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Interleukin-10 Stimulation of Phosphatidylinositol 3-Kinase and p70 S6 Kinase Is Required for the Proliferative but Not the Antiinflammatory Effects of the Cytokine*

Open AccessPublished:July 05, 1996DOI:https://doi.org/10.1074/jbc.271.27.16357
      Interleukin-10 (IL-10) is a powerful suppressor of the proinflammatory monokine production by lipopolysaccharide-stimulated monocytes as well as a T- and B-cell growth cofactor. The signal transduction cascades initiated by IL-10 ligation to its cognate receptor remain to be elucidated. Here, we demonstrate that in both primary monocytes and the D36 cell line, IL-10 rapidly and transiently stimulated phosphatidylinositol 3-kinase activity associated with the p85 subunit of the enzyme. IL-10 also activated p70 S6 kinase in both cell types. The activation of both of these kinases was sensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase. The activation of p70 S6 kinase was also inhibited by the immunosuppressive drug rapamycin. Both rapamycin and wortmannin inhibited the IL-10-induced proliferation of D36 cells but in contrast had no effect on the antiinflammatory effects of the cytokine on lipopolysaccharide-stimulated monocytes. Similar results on D36 proliferation and lipopolysaccharide-stimulated monocyte inhibition by IL-10 were obtained with another phosphatidylinositol 3-kinase inhibitor, LY294002. This suggests that the activation of phosphatidylinositol 3-kinase and p70 S6 kinase is involved in the proliferative functions of IL-10 and that other as yet uncharacterized pathways affect the suppressive effects on monocytes, indicating that multiple and distinct signaling pathways mediate the various pleiotropic activities of IL-10. Furthermore, these findings suggest that it may be possible in the future to modulate the antiinflammatory effects of IL-10 for therapeutic benefit without disrupting other functions of the cytokine.

      INTRODUCTION

      Interleukin-10 (IL-10),
      The abbreviations used are: IL
      interleukin
      LPS
      lipopolysaccharide
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PI-3P
      phosphatidylinositol 3-phosphate
      TNF-α
      tumor necrosis factor α
      MOPS
      3-(N-morpholino)propanesulfonic acid
      ELISA
      enzyme-linked immunosorbent assay
      mAb
      monoclonal antibody.
      originally identified as an inhibitor of cytokine synthesis by T-cells (
      • Fiorentino D.F.
      • Zlotnik A.
      • Mosmann T.R.
      • Howard M.
      • O'Garra A.
      ), was subsequently recognized to have a wide range of additional properties. It has potent antiinflammatory effects, suppressing the production of IL-1, tumor necrosis factor α (TNF-α), and γ-interferon in polymorphonuclear cells (
      • Cassatella M.A.
      • Meda L.
      • Bonora S.
      • Ceska M.
      • Constantin G.
      ) and monocytes (
      • de Waal Malefyt R.
      • Abrams J.
      • Bennett B.
      • Figdor C.G.
      • de Vries J.E.
      ) while increasing the release of soluble TNF receptor, a natural inhibitor of TNF-α function (
      • Joyce D.A.
      • Gibbons D.P.
      • Green P.
      • Steer J.H.
      • Feldmann M.
      • Brennan F.M.
      ). IL-10 down-regulates major histocompatibility complex class II expression (
      • de Waal Malefyt R.
      • Haanen J.
      • Spits H.
      • Roncarolo M.-G.
      • e Velde A.
      • Figdor C.
      • Johnson K.
      • Katelstein R.
      • Yssel H.
      • de Vries J.E.
      ) and inhibits the expression of other cell surface markers on macrophages (
      • Ding L.
      • Linsley P.S.
      • Huang L.Y.
      • Germain R.N.
      • Shevach E.M.
      ). It also acts as a cofactor for mast cell, T-cell, and B-cell growth (
      • Thompson-Snipes L.
      • Dhar V.
      • Bond M.W.
      • Mosmann T.R.
      • Moore K.W.
      • Rennick D.M.
      ,
      • MacNeil I.A.
      • Suda T.
      • Moore K.W.
      • Mosmann T.R.
      • Zlotnik A.
      ,
      • Fluckiger A.-C.
      • Garrone P.
      • Durand I.
      • Galizzi J.-P.
      • Banchereau J.
      ) and prevents apoptosis in IL-2-starved T-cells (
      • Taga K.
      • Cherney B.
      • Tosato G.
      ).
      IL-10 mediates these activities via a high affinity cell surface receptor structurally related to the interferon receptors (
      • Ho A.S.Y.
      • Liu Y.
      • Khan T.
      • Hsu D.-H.
      • Bazan J.F.
      • Moore K.W.
      ). However, little is known of the signaling mechanisms initiated by IL-10. Like many cytokines, IL-10 can induce phosphorylation and activation of members of the Janus kinase family, in this case Tyk2 and Jak1 (
      • Finbloom D.S.
      • Winestock K.D.
      ) and their effectors, the signal transducer and activator of transcription proteins, STAT1α and STAT3 (
      • Finbloom D.S.
      • Winestock K.D.
      ,
      • Lehmann J.
      • Seegert D.
      • Strehlow I.
      • Schindler C.
      • Lohmann-Matthes M.-L.
      • Decker T.
      ). IL-10 can activate NF-κB in T-cells (
      • Hurme M.
      • Henttinen T.
      • Karppelin M.
      • Varkila K.
      • Matikainen S.
      ), although inhibition of this transcription factor by this cytokine has been observed in monocytes (
      • Wang P.
      • Wu P.
      • Siegel M.I.
      • Egan R.W.
      • Billah M.M.
      ).
      The aim of this study was to investigate IL-10-mediated intracellular signaling mechanisms and their relationship to cellular functions of the cytokine. The study shows that IL-10 activates PI 3-kinase and p70 S6 kinase in both primary monocytes and a murine mast cell line, D36. IL-10-mediated proliferation in these cells was sensitive to wortmannin (
      • Kanai F.
      • Ito K.
      • Todaka M.
      • Hayashi H.
      • Kamohara S.
      • Ishii K.
      • Okada O.
      • Hazeki O.
      • Uli M.
      • Ebina Y.
      ,
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nomomura Y.
      • Matsuda Y.
      ,
      • Arcaro A.
      • Wymann M.P.
      ) and LY294002 (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ), inhibitors of PI 3-kinase, and rapamycin, which inhibits the activation of p70 S6 kinase (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ). However, PI 3-kinase and p70 S6 kinase activation was not required for IL-10 suppression of lipopolysaccharide (LPS)-induced TNF-α or soluble TNF receptor production. These data indicate a selective role for PI 3-kinase and p70 S6 kinase in IL-10 function and suggest the existence of multiple pathways with different functional end points induced by IL-10.

      DISCUSSION

      This study has shown that IL-10 can activate PI 3-kinase and p70 S6 kinase. Furthermore, the activation of these factors is required for the proliferative but not the antiinflammatory effects of IL-10, indicating that multiple signaling pathways are activated by this cytokine.
      The accumulation of the 3′-phosphorylated phospholipids due to the activation of PI 3-kinase correlates with the proliferative response to many growth factors and cytokines (
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ,
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ,
      • Merida I.
      • Diez E.
      • Gaulton G.N.
      ,
      • Wang L.M.
      • Keegan A.D.
      • Paul W.E.
      • Heideran M.A.
      • Gutkind J.S.
      • Pierce J.H.
      ,
      • Ventikaraman A.R.
      • Cowling R.J.
      ). However, unlike the receptors for these factors, the IL-10 receptor is related to the receptors for the interferons (
      • Ho A.S.Y.
      • Liu Y.
      • Khan T.
      • Hsu D.-H.
      • Bazan J.F.
      • Moore K.W.
      ). This paper demonstrates that PI 3-kinase is activated via a receptor of this family, although the delineation of the precise structure of the 3′-phosphorylated lipids induced by IL-10 was beyond the limit of this study.
      The PI 3-kinase enzyme is composed of two subunits, the catalytic p110 and regulatory p85. Whether the observed increase in PI 3-kinase activity in anti-p85 immunoprecipitates really does represent a bulk activation of PI 3-kinase is unclear. More likely, the observed modest activation of kinase activity would suggest that some subpopulation of the cellular PI 3-kinase pool is being activated. The mechanisms of activation of PI 3-kinase remain largely undetermined, as is the case here.
      Previous studies have generally measured factors activating PI 3-kinase by precipitating the enzyme with anti-phosphotyrosine antibodies or when associated with other signaling molecules (e.g. insulin receptor substrate 1 (
      • Myers Jr., M.G.
      • Grammer T.C.
      • Wang L.M.
      • Sun X.J.
      • Pierce J.H.
      • Blenis J.
      • White M.F.
      )). In addition, the p85 subunit is often tyrosine phosphorylated in response to cytokine activation (
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ,
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Augustine J.A.
      • Sutor S.L.
      • Abraham R.T.
      ,
      • Dadi H.
      • Ke S.
      • Roifman C.M.
      ). However, thus far we have been unable to demonstrate IL-10-induced activity in anti-phosphotyrosine or anti-insulin receptor substrate 1 immunoprecipitates (results not shown), although further studies in this area are still in progress.
      Although the downstream effects of the 3′-labeled PI second messengers are still elusive, in vitro they can activate protein kinase C ζ (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ), and the serine/threonine protein kinase Akt (
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ). Furthermore, in platelet-derived growth factor and insulin-stimulated HepG2 cells (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ) or in IL-2-stimulated CTLL T-cells (
      • Monfar M.
      • Lemon K.P.
      • Grammer T.C.
      • Cheatham L.
      • Chung J.
      • Vlathos C.J.
      • Blenis J.
      ) and CD28-stimulated T-cells (
      • Pages F.
      • Ragueeau M.
      • Rottapel R.
      • Truneh A.
      • Nunes J.
      • Imbert J.
      • Olive D.
      ,
      • Pai S.-Y.
      • Calvo V.
      • Wood M.
      • Bierer B.E.
      ), PI 3-kinase activity has been correlated with an increase in p70 S6 kinase activity. Our studies showed that IL-10 can also induce p70 S6 kinase activation, and the observations with wortmannin and LY294002 also demonstrate a possible causal link between the activation of PI 3-kinase and p70 S6 kinase, this time by IL-10. However, recent studies (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Kotani K.
      • Kotani K.
      • Kojima A.
      • Waterfields M.D.
      • Kasuga M.
      ) on insulin signaling have dissociated the activation of the two enzymes, suggesting that other wortmannin-sensitive targets may exist. Such a hypothesis is supported by the recent observation that the catalytic subunit of DNA dependent kinase, a molecule with a PI 3-kinase-like domain, is also sensitive to wortmannin (
      • Hartley K.O.
      • Gell D.
      • Smith G.C.M.
      • Zhang H.
      • Divecha N.
      • Connelly M.A.
      • Admon A.
      • Lees-Miller S.P.
      • Anderson C.W.
      • Jackson S.P.
      ).
      As for IL-2- (
      • Dumont F.J.
      • Staruch M.J.
      • Koprak S.L.
      • Melino M.R.
      • Sigal N.H.
      ), IL-3- (
      • Calvo V.
      • Wood M.
      • Gjertson C.
      • Vik T.
      • Bierer B.E.
      ), and IL-4-induced
      D. Taylor-Fishwick, unpublished data.
      p70 S6 kinase activation, rapamycin inhibited IL-10 activation of this enzyme with a concomitant inhibition of IL-10-induced proliferation of D36 cells. The intracellular target of rapamycin appears to be a family of molecules identified variously as a target of rapamycin in yeast (
      • Kunz J.
      • Henriquez R.
      • Schneider U.
      • Deuter-Reinhard M.
      • Movva N.R.
      • Hall M.N.
      ), rapamycin and FKBP targets in rats (
      • Chui M.
      • Katz H.
      • Berlin V.
      ,
      • Sabatini D.M.
      • Erdjument-Bromage H.
      • Lui M.
      • Tempst P.
      • Synder S.H.
      ), and FKBP12-rapamycin-associated protein in humans (
      • Brown E.J.
      • Albers M.W.
      • Shin T.B.
      • Ichikawa K.
      • Keith C.
      • Lane W.S.
      • Schreiber S.L.
      ). FKBP12-rapamycin-associated protein has recently been shown to lie proximal of the activation of p70 S6 kinase (
      • Brown E.
      • Beal P.A.
      • Keith C.T.
      • Chen J.
      • Shin B.-L.
      • Schreiber S.L.
      ). The precise role of these molecules in cell physiology is unknown. Although like DNA-dependent kinase they have some structural homology to the kinase domain of PI 3-kinase, FKBP12-rapamycin-associated protein, at least, is not a target for wortmannin (
      • Brown E.
      • Beal P.A.
      • Keith C.T.
      • Chen J.
      • Shin B.-L.
      • Schreiber S.L.
      ). A recent study has identified a rapamycin-sensitive step in the induction of the key cell survival factor Bcl-2 by IL-2 (
      • Miyazaki T.
      • Liu Z.-J.
      • Kawahara A.
      • Minami Y.
      • Yamada K.
      • Tsujimoto Y.
      • Barsoumian E.L.
      • Perlmutter R.M.
      • Taniguchi T.
      ). Because IL-10 can induce the Bcl-2 protein (
      • Levy Y.
      • Brouet J.-C.
      ), one could speculate that rapamycin inhibits this function of IL-10. This is currently under investigation.
      The inhibitory effects of wortmannin and rapamycin on IL-10-induced proliferation demonstrated the functional relevance of PI 3-kinase and p70 S6 kinase activation and agree with many previous studies indicating a role for these enzymes in cytokine and growth factor-induced mitogenesis. The possibility that other drug targets are mediating the inhibitory effects of wortmannin is unlikely, since LY294002 had similar effects on IL-10-induced proliferation. Its spectrum of cellular targets other than PI 3-kinase is likely to different, as the data obtained with monocytes suggest. However, until the effects of wortmannin, LY294002, and rapamycin are fully characterized, such a possibility cannot be totally excluded.
      In contrast, none of the drugs had any inhibitory effect on two of the key antiinflammatory functions of IL-10 in monocytes, the inhibition of LPS-induced TNF-α production and the release of soluble TNF receptor release, indicating that neither PI 3-kinase nor p70 S6 kinase participates in this function of IL-10. Similar results were also obtained when monocyte culture supernatants were assayed for IL-1β and IL-6 (data not shown). Interestingly, wortmannin actually synergized with LPS to increase TNF expression by as much as 3-fold. Furthermore, even for shorter assays of TNF production (6 h), wortmannin still had no effect on IL-10 function. This observation would indicate that the lack of effect of wortmannin on IL-10 function in monocytes is not due to the short half-life of the drug but may be due to other effects of the drug. The fact that LY294002 had the opposite effect on TNF-α production would support this conclusion. A recent publication has shown that wortmannin can stimulate the stress-activated protein kinase pathway (
      • Kharbanda S.
      • Saleem A.
      • Shafman T.
      • Emoto Y.
      • Taneja N.
      • Rubin E.
      • Weichselbaum R.
      • Woodgett J.
      • Avruch J.
      • Kyriakis J.
      • Kufe D.
      ), and this may have a positive effect on TNF-α production.
      In summary, this study shows that IL-10 can activate two key intracellular signaling enzymes, PI 3-kinase and p70 S6 kinase. The effects of wortmannin, LY294002, and rapamycin implicate these molecules in IL-10-induced proliferation in the D36 mast cell line, but not in the antiinflammatory effects of the cytokine in monocytes. These data would indicate that there are multiple signaling mechanisms activated by IL-10, involved in different functions of the cytokine.

      Acknowledgments

      We are grateful to Drs. G. Panayotou and Dr. R. Stein (Ludwig Institute, London) and also to Dr. T. Page for critical review of the manuscript.

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