Cytokines.docx

Chapter 11 :: Cytokines :: Ifor R. Williams & Thomas S. Kupper THE CONCEPT OF CYTOKINES When cells and tissues in complex organisms need to communicate over distances greater than one cell diameter, soluble factors must be employed. A subset of these factors is most important when produced or released transiently under emergent conditions. When faced with an infection- or injury-related challenge, the host must orchestrate a complex and carefully choreographed series of steps. It must mobilize certain circulating white blood cells precisely to the relevant injured area (but not elsewhere) and guide other leukocytes involved in host defense, particularly T and B cells, to specialized lymphatic tissue remote from the infectious lesion but sufficiently close to contain antigens from the relevant pathogen. After a limited period of time in this setting (i.e., lymph node), antibodies produced by B cells and effectormemory T cells, can be released into the circulation and will localize at the site of infection. Soluble factors produced by resident tissue cells at the site of injury, by leukocytes and platelets that are recruited to the site of injury, and by memory T cells ultimately recruited to the area, all conspire to generate an evolving and effective response to a challenge to host defense. Most important, the level of this response must be appropriate to the challenge and the duration of the response must be transient; that is, long enough to decisively eliminate the pathogen, but short enough to minimize damage to healthy host tissues. Much of the cell-tocell communication involved in the coordination of this response is accomplished by cytokines. Cytokines (which include the large family of chemokines, discussed in Chapter 12) are soluble polypeptide mediators that play pivotal roles in communication between cells of the hematopoietic system and other cells in the body.1 Cytokines influence many aspects of leukocyte function including differentiation, growth, activation, and migration. While many cytokines are substantially upregulated in response to injury to allow a rapid and potent host response, cytokines also play important roles in the development of the immune system and in homeostatic control of the immune system under basal conditions. The growth and differentiation effects of cytokines are not limited to leukocytes, although we will not discuss soluble factors that principally mediate cell growth and differentiation of cells other than leukocytes in this chapter. The participation of cytokines in many parts of immune and inflammatory

responses has prompted the examination of a variety of cytokines or cytokine antagonists (primarily antibodies and fusion proteins) as agents for pharmacologic manipulation of immune-mediated diseases. Only a few classes of effective cytokine drugs have emerged from the lengthy pathway of clinical trials to achieve FDA approval and widespread therapeutic use, but some of these drugs are now valuable therapeutics in dermatology. This chapter discusses these approved drugs and other promising biological agents still in clinical trials. General features of cytokines are their pleiotropism and redundancy. Before the advent of a systematic nomenclature for cytokines, most newly identified cytokines were named according to the biologic assay that was being used to isolate and characterize the active molecule (e.g., T-cell growth factor for the molecule that was later renamed interleukin 2, or IL-2). Very often, independent groups studying quite disparate bioactivities isolated the same molecule that revealed the pleiotropic effects of these cytokines. For example, before being termed interleukin 1 (IL-1), this cytokine had been variously known as endogenous pyrogen, lymphocyte-activating factor, and leukocytic endogenous mediator. Many cytokines have a wide range of activities, causing multiple effects in responsive cells and a different set of effects in each type of cell capable of responding. The redundancy of cytokines typically means that in any single bioassay (such as induction of T-cell proliferation), multiple cytokines will display activity. In addition, the absence of a single cytokine (such as in mice with targeted mutations in cytokine genes) can often be largely or even completely compensated for by other cytokines with overlapping biologic effects.

CLASSIFICATIONS OF CYTOKINES The first cytokines described had distinct and easily recognizable biological activities, exemplified by IL-1, IL-2, and the interferons (IFNs). The term cytokine was first coined by Cohen in 1975, to describe several such activities released into the supernatant of an epithelial cell line.2 Prior to this, such activities had been thought to be the exclusive domain of lymphocytes (lymphokines) and monocytes (monokines) and were considered a function of the immune system. Keratinocyte cytokines were first discovered in 1981,3 and the list of cytokines produced by this epithelial cell rivals nearly any other cell type in the body.4,5 The number of molecules that can be legitimately termed cytokines continues to expand and has brought under the cytokine rubric molecules with a broad range of distinct biological activities. The progress in genomic approaches has led to identification of novel cytokine genes based on homologies to known cytokine genes. Making sense of this plethora of

mediators is more of a challenge than ever, and strategies to simplify the analysis of the cytokine universe are sorely needed.

PRIMARY AND SECONDARY CYTOKINES A simple concept that continues to be extremely useful for discussion of cytokine function is the concept of “primary” and “secondary” cytokines.6 Primary cytokines are those cytokines that can, by themselves, initiate all the events required to bring about leukocyte infiltration in tissues. IL-1 (both α and β forms) and tumor necrosis factor (TNF; includes both TNF-α and TNF-β) function as primary cytokines, as do certain other cytokines that signal through receptors that trigger the nuclear factor κB (NF-κB) pathway. IL-1 and TNF are able to induce cell adhesion molecule expression on endothelial cells [selectins as well as immunoglobulin superfamily members such as intercellular adhesion molecule 1 (ICAM-1) and vascular cellular adhesion molecule 1 (VCAM-1)], to stimulate a variety of cells to produce a host of additional cytokines, and to induce expression of chemokines that provide a chemotactic gradient allowing the directed migration of specific leukocyte subsets into a site of inflammation (see Chapter 12). Primary cytokines can be viewed as part of the innate immune system (see Chapter 10), and in fact share signaling pathways with the so-called Toll-like receptors (TLRs), a family of receptors that recognize molecular patterns characteristically associated with microbial products.7 Although other cytokines sometimes have potent inflammatory activity, they do not duplicate this full repertoire of activities. Many qualify as secondary cytokines whose production is induced after stimulation by IL-1 and/or TNF family molecules. The term secondary does not imply that they are less important or less active than primary cytokines; rather, it indicates that their spectrum of activity is more restricted.

T-CELL SUBSETS DISTINGUISHED BY PATTERN OF CYTOKINE PRODUCTION Another valuable concept that has withstood the test of time is the assignment of many Tcell-derived cytokines into groups based on the specific helper T-cell subsets that produce them (Fig. 11-1). The original two helper T-cell subsets were termed Th1 and Th2.8 Commitment to one of these two patterns of cytokine secretion also occurs with CD8 cytotoxic T cells and γ/δ T cells. Dominance of type 1 or type 2 cytokines in a T-cell immune response has profound consequences for the outcome of immune responses to certain pathogens and extrinsic proteins capable of serving as allergens. Over two decades after the original description of the Th1 and Th2 subsets, strong evidence has emerged that there are

other functionally significant patterns of cytokine secretion by T cells. Most prominent among these newer T-cell lineages are Th17 cells and regulatory T cells (or Treg cells for short). The Th17 subset is distinguished by production of a high level of IL-17, but many Th17 cells also secrete IL-21 and IL-22. Th17 cells promote inflammation, and there is consistent evidence from human autoimmune diseases and mouse models of these diseases that IL-17-producing cells are critical effectors in autoimmune disease.9 A subset of T cells known as Treg cells has emerged as a crucial subset involved in the maintenance of peripheral selftolerance. 10 Two of the most distinctive features of Treg cells are their expression of the FoxP3 transcription factor and production of transforming growth factor-β (TGF-β), a cytokine that appears to be required for Treg cells to limit the excess activity of the proinflammatory T-cell subsets.11 IL-10 is also a significant contributor to the suppressive activity of Treg cells, particularly at some mucosal interfaces.12 Additional proposed helper T-cell subsets are follicular helper T cells (Tfh) that specialize in providing B cell help in germinal centers, Th9 cells distinguished by high levels of IL-9 production that function in antiparasite immunity along with Th2 cells, and Th22 cells associated with skin inflammation that produce Th22, but not other Th17-associated cytokines. Not only does each of these T-cell subsets exhibit distinctive patterns of cytokine production, cytokines are key factors in influencing the differentiation of naive T cells into these subsets. IL-12 is the key Th1-promoting factor, IL-4 is required for Th2 differentiation, and IL-6, IL-23, and TGFβ are involved in promoting Th17 development.

STRUCTURAL CLASSIFICAT ION OF CYTOKINES Not all useful classifications of cytokines are based solely on analysis of cytokine function. Structural biologists, aided by improved methods of generating homogenous preparations of proteins and establishment of new analytical methods (e.g., solution magnetic resonance spectroscopy) that complement the classical X-ray crystallography technique, have determined the three-dimensional structure of many cytokines. These efforts have led to the identification of groups of cytokines that fold to generate similar three-dimensional structures and bind to groups of cytokine receptors that also share similar structural features. For example, most of the cytokine ligands that bind to receptors of the hematopoietin cytokine receptor family are members of the four-helix bundle group of proteins. Four-helix bundle proteins have a shared tertiary architecture consisting of four antiparallel α-helical stretches separated by short connecting loops. The normal existence of some cytokines as oligomers rather than monomers was discovered in part as the result of structural investigations. For

example, interferon-γ (IFN-γ) is a four-helix bundle cytokine that exists naturally as a noncovalent dimer. The bivalency of the dimer enables this ligand to bind and oligomerize two IFN-γ receptor complexes, thereby facilitating signal transduction. TNF-α and TNF-β are both trimers that are composed almost exclusively of β-sheets folded into a “jelly roll” structural motif. Ligand-induced trimerization of receptors in the TNF receptor family is involved in the initiation of signaling.

SIGNAL TRANSDUCTION PATHWAYS SHARED BY CYTOKINES To accomplish their effects, cytokines must first bind with specificity and high affinity to receptors on the cell surfaces of responding cells. Many aspects of the pleiotropism and redundancy manifested by cytokines can be understood through an appreciation of shared mechanisms of signal transduction mediated by cell surface receptors for cytokines. In the early years of the cytokine biology era, the emphasis of most investigative work was the purification and eventual cloning of new cytokines and a description of their functional capabilities, both in vitro and in vivo. Most of the cytokine receptors have now been cloned, and many of the signaling cascades initiated by cytokines have been described in great detail. The vast majority of cytokine receptors can be classified into a relatively small number of families and superfamilies (Table 11-1), the members of which function in an approximately similar fashion. Table 11-2 lists the cytokines of particular relevance for cutaneous biology, including the major sources, responsive cells, features of interest, and clinical relevance of each cytokine. Most cytokines send signals to cells through pathways that are very similar to those used by other cytokines binding to the same class of receptors. Individual cytokines often employ several downstream pathways of signal transduction, which accounts in part for the pleiotropic effects of these molecules. Nevertheless, we propose here that a few major signaling pathways account for most effects attributable to cytokines. Of particularly central importance are the NF-κB pathway and the Jak/STAT pathway, described in the following sections.

NUCLEAR FACTOR kB, INHIBITOR OF kB, AND PRIMARY CYTOKINES A major mechanism contributing to the extensive overlap between the biologic activities of the primary cytokines IL-1 and TNF is the shared use of the NF-κB signal transduction pathway. IL-1 and TNF use completely distinct cell surface receptor and proximal signaling pathways, but these pathways converge at the activation of the NF-κB transcription factor. NF-κB is of central importance in immune and inflammatory processes because a large

number of genes that elicit or propagate inflammation have NF-κB recognition sites in their promoters.13 NF-κB-regulated genes include cytokines, chemokines, adhesion molecules, nitric oxide synthase, cyclooxygenase, and phospholipase A2. In unstimulated cells, NF-κB heterodimers formed from p65 and p50 subunits are inactive because they are sequestered in the cytoplasm as a result of tight binding to inhibitor proteins in the IκB family (Fig. 11-2). Signal transduction pathways that activate the NF-κB system do so through the activation of an IκB kinase (IKK) complex consisting of two kinase subunits (IKKα and IKKβ) and a regulatory subunit (IKKγ). The IKK complex phosphorylates IκBα and IκBβ on specific serine residues, yielding a target for recognition by an E3 ubiquitin ligase complex. The resulting polyubiquitination marks this IκB for rapid degradation by the 26S proteasome complex in the cytoplasm. Once IκB has been degraded, the free NF-κB (which contains a nuclear localization signal) is able to pass into the nucleus and induce expression of NFκBsensitive genes. The presence of κB recognition sites in cytokine promoters is very common. Among the genes regulated by NF-κB are IL-1β and TNF-a. This endows IL-1b and TNF-a with the capacity to establish a positive regulatory loop that favors persistent inflammation. Cytokines besides IL-1 and TNF that activate the NF-κB pathway as part of their signal transduction mechanisms include IL-17 and IL-18. Proinflammatory cytokines are not the only stimuli that can activate the NF-κB pathway. Bacterial products (e.g., lipop olysaccharide, or LPS), oxidants, activators of protein kinase C (e.g., phorbol esters), viruses, and ultraviolet (UV) radiation are other stimuli that can stimulate NF-κB activity. TLR4 is a cell surface receptor for the complex of LPS, LPS-binding protein, and CD14. The cytoplasmic domain of TLR4 is similar to that of the IL-1 receptor type 1 (IL-1R1) and other IL-1R family members and is known as the TIR domain (for Toll/IL-1 receptor).14 When ligand is bound to a TIR domain-containing receptor, one or more adapter proteins that also contain TIR domains are recruited to the complex. MyD88 was the first of these adapters to be identified; the other known adapters are TIRAP (TIR domain-containing adapter protein), TRIF (TIR domain-containing adapter inducing IFN-β), and TRAM (TRIF-related adapter molecule). Engagement of the adapter, in turn, activates one or more of the IL-1Rassociated kinases (IRAK1 to IRAK4) that then signal through TRAF6, a member of the TRAF (TNF receptor-associated factor) family, and TAK1 (TGF- β-activated kinase) to activate the IKK complex.15

JAK/STAT PAT HWAY A major breakthrough in the analysis of cytokinemediated signal transduction was the identification of a common cell surface to nucleus pathway used by the majority of cytokines. This Jak/STAT pathway was first elucidated through careful analysis of signaling initiated by IFN receptors (Fig. 11-3), but was subsequently shown to play a role in signaling by all cytokines that bind to members of the hematopoietin receptor family.16 The Jak/STAT pathway operates through the sequential action of a family of four nonreceptor tyrosine kinases (the Jaks or Janus family kinases) and a series of latent cytosolic transcription factors known as STATs (signal transducers and activators of transcription). The cytoplasmic portions of many cytokine receptor chains are noncovalently associated with one of the four Jaks [Jak1, Jak2, Jak3, and tyrosine kinase 2 (Tyk2)]. The activity of the Jak kinases is upregulated after stimulation of the cytokine receptor. Ligand binding to the cytokine receptors leads to the association of two or more distinct cytokine receptor subunits andbrings the associated Jak kinases into close proximity with each other. This promotes crossphosphorylation or autophosphorylation reactions that in turn fully activate the kinases. Tyrosines in the cytoplasmic tail of the cytokine receptor as well as tyrosines on other associated and newly recruited proteins are also phosphorylated. A subset of the newly phosphorylated tyrosines can then serve as docking points for attachment of additional signaling proteins bearing Src homology 2 (SH2) domains. Cytoplasmic STATs possess SH2 domains and are recruited to the phosphorylated cytokine receptors via this interaction. Homodimeric or heterodimeric STAT proteins are phosphorylated by the Jak kinases and subsequently translocate to the nucleus. In the nucleus they bind recognition sequences in DNA and stimulate transcription of specific genes, often in cooperation with other transcription factors. The same STAT molecules can be involved in signaling by multiple different cytokines. The specificity of the response in these instances may depend on the formation of complexes involving STATs and other transcription factors that then selectively act on a specific set of genes.

INTERLEUKIN 1 FAMILY OF CYTOKINES (INTERLEUKINS 1a, 1b, 18, 33) IL-1 is the prototype of a cytokine that has been discovered many times in many different biologic assays. Distinct genes encode the α and β forms of human IL-1, with only 26% homology at the amino acid level. Both IL-1s are translated as 31-kDa molecules that lack a signal peptide, and both reside in the cytoplasm. This form of IL-1α is biologically active, but 31-kDa IL-1b must be cleaved by caspase 1 (initially termed interleukin-1b-converting

enzyme) in a multiprotein cytoplasmic complex called the inflammasome to generate an active molecule.17 In general, IL-1β appears to be the dominant form of IL-1 produced by monocytes, macrophages, Langerhans cells, and dendritic cells, whereas IL-1α predominates in epithelial cells, including keratinocytes. This is likely to relate to the fact that epithelial IL1α is stored in the cytoplasm of cells that comprise an interface with the external environment. Such cells, when injured, release biologically active 31-kDa IL-1α and, by doing so, can initiate inflammation.6 However, if uninjured, these cells will differentiate and ultimately release their IL-1 contents into the environment. Leukocytes, including dendritic and Langerhans cells, carry their cargo of IL-1 inside the body, where its unregulated release could cause significant tissue damage. Thus, biologically active IL-1β release from cells is controlled at several levels: IL-1β gene transcription, caspase 1 gene transcription, and availability of the adapter proteins that interact with caspase 1 in the inflammasome to allow the generation of mature IL-1β. IL-1β stimulates the egress of Langerhans cells from the epidermis during the initiation of contact hypersensitivity, a pivotal event that leads to accumulation of Langerhans cells in skindraining lymph nodes. Studies of mice deficient in IL-1α and IL-1β genes suggest that both molecules are important in contact hypersensitivity, but that IL-1α is more critical. Active forms of IL-1 bind to the IL-1R1 or type 1 IL-1 receptor.14 This is the sole signal-transducing receptor for IL-1, and its cytoplasmic domain has showing greatest homology with the Toll gene product identified in Drosophila. A second cell surface protein, the mIL-1R accessory protein, or IL-1RAcP, must associate with IL-1R1 for signaling to occur. When IL-1 engages the IL-1R1/IL-1RAcP complex, recruitment of the MyD88 adapter occurs, followed by interactions with one or more of the IRAKs. These kinases in turn associate with TRAF6. Stepwise activation and recruitment of additional signaling molecules culminate in the induction of IKK activity. The net result is the activation of a series of NF-κB-regulated genes. A molecule known as the IL-1 receptor antagonist, or IL1ra, can bind to IL-1R1 but does not induce signaling through the receptor. This IL-1ra exists in threemalternatively spliced forms, and an isoform produced in monocytes is the only ligand for the IL-1R1 that both contains a signal peptide and is secreted from cells. Two other isoforms of IL-1ra, both lacking signal peptides, are contained within epithelial cells. The function of IL-1ra seems to be as a pure antagonist of IL-1 ligand binding to IL-1R1, and binding of IL-1ra to IL-1R1 does not induce the mobilization of IL-1RAcP. Consequently, although both IL-1α/β and

IL1ra bind with equivalent affinities to IL-1R1, the association of IL-1R1 with IL-1RAcP increases the affinity forIL-1α/β manyfold while not affecting the affinity for IL-1ra. This is consistent with the observation that a vast molar excess of IL-1ra is required to fully antagonize the effects of IL-1. The biologic role of IL-1ra is likely to be in the quenching of IL-1-mediated inflammatory responses, and mice deficient in IL-1ra show exaggerated and persistent inflammatory responses. A second means of antagonizing IL-1 activity occurs via expression of a second receptor for IL-1, IL-1R2. This receptor has a short cytoplasmic domain and serves to bind IL-1α/β efficiently, but not IL-1ra. This 68-kDa receptor can be cleaved from the cell surface by an unknown protease and released as a stable, soluble 45kDa molecule that retains avid IL-1-binding function. By binding the functional ligands for IL-1R1, IL-1R2 serves to inhibit IL-1-mediated responses. It is likely that IL-1R2 also inhibits IL-1 activity by associating with IL-1RAcP atd the cell surface and removing and sequestering it from the pool available to associate with IL-1R1. Thus, soluble IL-1R2 binds to free IL-1, whereas cell surface IL-1R2 sequesters IL-1RAcP. Expression of IL-1R2 can be upregulated by a number of stimuli, including corticosteroids and IL-4. However, IL-1R2 can also be induced by inflammatory cytokines, including IFN-γ and IL-1, probably as a compensatory signal designed to limit the scale and duration of the inflammatory response. Production of IL-1R2 serves to make the producing cell and surrounding cells resistant to IL1-mediated activation. Interestingly, some of the most efficient IL-1-producing cells are also the best producers of the IL-1R2. IL-18 was first identified based on its capacity to induce IFN-γ. One name initially proposed for this cytokine was IL-1γ, because of its homology with IL-1α and IL-1β. Like IL-1β, it is translated as an inactive precursor molecule of 23 kDa and is cleaved to an active 18-kDa species by caspase 1. It is produced by multiple cell types in skin, including keratinocytes, Langerhans cells, and monocytes. IL-18 induces proliferation, cytotoxicity, and cytokine production by Th1 and natural killer (NK) cells, mostly synergistically with IL-12. The IL-18 receptor bears striking similarity to the IL-1 receptor.14 The binding chain (IL-18R) is an IL1R1 homolog, originally cloned as IL-1Rrp1. IL-18R alone is a low-affinity receptor that must recruit IL-18RacP (a homolog of IL-1RAcP). As for IL-1, both chains of the IL-18 receptor are required for signal transduction. Although there is no IL-18 homolog of IL-1ra, a molecule known as IL-18-binding protein binds to soluble mature IL-18 and prevents it from binding to the IL- 18R complex. More recently, it has become clear that there is a family of receptors homologous to the IL-1R1 and IL-18R molecules,14 having in common a TIR motif (Fig. 11-4).

All of these share analogous signaling pathways initiated by the MyD88 adapter molecule. One of these receptors, originally known as ST2, was initially characterized as a gene expressed by Th2 cells, but not by Th1 cells. The description of a natural ligand for ST2 designated IL-33 has added a new member to the IL-1 family that shares characteristic features of other cytokines in the family, such as a requirement for processing by caspase 1 to release a mature form of the ligand.18 IL-33 stimulation of Th2 cells promotes their production of the characteristic Th2 cytokines IL-4, IL-5, and IL-10.19 IL-1R1, IL-18R, IL-33R (ST2), the TLRs, and their ligands are all best viewed as elements of the innate immune system that signal the presence of danger or injury to the host. When IL-1 produced by epidermis was originally identified, it was noted that both intact epidermis and stratum corneum contained significant IL-1 activity, which led to the concept that epidermis was a shield of sequestered IL-1 surrounding the host, waiting to be released on injury. More recently, it was observed that high levels of the IL-1ra coexist within keratinocytes; however, repeated experiments show that in virtually all cases, the amount of IL-1 present is sufficient to overcome any potential for inhibition mediated by IL-1ra. Studies have now shown that mechanical stress to keratinocytes permits the release of large amounts of IL-1 in the absence of cell death. Release of IL-1 induces expression of endothelial adhesion molecules, including E-selectin, ICAM-1, and VCAM-1, as well as chemotactic and activating chemokines. This attracts not only monocytes and granulocytes but a specific subpopulation of memory T cells that bear cutaneous lymphocyte antigen on their cell surface. Memory T cells positive for cutaneous lymphocyte antigen are abundant in inflamed skin, comprising the majority of T cells present. Therefore, any injury to the skin, no matter how trivial, releases IL-1 and attracts this population of memory T cells. If they encounter their antigen in this microenvironment, their activation and subsequent cytokine production will amplify the inflammatory response. This has been proposed as the basis of the clinical observation of inflammation in response to trauma, known as the Koebner reaction. Several biologics that act by inhibiting IL-1 function have been developed for clinical use including recombinant IL-1Ra (anakinra), antibody to IL-1β (canakinumab), and an IgG Fc fusion protein that includes the ligand binding domains of the type I IL-1R and IL1RAcP (rilonacept, also known as IL-1 Trap). All of these agents are efficacious in countering the IL-1-induced inflammation associated with a group of rare autoinflammatory diseases called the cryopyrin-associated periodic syndromes (CAPS). Anakinra was initially US Food and Drug Administration (FDA) approved as a therapy for adult rheumatoid

arthritis. IL-1 inhibition is also being tested as a therapy for gout, an inflammatory arthritis triggered by uric acid-mediated activation of inflammasomes that generate IL-1β.

TUMOR NECROSIS FACTOR: THE OTHER PRIMARY CYTOKINE TNF-α is the prototype for a family of related signaling molecules that mediate their biologic effects through a family of related receptor molecules. TNF-α was initially cloned on the basis of its ability to mediate two interesting biologic effects: (1) hemorrhagic necrosis f malignant tumors, and (2) inflammation-associated cachexia. Although TNF-α exerts many of its biologically important effects as a soluble mediator, newly synthesized TNF-α exists as a transmembrane protein on the cell surface. A specific metalloproteinase known as TNF-α-converting enzyme (TACE) is responsible for most TNF-α release by T cells and myeloid cells. The closest cousin of TNF-α is TNF-β, also known as lymphotoxin α (LT-α). Other related molecules in the TNF family include lymphotoxin β (LTβ) that combines with LT-α to form the LT-α1β2 heterotrimer; Fas ligand (FasL); TNFrelated apoptosis-inducing ligand (TRAIL); receptor activator of NF-κB ligand (RANKL); and CD40 ligand (CD154). Although some of these other TNF family members have not been traditionally regarded as cytokines, their structure (all are type II membrane proteins with an intracellular N-terminus and an extracellular C-terminus) and signaling mechanisms are closely related to those of TNF. The soluble forms of TNF-α, LT-α, and FasL are homotrimers, and the predominant form of LT-β is the membrane-bound LT-α1β2 heterotrimer. Trimerization of TNF receptor family members by their trimeric ligands appears to be required for initiation of signaling and expression of biologic activity. The initial characterization of TNF receptors led to the discovery of two receptor proteins capable of binding TNF-α with high affinity. The p55 receptor for TNF (TNFR1) is responsible for most biologic activities of TNF, but the p75 TNF receptor (TNFR2) is also capable of transducing signals (unlike IL-1R2, which acts solely as a biologic sink for IL-1). TNFR1 and TNFR2 have substantial stretches of close homology and are both present on most types of cells. Nevertheless, there are some notable differences between the two TNFRs. Unlike cytokine receptors from several of the other large families, TNF signaling does not involve the Jak/STAT pathway. TNF-α evokes two types of responses in cells: (1) proinflammatory effects, and (2) induction of apoptotic cell death (Fig. 11-5).

The proinflammatory effects of TNF-α that include upregulation of adhesion molecule expression and induction of secondary cytokines and chemokines, stem in large part from activation of NF-κB and can be transduced through both TNFR1 and TNFR2. Induction of apoptosis by signaling through TNFR1 depends on a region known as a death domain that is absent in TNFR2, as well as interactions with additional proteins with death domains within the TNFR1 signaling complex. Signaling initiated by ligand binding to TNFR1, Fas, or other death domain-containing receptors in the TNF family eventually leads to activation of caspase 8 or 10 and the nuclear changes and DNA fragmentation characteristic of apoptosis. At least two TNFR family members (TNFR1 and the LT-β receptor) also contribute to the normal anatomic development of the lymphoid system. Mice deficient in TNF-α lack germinal centers and follicular dendritic cells. TNFR1 mutant mice show the same abnormalities plus an absence of Peyer’s patches. Mice with null mutations in LT-α or LT-β have further abnormalities in lymphoid organogenesis and fail to develop peripheral lymph nodes. TNF-α is an important mediator of cutaneous inflammation, and its expression is induced in the course of almost all inflammatory responses in skin. Normal human keratinocytes and keratinocyte cell lines produce substantial amounts of TNF-α after stimulation with LPS or UV light. Cutaneous inflammation stimulated by irritants and contact sensitizers is associated with strong induction of TNF-α production by keratinocytes. Exposure to TNF-α promotes Langerhans cell migration to draining lymph nodes, allowing for sensitization of naive T cells. One molecular mechanism that may contribute to TNF-αinduced migration of Langerhans cells toward lymph nodes is reduced expression of the Ecadherin adhesion molecule after exposure to TNF-α. Induction of CC chemokine receptor 7 on both epidermal and dermal antigen-presenting cells correlates with movement into the draining lymphatics. The predominant TNFR expressed by keratinocytes is TNFR1. Autocrine signaling loops involving keratinocyte-derived TNF-α and TNFR1 lead to keratinocyte production of a variety of TNF-inducible secondary cytokines. The central role of TNF-α in inflammatory diseases, including rheumatoid arthritis and psoriasis, has mbecome evident from clinical studies. Clinical drugs that target the TNF pathway include the humanized anti-TNF-α antibody infliximab, the fully human anti-TNF-α antibody adalumimab, and the soluble TNF receptor etanercept. Drugs in this class are FDA approved for the treatment of several autoimmune and inflammatory diseases, including Crohn’s disease and rheumatoid arthritis. These three anti-TNF drugs are also FDA approved for the treatment of psoriasis and psoriatic arthritis (see Chapter 234). This class of drugs also has the potential to be valuable in the treatment of other inflammatory dermatoses. Paradoxically,

they are not effective against all autoimmune diseases—multiple sclerosis appears to worsen slightly after treatment with these agents. The TNF antagonists are powerful immunomodulating drugs, and appropriate caution is required in their use. Cases of cutaneous T-cell lymphoma initially thought to represent psoriasis have rapidly progressed to fulminant disease after treatment with TNF antagonists. TNFantagonists can also allow the escape of latent mycobacterial infections from immune control, with a potentially lethal outcome for the patient.

IL-17 FAMILY OF CYTOKINES IL-17 (also known as IL-17A) was the first described member of a family of related cytokines that now includes IL-17B through F. IL-17A and IL-17F have similar proinflammatory activities, bind to the same heterodimeric receptor composed of the IL17RA and IL-17RC receptor chains, and act to promote recruitment of neutrophils and induce production of antimicrobial peptides. These IL-17 species normally function in immune defense against pathogenic species of extracellular bacteria and fungi. Signaling by IL-17A and IL-17F depends on STAT3; mutations in STAT3 associated with the hyper-IgE syndrome block IL-17 signaling and lead to recurrent skin infections with Staphylococcus aureus and Candida albicans. Less is currently known about the actions of IL-17B, C, and D. IL-17E, also known as IL-25, is a product of Th2 cells and mast cells that signals through IL-17RB. A total of five receptor chains for IL-17 family cytokines have been identified, but how each of these individual receptor chains associates to form receptors for all the members of the IL-17 family remains to be worked out. These IL-17 receptor chains are homologous to each other, but display very limited regions of homology to the other major families of cytokine receptors. Recent expansion of interest in Th17 cells and the entire IL-17 family is closely linked to observations that the immunopathology of autoimmune disease in human patients and mouse models is often associated with an inappropriate expansion of Th17 cells. Thus, the cytokines produced by Th17 cells and the receptors that transduce these signals may turn out to be useful targets for therapies designed to dampen autoimmunity.

LIGANDS OF THE CLASS I (HEMATOPOIET IN RECEPTOR) FAMILY OF CYTOKINE RECEPTORS The hematopoietin receptor family (also known as the class I cytokine receptor family) is the largest of the cytokine receptor families and comprises a number of structurally related type I membrane-bound glycoproteins. The cytoplasmic domains of these receptors associate with

nonreceptor tyrosine kinase molecules, including the Jak kinases and src family kinases. After ligand binding and receptor oligomerization, these associated nonreceptor tyrosine kinases phosphorylate intracellular substrates, which leads to signal transduction. Most of the multiple-chain receptors in the hematopoietin receptor family consist of a cytokine-specific α chain subunit paired with one or more shared receptor subunits. Five shared receptor subunits have been described to date: (1) the common γ chain (γc), (2) the common β chain shared between the IL-2 and IL-15 receptors; (3) a distinct common β chain shared between the granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3, and IL-5 receptors; (4) the IL-12Rβ2 chain shared by the IL-12 and IL-23receptors; and (5) finally the glycoprotein 130 (gp130) molecule, which participates in signaling by IL-6 and related cytokines.

CYTOKINES WITH RECEPTORS THAT INCLUDE THE gc CHAIN The receptor complexes using the γc chain are the IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21 receptors. Two of these receptors, IL-2R and IL-15R, also use the IL-2Rβc chain. The γc chain is physically associated with Jak3, and activation of Jak3 is critical to most signaling initiated through this subset of cytokine receptors.20

INTERLEUKIN 2 AND INTERLEUKIN 15. IL-2 and IL-15 can each activate NK cells and stimulate proliferation of activated T cells. IL2 is a product of activated T cells, and IL-2R is largely restricted to lymphoid cells. The IL-15 gene is expressed by nonlymphoid tissues, and its transcription is induced by UVB radiation in keratinocytes and fibroblasts and by LPS in monocytes and dendritic cells. Multiple isoforms of IL-15Rα are found in various hematopoietic and nonhematopoietic cells. The IL2R and IL-15R complexes of lymphocytes incorporate up to three receptor chains, whereas most other cytokine receptor complexes have two. The affinities of IL-2R and IL-15R for their respective ligands can be regulated, and to some extent, IL-2 and IL-15 compete with each other. The highest affinity receptor complexes for each ligand (approximately 10−11 M) consist of the IL-2Rβc and γc chains, as well as their respective α chains (IL-2Rα, also known as CD25, and IL-15Rα). γc and IL-2Rβc without the α chains form a functional lower affinity receptor for either ligand (10−8 to 10−10 M). Although both ligands transmit signals through the γc chain, those signals elicit overlapping but distinct responses in various cells. Activation

of naive CD4 T cells by T-cell receptor and costimulatory molecules induces expression of IL-2, IL-2Rα, and IL-2Rβc, which leads to vigorous proliferation. Prolonged stimulation of T-cell receptor and IL-2R leads to expression of FasL and activation-induced cell death. Although IL-2 signaling facilitates the death of CD4 T cells in response to sustained exposure to antigen, IL-15 inhibits IL-2-mediated activation-induced cell death as it stimulates growth. Similarly, IL-15 promotes proliferation of memory CD8 T cells, whereas IL-2 inhibits it. IL15 is also involved in the homeostatic survival of memory CD8 T cells, NK cells, and NK T cells. These contrasting biologic roles are illustrated by mice deficient in IL-2 or IL-2Rα that develop autoimmune disorders, and mice deficient in IL-15 or IL-15Rα, which have lymphopenia and immune deficiencies. Thus, IL-15 appears to have an important role in promoting effector functions of antigen-specific T cells, whereas IL-2 is involved in reining in autoreactive T cells.21

INTERLEUKIN 4 AND INTERLEUKIN 13. IL-4 and IL-13 are products of activated Th2 cells that share limited structural homology (approximately 30%) and overlapping but distinct biologic activities. A specific receptor for IL-4, which does not bind IL- 13, is found on T cells and NK cells. It consists of IL-4Rα (CD124) and γc and transmits signals via Jak1 and Jak3. A second receptor complex that can bind either IL-4 or IL-13 is found on keratinocytes, endothelial cells, and other nonhematopoietic cells. It consists of IL-13Rα1 and IL-4Rα and transmits signals via Jak1 and Jak2. These receptors are expressed at low levels in resting cells, and their expression is increased by various activating signals. Curiously, exposure of monocytes to IL-4 or IL-13 suppresses expression of IL-4Rα and IL-13Rα1, whereas the opposite effect is observed in keratinocytes. Both signal transduction pathways appear to converge with the activation of STAT6, which is both necessary and sufficient to drive Th2 differentiation. IL-13Rα2 is a cell surface receptor homologous to IL-13Rα1 that specifically binds to IL-13 but is not known to transmit any signals.20 The biologic effects of engagement of the IL-4 receptor vary depending on the specific cell type, but most pertain to its principal role as a growth and differentiation factor for Th2 cells. Exposure of naive T cells to IL-4 stimulates them to proliferate and differentiateinto Th2 cells, which produce more IL-4, which in turn leads to autocrine stimulation that prolongs Th2 responses. Thus the expression of IL-4 early in the immune response can initiate a cascade of Th2 cell development that results in a predominately Th2 response. The genes encoding IL-4 and IL-13 are located in a cluster with IL-5 that undergoes structural changes during Th2 differentiation that are associated with

increased expression. Although naive T cells can make low levels of IL-4 when activated, IL4 is also produced by activated NK T cells. Mast cells and basophils also release preformed IL-4 from secretory granules in response to FcεRI-mediated signals. A prominent activity of IL-4 is the stimulation of class switching of the immunoglobulin genes of B cells. Nuocytes and natural helper cells are recently identified populations of innate immune effector cells that provide an early source of IL-13 during helminth infection. As critical factors in Th2 differentiation and effector function, IL-4 and IL-13 are mediators of atopic immunity. In addition to controlling the behavior of effector cells they also act directly on resident tissue cells, such as in inflammatory airway reactions.22

INTERLEUKIN 9 AND INTERLEUKIN 21. IL-9 is a product of activated Th2 cells exposed to TGF-β that acts as an autocrine growth factor as well as a mediator of inflammation.23 It is also produced by mast cells in response to IL-10 or stem cell factor. It stimulates proliferation of T and B cells and promotes expression of immunoglobulin E by B cells. It also exerts proinflammatory effects on mast cells and eosinophils. IL-9-deficient mice exhibit deficits in mast cell and goblet cell differentiation. IL-9 can be grouped with IL-4 and IL-13 as cytokines that function as effectors of allergic inflammatory processes and may play an important role in asthma and allergic disorders. IL-21 is also a product made by the Th2, Th17, and Tfh lineages that signals through a receptor composed of a specific α chain (IL-21R) homologous to the IL-4R α chain and γc.24 Absence of an intact IL-21 receptor is associated with impaired Th2 responses.25

INTERLEUKIN 7 AND THYMIC STROMAL LYMPHOPOIETIN. Mutations abrogating the function of IL-7, IL-7Rα (CD127), γc, or Jak3 in mice or humans cause profound immunodeficiency as a result of T- and NK-cell depletion.20 This is principally due to the indispensable role of IL-7 in promoting the expansion of lymphocytes and regulating the rearrangement of their antigen receptor genes. IL-7 is a potent mitogen and survival factor for immature lymphocytes in the bone marrow and thymus. The second function of IL-7 is as a modifier of effector cell functions in the reactive phase of certain immune responses. IL-7 transmits activating signals to mature T cells and certain activated B cells. Like IL-2, IL-7 has been shown to stimulate proliferation of cytolytic T cells and lymphokine-activated killer cells in vitro and to enhance their activities in vivo. IL-7 is a particularly significant cytokine for lymphocytes in the skin and other epithelial tissues. It is

expressed by keratinocytes in a regulated fashion, and this expression is thought to be part of a reciprocal signaling dialog between dendritic epidermal T cells and keratinocytes in murine skin. Keratinocytes release IL-7 in response to IFN-γ, and dendritic epidermal T cells secrete IFN-γ in response to IL-7. An IL-7-related cytokine using one chain of the IL-7 receptor as part of its receptor is thymic stromal lymphopoietin (TSLP). TSLP was originally identified as a novel cytokine produced by a thymic stromal cell line that could act as a growth factor for B- and T-lineage cells. The TSLP receptor consists of the IL-7Rα and a second receptor chain (TSLPR) homologous to but distinct from the γc chain. TSLP has attracted interest because of its ability to prime dendritic cells to become stronger stimulators of Th2 cells. This activity may permit TSLP to foster the development of some types of allergic diseases.26,27

CYTOKINES WITH RECE PTORS USING THE INTERLEUKIN 3 RECEPTOR b CHAIN The receptors for IL-3, IL-5, and GM-CSF consist of unique cytokine-specific α chains paired with a common β chain known as IL-3Rβ or βc (CD131). Each of these factors acts on subsets of early hematopoietic cells.28 IL-3, which was previously known as multilineage colony-stimulating factor, is principally a product of CD4+ T cells and causes proliferation, differentiation, and colony formation of various myeloid cells from bone marrow. IL-5 is a product of Th2 CD4+ cells and activated mast cells that conveys signals to B cells and eosinophils. IL-5 has a costimulatory effect on B cells in that it enhances their proliferation and immunoglobulin expression when they encounter their cognate antigen. In conjunction with an eosinophilattracting chemokine known as CC chemokine ligand 11 or eotaxin, IL-5 plays a central role in the accumulation of eosinophils that accompanies parasitic infections and some cutaneous inflammatory processes. IL-5 appears to be required to generate a pool of eosinophil precursors in bone marrow that can be rapidly mobilized to the blood, whereas eotaxin’s role is focused on recruitment of these eosinophils from blood into specific tissue sites. GM-CSF is a growth factor for myeloid progenitors produced by activated T cells, phagocytes, keratinocytes, fibroblasts, and vascular endothelial cells. In addition to its role in early hematopoiesis, GM-CSF has potent effects on macrophages and dendritic cells. In vitro culture of fresh Langerhans cells in the presence of GM-CSF promotes their transformation into mature dendritic cells with maximal immunostimulatory potential for naive T cells. The effects of GM-CSF on dendritic cells probably account for the dramatic ability of GM-CSF to evoke therapeutic antitumor immunity when tumor cells are engineered to express it.29,30

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6

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GLYCOPROTEIN 130 Receptors for a group of cytokines including IL-6, IL-11, IL-27, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1 interact with a hematopoietin receptor family member, gp130, which does not appear to interact with any ligand by itself. The gp130 molecule is recruited into signaling complexes with other receptor chains when they engage their cognate ligands. IL-6 is the most thoroughly characterized of the cytokines that use gp130 for signaling and serves as a paradigm for discussion of the biologic effects of this family of cytokines. IL-6 is yet another example of a highly pleiotropic cytokine with multiple effects. A series of different names (including IFN-β2, B-cell stimulatory factor 2, plasmacytoma growth factor, cytotoxic T cell differentiation factor, and hepatocytestimulating factor) were used for IL-6 before it was recognized that a single molecular species accounts for all of these activities. IL-6 acts on a wide variety of cells of hematopoietic origin. IL-6 stimulates immunoglobulin secretion by B cells and has mitogenic effects on B lineage cells and plasmacytomas. IL-6 also promotes maturation of megakaryocytes and differentiation of myeloid cells. Not only does it participate in hematopoietic development and reactive immune responses, but IL-6 is also a central mediator of the systemic acute-phase response. Increases in circulating IL-6 levels stimulate hepatocytes to synthesize and release acute-phase proteins. There are two distinct signal transduction pathways triggered by IL-6. The first of these is mediated by the gp130 molecule when it dimerizes on engagement by the complex of IL-6 and IL-6Rα. Homodimerization of gp130 and its associated Jak kinases (Jak1, Jak2, Tyk2) leads to activation of STAT3. A second pathway of gp130 signal transduction involves Ras and the mitogen-activated protein kinase cascade and results in phosphorylation and activation of a transcription factor originally designated nuclear factor of IL-6. IL-6 is an important cytokine for skin and is subject to dysregulation in several human diseases, including some with skin manifestations. IL-6 is produced in a regulated fashion by keratinocytes, fibroblasts, and vascular endothelial cells as well as by leukocytes infiltrating the skin. IL-6 can stimulate the proliferation of human keratinocytes under some conditions. Psoriasis is one of several inflammatory skin diseases in which elevated expression of IL-6 has been described. Human

herpesvirus 8 produces a viral homolog of IL-6 that may be involved in the pathogenesis of human herpes virus-8-associated diseases, including Kaposi sarcoma and body cavity-based lymphomas. The other cytokines using gp130 as a signal transducer have diverse bioactivities. IL-11 inhibits production of inflammatory cytokines and has shown some therapeutic activity in patients with psoriasis. Exogenous IL-11 also stimulates platelet production and has been used to treat thrombocytopenia occurring after chemotherapy. IL-27 is discussed in the next section with the IL-12 family of cytokines.

INTERLEUKIN 12, INTERLEUKIN 23,INTERLEUKIN 27, AND INTERLEUKIN 35: PIVOTAL CYTOKINES REGULATING T HELPER 1 AND T HELPER 17 RESPONSES IL-12 is different from most other cytokines in that its active form is a heterodimer of two proteins, p35 and p40. IL-12 is principally a product of antigenpresenting cells such as dendritic cells, monocytes, macrophages, and certain B cells in response to bacterial components, GM-CSF, and IFN-γ. Activated keratinocytes are an additional source of IL-12 in skin. Human keratinocytes constitutively make the p35 subunit, whereas expression of the p40 subunit can be induced by stimuli including contact allergens, phorbol esters, and UV radiation. IL-12 is a critical immunoregulatory cytokine that is central to the initiation and maintenance of Th1 responses. Th1 responses that are dependent on IL-12 provide protective immunity to intracellular bacterialpathogens. IL-12 also has stimulatory effects on NK cells, promoting their proliferation, cytotoxic function, and the production of cytokines, including IFN-γ. IL-12 has been shown to be active in stimulating protective antitumor immunity in a number of animal models.31 Two chains that are part of the cell surface receptor for IL-12 have been cloned. Both are homologous to other β chains in the hematopoietin receptor family and are designated β1 and β2. The β1 chain is associated with Tyk2 and the β2 chain interacts directly with Jak2. The signaling component of the IL-12R is the β2 chain. The β2 chain is expressed in Th1 but not Th2 cells and appears to be critical for commitment of T cells to production of type 1 cytokines. IL-12 signaling induces the phosphorylation of STAT1, STAT3, and STAT4, but it is STAT4 that is essential for induction of a Th1 response. IL-23 is a heterodimeric cytokine in the IL-12 family that consists of the p40 chain of IL-12 in association with a distinct p19 chain. IL-23 has overlapping activities with IL-12, but also induces proliferation of memory T cells. Interest in IL-23 has been sparked by the observation that IL-23 promotes the differentiation of T cells producing IL-17 (Th17 subset). The IL-23 receptor consists of two chains: (1) the IL-12Rβ1 chain that forms part of the IL12 receptor and (2) a specific IL-23 receptor.32 The third member of the IL-12 family to be

discovered was IL-27. IL-27 is also a heterodimer and consists of a subunit called p28 that is homologous to IL-12 p35 and a second subunit known as EBI3 that is homologous to IL-12 p40. IL-27 plays a role in the early induction of the Th1 response. The IL-27 receptor consists of a receptor called WSX-1 that associates with the shared signal-transducing molecule gp130.32,33 The newest member of the IL-12 family is IL-35. The IL-35 heterodimer is composed of the p35 chain of IL-12 associated with the IL-27β chain EBI3. In contrast to the other IL-12 family cytokines, IL-35 is selectively made by Treg cells, promotes the growth of Treg cells, and suppresses the activity of Th17 cells.34 The IL-12 family of cytokines has emerged as a promising new target for anticytokine pharmacotherapy. The approach that has been developed the furthest to date is targeting both IL-12 and IL-23 with monoclonal antibodies directed against the p40 subunit that is part of both cytokines. Ustekinumab is an antihuman p40 monoclonal antibody that has shown therapeutic activity against psoriasis comparable to that of TNF inhibitors and has received FDA approval for the treatment of psoriasis.35 The development of anti-p40 therapies is several years behind anti-TNF-α drugs, but development of additional anti-p40 biologics for clinical use is anticipated.

LIGANDS OF THE CLASS II FAMILY OF CYTOKINE RECEPTORS A second major class of cytokine receptors with common features includes two types of receptors for IFNs, IL-10R, and the receptors for additional IL-10- related cytokines including IL-19, IL-20, IL-22, IL-24, and IL-26.

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A

JAK/STAT PAT HWAY IFNs were one of the first families of cytokines to be characterized in detail. The IFNs were initially subdivided into three classes: (1) IFN-α (the leukocyte IFNs), (2) IFN-β (fibroblast IFN), and (3) IFN-γ (immune IFN). The α and β IFNs are collectively called type I IFNs, and all of these molecules signal through the same two-chain receptor (the IFN-αβ receptor).36 The second IFN receptor is a distinct two-chain receptor specific for IFN-γ. Both of these IFN receptors are present on many cell types within skin as well as in other tissues. Each of the chains comprising the two IFN receptors is associated with one of the Jak kinases (Tyk2 and Jak1 for the IFN-αβR, and Jak1 and Jak2 for the IFN-γR). Only in the presence of both chains and two functional Jak kinases will effective signal transduction occur after IFN binding. A new class of IFNs known as IFN-γ or type III IFNs has now been identified that has a low degree of homology with both type I IFNs and IL-10.37 The current members of

this class are IL-28A, IL-28B, and IL-29. Although the effects of these cytokines are similar to those of the type I IFNs, they are less potent. These type III IFNs use a shared receptor that consists of the β chain of the IL-10 receptor associated with an IL-28 receptor α chain. Viruses, double-stranded RNA, and bacterial products are among the stimuli that elicit release of the type I IFNs from cells. Plasmacytoid dendritic cells have emerged as a particularly potent cellular source of type I IFNs. Many of the effects of the type I IFNs directly or indirectly increase host resistance to the spread of viral infection. Additional effects mediated through IFN-αβR are increased expression of major histocompatibility complex (MHC) class I molecules and stimulation of NK cell activity. Not only does it have well-known antiviral effects, but IFN-α also can modulate T-cell responses by favoring the development of a Th1 type of T-cell response. Finally, the type I IFNs also inhibit the proliferation of a variety of cell types, which provides a rationale for their use in the treatment of some types of cancer. Forms of IFN-α enjoy considerable use clinically for indications ranging from hairy cell leukemia, various cutaneous malignancies, and papillomavirus infections (see Chapter 196). Some of the same conditions that respond to therapy with type I IFNs also respond to topical immunomodulatory agents like imiquimod. This synthetic imidazoquinoline drug is an agonist for the TLR7 receptor, whose natural ligand is single-stranded RNA. Imiquimod stimulation of cells expressing TLR7 elicits local release of large amounts of type I IFNs from plasmacytoid dendritic cells, which can trigger clinically useful antiviral and tumor inhibitory effects against genital warts, superficial basal cell carcinoma, and actinic keratoses. Resiquimod is a related synthetic compound that activates both TLR7 and TLR8, eliciting a slightly different spectrum of cytokines.38 Production of IFN-γ is restricted to NK cells, CD8 T cells, and Th1 CD4 T cells. Th1 cells produce IFN-γ after engagement of the T-cell receptor, and IL-12 can provide a strong costimulatory signal for T-cell IFN-γ production. NK cells produce IFN-γ in response to cytokines released by macrophages, including TNF-γ, IL-12, and IL-18. IFN-γ has antiviral activity, but it is a less potent mediator than the type I IFNs for induction of these effects. The major physiologic role of IFN-γ is its capacity to modulate immune responses. IFN-γ induces synthesis of multiple proteins that play essential roles in antigen presentation to T cells, including MHC class I and class II glycoproteins, invariant chain, the Lmp2 and Lmp7 components of the proteasome, and the TAP1 and TAP2 intracellular peptide transporters. These changes increase the efficiency of antigen presentation to CD4 and CD8 T cells. IFN-γ is also required for activation of macrophages to their full antimicrobial potential, enabling them to eliminate microorganisms capable of intracellular growth. Like type I IFNs, IFN-γ

also has strong antiproliferative effects on some cell types. Finally, IFN-γ is also an inducer of selected chemokines (CXC chemokine ligands 9 to 11) and an inducer of endothelial cell adhesion molecules (e.g., ICAM-1 and VCAM-1). Because of the breadth of IFN-γ’s activities, it comes the closest of the T-cell cytokines to behaving as a primary cytokine. INTERLEUKIN 10: AN “ANTIINFLAMMAT ORY” CYTOKINE IL-10 is one of several cytokines that primarily exert regulatory rather than stimulatory effects on immune responses. IL-10 was first identified as a cytokine produced by Th2 T cells that inhibited cytokine production after activation of T cells by antigen and antigenpresenting cells. IL-10 exerts its action through a cell surface receptor found on macrophages, dendritic cells, neutrophils, B cells, T cells, and NK cells. The ligand-binding chain of the receptor is homologous to the receptors for IFN-α/β and IFN-γ, and signaling events mediated through the IL-10 receptor use a Jak/STAT pathway. IL-10 binding to its receptor activates the Jak1 and Tyk2 kinases and leads to the activation of STAT1 and STAT3. The effects of IL-10 on antigenpresenting cells such as monocytes, macrophages, and dendritic cells include inhibition of expression of class II MHC and costimulatory molecules (e.g., B7–1, B7–2) and decreased production of T cell-stimulating cytokines (e.g., IL-1, IL-6, and IL-12). At least four viral genomes harbor viral homologs of IL-10 that transmit similar signals by binding to the IL-10R.39 A major source of IL-10 within skin is epidermal keratinocytes. Keratinocyte IL-10 production is upregulated after activation; one of the best-characterized activating stimuli for keratinocytes is UV irradiation. UV radiation-induced keratinocyte IL-10 production leads to local and systemic effects on immunity. Some of the well-documented immunosuppressive effects that occur after UV light exposure are the result of the liberation of keratinocyte-derived IL-10 into the systemic circulation. IL-10 also plays a dampening role in other types of cutaneous immune and inflammatory responses, because the absence of IL-10 predisposes mice to exaggerated irritant and contact sensitivity responses.

NOVEL INTERLEU KIN 10-RELATE D CYTOKINE S: INTERLEUKINS 19, 20, 22, 24, AND 26 A series of cytokines related to IL-10 have been identified and shown to engage a number of receptor complexes with shared chains.40 IL-19, IL-20, and IL-24 transmit signals via a complex consisting of IL-20Rα and IL-20Rβ. IL-22 signals through a receptor consisting of IL-22R and IL-10Rβ. The receptors for these IL-20 family cytokines are preferentially expressed on epithelial cells including keratinocytes. Increased expression of these cytokines and their receptors is associated with psoriasis. The IL-20 family cytokines have profound

effects on the proliferation and differentiation of human keratinocytes in culture.41 Transgenic mice overexpressing IL-20, IL-22, or IL-24 develop epidermal hyperplasia and abnormal keratinocyte differentiation. 42 All of these findings point to a significant role for these cytokines in the epidermal changes associated with cutaneous inflammation. T cells producing IL-22 that elaborate a distinct set of cytokines from Th1, Th2 and Th17 cells have been isolated from the epidermis of patients with psoriasis and other inflammatory skin disorders. The IL-22 produced by these T cells promotes keratinocyte proliferation and epidermal acanthosis.43,44

TRANSFORMING GROWTH FACTOR-b FAMILY AND ITS RECEPTORS TGF-β1 was first isolated as a secreted product of virally transformed tumor cells capable of inducing normal cells in vitro to show phenotypic characteristics associated with transformation. Over 30 additional members of the TGF-β family have now been identified. They can be grouped into several families: the prototypic TGF-βs (TGF-β1 to TGF-β3), the bone morphogenetic proteins, the growth/differentiation factors, and the activins. The TGF name for this family of molecules is somewhat of a misnomer, because TGF-β has antiproliferative rather than proliferative effects on most cell types. Many of the TGF-β family members play an important role in development, influencing the differentiation of uncommitted cells into specific lineages. TGF-β family members are made as precursor proteins that are biologically inactive until a large prodomain is cleaved. Monomers of the mature domain of TGF-β family members are disulfide linked to form dimers that strongly resist denaturation. Participation of at least two cell surface receptors (type I and type II) with serine/threonine kinase activity is required for biologic effects of TGF-β.45 Ligand binding by the type II receptor (the true ligand-binding receptor) is associated with the formation of complexes of type I and type II receptors. This allows the type II receptor to phosphorylate and activate the type I receptor, a “transducer” molecule that is responsible for downstream signal transduction. Downstream signal transmission from the membrane-bound receptors in the TGF-β receptor family to the nucleus is primarily mediated by a family of cytoplasmic Smad proteins that translocate to the nucleus and regulate transcription of target genes. TGF-β has a profound influence on several types of immune and inflammatory processes. An immunoregulatory role for TGF-β1 was identified in part through analysis of TGF-β1 knockout mice that develop a wasting disease at 20 days of age associated with a mixed inflammatory cell infiltrate involving many internal organs. This phenotype is now

appreciated to be a result in part of the compromised development of regulatory T cells when TGF-β1 is not available. Development of cells in the dendritic cell lineage is also perturbed in the TGF-β1-deficient mice, as evidenced by an absence of epidermal Langerhans cells and specific subpopulations of lymph node dendritic cells. TGF-β-treated fibroblasts display enhanced production of collagen and other extracellular matrix molecules. In addition, TGF-β inhibits the production of metalloproteinases by fibroblasts and stimulates the production of inhibitors of the same metalloproteinases (tissue inhibitors of metalloproteinase, or TIMPs). TGF-β may contribute to the immunopathology of scleroderma through its profibrogenic effects.46

CHEMOKINES:

SECONDARY

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CENTRAL

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LEUKOCYTE

MOBILIZATION Chemokines are a large superfamily of small cytokines that have two major functions. First, they guide leukocytes via chemotactic gradients in tissue. Typically, this is to bring an effector cell to where its activities are required. Second, a subset of chemokines has the capacity to increase the binding of leukocytes via their integrins to ligands at the endothelial cell surface, which facilitates firm adhesion and extravasation of leukocytes in tissue. The activities of this important class of cytokines are sufficiently complex that they are the subject of a separate chapter (Chapter 12).

CYTOKINE NETWORK—THERAPEUTIC IMPLICATIONS AND APPLICATIONS This chapter has attempted to bring some degree of order and logic to the analysis of a field of human biology that continues to grow at a rapid rate. Although many things may change in the world of cytokines, certain key concepts have stood the test of time. Principal among them is the idea that cytokines are emergency molecules, designed to be released locally and transiently in tissue microenvironments. When cytokines are released persistently, the result is typically chronic disease. One potential way to treat such diseases is with cytokine antagonists or other drugs that target cytokines or cytokine-mediated pathways. Cytokines and cytokine antagonists are being used therapeutically by clinicians, and development of additional agents continues. With certain notable exceptions, systemic cytokine therapy has been disappointing and is often accompanied by substantial morbidity. In contrast, local and transient administration of cytokines may yield more promising results. An example of this approach is the transduction of tumor cells to express GM-CSF to create the therapeutic cancer vaccines that are capable of boosting antitumor immune responses.30 Conversely,

multiple biologics that specifically block cytokine activity have been developed and approved for clinical use. Antibodies and TNF receptor–Fc fusion proteins are FDA-approved antagonists of TNF-α activity that are highly effective at inducing durable remissions in psoriasis (see Chapters 18 and 234). Antibodies against the p40 subunit shared by IL-12 and IL-23 are also active in treating psoriasis. An IL-1 receptor-Fc fusion protein, an antibody to IL-1β, and recombinant IL-1Ra are all effective therapy for patients with the cryopyrinassociated periodic syndromes. IL-1Ra is FDA-approved for treatment of adult rheumatoid arthritis. A class of pharmacologic agents that inhibits the production of multiple T cellderived cytokines is the calcineurin inhibitors. Tacrolimus and pimecrolimus both bind to the immunophilin FK-506 binding protein-12 (FKBP-12), producing complexes that bind to calcineurin, a calcium-dependent phosphatase that acts on proteins in the nuclear factor of activated T-cells (NFAT) family to promote their nuclear translocation and activation of cytokine genes (including IL-2, IL-4, and IFN-γ)47 (see Chapters 221 and 233). Finally, fusion toxins linked to cytokines, such as the IL-2 fusion protein denileukin diftitox, exploit the cellular specificity of certain cytokine–receptor interactions to kill target cells (see Chapter 234). Denileukin diftitox is FDA approved for the treatment of cutaneous T-cell lymphoma and has also shown therapeutic activity in other types of lymphoid malignancies.48 Each of the aforementioned approaches is still relatively new and open to considerable future development. An understanding of cytokines by clinicians of the future is likely to be central to effective patient care.

KEY REFERENCES Full reference list available at www.DIGM8.com DVD contains references and additional content 1. Oppenheim JJ: Cytokines: Past, present, and future. Int J Hematol 74:3, 2001 3. Luger TA et al: Epidermal cell (keratinocyte)-derived thymocyte-activating factor (ETAF).J Immunol 127:1493, 1981 4. Kupper TS: The activated keratinocyte: A model for inducible cytokine production by non bone marrow-derived cells in cutaneous inflammatory and immune responses. J Invest Dermatol 94:146S, 1990 5. Albanesi C, Pastore S: Pathobiology of chronic inflammatory skin diseases: Interplay between keratinocytes and immune cells as a target for anti-inflammatory drugs. Curr Drug Metab 11:210, 2010

6. Kupper TS: Immune and inflammatory processes in cutaneous tissues. Mechanisms and speculations. J Clin Invest 86:1783, 1990 7. Beutler B: Microbe sensing, positive feedback loops, and the pathogenesis of inflammatory diseases. Immunol Rev 227:248, 2009 9. O’Quinn DB et al: Emergence of the Th17 pathway and its role in host defense. Adv Immunol 99:115, 2008 10. Josefowicz SZ, Rudensky A: Control of regulatory T cell lineage commitment and maintenance. Immunity 30:616, 2009 15. Kawai T, Akira S: The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors. Nat Immunol 11:373, 2010 16. O’Shea JJ, Murray PJ: Cytokine signaling modules in inflammatory responses. Immunity 28:477, 2008 17. Martinon F, Mayor A, Tschopp J: The inflammasomes: Guardians of the body. Annu Rev Immunol 27:229, 2009 27. Ziegler SF, Artis D: Sensing the outside world: TSLP regulates barrier immunity. Nat Immunol 11:289, 2010 35. Griffiths CE et al: Comparison of ustekinumab and etanercept for moderate-to-severe psoriasis. N Engl J Med 362:118, 2010 43. Eyerich S et al: Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest 119:3573, 2009 44. Fujita H et al: Human Langerhans cells induce distinct IL-22-producing CD4+ T cells lacking IL-17 production. Proc Natl Acad Sci U S A 106:21795, 2009