Update on cytokines

Continuing Medical Education This continuing medical education self-assessment program is sponsored by The American Academy of Allergy Asthma and Immunology,

Update on cytokines Larry Borish, MD, and Lanny J. Rosenwasser, MD Denver, Colo.

Cytokines are secreted proteins that regulate and determine the nature of immune responses. 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 responses. The particular cytokines that are produced in response to an immune insult will determine whether an immune response develops and whether that response will be humoral, cell-mediated, or allergic. Confounding any discussion of the cytokine network is a tremendous redundancy both in variety of cell sources and range of biologic activities for each eytokine. This redundancy extends to cytokine receptors, with many receptor families having homologous peptide sequences or shared components. The ability of a cytokine to induce synthesis of many other cytokines--and frequently even its own--further confuses any attempt to specify biologic roles for a given cytokine. It is impossible to accurately group eytokines according to unique tissue sources or biologic activities. For the purpose of this review, however, cytokines will be grouped according to those that are predominantly mononuclear phagocyte-derived (monokines) or T lymphocyte-derived (lymphokines) and then according to those that predominantly mediate humoral, cell-mediated, or allergic immunity or, alternatively, are immunosuppressive.

From National Jewish Center for Immunology and Respiratory Medicine, University of Colorado Health Sciences Center, Denver. Reprint requests: Larry Borish, MD, National Jewish Center for Immunology and Respiratory Medicine, University of Colorado Health Sciences Center, 1400 Jackson St., Denver, CO 80206. J ALLERGYCLINIMMUNOL1996;97:719-34. Copyright 9 1996 by Mosby-Year Book, Inc. 0091-6749/96 $5.00 + 0 1/2/70628

Abbreviations used GM-CSF: Granulocyte-macrophage colonystimulating factor ICAM: Intercellular adhesion molecule IFN: Interferon LAK: Lymphokine-activated killer Monocyte chemotactic peptide MCP: Major histocompatibility complex MHC: MIP: Macrophage inflammatory protein NK: Natural killer TGF: Transforming growth factor TNF: Tumor necrosis factor

MONONUCLEAR PHAGOCYTE-DERIVED CYTOKINES Cytokines that are primarily derived from mononuclear phagocytes are particularly effective in promoting the cellular infiltration and damage to resident tissue, which are characteristic of inflammation. The processing of antigens as they are taken up by mononuclear phagocytes, metabolized, bound to major histocompatibility complex (MHC) class II molecules, and presented to T H lymphocytes provides one pathway for cytokine (monokine) production. Alternatively, monocytes may be directly triggered to produce cytokines in an antigen-independent fashion, such as through the interaction of endotoxin with CD14.1 The cytokines predominantly produced by monocytes include tumor necrosis factor (TNF), IL-1, IL-6, IL-8, and other members of the small cytokine (chemokine) family, IL-12 and IL-15. Tumor necrosis factor TNF represents two homologous 17 kd proteins, which are primarily derived from mononuclear phagocytes (TNF-o0 and lymphocytes (TNF-[3). 2' 3 In addition to mononuclear phagocytes, TNF-~ may be produced by neutrophils, activated lymphocytes, natural killer (NK) cells, endothelial cells, 719

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and smooth muscle cells. The most potent inducer of TNF by monocytes is lipopolysaccharide. Numerous cytokines including IL-1, IL-3, granulocyte-macrophage colony-stimulating factor (GMCSF), and interferon (IFN)-~/ may also induce TNF transcription. TNF-oL is processed as a membrane-bound protein from which the soluble active factor is derived through cleavage of the extracellular domain. 4 TNF-[~ is synthesized and processed as a typical secretory protein? The active form of both cytokines is a noncovalently linked trimer. TNF-oL and TNF-[3 have approximately 30% amino acid residue homology but bind to the same two distinct cell surface receptors with similar affinities and produce similar, although not identical, effects. 6 Virtually all cells studied demonstrate the presence of one or both of these receptors. As their name implies, TNFs were originally discovered as secreted peptides that induce antitumor immunity. 2-4 TNFs are directly cytotoxic toward cancerous cells, stimulate antitumor immune responses by immune cells, and when administered in vivo, induce hemorrhagic necrosis of tumors. A major proinflammatory activity of TNFs is their ability to interact with endothelial cells to induce intercellular adhesion molecule (ICAM)-I, permitting the egress of granulocytes into inflammatory loci. 7 Additional activities of TNF include stimulating bone resorption, cartilage degradation, and B-cell and monocyte activation. TNF augments the capacity of monocytes to produce inflammatory mediators, including IL-6 and IL-8. TNF is a potent activator of neutrophils, mediating adherence, chemotaxis, degranulation, and the respiratory burst, s Like the IFNs, TNF has antiviral activity and stimulates MHC class I and II expression. Enthusiasm for the potential therapeutic value of TNF has been tempered by its severe side effects. TNF is responsible for the severe wasting that occurs in chronic infections and cancer, with cachexia occurring as the result of inhibition by TNF of the enzyme lipoprotein lipase. 2 Furthermore, TNF induces vascular leakage, has negative inotropic effects, and is thought to be the endogenous mediator of toxic shock and sepsis. 9 Interleukin-1 The IL-1 family represents three peptides (IL1~, IL-113, and the IL-1 receptor antagonist [IL-lra or IL-I~]). 1~ 11 Despite the minimal sequence similarity ( - 2 5 % amino acid homology), IL-la and IL-113 have similar biologic activities, and all three proteins interact with similar affinities to the two IL-1 receptors. 12Type I receptors have been found

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on T cells, fibroblasts, endothelial cells, hepatocytes, and numerous other cells. Type I receptors have an extensive cytoplasmic domain, and after ligand binding, transduce the biologic effects attributed to IL-1.13 These are in contrast to type II receptors, which have been identified on 13 cells, neutrophils, and bone marrow cells and which have a minimal intracellular domain. The "capture" and sequestration of IL-1 by these inactive type II receptors may subserve an antiinflammatory function, and hence these receptors are sometimes referred to as "decoy" receptors. Type II receptors are believed to function as the precursor for the soluble IL-l-binding factors, which when shed, antagonize IL-1 activity) 3,14 The capacity of ILlra to bind to the type I (proinflammatory) IL-1 receptor without transducing biologic activities is the basis for its capacity to function as an antiinflammatory cytokine antagonist. 15-t7 IL-1 is primarily produced by cells of the mononuclear phagocytic lineage but is also produced by endothelial cells, keratinocytes, synovial cells, astrocytes, osteoblasts, neutrophils, neuroglial cells, and numerous other cells. IL-I production may be stimulated by a variety of agents including endotoxins, other cytokines, microorganisms, and other antigens. Both IL-I~ and IL-113 are synthesized without a hydrophobic leader sequence as a less active 31 kd precursor) 8 The mechanism for IL-1 secretion is unknown, but it is dependent on its cleavage by a specific converting enzyme into the active 17 kd form 19 and may use polypeptide transporters with unique signal recognition particles. 2~ One of the most important biologic activities of IL-1 is its function as a lymphocyte activating factor. T Hcell activation requires the interaction of antigen-MHC complex with the T-cell receptor. However, an additional, noncognate signal is also required for optimal T-cell activation and proliferation, and this is generally provided by IL-1. IL-1 enhances the production of T lymphocyte-derived cytokines, such as IL-2 and IL-2 receptors. In the absence of IL-1, either no immune response or a state of tolerance develops. In addition to these effects on T lymphocytes, IL-1 can augment B-cell proliferation and increase immunoglobulin synthesis. IL-] synergizes with various colony-stimulating factors to stimulate early bone marrow hematopoietic progenitor cell proliferation. IL-1 administration produces a bone marrow-derived neutrophilia. The production of IL-1 during the immune response produces a spectrum of changes associ-

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ated with illness. IL-1 interacting with the central nervous system is responsible for producing fever, lethargy, slow wave sleep, anorexia, and the release of corticotropin-releasing factor. An IL-l-hepatocyte interaction inhibits production of"housekeeping" proteins (e.g., albumin) and stimulates in turn the synthesis of acute-phase response peptides (e.g., amyloid peptide, C-reactive peptide, complement). To provide the amino acids for this new protein synthesis, IL-1 stimulates the catabolism of muscle. IL-1 activity in the joints stimulates synovial cell proliferation, cartilage and bone resorption, and collagen deposition. These effects of IL-1 on muscles and joints contribute to the myalgias and arthralgias associated with illness. IL-1 stimulates endothelial cell adherence of leukocytes through the upregulation of ICAM. 7 IL-1 also acts on blood vessels to induce vasodilation, contributing to the hypotension of septic shock. Much of the proinflammatory activity of IL-1 relates to its ability to induce arachidonate metabolism with many eicosanoids, such as prostaglandin E 2 and leukotriene B4, which function as second messengers. IL-1 induces synthesis of additional cytokines including TNF, IL-6, GM-CSF, and--as a positive feedback mechanism--additional IL-1. Finally, like TNF, IL-1 is directly cytotoxic to cancerous and virus-infected cells. TNF and IL-1 share numerous biologic activities, the major distinction being that TNF has no direct effect on lymphocyte proliferation. (A comparative listing of the biologic activities of mononuclear cell-derived cytokines is displayed in Table I.) The IL-1 receptor antagonist (IL-lra or IL-1-/) is secreted naturally in inflammatory processes. Its production is upregulated by many cytokines including IL-4, IL-13, transforming growth factor (TGF)-[3, and IL-1 itself. Production of IL-lra is believed to modulate the potentially deleterious effects of IL-1 in the natural course of inflammation. IL-ra has been used experimentally as an antiinflammatory agent. In rabbits IL-lra prevents death caused by lipopolysaccharide-mediated septic shock 21 and modulates immune complex-induced colitis. 22

Interleukin-6 Mononuclear phagocytic cells are probably the most important source of IL-6; however, IL-6 is also produced by T and B lymphocytes, fibroblasts, keratinocytes, hepatocytes, neuroglial cells, and bone marrow stromal cells.23,24 Its synthesis is induced by IL-1, IL-2, TNF, and IFNs; and it is inhibited by IL-4 and IL-13. An important biologic

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activity of IL-6 is its ability to stimulate the final stages of B-lymphocyte maturation. Under the influence of IL-6, B lymphocytes differentiate into mature plasma cells and are stimulated to secrete immunoglobulins. IL-6 mediates T-cell activation, growth, and differentiation, including the differentiation of cytotoxic T cells. In addition to costimulating lymphocyte proliferation, IL-6 shares several activities with IL-1, including the induction of pyrexia and the acute-phase response (Table I). IL-6 is considered the most important inducer of hepatocyte synthesis of acute-phase proteins such as C-reactive protein, fibrinogen, haptoglobin, and amyioid protein. IL-6 may have antiviral activity and a capacity, shared with IFNs, to induce class I MHC expression. Like TNF and IL-1, IL-6 may display antitumor effects. Finally, IL-6 is a neutrophil activator and synergizes with other cytokines to support bone marrow stem cell maturation.

Interleukin-8 and the small cytokine family (chemokines) The chemokines are a family of structurally and functionally related proteins that include at least 14 distinct members? 5-27 In addition to mononuclear phagocytic cells, chemokines are secreted by T lymphocytes, NK cells, neutrophils, keratinocytes, hepatocytes, fibroblasts, endothelium, and epithelial cells. The peptides are small (8 to 10 kd) secreted proinflammatory cytokines that exhibit between 20% and 50% homology in their amino acid sequences. The chemokines are characterized by the presence of four conserved cysteine residues and are subdivided into two families on the basis of positioning of these cysteines (Table II). The C-X-C subfamily is located on chromosome 4q, and its members are characterized by the separation of the first two cysteines by a variable amino acid. The C-C subfamily has the first two cysteine residues adjacent to each other, and its genes are located on chromosome 17q. These two families may also be distinguished by their primary target cells: the C-X-C subfamily primarily targets neutrophils, and the C-C family targets monocytes and T cells. Recently, a new family of chemokines, which lacks the first and third C, has been identified and is referred to as the "C" subfamily. The most extensively studied member of the chemokine superfamily is IL-8Y, 26,28 IL-8 is primarily derived from mononuclear phagocytes and endothelial cells but also from T cells, eosinophils, neutrophils, fibroblasts, keratinocytes, hepatocytes, and chondrocytes. IL-8 synthesis may be induced by LPS, IL-1, TNF, and viruses. IL-8 on a

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TABLE I. Mononuclear phagocytic cell-derived cytokines IL-1

IL-6

TNF

IL-8

T-cell effects

T-cell activation; increased IL-2 and IL-2r expression

T-cell activation; inT-cell activation; increased IL-2 and creased IL-2 and IL-2 IL-2r expression; cyto- receptor expression; toxic T-cell activation cytotoxicT-cell activa-

T-cell chemotaxis

B-cell effects Neutrophil effects

Proliferation and increased antibody production LTB4 synthesis

NK cells

Activation

Differentiation into plasma cells and antibody secretion Degranulation; respiratory burst Activation

Proliferation and increased antibody production Chemoattraction and activation Activation

Tumoricida] Induction of acutephase responses Cachexia Adhesion molecule expression

Yes Yes

No Yes

Yes Yes

No No

No ICAM, VCAM- 1, E-selectin

No No

No CD18/CD1 la+b

Mononuclear phagocytic cells

Increased cytokine production

Differentiation

Yes ICAM, V C A M - 1 , CD18/CDllb+c, P/Eselectin Chemoattraction; increased cytokine production

Hematopoiesis

Stimulation of progenitor cells

Megakaryocyte maturation and hematopoietic cell proliferation

CNS effects

Fever induction; induction of slow wave sleep; corticotropin release

tion

L T B 4,

Chemoattraction

Chemoattraction

Inhibition Fever induction; induction of slow wave sleep

LeukotrieneB4; CNS, central nervous system.

molar basis is one of the most potent chemoattractants for neutrophils. It also stimulates polymorphonuclear neutrophil degranulation, the respiratory burst, and adherence to endothelial cells through CDllb/CD18. During the inflammatory response, IL-8 appears relatively late in comparison with other chemoattractants. For example, leukotriene B 4 appears within minutes of cellular activation, and its concentration peaks at 3 hours. As leukotriene B 4 concentrations decline, the newly synthesized IL-8 begins to be secreted, and secretion persists for at least 24 hours. Other members of the chemokine family--including platelet factor-4, macrophage inflammatory protein (MIP) ltx, and neutrophil-activating protein2--share to a Iesser extent a capacity to activate polymorphonuclear neutrophils. Other members of the chemokine C-C family, including RANTES, MIP, and monocyte chemotactic peptide (MCP)-I

and MCP-3 have unique contributions toward allergic inflammation and will be discussed later. Interleukin-12

IL-12 is a mononuclear phagocytic cell-derived peptide originally described as NK stimulatory factor.29, 3o It is a heterodimer composed of two unrelated subunits. The larger subunit (p40) is homologous to the soluble receptor for IL-6, whereas the smaller subunit (p35) is homologous to both IL-6 and GM-CSF. IL-12 is derived from monocytes and macrophages but also from B cells and connective tissue type mast cells. IL-12 activates and induces proliferation of NK cells, stimulates their cytotoxicity, causes secretion of IFN-~, and TNF-a, and increases expression of CD56, CD2, and C D l l a . Like IL-2, tL-12 stimulates the differentiation of NK cells into lymphokine-activated killer (LAK) cells. Other activities attributed

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IL-IO

IL-12

Inhibition of cytokine production; tolerance induction; cytotoxic T-cell function

Increased TNF-c~, IFN--/synthesis; activation of helper and cytotoxic cells; T m differentiation Inhibition of IL-4-mediated IgE secretion

Proliferation and increased antibody production Inhibition of cytokine production No No

Proliferation, activation, and differentiation into LAK cells No No

No

No

No

No

Inhibition of cytokine production; MHC II expression; antigen processing Increased mast cells

No

Stimulation of progenitor cells

to IL-12 include proliferation of CD4 and CD8 lymphocytes and stimulation of hematopoiesis. 31 Its counterregulatory role in allergic inflammation is discussed later. Interleukin-15

IL-15 is a 15 kd peptide that has activity similar to that of IL-2 and interacts with a heterotrimeric receptor, two components of which are also used by IL-2 (the IL-2R13 and ~ chains). 3z IL-15 competes for binding sites with IL-2, and anti-IL-2 receptor antibodies block binding of IL-15. Mononuclear phagocytic cells, epithelium, fibroblasts, muscle, and placenta are the best sources of IL15.33 In one study activated T lymphocytes, the most important source for IL-2, were not found to express detectable IL-15. 33 Like IL-2, as discussed below, IL-15 is a T-cell growth factor, is chemotactic for T lymphocytes, differentiates NK cells into LAK cells, and stimulates B-cell growth and dif-

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ferentiation. The significance of IL-15 is that this peptide represents an additional mechanism by which mononuclear phagocytic cells regulate T-cell and NK cell proliferation and function. After induction of T-cell activation and proliferation, depending on the repertoire of cytokines produced, either cell-mediated, humoral, or allergic immune responses will occur. CELL-MEDIATED I M M U N I T Y Interleukin-2

Stimulation of T cells by antigen in the additional presence of IL-1 induces the simultaneous secretion of IL-2 and the expression of high-affinity IL-2 receptors. 34, 35 Subsequently, the binding of secreted IL-2 to these IL-2r-positive T cells induces clonal T-cell proliferation. The requirement for both IL-2 production and IL-2r expression for T cells to proliferate assures that only T cells specific for the antigen inciting the immune response will become activated. IL-2 receptors consist of three chains? 6 IL-2m (or Tac antigen and CD25) is a 55 kd transmembrane glycoprotein with only 13 of 351 amino acids located within the cytoplasm. 37 IL-2r13 is a 75 kd glycoprotein with a large intracytoplasmic component and therefore is critical for signal transduction. 3s More recently, a third chain, IL-2r-/, has been identified and shown to be essential for high-affinity binding, ligand internalization, and--when approximated to the 13 chain--signaling. 39,4~ IL-4, IL-7, IL-9, IL-13, and IL-15 all make use of the ~/chain as part of their receptor complexes. All three chains are in contact with the IL-2 ligand in the high-affinity form of the receptor. Alone, IL-2m has low affinity for IL-2 and is unable to transduce a signal. The ~13 chains in combination produce a receptor with intermediate affinity but are still unable to activate the cell. Absence of -/chains is responsible for one form of X-linked severe combined immunodeficiency,41 reflecting its importance in signaling by at least six cytokines. IL-2 receptors are also found on NK cells through which IL-2 induces a more potent cytotoxic response, differentiating the NK cells into LAK cells. 42,43 IL-2 also functions as a B-cell, 44 cytotoxic T-cell, and macrophage activator 45 and may have neurotransmitter functions. Interferon-~

The most important cytokine responsible for macrophage activation, and therefore for cellmediated immunity, is IFN-'y.46 It is primarily produced by T . lymphocytes but may also be

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TABLE II. Small cytokine family Cytokine

C-X-C subfamily IL-8 (NAP-l) GRO-~x, -[3, -~/ NAP-2 Platelet factor 4 Platelet basic protein (CTAP-III, [3-thromboglobulin) ~IP-10 GCP-2 ENA-78 C-C subfamily MIP-lc~ MIP-I[3 MCP-1 MCP-2 MCP-3 RANTES 1-309

Chromosomal location

Primary target(s)

4q Neutrophils, T cells, eosinophils, endothelial cells, basophils Neutrophils, basophils Neutrophils Fibroblasts, neutrophils, endothelial cells, T cells, monocytes Fibroblasts, neutrophils, basophils Monocytes, T cells Neutrophils Neutrophils 17q Monocytes, neutrophils, T cells, basophils, eosinophils Monocytes, T cells Monocytes, basophils Monocytes Monocytes, basophils, eosinophils Monocytes, T cells, eosinophils, basophils Monocytes

NAP, Neutrophil-activating protein; GRO, growth-regulated protein; CTAP, connective tissue activating peptide; IP, inducible protein; GCP, granulocytechernotacticprotein; ENA, epithelial cell-derived neutrophil-activating protein.

derived from cytotoxic T cells, ~/~ cells, and NK cells. IFN-~ mediates increased class I and II MHC and IgG receptor expression. IFN-~ stimulates antigen presentation and cytokine production by monocytes and also stimulates monocyte effector functions including adherence, phagocytosis, secretion, the respiratory burst, nitric oxide production, and microbicidal and tumoricidal activity. The net result is the accumulation of macrophages at the site of cellular immune responses, and their activation into macrophages makes them capable of killing intracellular pathogens. In addition to its effects on mononuclear phagocytes, IFN-~ stimulates killing by NK cells and neutrophils. It stimulates adherence of granulocytes to endothelial cells through the induction of ICAM-1, an activity shared with IL-1 and TNF. 7 IFN-~/ stimulates antigen-specific B-cell proliferation and differentiation. Finally, as with other IFNs, IFN-~ inhibits viral replication. As discussed later, IFN-~ has been proposed as an inhibitor of allergic responses through its capacity to inhibit IL-4-mediated effects on B cells, thereby inhibiting IgE secretion and expression of low-affinity IgE receptors. An additional cytokine secreted by T. lymphocytes, which contributes to cell-mediated immunity, is TNF-[3, as previously discussed.

HUMORAL

IMMUNITY

At least two cytokines contribute to B-lymphocyte maturation in the bone marrow, the lymphoid stem cell growth factors IL-7 and IL-11. Once B cells egress from the bone marrow, isotype switching, the activation of mature B cells into immunoglobulin-secreting B cells, and their final differentiation into plasma cells are processes that are under T-cell control. 47 Switching mechanisms involve at least two factors: T cell-B cell cognate interactions and secretion of interleukin molecules. These interleukins make accessible the 5' switch regions of the heavy chain DNA sequence so that the ~/, e, or a genes can be transcribed. Contact signals provided by the T cells are triggered through CD40 on the surface of the B cells by the CD40 ligand on T cells. Cytokines that trigger the isotype switch include IL-4 and IL-13, which induce switching to the IgE isotype, and TGF-[3, which triggers the IgA isotype switch. IL-10 contributes to generation of IgG isotypes. Other cytokines that influence B-cell maturation but are not involved in isotype switching are IFN-~/, IL-1, IL-2, IL-5, and the recently described B-cell growth factor IL-14. IL-6, IL-12, and IL-15 (as discussed previously) are also involved in humoral immunity.

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ALLERGIC IMMUNITY A third possible outcome of T-cell activation is the development of allergic immunity. Several pathophysiologic features that are specifically associated with the asthmatic state are regulated by cytokines. These include the regulation of IgE, eosinophilia, and mast cell proliferation.

REGULATION OF IgE The inappropriate production of IgE in response to allergen defines atopy. Recent investigations have demonstrated that the regulation of IgE is primarily a function of the relative activities of IL-4, IL-13, and IFN-y.

Interleukin-4 IL-4 was originally identified as a B-cell growth factor, which drives the optimal stimulation of B cells by antigen. 48,49 In addition to T Hlymphocytes, IL-4 may be derived from cytotoxic T cells, mast cells, and basophils. In addition to its stimulatory activity, IL-4 stimulates MHC class II antigen, B7.1 (CD80), B7.2 (CD86), CD40, surface IgM, and low-affinity IgE receptor expression by B cells, resulting in enhanced antigen-presenting capacity of B cells, s~ However, IL-4 also induces the immunoglobulin isotype switch from IgM to IgE. 49,51-53 IgE production occurs after the IL-4-mediated initiation of transcription of e heavy chain transcripts. In the additional presence of signals triggered through CD40, genomic splicing occurs and mature e heavy chain transcripts are synthesized. The additional presence of B-cell-activating cytokines, including IL-2, IL-5, and IL-6, synergizes with IL-4 to increase the secretion of IgE. 54 That IL-4 is absolutely essential for IgE production has been demonstrated by the ability of anti-IL-4 antibodies to abrogate the capacity of Nippostrongylus infection to induce IgE in mice. 55 Similarly, mice that have been genetically engineered to lack the IL-4 gene (IL-4 "knockouts") are unable to synthesize IgE. 56 In contrast to these experiments, mice that have been genetically engineered to secrete excessive IL-4 gene demonstrate elevated serum IgE levels.57 In addition, they have a conjunctivitis, which on pathologic examination, has the appearance of an allergic reaction with increased eosinophilia and infiltration with mast cells. Evidence supporting a role for IL-4 regulation of IgE synthesis in human beings is derived from several observations. IL-4 induces the synthesis of IgE by B lymphocytes, and the capacity of T-cell clones to support IgE production is directly proportional to their IL-4 production. 53

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In addition to these effects on B cells, IL-4 is a growth factor for T cells, the function in allergic inflammation of which is discussed later. It enhances the production of cytotoxic T cells and activates NK and LAK cells. It has some macrophage-activating activities, enhancing expression of MHC class I and II molecules, leukocyte function associated antigen-i, and low-affinity IgE receptors9 In contrast to these proinflammatory effects on monocytes, IL-4 inhibits antibody-dependent cellular cytotoxicity, inhibits expression of Fc~/ receptors, and downregulates production of nitric oxide, IL-1, IL-6, and TNF-c~ while stimulating production of IL-lra. 55 Another potentially important activity of IL-4 in allergic inflammation is its ability to induce expression of vascular cell adhesion molecule-1 on endothelial cells. This will produce the enhanced adhesiveness of endothelium for T cells, eosinophils, basophils, and monocytes--but not neutrophils--which is characteristic of allergic reactions. 59

Interleukin-13 IL-13 has approximately 30% homology to IL-4 and shares much of IL-4's biologic activities on mononuclear phagocytic cells and B cells. Thus IL-13 inhibits monocyte-mediated antibody-dependent cell-mediated cytotoxicity, proinflammatory cytokine production, and nitric oxide secretion and induces the IgE isotype switch. 6~ 61 The functional IL-13 receptor is believed to represent a heterotrimer containing the IL-4r, the IL-2r~ chain, and a unique IL-13-binding component. IL-13 is distinguished from IL-4 by different mechanisms of transcriptional regulation. IL-4 tends to be an earlier and more transient signal in comparison with IL-13. IL-13 is also distinguished from IL-4 by an absence of effects on T lymphocytes. As discussed below, IL-4 may be particularly important in allergic diseases through its ability to induce the differentiation of IL-4-producing T , lymphocytes, an activity not shared by IL-13. The third cytokine that is critically important in the regulation of IgE synthesis is IFN-y. IFN-~ functions as an inhibitor of allergic responses through its capacity to inhibit IL-4-mediated expression of low-affinity IgE receptors and the isotype switch to IgE. The capacity of T-lymphocyte clones to support IgE production is inversely proportional to their IFN--/production, and clones producing both IL-4 and IFN-~ will support IgE production only in the additional presence of anti-IFN--y antibodies. 5a,52,54 Additional cytokines

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that have been reported to inhibit IL-4-induced IgE production are IL-12, TGF-13, and IL-8.62,63 In summary, IgE production represents a combination of excessive IL-4 and IL-I3 occurring in the relative absence of IFN-% It is therefore believed that the immune response to allergens in atopic subsets selectively activates T, lymphocytes to secrete IL-4 and IL-13 as opposed to IFN-% In support of this are experiments with a series of T-lymphocyte clones directed against the dust mite allergen.64 These clones were developed from atopic and nonatopic subjects, and the clones were then assayed for their capacity to produce IFN-% IL-4, and IL-2. Most of the clones produced IL-2. However, allergen-specific T-lymphocyte clones obtained from the atopic subjects produced IL-4, whereas similar clones generated to the same allergen, but obtained from nonatopic subjects, produced IFN-%

EOSINOPHILIA Another characteristic feature of allergic diseases is the presence of increased numbers of circulating eosinophils. These eosinophils can be shown to be in an activated, hypodense state. Eosinophilia is a Tlymphocyte-mediated response, as demonstrated by its absence in athymic or T-cell-depleted animals.

Interleukin-5 As with IL-4, IL-5 was originally identified as a B-cell-stimulating factor, which mediates proliferation and differentiation of B lymphocytes and induces secretion of IgM. 65"67 In addition to T. lymphocytes, potentially more important sources for IL-5 in allergic inflammation may be eosinophils and mast cells. IL-5 interacts with specific IL-5 receptors, which consist of a heterodimer made up of IL-5m and IL-5r13 chains.68 The 13 subunit is shared with GM-CSF and IL-3 receptors. After cloning and generating purified protein, human IL-5 was demonstrated to have minimal activities toward B cells. However, IL-5 has become very important to the understanding of allergic inflammation because it is the most important eosinophilopoietin. IL-5 induces maturation of a homogeneous population of eosinophils when exposed to bone marrow precursors, 69 and mice transgenic for constitutive IL-5 expression develop eosinophilia.7~ In addition to stimulating eosinophil production, IL-5 is chemotactic for eosinophils and activates mature eosinophils, inducing eosinophil secretion and enhanced antibody-dependent cytotoxicity. IL-5 is responsible for the

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generation of hypodense eosinophils, and it prolongs eosinophil survival while limiting apoptosis. 71 Mice injected with Nippostrongylus organisms and anti-IL-5 antibodies fail to develop eosinophilia,72 and anti-IL-5 therapy abrogates the development of eosinophilic bronchitis in animal models of asthma. 73 The importance of IL-5 in human allergic disorders is supported by the demonstration of increased serum IL-5 levels in the hypereosinopbilic syndrome,74 in eosinophilia-myalgia syndrome, 75 and after endobronchial allergen challenges.76 Additional activities of IL-5 include an interaction with T lymphocytes to mediate maturation of cytotoxic T lymphocytes and basophilic differentiation.77 In addition to IL-5, two colony-stimulating factors, IL-37a and GM-CSF, 79 contribute to the activity of eosinophils in allergic inflammation through their capacities to prolong eosinophil survival and to generate activated, hypodense eosinophils. Additional factors that are chemotactic and activating for eosinophils are members of the chemokine family including RANTES, MIP-I~, MCP-3, and eotaxin-A.

MAST CELL PROLIFERATION Increased numbers of mast cells characterize allergic diseases, and as with elevated IgE concentrations and eosinophilia, this is a T cell-dependent process. Mast cell proliferation may result from the activities of the cytokines IL-3, IL-9, IL-10, nerve growth factor, and stem cell factor. IL-3 supports the differentiation of megakaryocytes, monocytes, erythrocytes, and granulocytes,a~ However, a prolonged exposure of human bone marrow cells to IL-3 results in the predominance of mast cells and basophils. This IL-3-stimulated mast cell proliferation is inhibited by GM-CSF. sl IL-9 is a T. lymphocyte-derived T-cell growth factor, which in mice has been shown to function as a mast cell growth factor.82 IL-10 is discussed below, but in mice it is also a cofactor for the proliferation of mast cell lines, a3 Additional mast cell growth factors are nerve growth factora4 and hematopoietic stem cell factor, as Hematopoietic stem cell factor is a connective tissue-derived protein, which may be a particularly important mast cell growth factor. It is responsible for the differentiation of mast cells into the connective tissue phenotype. The importance of this factor in vivo is supported by clinical observations in which the administration of stem cell factor was associated with cutaneous mast cell proliferation and chronic urticaria, a6

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In addition to those factors that stimulate mast cell proliferation, several cytokines have been demonstrated to induce histamine release from basophils and possibly mast cells. These histaminereleasing factors include IL-3 and several members of the chemokine family including RANTES, MIPlc~, MCP-1, MCP-3, and connective tissue activating peptide-III. Of these, MCP-1 and RANTES are the most potent histamine-releasing factors, and RANTES is the most effective as a basophil chemoattractant.27, 87,88 IL-8 inhibits cytokine-mediated histamine release by basophils, s9

ANTIINFLAMMATORY CYTOKINES In addition to those cytokines that mediate cellular, humoral, and allergic inflammation, two cytokines have predominantly antiinftammatory effects, TGF-[3 and IL-10.

Transforming growth factor-13 TGF-[3 represents a family of at least five peptides that regulate cell growth, having both stimulatory and inhibitory effects on different cell types.90 It is produced by numerous cell types but primarily by mesenchymal cells, including platelets and osteocytes. It is synthesized as an inactive precursor that requires proteolytic cleavage to become active. Receptors for TGF-[3 are present on virtually all cells, and it mediates a wide range of biologic activities. In general, it is an important stimulant of fibrosis, inducing formation of the extracellular matrix. In immunity it is generally found to be inhibitory for B lymphocytes and T helper and cytotoxic lymphocytes. It inhibits immunoglobulin secretion by B lymphocytes and cytotoxicity of mononuclear phagocytes and NK or LAK cells. In contrast to these antiinflammatory effects, TGF-[~ is chemoattractive for macrophages and supports the ~ isotype switch to IgA by B cells. 91,92 TGF-[3 knockout mice do n o t survive because of diffuse inflammation of numerous organs by mononuclear cells. 93 In allergic inflammation the expression of TGF-[3 may be associated with the fibrosis observed in long-standing asthma and the subendocardial fibrosis associated with the hypereosinophilic syndrome. TGF-[3 may lessen allergic inflammation through a capacity to inhibit germline transcription and IgE synthesis in IL4-treated human B cells and through inhibition of mast cell proliferation.

Interleukin-lO IL-10, or cytokine synthesis inhibitory factor, was initially identified in mice, in which it is a T,z

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lymphocyte product (discussed below) that inhibits both T m proliferation and production of IFN-~/ and IL-2. 94 Subsequent studies have extended these activities to human beings and have shown that IL-10 is also a product of various other cells including TH1 lymphocytes,9s, 96 cytotoxic T cells, B lymphocytes, mast cells, and mononuclear phagocytic cells. In human beings monocytes may be the major source of IL-10. 97 In addition to its effects on TH1 cytokines, IL-10 acts to inhibit production of IL-I[3, IL-6, IL-8, IL-12, and TNF-~ by mononuclear phagocytes,98-w~ IL-4 and IL-5 by TH2 lymphocytes,95 and IFN-~ and TNF-~ by NK cells, lm In addition to the effects of IL-10 on monocytederived cytokines, IL-10 also inhibits monocyte MHC class II expression and accessory cell function. Expression of IL-10 by antigen-presenting cells may have a role in the induction and maintenance of allergen-specific tolerance in allergic disorders? ~ Support for a modulator role for IL-10 in human atopic disease is further derived from observations that IL-10 inhibits eosinophil survival 1~ and IL-4-induced IgE synthesis?~ These inhibitory effects of IL-10 are in contrast to its effect on B lymphocytes where it functions as an activating factor, stimulating cell proliferation and immunoglobulin secretion, m5 IL-10 contributes to isotype switching to IgG 1 and IgG3.1~ IL-10 also functions as a growth cofactor for immature T cells and is a differentiating factor for cytotoxic T cells. 1~ Thus IL-10 inhibits cytokines associated with cellular immunity and allergic inflammation while stimulating humoral and cytotoxic immune responses. The kinetics of IL-10 production differ from those of other cytokines.96 Although messenger RNA for IL-l[3, IL-2, IL-4, and IFN--/can be identified as rapidly as 15 minutes and peaks 2 to 4 hours after stimulation, IL-10 mRNA does not appear until 8 hours after stimulation and is maximal only at 12 to 24 hours. The appearance of IL-10 thus correlates with and may contribute to the downregulation of the proinflammatory cytokines. Secretion of these proinflammatory cytokines may be a signal for IL-10 synthesis.97 TNF-o~ is a potent stimulus for IL-10 secretion. These studies suggest a homeostatic mechanism whereby an inflammatory stimulus induces TNF-a secretion, which in turn stimulates IL-10 secretion, which feeds back to turn off TNF-~ synthesis. Support for an antiinflammatory mechanism for IL-10 is suggested by knockout mice, which develop an inflammatory bowel disease believed to be due to uncontrolled immune responses to enteric antigens? ~

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TABLE III. Cytokine profiles of human and murine tymphocytes T-helper subtypes

T.1

T.2

Human

IFN-y, IL-4, IL-5, TNF-[3 IL-9

Murine

IFN-% IL-4, IL-5, TNF-[3, IL-6,IL-9, IL-2 IL-10, IL-13

Both

GM-CSF, IL-2, IL-3, IL-10, IL-13 GM-CSF, IL-3

T. LYMPHOCYTE FAMILIES T lymphocytes do not necessarily produce all of their cytokines on activation. Subclasses of T helper lymphocytes can therefore be identified among antigen-specific T cells on the basis of their repertoire of cytokines (Table III). 6< 109 In human beings type 1 helper cells produce IFN-y and TNF-[3 but not IL-4 and IL-5. Type 2 helper cells produce IL-4, IL-5, and IL-9 but not IFN-~ or TNF-[3. Both classes produce GM-CSF, IL-2, IL-3, IL-10, and IL-13. These observations on human T-cell clones are in contrast to the mouse, insofar as production of IL-2, IL-10, and IL-13 is shared by both classes of T helper cells. Although T. lymphocyte subclasses may be less apparent in human cells than in murine cells, there is an inverse relationship between the tendency of a T-cell clone to produce IFN-y as opposed to IL-4 or IL-5. Undifferentiated 3".0 cells primarily produce IL-2 but may also synthesize cytokines characteristic of both T m and TH2 lymphocytes. There are, however, a number of caveats concerning this differentiation of type 1 versus type 2 T helper lymphocytes. First of all, no firm differentiation markers exist that distinguish a T m cell from a T,2 cell in either mice or human beings. Therefore functional characterization of enhanced cytokine production may not represent a firm state of differentiation but rather the functional consequences of the milieu in which the cell is being stimulated. The second caveat is that there are circumstances in which the analogy of cytokine profile between human and murine 3". type lymphocytes and T m type lymphocytes are different (Table III). For example, in human beings both Tm and YiJ2, as well as T~0 subsets produce IL-2, IL-10, and IL-13 in addition to GM-CSF and IL-3. Published information clearly demonstrates the enhanced and significant production of IL-2 in a patient with Sdzary syndrome and a predominant T.z-like pattern to the S6zary cell cytokine produc-

tion, n~ in T.2-1ike T-cell clones taken from the blood of atopic mite-sensitive individuals, 111 and finally, even in the bronchoalveolar lavage of patients with allergic asthma. 112 In addition, IL-13 in human beings is not solely a product of T.2 cells but can be made by T.1 and T.0 cells as well. In human beings IL-10 is predominantly a product of macrophages but can be made by both TH1- and T.z-like cells. 95-97 Hence significant limitations concerning the clear distinction between the cytokine profiles made by T. 1 and TH2 cells in human beings need to be kept in mind. Type 1 T. lymphocytes, through their production of IL-2, TNF-[3, and IFN-y, activate T cells and monocytes and therefore promote cell-mediated immune responses. T. lymphocytes, which produce IFN-y, IL-2, IL-6, IL-10, and IL-14, induce B-cell maturation and are important in antibody-dependent immunity. Type 2 helper lymphocytes, which produce IL-4, IL-5, and IL-13 and which function in the relative absence of IFN-y, induce allergic immune responses. Support for the concept that TH2-1ike cells are important in mediating allergic diseases is provided by the observations discussed previously that mite-specific T cell clones obtained from atopic subjects were TH2-1ike, whereas clones derived from a nonallergic subject were Tin-like. m M E C H A N I S M S OF T.1/T.2 DIFFERENTIATION

One of the most important issues in understanding the cause of allergic disorders is therefore to determine the basis for Tm/TH2 differentiation in response to allergen. A detailed discussion of this topic is beyond the scope of this review. Factors including genetic predisposition, specific physiochemical nature of the offending allergen, different antigen-processing and presenting pathways and antigen-presenting cell type, as well as usage of unique MHC restriction elements or T-cell receptor V region usage have all been proposed as having a role (Table IV). However, one of the most critical elements in determining 3". differentiation is the cytokine milieu in which the T lymphocyte is activated. The major determinant of T.2 differentiation is the cytokine IL-4. ~13,114 This has been best established in T-cell receptor transgenic mice in which the concomitant exposure of the animal to IL-4 and the relevant antigen induces, in a dosedependent fashion, increasing production of IL-4 in the responding T cells. Neutralizing antibodies to IL-4 preclude development of IL-4-producing T lymphocytes. More recently, a mechanism for T m differentiation has been shown to be IL-12.

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TABLE IV. Mechanisms of T.1/T.2 differentiation

Familial predisposition toward development of atopy Allergens vs antigens, allergenic epitopes, physiochemistry, dose, route of administration Antigen processing pathways, antigen processing cell, expression of adhesion, accessory, or homing molecules

Genetic component Allergen-specific factors Antigen processing and presentation MHC restriction elements/Tcell receptor V region usage Cytokine milieu

IL-4, IL-12

TABLE V. Cytokines and allergy Cytokine

IgE regulation

IL-4, IL-13 IL-4 IL-2, IL-5, IL-6 IFN--r TGF-t3 IL-12

IgA regulation Eosinophilia

Mast cell development and activation

T-cell growth and activation Inflammation

Antiinflammatory

TGF-13 IL-5, IL-3, GM-CSF RANTES, MIP-lc~, eotaxin, MCP-3 IL-1, TNF IL-3, IL-9, IL-10, nerve growth factor, hematopoietic stem cell factor GM-CSF MIP-la, MCP-1, MCP-3, RANTES IL-8 IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15 IFN-~/, GM-CSF, G-CSF, TNFs, IL-I, IL-4, IL-6, IL-8 GM-CSF, TNFs, IL-1, IL-3, IL-5, RANTES IFN-~, GM-CSF, M-CSF, TNFs, IL-1, IL-2, IL-3, IL-4 IL-10, TGF-[3

Thus IL-12 induces, in a dose-dependent fashion, increasing concentrations of IFN-~ and decreasing concentrations of IL-4 in the responding T-ceU receptor antigen-specific T lymphocytes.115 Similarly, concomitant infection of mice with Leishmania species and exposure to murine IL-12 leads to the development of IFN-~-producing T . cells and a decrease in IL-4 production within lymph node cellsJl6,117 IL-12-mediated IFN-~/production may be required for the full expression of the T.1

Activity

isotype switch Generation of IL-4 producing T lymphocytes Synergize with IL-4, IL-13 Inhibit IL-4, IL-13 Stimulates IFN-~ production by NK cells and T cells Inhibits differentiation of IL-4producing T lymphocytes isotype switch Eosinophilopoietins Eosinophil chemotaxis and activation Eosinophil activation Mast cell growth factors Inhibits mast cell proliferation Basophil chemotaxis and histamine release Inhibition of histamine release T-cell growth factors - direct or costimulation Neutrophil activating factors Eosinophil activating factors Macrophage activating factors Inhibit cytokine production, monocytes/T-celt functions

phenotype. 11s In one study IL-12 costimulated antigen-specific proliferation of established T.a clones but not T~o or T.~ clones, n9 Insofar as mononuclear phagocytes are the major source of IL-12, this suggests a mechanism whereby antigens more likely to be processed by macrophages, including bacterial antigens and intracellular pathogens, produce T.1 responses. The Tin-inducing effects of IL-12 can be overcome in the additional presence of IL-4; thus, the IL-4 effects on undif-

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ferentiated T.0 cells are dominant, lz~ 121 The original source of the IL-4 responsible for T,2 differentiation remains unclear. The net result, however, is that in a milieu in which allergic inflammation is present (e.g., nasal and bronchial lymphatics), more and more extensive allergenic responses against bystander antigens will be expected to develop. SUMMARY

Cytokines that are important in the pathophysiology of allergic disorders are summarized on Table V. The IgE isotype switch results from the activities of IL-4 and IL-13 and is inhibited by IFN-~ and TGF-[3. IL-2, IL-5, and IL-6 synergize with IL-4 and IL-13 to enhance IgE secretion. IL-4 is responsible for the differentiation of IL-4-producing lymphocytes, whereas IL-12 inhibits the differentiation of IL-4-producing T cells. IL-5 is the most important eosinophilopoietin, and together with GM-CSF and IL-3, prolongs the survival of and activates mature eosinophils. These three cytokines are responsible for the generation of the hypodense eosinophils, which characterize the asthmatic state. Eosinophilia may also result from selective recruitment by the eosinophil chemotactic factors RANTES, MIP-I~, and eotaxin. Mast cell proliferation results from the activities of IL-3, IL-9, IL-10, nerve growth factor, and stem cell factor. Finally, several cytokines contribute to the inflammatory state of allergic disorders. IL-1, TNF, and IFN--/increase expression of endothelial cell adhesion molecules and support the egress of mononuclear ceils, neutrophils, and eosinophils into the lungs. Induction of vascular cell adhesion molecule-1 by IL-4 may promote the selective recruitment of eosinophils, basophils, and lymphocytes. Many cytokines may then contribute to the activation of these leukocytes once they have reached the airways. REFERENCES

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