Elsevier

Cytokine

Volume 60, Issue 2, November 2012, Pages 575-582
Cytokine

IL-6 activates STAT5 in T cells

https://doi.org/10.1016/j.cyto.2012.07.002Get rights and content

Abstract

Background

IL-6 is a pleiotropic cytokine which emerged recently as a key regulator of CD4 T cell function. IL-6 alone or in combination with other cytokines promotes T helper 1, T helper 17 and T follicular helper cell differentiation whilst inhibiting the induction of regulatory T cell generation. IL-6 activates multiple pathways among which JAK/STAT3 is the most clearly validated in the control of CD4 T helper differentiation. Activation of STAT5 by cytokines such as IL-2 can counteract IL-6-induced T helper 17 and T follicular helper cell differentiation and promote the induction of regulatory T cell generation. STAT5 and STAT3 are known to compete for promoter binding sites in CD4 T cells and the two transcription factors are believed to have opposite functions in the control of CD4 T cell differentiation.

Methods

We analyzed IL-6-induced STAT1, 3 and 5 activation by flow cytometry (phosflow) in mouse mononuclear cells and its effect on the level of the mRNA coding for cytokine-inducible SH2-containing protein (CIS).

Results

The results show that IL-6 also induces STAT5 activation in both CD4 and CD8 T as well as NK cells. Analysis of STAT5 phosphorylation in CD4 T cells indicates that it is transient and requires higher cytokine concentrations than that of STAT3. CD4 T cell stimulation with IL-6 induces the synthesis of CIS, which is encoded by a gene known to be regulated by STAT5.

Conclusions

Thus, IL-6 at concentrations corresponding to levels observed in the serum during inflammation may activate, in CD4 T cells, a STAT5-negative feedback loop which alters the balance between STAT3-dependent pro-inflammatory helper T cells and STAT5-induced T regulatory cells. STAT5 activation may modulate the differentiation of T helper cells through attenuation of TGF-β stability and production. Since STAT5 is directly activated by Janus kinases, therapeutic approaches designed to inhibit STAT3 activation or to recruit STAT3 phosphatases may be useful in altering the balance of activated STAT3 and STAT5 in favor a profile that would be beneficial in pathologies involving IL-6.

Highlights

► IL-6 induces STAT5 phosphorylation in CD4 T cells. ► IL-6 up-regulates the STAT5-regulated CIS mRNA level in CD4 T cells. ► “Inflammatory” IL-6 levels are required to induce STAT5 activation.

Introduction

IL-6 is a cytokine extensively characterized for its role in the maturation of B cells [1] and the induction of the acute phase response [2], [3]. This role has been illustrated in IL-6-deficient mice that have a markedly impaired response to infection and lack the liver-induced acute phase response to infection and trauma [4]. During acute inflammation, IL-6 has a unique function in driving the transition from the early, predominantly neutrophilic innate response to the mononuclear adaptive response [5]. Recently, IL-6 has also emerged as a signal promoting the development of pro-inflammatory CD4 T cell helper (Th), Th17 [6], [7], [8], [9], [10] and follicular T helper (Tfh) cells [11], [12] and as an inhibitory signal for the differentiation of Th1 [13] and CD4 T regulatory (Treg) cell subsets [6], [14]. Besides their important roles in host defense, Th17 cells have been associated with several autoimmune and inflammatory disorders such as rheumatic diseases, multiple sclerosis and inflammatory bowel diseases [7], [15]. Tregs are central to the maintenance of peripheral immune tolerance and deficiencies in this population lead to devastating autoimmunity in both mice and human [16], [17].

Inhibition of IL-6 is a promising therapeutic avenue which has been extensively investigated in preclinical models of inflammatory and auto-immune diseases [18], [19], [20]. This has led to the development of humanized and fully human antibodies (Abs) directed against both IL-6 [21] and IL-6Rα such as tocilizumab [22], [23]. This antibody has been tested in clinical trials for the treatment of Castelman’s disease [24], rheumatoid arthritis [25], [26], [27], juvenile idiopathic arthritis [28] and Crohn’s disease [29]. It has been approved by Japanese, European and US regulatory agencies [23], [30].

IL-6 signals through a receptor comprising the non-signaling IL-6Rα chain and gp130 [31]. This leads to the activation of several signaling pathways among which Janus kinase (JAK)/signal transducers and activators of transcription 3 (STAT3) is the most validated in the control of CD4 Th differentiation [32], [33], [34], [35] and in IL-6 mediated pathologies [31], [36], [37], [38], [39]. The IL-6 receptor α chain is shared by IL-6 and ciliary neurotrophic factor while gp130 is a common signaling component in other cytokine receptors such as IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin-1, oncostatin M, cardiotrophin-like cytokine, neuropoietin, IL-27 and IL-35 [31], [40], [41], [42].

Soluble forms of IL-6Rα have been found in various body fluids [20], [31]. They are generated by metalloproteinase cleavage of the membrane form of IL-6Rα or by the translation of an mRNA encoding a secreted IL-6Rα generated by alternative splicing [20], [43], [44]. These soluble forms bind IL-6 and activate cells expressing only gp130, a process known as trans-signaling [20], [31]. As expression of IL-6Rα is very restricted whereas the expression of gp130 is ubiquitous, IL-6 trans-signaling plays a crucial role in mediating IL-6 functions during inflammation [20], [31], [45].

The IL-6-mediated induction of Th17 and Tfh differentiation can be inhibited by IL-2 via STAT5 [46], [47]. STAT5 is believed to compete with STAT3 and to repress STAT3 target promoters [48], [49]. Surprisingly, we observed that IL-6 induces STAT5 activation in mouse T cells in vitro. In CD4 T cells this was paralleled by the up-regulation of cytokine-inducible SH2-containing protein (CIS) mRNA which is encoded by a gene regulated by STAT5. These results suggest an unexpected level of complexity in the activation of the STAT signaling pathways activated by IL-6 in CD4 T cells.

Section snippets

Experimental animals

All procedures conformed to the Canadian Council on Animal Care guidelines and were approved by the Animal Ethics Committee of the Université de Montréal. Six- to eight-week-old female C57BL/6 mice were purchased from Jackson Laboratory, Bar Harbor, ME.

Expression and isolation of recombinant proteins

The construct for the expression of mouse hyper-IL-6 was described previously [50]. Recombinant proteins were expressed and purified under conditions ensuring the absence of endotoxin contamination [50] and quantified by Western blot analysis

IL-6 induces STAT5 phosphorylation in resting T cells

IL-6 is known to induce STAT1 and STAT3 phosphorylation in IL-6Rα expressing immune cells [31], [54]. Unexpectedly, we observed that this cytokine can also induce STAT5 phosphorylation in a fraction of mouse spleen mononuclear cells (Fig. 1A). Hyper-IL-6, a soluble IL-6Rα-IL6 fusion protein, can be used to induce IL-6 trans-signaling, which is mediated by soluble IL-6Rα-IL6 complexes binding and activating cells expressing only gp130 [20], [31], [54]. STAT5 phosphorylation was induced in a

Discussion

We observed that incubation of resting or activated CD4 T cells with IL-6 induces the phosphorylation of STAT5. Since STAT5 activation by IL-2 is believed to be a key signaling event for constraining the role of IL-6 in Tfh and Th17 differentiation, our observation was unexpected [46], [47], [48], [49], [56]. IL-6 induced the up-regulation of CIS mRNA, a gene which is known to be regulated by STAT5 [62], [63], [64], indicating that STAT5 phosphorylation results in the functional activation of

Acknowledgments

We thank S. Sénechal (Université de Montréal, Montreal, Canada) for help with flow cytometry and the IRIC genomic platform (Université de Montréal, Montreal, Canada) for the real time PCR assays. This work was supported by the Canadian Institutes of Health Research (MOP-57832).

References (86)

  • H. Ito et al.

    A pilot randomized trial of a human anti-interleukin-6 receptor monoclonal antibody in active Crohn’s disease

    Gastroenterology

    (2004)
  • X.O. Yang et al.

    STAT3 regulates cytokine-mediated generation of inflammatory helper T cells

    J Biol Chem

    (2007)
  • S. Grivennikov et al.

    IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer

    Cancer Cell

    (2009)
  • J. Bollrath et al.

    Gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis

    Cancer Cell

    (2009)
  • B. Schuster et al.

    Signaling of human ciliary neurotrophic factor (CNTF) revisited. The interleukin-6 receptor can serve as an alpha-receptor for CTNF

    J Biol Chem

    (2003)
  • J.A. Lust et al.

    Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor

    Cytokine

    (1992)
  • V. Matthews et al.

    Cellular cholesterol depletion triggers shedding of the human interleukin-6 receptor by ADAM10 and ADAM17 (TACE)

    J Biol Chem

    (2003)
  • A. Laurence et al.

    Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation

    Immunity

    (2007)
  • L. Wei et al.

    New insights into the roles of Stat5a/b and Stat3 in T cell development and differentiation

    Semin Cell Dev Biol

    (2008)
  • A.R. Kimmel et al.

    Preparation of cDNA and the generation of cDNA libraries: overview

    Methods Enzymol

    (1987)
  • C.E. Hack et al.

    Increased plasma levels of interleukin-6 in sepsis

    Blood

    (1989)
  • M.R. Pinsky et al.

    Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality

    Chest

    (1993)
  • S. Haan et al.

    Multiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation

    Cell Signal

    (2005)
  • G.M. Feldman et al.

    STAT5A-deficient mice demonstrate a defect in granulocyte-macrophage colony-stimulating factor-induced proliferation and gene expression

    Blood

    (1997)
  • A. Matsumoto et al.

    CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation

    Blood

    (1997)
  • J.X. Lin et al.

    The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15

    Immunity

    (1995)
  • S. Pflanz et al.

    IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4(+) T cells

    Immunity

    (2002)
  • K. Yoshizaki et al.

    Pathogenic significance of interleukin-6 (IL-6/BSF-2) in Castleman’s disease

    Blood

    (1989)
  • H. Nakajima et al.

    An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor alpha chain induction

    Immunity

    (1997)
  • Z. Yao et al.

    Nonredundant roles for Stat5a/b in directly regulating Foxp3

    Blood

    (2007)
  • M.O. Li et al.

    T cell-produced transforming growth factor-beta1 controls T cell tolerance and regulates Th1- and Th17-cell differentiation

    Immunity

    (2007)
  • C. Becker et al.

    TGF-beta suppresses tumor progression in colon cancer by inhibition of IL-6 trans-signaling

    Immunity

    (2004)
  • C.F. Lai et al.

    STAT3 and STAT5B are targets of two different signal pathways activated by hematopoietin receptors and control transcription via separate cytokine response elements

    J Biol Chem

    (1995)
  • J.A. Ripperger et al.

    Transcription factors Stat3 and Stat5b are present in rat liver nuclei late in an acute phase response and bind interleukin-6 response elements

    J Biol Chem

    (1995)
  • T. Hirano et al.

    Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin

    Nature

    (1986)
  • J. Gauldie et al.

    Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells

    Proc Natl Acad Sci USA

    (1987)
  • M. Kopf et al.

    Impaired immune and acute-phase responses in interleukin-6-deficient mice

    Nature

    (1994)
  • E. Bettelli et al.

    Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells

    Nature

    (2006)
  • E. Bettelli et al.

    T(H)-17 cells in the circle of immunity and autoimmunity

    Nat Immunol

    (2007)
  • M.J. McGeachy et al.

    TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology

    Nat Immunol

    (2007)
  • P.R. Mangan et al.

    Transforming growth factor-beta induces development of the T(H)17 lineage

    Nature

    (2006)
  • D. Eto et al.

    IL-21 and IL-6 are critical for different aspects of B cell immunity and redundantly induce optimal follicular helper CD4 T cell (Tfh) differentiation

    PLoS One

    (2011)
  • C. Pasare et al.

    Toll pathway-dependent blockade of CD4 + CD25 + T cell-mediated suppression by dendritic cells

    Science

    (2003)
  • Cited by (0)

    1

    Present address: Wittycell, 4 rue Pierre Fontaine, F-91000 Evry, France.

    View full text