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Th2 differentiation
Molecular Immunology 39 (2002) 531–536
Review
The two faces of IL-6 on Th1/Th2 differentiation Sean Diehl, Mercedes Rincón∗ Immunobiology Program, Department of Medicine, University of Vermont, Given Medical Building D305, Burlington, VT 05405, USA Received 31 January 2002; accepted 2 February 2002
Abstract Interleukin (IL)-6 is a cytokine produced by several cell types including antigen presenting cells (APC) such as macrophages, dendritic cells, and B cells. IL-6 is involved in the acute phase response, B cell maturation, and macrophage differentiation. Here, we discuss a novel function of IL-6: the control of T helper (Th) 1/Th2 differentiation. IL-6 promotes Th2 differentiation and simultaneously inhibits Th1 polarization through two independent molecular mechanisms. IL-6 activates transcription mediated by nuclear factor of activated T cells (NFAT) leading to production of IL-4 by na¨ıve CD4+ T cells and their differentiation into effector Th2 cells. While the induction of Th2 differentiation by IL-6 is dependent upon endogenous IL-4, inhibition of Th1 differentiation by IL-6 is IL-4- and NFAT-independent. IL-6 inhibits Th1 differentiation by upregulating supressor of cytokine signaling (SOCS)-1 expression to interfere with IFN␥ signaling and the development of Th1 cells. Since IL-6 is abundantly produced by APC, it is a likely source of early Th1/Th2 control during CD4+ T cell activation. Thus, by using two independent molecular mechanisms, IL-6 plays a dual role in Th1/Th2 differentiation. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Th1/Th2 differentiation; Interleukin; Cytokine signaling
1. Introduction Activation of na¨ıve CD4+ helper T (Th) cells through the TcR causes these cells to proliferate and differentiate into effector T helper cells. Two major subsets of effector Th cells have been defined on the basis of their distinct cytokine secretion patterns and their immunomodulatory effects. Th1 cells produce primarily interferon-gamma (IFN␥) and tumor necrosis factor  (TNF), which are required for cell-mediated inflammatory reactions; Th2 cells secrete interleukin (IL)-4, IL-5, IL-10 and IL-13, which mediate B cell activation and antibody production (for review, see Swain, 1995; O’Garra, 1998; Murphy et al., 2000). In general, an efficient clearance of intracellular pathogens is based on innate cell activation, while antibody responses are best suited for extracellular infections. The decision of na¨ıve CD4+ T cells to become Th1 and Th2 has important consequences in the success of an immune response and the progression of diseases. A Th1 response against Leishmania major results in the resolution of disease, while a Th2-type response allows the progression of disease. In contrast, a predominant Th1 response has been observed in several autoimmune diseases, such as rheumatoid arthritis,
∗
Corresponding author. Tel.: +1-802-656-0937; fax: +1-802-656-3854. E-mail address: [email protected] (M. Rinc´on).
experimental autoimmune encephalomyelitis (EAE) and insulin-dependent diabetes mellitus. The selective differentiation of precursor CD4+ T cells into effector Th1 and Th2 cells is established during the initial priming of these cells and is influenced by a variety of extracellular factors, such as the cytokine environment, the dose of antigen and the source of costimulation (Constant and Bottomly, 1997). Among these, the most effective polarizing factor is the cytokine environment. The presence of IL-4 during activation drives the differentiation of precursor CD4+ T cells into Th2 cells, whereas the presence of IL-12, IL-18 and IFN␥ promotes differentiation into Th1 cells (Hsieh et al., 1993; Le Gros et al., 1990; Nakanishi et al., 2001; Seder et al., 1993; Swain et al., 1990). While IL-12 is secreted by professional antigen presenting cells (APCs), IL-4 is not. IL-6 is a cytokine produced by a number of cell types including fibroblasts, macrophages, dendritic cells, T and B lymphocytes, endothelial cells, glial cells and keratinocytes in response to a variety of external stimuli (e.g. IL-1, TNF, and PDGF). IL-6 induces the synthesis of acute phase response proteins in hepatocytes, terminal differentiation of B cells to antibody producing plasma cells, differentiation of monocytes to macrophages, and growth of hematopoetic stem cells (for review see Hirano, 1998). IL-6 binds to the surface IL-6 receptor (IL-6R)␣, leading to the dimerization of gp130/IL-6R (Taga et al., 1989). Dimerization of gp130
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by IL-6 causes the activation of two signaling pathways: (1) the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway; and (2) the CCAAT/enhancer binding protein (C/EBP) pathway. Activation of the extracellular signal-regulated kinase (ERK) pathway by IL-6 appears to mediate phosphorylation and activation of C/EBP (NF-IL6; Akira et al., 1990; Daeipour et al., 1993; Poli et al., 1990). IL-6 also induces the expression of C/EBP␦ (NF-IL6), another member of the C/EBP family of transcription factors (Ramji et al., 1993). C/EBP␦ together with C/EBP regulate Type-1 IL-6 responsive genes (Kinoshita et al., 1992; Poli, 1998). Activation of Jak1, 2, and Tyk2 by IL-6 results in the phosphorylation and activation of STAT3 and, to a much lesser extent, STAT1, leading to the induction of Type-2 IL-6 responsive gene expression (Akira et al., 1994; Lutticken et al., 1994; Nakajima et al., 1995; Stahl et al., 1994). The production of IL-6 by both lymphoid and nonlymphoid cells makes this cytokine relevant for different aspects of the immune response. Here, we describe the regulatory role of IL-6 on the decision of CD4+ T cells to become Th1 or Th2 effector cells.
2. IL-6 promotes Th2 differentiation by inducing the expression of IL-4 gene during activation of CD4+ T cells To test a potential involvement of IL-6 in Th1/Th2 differentiation we examined the effect of IL-6 on IL-4-induced Th2 differentiation or IL-12-induced Th1 differentiation. Interestingly, even in the absence of any polarizing cytokine, IL-6 directed the differentiation of the CD4+ cells to a Th2 phenotype, since the cells differentiated in the presence of IL-6 produce high amounts of IL-4, but not IFN␥, after restimulation (Rincón et al., 1997). IL-6 did not modify the differentiation of the Th2 cells directed by IL-4. IL-6, however, impaired Th1 differentiation (see Section 3). IL-6, like IL-2, has been described to be a growth factor for a number of cells. However, only IL-6 but not IL-2 was able to modify the polarization of the CD4+ T cells to a Th2 phenotype, indicating that IL-6 is involved in differentiation rather than growth of T cells. We also analyzed the role of IL-6 in the differentiation of purified na¨ıve CD4+ T cells isolated from T cell receptor (TcR) transgenic mice, which express the ␣ and  chain of the TcR that recognizes a pigeon cytochrome c (Cyt c) peptide (Kaye et al., 1989). IL-6 could promote the antigen-specific differentiation of the na¨ıve CD4+ cells to IL-4 producing cells (Rincón et al., 1997). We observed that during the activation of CD4+ T cells in the presence of APC, high levels of IL-6 were secreted by APC (Rincón and Flavell, 1997). B cells appears to be the primary source of IL-6 in the cultures, but macrophages also have an important contribution (Rincón et al. unpublished data). To determine whether physiological levels of IL-6 secreted by APC were relevant for Th2 cell differentiation
we examined the function of APC from IL-6−/− mice. We first purified CD4+ T cells from WT mice and stimulated them for 4 days with ConA in the presence of APC from WT mice or IL-6−/− mice in the absence of any added exogenous cytokines. Restimulation with ConA resulted in significantly less IL-4 production in cells differentiated in the presence of IL-6−/− than in WT APC. We also examined the in vitro differentiation of CD4+ T cells from IL-6 deficient mice. IL-6−/− CD4+ T cells stimulated with ConA in the presence of WT APC, which can provide the IL-6 required, produced IL-4, but this IL-4 production was impaired when the IL-6 pathway was blocked by the addition of anti-IL-6 mAb (Rincón et al., 1997). Moreover, high IL-4 production was restored in cells that were differentiated in the presence of IL-6−/− APC together with an exogenous source of IL-6. Thus, IL-6 production by APC during activation of CD4+ T cells plays a major role in determining the type of cytokines that effector CD4+ T cells will secrete. To further prove that the effect of IL-6 was on T cells directly, rather than APC, we differentiated CD4+ T cells with immobilized anti-CD3 mAb plus anti-CD28 mAb in the complete absence of APC, and then restimulated these cells with anti-CD3 mAb alone. The presence of IL-6 (or IL-4 as a control) during the first culture resulted in increased IL-4 production and reduced IFN␥ production by the cells that were elicited. Thus, IL-6 directly favors the polarization of na¨ıve CD4+ T cells to Th2 cells (Diehl et al., 2002). IL-4 is a critical differentiation factor for Th2 cells, which acts by promoting the secretion of more IL-4 by T cells (Le Gros et al., 1990; Swain et al., 1990). It was therefore possible that IL-6 could directly upregulate the synthesis of IL-4 by T cells and, consequently that the IL-6 effect on the differentiation of Th2 cells was mediated through IL-4. To address this hypothesis, CD4+ T cells were differentiated with IL-6 in the presence or absence of neutralizing anti-IL-4 mAb, and after 4 days the cells were washed and restimulated. The ability of IL-6 to polarize CD4+ T cells towards the Th2 phenotype was blocked by anti-IL-4 mAb, since the cells were unable to produce IL-4 upon restimulation. These results indicated that the differentiation of Th2 cells by IL-6 is dependent on the endogenous production of IL-4 by CD4+ T cells. It was therefore possible that IL-6 could trigger the initial IL-4 production by CD4+ T cells during activation and this early IL-4 could promote further Th2 differentiation. To address this hypothesis we examined the effect of IL-6 on IL-4 production during the activation of CD4+ T cells. Increased levels of IL-4 were produced by CD4+ T cells activated with anti-CD3 and anti-CD28 in the presence of IL-6 compared with those in the absence of IL-6. Moreover, increased levels of IL-4 mRNA were detected in CD4+ T cells stimulated in the presence of IL-6 (Diehl et al., 2002). To show that early IL-4 production induced by IL-6 was not dependent on endogenous IL-4, we examined the regulation of IL-4 gene expression in the presence of a blocking anti-IL-4 mAb. Upregulation of IL-4 mRNA levels by IL-6 was also observed
S. Diehl, M. Rinc´on / Molecular Immunology 39 (2002) 531–536
Fig. 1. IL-6 promotes Th2 differentiation by activation of NFAT and induction of early IL-4 gene expression.
in the presence of the anti-IL-4 mAb early during activation (Diehl et al., 2002). Thus, IL-6 upregulates IL-4 gene and protein expression during the initial stages of differentiation. IL-4 gene transcription is regulated by several transcription factors (e.g. nuclear factor of activated T cells (NFAT), GATA-3, and c-maf; Murphy et al., 2000). We found that IL-6 increased the levels of GATA-3 in CD4+ T cells. However, this increased on GATA-3 expression was dependent of endogenous IL-4, indicating that this effect of IL-6 was an indirect effect caused by the upregulation of IL-4 production. In contrast, IL-6 activated NFAT transcriptional activity independently of endogenous IL-4 (Diehl et al., 2002). Activation of NFAT by IL-6 occurs through upregulation of NFATc2 expression (Diehl et al., 2002). Even more, IL-4 failed to upregulate NFAT activity. Thus, NFAT is regulated by specific signals delivered by IL-6. We generated transgenic mice that express a mutant form of NFAT that acts as a dominant negative mutant inhibiting transcription by all four members of the NFAT family (Chow et al., 1999). We examined the ability of IL-6 to promote Th2 differentiation in CD4+ T cells from the dnNFAT transgenic mice. Inhibition of NFAT completely abrogated the induction of IL-4 gene expression and Th2 differentiation by IL-6 (Diehl et al., 2002). Thus, IL-6 promotes Th2 differentiation by activation of NFAT and induction of early IL-4 gene expression (Fig. 1). We have also shown that IL-6 inhibits IFN␥ production and Th1 differentiation (see Section 3). Interestingly, the presence of the dnNFAT did not prevent the inhibitory effect of IL-6 on IFN␥ gene expression. 3. IL-6 inhibits Th1 differentiation by inducing the expression of SOCS-1 and IFN␥ during the activation of CD4+ T cells We have previously observed that the presence of IL-6 during the differentiation of CD4+ T cells with ConA, APC
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and IL-12 caused a dramatic inhibition of IFN␥ production after restimulation (Rincón et al., 1997). IL-6 also inhibited Th1 differentiation induced with anti-CD3 and IL-12 (Diehl et al., 2002). We also tested whether IL-6 could prevent the differentiation of na¨ıve CD4+ T cells into effector Th1 cells. CD4+ T cells from cytochrome c TcR transgenic mice were unable to differentiate into high-IFN␥ producing Th1 effector cells when IL-6 was present (Diehl et al., 2000). Moreover, we have examined the role of IL-6 in vivo Th1 differentiation. Borrelia burgdoferi infection induces a Th1 response in C3H/HeN mice, resulting in Lyme arthritis (Anguita et al., 1996; Keane-Myers and Nickell, 1995; Matyniak and Reiner, 1995). IL-b-defficient mice exhibit increased incidence of Lyme arthritis (Anguita et al., 1997). We have also shown that IL-6 is able to inhibit in vivo Th1 differentiation since administration of IL-6 in vivo during infection with B. burgderfori prevents the generation of antigen-specific Th1 effector CD4+ T cells (Diehl et al., 2000). Together, these results indicated that IL-6, in addition to promoting Th2 polarization, was able to inhibit Th1 differentiation. IL-4 has been reported to have a negative effect on Th1 differentiation in vitro (Seder et al., 1992). Given the fact that IL-6 can induce IL-4 production, we tested whether the effect of IL-6 was simply dependent on IL-4. We showed that IL-6 was still able to inhibit Th1 differentiation even in the presence of a neutralizing anti-IL-4 mAb, suggesting that IL-6 was able to inhibit Th1 differentiation in an IL-4-independent manner (Diehl et al., 2000). The presence of IL-12 during the activation of CD4+ T cells highly enhances IFN␥ production and promotes Th1 differentiation (Hsieh et al., 1993; Seder et al., 1993). However, it has also been shown that effector CD4+ T cells differentiated in the absence of IL-12 produced significant amounts of IFN␥ (Bradley et al., 1996). We also observed that stimulation with anti-CD3 and anti-CD28 mAbs in the absence of IL-12 gave rise to effector cells that produced significant levels of IFN␥ upon restimulation. Interestingly, CD4+ T cells differentiated with anti-CD3 and anti-CD28 mAbs in the presence of IL-6 produced low levels of IFN␥ upon restimulation compared with those differentiated in the absence of IL-6. Thus, IL-6 could inhibit Th1 differentiation independently of IL-12. In the absence of IL-12 endogenous IFN␥ is the major factor to promote Th1 differentiation. During their activation, na¨ıve CD4+ T cells produce significant levels of IFN␥. It was therefore possible that IL-6 inhibited IFN␥ production by CD4+ T cells during activation and, therefore, prevented the generation of Th1 effector cells. We found that IL-6 potently suppressed IFN␥ production by CD4+ T cells early during their activation. Analysis of cytokine mRNA levels during this period revealed that IL-6 also inhibited IFN␥ gene expression (Diehl et al., 2000). Furthermore, the presence of an anti-IL-4 mAb did not affect the decrease in IFN␥ gene expression caused by IL-6. Together these results demonstrated that IL-6 could inhibit IFN␥ gene
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expression during the activation of CD4+ T cells independently of IL-4. However, even in the presence of exogenous IFN␥, IL-6 was still able to inhibit Th1 differentiation, suggesting that IL-6 could interfere with the IFN␥ receptor (IFN␥R) signals that lead to an autoregulation of IFN␥ gene expression. We therefore analyzed the effect of IL-6 on Th1 differentiation in IFN␥R−/− CD4+ T cells. IL-6 suppressed the differentiation of wild type CD4+ T cells into effector Th1 cells that, while IL-6 did not affect the levels of IFN␥ produced by IFN␥R−/− Th1 cells (Diehl et al., 2000). Together, these results indicated that IL-6 inhibited signaling through the IFN␥R, which is critical for complete Th1 polarization. Signaling through the IFN␥R (as for many other cytokine receptors) utilizes the Jak/STAT pathway. Currently, there are four mammalian JAKs (Jak1, Jak2, Jak3 and Tyk2) and seven STATs (Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b and Stat6; for review see (Leonard and O’Shea, 1998). Binding of IFN␥ to its receptor induces receptor dimerization and activation of Jak1 and Jak2 that recruit and phosphorylate STAT1 (Bach et al., 1997). To determine whether IL-6 targeted the Jak/STAT1 pathway triggered by IFN␥ we analyzed STAT1 tyrosine phosphorylation. IFN␥-induced Tyr phosphorylation of STAT1 in cells cultured in the absence of IL-6, but not in cells that were activated in the presence of IL-6 (Diehl et al., 2000). We explored the possibility that IL-6 could affect negative regulators of cytokine signaling to interfere with IFN␥ signaling. The suppressors of cytokine signaling (SOCS; also known as STAT-induced STAT inhibitors (SSIs)) proteins are negative regulators of cytokine signaling (reviewed by Alexander et al., 1999b; Naka et al., 1999). There are eight members of the SOCS family (SOCS1–SOCS7 and CIS; cytokine inducible SH2 containing protein) and they regulate signaling through cytokine receptors and some tyrosine kinase growth factor receptors such as insulin-like growth factor-1. SOCS1 was initially found to be induced by IL-6 in the M1 monocytic leukemia cell line (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997). A critical role for SOCS1 in IFN␥ signaling in vivo was highlighted through the generation of SOCS1-deficient mice (SOCS1−/− ). SOCS1−/− mice die perinatally due to IFN␥ overproduction and IFN␥ hypersensitivity that result on severe liver damage (Alexander et al., 1999a; Bullen et al., 2001; Marine et al., 1999; Naka et al., 1998; Starr et al., 1998). We therefore examined whether IL-6 was able to induce SOCS1 expression to inhibit IFN␥ signaling in CD4+ T cells. We have shown that CD4+ T cells activated in the presence of IL-6 contain higher levels of SOCS1 mRNA. Thus, IL-6 was able to upregulate the expression of SOCS1 in CD4+ T cells. To determine whether SOCS1 was required for IL-6 mediated Th1 inhibition, we analyzed the effect of IL-6 on IFN␥ production in CD4+ T cells from SOCS1−/− mice. While differentiation of wild type CD4+ T cells into Th1 effector cells was effectively inhibited by IL-6, differentiation of SOCS1−/− CD4+ T cells into Th1 cells was not affected by
Fig. 2. Requirement of SOCS1 for IL-6 to inhibit IFN␥ signaling and Th1 differentiation.
IL-6 (Diehl et al., 2000). Furthermore, we have demonstrated that IL-6 did not inhibit IFN␥-induced STAT1 phosphorylation in cells lacking SOCS1. SOCS1 is therefore required for IL-6 to inhibit IFN␥ signaling and Th1 differentiation (Fig. 2). SOCS1, however, was not required for IL-6 to promote Th2 differentiation (Diehl et al., 2000), indicating that this cytokine uses two independent signaling pathways to promote Th2 differentiation and inhibit Th1 differentiation.
4. Conclusive remarks and future directions Our studies clearly show that IL-6 produced by APCs can modulate the differentiation of CD4+ T cells into effector Th1 or Th2. The presence of IL-6 shifted the Th1/Th2 balance toward the Th2 direction using two independent approaches: (1) promoting IL-4 production and Th2 differentiation; and (2) inhibiting IFN␥ production and Th1 differentiation (Fig. 3). While differentiation of Th2 by IL-6 is dependent on endogenous production of IL-4, inhibition of Th1 differentiation by IL-6 is not. IL-6 inhibits Th1 differentiation by interfering with IFN␥ signaling through upregulation of SOCS1. However, IL-6 promotes Th2 differentiation by activating NFAT, which induces endogenous IL-4 gene expression. Activation of NFAT is not required for inhibition of IFN␥ gene expression and Th1 differentiation by IL-6. In the absence of SOCS1, IL-6 fails to inhibit Th1 differentiation, but it retains its ability to drive Th2 differentiation. Thus, this is an example of how a cytokine uses two different signaling pathways to regulate two independent functions. In addition to APC IL-6 is also produced by other non-lymphoid cells including astrocytes, fibroblasts and tumor cells. Future studies will be carried out to examine the contribution of IL-6 produced by these cells on the differentiation of CD4+ T cells into Th1 and Th2. It is possible that the production of IL-6 by tumor cells may prevent the development of an anti-tumor Th1 response, or the production of IL-6 by the lung environment during asthma may be a determinant factor for the development of
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Fig. 3. IL-b shifts the Th1/Th2 balance towards Th2 by inhibiting Th1 and promoting Th2.
Th2 responses. Moreover, further experimental approaches need to be developed to examine the contribution of IL-6 derived from any of these source in vivo Th1/Th2 balance (e.g. IL-6 conditional knock outs). The pleiotropic character of IL-6 has made difficult to obtain a clear answer for the role of this cytokine in vivo models
Acknowledgements Support for the studies performed in this laboratory was received by the National Institute of Health (PO1 AI45666), the COBRE Program of the National Center for Research Resources (P20 RR15557), the Arthritis Foundation, and the Gustavus and Louise Pfeiffer Foundation. S.D. is supported by an Environmental Pathology Training Grant from the National Institute of Environmental Health Science (T32ES07122).
References Akira, S., Issihiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., Kishimoto, T., 1990. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9, 1897–1906. Akira, S., Nishio, Y., Inoue, M., Wang, X.J., Wei, S., Matsusaka, T., Yoshida, K., Sudo, T., Naruto, M., Kishimoto, T., 1994. Molecular cloning of APRF a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 77, 63–71. Alexander, W.S., Starr, R., Fenner, J.E., Scott, C.L., Handman, E., Sprigg, N.S., Corbin, J.E., Cornish, A.L., Darwiche, R., Owczarek, C.M., Kay, T.W., Nicola, N.A., Hertzog, P.J., Metcalf, D., Hilton, D.J., 1999a. SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98, 597–608. Alexander, W.S., Starr, R., Metcalf, D., Nicholson, S.E., Farley, A., Elefanty, A.G., Brysha, M., Kile, B.T., Richardson, R., Baca, M., Zhang, J.G., Willson, T.A., Viney, E.M., Sprigg, N.S., Rakar, S., Corbin, J., Mifsud, S., DiRago, L., Cary, D., Nicola, N.A., Hilton, D.J., 1999b. Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction. J. Leukoc. Biol. 66, 588–592. Anguita, J., Persing, D., Rincón, M., Barthold, S.W., Fikrig, E., 1996. Effect of anti-interleukin 12 treatment on murine Lyme borreliosis. J. Clinic. Invest. 97, 1028–1034.
Anguita, J., Rincón, M., Samanta, S., Barthold, S.W., Flavell, R.A., Fikrig, E., 1997. Borrelia burgdorferi-infected, interleukin-6-deficient mice have decreased Th2 responses and increased Lyme arthritis. J. Infect. Dis. 178, 1512–1515. Bach, E.A., Aguet, M., Schreiber, R.D., 1997. The IFN␥ receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15, 563–591. Bradley, L.M., Dalton, D.K., Croft, M., 1996. A direct role for IFN␥ in regulation of Th1 cell development. J. Immunol. 157, 1350–1358. Bullen, D.V., Darwiche, R., Metcalf, D., Handman, E., Alexander, W.S., 2001. Neutralization of interferon-gamma in neonatal SOCS1−/− mice prevents fatty degeneration of the liver but not subsequent fatal inflammatory disease. Immunology 104, 92–98. Chow, C.-W., Rincón, M., Davis, R.J., 1999. Requirement for transcription factor NFAT in interleukin-2 expression. Mol. Cell. Biol. 19, 2300– 2307. Constant, S.L., Bottomly, K., 1997. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15, 297–322. Daeipour, M., Kumar, G., Amaral, M.C., Nel, A.E., 1993. Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line, AF-10. J. Immunol. 150, 4743–4753. Diehl, S., Anguita, J., Hoffmeyer, A., Zapton, T., Ihle, J.N., Fikrig, E., Rincón, M., 2000. Inhibition of Th1 differentiation by IL-6 is mediated by SOCS1. Immunity 13, 805–815. Diehl, S., Chow, C.W., Weiss, L., Palmetshofer, A., Twardzik, T., Rounds, L., Serfling, E., Davis, R.J., Anguita, J., Rincon, M., 2002. Induction of NFATc2 expression by interleukin 6 promotes T helper type 2 differentiation. J. Exp. Med. 196, 39–49. Endo, T.A., Masura, M., Yokouchi, M., Suzuki, R., Sakamoto, H., Mitsui, K., Matsumoto, A., Tanimura, S., Ohtsubo, M., Misawa, H., Miyazaki, T., Leonor, N., Taniguchi, T., Fujita, T., Kanakura, Y., Komiya, S., Yoshimura, A., 1997. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387, 921–924. Hirano, T., 1998. Interleukin 6 and its receptor: 10 years later. Int. Rev. Immunol. 16, 249–284. Hsieh, C.S., Macatonia, S.E., Tripp, C.S., Wolf, S.F., O’Garra, A., Murphy, K.M., 1993. Development of Th1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260, 547–549. Kaye, J., Hsu, M.-L., Sauron, M.-E., Jameson, S.C., Gascoigne, N.R.J., Hedrick, S.M., 1989. Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor. Nature 341, 746–749. Keane-Myers, A., Nickell, S.P., 1995. Role of IL-4 and IFN␥ in modulation of immunity to Borrelia burgdorferi in mice. J. Immunol. 155, 2020– 2028. Kinoshita, S., Akira, S., Kishimoto, T., 1992. A member of the C/EBP family, NF-IL6, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc. Natl. Acad. Sci. U.S.A. 89, 1473–1476. Le Gros, G., Ben-Sasson, S.Z., Seder, R., Finkelman, F.D., Paul, W.E., 1990. Generation of interleukin 4 (IL-4)-producing cells in vivo and
536
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in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4 producing cells. J. Exp. Med. 172, 921–929. Leonard, W.J., O’Shea, J.J., 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16, 293–322. Lutticken, C., Wegenka, U.M., Yuan, J., Buschmann, J., Schindler, C., Ziemiecki, A., Harpur, A.G., Wilks, A.F., Yasukawa, K., Taga, T., Kismimoto, T., Barbieri, G., Pellegrini, S., Sendtner, M., Heinrich, P.C., Horn, F., 1994. Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science 263, 89–92. Marine, J.C., Topham, D.J., McKay, C., Wang, D., Parganas, E., Stravopodis, D., Yoshimura, A., Ihle, J.N., 1999. SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell 98, 609–616. Matyniak, J.E., Reiner, S.L., 1995. T helper phenotype and genetic susceptibility in experimental Lyme disease. J. Exp. Med. 181, 1251– 1254. Murphy, K.M., Ouyang, W., Farrar, J.D., Yang, J., Ranganath, S., Asnagli, H., Afkarian, M., Murphy, T.L., 2000. Signaling and transcription in T helper development. Annu. Rev. Immunol. 18, 451–594. Naka, T., Fujimoto, M., Kishimoto, T., 1999. Negative regulation of cytokine signaling: STAT-induced STAT inhibitor. Trends Biochem. Sci. 24, 394–398. Naka, T., Matsumoto, T., Narazaki, M., Fujimoto, M., Morita, Y., Ohsawa, Y., Saito, H., Nagasawa, T., Uchiyama, Y., Kishimoto, T., 1998. Accelerated apoptosis of lymphocytes by augmented induction of Bax in SSI-1 (STAT-induced STAT inhibitor-1) deficient mice. Proc. Natl. Acad. Sci. U.S.A. 95, 15577–15582. Naka, T., Narazaki, M., Hirata, M., Matsumoto, T., Minamoto, S., Aono, A., Nishimoto, N., Kajita, T., Taga, T., Yoshizaki, K., Akira, S., Kishimoto, T., 1997. Structure and function of a new STAT-induced STAT inhibitor. Nature 387, 924–929. Nakajima, K., Matsuda, T., Fujitani, Y., Kojima, H., Yamanaka, Y., Nakae, K., Takeda, T., Hirano, T., 1995. Signal transduction through IL-6 receptor: involvement of multiple protein kinases, stat factors, and a novel H7-sensitive pathway. Ann N.Y. Acad. Sci. 762, 55–70. Nakanishi, K., Yoshimoto, T., Tsutsui, H., Okamura, H., 2001. Interleukin-18 regulates both Th1 and Th2 responses. Annu. Rev. Immunol. 19, 423–474. O’Garra, A., 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8, 275–283. Poli, V., 1998. The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J. Biol. Chem. 273, 29279–29282.
Poli, V., Mancini, F.P., Cortese, R., 1990. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell 63, 643–653. Ramji, D.P., Vitelli, A., Tronche, F., Cortese, R., Ciliberto, G., 1993. The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6, are induced by IL-6 to promote acute phase gene transcription via different mechanisms. Nucleic Acids Res. 21, 289–294. Rincón, M., Anguita, J., Nakamura, T., Fikrig, E., Flavell, R.A., 1997. IL-6 directs the differentiation of IL-4-producing CD4+ T cells. J. Exp. Med. 185, 461–469. Rincón, M., Flavell, R.A., 1997. Transcriptional control of Th1/Th2 polarization. Curr. Biol. 7, R729–R732. Seder, R.A., Paul, W.E., Davis, M.M., 1992. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176, 1091–1098. Seder, R.A., Gazzinelli, R., Sher, A., Paul, W.E., 1993. IL-12 acts directly on CD4+ T cells to enhance priming for IFN␥ production and diminish IL-4 inhibition of such priming. Proc. Natl. Acad. Sci. U.S.A. 90, 10188–10192. Stahl, N., Boulton, T.G., Farruggella, T., Ip, N.Y., Davis, S., Witthuhn, B.A., Quelle, F.W., Silvennoinen, O., Barbieri, G., Pellegrini, S., Ihle, J.N., Yancopoulos, G.D., 1994. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 receptor components. Science 263, 92–95. Starr, R., Metcalf, D., Elefanty, A.G., Brysha, M., Willson, T.A., Nicola, N.A., Hilton, D.J., Alexander, W.S., 1998. Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1. Proc. Natl. Acad. Sci. U.S.A. 95, 14395– 14399. Starr, R., Willson, T.A., Viney, E., Murray, L.J.L., Rayner, J.R., Jenkins, B.J., Gonda, T.J., Alexander, W.S., Metcalf, D., Nicola, N.A., Hilton, D.J., 1997. A family of cytokine-inducible inhibitors of signaling. Nature 387, 917–921. Swain, S.L., Weinberg, A.D., English, M., Huston, G., 1990. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145, 3796– 3806. Swain, S.L., 1995. Who does the polarizing? Curr. Biol. 5, 849–851. Taga, T., Hibi, M., Hirata, Y., Yamasaki, K., Yasukawa, K., Matsuda, T., Hirano, T., Kishimoto, T., 1989. Interleukin-6-triggers the association of its receptor with a possible signal transducer, gp130. Cell 58, 573– 581.
Review
The two faces of IL-6 on Th1/Th2 differentiation Sean Diehl, Mercedes Rincón∗ Immunobiology Program, Department of Medicine, University of Vermont, Given Medical Building D305, Burlington, VT 05405, USA Received 31 January 2002; accepted 2 February 2002
Abstract Interleukin (IL)-6 is a cytokine produced by several cell types including antigen presenting cells (APC) such as macrophages, dendritic cells, and B cells. IL-6 is involved in the acute phase response, B cell maturation, and macrophage differentiation. Here, we discuss a novel function of IL-6: the control of T helper (Th) 1/Th2 differentiation. IL-6 promotes Th2 differentiation and simultaneously inhibits Th1 polarization through two independent molecular mechanisms. IL-6 activates transcription mediated by nuclear factor of activated T cells (NFAT) leading to production of IL-4 by na¨ıve CD4+ T cells and their differentiation into effector Th2 cells. While the induction of Th2 differentiation by IL-6 is dependent upon endogenous IL-4, inhibition of Th1 differentiation by IL-6 is IL-4- and NFAT-independent. IL-6 inhibits Th1 differentiation by upregulating supressor of cytokine signaling (SOCS)-1 expression to interfere with IFN␥ signaling and the development of Th1 cells. Since IL-6 is abundantly produced by APC, it is a likely source of early Th1/Th2 control during CD4+ T cell activation. Thus, by using two independent molecular mechanisms, IL-6 plays a dual role in Th1/Th2 differentiation. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Th1/Th2 differentiation; Interleukin; Cytokine signaling
1. Introduction Activation of na¨ıve CD4+ helper T (Th) cells through the TcR causes these cells to proliferate and differentiate into effector T helper cells. Two major subsets of effector Th cells have been defined on the basis of their distinct cytokine secretion patterns and their immunomodulatory effects. Th1 cells produce primarily interferon-gamma (IFN␥) and tumor necrosis factor  (TNF), which are required for cell-mediated inflammatory reactions; Th2 cells secrete interleukin (IL)-4, IL-5, IL-10 and IL-13, which mediate B cell activation and antibody production (for review, see Swain, 1995; O’Garra, 1998; Murphy et al., 2000). In general, an efficient clearance of intracellular pathogens is based on innate cell activation, while antibody responses are best suited for extracellular infections. The decision of na¨ıve CD4+ T cells to become Th1 and Th2 has important consequences in the success of an immune response and the progression of diseases. A Th1 response against Leishmania major results in the resolution of disease, while a Th2-type response allows the progression of disease. In contrast, a predominant Th1 response has been observed in several autoimmune diseases, such as rheumatoid arthritis,
∗
Corresponding author. Tel.: +1-802-656-0937; fax: +1-802-656-3854. E-mail address: [email protected] (M. Rinc´on).
experimental autoimmune encephalomyelitis (EAE) and insulin-dependent diabetes mellitus. The selective differentiation of precursor CD4+ T cells into effector Th1 and Th2 cells is established during the initial priming of these cells and is influenced by a variety of extracellular factors, such as the cytokine environment, the dose of antigen and the source of costimulation (Constant and Bottomly, 1997). Among these, the most effective polarizing factor is the cytokine environment. The presence of IL-4 during activation drives the differentiation of precursor CD4+ T cells into Th2 cells, whereas the presence of IL-12, IL-18 and IFN␥ promotes differentiation into Th1 cells (Hsieh et al., 1993; Le Gros et al., 1990; Nakanishi et al., 2001; Seder et al., 1993; Swain et al., 1990). While IL-12 is secreted by professional antigen presenting cells (APCs), IL-4 is not. IL-6 is a cytokine produced by a number of cell types including fibroblasts, macrophages, dendritic cells, T and B lymphocytes, endothelial cells, glial cells and keratinocytes in response to a variety of external stimuli (e.g. IL-1, TNF, and PDGF). IL-6 induces the synthesis of acute phase response proteins in hepatocytes, terminal differentiation of B cells to antibody producing plasma cells, differentiation of monocytes to macrophages, and growth of hematopoetic stem cells (for review see Hirano, 1998). IL-6 binds to the surface IL-6 receptor (IL-6R)␣, leading to the dimerization of gp130/IL-6R (Taga et al., 1989). Dimerization of gp130
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by IL-6 causes the activation of two signaling pathways: (1) the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway; and (2) the CCAAT/enhancer binding protein (C/EBP) pathway. Activation of the extracellular signal-regulated kinase (ERK) pathway by IL-6 appears to mediate phosphorylation and activation of C/EBP (NF-IL6; Akira et al., 1990; Daeipour et al., 1993; Poli et al., 1990). IL-6 also induces the expression of C/EBP␦ (NF-IL6), another member of the C/EBP family of transcription factors (Ramji et al., 1993). C/EBP␦ together with C/EBP regulate Type-1 IL-6 responsive genes (Kinoshita et al., 1992; Poli, 1998). Activation of Jak1, 2, and Tyk2 by IL-6 results in the phosphorylation and activation of STAT3 and, to a much lesser extent, STAT1, leading to the induction of Type-2 IL-6 responsive gene expression (Akira et al., 1994; Lutticken et al., 1994; Nakajima et al., 1995; Stahl et al., 1994). The production of IL-6 by both lymphoid and nonlymphoid cells makes this cytokine relevant for different aspects of the immune response. Here, we describe the regulatory role of IL-6 on the decision of CD4+ T cells to become Th1 or Th2 effector cells.
2. IL-6 promotes Th2 differentiation by inducing the expression of IL-4 gene during activation of CD4+ T cells To test a potential involvement of IL-6 in Th1/Th2 differentiation we examined the effect of IL-6 on IL-4-induced Th2 differentiation or IL-12-induced Th1 differentiation. Interestingly, even in the absence of any polarizing cytokine, IL-6 directed the differentiation of the CD4+ cells to a Th2 phenotype, since the cells differentiated in the presence of IL-6 produce high amounts of IL-4, but not IFN␥, after restimulation (Rincón et al., 1997). IL-6 did not modify the differentiation of the Th2 cells directed by IL-4. IL-6, however, impaired Th1 differentiation (see Section 3). IL-6, like IL-2, has been described to be a growth factor for a number of cells. However, only IL-6 but not IL-2 was able to modify the polarization of the CD4+ T cells to a Th2 phenotype, indicating that IL-6 is involved in differentiation rather than growth of T cells. We also analyzed the role of IL-6 in the differentiation of purified na¨ıve CD4+ T cells isolated from T cell receptor (TcR) transgenic mice, which express the ␣ and  chain of the TcR that recognizes a pigeon cytochrome c (Cyt c) peptide (Kaye et al., 1989). IL-6 could promote the antigen-specific differentiation of the na¨ıve CD4+ cells to IL-4 producing cells (Rincón et al., 1997). We observed that during the activation of CD4+ T cells in the presence of APC, high levels of IL-6 were secreted by APC (Rincón and Flavell, 1997). B cells appears to be the primary source of IL-6 in the cultures, but macrophages also have an important contribution (Rincón et al. unpublished data). To determine whether physiological levels of IL-6 secreted by APC were relevant for Th2 cell differentiation
we examined the function of APC from IL-6−/− mice. We first purified CD4+ T cells from WT mice and stimulated them for 4 days with ConA in the presence of APC from WT mice or IL-6−/− mice in the absence of any added exogenous cytokines. Restimulation with ConA resulted in significantly less IL-4 production in cells differentiated in the presence of IL-6−/− than in WT APC. We also examined the in vitro differentiation of CD4+ T cells from IL-6 deficient mice. IL-6−/− CD4+ T cells stimulated with ConA in the presence of WT APC, which can provide the IL-6 required, produced IL-4, but this IL-4 production was impaired when the IL-6 pathway was blocked by the addition of anti-IL-6 mAb (Rincón et al., 1997). Moreover, high IL-4 production was restored in cells that were differentiated in the presence of IL-6−/− APC together with an exogenous source of IL-6. Thus, IL-6 production by APC during activation of CD4+ T cells plays a major role in determining the type of cytokines that effector CD4+ T cells will secrete. To further prove that the effect of IL-6 was on T cells directly, rather than APC, we differentiated CD4+ T cells with immobilized anti-CD3 mAb plus anti-CD28 mAb in the complete absence of APC, and then restimulated these cells with anti-CD3 mAb alone. The presence of IL-6 (or IL-4 as a control) during the first culture resulted in increased IL-4 production and reduced IFN␥ production by the cells that were elicited. Thus, IL-6 directly favors the polarization of na¨ıve CD4+ T cells to Th2 cells (Diehl et al., 2002). IL-4 is a critical differentiation factor for Th2 cells, which acts by promoting the secretion of more IL-4 by T cells (Le Gros et al., 1990; Swain et al., 1990). It was therefore possible that IL-6 could directly upregulate the synthesis of IL-4 by T cells and, consequently that the IL-6 effect on the differentiation of Th2 cells was mediated through IL-4. To address this hypothesis, CD4+ T cells were differentiated with IL-6 in the presence or absence of neutralizing anti-IL-4 mAb, and after 4 days the cells were washed and restimulated. The ability of IL-6 to polarize CD4+ T cells towards the Th2 phenotype was blocked by anti-IL-4 mAb, since the cells were unable to produce IL-4 upon restimulation. These results indicated that the differentiation of Th2 cells by IL-6 is dependent on the endogenous production of IL-4 by CD4+ T cells. It was therefore possible that IL-6 could trigger the initial IL-4 production by CD4+ T cells during activation and this early IL-4 could promote further Th2 differentiation. To address this hypothesis we examined the effect of IL-6 on IL-4 production during the activation of CD4+ T cells. Increased levels of IL-4 were produced by CD4+ T cells activated with anti-CD3 and anti-CD28 in the presence of IL-6 compared with those in the absence of IL-6. Moreover, increased levels of IL-4 mRNA were detected in CD4+ T cells stimulated in the presence of IL-6 (Diehl et al., 2002). To show that early IL-4 production induced by IL-6 was not dependent on endogenous IL-4, we examined the regulation of IL-4 gene expression in the presence of a blocking anti-IL-4 mAb. Upregulation of IL-4 mRNA levels by IL-6 was also observed
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Fig. 1. IL-6 promotes Th2 differentiation by activation of NFAT and induction of early IL-4 gene expression.
in the presence of the anti-IL-4 mAb early during activation (Diehl et al., 2002). Thus, IL-6 upregulates IL-4 gene and protein expression during the initial stages of differentiation. IL-4 gene transcription is regulated by several transcription factors (e.g. nuclear factor of activated T cells (NFAT), GATA-3, and c-maf; Murphy et al., 2000). We found that IL-6 increased the levels of GATA-3 in CD4+ T cells. However, this increased on GATA-3 expression was dependent of endogenous IL-4, indicating that this effect of IL-6 was an indirect effect caused by the upregulation of IL-4 production. In contrast, IL-6 activated NFAT transcriptional activity independently of endogenous IL-4 (Diehl et al., 2002). Activation of NFAT by IL-6 occurs through upregulation of NFATc2 expression (Diehl et al., 2002). Even more, IL-4 failed to upregulate NFAT activity. Thus, NFAT is regulated by specific signals delivered by IL-6. We generated transgenic mice that express a mutant form of NFAT that acts as a dominant negative mutant inhibiting transcription by all four members of the NFAT family (Chow et al., 1999). We examined the ability of IL-6 to promote Th2 differentiation in CD4+ T cells from the dnNFAT transgenic mice. Inhibition of NFAT completely abrogated the induction of IL-4 gene expression and Th2 differentiation by IL-6 (Diehl et al., 2002). Thus, IL-6 promotes Th2 differentiation by activation of NFAT and induction of early IL-4 gene expression (Fig. 1). We have also shown that IL-6 inhibits IFN␥ production and Th1 differentiation (see Section 3). Interestingly, the presence of the dnNFAT did not prevent the inhibitory effect of IL-6 on IFN␥ gene expression. 3. IL-6 inhibits Th1 differentiation by inducing the expression of SOCS-1 and IFN␥ during the activation of CD4+ T cells We have previously observed that the presence of IL-6 during the differentiation of CD4+ T cells with ConA, APC
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and IL-12 caused a dramatic inhibition of IFN␥ production after restimulation (Rincón et al., 1997). IL-6 also inhibited Th1 differentiation induced with anti-CD3 and IL-12 (Diehl et al., 2002). We also tested whether IL-6 could prevent the differentiation of na¨ıve CD4+ T cells into effector Th1 cells. CD4+ T cells from cytochrome c TcR transgenic mice were unable to differentiate into high-IFN␥ producing Th1 effector cells when IL-6 was present (Diehl et al., 2000). Moreover, we have examined the role of IL-6 in vivo Th1 differentiation. Borrelia burgdoferi infection induces a Th1 response in C3H/HeN mice, resulting in Lyme arthritis (Anguita et al., 1996; Keane-Myers and Nickell, 1995; Matyniak and Reiner, 1995). IL-b-defficient mice exhibit increased incidence of Lyme arthritis (Anguita et al., 1997). We have also shown that IL-6 is able to inhibit in vivo Th1 differentiation since administration of IL-6 in vivo during infection with B. burgderfori prevents the generation of antigen-specific Th1 effector CD4+ T cells (Diehl et al., 2000). Together, these results indicated that IL-6, in addition to promoting Th2 polarization, was able to inhibit Th1 differentiation. IL-4 has been reported to have a negative effect on Th1 differentiation in vitro (Seder et al., 1992). Given the fact that IL-6 can induce IL-4 production, we tested whether the effect of IL-6 was simply dependent on IL-4. We showed that IL-6 was still able to inhibit Th1 differentiation even in the presence of a neutralizing anti-IL-4 mAb, suggesting that IL-6 was able to inhibit Th1 differentiation in an IL-4-independent manner (Diehl et al., 2000). The presence of IL-12 during the activation of CD4+ T cells highly enhances IFN␥ production and promotes Th1 differentiation (Hsieh et al., 1993; Seder et al., 1993). However, it has also been shown that effector CD4+ T cells differentiated in the absence of IL-12 produced significant amounts of IFN␥ (Bradley et al., 1996). We also observed that stimulation with anti-CD3 and anti-CD28 mAbs in the absence of IL-12 gave rise to effector cells that produced significant levels of IFN␥ upon restimulation. Interestingly, CD4+ T cells differentiated with anti-CD3 and anti-CD28 mAbs in the presence of IL-6 produced low levels of IFN␥ upon restimulation compared with those differentiated in the absence of IL-6. Thus, IL-6 could inhibit Th1 differentiation independently of IL-12. In the absence of IL-12 endogenous IFN␥ is the major factor to promote Th1 differentiation. During their activation, na¨ıve CD4+ T cells produce significant levels of IFN␥. It was therefore possible that IL-6 inhibited IFN␥ production by CD4+ T cells during activation and, therefore, prevented the generation of Th1 effector cells. We found that IL-6 potently suppressed IFN␥ production by CD4+ T cells early during their activation. Analysis of cytokine mRNA levels during this period revealed that IL-6 also inhibited IFN␥ gene expression (Diehl et al., 2000). Furthermore, the presence of an anti-IL-4 mAb did not affect the decrease in IFN␥ gene expression caused by IL-6. Together these results demonstrated that IL-6 could inhibit IFN␥ gene
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expression during the activation of CD4+ T cells independently of IL-4. However, even in the presence of exogenous IFN␥, IL-6 was still able to inhibit Th1 differentiation, suggesting that IL-6 could interfere with the IFN␥ receptor (IFN␥R) signals that lead to an autoregulation of IFN␥ gene expression. We therefore analyzed the effect of IL-6 on Th1 differentiation in IFN␥R−/− CD4+ T cells. IL-6 suppressed the differentiation of wild type CD4+ T cells into effector Th1 cells that, while IL-6 did not affect the levels of IFN␥ produced by IFN␥R−/− Th1 cells (Diehl et al., 2000). Together, these results indicated that IL-6 inhibited signaling through the IFN␥R, which is critical for complete Th1 polarization. Signaling through the IFN␥R (as for many other cytokine receptors) utilizes the Jak/STAT pathway. Currently, there are four mammalian JAKs (Jak1, Jak2, Jak3 and Tyk2) and seven STATs (Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b and Stat6; for review see (Leonard and O’Shea, 1998). Binding of IFN␥ to its receptor induces receptor dimerization and activation of Jak1 and Jak2 that recruit and phosphorylate STAT1 (Bach et al., 1997). To determine whether IL-6 targeted the Jak/STAT1 pathway triggered by IFN␥ we analyzed STAT1 tyrosine phosphorylation. IFN␥-induced Tyr phosphorylation of STAT1 in cells cultured in the absence of IL-6, but not in cells that were activated in the presence of IL-6 (Diehl et al., 2000). We explored the possibility that IL-6 could affect negative regulators of cytokine signaling to interfere with IFN␥ signaling. The suppressors of cytokine signaling (SOCS; also known as STAT-induced STAT inhibitors (SSIs)) proteins are negative regulators of cytokine signaling (reviewed by Alexander et al., 1999b; Naka et al., 1999). There are eight members of the SOCS family (SOCS1–SOCS7 and CIS; cytokine inducible SH2 containing protein) and they regulate signaling through cytokine receptors and some tyrosine kinase growth factor receptors such as insulin-like growth factor-1. SOCS1 was initially found to be induced by IL-6 in the M1 monocytic leukemia cell line (Endo et al., 1997; Naka et al., 1997; Starr et al., 1997). A critical role for SOCS1 in IFN␥ signaling in vivo was highlighted through the generation of SOCS1-deficient mice (SOCS1−/− ). SOCS1−/− mice die perinatally due to IFN␥ overproduction and IFN␥ hypersensitivity that result on severe liver damage (Alexander et al., 1999a; Bullen et al., 2001; Marine et al., 1999; Naka et al., 1998; Starr et al., 1998). We therefore examined whether IL-6 was able to induce SOCS1 expression to inhibit IFN␥ signaling in CD4+ T cells. We have shown that CD4+ T cells activated in the presence of IL-6 contain higher levels of SOCS1 mRNA. Thus, IL-6 was able to upregulate the expression of SOCS1 in CD4+ T cells. To determine whether SOCS1 was required for IL-6 mediated Th1 inhibition, we analyzed the effect of IL-6 on IFN␥ production in CD4+ T cells from SOCS1−/− mice. While differentiation of wild type CD4+ T cells into Th1 effector cells was effectively inhibited by IL-6, differentiation of SOCS1−/− CD4+ T cells into Th1 cells was not affected by
Fig. 2. Requirement of SOCS1 for IL-6 to inhibit IFN␥ signaling and Th1 differentiation.
IL-6 (Diehl et al., 2000). Furthermore, we have demonstrated that IL-6 did not inhibit IFN␥-induced STAT1 phosphorylation in cells lacking SOCS1. SOCS1 is therefore required for IL-6 to inhibit IFN␥ signaling and Th1 differentiation (Fig. 2). SOCS1, however, was not required for IL-6 to promote Th2 differentiation (Diehl et al., 2000), indicating that this cytokine uses two independent signaling pathways to promote Th2 differentiation and inhibit Th1 differentiation.
4. Conclusive remarks and future directions Our studies clearly show that IL-6 produced by APCs can modulate the differentiation of CD4+ T cells into effector Th1 or Th2. The presence of IL-6 shifted the Th1/Th2 balance toward the Th2 direction using two independent approaches: (1) promoting IL-4 production and Th2 differentiation; and (2) inhibiting IFN␥ production and Th1 differentiation (Fig. 3). While differentiation of Th2 by IL-6 is dependent on endogenous production of IL-4, inhibition of Th1 differentiation by IL-6 is not. IL-6 inhibits Th1 differentiation by interfering with IFN␥ signaling through upregulation of SOCS1. However, IL-6 promotes Th2 differentiation by activating NFAT, which induces endogenous IL-4 gene expression. Activation of NFAT is not required for inhibition of IFN␥ gene expression and Th1 differentiation by IL-6. In the absence of SOCS1, IL-6 fails to inhibit Th1 differentiation, but it retains its ability to drive Th2 differentiation. Thus, this is an example of how a cytokine uses two different signaling pathways to regulate two independent functions. In addition to APC IL-6 is also produced by other non-lymphoid cells including astrocytes, fibroblasts and tumor cells. Future studies will be carried out to examine the contribution of IL-6 produced by these cells on the differentiation of CD4+ T cells into Th1 and Th2. It is possible that the production of IL-6 by tumor cells may prevent the development of an anti-tumor Th1 response, or the production of IL-6 by the lung environment during asthma may be a determinant factor for the development of
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Fig. 3. IL-b shifts the Th1/Th2 balance towards Th2 by inhibiting Th1 and promoting Th2.
Th2 responses. Moreover, further experimental approaches need to be developed to examine the contribution of IL-6 derived from any of these source in vivo Th1/Th2 balance (e.g. IL-6 conditional knock outs). The pleiotropic character of IL-6 has made difficult to obtain a clear answer for the role of this cytokine in vivo models
Acknowledgements Support for the studies performed in this laboratory was received by the National Institute of Health (PO1 AI45666), the COBRE Program of the National Center for Research Resources (P20 RR15557), the Arthritis Foundation, and the Gustavus and Louise Pfeiffer Foundation. S.D. is supported by an Environmental Pathology Training Grant from the National Institute of Environmental Health Science (T32ES07122).
References Akira, S., Issihiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., Kishimoto, T., 1990. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 9, 1897–1906. Akira, S., Nishio, Y., Inoue, M., Wang, X.J., Wei, S., Matsusaka, T., Yoshida, K., Sudo, T., Naruto, M., Kishimoto, T., 1994. Molecular cloning of APRF a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 77, 63–71. Alexander, W.S., Starr, R., Fenner, J.E., Scott, C.L., Handman, E., Sprigg, N.S., Corbin, J.E., Cornish, A.L., Darwiche, R., Owczarek, C.M., Kay, T.W., Nicola, N.A., Hertzog, P.J., Metcalf, D., Hilton, D.J., 1999a. SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98, 597–608. Alexander, W.S., Starr, R., Metcalf, D., Nicholson, S.E., Farley, A., Elefanty, A.G., Brysha, M., Kile, B.T., Richardson, R., Baca, M., Zhang, J.G., Willson, T.A., Viney, E.M., Sprigg, N.S., Rakar, S., Corbin, J., Mifsud, S., DiRago, L., Cary, D., Nicola, N.A., Hilton, D.J., 1999b. Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction. J. Leukoc. Biol. 66, 588–592. Anguita, J., Persing, D., Rincón, M., Barthold, S.W., Fikrig, E., 1996. Effect of anti-interleukin 12 treatment on murine Lyme borreliosis. J. Clinic. Invest. 97, 1028–1034.
Anguita, J., Rincón, M., Samanta, S., Barthold, S.W., Flavell, R.A., Fikrig, E., 1997. Borrelia burgdorferi-infected, interleukin-6-deficient mice have decreased Th2 responses and increased Lyme arthritis. J. Infect. Dis. 178, 1512–1515. Bach, E.A., Aguet, M., Schreiber, R.D., 1997. The IFN␥ receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15, 563–591. Bradley, L.M., Dalton, D.K., Croft, M., 1996. A direct role for IFN␥ in regulation of Th1 cell development. J. Immunol. 157, 1350–1358. Bullen, D.V., Darwiche, R., Metcalf, D., Handman, E., Alexander, W.S., 2001. Neutralization of interferon-gamma in neonatal SOCS1−/− mice prevents fatty degeneration of the liver but not subsequent fatal inflammatory disease. Immunology 104, 92–98. Chow, C.-W., Rincón, M., Davis, R.J., 1999. Requirement for transcription factor NFAT in interleukin-2 expression. Mol. Cell. Biol. 19, 2300– 2307. Constant, S.L., Bottomly, K., 1997. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu. Rev. Immunol. 15, 297–322. Daeipour, M., Kumar, G., Amaral, M.C., Nel, A.E., 1993. Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line, AF-10. J. Immunol. 150, 4743–4753. Diehl, S., Anguita, J., Hoffmeyer, A., Zapton, T., Ihle, J.N., Fikrig, E., Rincón, M., 2000. Inhibition of Th1 differentiation by IL-6 is mediated by SOCS1. Immunity 13, 805–815. Diehl, S., Chow, C.W., Weiss, L., Palmetshofer, A., Twardzik, T., Rounds, L., Serfling, E., Davis, R.J., Anguita, J., Rincon, M., 2002. Induction of NFATc2 expression by interleukin 6 promotes T helper type 2 differentiation. J. Exp. Med. 196, 39–49. Endo, T.A., Masura, M., Yokouchi, M., Suzuki, R., Sakamoto, H., Mitsui, K., Matsumoto, A., Tanimura, S., Ohtsubo, M., Misawa, H., Miyazaki, T., Leonor, N., Taniguchi, T., Fujita, T., Kanakura, Y., Komiya, S., Yoshimura, A., 1997. A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387, 921–924. Hirano, T., 1998. Interleukin 6 and its receptor: 10 years later. Int. Rev. Immunol. 16, 249–284. Hsieh, C.S., Macatonia, S.E., Tripp, C.S., Wolf, S.F., O’Garra, A., Murphy, K.M., 1993. Development of Th1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science 260, 547–549. Kaye, J., Hsu, M.-L., Sauron, M.-E., Jameson, S.C., Gascoigne, N.R.J., Hedrick, S.M., 1989. Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor. Nature 341, 746–749. Keane-Myers, A., Nickell, S.P., 1995. Role of IL-4 and IFN␥ in modulation of immunity to Borrelia burgdorferi in mice. J. Immunol. 155, 2020– 2028. Kinoshita, S., Akira, S., Kishimoto, T., 1992. A member of the C/EBP family, NF-IL6, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc. Natl. Acad. Sci. U.S.A. 89, 1473–1476. Le Gros, G., Ben-Sasson, S.Z., Seder, R., Finkelman, F.D., Paul, W.E., 1990. Generation of interleukin 4 (IL-4)-producing cells in vivo and
536
S. Diehl, M. Rinc´on / Molecular Immunology 39 (2002) 531–536
in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4 producing cells. J. Exp. Med. 172, 921–929. Leonard, W.J., O’Shea, J.J., 1998. Jaks and STATs: biological implications. Annu. Rev. Immunol. 16, 293–322. Lutticken, C., Wegenka, U.M., Yuan, J., Buschmann, J., Schindler, C., Ziemiecki, A., Harpur, A.G., Wilks, A.F., Yasukawa, K., Taga, T., Kismimoto, T., Barbieri, G., Pellegrini, S., Sendtner, M., Heinrich, P.C., Horn, F., 1994. Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science 263, 89–92. Marine, J.C., Topham, D.J., McKay, C., Wang, D., Parganas, E., Stravopodis, D., Yoshimura, A., Ihle, J.N., 1999. SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell 98, 609–616. Matyniak, J.E., Reiner, S.L., 1995. T helper phenotype and genetic susceptibility in experimental Lyme disease. J. Exp. Med. 181, 1251– 1254. Murphy, K.M., Ouyang, W., Farrar, J.D., Yang, J., Ranganath, S., Asnagli, H., Afkarian, M., Murphy, T.L., 2000. Signaling and transcription in T helper development. Annu. Rev. Immunol. 18, 451–594. Naka, T., Fujimoto, M., Kishimoto, T., 1999. Negative regulation of cytokine signaling: STAT-induced STAT inhibitor. Trends Biochem. Sci. 24, 394–398. Naka, T., Matsumoto, T., Narazaki, M., Fujimoto, M., Morita, Y., Ohsawa, Y., Saito, H., Nagasawa, T., Uchiyama, Y., Kishimoto, T., 1998. Accelerated apoptosis of lymphocytes by augmented induction of Bax in SSI-1 (STAT-induced STAT inhibitor-1) deficient mice. Proc. Natl. Acad. Sci. U.S.A. 95, 15577–15582. Naka, T., Narazaki, M., Hirata, M., Matsumoto, T., Minamoto, S., Aono, A., Nishimoto, N., Kajita, T., Taga, T., Yoshizaki, K., Akira, S., Kishimoto, T., 1997. Structure and function of a new STAT-induced STAT inhibitor. Nature 387, 924–929. Nakajima, K., Matsuda, T., Fujitani, Y., Kojima, H., Yamanaka, Y., Nakae, K., Takeda, T., Hirano, T., 1995. Signal transduction through IL-6 receptor: involvement of multiple protein kinases, stat factors, and a novel H7-sensitive pathway. Ann N.Y. Acad. Sci. 762, 55–70. Nakanishi, K., Yoshimoto, T., Tsutsui, H., Okamura, H., 2001. Interleukin-18 regulates both Th1 and Th2 responses. Annu. Rev. Immunol. 19, 423–474. O’Garra, A., 1998. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 8, 275–283. Poli, V., 1998. The role of C/EBP isoforms in the control of inflammatory and native immunity functions. J. Biol. Chem. 273, 29279–29282.
Poli, V., Mancini, F.P., Cortese, R., 1990. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell 63, 643–653. Ramji, D.P., Vitelli, A., Tronche, F., Cortese, R., Ciliberto, G., 1993. The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6, are induced by IL-6 to promote acute phase gene transcription via different mechanisms. Nucleic Acids Res. 21, 289–294. Rincón, M., Anguita, J., Nakamura, T., Fikrig, E., Flavell, R.A., 1997. IL-6 directs the differentiation of IL-4-producing CD4+ T cells. J. Exp. Med. 185, 461–469. Rincón, M., Flavell, R.A., 1997. Transcriptional control of Th1/Th2 polarization. Curr. Biol. 7, R729–R732. Seder, R.A., Paul, W.E., Davis, M.M., 1992. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176, 1091–1098. Seder, R.A., Gazzinelli, R., Sher, A., Paul, W.E., 1993. IL-12 acts directly on CD4+ T cells to enhance priming for IFN␥ production and diminish IL-4 inhibition of such priming. Proc. Natl. Acad. Sci. U.S.A. 90, 10188–10192. Stahl, N., Boulton, T.G., Farruggella, T., Ip, N.Y., Davis, S., Witthuhn, B.A., Quelle, F.W., Silvennoinen, O., Barbieri, G., Pellegrini, S., Ihle, J.N., Yancopoulos, G.D., 1994. Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 receptor components. Science 263, 92–95. Starr, R., Metcalf, D., Elefanty, A.G., Brysha, M., Willson, T.A., Nicola, N.A., Hilton, D.J., Alexander, W.S., 1998. Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1. Proc. Natl. Acad. Sci. U.S.A. 95, 14395– 14399. Starr, R., Willson, T.A., Viney, E., Murray, L.J.L., Rayner, J.R., Jenkins, B.J., Gonda, T.J., Alexander, W.S., Metcalf, D., Nicola, N.A., Hilton, D.J., 1997. A family of cytokine-inducible inhibitors of signaling. Nature 387, 917–921. Swain, S.L., Weinberg, A.D., English, M., Huston, G., 1990. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145, 3796– 3806. Swain, S.L., 1995. Who does the polarizing? Curr. Biol. 5, 849–851. Taga, T., Hibi, M., Hirata, Y., Yamasaki, K., Yasukawa, K., Matsuda, T., Hirano, T., Kishimoto, T., 1989. Interleukin-6-triggers the association of its receptor with a possible signal transducer, gp130. Cell 58, 573– 581.