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

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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 monocytic 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.

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 evidence 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). However, the role of MAPK in IL-10-mediated effects is cytometry as previously described (30). Briefly, cells were stained with not clear at present. PE-conjugated anti-CD14 mAbs (BD Biosciences). Autofluorescence and In contrast to the IL-10-mediated inhibitory effects, there is little isotype-matched control Abs, IgG2b (BD Biosciences), were also included. information regarding the molecular mechanism by which IL-10 The gates were set in accordance with the gates obtained with the isotype- Downloaded from exerts its stimulatory effects. We have previously demonstrated matched control Abs. Data were acquired on a BD Biosciences FACScan flow cytometer and analyzed using the WinMDI version 2.8 software pack- that LPS, a component of Gram-negative bacterial cell walls, in- age (J. Trotter, Scripps Institute, San Diego, CA). duced CD14 expression (30). CD14 is the primary LPS receptor expressed on cells of the monomyelocytic lineage (31). The inter- Western blot analysis action of LPS with CD14 on monocytic cells plays a critical role Phosphorylation of p38, p42/44 ERK and JNK MAPKs, STAT-3, STAT-1, in the activation of these cells during inflammation and sepsis (31). and Akt was determined by Western blot analysis using phospho-MAPKs, http://www.jimmunol.org/ CD14 is one of the few surface receptors whose expression is phospho-STAT, and phospho-Akt specific Abs, respectively, as previously described (37, 38). Primary human monocytes and HL-60 cells were pre- enhanced by IL-10 on human monocytes (30). In this study, we treated with indicated concentrations of inhibitors followed by stimulation investigated the molecular mechanism by which IL-10 induces up- at 37°C for 0–60 min with either 1 ␮g/ml LPS or 10 ng/ml IL-10. regulation of surface CD14 by using primary human monocytes Subsequently, cells were immediately placed in ice followed by lysing the cell and promyelocytic HL-60 cells as model systems. We show for the pellets at 4°C for 45 min with lysis buffer. The protein concentration of the first time that the stimulatory effect of IL-10 on CD14 expression supernatants was determined as per the Bradford method by using the Bio-Rad protein determination assay kit. Total cell proteins were subjected to electro- in monocytic cells does not require STAT-3 activation but rather is phoresis on 10% polyacrylamide SDS gels. The polyvinylidene difluoride regulated by a STAT-1-dependent pathway. Furthermore, IL-10- membranes (Bio-Rad) were probed with either rabbit anti-human phospho-p38 induced STAT-1 activation is mediated by the upstream PI3K ac- (Cell Signaling Technology), mouse anti-human-phospho-p42/44 (Santa Cruz by guest on September 30, 2021 tivity either alone or in concert with ERK MAPK. Biotechnologies), rabbit anti-human phospho-JNK, rabbit anti-human phospho-Akt, rabbit anti-human phospho-STAT-1, and rabbit anti-human phospho-STAT-3 (Cell Signaling Technology) Abs followed by HRP-conjugated Materials and Methods goat anti-rabbit or goat anti-mouse polyclonal Abs (Bio-Rad). The membranes Cell lines, cell culture, and reagents were stripped of the primary Abs and reprobed with rabbit polyclonal Abs specific for the unphosphorylated forms of either p38, p42/44, JNK MAPKs HL-60, a promyelocytic cell line derived from a human acute promyelo- (Santa Cruz), STAT-1, STAT-3, and Akt (Cell Signaling Technology) as cytic leukemia patient, was obtained from the American Type Culture Col- described (37, 38). All immunoblots were visualized by ECL (Amersham lection. Cells were cultured in IMDM (Sigma-Aldrich) supplemented with Biosciences). 10% FBS (Invitrogen Life Technologies), penicillin (100 U/ml), gentami- cin (100 ␮g/ml), HEPES (100 mM), and glutamine (2 mM). PD98059, an Transfection with short interfering RNAs (siRNA) plasmids inhibitor of mitogen-activated protein/ERK kinase-1 kinase that selectively blocks the activity of ERK MAPK (32, 33), was purchased from Calbio- HL-60 cells (0.25 ϫ 106 cells/ml) were cultured in 12-well plates (Falcon) chem. The pyridinyl imidazole, SB202190, a potent and specific inhibitor in serum-free IMDM. Cells were transfected with either 1 ␮g of siRNA of p38 MAPK (33, 34), was also purchased from Calbiochem. SP600125, specific for STAT-1 or control vector (Panomics) using Fugene 6 (Roche a specific JNK inhibitor (BIOMOL), is a reversible ATP competitive in- Applied Biosciences) as described by the manufacturer. The siRNA for hibitor with Ͼ300-fold selectivity over related MAPKs including ERK1 STAT-1 was a mixture of up to three different plasmids. Briefly, the cells and p38 as well as protein kinase A and I␬B kinase-2 (35). LY294002, a were washed once in PBS and then resuspended in serum-free IMDM. potent and specific PI3K inhibitor (36), was also obtained from Calbio- Fugene 6 was allowed to complex with 1 ␮g of the siRNA plasmids in a chem. LPS derived from Escherichia coli 0111:B4 (Sigma-Aldrich), and total volume of 100 ␮l of serum-free IMDM for 15 min before dropwise rIL-10 and IL-10R␣ Abs (R&D Systems) were also purchased. All other addition to the cell culture. Following transfection, cells were incubated in chemicals used for Western blotting were obtained from Sigma-Aldrich. serum-free medium for 6 h and were cultured overnight for an additional 16 h with 2% FCS. IL-10 (10 ng/ml) was added 24 h after transfection, and Monocyte isolation and cell stimulation following an additional 24-h period, the cells were harvested and analyzed PBMCs were prepared from blood obtained from healthy adult volunteers for CD14 expression by flow cytometry. following approval of the protocol by the Ethics Review Committee of the EMSA Children’s Hospital of Eastern Ontario (Ottawa, Ontario, Canada). The PBMCs were isolated by density gradient centrifugation over Ficoll The EMSA was performed as described earlier (28, 37). Briefly, cells har- Hypaque (Amersham Biosciences), as previously described (28). Briefly, vested in Tris-EDTA-saline buffer (pH 7.8) were lysed followed by col- the cell layer containing mononuclear cells was collected and washed three lection of nuclear extracts essentially as described earlier (28, 37). Nuclear times in PBS. Purified, nonactivated monocytes were isolated by negative proteins (5 ␮g) were mixed for 20 min at room temperature with either selection by depletion of T cells and B cells using magnetic polystyrene 32P-labeled STAT-1 or STAT-3 oligonucleotide probes, and the complexes M-450 Dynabeads coated with Abs specific for CD2 (T cells, NK cells) and were subjected to nondenaturing 5% PAGE. The oligonucleotide sequence CD19 (B cells) (Dynal Biotech), as described earlier (28, 37). for STAT-3 was as follows: 5Ј-GAT CCT TCT GGG AAT TCC TAG CD2ϩCD19ϩ cells were separated magnetically from the CD2ϪCD19Ϫ ATC-3Ј (Santa Cruz). To illustrate specificity of NF binding, parallel cells. CD2ϪCD19Ϫ cells were incubated at 37°C for 2 h, following which EMSA reactions were incubated with 50- to 200-fold excess of cold un- nonadherent cells were removed. The adherent mononuclear cells obtained labelled probe. The supershift analyses were also performed to identify the The Journal of Immunology 7825

bands containing STAT-3 by using specific mouse anti-STAT-3 (Santa Cruz) Abs. Briefly, nuclear extracts were incubated with the STAT-3 oligonucleotides in the presence of anti-STAT-3, or control Abs at a final concentration of 0.5–2 ␮g/ml. The bound and unbound 32P-labeled oligonucleotides were resolved by gel electrophoresis as described above. The gel was dried and exposed to x-ray film (Kodak). Results IL-10 mediates LPS-induced CD14 expression in normal human monocytes We confirmed our previous observations (30) that stimulation of primary monocytes with LPS enhanced CD14 expression (Fig. 1). To determine whether endogenously produced IL-10 regulated LPS-induced CD14 expression, we used Abs specific for IL-10R␣ (0.1 ␮g/ml) that are capable of neutralizing the biological activity of IL-10 (10 ng/ml), as described previously (28). Anti-IL-10R␣ Abs inhibited the LPS-induced CD14 expression (Fig. 1), suggesting that endogenously produced IL-10 may mediate LPS-induced CD14 expression in monocytic cells. We have previously demonstrated that LPS-induced IL-10 production is regulated via the ac- Downloaded from tivation of p38 MAPKs (38). To investigate whether endogenously produced IL-10 following LPS stimulation plays a role in up-regulating CD14 expression, IL-10 production was inhibited by using the p38 inhibitor SB202190. As shown previously (38), LPS strongly induced p38 phosphorylation in monocytes, which could be abolished by pretreatment of cells with SB202190 (Fig. 2A). http://www.jimmunol.org/ Pretreatment of monocytes with SB202190 inhibited CD14 expression (Fig. 2B) and concomitant IL-10 production (C) in a dose- dependent manner. In addition, stimulation of normal monocytes FIGURE 2. LPS-induced CD14 expression is mediated by endog- with exogenous IL-10 enhanced CD14 expression (Fig. 3). Un- enously produced IL-10 that is regulated by p38 MAPK activation. A, stimulated cells expressed CD14 at considerable levels, and IL-10 Purified monocytes (1 ϫ 106/ml) were pretreated with varying doses of ␮ stimulation was able to further up-regulate its expression (Fig. 3). SB202190 for 2 h before stimulation with LPS (1 g/ml) for 10 min fol- Taken together, these observations suggest that endogenously pro- lowed by analysis of p38 phosphorylation by Western blotting using anti- phospho-p38 Abs. The membrane was stripped and reprobed with total

anti-p38 Abs to control for equal loading. B, SB202190-treated cells were by guest on September 30, 2021 stimulated with LPS (1 ␮g/ml) for 24 h followed by analysis of CD14 expression by ﬂow cytometry. C, The supernatants harvested from LPS- stimulated cells were analyzed for IL-10 production by ELISA. The results shown are representative of three different experiments performed.

duced IL-10 plays an important role in LPS-induced CD14 expression in monocytic cells.

Role of MAPK in IL-10-induced up-regulation of CD14 expression on primary monocytes and HL-60 cells To understand the molecular mechanism involved in the regulation of IL-10-induced CD14 expression, we examined a number of human monocytic cell lines including THP-1, U937, and HL-60, with a view to reproduce the stimulatory effect of IL-10 on CD14 expression, which could serve as a model system. HL-60 cells alone were found to be responsive to IL-10 and therefore were used to study IL-10-mediated CD14 up-regulation (Fig. 3). It may be noted that HL-60 cells were heterogeneous with respect to CD14 expression and only 5–10% of cells responded by up-regulating CD14 expression in response to stimulation with IL-10 (Fig. 3). The MAPKs play a major role in LPS- and cytokine-mediated induction of several cell surface receptors (39). We have previously demonstrated that IL-10 selectively induced the activation of p42/44 ERK MAPK in human monocytes (28, 37, 38). It was, FIGURE 1. Endogenously produced IL-10 mediates LPS-induced CD14 expression in human monocytes. Purified monocytes (1 ϫ 106/ml) therefore, of interest to identify the members of the MAPK family were stimulated with either LPS (1 ␮g/ml) alone or LPS plus anti-IL-10R␣ that may be involved in IL-10-induced CD14 expression. We first Abs or isotype-matched control Abs (0.1 ␮g/ml) for 24 h followed by flow investigated the involvement of p38 and p42/44 MAPKs by ex- cytometric analysis of CD14 expression. The results shown are represen- amining their activation following stimulation of both monocytes tative of three independent experiments performed. and HL-60 cells with IL-10. IL-10 induced the phosphorylation of 7826 STAT-1 REGULATES IL-10-INDUCED CD14 EXPRESSION

stream substrate for PI3K, and if so, whether the PI3K-speciﬁc inhibitor, LY294002, can inhibit IL-10-induced Akt activation. The results show that IL-10 induced strong phosphorylation of Akt in both monocytes and HL-60 cells at 60 min poststimulation that was inhibited by LY294002 in a dose-dependent manner (Fig. 5, A and B). To determine the role of PI3K in IL-10-induced CD14 expression, both monocytes and HL-60 cells were treated with LY294002 for 2 h before stimulation with IL-10 for 24 h followed by analysis of CD14 expression. Treatment of monocytes and HL-60 cells with increasing concentrations of LY294002 only partially inhibited IL-10-induced CD14 expression even though LY294002 completely inhibited Akt phosphorylation (Fig. 5, B and C). Because partial inhibition of CD14 expression was observed with both ERK- and PI3K-speciﬁc inhibitors, we hypothesized that ERK and PI3K may cooperate to regulate IL-10-induced CD14 expression. For this, both monocytes and HL-60 cells were pretreated with varying concentrations of LY294002 either

alone or in combination with PD98059 for 2 h before stimulation Downloaded from with IL-10 followed by analysis of Akt phosphorylation and CD14 expression. As expected, LY294002 inhibited Akt phosphorylation at low doses (10 mM), and PD98050 did not inﬂuence Akt activation (Fig. 5, B and C). Interestingly, the combination of ERK and PI3K inhibitors even at low concentrations completely inhib-

ited the increase in levels of CD14 expression (Fig. 5C), suggest- http://www.jimmunol.org/ ing that IL-10-induced CD14 expression may be regulated by PI3K and ERK MAPK in a synergistic manner. It may be noted that treatment of cells with a combination of the inhibitors LY294002 and PD98059 at concentrations used did not affect cell viability as determined by trypan blue staining (data not shown). IL-10-induced CD14 expression is not regulated via the FIGURE 3. IL-10 induces CD14 expression in human monocytes and in activation of STAT-3 6 6

ϫ ϫ by guest on September 30, 2021 HL-60 cells. Purified monocytes (1 10 /ml) and HL-60 cells (1 10 / IL-10 has been shown to regulate its effects primarily through the ml) were stimulated with IL-10 (10 ng/ml) for 24 h followed by analysis of activation of STAT-3 (13, 14, 16, 17). Therefore, it was important CD14 expression by flow cytometry. The results shown are representative of five different experiments performed. to determine whether IL-10-induced CD14 expression is regulated by STAT-3 activation directly or indirectly through the ERK/ PI3K-mediated activation. For this, cells were treated with various ERK MAPK from 10 to 30 min poststimulation in monocytes and concentrations of PD98059 either alone or in combination with HL-60 cells (Fig. 4 and data not shown). Furthermore, the ERK- different concentrations of LY294002 for 2 h before stimulation specific inhibitor PD98059 completely inhibited IL-10-induced ac- with IL-10. PD98059 either alone or in combination with tivation of p42/44 MAPKs (Fig. 4). In contrast, IL-10 failed to LY294002 at any concentration did not down-regulate STAT-3 induce phosphorylation to a significant level of either p38 (Fig. 4B) phosphorylation in either monocytes or HL-60 cells (Fig. 6). or JNK MAPKs (data not shown) in both monocytes and HL-60 To determine the involvement of STAT-3 in IL-10-induced cells. CD14 expression, we used a specific peptide inhibitor (PpYLKTK- To determine the role of ERK MAPK in IL-10-induced CD14 mts) of STAT-3. The PpYLKTK-mts peptide mediates its effect by expression, monocytes and HL-60 cells were pretreated with preventing dimerization of STAT-3 proteins and subsequent trans- PD98059 for 2 h before IL-10 stimulation for 24 h followed by location into the nucleus (40). The biological activity of the analysis of CD14 expression. PD98059 partially inhibited CD14 PpYLKTK-mts peptide was measured by examining the STAT-3 expression in monocytes (mean channel fluorescence from 347 to translocation into the nucleus following stimulation with IL-10 by 320) and in HL-60 cells (mean channel fluorescence from 248 to performing a gel shift assay. IL-10 induced maximum STAT-3 214) (Fig. 4). As a negative control, treatment with SB202190 did activation at 2–4 h poststimulation (Fig. 7A). IL-10 stimulation not affect CD14 expression in monocytes (Fig. 4A) and HL-60 resulted in the induction of a single band whose specificity was cells (data not shown). confirmed by cold competition with STAT-3 oligonucleotides, which specifically inhibited this band, whereas unlabeled nonspe- Synergistic action of PI3K and ERK MAPK in IL-10-induced cific oligonucleotides did not affect the intensity of this band. In CD14 expression addition, treatment of nuclear extracts obtained from IL-10-stim- The above observations suggest that IL-10-induced CD14 expres- ulated cells with anti-STAT-3 Abs resulted in abrogation of this sion may be regulated by signaling molecules other than the ERK band. HL-60 cells were pretreated with the PpYLKTK-mts peptide MAPK either independently or in synergy with the ERK MAPKs. inhibitor for 2 h before stimulation with IL-10 for 2 h followed by IL-10 has been shown to activate the PI3K pathway, which has analysis of STAT-3 induction. The PpYLKTK-mts peptide inhib- been suggested to regulate the biological effects of IL-10 (22, 23). ited STAT-3 induction following IL-10 stimulation in a dose-de- To investigate the role of PI3K in IL-10-induced CD14 expression, pendent manner (Fig. 7A). To determine the effect of PpYLKTK- we first determined whether IL-10 can activate Akt, the down- mts peptide on IL-10-induced CD14 expression, HL-60 cells were The Journal of Immunology 7827

FIGURE 4. IL-10-induced CD14 expression is partially dependent on p42 ERK MAPK activation. A, Pu- riﬁed monocytes (1 ϫ 106/ml) were pretreated with varying doses of either SB202190 (SB) or PD98059 (PD) for 2 h before stimulation with IL-10 (10 ng/ml) for 10 min. Cell lysates were analyzed for phosphorylation of p38 or p42 MAPK by Western blot analysis by anti-phospho-p38 or -p42/44 Abs, respectively, as described in the legend to Fig. 2 (upper panel). The cells pretreated with MAPK inhibitors were also stimulated with IL-10 (10 ng/ml) for 24 h and analyzed for CD14 expression by ﬂow cytometry (lower panel). B, HL-60 cells were stimulated with IL-10 for 5–60 min, and the

cell lysates were analyzed for p38 and p42 MAPK ac- Downloaded from tivation by Western blotting with phosphospeciﬁc Abs (upper left panel). HL-60 cells were pretreated with varying doses of PD98059 (10–50 ␮M) for 2 h before IL-10 stimulation for 10 min followed by Western blotting analysis for phospho-p42 ERK (lower left panel). Cells pretreated with PD98059 were also analyzed for

CD14 expression by flow cytometry after IL-10 stimu- http://www.jimmunol.org/ lation for 24 h (right panel). The results shown are representative of three different experiments performed. by guest on September 30, 2021 treated with varying concentrations of PpYLKTK-mts peptide for phosphorylation in response to IL-10, even though STAT-3 phos- 2 h before stimulation with IL-10. The PpYLKTK-mts peptide did phorylation was observed in the same lysates (Fig. 8B). not affect CD14 expression at any concentration in HL-60 cells To more directly determine whether IL-10 stimulates CD14 ex- (Fig. 7B) or monocytes (data not shown). pression through STAT-1 activation, HL-60 cells were transfected with either STAT-1 siRNA plasmids or a control plasmid. First, IL-10-induced CD14 expression involves the activation of STAT-1 we determined whether transfection with STAT-1 siRNA plasmid In addition to STAT-3, IL-10 has been shown to activate STAT-1 affected the expression and the phosphorylation of STAT-1 in IL- through the JAK signaling pathway (14, 15). However, the role of 10-stimulated cells. Cells transfected and cultured for 24 h were STAT-1 in IL-10-mediated biological effects is not known. Be- stimulated with IL-10 for 30 min and analyzed for STAT-1 ex- cause IL-10-induced CD14 expression was not found to be regu- pression and STAT-1 phosphorylation. Transfection of HL-60 lated by STAT-3 activation, we hypothesized that IL-10 may stim- cells with STAT-1 siRNA plasmids inhibited both IL-10-induced ulate CD14 expression through ERK/PI3K-mediated activation of STAT-1 phosphorylation as well as STAT-1 expression compared STAT-1. Using a similar experimental design, cells were treated with the cells transfected with a control plasmid (Fig. 8C). As a with various concentrations of PD98059 either alone or in combi- loading control and to determine specificity, transfection of cells nation with different concentrations of LY294002 for 2 h before with STAT-1 siRNA did not reduce STAT-3 expression. To de- stimulation with IL-10, following which cells were analyzed for termine whether STAT-1 inactivation influenced CD14 expres- STAT-1 phosphorylation. LY294002 inhibited IL-10-induced sion, cells were analyzed for CD14 expression 24 h after transfec- STAT-1 phosphorylation in both monocytes and HL-60 cells. Fur- tion by flow cytometry. IL-10 failed to stimulate CD14 expression thermore, STAT-1 phosphorylation was completely inhibited in cells transfected with STAT-1 siRNA plasmid compared with when LY294002 was used in combination with PD98059 in a the cells transfected with the control plasmid (Fig. 8D). dose-dependent manner (Fig. 8A). These results suggest that IL- 10-induced STAT-1 activation may be mediated by the synergistic Discussion action of PI3K and ERK. In general, IL-10-mediated inhibitory effects on cell proliferation Recently, phosphorylation of STAT-1 on serine residue 727 was and synthesis of proinflammatory cytokines have been shown to be implicated in STAT-1-mediated effects (41, 42). To determine regulated through STAT-3 activation (13, 14, 16, 17). However, in whether serine 727 phosphorylation was induced by IL-10 in contrast to STAT-3, the role of STAT-1 in regulating IL-10-me- monocytic cells, we treated purified monocytes and HL-60 cells diated biological effects remains to be established. In this study, we with IL-10 for 0–60 min. Neither monocytes (left panel) nor investigated the signaling pathways involved in the IL-10-induced HL-60 cells (right panel) showed any induction of serine 727 stimulatory effect on CD14 expression in human monocytic cells. 7828 STAT-1 REGULATES IL-10-INDUCED CD14 EXPRESSION

FIGURE 5. PI3K and p42-MAPK synergistically induce CD14 expression in response to IL-10 in monocytic cells. A, Puriﬁed monocytes (1 ϫ 106/ml) and HL-60 cells (0.5 ϫ 106/ml) were stimulated with IL-10 (10 ng/ml) for 5–60 min. B and C, Monocytes (B) and HL-60 cells (C) were pretreated with varying doses of

LY294002 followed by stimulation with IL-10 for 10 Downloaded from min in monocytes and 30 min in HL-60 cells (upper blots in B and C). Cell lysates were analyzed for Akt phosphorylation by using anti-phospho-Akt Abs as described in the legend to Fig. 2. To determine the synergistic effect of PI3K and p42 ERK MAPK activation, cells were pretreated with either LY294002 or PD98059 alone or in combination for 2 h followed by IL-10 (10 http://www.jimmunol.org/ ng/ml) stimulation for 24 h. CD14 expression was analyzed by ﬂow cytometry (lower panel in B and C). The results shown are representative of three different experiments performed. by guest on September 30, 2021

Our results show for the first time that IL-10-induced CD14 ex- vations that IL-10 enhances sCD14 levels (30), LPS-induced pression is mediated by a mechanism involving STAT-1 activation CD14 expression through endogenously produced IL-10 suggests independent of STAT-3. Furthermore, IL-10-activated STAT-1 is that IL-10 may act as an anti-inflammatory agent by neutralizing regulated through PI3K activation either alone or in concert with endotoxin, and hence decreasing host susceptibility to bacterial the ERK MAPKs. infections (49). LPS stimulates monocytic cells through its association with the The signaling molecules involved in IL-10-induced CD14 ex- plasma LPS-binding protein, following which the LPS/LPS-bind- pression are not known. Interaction of IL-10 with its receptor com- ing protein complex interacts with the CD14/Toll receptor complex initiates a cascade of events leading to the tyrosine phosphor- plex expressed on surface membrane (43). The levels of CD14 ylation of JAK1 and Tyk2 tyrosine kinases that is followed by the expression on monocytic cells may determine their responsiveness recruitment and phosphorylation of STAT-3 and STAT-1 (13, 14). to the synthesis of proinflammatory cytokines and costimulatory Binding of STAT-3 to the phosphotyrosine residues of the acti- molecules. CD14 transgenic mice were found to be hypersensitive vated IL-10R complex is required for STAT-3 activation and to endotoxins (44), suggesting a critical role for CD14 in the im- dimerization, and consequent manifestation of a number of bio- munopathogenesis of sepsis. In addition, elevated levels of circu- logical effects of IL-10 (14, 16, 17). We provide evidence that lating soluble CD14 (sCD14) were observed in inflammatory con- IL-10-induced CD14 expression is mediated not through STAT-3, ditions such as systemic lupus erythematosus, chronic active but through STAT-1 activation in monocytic cells. STAT-1 plays hepatitis, and septic shock (45–47). Enhanced levels of sCD14 in a critical role in IFN-␥-mediated biological effects such as anti- plasma and other body fluids may neutralize the endotoxin content proliferative responses, immune surveillance, and tumor suppres- in septic patients (48). In this study, we show that LPS-induced sion (50). The role of STAT-1 in biological responses has been CD14 expression on monocytic cells may be regulated, at least in studied by using STAT-1Ϫ/Ϫ mice. Although STAT-1Ϫ/Ϫ mice part, by the endogenously produced IL-10. In view of the obser- develop normally, they fail to elicit biological responses to IFN-␥ The Journal of Immunology 7829 Downloaded from

FIGURE 6. p42 ERK MAPK- or PI3K-specific inhibitors do not affect IL-10-induced STAT-3 activation. Purified monocytes (A) and HL-60 cells (B) were pretreated with varying doses of either LY294002 or PD98059 alone or in combination for 2 h before stimulation with IL-10 (10 ng/ml) for 10 min in monocytes and 30 min in HL-60 cells. Cell lysates were http://www.jimmunol.org/ analyzed for STAT-3 phosphorylation by using anti-phospho-STAT-3 Abs as described in the legend to Fig. 2. The results shown are representative of three different experiments performed or IFN-␣ (21, 51), exhibit impaired development of regulatory T cells (52), and develop tumors rapidly and with greater frequency following challenge with chemical carcinogens (53). However, macrophages from STAT-1Ϫ/Ϫ mice were shown to be responsive by guest on September 30, 2021 to IL-10 (21), and overexpression of dominant-negative STAT-1 did not block IL-10-induced effects in a macrophage cell line (17), suggesting that STAT-1 may not be involved in IL-10-mediated biological effects. However, these studies did not fully address the role of STAT-1 in IL-10-mediated biological effects. Our results show that IL-10-induced up-regulation of CD14 is mediated by STAT-1 through the activation of PI3K in monocytic cells. This was demonstrated first by using the PI3K and ERK MAPK inhibitors, LY294002 and PD98059, respectively, which inhibited IL- 10-induced STAT-1 phosphorylation without affecting STAT-3 FIGURE 7. IL-10-induced CD14 expression is not regulated via activation and consequently CD14 expression. Second, STAT-1 STAT-3 activation. A, HL-60 cells were treated with IL-10 (10 ng/ml) for siRNA inhibited IL-10-induced CD14 expression. Finally, 0–240 min, and the nuclear extracts were analyzed for STAT-3 activation LY294002 and PD98059 inhibited STAT-1 binding to its binding by EMSA (left panel). The nuclear extracts were incubated with unlabelled site in the CD14 promoter (data not shown). STAT-3-specific (CC) or with nonspecific (NS) oligonucleotides (middle Recently, it has been shown that JAK proteins can also activate left panel). Nuclear extracts were also incubated with anti-STAT-3 Ab for other signaling molecules especially from the PI3K family. For supershift analysis (middle right panel). HL-60 cells were pretreated with a STAT-3-specific inhibitor peptide (PpYLKTK-mts) at two different con- example, JAK1 activated by either IL-4, IFN-␣, IL-10, or Oncosta- centrations for 2 h before IL-10 (10 ng/ml) stimulation for2h(right panel). tin-M activates the p85 subunit of PI3K through tyrosine phos- The STAT-3-specific band is indicated by the arrow. B, HL-60 cells were phorylation of the IRS-1 docking molecule (23, 24). In this study, pretreated with a STAT-3-specific inhibitor peptide (PpYLKTK-mts) at IL-10 stimulation of monocytic cells induced the activation of the varying doses for 2 h before IL-10 (10 ng/ml) stimulation for 24 h. CD14 survival enzyme, Akt-1, the signaling molecule downstream of expression was analyzed by flow cytometry. The results are expressed as PI3K. It has been suggested that Akt activation by IL-10 is re- mean channel fluorescence of two experiments performed. quired to promote cell proliferation and cell survival, but not to suppress the synthesis of the proinflammatory cytokine TNF-␣ (22, 26). Our studies showing the involvement of PI3K in IL-10- role of PI3K and/or STAT-1 as a key mediator of IL-10-induced induced CD14 expression suggest that PI3K may be involved in stimulatory effects. the stimulatory effects of IL-10 and further support the notion that The observations that IL-10 induced STAT-1 activation via IL-10 stimulation may involve the activation of distinct signaling PI3K suggest that IL-10-induced PI3K may directly interact and pathways that may determine either an inhibitory or a stimulatory contribute to STAT-1 activation. Similar interactions have been outcome. However, additional studies are needed to address the reported for activation of STAT-1 and STAT-3 by PI3K in IFN 7830 STAT-1 REGULATES IL-10-INDUCED CD14 EXPRESSION

signaling. The p85 subunit of PI3K was shown to bind tyrosine- phosphorylated STAT-3 through p85 SH2 domains in IFN-␣-activated cells (54). STAT-3 bound a conserved sequence at Tyr527 and Tyr538 of IFN-R type I and underwent IFN-dependent phosphorylation at residues Tyr656 and Tyr705. The p85 subunit bound to the phosphorylated STAT-3 recruited the catalytic p110 subunit of PI3K, an enzyme known to activate a series of serine kinases. This interaction induces activation of PI3K and promotes serine phosphorylation of STAT-3, which is critical for the formation of stable STAT-3 homodimers and their translocation into the nuclei (54). Similarly, PI3K and its effector kinase, Akt, have been shown to play an important role in the serine phosphorylation of STAT-1 (serine 727) and in the activation of IFN-␥-responsive genes (55). The ﬁnding that IL-10 selectively induced ERK MAPK activation in both monocytes and HL-60 cells was unexpected. Whether IL-10-induced activation of ERK MAPKs has a broader role in IL-10 signaling is not clear. IL-10 has been shown to activate p38 and ERK MAPKs in murine macrophages that were implicated in

the expression of heme oxygenase (27) and apoptosis (23), respec- Downloaded from tively. The upstream signaling events required in the activation of ERK MAPK by IL-10 are not known. The signaling molecules and the transcription factors involved in the regulation of CD14 transcription are not well understood. The human CD14 promoter has been cloned and the sequence

responsible for transcription in response to LPS have been eluci- http://www.jimmunol.org/ dated (56). CD14 expression was shown to be regulated primarily by the Sp-1 transcription factor in human monocytic cells (56). Analysis of the CD14 promoter sequence revealed the presence of a consensus STAT-1 binding site (56). Consistent with this analysis, our results suggest that IL-10-induced CD14 expression is regulated by the STAT-1 transcription factor (data not shown). Although we demonstrate a role for STAT-1 in IL-10-induced CD14 induction, the role of other transcription factors, particularly Sp-1, cannot be ruled out. STAT-1 has been shown to cooperate by guest on September 30, 2021 physically and functionally with coactivator proteins such as CBP/ p300, BRCA1, Sp-1, AP-1, and NF␬B (50, 57–59). Whether STAT-1 induces CD14 expression in response to IL-10 stimulation through cooperative action with Sp-1 remains to be understood. In summary, we demonstrate a previously unrecognized role of STAT-1 in IL-10-induced signaling in human monocytic cells. IL-10 has been shown to mediate its inhibitory effects in general through the activation of STAT-3. However, there is emerging evidence from several laboratories that the STAT-3-dependent pathway is not sufficient to mediate the anti-inflammatory effects of IL-10. Furthermore, the molecular mechanisms regulating the stimulatory effects of IL-10 on various cell types including B cells and monocytic cells are poorly understood. Taken together, our results point to the key role for STAT-1 and its activation via the PI3K/ERK MAPKs in the regulation of IL-10 stimulatory effects on CD14 expression in human monocytic cells. These studies may provide a basis for the identification of molecular players in IL-10 signaling pathways that may help in designing targeted drug ther- FIGURE 8. IL-10-induced CD14 expression is dependent on PI3K- and apy for inflammatory diseases. ERK MAPK-induced STAT-1 activation. A, Purified monocytes (upper panel) and HL-60 cells (lower panel) were pretreated with varying doses of either LY294002 or PD98059 alone or in combination for 2 h before stimulation with IL-10 (10 ng/ml) for 10 min in monocytes and 30 min in for STAT-1 or control plasmids. Following 24 h of transfection, cells were HL-60 cells. Cell lysates were analyzed for STAT-1 phosphorylation using stimulated with IL-10 (10 ng/ml) for 30 min, following which lysates were anti-phospho-STAT-1 Abs as described above. B, Purified monocytes (left analyzed for STAT-1 phosphorylation and expression of STAT-1 and panel) or HL-60 cells (right panel) were treated with IL-10 for 0–60 min. STAT-3 by Western blotting by using anti-phospho-STAT-1, anti- Cell lysates were analyzed for serine 727 phosphorylation through the use STAT-1, and anti-STAT-3 Abs, respectively. D, HL-60 cells were tran- of rabbit anti-phospho-STAT-1 serine 727-specific Abs. The blots were siently transfected with vectors containing STAT-1 siRNAs or control vec- stripped and reprobed with anti-phospho-STAT-3 Abs as a control for tors. Following 24 h of transfection, cells were stimulated with IL-10 (10 IL-10 activity and with anti-STAT-3 Abs as a loading control. C, HL-60 ng/ml) followed by analysis of CD14 expression. The results shown are cells were transiently transfected with vectors containing siRNAs specific representative of three different experiments performed. The Journal of Immunology 7831

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