinical trials have been reported. Antihistamines should be given for at least 2 weeks before a judgment is made as to their usefulness. Antihistamines may act synergistically with glucocorticoids and/or fatty acids in pruritic horses. Antihistamine side effects are rare in the horse and include sedation, lethargy, and behavioral changes.

TABLE 8-12 Antihistamines and Antidespressants Used in Allergic Horses*

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