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Monocytic Cells IL-10 on CD14 Expression in Human STAT-1 STAT-1 Mediates the Stimulatory Effect of IL-10 on CD14 Expression in Human Monocytic Cells This information is current as Ali Akbar Rahim Rahimi, Katrina Gee, Sasmita Mishra, of September 30, 2021. Wilfred Lim and Ashok Kumar J Immunol 2005; 174:7823-7832; ; doi: 10.4049/jimmunol.174.12.7823 http://www.jimmunol.org/content/174/12/7823 Downloaded from References This article cites 59 articles, 41 of which you can access for free at: http://www.jimmunol.org/content/174/12/7823.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 30, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology STAT-1 Mediates the Stimulatory Effect of IL-10 on CD14 Expression in Human Monocytic Cells1 Ali Akbar Rahim Rahimi,† Katrina Gee,† Sasmita Mishra,† Wilfred Lim,† and Ashok Kumar2*†‡ IL-10, an anti-inflammatory cytokine, has been shown to exhibit stimulatory functions including CD14 up-regulation on human monocytic cells. CD14-mediated signaling following LPS stimulation of monocytic cells results in the synthesis of proinflammatory cytokines. Our results show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. To investigate the molecular mechanism by which IL-10 enhances CD14 expression, both human monocytes and the promyelocytic HL-60 cells were used as model systems. IL-10 induced the phosphorylation of PI3K and p42/44 ERK MAPK. By using specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), we demonstrate that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT-1 and consequently CD14 expression. However, Downloaded from IL-10-induced STAT-3 phosphorylation remained unaffected under these conditions. Finally, STAT-1 interfering RNA inhibited IL-10-induced CD14 expression. Taken together, these results suggest that IL-10-induced CD14 up-regulation in human mono- cytic cells may be mediated by STAT-1 activation through the activation of PI3K either alone or in concert with the ERK MAPK. The Journal of Immunology, 2005, 174: 7823–7832. ϩ nterleukin-10, initially described as a cytokine synthesis in- an autocrine growth factor for Ly-1 B cells, which are important http://www.jimmunol.org/ hibitory factor (1), is a pleiotropic cytokine whose effects in murine models of autoimmune diseases (12). I primarily include the inhibition of APC-dependent cytokine The molecular mechanism underlying IL-10-mediated inhibi- synthesis by Th1 cells and associated inflammatory responses (2– tory effects has been studied in a number of model systems. IL-10 5). IL-10 is produced by a wide variety of cell types, including interacts with its high-affinity receptor complex, which is ex- CD4ϩ Th0 and Th2 cells, CD8ϩ T cells, regulatory T cells, B pressed on most hemopoietic cell types (3, 13). The IL-10R is cells, and monocytic cells (6–8). IL-10 inhibits the Ag-driven ac- composed of two subunits, IL-10R1, the ligand binding subunit, tivity of both Th1 and Th2 subsets, and hence is not strictly a and IL-10R2, the accessory subunit (3, 13). Engagement of the Th2-type cytokine, although it facilitates the induction of Th2 cell IL-10R complex with IL-10 activates the JAK-STAT signaling types (5, 7). IL-10 has been shown to down-regulate the release of pathway. JAK-1 and Tyk-2 tyrosine kinases are constitutively as- by guest on September 30, 2021 reactive oxygen, nitrogen intermediates, and TNF-␣, resulting in sociated with IL-10R1 and IL-10R2, respectively (3, 14). IL-10 macrophage deactivation which may allow the growth of tumor induces tyrosine phosphorylation and activation of the latent tran- cells and intracellular microbes (5, 9). The potent inhibitory action scription factors STAT-3 and STAT-1, and in nonmacrophage of IL-10 on macrophages, particularly at the level of cytokine pro- cells, STAT-5 (14, 15). STAT-3 is recruited directly to the IL-10R duction, supports an important role for IL-10 in the regulation of complex via either of two tyrosine residues present in the IL-10R1 T cell responses and acute inflammation (5). In addition, IL-10 has cytoplasmic domain (14). Dimers of phosphorylated STAT-3 mol- been shown to stimulate a variety of biological functions such as ecules translocate to the nucleus, resulting in gene regulation. costimulation of thymocyte growth in the presence of IL-2 and/or IL-10 mediates its inhibitory effects on macrophage proliferation IL-4, and B cell growth and differentiation (5, 6, 10). Naive IgDϩ and TNF-␣ production through STAT-3 activation (13, 14). IL-10 B cells, when activated with anti-CD40 Abs, secrete IgG1 and has been shown to increase the expression of the cyclin-dependent IgG3 in the presence of IL-10 (6, 11). Furthermore, IL-10 acts as kinase inhibitor p19INK4D in macrophages. The expression of p19INK4D was later shown to be dependent on STAT-3 activation (16, 17). Recently, the inhibitory effects of IL-10 have also been Departments of *Pathology and Laboratory Medicine, and †Biochemistry, Microbi- shown to involve the activation of the suppressor of cytokine sig- ology, and Immunology, University of Ottawa, and ‡Division of Virology and Mo- naling (SOCS)3 proteins, in particular, SOCS1 and SOCS3, in lecular Immunology, Research Institute, Children’s Hospital of Eastern Ontario, Ot- ␥ tawa, Ontario, Canada LPS- and IFN- -stimulated monocytic cells (18, 19). The induc- Received for publication September 2, 2004. Accepted for publication April 12, 2005. tion of the SOCS3 gene has been attributed to STAT-3 activation (20), indicating that STAT-3 constitutes an important component The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance of the mechanism by which IL-10 exerts its inhibitory effects. In with 18 U.S.C. Section 1734 solely to indicate this fact. contrast to STAT-3, the role of STAT-1 in the regulation of IL- 1 This work was supported by grants from the Ontario HIV Treatment Network 10-mediated biological effects is not known. Macrophages from (OHTN) and the Canadian Institute of Health Research (CIHR) (to A.K.). A.K. is a STAT-1 knockout mice were found to be responsive to IL-10 (21), recipient of the Career Scientist Award from the OHTN. K.G. was supported by a fellowship from the OHTN and a scholarship from the CIHR. W.L. and S.M. were and dominant-negative STAT-1 did not block IL-10-mediated ef- supported by fellowships from the Ontario Graduate Scholarship program and the fects in monocytic cells (17). Ontario Graduate Scholarships in Science and Technology program, respectively. A.A.R.R. was supported by the Ministry of Health, Government of Iran. 2 Address correspondence and reprint requests to Dr. Ashok Kumar, Division of Vi- rology, Research Institute, Children’s Hospital of Eastern Ontario, 401 Smyth Road, 3 Abbreviations used in this paper: SOCS, suppressor of cytokine signaling; IRS-1, Ottawa, Ontario, Canada, K1H 8L1. E-mail address: [email protected] insulin receptor substrate-1; siRNA, short interfering RNA; s, soluble. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 7824 STAT-1 REGULATES IL-10-INDUCED CD14 EXPRESSION Recently, IL-10 was shown to regulate cytokine production by contained Ͻ1% CD2ϩ T cells and CD19ϩ B cells as determined by flow monocytic cells through signaling pathways other than the JAK/ cytometry (28, 37). STAT pathway. For example, IL-10 induced the activation of Cell stimulation PI3K (22, 23) possibly through JAK-1 activation. There is evi- dence to suggest that JAK1 phosphorylates the insulin receptor To determine the effect of p38, p42/44 ERKs, JNK, STAT-3, and PI3K specific inhibitors on IL-10-induced CD14 expression, monocytes (1.0 ϫ substrate-1 (IRS-1) docking molecule following interaction of 106 cells/ml) and HL-60 cells (0.5–1 ϫ 106 cells/ml) were incubated with IL-2, IL-4, IL-10, and IFN-␣ with their corresponding receptors various concentrations of the MEK-1 inhibitor PD98059, the p38 MAPK (23, 24). Subsequently, tyrosine-phosphorylated IRS-1 proteins inhibitor SB202190, the JNK inhibitor SP600125, a peptide (PpYLKTK- may recruit the regulatory p85 subunit of PI3K to the plasma mem- mts) inhibitor specific for STAT-3 (Calbiochem), or the PI3K inhibitor LY294002, for2hin24-well culture plates (Falcon; BD Biosciences). brane through phospho-tyrosine-SH2 domain interactions (25). It Cells were left untreated or stimulated with LPS (1 ␮g/ml) or IL-10 (10 was reported that specific inhibitors of PI3K inhibited the prolif- ng/ml) for 24 h in the presence or the absence of the above-mentioned erative but not the anti-inflammatory activities of IL-10 (22). IL-10 inhibitors. The cells were then analyzed for CD14 expression by flow was also shown to enhance the survival of astrocytes through PI3K cytometry. activation (26). In addition, there is evidence to suggest that IL-10 Flow cytometric analysis can induce the activation of p38 and p42/44 ERK MAPK (23, CD14 expression on monocytes and HL-60 cells was determined by flow 27–29).
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