Signaling and IL-6 Release 14/16 Α Pertussis Toxin-Insensitive G THP-1

CCR1-Mediated STAT3 Tyrosine Phosphorylation and CXCL8 Expression in THP-1 Macrophage-like Cells Involve Pertussis Toxin-Insensitive G α14/16 This information is current as Signaling and IL-6 Release of September 25, 2021. Maggie M. K. Lee, Ricky K. S. Chui, Issan Y. S. Tam, Alaster H. Y. Lau and Yung H. Wong J Immunol published online 2 November 2012 http://www.jimmunol.org/content/early/2012/10/31/jimmun Downloaded from ol.1103359

Supplementary http://www.jimmunol.org/content/suppl/2012/11/02/jimmunol.110335 http://www.jimmunol.org/ Material 9.DC1

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 2, 2012, doi:10.4049/jimmunol.1103359 The Journal of Immunology

CCR1-Mediated STAT3 Tyrosine Phosphorylation and CXCL8 Expression in THP-1 Macrophage-like Cells Involve Pertussis Toxin-Insensitive Ga14/16 Signaling and IL-6 Release

Maggie M. K. Lee,*,† Ricky K. S. Chui,*,† Issan Y. S. Tam,‡ Alaster H. Y. Lau,‡ and Yung H. Wong*,†,x

Agonists of CCR1 contribute to hypersensitivity reactions and atherosclerotic lesions, possibly via the regulation of the transcrip-

tion factor STAT3. CCR1 was demonstrated to use pertussis toxin-insensitive Ga14/16 to stimulate phospholipase Cb and NF-kB, whereas both Ga14 and Ga16 are also capable of activating STAT3. The coexpression of CCR1 and Ga14/16 in human THP-1 macrophage-like cells suggests that CCR1 may use Ga14/16 to induce STAT3 activation. In this study, we demonstrated that 705 727 a CCR1 agonist, leukotactin-1 (CCL15), could indeed stimulate STAT3 Tyr and Ser phosphorylation via pertussis toxin- Downloaded from insensitive G proteins in PMA-differentiated THP-1 cells, human erythroleukemia cells, and HEK293 cells overexpressing CCR1 705 727 and Ga14/16. The STAT3 Tyr and Ser phosphorylations were independent of each other and temporally distinct. Subcellular fractionation and confocal microscopy illustrated that Tyr705-phosphorylated STAT3 translocated to the nucleus, whereas Ser727-

phosphorylated STAT3 was retained in the cytosol after CCR1/Ga14 activation. CCL15 was capable of inducing IL-6 and IL-8 (CXCL8) production in both THP-1 macrophage-like cells and HEK293 cells overexpressing CCR1 and Ga14/16. Neutralizing Ab 705 to IL-6 inhibited CCL15-mediated STAT3 Tyr phosphorylation, whereas inhibition of STAT3 activity abolished CCL15- http://www.jimmunol.org/

activated CXCL8 release. The ability of CCR1 to signal through Ga14/16 provides a linkage for CCL15 to regulate IL-6/ STAT3–signaling cascades, leading to expression of CXCL8, a cytokine that is involved in inﬂammation and the rupture of atherosclerotic plaque. The Journal of Immunology, 2012, 189: 000–000.

hemokines are a large family of low-molecular-weight mature cells. CCR1 knockout mice are defective in the trafﬁcking cytokines that are characterized by their ability to direct and proliferation of myeloid progenitor cells (4). the migration of leukocytes from the bloodstream to sites of Hematopoiesis and angiogenesis require transcriptional activa-

C by guest on September 25, 2021 inflammation (1). Leukotactin-1 (CCL15) belongs to the CC sub- tion, which can be mediated by STAT3. STAT3 is involved in T cell family, one of four chemokine groups (CXC, CC, C, and CX3C), as proliferation induced by IL-6 (11), c-Kit–mediated stem cell factor defined by their primary structures. CCL15 exerts its effect mainly (SCF)-independent proliferation in human leukemia cells (12), via CCR1 (2, 3), which is a G protein-coupled receptor. In addition and vessel formation triggered by GM-CSF (13). Deletion of to mediating chemotaxis, CCL15 and CCR1 were shown to regulate STAT3 is embryonic lethal (14). In addition, STAT3 acts as a hematopoiesis (4), angiogenesis (5), mast cell activation (6), and negative regulator of inflammatory responses in hematopoietic inflammatory diseases, including atherosclerosis (7, 8). CCR1 is cells. Tissue-specific deletion of STAT3 in macrophages enhances expressed in myeloid progenitor cells (9) and endothelial cells (10), the production of inflammatory cytokines (15), whereas disruption both of which are capable of proliferating and differentiating into of STAT3 during hematopoiesis leads to severe inflammatory bowel disease (16). In addition, expression of RANTES/CCL5 (a CCR1 agonist) is regulated, in part, by a transcription complex of STAT3 and NF-kB (17). *Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China; †Biotechnology Research Institute, Hong Although chemokine receptors are typically characterized as Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Gi-coupled receptors, there is substantial evidence to suggest that ‡ China; School of Biomedical Sciences, Faculty of Medicine, Chinese University of chemokines may be able to stimulate STAT3 activity through Hong Kong, Hong Kong, China; and xState Key Laboratory of Molecular Neurosci- ence, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, pertussis toxin (PTX)-insensitive G proteins. CCR1 agonists were Hong Kong, China found to induce gene expression of the STAT-inducible proto- Received for publication November 23, 2011. Accepted for publication September oncogene, c-fos (18). c-Fos expression and transcriptional acti- 28, 2012. vation is induced upon Ga16 activation (19), and constitutively This work was supported in part by grants from the Research Grants Council of Hong active mutants of Ga14 and Ga16 were demonstrated to enhance Kong (HKUST 1/06C, 663108, and AoE/B-15/01) and the Hong Kong Jockey Club. the activity of STAT3 in cotransfection systems (20, 21). The Address correspondence and reprint requests to Prof. Yung H. Wong, Division of Life coexistence of Ga and CCR1, as well as their demonstrated Science, Hong Kong University of Science and Technology, Clear Water Bay, Kow- 14/16 loon, Hong Kong, China. E-mail address: [email protected] functional coupling in THP-1 macrophage-like cells (2, 22–26), The online version of this article contains supplemental material. suggests that CCR1 may use Ga14/16 to stimulate STAT3. There is Abbreviations used in this article: bFGF, basic fibroblast growth factor; EGF, epider- increasing evidence to support a role for STAT3 activation in mal growth factor; HEL, human erythroleukemia; MMP, matrix metalloproteinase; CCR1-mediated cellular responses. In human macrophages and PLCb, phospholipase Cb; PTX, pertussis toxin; SCF, stem cell factor; siRNA, small macrophage-derived foam cells, CCL15 promotes the release of interfering RNA. matrix metalloproteinase (MMP)-9 (27), which is implicated in Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 the progression of atherosclerosis and whose expression is regu-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103359 2 INDUCTION OF CXCL8 BY CCR1/Ga14/16 REQUIRES IL-6/STAT3 lated by STAT3 (28). Moreover, CCR1 was shown to mediate IL-6 Cells were transiently transfected with cDNAs encoding p-STAT3–TA– production in marrow stromal cells upon stimulation by human Luc, STAT3 or its phosphorylation-resistant mutants (500 ng) or wild-type or constitutively active forms of Ga (625 ng) using Lipofectamine Plus myeloma cells (29), whereas MCP-1/CCL2 (a CCR2 agonist) reagent. enhances IL-6 production in fibroblast-like synoviocytes from patients with rheumatoid arthritis, and the response is mediated, in Luciferase reporter assay part, via PTX-insensitive G proteins (30). In addition, in THP-1 After 24 h of transfection, cells were serum starved with 100 ng/ml PTX for macrophage-like cells, lipoprotein (31) and human placenta 4 h and then stimulated in the absence or presence of 10 nM CCL15 for extracts (32) were demonstrated to promote the expression of another 24 h. Subsequently, the assay medium (culture medium without FBS) was removed and replaced by 150 ml lysis buffer provided in the IL-8 (CXCL8), which is capable of inducing MMP expression Luciferase Reporter Gene Assay Kit (Roche Applied Science, Penzberg, (33). Interestingly, both IL-6 (34) and CXCL8 (35) are STAT3- Upper Bavaria, Germany). The 6-well plate was shaken at 4˚C for 30 min, regulated cytokines, and we recently demonstrated that activa- and 25 ml lysate was transferred to white 96-well microplates designed for tion of Ga can lead to STAT3 activation and upregulation of luminescent work (Nunc, Roskilde, Denmark). An additional 25 ml lysis 16 buffer and 25 ml luciferin substrate were added to each well to initiate the CXCL8 via IL-6 autocrine signaling in HEK293 and human Jurkat reaction. Luciferase activity was determined using a microplate lumino- T cells (36). Given the importance of STAT3 in hematopoiesis and meter LB96V (EG&G Berthold, Bad Wildbad, Germany) (20, 21). its purported involvement in inflammatory diseases, we explored Assay for STAT3 phosphorylation whether CCR1 can indeed induce STAT3 phosphorylation and the production of IL-6 and CXCL8 through Ga -mediated sig- HEL cells, as well as THP-1 and U-937 macrophage-like cells, were seeded 14/16 3 6 naling. Mapping of such a pathway will help to elucidate the in- at 1 10 cells in assay medium (culture medium containing 0.1% BSA instead of FBS) and cultured or not with 100 ng/ml PTX for 16 h. Cells tricate interplay between chemokines and cytokines in their were treated with cycloheximide for 2 h or with Stattic, IL-6, or CXCL8- Downloaded from regulation of complex diseases, such as atherosclerosis and neutralizing Ab for 30 min and then incubated in the absence or presence rheumatoid arthritis. of chemokines at 10 nM for specific durations at 37˚C. Subsequently, cells were lysed in 100 ml lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM Materials and Methods NaCl, 5 mM EDTA, 40 mM NaP2O7, 1% Triton X-100, 1 mM DTT, 200 mMNa3VO4, 200 mM PMSF, 4 mg/ml aprotinin, and 0.6 mg/ml leupeptin) Materials and then shaken at 4˚C for 30 min. Supernatants were collected by cen-

3 http://www.jimmunol.org/ The cDNAs encoding human CCR1, wild-type and constitutively active trifugation at 16,000 g for 8 min. HEK293 cells were seeded on six-well 3 5 forms of Ga, and STAT3, STAT3Y705F, STAT3S727A, and p-STAT3–TA– plates at a density of 5 10 cells/well, serum-starved in MEM, and lysed Luc were obtained as previously described (20, 21, 25, 26, 37). HEK293 in 250 ml lysis buffer. Protein concentration was determined by DC protein cells (ATCC CRL-1573), as well as human monocytic THP-1 (ATCC TIB- assay kit (Bio-Rad, Hercules, CA). Eighty micrograms of proteins of each 202) and U-937 cells (ATCC CRL-1593.2), were purchased from Ameri- lysate was resolved by 12% SDS-PAGE and transferred to nitrocellulose can Type Culture Collection (Rockville, MD). Human erythroleukemia membrane by electroblotting. Abs against phosphorylated STAT3 were (HEL) cells (ACC 11) were from the German Collection of Micro- used for the recognition of their respective phosphorylations. Fluoro- organisms and Cell Cultures (Braunschweig, Germany). Lipofectamine graphs were visualized with a chemiluminescence detection kit (Amersham Plus reagent, Alexa Fluor 488 anti-mouse IgG, Alexa Fluor 555 anti-rabbit Pharmacia Biotech, Piscataway, NJ). Signal intensities of the immunoreactive bands were quantified using Image J software, version 1.38x (Na- IgG, Stealth Select RNAi for Ga14 or Ga16, and Stealth RNAi negative control were purchased from Invitrogen (Carlsbad, CA). PTX was obtained tional Institutes of Health, Bethesda, MD). by guest on September 25, 2021 from List Biological Laboratories (Campbell, CA). CCL15 (68 aa), mye- Nuclear/cytosol fractionation loid progenitor inhibitory factor-1 (CCL23) (75 aa), and human IL-6 and human CXCL8 neutralizing Abs were purchased from R&D Systems CCR1/293 cells, CCR1/Ga14/293 cells, or HEK293 cells transiently (Minneapolis, MN). Cycloheximide, STAT3 inhibitor V (Stattic), and transfected with wild-type or constitutively active forms of Ga were col- MEK1/2 kinase inhibitor U0126 and its inactive analog U0124 were ob- lected, and whole-cell lysates were subjected to nuclear/cytosol fraction- tained from Calbiochem (San Diego, CA). Abs used in the study were ation according to the manufacturer’s protocol (Biovision, Mountain View, products of Cell Signaling Technology (Beverly, MA). CA). Briefly, 1 3 107 cells was resuspended in cytosol extraction buffer 3 Cell culture and centrifuged at 16,000 g for 5 min to collect the supernatant (cytosolic fraction). The resulting pellet was resuspended in nuclear extraction U-937 and HEL cells were cultured in RPMI 1640 medium supplemented buffer and centrifuged again at 16,000 3 g for 10 min to collect the su- with 10% FBS, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM L-glu- pernatant representing the nuclear fraction. The concentration of protein tamine, and 1 mM sodium pyruvate. THP-1 cells were cultured in the same was determined using a DC protein assay kit (Bio-Rad). Eighty micro- medium supplemented with 55 mM 2-ME. THP-1 and U-937 cells were grams of proteins was separated in 12% SDS-PAGE and transferred to differentiated into macrophage-like cells by treatment with 20 nM PMA nitrocellulose membrane by electroblotting. Abs against caspase-3 and for 48 h. HEK293 cells were cultured in MEM supplemented with 10% CREB were used as markers for cytosolic and nuclear compartments, re- FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin. Primary human mast spectively. Fluorographs were visualized with a chemiluminescence de- cell cultures were prepared from peripheral blood CD34+ progenitor using tection kit (Amersham Pharmacia Biotech). a modified protocol of Lappalainen et al. (38). Briefly, fresh peripheral 705 blood buffy coat of healthy adult donors was provided by the Hong Kong Confocal microscopy of STAT3 Tyr phosphorylation Red Cross. Mononuclear cells were separated from peripheral blood buffy CCR1/293 and CCR1/Ga /293 cells grown on coverslips were fixed with coat by density-gradient centrifugation through Ficoll-Paque Plus (GE 14 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, labeled Medical System). CD34+ progenitors were isolated from the mononuclear with rabbit phosphorylated Tyr705 STAT3 Ab and mouse STAT3 Ab, and cell suspension using a MACS system (Miltenyi Biotec), according to the stained with Alexa Fluor 555 anti-rabbit IgG and Alexa Fluor 488 anti- manufacturer’s instructions. The CD34+ progenitors were cultured in a mouse IgG. Nuclei were stained with DAPI. Confocal microscopy was medium based on IMDM containing SCF for $6 wk, with IL-3, IL-9, IL-6 performed with a Zeiss LSM 510 META, and images were deconvoluted and IL-4 added to the culture medium sequentially for different periods of with LSM Image Browser Rel. 4.2 (Carl Zeiss, Oberkochen, Germany). time. Cells were incubated at 37˚C in a humidified atmosphere of 5% CO2 and 95% air. Cytokine detection Establishment of stable cell lines and transfections Culture supernatants from THP-1 macrophage-like cells or HEK293 stable HEK293 cells overexpressing CCR1 alone (CCR1/293) or expressing cell lines stimulated with CCL15 were harvested, and the presence of CCR1 together with Ga14 (CCR1/Ga14/293) or Ga16 (CCR1/Ga16/293) cytokines, including basic fibroblast growth factor (bFGF), epidermal were generated as described previously (23, 26). Knockdown of Ga14 and growth factor (EGF), G-CSF, GM-CSF, IFNs (IFN-a, IFN-b, IFN-g), IL-2, Ga16 in THP-1 macrophage-like cells by transfection of small interfering IL-4, IL-6, CXCL8, and TNF-a, were analyzed using the Procarta cytokine RNA (siRNA) against Ga14 and Ga16 was performed as described previ- assay kit (Affymetrix, Santa Clara, CA). Briefly, 50 ml Ab beads was ously (25). HEK293 cells, CCR1/Ga14/293 cells, and CCR1/Ga16/293 added to the prewet 96-well microtiter plate. Then, 50 ml each standard cells were seeded on six-well plates at a density of 3 3 105 cells/well. or test sample was added to the wells, followed by 25 ml detection Ab The Journal of Immunology 3 mixture. The samples were then incubated for 30 min at 25˚C with gentle shaking, followed by three rounds of gentle rinsing to wash away unbound Abs. Finally, 50 ml streptavidin-PE was added and incubated for 30 min. The beads were then washed again and resuspended in 120 ml reading buffer. Analysis was performed with a Bio-Plex 200 system (Bio-Rad) using the Bio-Plex manager software (version 5.0). A minimum of 100 beads/region was analyzed. A curve fit was applied to each standard curve, according to the manufacturer’s manual, and sample concentrations were interpolated from the standard curves. For detection of CXCL8 release from human mast cells, cells cultured for $6 wk were counted and seeded in IMDM containing SCF overnight before sensitization with 0.5 mg/ml human myeloma IgE for 24 h. IgE-sensitized mast cells were then washed and resuspended in IMDM containing SCF. CCR1 was activated by preincubating the cells with 100 nM CCL15 for 10 min before challenging the sensitized cells with 0.25 mg/ml anti-human IgE (Sigma) for 6 h. Culture medium was harvested, and cells were removed by centrifugation at 4˚C. The amount of CXCL8 released into the culture medium by human mast cells or CCL15-stimulated U-937 macrophage-like cells was measured by ELISA (BD). Data analysis The data shown in each figure represent mean 6 SEM of determinations from three or more separate experiments. Statistical analyses were per- Downloaded from formed by ANOVA, followed by the Dunnett test or paired t test.

Results 705 CCR1/Ga14/16-mediated STAT3 phosphorylations at Tyr and Ser727 exhibit distinct temporal patterns http://www.jimmunol.org/

The coexistence of CCR1 (2, 25), Ga14 (26), and Ga16 (22, 25), as well as their functional coupling in THP-1 macrophage-like cells (23, 25), suggests that CCR1 may use Ga14/16 to stimulate STAT3. We began the study by determining whether CCR1 can use PTX- insensitive G proteins to induce STAT3 Tyr705 and Ser727 phosphorylation in PMA-differentiated THP-1 cells. PMA-differentiated THP-1 cells have been widely used as an in vitro model of human macrophages, and lipid-laden macrophages are one of the major cell types involved in atherosclerosis. Upon PMA differ- by guest on September 25, 2021 FIGURE 1. CCL15-induced STAT3 phosphorylations at Tyr705 and Ser727 a a entiation for 48 h, G 16, but not G 14, was upregulated by 2-fold occur in a temporally distinct pattern. (A) PMA-differentiated THP-1 mac- (data not shown); the expression of CCR1 remained unaffected rophage-like cells or HEL cells were stimulated in the absence or presence of (data not shown), which is in agreement with the literature (39). 10 nM CCL15 for various durations (15 min–24 h) and with PTX pretreat- PMA-differentiated THP-1 cells were pretreated with PTX and ment (100 ng/ml; 4 h). (B) PMA-differentiated THP-1 cells were transfected challenged with 10 nM CCL15 for different periods of time (15 with Stealth Select RNAi against Ga14 and Ga16 or Stealth RNAi negative min–24 h), and the STAT3 Tyr705 and Ser727 phosphorylation control and stimulated in the absence or presence of 10 nM CCL15 for 6 h status was determined using p-STAT3–Tyr705 or p-STAT3–Ser727 and with PTX pretreatment (100 ng/ml; 16 h). (C) HEK293 stable cell lines antiserum. STAT3 Ser727 phosphorylation occurred rapidly (within were stimulated in the absence or presence of 10 nM CCL15 for various 15 min) upon application of CCL15, and the response faded away durations (5 min–24 h). Phosphorylated STAT3 was detected by phospho- after 6 h, whereas STAT3 Tyr705 phosphorylation started after 2 h specific Abs, followed by subsequent quantification by densitometry. Nu- merical values shown above the immunoreactive bands represent mean rel- left panels of CCL15 treatment (Fig. 1A, ). The total amount of ative intensities of STAT3 phosphorylation expressed as a ratio of the basal STAT3 did not change in response to CCL15 challenge (Fig. 1A), level (set as 1.0) from three or more separate experiments; they were in- 705 indicating that the increase in STAT3 phosphorylation at Tyr or creased significantly compared with the basal level. p , 0.05, Dunnett test. Ser727 was not due to variations in the abundance of the protein. 705 727 Similar kinetics of CCL15-induced STAT3 Tyr and Ser Ga14 and Ga16 expression by siRNA completely abrogated the phosphorylation were observed in pluripotent HEL cells (Fig. 1A, CCL15-mediated STAT3 Tyr705 phosphorylation (Fig. 1B). These right panels), which endogenously express CCR1 (40), Ga14, and results suggest that the PTX-insensitive portion of the CCR1- 705 Ga16 (20, 21). These results demonstrate that, in cells that en- induced STAT3 Tyr phosphorylation in THP-1 cells was me- dogenously express both CCR1 and Ga14/16, CCL15 can stimulate diated via Ga14 and Ga16. 705 727 STAT3 Tyr and Ser phosphorylation in PTX-insensitive and To confirm the role of Ga14 and Ga16 in CCR1-induced STAT3 temporally distinct manners. To confirm the involvement of Ga14 phosphorylation, we used previously established HEK293 cells and Ga16 in regulating CCR1-mediated signaling, THP-1 macro- (23, 26) stably expressing CCR1 alone (CCR1/293) or expressing phage-like cells were transfected with siRNA against Ga14 and CCR1 together with Ga14 (CCR1/Ga14/293) or Ga16 (CCR1/ 705 Ga16 and assayed for CCL15-induced STAT3 Tyr phosphory- Ga16/293). Ga14 and Ga16 were prominently expressed in CCR1/ lation. Compared with cells transfected with control siRNA, in- Ga14/293 and CCR1/Ga16/293 cells, respectively, but they were troduction of siRNA against Ga14 and Ga16 into THP-1 cells absent in HEK293 cells (23, 26). The expression of CCR1 was reduced the level of Ga14 and Ga16 expression by ∼65 and ∼70%, confirmed previously by both Western blotting analysis (26) and respectively, whereas there was no detectable change in the ex- immunofluorescence microscopy (25), whereas the coupling of pression of other G proteins (data not shown). After eliminating CCR1 to Ga14 or Ga16 was verified by agonist-induced phos- the Gi-regulated pathways by PTX treatment, the reduction in pholipase Cb (PLCb) stimulation (23). Thus, these cell lines were 4 INDUCTION OF CXCL8 BY CCR1/Ga14/16 REQUIRES IL-6/STAT3 challenged with 10 nM CCL15 for various durations. In cells expressing CCR1 alone, CCL15 stimulated STAT3 Ser727 phosphorylation transiently (15–45 min), whereas the STAT3 Tyr705 phosphorylation level was unaffected by CCL15 during the entire duration of the experiment (Fig. 1C), indicating that CCR1 could not stimulate STAT3 phosphorylation at Tyr705 via endogenous G proteins. The incorporation of Ga14 or Ga16 strengthened the STAT3 Ser727 phosphorylation response (Fig. 1C); the duration of the response was lengthened in CCR1/Ga14/293 cells. STAT3 Tyr705 phosphorylation was also detected in cells coexpressing CCR1 and Ga14 or Ga16, with stimulations of ∼3.5–4-fold, but these events occurred much later, at $4 h (Fig. 1C). The cell lines were then pretreated with PTX to eliminate possible Gi-mediated STAT3 phosphorylation. Application of CCL15 for 15 min weakly stimulated Ser727 phosphorylation of STAT3 in cells expressing CCR1 alone, which was sensitive to PTX treatment (Fig. 2A); PTX sensitivity suggests the involvement of endogenous Gi proteins in HEK293 cells. Coexpression of Ga14 or Ga16 with

CCR1 enhanced the CCL15-induced STAT3 Ser727 phosphoryla- Downloaded from tion, with the stimulations increased to ∼2.5-fold (Fig. 2A). Ser727 phosphorylation of STAT3 was resistant to PTX in CCR1/Ga14/293 and CCR1/Ga16/293 cells, demonstrating that CCR1 can mediate 727 STAT3 Ser phosphorylation via Ga14 and Ga16 because they are the only two PTX-insensitive G proteins known to be recognized by

CCR1 (25, 26). To investigate CCR1/Ga14/16-stimulated STAT3 http://www.jimmunol.org/ Tyr705 phosphorylation, the cell lines were pretreated with PTX and stimulated with CCL15 for 6 h. In cells coexpressing CCR1 and 705 Ga14 or Ga16, CCL15-induced STAT3 Tyr phosphorylation was resistant to PTX (Fig. 2A). Another CCR1 agonist, myeloid progenitor inhibitory factor-1 (CCL23), induced STAT3 phosphoryla- 727 705 tion at Ser within 15 min, but STAT3 phosphorylation at Tyr FIGURE 2. CCL15-induced STAT3 phosphorylations at Tyr705 and was detected 6 h after drug treatment, and the response was not Ser727 were independent. (A) HEK293 stable cell lines were stimulated in sensitive to PTX (Fig. 2B). These results indicated that CCR1/ the absence (basal) or presence of 10 nM CCL15 for 15 min or 6 h, with or

705 727 6 by guest on September 25, 2021 Ga14/16-induced phosphorylation of STAT3 at Tyr and Ser without PTX pretreatment (100 ng/ml; 16 h). Data represent mean SEM have distinct kinetics. It should be noted that CCL15-induced from at least three separate experiments. *p , 0.05, CCL15 stimulation B STAT3 phosphorylation at Tyr705 was a delayed response because versus no stimulation, Dunnett test. ( ) HEK293 stable cell lines were stimulated in the absence (basal) or presence 10 nM CCL23 for 15 min or it remained detectable at 6 h, even when the agonist was washed 6 h and with PTX pretreatment (100 ng/ml; 16 h). Three individual ex- out 15 min after application (data not shown). periments showed similar results. (C) CCR1/Ga14/293 and CCR1/Ga16/ Next, luciferase reporter assay was used to demonstrate that CCR1/ 293 cells were transfected with cDNAs encoding p-STAT3–TA–Luc Ga14/16-mediated STAT3 phosphorylation can indeed lead to STAT3 (500 ng), pretreated with 100 ng/ml PTX for 4 h, and then stimulated in the transcriptional activity. CCR1/Ga14/293 cells were transfected with absence (basal) or presence of 10 nM CCL15 for 24 h. Luciferase activity cDNAs encoding STAT3-driven luciferase reporter, pretreated with was analyzed and expressed as fold stimulation of the basal STAT3 activity PTX, and challenged with 10 nM CCL15. Consistent with the re- for each cell lines. Data represent mean 6 SEM of at least three experi- sults for STAT3 Tyr705 phosphorylation, CCL15 signiﬁcantly stimu- ments. *p , 0.05, CCL15 stimulation versus no stimulation, Dunnett test. D lated the STAT3-driven luciferase activity (Fig. 2C). Similar results ( ) CCR1/Ga14/293 cells were transfected with cDNAs encoding vector plasmid STAT3, STAT3Y705F, or STAT3S727A (500 ng), pretreated with were obtained in STAT3-Luc–expressing CCR1/Ga16/293 cells 705 100 ng/ml PTX for 16 h, and then stimulated in the absence (basal) or (Fig. 2C). To investigate whether CCR1/Ga14-mediated Tyr and 727 presence of 10 nM CCL15 for 15 min or 6 h. Three individual experiments Ser phosphorylation of STAT3 can occur independently, CCR1/ showed similar results. Ga14/293 cells were transfected with vector plasmid, wild-type STAT3 or its phosphorylation-resistant mutants (STAT3Y705F and STAT3S727A), pretreated with PTX, and then challenged regulate gene expression. The ability of CCL15 to stimulate 727 705 with 10 nM CCL15 for 15 min or 6 h. CCR1/Ga14-induced STAT3 STAT3 Ser phosphorylation in the absence of Tyr phos- Tyr705 phosphorylation was attenuated in STAT3Y705F-expressing phorylation is intriguing, because the Ser727 site is generally be- cells but not in STAT3S727A-expressing cells (Fig. 2D, left panels). lieved to enhance STAT3 transcriptional activity, which requires 727 705 In contrast, CCR1/Ga14-mediated STAT3 Ser phosphorylation Tyr phosphorylation (41). Nuclear/cytosol fractionation was was abolished by STAT3S727A overexpression, whereas it was performed to investigate whether CCR1/Ga14 activation can lead unaffected by STAT3Y705F overexpression (Fig. 2D, right panels). to the translocation of Tyr705- or Ser727-phosphorylated STAT3 to 705 These results implied that CCR1/Ga14 activated STAT3 Tyr and the nucleus. Consistent with the results shown in Fig. 2A, the level Ser727 phosphorylations independently. of STAT3 Tyr705 and Ser727 phosphorylations could not be stim-

705 ulated by CCL15 in PTX-treated CCR1/293 cells (Fig. 3A, upper CCR1/Ga14 induces phosphorylation of STAT3 at Tyr in 727 right panels). Subcellular fractionation also indicated that no nucleus and Ser in cytosol detectable change in STAT3 Tyr705 or Ser727 phosphorylation was As a latent cytoplasmic transcription factor, STAT3 is activated by observed in cytosolic or nuclear compartments. Coexpression of cell surface receptors and translocates from cytosol to nucleus to Ga14 with CCR1/293 cells allowed CCL15 to stimulate STAT3 The Journal of Immunology 5

phosphorylation in CCR1/293 cells (Fig. 4). However, the intro- duction of Ga14 to CCR1/293 cells supported the CCL15-induced STAT3 Tyr705 phosphorylation, which was primarily localized to the nucleus (Fig. 4). Thus, these results demonstrated that Tyr705- phosphorylated STAT3, but not Ser727-phosphorylated STAT3, was translocated to the nucleus after CCR1/Ga14 activation. We demonstrated previously that members of the Gq subfamily can induce STAT3 Tyr705 phosphorylation (37). Thus, we investigated 705 whether the activation of Ga14 or Ga16 could translocate Tyr - phosphorylated STAT3 into the nucleus. In line with the previous study, active mutants of Ga14 and Ga16 could induce STAT3 Tyr705 phosphorylation, which was primarily localized to the nucleus (Fig. 3B). 705 CCR1/Ga14/16-mediated STAT3 Tyr phosphorylation involves protein synthesis 705 Because CCR1/Ga14/16-induced STAT3 Tyr phosphorylation occurred after prolonged treatment with CCL15 (Fig. 1), this may

imply that protein synthesis of a STAT3 activator may be required. Downloaded from To address this possibility, HEK293 stable cell lines were pre- http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. CCL15 induced STAT3 phosphorylations at Tyr705 in nu- 727 cleus and Ser in cytosol. (A) CCR1/293 and CCR1/Ga14/293 cells were pretreated with 100 ng/ml PTX for 16 h and then stimulated in the absence (basal) or presence of 10 nM CCL15 for 15 min, 4 h, or 6 h. Total cell lysates (right panels) of HEK293 stable cell lines were fractionated into cytosolic (left panels) and nuclear fractions (middle panels). Three individual experiments showed similar results. (B) HEK293 cells were transfected with cDNA encoding wild-type and active mutant of Ga14 or Ga16. Total cell lysates (right panels) of transfected HEK293 cells were fractionated into cytosolic (left panels) and nuclear fractions (middle panels). Three individual experiments showed similar results.

Tyr705 and Ser727 phosphorylation, even in the presence of PTX (Fig. 3A, lower right panels). Ser727 phosphorylation of STAT3 was increased after treatment with CCL15 for 15 min, and it gradually decreased to near basal levels by 6 h. Similar kinetics of CCL15-triggered STAT3 Ser727 phosphorylation were detected in the cytosolic compartment. However, CCL15-activated Ser727- phosphorylated STAT3 was not detectable in the nuclear compartments (Fig. 3A, lower panel). Tyr705 phosphorylation of STAT3 was elevated after treatment with CCL15 for 4 h and gradually increased to the maximal level by 6 h; a similar profile 705 was observed in the nuclear compartment. However, CCL15- FIGURE 4. CCL15 induced STAT3 phosphorylation at Tyr in nucleus. activated Tyr705-phosphorylated STAT3 was not detectable in CCR1/293 and CCR1/Ga14/293 cells were pretreated with 100 ng/ml PTX for 16 h and then stimulated in the absence (basal) or presence of 10 nM the cytosolic compartments (Fig. 3A, lower panel). Abs against CCL15 for 15 min or 6 h. Cells were fixed, permeabilized, labeled with caspase-3 and CREB were used as markers for the cytosolic and rabbit Tyr705-phosphorylated STAT3 Ab and mouse STAT3 Ab, and then nuclear compartments, respectively. These results were further stained with Alexa Fluor 555 anti-rabbit IgG for Tyr705-phosphorylated confirmed by confocal microscopy to reveal the subcellular lo- STAT3 detection (red) and Alexa Fluor 488 anti-mouse IgG for STAT3 705 calization of STAT3 Tyr phosphorylation. In line with the detection (green). Nuclei were stained with DAPI (blue). Three individual results illustrated in Fig. 3A, CCL15 did not elicit STAT3 Tyr705 experiments showed similar results. Original magnification 363. 6 INDUCTION OF CXCL8 BY CCR1/Ga14/16 REQUIRES IL-6/STAT3 treated with cycloheximide (a transcription/translation inhibitor) for 2 h and then incubated with CCL15 for 15 min or 6 h. Cy- cloheximide treatment of cells prior to CCL15 treatment led to 705 a complete abrogation of CCR1/Ga14/16-induced STAT3 Tyr phosphorylation without affecting the cell viability, whereas no inhibitory effect was observed on Ser727 phosphorylation (Fig. 5A). The same inhibitory effect on CCL15-induced STAT3 Tyr705 phosphorylation was observed in PMA-differentiated THP-1 cells (Fig. 5B). CCL15-activated Ser727 phosphorylation of STAT3 in HEK293 stable cell lines (Supplemental Fig. 1A) and THP-1 cells (Supplemental Fig. 1B) required ERK, as demonstrated by the use of an MEK1/2 kinase inhibitor (U0126) and its inactive analog (U0124). Therefore, the late and robust activation of STAT3 Tyr705 phosphorylation induced by CCR1/Ga14/16 may require the synthesis of STAT3 activators. We then investigated whether the conditioned medium from CCL15-stimulated CCR1/Ga14/293 or CCR1/Ga16/293 cells was sufficient to phosphorylate STAT3 at 705 Tyr . Conditioned medium was collected from CCR1/Ga14/293 or CCR1/Ga16/293 cells stimulated with CCL15 for 15 min, 4 h, Downloaded from or 6 h, transferred to parental HEK293 cells, and incubated for 30 min; STAT3 Tyr705 phosphorylation was observed in the parental HEK293 cells treated with conditioned medium of CCR1/Ga14/ 293 or CCR1/Ga16/293 cells with 6 h of CCL15 treatment (Fig. 6A). Similarly, STAT3 Tyr705 phosphorylation was detected in FIGURE 6. Conditioned medium from CCL15-stimulated CCR1/Ga14/ THP-1 cells treated with conditioned medium of THP-1 cells with http://www.jimmunol.org/ 293 cells, CCR1/Ga16/293 cells, and THP-1 macrophage-like cells in- 6 h of CCL15 treatment (Fig. 6B). The same phenomenon was duced STAT3 phosphorylation at Tyr705. Conditioned medium from observed when the conditioned medium was collected from HEK293 stable cells that were pretreated with 100 ng/ml PTX for 16 h and HEK293 cells overexpressing active mutants of Gq subfamily then stimulated in the absence (basal) or presence of 10 nM CCL15 for members (Ga14,Ga16,Gaq, and Ga11) but not Gaz (the PTX- 15 min, 4 h, or 6 h (A), THP-1 cells that were pretreated with 100 ng/ml insensitive Gi subfamily members) (Fig. 6C). These results indi- PTX for 16 h and then stimulated in the absence (basal) or presence of cated that there were cytokines or growth factors in the condi- 10 nM CCL15 for 15 min, 2 h, or 6 h (B), or HEK293 cells transfected with 705 tioned medium that could induce STAT3 Tyr phosphorylation. cDNA encoding wild-type and active mutant of Ga14,Ga16,Gaq,Ga11,or Gaz (C) was collected, transferred to HEK293 cells (A, C) or THP-1 cells B ( ) and incubated for 30 min. Numerical values shown above the immu- by guest on September 25, 2021 noreactive bands represent mean relative intensities of STAT3 phosphorylation expressed as a ratio of the basal level (set as 1.0) from at least three experiments; they were increased significantly compared with the basal level (p , 0.05, Dunnett test). For HEK293 cells transfected with cDNA encoding wild-type or active mutant of Ga subunits, three individual experiments showed similar results.

IL-6, but not CXCL8, is required for CCR1/Ga14/16-mediated STAT3 Tyr705 phosphorylation Many cytokines and growth factors are either STAT3 activators or their transcriptions are regulated by STAT3 activation; they in- clude bFGF, EGF, G-CSF, GM-CSF, IFN-a, IFN-b, IFN-g, IL-2, IL-4, IL-6, CXCL8, and TNF-a. Multiplex cytokine detection analysis was performed to screen for the aforementioned STAT3 activators or STAT3-regulated cytokines or growth factors in the conditioned medium of CCL15-stimulated CCR1/Ga14/293 and CCR1/Ga16/293 cells. After 4 h of drug incubation, CCL15 significantly stimulated IL-6 production in CCR1/Ga14/293 and CCR1/Ga16/293 cells in a PTX-insensitive manner, with maximal stimulations of 11- and 8-fold, respectively (Fig. 7A). No signif- FIGURE 5. CCL15-induced STAT3 phosphorylation at Tyr705 required icant CCL15-induced IL-6 secretion was observed in CCR1/293 protein synthesis. (A) HEK293 stable cell lines were pretreated with 100 ng/ cells, indicating that CCR1 used Ga14 and Ga16 to stimulate IL-6 ml PTX for 16 h and 50 mM cycloheximide for 2 h and then stimulated in expression. CXCL8, a proinflammatory CXC chemokine, was also B the absence (basal) or presence of 10 nM for 15 min or 6 h. ( ) PMA-dif- detected in the conditioned medium of CCR1/Ga /293 and ferentiated THP-1 macrophage-like cells were pretreated with 100 ng/ml 14 CCR1/Ga /293 cells with a 4-h treatment with CCL15. CCL15 PTX for 16 h and 50 mM cycloheximide for 2 h and then stimulated in the 16 absence (basal) or presence of 10 nM CCL15 for 15 min, 2 h, or 6 h. Nu- induced CXCL8 production by 22.5-fold in CCR1/Ga14/293 cells, merical values shown above the immunoreactive bands represent mean rel- whereas the production was increased 12-fold in CCR1/Ga16/293 ative intensities of STAT3 phosphorylation expressed as a ratio of the basal cells (Fig. 7B). No significant CCL15-induced CXCL8 secretion level (set as 1.0) from at least three separate experiments; they were in- was detected in CCR1/293 cells. In contrast, CCL15 treatment creased significantly compared with the basal level (p , 0.05, Dunnett test). was unable to stimulate bFGF, EGF, G-CSF, GM-CSF, IFN-a, The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 8. IL-6, but not CXCL8, was required for CCL15-induced STAT3 phosphorylation at Tyr705.(A) HEK293 stable cell lines were pretreated with 100 ng/ml PTX for 16 h and 10 mg/ml IL-6–neutralizing Ab for 30 min and then stimulated in the absence (basal) or presence of 10 nM CCL15 for 6 h. (B) HEK293 stable cell lines were pretreated with 100 ng/ml PTX for 16 h and 10 mg/ml CXCL8-neutralizing Ab for 30 min and then stimulated in the absence (basal) or presence of 10 nM CCL15 for by guest on September 25, 2021 15 min or 6 h. (C) PMA-differentiated THP-1 macrophage-like cells were FIGURE 7. CCL15 induced IL-6 and CXCL8 production. HEK293 pretreated with 100 ng/ml PTX for 16 h and10 mg/ml IL-6– or CXCL8– stable cell lines (A, B) or PMA-differentiated THP-1 macrophage-like cells neutralizing Ab for 30 min and then stimulated in the absence (basal) or (C, D) were stimulated in the absence (basal) or presence of 10 nM CCL15 presence of 10 nM CCL15 for 15 min, 2 h, or 6 h. (D) PMA-differentiated for various durations (15 min–24 h) and with PTX pretreatment (100 ng/ U-937 macrophage-like cells were pretreated with 100 ng/ml PTX for 16 h ml; 4 h). (E) PMA-differentiated THP-1 cells were also transfected with and 10 mg/ml IL-6–neutralizing Ab for 30 min and then stimulated in the

Stealth Select RNAi against Ga14 and Ga16 or Stealth RNAi negative absence (basal) or presence of 10 nM CCL15 for 6 h. Numerical values control and then were stimulated in the absence or presence of 10 nM shown above the immunoreactive bands represent mean relative intensities CCL15 for 6 h and with PTX pretreatment (100 ng/ml; 16 h). Conditioned of STAT3 phosphorylation expressed as a ratio of the basal level (set as medium was collected, and the protein concentration of IL-6 (A, C)or 1.0) from at least three separate experiments; they were increased signif- CXCL8 (B, D, E), was measured by multiplex cytokine-detection analysis. icantly compared with the basal level (p , 0.05, Dunnett test). Data represent mean 6 SEM of at least three separate experiments. *p , 0.05, CCL15 stimulation versus no stimulation, Dunnett test.

phorylation was observed in CCR1/Ga16/293 cells (Fig. 8A). However, pretreatment with CXCL8-neutralizing Ab did not af- IFN-b, IFN-g, IL-2, IL-4, or TNF-a expression in all three cell fect Tyr705 phosphorylation of STAT3 in HEK293 cells over- lines. The CCL15-stimulated THP-1 cells also produced IL-6 and expressing CCR1 and Ga14 or CCR1 and Ga16 (Fig. 8B), dem- CXCL8 in 2 and 6 h, respectively (Fig. 7C, 7D). Moreover, the onstrating that CXCL8 expression was not involved in CCR1/ 705 reduction in Ga14 and Ga16 expression by siRNA completely Ga14 or Ga16-mediated STAT3 Tyr phosphorylation. The abrogated CCL15-mediated CXCL8 production in THP-1 cells STAT3 Tyr705 phosphorylation level was unaffected by CCL15 (Fig. 7E). This demonstrates the capability of CCL15 to express after the addition of IL-6– or CXCL8-neutralizing Ab to CCR1/ IL-6 and CXCL8 via Ga14 and Ga16 in a native cellular envi- 293 cells. The requirement of IL-6, but not CXCL8, for CCL15- ronment. We further investigated whether IL-6 expression was induced STAT3 Tyr705 phosphorylation was also illustrated in 705 required for CCR1/Ga14-orGa16-mediated STAT3 Tyr phos- THP-1 cells (Fig. 8C). To further conﬁrm the involvement of IL-6 705 phorylation. CCR1/Ga14/293 cells were pretreated with IL-6– in CCL15-mediated STAT3 Tyr phosphorylation in macro- neutralizing Ab for 30 min before the addition of CCL15. CCR1/ phages, U-937 macrophage-like cells were treated with IL-6– 705 Ga14-induced STAT3 Tyr phosphorylation could be inhibited neutralizing Ab for 30 min before the addition of CCL15. PMA- by IL-6–neutralizing Ab (Fig. 8A), showing the requirement for differentiated U-937 cells, which endogenously express CCR1 705 IL-6 in the phosphorylation of STAT3 at Tyr . The same in- (42) as well as Ga14 and Ga16 (23), are another widely used hibitory effect of IL-6–neutralizing Ab on STAT3 Tyr705 phos- in vitro model of human macrophages (43). As shown in Fig. 8D, 8 INDUCTION OF CXCL8 BY CCR1/Ga14/16 REQUIRES IL-6/STAT3

CCL15-induced STAT3 Tyr705 phosphorylation was inhibited by treated with Stattic. For THP-1 cells, pretreatment with Stattic also IL-6–neutralizing Ab, illustrating the requirement for IL-6 in the diminished CCL15-induced STAT3 Tyr705 phosphorylation but phosphorylation of STAT3 at Tyr705. not CCL15-triggered STAT3 Ser727 phosphorylation (Fig. 10A). The requirement of STAT3 Tyr705 phosphorylation was also demonstrated STAT3 Tyr705 phosphorylation is necessary for CCR1/Ga 14/ in CCL15-stimulated CXCL8 production (Fig. 10B), but not CCL15- -mediated CXCL8 expression 16 activated IL-6 release (Fig. 10B), in THP-1 cells. In U-937 cells, Because the expression of cytokines could be regulated by STAT3 application of Stattic attenuated CCL15-mediated STAT3 Tyr705 705 activation, we investigated whether STAT3 Tyr phosphorylation phosphorylation (data not shown) and CXCL8 release (Fig. 10C). was required for CCR1/Ga14/16-mediated IL-6 or CXCL8 pro- Pretreatment with IL-6–neutralizing Ab also abolished CCL15- duction using STAT3 inhibitor V (Stattic), which selectively inhibits induced CXCL8 production (Fig. 10C), showing the involvement the activation, dimerization, and nuclear translocation of STAT3 of STAT3 Tyr705 phosphorylation and IL-6 in CCL15-triggered (44). Application of Stattic abolished CCL15-stimulated STAT3 CXCL8 release. The putative mechanism is depicted in Fig. 11. 705 Tyr phosphorylation in both CCR1/Ga14/293 and CCR1/ Lastly, to test whether chemokine-induced CXCL8 can be ob- 727 Ga16/293 cells (Fig. 9A), but it did not affect Ser phosphoryla- served in other hematopoietic cells, we used isolated human mast tion of STAT3 in these cells. The level of STAT3 phosphorylations at Tyr705 and Ser727 was unaffected by CCL15 after pretreatment of Stattic in CCR1/293 cells. CCL15-mediated CXCL8 secretion was abrogated by Stattic in HEK293 cells overexpressing CCR1 and

Ga14, but no inhibitory effect on CCL15-activated IL-6 production Downloaded from was observed (Fig. 9B, 9C); similar results were obtained with CCL15-treated CCR1/Ga16/293 cells. No signiﬁcant CCL15-induced IL-6 and CXCL8 secretion was detected in CCR1/293 cells pre- http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 10. CCL15 induced CXCL8 production in THP-1 macrophage-like cells, U-937 macrophage-like cells, and human mast cells. (A and B) PMA-differentiated THP-1 cells were pretreated with 100 ng/ml PTX for 16 h and 20 mM Stattic for 30 min and then stimulated in the absence (basal) or presence of 10 nM CCL15 for 15 min, 2 h, or 6 h. (A) Cell lysates were collected for detection of p-STAT3. (B) Conditioned medium was collected, and the protein concentration of IL-6 or CXCL8 was measured by multiplex cytokine-detection analysis. Numerical values shown above the immunoreactive bands represent mean relative intensities of STAT3 phosphorylation expressed as a ratio of the basal level (set as 1.0) from at least three separate experiments; they were increased signif- FIGURE 9. STAT3 phosphorylation at Tyr705 was required for CCL15- icantly increased compared with the basal level (p , 0.05, Dunnett test). induced CXCL8 production in HEK293 stable cell lines. HEK293 stable For multiplex cytokine-detection analysis, data represent mean 6 SEM cell lines were pretreated with 100 ng/ml PTX for 16 h and 20 mM Stattic from at least three separate experiments. *p , 0.05, CCL15 stimulation for 30 min and then stimulated in the absence (basal) or presence of 10 nM versus no stimulation, Dunnett test. (C) PMA-differentiated U-937 cells CCL15 for 15 min or 6 h. (A) Cell lysates were collected for detection of were pretreated with 100 ng/ml PTX for 16 h and 20 mM Stattic or 10 mg/ p-STAT3. Conditioned medium was collected, and the protein concentra- ml IL-6–neutralizing Ab for 30 min and then stimulated in the absence tion of IL-6 (B) or CXCL8 (C) was measured by multiplex cytokine-de- (basal) or presence of 10 nM CCL15 for 6 h. (D) Human mast cells cul- tection analysis. Numerical values shown above the immunoreactive bands tured for $6 wk were sensitized with 0.5 mg/ml human myeloma IgE for represent mean relative intensities of STAT3 phosphorylation expressed as 24 h. CCR1 was activated by preincubating the cells with 100 nM CCL15 a ratio of the basal level (set as 1.0) from at least three separate experi- for 10 min before challenging the sensitized cells with 0.25 mg/ml anti- ments; they were increased signiﬁcantly compared with the basal level human IgE for 6 h. Conditioned medium was collected, and the protein (p , 0.05, Dunnett test). For multiplex cytokine-detection analysis, data concentration of CXCL8 was measured. Data represent mean 6 SEM of at represent mean 6 SEM from at least three separate experiments. *p , least three separate experiments. *p , 0.05, CCL15 stimulation versus no 0.05, CCL15 stimulation versus no stimulation, Dunnett test. stimulation, paired t test. The Journal of Immunology 9

STAT3 Tyr705 phosphorylation, which is required for the subsequent CXCL8 production. Although CCL15 stimulated both CCR1 and CCR3 endogenously expressed in THP-1 cells (50), CCR3 is downregulated upon maturation of the monocytes to macrophages, whereas the expression of CCR1 remains unaffected (39). Thus, in the differentiated THP-1 and U-937 cells used in the current study, CCL15-mediated STAT3 phosphorylations at Tyr705 and Ser727 are most likely to be contributed by CCR1. Our study clearly demonstrated that CCL15-stimulated CCR1 is capable of inducing PTX-insensitive STAT3 phosphorylations at 705 727 Tyr and Ser only in the presence of Ga14 or Ga16. Moreover, the chemokine-induced phosphorylations of STAT3 at Tyr705 and Ser727 occurred independently (Fig. 2D) and in temporally distinct manners (Fig. 1), with Tyr705-phosphorylated STAT3 translocated to the nucleus and Ser727-phosphorylated STAT3 remaining in the cytosol (Fig. 3A). Apparently, CCR1-mediated STAT3 activation is a delayed response that requires the induction of IL-6 (Fig. 8A,

8C, 8D). In THP-1 cells, U-937 cells, and HEK293 cells, the bi- Downloaded from ological consequence of CCR1-mediated STAT3 activation is the upregulation of yet another chemokine, CXCL8 (Figs. 9C, 10B, FIGURE 11. A proposed model of CCR1/Ga14/16-mediated IL-6/STAT3/ 10C). Fig. 11 shows a putative model of CCR1/Ga14/16-induced CXCL8 activation. CCL15-bound CCR1 activates Ga14 (or Ga16)and 705 727 stimulates PLCb and PKC/c-Src pathways, leading to the activation of Ras/ STAT3 Tyr and Ser phosphorylation, in which the present MEK/ERK cascades. These activation signals lead to STAT3 Ser727 phos- findings are incorporated with established pathways. Upon bind- phorylation. For prolonged pretreatment with CCL15, the activated MEK/ ing of CCL15 to CCR1, Ga14/16 becomes activated and, in turn, http://www.jimmunol.org/ ERK may also phosphorylate transcription factors, leading to the upregulation stimulates PLCb, which hydrolyzes phosphatidylinositol 4,5- of IL-6 and transactivation of IL-6R. The secreted IL-6 induces STAT3 bisphosphate to diacylglycerol, subsequently leading to activation phosphorylation at Tyr705 in an autocrine fashion, resulting in the dimerization of the PKC/c–Src pathways. The coupling of tetratricopeptide and nuclear translocation of Tyr705-phosphorylated STAT3 and subsequent repeat 1 to Ga14/16 (51) activates Ras and MEK/ERK, which then CXCL8 transcription. Solid arrows illustrate findings based on previous induces STAT3 Ser727 phosphorylation (20, 21). Upon prolonged studies; dashed arrows indicate putative interactions. DAG, Diacylglycerol; pretreatment with CCL15, activated MEK/ERK may phosphory- MEK, MAPK kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; TF, transcription factor; TPR1, tetratricopeptide repeat 1. late transcription factors, such as Sp-1 (52), leading to the translocation of transcription factors to the nucleus, upregulation of IL- 6, and transactivation of IL-6R. The secreted IL-6 induces STAT3 by guest on September 25, 2021 cells to examine the ability of CCL15 to stimulate the production phosphorylation at Tyr705 in an autocrine fashion, resulting in of CXCL8. Costimulation with CCR1 enhanced anti-IgE–induced dimerization and nuclear translocation of Tyr705-phosphorylated mast cell activation and degranulation (45). In the unstimulated STAT3 and subsequent CXCL8 transcription. state, human mast cells did not release CXCL8 at detectable We showed previously that activation of Ga14/16 by a variety of levels. Sensitization of human mast cells by human myeloma IgE G protein-coupled receptors, including the d-opioid, C5a, formyl and anti-human IgE resulted in the release of CXCL8; this re- peptide, and opioid receptor-like receptors (20, 21, 53), can lead to sponse was enhanced significantly in the presence of 100 nM STAT3 Tyr705 phosphorylation within 15 min in HEK293 cells. 705 CCL15 (Fig. 10D). Anti-IgE–induced CXCL8 production in mast In contrast, CCR1/Ga14/16-mediated Tyr phosphorylation of cells was completely abolished in the presence of Stattic, sug- STAT3 was only detected after prolonged drug pretreatment. This gesting the involvement of STAT3 (data not shown). Pretreatment discrepancy is puzzling, especially because the receptors were with IL-6–neutralizing Ab diminished CCL15-mediated CXCL8 expressed in the same cellular background. Although we do not release (Fig. 10D), illustrating the requirement of IL-6 in CCL15- have a plausible explanation, it should be noted that delayed activated CXCL8 production. STAT3 Tyr705 phosphorylation is not unique to CCR1; it has been similarly observed with G16-coupled melatonin MT1 and MT2 Discussion receptors (36). Moreover, distinct temporal patterns of STAT3 Although activation of STAT3 Tyr705 phosphorylation was shown phosphorylations at Tyr705 and Ser727 have been documented. In to be mediated by several chemokines, including CCL5, CCL2, murine macrophage-like RAW 264.7 cells, STAT3 Ser727 phos- and SDF-1a (CXCL12) (18, 46–48), the biochemical linkage phorylation induced by LPS can be observed at 5 min, whereas between heterotrimeric G protein activation and STAT3 in this STAT3 Tyr705 phosphorylation requires 2 h of treatment (54). pathway remains poorly defined. Several reports suggest that Likewise, two groups demonstrated that angiotensin II induces chemokines induce receptor dimerization, resulting in the activa- delayed STAT3 Tyr705 phosphorylation in rat aortic smooth tion of the JAK–STAT pathway (reviewed in Ref. 49). However, muscle cells (55) and rat cardiomyocytes (56). Fukuda and co- CCR1 oligomerization has not been unequivocally established. In workers (57) also showed that the delayed STAT3 Tyr705 phos- addition, the ability of chemokine receptors to regulate STAT3 phorylation stimulated by angiotensin II is mediated by autocrine/ Ser727 phosphorylation has not been documented. By examining paracrine secretion of IL-6. the role of Ga14/16 in transfected HEK293 cells, native HEL cells, The induction of IL-6 and CXCL8 by CCR1/Ga14/16 signaling and native THP-1 and U-937 macrophage-like cells, this study has several implications. IL-6 is a potent inflammatory cytokine provided evidence that STAT3 Tyr705 phosphorylation is not that directly activates STAT1 and STAT3. The production of IL-6 a prerequisite for Ser727 phosphorylation, and IL-6 autocrine in CCL15-stimulated THP-1 cells and transfected HEK293 cells signaling is apparently involved in CCR1/Ga14/16-mediated implied that CCR1/Ga14/16 could also stimulate STAT1, which is in 10 INDUCTION OF CXCL8 BY CCR1/Ga14/16 REQUIRES IL-6/STAT3 line with our previous study showing that the constitutively active 2. Neote, K., D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall. 1993. Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine mutant of Ga16 can enhance the activity of STAT1 (19). Our 705 receptor. Cell 72: 415–425. demonstration of CCR1/Ga14/16-mediated STAT3 Tyr phosphor- 3. Youn, B. S., S. M. Zhang, E. K. Lee, D. H. Park, H. E. Broxmeyer, P. M. Murphy, ylation leading to subsequent CXCL8 production is in agreement M. Locati, J. E. Pease, K. K. Kim, K. Antol, and B. S. Kwon. 1997. Molecular cloning of leukotactin-1: a novel human b-chemokine, a chemoattractant for with the report that CXCL8 expression is transcriptionally upregu- neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine lated by STAT3 in human melanoma cells (58). In addition, it was receptors 1 and 3. J. Immunol. 159: 5201–5205. reported that JAK/STAT3 is involved in thrombin-induced CXCL8 4. Gao, J. L., T. A. Wynn, Y. Chang, E. J. Lee, H. E. Broxmeyer, S. Cooper, H. L. Tiffany, H. Westphal, J. Kwon-Chung, and P. M. Murphy. 1997. Impaired secretion in human dermal fibroblasts (59). CXCL8 was reported to host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 activate MMP expression (33) and suppress tissue inhibitor of cytokine balance in mice lacking CC chemokine receptor 1. J. Exp. Med. 185: metalloproteinases expression (60), and these regulations by CXCL8 1959–1968. 5. Hwang, J., C. W. Kim, K. N. Son, K. Y. Han, K. H. Lee, H. K. Kleinman, J. 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STAT3 Ser phos- by human CCR3+ endothelial cells. J. Immunol. 166: 7571–7578. http://www.jimmunol.org/ phorylation is essential for postnatal survival and growth, as well as 11. Takeda, K., T. Kaisho, N. Yoshida, J. Takeda, T. Kishimoto, and S. Akira. 1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through thymocyte proliferation, as shown in knock-in mice with alanine preventing apoptosis: generation and characterization of T cell-specific Stat3- 727 substituted for Ser in STAT3 (66). A recent study in mouse pro- deficient mice. J. Immunol. 161: 4652–4660. B cells showed that mitochondrial STAT3 Ser727 phosphorylation is 12. Ning, Z. Q., J. Li, and R. J. Arceci. 2001. 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Barnes, P. E. Auron, and G. R. Stark. 2007. tigation is warranted to examine the biological function of CCR1/ Unphosphorylated STAT3 accumulates in response to IL-6 and activates tran- Ga -mediated STAT3 Ser727 phosphorylation. scription by binding to NFkappaB. Genes Dev. 21: 1396–1408. 14/16 18. Wong, M., and E. N. Fish. 1998. RANTES and MIP-1a activate stats in T cells. In summary, IL-6 autocrine signaling is apparently involved in J. Biol. Chem. 273: 309–314. 705 CCR1/Ga14/16-mediated STAT3 Tyr phosphorylation, which is 19. Lo, R. K., and Y. H. Wong. 2006. Transcriptional activation of c-Fos by con- required for the subsequent production of CXCL8. In turn, CXCL8 stitutively active Galpha(16)QL through a STAT1-dependent pathway. Cell. 705 Signal. 18: 2143–2153. may activate CXCR2, leading to further STAT3 Tyr phosphor- 20. Lo, R. K., and Y. H. Wong. 2004. Signal transducer and activator of transcription ylation, which synergizes with the effect of CCR1 agonists. 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CCR1-mediated activation of Nuclear Factor-kappaB in THP-1 monocytic cells involves Pertussis Toxin-insensitive Acknowledgments Galpha(14) and Galpha(16) signaling cascades. J. Leukoc. Biol. 86: 1319–1329. 24. Su, Y., M. K. Ho, and Y. H. Wong. 2009. A hematopoietic perspective on the We thank Dr. Rodger A. Allen for kindly providing the human CCR1 cDNA. promiscuity and specificity of Galpha16 signaling. Neurosignals 17: 71–81. 25. Tian, Y. J., M. M. Lee, L. Y. Yung, R. A. Allen, P. M. Slocombe, B. M. Twomey, and Y. H. Wong. 2008. Differential involvement of Galpha16 in CC chemokine- Disclosures induced stimulation of phospholipase Cbeta, ERK, and chemotaxis. Cell. Signal. The authors have no financial conflicts of interest. 20: 1179–1189. 26. Tian, Y., D. C. New, L. Y. Yung, R. A. Allen, P. M. Slocombe, B. M. Twomey, M. M. Lee, and Y. H. Wong. 2004. 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