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    • © 2001 Macmillan Magazines Ltd Autoimmune diseases, which affect approximately 5% of the population and disproportionately affect women, comprise a heterogeneous group of poorly understood disorders1,2 . In general, these illnesses are characterized by the presence of abnormal lymphocyte activation, although we now know that non-lymphoid cells, especially antigen-presenting cells such as macrophages and dendritic cells (DCs), also have important contributions to disease pathogenesis. It is well established that cytokines have essential roles in immune cell development, immunoregulation and immune effector functions. Accordingly, the role of cytokines has been extensively studied in the pathogene- sis of autoimmune disease3 .The surprise has been that alteration of cytokine levels and responsiveness does not necessarily have the expected effect — cytokines thought to promote inflammatory and immune responsiveness have unexpected, but essential, immunosuppressive, actions.The idea that pro-inflammatory cytokines pro- mote autoimmune disease,whereas regulatory cytokines suppress it, is too simple to explain the mechanisms underlying autoimmunity.Emerging data indicate that, as the Dave Mason song goes,“there ain’t no good guys, there ain’t no bad guys”, but rather a combination of cytokines exert variable effects at different times during the evolution of autoimmune disease.The complex role of cytokines in autoimmunity,especially their unantici- pated immunosuppressive functions,is the focus of the this review.We have focused on interleukin-2 (IL-2),but will also draw on lessons learned from interferons (IFNs) and tumour-necrosis factor (TNF) to illustrate these points. A simple, but flawed, view of cytokines It is recognized that T-lymphocyte function contributes to the pathogenesis of many autoimmune diseases.The accepted model is that naive T cells become activated by antigen and produce IL-2,which,in turn,causes clonal expansion and induces the production of other pro- inflammatory cytokines, such as TNF.Administration of IL-2 to humans results in expansion of lymphoid cells,but its clinical application is limited by its toxicity. IL-2 administration is also associated with a variety of autoimmune disorders, including immune thyroiditis, rheumatoid arthritis and other arthropathies4 . CD4+ T cells differentiate into at least two subsets of helper cells, T helper 1 (TH 1) and TH 2 cells5 (FIG. 1). The former produce IFN-γ (also known as type 2 IFN) and lymphotoxin-α. This differentiation is regulated by IL-12, which activates the transcription factor STAT4 (signal transducer and activator of transcrip- tion 4) in lymphocytes6 . TH 1 responses have been implicated in many autoimmune disorders, as shown by elevated levels of IFN-γ in tissues, by the ameliora- tion of disease with anti-cytokine treatment, and from studies of IFN-γ-, IL-12- and Stat4-deficient mice. In addition, a promoter polymorphism associated with overexpression of an IFN-inducible gene (Ifi202) CYTOKINES AND AUTOIMMUNITY John J. O’Shea*, Averil Ma‡ and Peter Lipsky* Cytokines have crucial functions in the development, differentiation and regulation of immune cells. As a result, dysregulation of cytokine production or action is thought to have a central role in the development of autoimmunity and autoimmune disease. Some cytokines, such as interleukin-2, tumour-necrosis factor and interferons — ostensibly, the ‘bad guys’ in terms of disease pathogenesis — are well known for the promotion of immune and inflammatory responses. However, these cytokines also have crucial immunosuppressive functions and so, paradoxically, can also be ‘good guys’. The balance between the pro-inflammatory and immunosuppressive functions of these well-known cytokines and the implications for the pathogenesis of autoimmune disease is the focus of this review. NATURE REVIEWS | IMMUNOLOGY VOLUME 2 | JANUARY 2002 | 37 *Lymphocyte Cell Biology Section,Arthritis and Rheumatism Branch and Autoimmunity Branch, NationalInstituteofArthritis andMusculoskeletaland SkinDiseases, NationalInstitutesof Health, Building 10 Room 9N252, 10 Center Drive, MSC-1820,Bethesda, Maryland 20892,USA. ‡ Departments of Medicine and Ben May Institute for Cancer Research, University of Chicago, Chicago,Illinois 60637,USA. Correspondence to J.J.O. e-mail: osheaj@arb. niams.nih.gov DOI: 10.1038/nri702 R E V I E W S
    • © 2001 Macmillan Magazines Ltd CROHN’S DISEASE One of the two predominant forms of inflammatory bowel disease that afflicts human patients.The pathophysiology is unknown,but is presumed to be autoimmune in nature. TOLERANCE Denotes lymphocyte non- responsiveness to antigen,but implies an active process,not simply a passive lack of response. PERIPHERAL TOLERANCE This form of tolerance refers to the lack of responsiveness of mature lymphocytes. 38 | JANUARY 2002 | VOLUME 2 www.nature.com/reviews/immunol R E V I E W S responses might contribute to disease.Additionally,our conclusions regarding the role of cytokines in autoim- munity largely arise from animal models of disease,and the extent to which these models accurately reflect mechanisms in human disease varies. More intriguing, however, is the fact that the cytokines themselves have complex actions. It is well known that cytokines are pleiotropic, redundant to some degree, and induce the production of other cytokines. More pertinent for this review, however, is that they have both immunostimu- latory and immunosuppressive actions; cytokines such as IL-2,IFN-γ and TNF really are double-edged swords, both enhancing and limiting autoimmune disease.This is not to say that these are the only cytokines that have such complex actions. The actions of other cytokines are also not simple. However, because the positive effects of IL-2, IFN-γ and TNF on immune responses are so well appreciated, we have focused on just these three cytokines. Autoimmunity associated with IL-2 deficiency The pathology in IL-2 and IL-2 receptor (IL-2R) knockout mice necessitated a major revision in our thinking about the function of IL-2 (REFS 11,12 andFIG.2). Because of its well-known role in lymphoid prolifera- tion, it was anticipated that the main phenotype associ- ated with impaired IL-2 signalling would primarily be a form of immunodeficiency. Instead, the mice exhibit lymphoid hyperplasia and produce high levels of multi- ple autoantibodies, including anti-DNA antibodies. About half of the mice die of autoimmune haemolytic anaemia before 2 months of age, and the survivors develop inflammatory bowel disease. Similar to CROHN’S DISEASE, the cytokines present in the colitis associated with IL-2 deficiency are typical of a TH 1 response, and disease is attenuated by interfering with IL-12 and IFN-γ. Importantly, the pathology is also corrected by the addition of exogenous IL-2 and adoptive transfer of lymphocytes capable of producing IL-2- or IL-2-treated cells from IL-2-deficient mice12 .Although the disorder is improved when IL-2-deficient mice are kept in germ- free conditions, colitis does not occur in more pro- foundly immunodeficient, recombination-activating gene (Rag) knockout animals, which indicates that the inflammatory bowel disease is not solely the result of impaired host defence or an opportunistic infection. Instead, the autoimmune disease in Il2–/– mice indicates that,despite the positive effects of this cytokine on lym- phocyte proliferation in vitro, the non-redundant role of IL-2 in vivo is to constrain lymphoid growth and maintain PERIPHERAL TOLERANCE.What,then,is the mecha- nism(s) through which IL-2 maintains TOLERANCE, and what is the relevance to human disease? IL-2 and activation-induced cell death. One mechanism that has been pursued is the ability of IL-2 to promote programmed cell death13 .Apoptosis of lymphocytes has emerged as a principal means of controlling immune response,but there are two biochemically distinct path- ways that contribute to this process. The first type of apoptosis, referred to as passive cell death,results from has been shown to be associated with mouse lupus, and a polymorphism in the IL-12p40 gene has been reported as a disease-associated allele in type I diabetes in humans7,8 . Conversely, TH 2 cells, which produce IL-4, promote allergic responses and constrain cell-mediated immu- nity. Accordingly, TH 2 cytokines can inhibit autoim- mune disease. Two important immunosuppressive cytokines, IL-10 and transforming growth factor-β (TGF-β) are produced by both subsets of TH cells, although T cells that are the main producers of these cytokines have also been denoted as regulatory T cells; interfering with these cytokines results in severe inflam- mation and autoimmune disease.IL-10 activates Stat3, and absence of Stat3 in myeloid cells also leads to autoimmune disease9 .Therefore,it would seem reason- able to speculate that the balance between the‘bad guys’ (IL-2, IFN-γ and TNF) and the‘good guys’(IL-4, IL-10 and TGF-β) would determine the propensity for the development of autoimmune disease.If only this simple model were true — unfortunately, this scenario is too simple to reflect reality.Indeed,if it were true,then one might expect that the rising incidence of TH 2-driven allergic diseases in developed countries would be associ- ated with a concomitant reduction in TH 1-diseases,and this is evidently not the case10 . There are several reasons underlying this complexity. First, not all autoimmune diseases are strictly TH 1 dis- eases; ulcerative colitis,and to an extent,systemic lupus erythematosus, are two examples in which TH 2 IL-12 IL-4 IL-5 IL-6 IL-10 IL-13 B cell Macrophage LT IFN-γ Cell-mediated immunity Allergic responses Anti-helminth responses IL-12STAT4 STAT6 IL-4 TH TH1 TH2 Eosinophil IgE Figure 1 | Cytokines and helper T-cell differentiation. In the standard model, helper T cells are polarized to one of two subsets, TH 1 and TH 2, by the cytokines interleukin (IL)-12 and IL-4, respectively. TH 1 cells promote cell-mediated immunity and host defence against intracellular organisms, but also contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, Crohn’s disease and multiple sclerosis. TH 2 cytokines, especially IL-4, antagonize TH 1 differentiation and attenuate cell-mediated immunity but promote allergic and anti-helminth responses. Although this model has been extremely useful and explains a great deal, the role of cytokines in autoimmune disease is more complicated than is indicated by this simple model. IFN-γ, interferon-γ; IgE, immunoglobulin E; LT, lymphotoxin; STAT, signal transducer and activator of transcription.
    • © 2001 Macmillan Magazines Ltd NATURE REVIEWS | IMMUNOLOGY VOLUME 2 | JANUARY 2002 | 39 R E V I E W S These mice have profound LYMPHADENOPATHY, autoanti- body production and nephritis. No defects in thymic deletion were found in Fas-deficient mice; instead, this disorder is an example of impaired peripheral toler- ance. Fas also has an important role in the removal of autoreactive B cells. The human disease autoimmune lymphoproliferative disease (ALPS) is caused by mutations of Fas14 . Mutations of Fas and other com- ponents of this pathway have been sought, but have not been found in lupus or other common human autoimmune diseases. As mentioned above, the relevance of defects in the Fas pathway to the autoimmunity associated with IL-2 deficiency is that IL-2 promotes Fas-dependent AICD by upregulating Fas, FasL and FADD transcription15 . This is dependent on Stat5 (REF. 15). Conversely, FLIP (FLICE/caspase-8 inhibitory protein) interferes with Fas-mediated apoptosis, and IL-2 downregulates FLIP expression. Transfer of cells that constitutively express FLIP results in autoimmunity16 . IL-2R is a multimeric complex that comprises IL-2Rα (CD25),IL-2Rβ and a shared receptor cytokine subunit, termed the common γ-chain (γc ). Other cytokines that use γc include: IL-4, IL-7,IL-9, IL-15 and IL-21.IL-15,in fact,binds to two receptor subunits also bound by IL-2, IL-2Rβ and γc ; IL-2 and IL-15 use sepa- rate α-chains. One striking finding is that although all the γc cytokines induce lymphocyte proliferation, the promotion of AICD seems to be a unique action of IL-2,at least at physiological levels of cytokine.The lack of effect of IL-15 is particularly difficult to understand, given that no discernible differences in IL-2 and IL-15 signalling have been unearthed. Non-mutually exclu- sive factors that might account for the distinct activities of IL-15 and IL-2 include in vivo receptor and ligand bioavailability or the location of signalling events. These possibilities are appealing given the distinct tis- sue expression of the ligands.Alternatively, the ligands might differ in their relative strength of signalling. Despite the clear importance of Fas andAICD,is this the whole explanation for the autoimmunity associated with IL-2 deficiency? Evidently not,as the phenotype of Fas- and IL-2-deficient mice are quite distinct even in strain-matched mice.For instance,neither lpr mice nor patients with ALPS develop colitis. These differences indicate that Fas deficiency is not the sole explanation for the autoimmunity that occurs in the absence of IL-2. Furthermore, despite in vitro results, there are still no compelling in vivo data that establish IL-2-dependent AICD as the primary mechanism underlying the role of IL-2 in maintaining peripheral tolerance. Regulatory T cells — is there an IL-2 connection? A sec- ond possible mechanism underlying the role of IL-2 in maintaining peripheral tolerance relates to the genera- tion of regulatory cells17,18 .The controversial notion that ‘suppressor’ T cells are important in physiology and pathology has undergone a recent renaissance. Studies initiated more than 20 years ago on the generation of organ-specific autoimmunity by neonatal thymectomy have lead to the conclusion that regulatory T cells reside the lack of crucial pro-survival signals. Absence of cytokine signalling results in increased mitochondrial permeability and cytochrome c release, which, along with apoptotic protease-activating factor 1 (APAF1), activates caspase-9 and downstream effector caspases. Proteins in the BCL-2 family interfere with this mode of programmed cell death, and cytokines such as IL-7 (and IL-2) upregulate anti-apoptotic members of the BCL-2 family. The second type of apoptosis,which is probably more importantintermsof thepathogenesisof autoimmunity, is activation-induced cell death (AICD).This is mediated predominantlybythedeath-domain-containingreceptor Fas(CD95) and,importantly,IL-2 has essential functions in promoting Fas-dependent AICD (FIG. 2). Occupancy of Fas results in the recruitment of proteins containing death domains and death effector domains; for exam- ple, Fas-associated death domain (FADD), which recruit and activate caspase-8, which then activates downstream caspases, inducing programmed cell death12 . The importance of Fas/Fas ligand (FasL; CD178) was established by the demonstration of their deficiency in autoimmune lpr and gld mice,respectively. LYMPHADENOPATHY Enlargement of lymph nodes. TH1 Granzyme Perforin Apoptosis Cytotoxicity Perforin Granzymes TH2 cells Apoptosis Immunoregulatory T cells Cytokines Proliferation Cyclins Cell-cycle inhibitors FOS MYC Anti-apoptotic BCL-2 BCL-XL Fas FasL FLIP IFN-γ TNF Perforin IL-4 Immunostimulatory Immunosuppressive IL-10 TGF-β TH1 TH2 IL-2 CD25 CD152 CD4 Treg Figure 2 | Summary of the immunostimulatory and immunosuppressive effects of IL-2. Although interleukin-2 (IL-2) is well known for its ability to promote proliferation, to inhibit apoptosis and to induce cytokines, the main, non-redundant in vivo function of IL-2 is to limit lymphoid expansion and promote peripheral tolerance. IL-2 is also important for the differentiation of T-helper (TH )2 cells. The importance of CD4+ CD25+ regulatory T cells is increasingly recognized. These cells are absent in Il2–/– mice, but the exact role in the development and/or function of these cells is not known. FasL, Fas ligand; FLIP, FLICE/caspase-8 inhibitory protein; IFN-γ, interferon-γ; TGF-β; transforming growth factor-β; TNF, tumour-necrosis factor; Treg , T-regulatory cell.
    • © 2001 Macmillan Magazines Ltd 40 | JANUARY 2002 | VOLUME 2 www.nature.com/reviews/immunol R E V I E W S fatal lymphoproliferative disease that is more aggressive than either IL-2 or Fas deficiency21 .Another mechanism by which CD4+ CD25+ regulatory T cells mediate sup- pression is by the production of immunosuppressive cytokines such as IL-4, TGF-β and IL-10. In some, but not all, systems, the effect of the CD4+ CD25+ cells is reversed by anti-cytokine interventions,but the roles of these cytokines in suppression remain controversial and depend on the experimental systems used.IL-2 has been reported to be required for differentiation of CD4+ cells into IL-4-producing TH 2 cells11,22 .Additionally,it should be noted that IL-2 induces TNF and IFN-γ, which, as will be discussed,also have immunosuppressive actions in addition to the well-known pro-inflammatory effects. So, there are several mechanisms by which IL-2 sig- nals might negatively regulate activated T cells and immune responses. But why do IL-2-deficient mice specifically develop colitis? Recently,missense mutations of the NOD2 gene have been found to be associated with Crohn’s disease and BLAU SYNDROME 23–25 . But is there an IL-2/NOD2 connection? There are no data that address this issue presently, although both molecules can regulate apoptosis.Still,exactly how IL-2 deficiency predisposes to colitis remains uncertain.Nonetheless,it is clear that IL-2 has an essential immunoregulatory role and its absence is accompanied by autoimmune mani- festations (see BOX 1 for a discussion of the role of IL-2 and human autoimmune disease). Immunosuppression by TNF One of the most exciting breakthroughs for physicians caring for patients with autoimmune diseases is the development of agents that interfere with TNF, either monoclonal antibodies or soluble receptors. Inhibition of TNF has proven to be remarkably effective in treating rheumatoid arthritis and Crohn’s disease,and this strat- egy has a strong scientific basis given the abundance of literature substantiating the pro-inflammatory role of TNF.TNF activates macrophages,endothelial cells,syn- oviocytes and other cells,and induces the production of other pro-inflammatory cytokines (for example, IL-1 and IL-6) and chemokines (FIG. 3). Its overproduction within the CD4+ CD25+ T-cell population that exits the thymus within the first few days of life. Transfer of CD4+ cells that are depleted of CD25+ cells into immunodeficient recipients leads to a spectrum of autoimmune diseases17 , the phenotype of which depend on background genes. However, transfer of CD4+ CD25+ cells within 10 days of the transfer of CD4+ CD25– T cells prevents disease. CD4+ CD25+ lym- phocytes are present in the thymus,but it is not known whether the regulatory properties of this subset are acquired within the thymus or in the periphery, although there are data that indicate the former is the case17,18 . Importantly, these cells do not fit neatly into either the TH 1 or TH 2 category. Although the protective effect of this subpopulation of lymphocytes has been well documented, the mecha- nism by which these cells attenuate autoimmunity is unclear, and an unequivocal connection with IL-2 action has not been made.IL-2 has important functions in controlling the expression of its high-affinity receptor, but is CD25 a marker indicating that the cells have undergone activation or does IL-2 actually have an important role in the physiology of these cells? Several lines of evidence support the latter possibility.The sup- pression that is mediated by CD4+ CD25+ cells is associ- ated with inhibition of IL-2 production. Conversely, it can be overcome by exogenous IL-2. Finally, and per- haps of most relevance, CD4+ CD25+ cells are absent in IL-2-deficient mice.Transfer of normal T cells into IL-2 or IL-2Rβ knockout mice results in removal of the abnormally activated T cells present in these mice19,20 ; in the former case, transfer of CD4+ CD25+ cells sup- presses lymphoproliferation, despite the fact that these regulatory cells produce little IL-2. An interesting connection is that the CD4+ CD25+ subset is the only population in the normal mouse that constitutively expresses CTLA-4 (cytotoxic T-lympho- cyte-associated protein 4; CD152) and IL-2 is known to upregulate CTLA-4 expression20 . CTLA-4 binds B7.1 (CD80) and B7.2 (CD86) on antigen-presenting cells and,in contrast to CD28,has a vital negative regulatory role in T-cell function.CTLA-4-deficient mice develop a BLAU SYNDROME A rare, autosomal-dominant disorder characterized by granulomatous arthritis, uveitis, skin rash and cranial neuropathy. Box 1 | IL-2 and human autoimmune disease Although there is strong evidence supporting the role of interleukin (IL)-2 in maintaining lymphoid homeostasis and peripheral tolerance,there is a paucity of evidence indicating that IL-2 deficiency is the primary mechanism underlying common autoimmune diseases.Mutation in the common γ-chain (γc ) and Janus kinase 3 (JAK3; the tyrosine kinase that binds γc ) typically results in profound immunodeficiency,probably owing to the impairment in IL-7 signalling6 . Nonetheless,some patients with mutations in the IL-2 receptor and JAK3 have features of autoimmune disease41,42 . In a recently described JAK3-deficient patient with severe combined immunodeficiency (SCID),this was found to be associated with impaired expression of Fas ligand in the lymphocytes produced41–44 . Lymphocytes from these patients, and from the respective knockout mice,have activation markers,but have defective in vitro proliferation — a phenotype that is shared with STAT5-deficient cells44 .This apparent paradox of poor in vitro proliferation associated with evidence of in vivo activation is also found in cells from patients with systemic lupus erythematosus and rheumatoid arthritis45 . These cells typically fail to produce normal IL-2 levels in vitro,but there is no evidence that this is owing to an intrinsic abnormality that is a causal defect in these disorders.The IL2 gene has also been linked to type I diabetes and resides within the Idd3 locus identified in non-obese diabetic mice.Disease susceptibility and resistance are linked with distinct patterns of IL-2 glycosylation.Although some data indicate that this modification might alter the activity of IL-2,this has not been firmly established46,47 .
    • © 2001 Macmillan Magazines Ltd NATURE REVIEWS | IMMUNOLOGY VOLUME 2 | JANUARY 2002 | 41 R E V I E W S clear examples of the immunosuppressive functions of TNF.The issue,however,is not simply of academic inter- est.Despite the impressive positive effects of blockade of TNF in rheumatoid arthritis, and even the salutatory effects of TNF antagonism in experimental autoimmune encephalomyelitis (the mouse model for multiple sclero- sis),anti-TNF agents worsened disease in patients with multiple sclerosis and precipitated demyelination in oth- ers26 .Additionally,enhanced production of anti-nuclear, anti-DNA and anti-cardiolipin antibodies is associated with anti-TNF therapy,although systemic lupus erythe- matosus is still very uncommon.Therefore,understand- ing the counterintuitive consequences of inhibition of TNF is of very practical concern. There are several examples in which TNF amelio- rates animal models of lupus,but more unexpected was that TNF can also reduce the severity of prototypic models of TH 1 diseases,including diabetes,experimen- tal autoimmune encephalomyelitis and arthritis. TNF has been documented in rheumatoid arthritis,multiple sclerosis and in many animal models of autoimmune disease26,27 .Additionally, transgenic overexpression of TNF results in arthritis, demyelinating disease, inflam- matory bowel disease and diabetes26,27 . The arthritis associated with transgenic expression of TNF, which results in synovial proliferation and erosive arthritis, is particularly impressive as it occurs even in the absence of lymphocytes. However,about one-third of patients with rheuma- toid arthritis treated with anti-TNF agents do not respond.Interestingly,collagen-induced arthritis can also occur in TNF-deficient mice, albeit with somewhat reducedseverity28 .Intriguingly,however,thisarthropathy is associated with splenomegaly,lymphadenopathy and increased memory CD4+ T cells.There is also an increase in activated lymph node B cells in TNF-deficient mice, indicating immunosuppressive functions of TNF. In fact,there are a number of model systems that provide IL-6 Acute-phase response IL-1 Fever Activation of APCs MHC Adhesion molecules Shock iNOS Activation of neutrophils Activation of endothelial cells Apoptosis Inhibition of DCs Defective co-stimulation Inhibition of T-cell signalling Cytokine inductionCytokine induction Macrophage Endothelial cells O2 •– Immunostimulatory Immunosuppressive Leukotriene IL-6 IL-10 TGF-β Dendritic cell Lymphopaenia Suppression of proliferation TH2 TNF Figure 3 | Summary of immunosuppressive effects of TNF, the prototypic pro-inflammatory cytokine. The pro-inflammatory actions of tumour-necrosis factor (TNF) on haematopoietic and non-haematopoietic cells are well recognized; however, it is also clear that TNF can attenuate immune responses by inhibiting T-cell receptor signalling, promoting lymphoid T-cell apoptosis, inhibiting dendritic cell (DC) co-stimulation and inducing other cytokines that can inhibit cell-mediated immunity. APCs, antigen-presenting cells; IL, interleukin; iNOS, inducible nitric oxide synthase; MHC, major histocompatibility complex, O2 .– , superoxide; TGF-β, transforming growth factor-β.
    • © 2001 Macmillan Magazines Ltd 42 | JANUARY 2002 | VOLUME 2 www.nature.com/reviews/immunol R E V I E W S activates nuclear factor-κB (NF-κB),a key anti-apoptotic transcription factor.TNF has also been shown to inhibit T-cell-receptor signalling and even a short course of anti- TNF therapy can reverse this28 (FIG.3).TNF also induces the expression IL-6, a pro-inflammatory cytokine that also promotes TH 2 differentiation through IL-4 induc- tion.Additionally, IL-6 attenuates TH 1 differentiation, independent of IL-4 but dependent on suppressor of cytokine signalling 1 (SOCS1;see below). Finally,TNF also influences the function of antigen- presenting cells (APCs),but,again,its effects are compli- cated28 .In some circumstances,TNF can activate APCs, augment antigen-presentation capability and upregulate the expression of co-stimulatory molecules.However,it can also inhibit the function of mature DCs,and might induce their apoptosis and impair antigen presentation. So, although it is clear that TNF has profound pro- inflammatory effects, there is also no question that it can have equally impressive immunosuppressive effects in vivo and in vitro; precisely which effect will predominate is regulated by the timing and the duration of the exposure. Complex effects of IFNs on autoimmunity Both type 1 (IFN-α and IFN-β) and type 2 (IFN-γ) IFNs are used to treat infectious diseases33,34 , but can precipitate autoimmune disease (4–19% of patients treated with these cytokines)4 . Complications associ- ated with IFN treatment include autoimmune thy- roiditis, the development of antinuclear antibodies or lupus, rheumatoid arthritis and other arthropathies. Paradoxically,IFNs,especially type 1 IFNs are also used to treat autoimmune disease; in fact, IFN-β is a corner- stone of therapy in multiple sclerosis35 .In humans,type 1 IFNs can induce IFN-γ production,promote TH 1 differ- entiation and activate STAT4, although this is not the case in the mouse (TABLE 1)36 . Type 1 IFNs also induces IL-15,which promotes the differentiation and develop- ment of natural killer and memory T cells.Additionally, type 1 IFNs induce production of chemokines in APCs and enhance DC maturation. Notably, type 1 IFNs are made at substantial levels by some DC subsets37 . Moreover,type 1 IFNs enhance DC maturation and can increase antigen presentation.Why then, would such a cytokine be useful in ameliorating a putative TH 1 dis- ease? The predominant mechanism underlying the ther- apeutic activity of IFN-β in multiple sclerosis is not known, but it is notable that type 1 IFNs inhibit IL-12 expression as a transgene in pancreatic islet cells or by systemic administration can either precipitate diabetes or be protective.Specifically,TNF expression under the control of the rat insulin promoter (RIP) along with expression of CD80 in C57BL/6 mice caused dia- betes29,30 . In non-obese diabetic mice (also abbreviated to NOD, but this has nothing to do with NOD2, the Crohn’s susceptibility gene!),TNF expression in neona- tal islets alone resulted in acceleration of spontaneous diabetes.Early systemic administration of TNF in these mice also exacerbated disease. By contrast, transgenic expression of TNF or systemic administration of TNF in adult NOD mice protected against diabetes,illustrating the immunosuppressive actions of TNF and arguing for a role in promoting tolerance.The basis for these differ- ential effects of TNF has been attributed to factors such as the effect of background genes.However,more recent detailed studies indicate that both the timing and the duration of TNF expression are important in determin- ing pathogenic versus protective roles of TNF. Using transgenic mice in which pancreatic TNF expression is controlled by a tetracycline-responsive promoter, it was found that only if TNF is expressed for more than 21 days did CD8+ lymphocytes enter islets and destroy them26 . Importantly, this was the case in both neonatal and adult animals.A second model used TNF expres- sion controlled in the same manner in a model of insulitis in which lymphocytic choriomenigitis viral glycoprotein was expressed as an autoantigen under the control of RIP31 . Expression of TNF early in the disease enhanced inflammation, whereas late expression of the TNF transgene abrogated diabetes with a marked decrease in autoreactive CD8+ lymphocytes. A number of possible mechanisms for the immunosuppressive effects of TNF have emerged and lymphocytes have been implicated as targets of this immunosuppressive action32 .Administration of TNF can result in lymphopaenia and TNF can clearly induce apoptosis – similar to Fas,the TNF receptor 1 (TNFR1) is a death-domain-containing receptor.Ligand binding causes recruitment of the death-domain-containing adaptor protein TRADD,which recruits FADD,leading to caspase activation. Several groups have shown a role of TNF in lymphocyte apoptosis, especially for CD8+ cells.However,TNF,TNFRI and TNFR2 knockout mice do not show spontaneous lymphadenopathy as found in gld or lpr mice28 .Perhaps this is because,similar to IL-2, TNF has both pro- and anti-apoptotic actions; TNF Table 1 | Examples of the complex and even counterintuitive effects of interferons Cytokine IFN-α/β IFN-γ Immunostimulatory Antiviral; increased antigen presentation; Activation of macrophages; increased TH 1 differentiation*; induction of IL-15, which antigen presentation and expression promotes NK and memory T-cell development of MHC class II; promotion of TH 1 and differentiation; increased DC maturation. differentiation. Immunosuppressive Increased Fas; increased IL-10; decreased IL-12; Upregulation of SOCS1; anti-proliferative inhibition of IL-12-signalling; anti-proliferative effects on myeloid and lymphoid cells. effects; differentiation of TR cells. *IFN-α/β (Type I IFN) induces TH 1 differentiation of human cells, but not mouse cells. DC, dendritic cell; IFN-γ, interferon-γ; IL, interleukin; MHC, major histocompatibility complex, NK, natural killer; SOCS1, suppressor of cytokine signalling 1; TCR, T-cell receptor; TH 1, T helper 1; TR cells, T-regulatory cells.
    • © 2001 Macmillan Magazines Ltd NATURE REVIEWS | IMMUNOLOGY VOLUME 2 | JANUARY 2002 | 43 R E V I E W S exogenous IFN-γ can serve as a protective factor in models of autoimmune disease. Possible mechanisms by which IFN-γ can attenuate autoimmunity will there- fore be briefly considered. A principal immunosup- pressive action of IFN-γ and other cytokines relates to their ability to induce members of the SOCS family. These proteins contain Src homology 2 (SH2) domains and function as classic feedback inhibitors6 . They bind cytokine receptors and Janus-activated kinases (JAKs) to interfere with cytokine signal transduction, and might also promote degradation of activated signalling molecules by a ubiquitin/proteasome-dependent path- way. SOCS1 is of particular importance in attenuating the effects of IFN-γ. Mice in which the Socs1 gene has been knocked out have extensive IFN-γ-dependent pathology and show excessive IFN-γ responses6 . SOCS1 seems to be a key modulator of IFN-γ action production and enhance IL-10 secretion.Additionally, recombinant IFN can inhibit IL-12 signalling. The inhibitory effect is STAT1 dependent; in fact, in the absence of STAT1, IFN-α induces IFN-γ production. Furthermore,IFN-α with IL-10 can promote the differ- entiation of CD25+ CD4+ regulatory T cells38 . Clearly, more work needs to be done to explain these complex immunoregulatory effects of type 1 IFNs. IFN-γ,the cytokine that defines TH 1 differentiation, has been extensively studied in both mouse and man. IFN-γ activates APCs and promotes TH 1 differentiation by upregulating the transcription factor T-bet (TABLE 1)39 . Again, despite extensive evidence incriminating it as a mediator of disease,there is also evidence for protective roles; this seemingly conflicting information has been reviewed in REFS 27,40.Suffice to say,that despite the role of IFN-γ as the canonical TH 1 cytokine,endogenous and IL-4 IL-10 TGF-βIL-2 TNF IL-10 TGF-β a Traditional view b Revised view Pro-inflammatory Anti-inflammatory IL-4 IL-10 TGF-β IL-10IL-4 TGF-β TNF IFN-γ Pro-inflammatory Anti-inflammatory Pro-inflammatory Normal Autoimmunity Normal Autoimmunity Anti-inflammatory Pro-inflammatory Anti-inflammatory TNFIFN-γ IL-2 IL-2 IFN-γ IL-2 TNF IFN-γ IFN-α/β IFN-α/β ? IL-2 TNF IFN-γ IL-4 IFN-γ TNF Figure 4 | Cytokines in autoimmunity — good guys and bad guys switch sides! a | The traditional view of cytokines is depicted.The cytokines that promote inflammation and immune responses, including interleukin (IL)-2, tumour-necrosis factor (TNF) and interferon-γ (IFN-γ), are shown wearing red shirts. The good guys, IL-10, transforming growth factor-β (TGF-β) and IL-4, inhibit cell-mediated immune responses and are depicted wearing blue shirts. Normally, there is an equilibrium between these cytokines (left), but in autoimmune disease (right), the bad guys overpower the good guys. b | A more realistic view is that these cytokines are not especially loyal to their team; they switch sides, meaning that they do not have simple pro- or anti-inflammatory actions; this is shown by cytokines such as IL-2 and IFN-α/β wearing both red and blue shirts. The cartoon is meant to emphasize that many cytokines have complex actions. The same cytokine can promote immune and inflammatory responses in some circumstances and inhibit responses in other settings.
    • © 2001 Macmillan Magazines Ltd 44 | JANUARY 2002 | VOLUME 2 www.nature.com/reviews/immunol R E V I E W S sobering lesson that it is so easy to generate autoim- mune disease. Given the large numbers of apparently essential negative regulators, one might conclude that autoimmunity does not routinely occur only because of the constant operation of a variety of inhibitory mecha- nisms. Indeed, the immune response seems to be in a constant balancing act with itself. With the many layers of inhibitory mechanisms used by the vertebrate immune system,it comes as no surprise that cytokines,even those thought to promote immune responses, have essential inhibitory functions. Apparently,they push on both ends of the balance in this effort to maintain effective host defence mechanisms while preventing autoimmunity.As we rush to the clinic with new therapies in hand, we need to keep in mind that blocking cytokines are used to prevent autoimmune disease in most experimental systems,and are not used to treat established disease. Often paradoxical effects occur when cytokines are administered during the course of disease; the exact timing and duration of expo- sure to these cytokines can evidently have variable and unexpected effects. Apparently, cytokines can switch sides in the balancing act in surprising ways (FIG.4).Now that cytokine and anti-cytokine therapies are increasingly used, we need to learn in detail the complex action of these cytokines,including both their pro-inflammatory and immunosuppressive properties. Prior to Dave Mason,another Bard also put it well:“There is nothing either good or bad,but thinking makes it so”(HamletAct ii,Sc.2).Clearly,there is much left to be done to dissect the complex and highly interactive effects of cytokines such as IL-2, TNF and the IFNs — old players that one would have thought we understood very well. thatpermitstheprotectiveeffectsof thiscytokinetooccur without the risk of associated pathological responses. Once again,invoking the notion of timing and duration of cytokine exposure in the pathogenesis of autoimmune disease, one can imagine scenarios in which low-level exposure to IFN-γ or other cytokines might attenuate subsequent responsiveness to these cytokines.Therefore, the net effect would be inhibitory. Additionally, it has been noted that the variability of the effects of IFN-γ depend on the model used.In models in which complete Freund’s adjuvant (CFA) is used to induce disease,it has been proposed that IFN-γ can have protective effects by inhibitingCFA-inducedproliferationof myeloidcells.40 Conclusions Although thymic selection and the deletion of autoreac- tive clones (CENTRAL TOLERANCE) has been put forth as a principal regulator of autoimmunity,we now know that central tolerance is incomplete and that some degree of autoreactivity is normal. Conversely, there are many recent examples in which loss of any one of an array of inhibitory molecules results in autoimmune disease.For many of these inhibitors,their function in the periphery is of greater significance than their thymic function, which emphasizes the importance of peripheral toler- ance. Experiments with knockout mice indicate that many of these mechanisms are remarkably non-redun- dant because, despite the apparently vast number of inhibitory pathways, the absence of even one of these regulators results in inflammatory and autoimmune disease.Although it is conceivable that species differ- ences or the use of inbred mice might overestimate the propensity toward autoimmunity, it is nevertheless a CENTRAL TOLERANCE This form of tolerance refers to the lack of self-responsiveness found as lymphoid cells develop, and is associated with the deletion of autoreactive clones. For T cells, this occurs in the thymus. 1. Davidson, A. & Diamond, B. Autoimmune diseases. N. Engl. J. Med. 345, 340–350 (2001). 2. Marrack, P., Kappler, J. & Kotzin, B. L. Autoimmune disease: why and where it occurs. Nature Med. 7, 899–905 (2001). 3. Falcone, M. & Sarvetnick, N. Cytokines that regulate autoimmune responses. Curr. Opin. Immunol. 11, 670–676 (1999). 4. Ioannou, Y. & Isenberg, D. A. 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