Coupled Receptor Kinase 2 Translocation − Chemotaxis By

TLR4 Signaling Augments Monocyte Chemotaxis by Regulating G Protein− Coupled Receptor Kinase 2 Translocation

This information is current as Zheng Liu, Yong Jiang, Yuehua Li, Juan Wang, Liyan Fan, of September 29, 2021. Melanie J. Scott, Guozhi Xiao, Song Li, Timothy R. Billiar, Mark A. Wilson and Jie Fan J Immunol published online 14 June 2013 http://www.jimmunol.org/content/early/2013/06/14/jimmun

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 14, 2013, doi:10.4049/jimmunol.1300790 The Journal of Immunology

TLR4 Signaling Augments Monocyte Chemotaxis by Regulating G Protein–Coupled Receptor Kinase 2 Translocation

Zheng Liu,*,† Yong Jiang,* Yuehua Li,† Juan Wang,*,† Liyan Fan,‡ Melanie J. Scott,† Guozhi Xiao,x Song Li,{ Timothy R. Billiar,†,‖ Mark A. Wilson,†,# and Jie Fan†,‖,#

Monocytes are critical effector cells of the innate immune system that protect the host by migrating to inﬂammatory sites, differentiating to macrophages and dendritic cells, eliciting immune responses, and killing pathogenic microbes. MCP-1, also known as CCL2, plays an important role in monocyte activation and migration. The chemotactic function of MCP-1 is mediated by binding to the CCR2 receptor, a member of the G protein–coupled receptor (GPCR) family. Desensitization of GPCR chemokine receptors is

an important regulator of the intensity and duration of chemokine stimulation. GPCR kinases (GRKs) induce GPCR phosphor- Downloaded from ylation, and this leads to GPCR desensitization. Regulation of subcellular localization of GRKs is considered an important early regulatory mechanism of GRK function and subsequent GPCR desensitization. Chemokines and LPS are both present during Gram-negative bacterial infection, and LPS often synergistically exaggerates leukocyte migration in response to chemokines. In this study, we investigated the role and mechanism of LPS–TLR4 signaling on the regulation of monocyte chemotaxis. We demonstrate that LPS augments MCP-1–induced monocyte migration. We also show that LPS, through p38 MAPK signaling,

induces phosphorylation of GRK2 at serine 670, which, in turn, suppresses GRK2 translocation to the membrane, thereby http://www.jimmunol.org/ preventing GRK2-initiated internalization and desensitization of CCR2 in response to MCP-1. This results in enhanced monocyte migration. These ﬁndings reveal a novel function for TLR4 signaling in promoting innate immune cell migration. The Journal of Immunology, 2013, 191: 000–000.

onocytes are critical effector cells of the innate immune MCP-1, also known as CCL2, is a 13-kDa chemokine that plays system that protect the host by migrating to inflam- an important role in monocyte activation and migration (4). MCP-1 M matory sites, differentiating to macrophages and den- is released from both hematopoietic and nonhematopoietic cells, dritic cells, eliciting immune responses, and killing pathogenic including monocytes/macrophages, dendritic cells, astrocytes, microbes (1, 2). Monocyte migration is tightly regulated by sig- endothelial cells, and fibroblasts (5). MCP-1 derived from these by guest on September 29, 2021 naling mechanisms activated by chemokines, which act through MCP-1-produce cells contributes to the migration of monocytes chemokine receptors to direct monocyte migration along a con- into local tissue and organ during infection and inflammation (6). centration gradient (3). The physiological function of MCP-1 is mediated by binding to the CCR2 receptor, a member of the G protein–coupled receptor (GPCR) family (7). *Department of Pathophysiology, Southern Medical University, Guangzhou 510515, Desensitization of the GPCR family of chemokine receptors is China; †Department of Surgery, University of Pittsburgh School of Medicine, Pitts- burgh, PA 15213; ‡University of Pittsburgh School of Arts and Science, Pittsburgh, an important determinant of the intensity and duration of agonist PA 15213; xDepartment of Biochemistry, Rush University Medical Center, Chicago, stimulation (8, 9). Receptor desensitization regulates the number { IL 60612; Department of Pharmaceutical Sciences, Center for Pharmacogenetics, of migrating cells, as well as their motility and ability to stop upon University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261; ||McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219; contact with pathogens or target cells (8). G protein–coupled re- and #Research and Development, Veterans Affairs Pittsburgh Healthcare System, ceptor kinases (GRKs) induce GPCR phosphorylation and, thereby, Pittsburgh, PA 15240 signal GPCR desensitization (10). GRKs constitute a family of Received for publication March 22, 2013. Accepted for publication May 8, 2013. seven mammalian serine and threonine protein kinases (11, 12). This work was supported by National Institutes of Health Grant R01-HL-079669 (to GRK2 is a member of the GRK family that was shown to modulate J.F. and M.A.W.), National Institutes of Health Center Grant P50-GM-53789 (to T.R.B. and J.F.), National Institutes of Health Grant R01-GM-50441 (to T.R.B.), a Veterans a variety of functions in leukocytes (13). Activity of GRKs is tightly Affairs Merit Award (to J.F.), National Key Basic Research (973) Program of China regulated by three mechanisms: subcellular localization, alterations 2011CB510200 (to Y.J.), National Natural Science Foundation of China 81030055 (to in intrinsic kinase activity, and alterations in GRK expression (14). Y.J.), Natural Science Foundation of China-Guangdong Joint Fund U0632004 (to Y.J.), and Program for Changjiang Scholars and Innovative Research Team in University Therefore, regulation of GRK subcellular localization represents IRT0731 (to Y.J.). a logical means of regulating monocyte migration. However, the The contents do not represent the views of the Department of Veterans Affairs or the regulation of subcellular localization of GRKs in monocytes and U.S. Government. their effects on monocytes behavior remain unknown. Address correspondence and reprint requests to Dr. Jie Fan or Dr. Yong Jiang, De- LPS, a component of the outer membrane of Gram-negative partment of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (J.F.) or Department of Pathophysiology, Southern Medical University, Guang- bacteria, is a specific ligand for TLR4 and induces a range of in- zhou 510515, China (Y.J.). E-mail addresses: [email protected] (J.F.) or jiang48231@163. flammatory responses, including production of proinflammatory com (Y.J.) mediators and induction of migration of innate immune cells to the Abbreviations used in this article: BALF, bronchoalveolar lavage fluid; BMDM, bone site of infection (15). In this study, we investigated the role and marrow–derived monocyte; GPCR, G protein–coupled receptor; GRK, G protein– coupled receptor kinase; i.t., intratracheal(ly); siRNA, small interfering RNA; WT, mechanism of LPS–TLR4 signaling in regulating monocyte chemo- wild-type. taxis. We demonstrate that LPS augments MCP-1–induced monocyte

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300790 2 TLR4 ACTING THROUGH GRK2 AUGMENTS MONOCYTE CHEMOTAXIS migration. We also show that LPS, through p38 MAPK signaling, monocytes were preincubated with a variety of MAPK inhibitors: p38 induces phosphorylation of GRK2 at Ser670, which, in turn, sup- inhibitor SB203580 (10 mM; Cell Signaling Technology, Boston, MA), presses GRK2 translocation to the membrane, thereby preventing ERK inhibitor PD98059 (50 mM; Cell Signaling Technology) (19), or JNK inhibitor SP600125 (10 mM; Sigma-Aldrich) for 30 min (20). The chamber GRK2-initiated internalization and desensitization of CCR2 in re- was incubated in humidified air with 5% CO2 at 37˚C for the time indi- sponse to MCP-1. This results in enhanced monocyte migration. cated. At the end of experiment, the membrane between the upper and These findings reveal a novel function for TLR4 signaling in pro- lower wells was fixed and stained with Wright–Giemsa stain (Sigma- moting innate immune cell migration. Aldrich). Cell migration was determined by counting the number of cells that had migrated through the filter in three random high-power microscope fields (3400). A chemotactic index was used to express chemotactic ac- Materials and Methods tivity and was calculated as the number of monocytes that migrated across Animals the membrane/number of monocytes that migrated spontaneously toward blank medium in the control group. All experiments were performed in Male C57BL/6 wild-type (WT) mice were purchased from The Jackson triplicate. Laboratory (Bar Harbor, ME). TLR4-knockout (TLR42/2)miceona C57BL/6 background were bred in T.R.B.’s laboratory at the Univer- Measurement of cell surface CCR2 sity of Pittsburgh. All experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee of VA Monocytes and RAW264.7 cells were treated with p38 inhibitor SB203580 Pittsburgh Healthcare System and University of Pittsburgh. (10 mM), ERK inhibitor PD98059 (50 mM) (19), or JNK inhibitor SP600125 (10 mM) for 30 min at 37˚C and then stimulated with MCP-1 Cell culture (200 ng/ml) in the presence or absence of LPS (100 ng/ml) for the indicated time. The treatment was stopped by addition of 2 ml ice-cold FACS The preparation of murine bone marrow–derived monocytes (BMDMs) buffer (0.1% sodium azide, 2% BSA, PBS). The cells were then incubated was performed as previously described (16). Bone marrow cells were with CCR2 Ab for 1 h on ice, followed by incubation with FITC-labeled Downloaded from isolated by flushing femurs and tibias of 8–12-wk-old C57BL/6 mice or 2/2 secondary Ab (Abcam, Cambridge, MA) and PE-labeled CD11b Ab TLR4 mice with DMEM containing 10% FBS. Erythroid cells were (eBioscience) for 1 h on ice. The cells were washed and analyzed using lysed with RBC lysis buffer (eBioscience, San Diego, CA), washed twice a flow cytometer (FACSCalibur; Becton Dickinson). The cutoff to define 3 6 with DMEM, adjusted to a cell suspension of 1 10 cells/ml, and chemokine receptor–positive cells was set according to the staining with seeded in 6-cm ultra-low attachment surface plates (Corning Costar, the isotype control Ab. Monocytes were identified by their light-scatter Corning, NY). Cells were supplemented with 20 ng/ml recombinant properties and expression of CCR2 and CD11b. mouse M-CSF (Sigma-Aldrich, St. Louis, MO) and cultured in humidified http://www.jimmunol.org/ incubators at 37˚C and 5% CO2 for 5 d. BMDMs were collected from the GRK2 RNA interference nonadherent cell population by centrifugation of cell culture supernatant at 1000 rpm for 10 min. RAW 264.7 cells, a mouse macrophage cell line Accell small interfering RNA (siRNA; Dharmacon, Lafayette, CO) was obtained from the American Type Culture Collection (Rockville, MD), used to knock down GRK2 (21). Briefly, RAW264.7 cells (3 3 105/well) were also used in the experiments and were cultured in DMEM containing were seeded in 6-cm plates and incubated for 16 h. The growth media were 10% FBS. removed, and cells were transfected with 25 nM Adrbk1 Accell siRNA, according to the manufacturer’s instructions, and incubated for an addi- Cell-migration assay tional 48 h. Cells were then collected for detection of GRK2 protein expression. A cell-migration assay was performed in chemotaxis microchambers (Neuroprobe, Gaithersburg, MD) (15, 17). The lower well of the chemo-

Membrane-bound GRK2 assay by guest on September 29, 2021 taxis microchamber was ﬁlled with DMEM containing 0.2% BSA and different concentrations of recombinant mouse MCP-1 (BioLegend, San BMDMs were stimulated with MCP-1 (200 ng/ml) in the presence or Diego, CA). The upper well was loaded with monocytes in suspension absence of LPS (100 ng/ml) for up to 1 h. Cells incubated with DMEM only (50,000 cells), with or without LPS (100 ng/ml) (18). In some cases, were used as a negative control. In some cases, cells were treated with p38

FIGURE 1. LPS–TLR4 augments MCP- 1–induced monocyte migration. (A) Mono- cytes were loaded into the upper wells of the microchamber, with or without 100 ng/ml LPS. The lower wells were filled with MCP- 1 at concentration of 0, 2, 20, or 200 ng/ml. Cell migration was allowed for 2 h, and cells that crossed the filter were counted. (B) Monocytes isolated from C57BL/6 WT mice or TLR4 knockout (TLR42/2) mice were loaded into the upper wells of the microchamber, with or without 100 ng/ml LPS. The lower wells were filled with 200 ng/ml of MCP-1 or medium alone. Cell migration was induced for 2 h, and cells that crossed the filter were counted. (C)WT mice were given MCP-1 (50 ng/mouse; i.t.), and BALF was collected for cell counts. (D and E) WT mice and TLR42/2 mice were given MCP-1 (50 ng/mouse; i.t.) and/or LPS (25 ng/mouse; i.t.) at the same time, and BALF was collected after 6 h for cell counts. Data are mean and SEM of the percentage of control from three independent experiments. *p , 0.01 versus the groups not labeled with an asterisk. The Journal of Immunology 3 inhibitor SB203580 (10 mM), ERK inhibitor PD98059 (50 mM), or JNK Data presentation and statistical analysis inhibitor SP600125 (10 mM) for 30 min at 37˚C before MCP-1 treatment. 6 The reactions were quenched by addition of 10 ml ice-cold PBS, and cell The data are presented as mean SEM of the indicated number of ex- membrane proteins were extracted using a ProteoExtract Native Mem- periments. Statistical significance among group means was assessed by brane Protein Extraction Kit (22) and concentrated using a ProteoExtract ANOVA. The Student Newman–Keuls post hoc test was performed. Dif- , Protein Precipitation Kit (both from Millipore, Billerica, MA). The references were considered significant at p 0.01. sultant proteins were resuspended in 23 Laemmli buffer and boiled for 10 min for SDS-PAGE. Rabbit polyclonal Ab to GRK2 (Santa Cruz Bio- Results technology, Santa Cruz, CA) was used for detection of membrane-bound LPS–TLR4 augments MCP-1–induced monocyte migration GRK2. GRK2 and actin levels in the cell supernatant were also examined. The images presented are representatives of at least three independent Monocyte migration to the site of infection is an essential part of experiments. The densitometry analysis of blots was performed using first-line immune defenses. MCP-1 plays an important role in ini- National Institutes of Health–developed ImageJ software, and data are mean 6 SEM of the percentage changes in the ratio of membrane-bound tiating monocyte activation and migration (23). However, LPS pro- GRK2/cytosol-GRK2 in the monocytes, which were normalized by the motes innate immune cell migration through an unknown mechanism. density of actin, from three experiments. To address the influence of LPS on chemokine-induced monocyte In vivo monocyte-migration study migration, we used a Boyden chamber assay (24), in which monocyte migration was driven by MCP-1. As shown in Fig. 1A, within an MCP-1–induced monocyte migration into the lungs was studied in vivo. MCP-1 concentration range of 0–20 ng/ml, MCP-1 induced monocyte MCP-1 (50 ng/mouse) and/or LPS (25 ng/mouse) was injected intra- 2 2 migration in a dose-dependent manner, whereas a higher concentra- tracheally (i.t.) into WT or TLR4 / mice; at 6 h after MCP-1 and/or LPS injection, bronchoalveolar lavage fluid (BALF) was collected for mono- tion of 200 ng/ml decreased monocyte migration, possibly as a result cyte counts. of a rapid induction of chemokine receptor desensitization (15, 25). Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. LPS–TLR4 augments MCP-1–induced monocyte migration by modulating cell surface expression of CCR2. (A) Monocytes were incubated with 200 ng/ml of MCP-1, and the cell surface expression of CCR2 was measured using ﬂow cytometry. (B) Monocytes isolated from WT or TLR42/2 mice were stimulated with MCP-1 and/or LPS for 60 min, and the cell surface expression of CCR2 was measured by ﬂow cytometry. Results represent percentage of control; mean 6 SEM (n =3).*p , 0.01 versus the groups not labeled with an asterisk. 4 TLR4 ACTING THROUGH GRK2 AUGMENTS MONOCYTE CHEMOTAXIS

Importantly, although LPS alone failed to induce monocyte mi- receptor (26). Receptor internalization is a major mechanism of gration, LPS significantly augmented monocyte migration in re- receptor desensitization (7, 27). It was reported that MCP-1 in- sponse to MCP-1 in a concentration range of 2–200 ng/ml (Fig. 1A). duces CCR2 internalization and, therefore, regulates the desensi- This effect of LPS was mediated through TLR4, because TLR4 tization of its receptor in monocytes (28). To elucidate the effect deficiency completely prevented the LPS-enhanced monocyte mi- of LPS on MCP-1–induced CCR2 internalization, we detected cell gration in response to MCP-1 (Fig. 1B). surface CCR2 expression on monocytes following MCP-1 and/or The augmenting effects of LPS on MCP-1–induced chemotaxis LPS treatment using flow cytometry. As shown in Fig. 2A, MCP-1 seen in vitro were also noted in vivo in mice. I.t. instillation of induced a decrease in the cell surface expression of CCR2 in a MCP-1 in WT mice induced monocyte infiltration into the lungs in time-dependent manner. However, LPS prevented MCP-1–induced a time-dependent manner (Fig. 1C), and LPS given i.t. signifi- CCR2 internalization, as shown in Fig. 2B. This effect of LPS was cantly enhanced this MCP-1–induced monocyte infiltration in the blocked by TLR4 deficiency (Fig. 2B). The data suggest that LPS– lungs at 6 h after MCP-1 stimulation (Fig. 1D). Also, consistent TLR4 augments MCP-1–induced monocyte migration by mod- with the in vitro findings, genetic deletion of TLR4 prevented ulating the cell surface expression of CCR2. LPS-enhanced monocyte migration into the lungs (Fig. 1D). In GRK2 is a key molecule regulating MCP-1–induced CCR2 addition, we observed, using light microscopy, that LPS alone did internalization not induce monocyte infiltration in the lungs of WT mice, but instead induced neutrophil sequestration in the lungs (Fig. 1E). GRK2 is known to promote CCR2 desensitization through phos- Taken together, these results indicate that LPS signals via TLR4 to phorylation (29). To determine the role of GRK2 in mediating augment, but not directly induce, monocyte migration in response MCP-1–induced CCR2 internalization in monocytes/macrophages, Downloaded from to chemoattractants through a mechanism that is different from we used an RNA interference approach to knock down GRK2 in LPS-induced neutrophil migration. RAW264.7 cells and analyzed the influence of GRK2 knockdown on cell surface expression of CCR2 and the ability of monocytes LPS–TLR4 augments MCP-1–induced monocyte migration by to migrate. Transfection of Accell siRNA to GRK2 resulted in an modulating cell surface expression of CCR2 receptors 85% decrease in GRK2 protein expression in RAW264.7 cells (Fig.

Upon agonist activation of receptors, a rapid attenuation of recep- 3A). In these cells, the MCP-1–induced decrease in cell surface http://www.jimmunol.org/ tor responsiveness, called desensitization, occurs through feedback expression of CCR2 was markedly suppressed (Fig. 3B), which mechanisms and prevents acute and chronic overstimulation of the was associated with increased cell migration in response to MCP-1 by guest on September 29, 2021

FIGURE 3. GRK2 mediates the cross-talk between LPS–TLR4 and MCP-1 CCR2 signaling. (A) RAW264.7 cells were treated with Adrbk1 siRNA (siRNA to GRK2) for 48 h. The efficiency of GRK2 knockdown was measured by immunoblotting. The images are representatives of three independent experiments. (B) RAW264.7 cells or RAW264.7 cells with GRK2 knockdown (RNAi) were loaded into the upper wells of the microchamber in the presence or absence of 100 ng/ml of LPS. The lower wells were filled with 200 ng/ml of MCP-1 or medium only. Cell migration was induced for 6 h, and cells that crossed the filter were counted under the microscope. (C) RAW264.7 cells or RAW264.7 cells with GRK2 knockdown were stimulated with LPS and/ or MCP-1 for 60 min, and the cell surface expression of CCR2 was measured by flow cytometry. Results represent percentage of control; mean 6 SEM (n = 3). *p , 0.01 versus groups not labeled with an asterisk. The Journal of Immunology 5

(Fig. 3C). Of note, GRK2 knockdown also diminished the influ- MAPKs were reported to regulate GRK2 activity. In vitro and ence of LPS on cell surface CCR2 expression (Fig. 3B), as well as in situ experiments showed that ERK1 is able to phosphorylate prevented the enhancing effect of LPS on cell migration (Fig. 3C). recombinant GRK2 at Ser670, which lies within the Gbg-binding Collectively, the results demonstrate a critical role for GRK2 in domain of GRK2 (30). Another study showed that MAPK phos- mediating LPS/MCP-1 regulation of monocyte migration. phorylation at this site impairs the GRK2–Gbg interaction, thereby inhibiting GRK2 translocation to the cell membrane and subse- LPS-induced phosphorylation of GRK2 at serine 670 quent induction of kinase activity and GPCR regulation (31). To suppresses GRK2 translocation to cell membrane elucidate whether LPS–TLR4, through modification of GRK2 It was reported that GRK2 translocation to the cell membrane upon phosphorylation, regulates GRK2 mobility, we detected specific MCP-1 stimulation is a determinant for desensitization of the CCR2 phosphorylation of Ser670 in GRK2 in monocytes following stim- receptor in monocytes (7, 16). We hypothesized that LPS prevents ulation of BMDMs with MCP-1 and/or LPS. Western blotting for GRK2 subcellular translocation and so suppresses CCR2 inter- phospho-Ser670 on GRK2 showed that LPS or LPS plus MCP-1 nalization and subsequent desensitization in response to MCP-1, induced the phosphorylation of GRK2 at Ser670, whereas MCP-1 enhancing monocyte migration. To test this hypothesis, we treated alone failed to increase the phosphorylation level of Ser670 (Fig. 5). monocytes with MCP-1 and/or LPS for up to 60 min and then Furthermore, LPS or LPS plus MCP-1 was unable to induce the extracted membrane proteins from the monocytes for detection of phosphorylation of GRK2 on Ser670 in TLR4-deficient monocytes membrane-bound GRK2. As shown in Fig. 4A, MCP-1 increased (Fig. 5B). These data demonstrate that LPS–TLR4 signaling mod- the membrane-bound GRK2 in a time-dependent manner, which ifies GRK2 phosphorylation at Ser670. suggests the induction of translocation of GRK2 to the cell mem- Downloaded from brane. However, LPS prevented this increase in membrane-bound Opposite roles for p38 MAPK and ERK in regulating monocyte GRK2 in response to MCP-1 stimulation (Fig. 4A). Again, TLR4 migration induced by LPS and MCP-1 deficiency prevented LPS-mediated effects on MCP-1–mediated MAPKs, including ERK, JNK, and p38, are important regulatory translocation of GRK2 (Fig. 4B). These results suggest a critical kinases involved in cell migration (20, 32). MCP-1–induced mono- inhibitory role for LPS in GRK2 translocation to the cell membrane, cyte migration was reported to be dependent on p38 MAPK (19,

which is an important step in promoting CCR2 desensitization. 33). However, the role of MAPK in LPS-enhanced monocyte http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. The translocation of GRK2 is regulated through phosphorylation of GRK2 at Ser670 induced by LPS. (A) Monocytes isolated from FIGURE 4. LPS inhibits MCP-1–induced GRK2 translocation. (A and WT mice were stimulated with MCP-1 (200 ng/ml) or LPS (100 ng/ml) for B) Monocytes isolated from WT or TLR42/2 mice were incubated with the time indicated, and the phosphorylation of GRK2 at Ser670 (P-GRK2 MCP-1 (200 ng/ml) and/or LPS (100 ng/ml) for the time indicated, and Ser670) was detected by immunoblotting analysis. The images are repre- cell membrane protein and plasma proteins were extracted, separated, and sentatives of three independent experiments. The graph depicts the mean 6 subjected to Western blot analysis for detection of membrane-bound SEM of the percentage changes in the ratio of P-GRK2/total GRK2 in the GRK2 (M-GRK2) and cytosolic GRK2 (C-GRK2). The images are rep- monocytes from three experiments. (B) Monocytes isolated from WT or resentatives of three independent experiments. The densitometry analysis TLR42/2 mice were stimulated with MCP-1 (200 ng/ml) and/or LPS (100 of blots was performed using ImageJ software. The graphs depict the ng/ml) for 30 min, and the phosphorylation of GRK2 at Ser670 (P-GRK2 mean 6 SEM of the percentage changes in the ratio of M-GRK2/C-GRK2 Ser670) was detected by immunoblotting analysis. The graph depicts the in the monocytes isolated from WT and TLR42/2 mice, which were mean 6 SEM of the percentage changes in the ratio of P-GRK2/total normalized by the density of actin, from three experiments. *p , 0.01 GRK2 in the monocytes from three experiments. *p , 0.01 versus groups versus groups not labeled with an asterisk. not labeled with an asterisk. 6 TLR4 ACTING THROUGH GRK2 AUGMENTS MONOCYTE CHEMOTAXIS migration has not been elucidated. Therefore, we investigated the prevented LPS-induced phosphorylation of GRK2 at Ser670.How- role of ERK, JNK, and p38 in LPS-enhanced monocyte migration ever, ERK inhibitor PD98059 appeared to increase phosphorylation in response to MCP-1, using pharmacological inhibitor approaches. of GRK2 at Ser670. These data suggest that p38 enhances LPS- We found, using the Boyden chamber–migration assay, that ERK induced Ser670 phosphorylation, whereas ERK signaling nega- inhibitor PD98059 signiﬁcantly increased monocyte migration in- tively regulates Ser670 phosphorylation. These alterations in the duced by LPS/MCP-1 (Fig. 6A). In contrast, p38 inhibitor SB203580 phosphorylation of GRK2 at Ser670 are associated with consistent abrogated the LPS/MCP-1–induced monocyte migration (Fig. 6A), changes in the levels of cell membrane–bound GRK2 (Fig. 6C), as and JNK inhibitor SP600125 had no effect on the LPS/MCP-1– well as cell surface expression of CCR2 (Fig. 6D). In monocytes induced monocyte migration. pretreated with p38 inhibitor SB203580, which decreased the To further address the mechanism by which ERK and p38 regulate phosphorylation of GRK2 at Ser670, the membrane-bound level of monocyte migration, we determined the effect of ERK and p38 GRK2 was increased (Fig. 6C), and cell surface expression of on the phosphorylation level of GRK2 at Ser670. Monocytes were CCR2 was decreased (Fig. 6D). In contrast, in the cells pretreated pretreated with MAPK inhibitor for 30 min, followed by MCP-1 with ERK inhibitor PD98059, which increased Ser670 phosphory- and/or LPS stimulation for an additional 30 min. As shown in Fig. lation, the membrane-bound level of GRK2 was reduced (Fig. 6C), 6B, Western blotting demonstrated that p38 inhibitor SB203580 and cell surface expression of CCR2 was elevated (Fig. 6D). Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. ERK and p38 play opposite roles in regulating monocyte migration induced by LPS and MCP-1. (A) WT monocytes were pretreated with p38 inhibitor SB203580 (10 mM), ERK inhibitor PD98059 (50 mM), or JNK inhibitor SP600125 (10 mM) for 30 min and then loaded into the upper wells of the microchamber, with or without 100 ng/ml LPS. The lower wells were filled with MCP-1 at a concentration of 200 ng/ml. Cell migration was allowed for 2 h, and the cells that crossed the filter were counted. The graph shows the mean and SEM of the percentage of control from three independent experiments. (B–D) WT monocytes were pretreated with p38 inhibitor SB203580 (10 mM), ERK inhibitor PD98059 (50 mM), or JNK inhibitor SP600125 (10 mM) for 30 min and then stimulated with LPS (100 ng/ml) and/or MCP-1 (200 ng/ml) for an additional 30 min. (B) The phosphorylation of GRK2 at Ser670 was detected by immunoblotting. The images are representatives of three independent experiments. The graph depicts the mean and SEM of the percentage changes in the ratio of P-GRK2/total GRK2 in the monocytes from three experiments. (C) Cell membrane protein and plasma proteins were also extracted, separated, and subjected to immunoblotting analysis for detection of membrane-bound GRK2 (M-GRK2) and cytosolic GRK2 (C-GRK2). The images are representatives of three independent experiments. The graph depicts the mean 6 SEM of the percentage changes in the ratio of M-GRK2/C- GRK2 in the monocytes, which were normalized by the density of actin, from three experiments. (D) The cell surface expression of CCR2 was measured by flow cytometry. Results are presented as percentage of control; mean 6 SEM (n = 3). *p , 0.01 versus groups not labeled with an asterisk. The Journal of Immunology 7

Considered as a whole, phosphorylation of Ser670 of GRK2 is ulate multiple cellular responses in various physiological contexts a critical regulatory target for LPS–TLR4 regulation of GRK2 (41, 42). Based on the previous report showing an important role subcellular translocation, in which p38 mediates LPS–TLR4 sig- for GRK2 in regulating MCP-1–induced CCR2B receptor desen- naling; this is negatively regulated by ERK. sitization (3), we focused our study on the role of GRK2 in the cross-talk between TLR4 and CCR2. Subcellular localization Discussion of GRK represents a signiﬁcant early regulatory mechanism of In the current study, we observed that cross-talk between TLR4 and GPCR desensitization (31). Ser670 lies within the Gbg-binding CCR2 in monocytes results in signiﬁcant augmentation of MCP- domain of GRK2 (31, 40), and phosphorylation at this site impairs 1–driven monocyte migration. LPS activation of TLR4 induces the GRK2/Gbg interaction, thereby inhibiting the translocation of phosphorylation of Ser670 of GRK2 through a mechanism medi- the kinase and its subsequent catalytic kinase activity at receptor ated by p38 MAPK, which suppresses GRK2 translocation to the membrane substrates (31, 43). Therefore, this signaling pathway cell membrane and the subsequent initiation of CCR2 internaliza- represents a negative-feedback loop that inhibits the active pool of tion and desensitization. This leads to enhanced monocyte migra- GRK2 engaged in GPCR regulation (31), although the signaling tion (Fig. 7). Thus, the current study demonstrates a novel function that modulates phosphorylation of Ser670 on GRK2 was not fully for TLR4 signaling in driving innate immune responses that control elucidated in previous studies. ERK1 is known to be activated by monocyte migratory responses toward sites of infection by regu- MCP-1 (44), and previous studies implicated ERK1 in the in- lating the availability of cell surface chemokine receptors. duction of phosphorylation of GRK2 on Ser670 (30, 45). In the MCP-1 is a major chemokine that directs monocyte migration current study, we did not see MCP-1–induced phosphorylation of to the site of infection and activates monocyte functions, such GRK2 on Ser670, but we observed that LPS alone induced the Downloaded from as endocytosis (5, 23). Monocyte migration is the net result of phosphorylation of GRK2 at this site. We further found that p38 multiple mediators, including chemokines and LPS, which are plays a role in mediating this LPS–TLR4–induced phosphoryla- both present during Gram-negative bacterial infection. Although tion of GRK2 on Ser670, with ERK playing a negative regulatory LPS alone fails to induce monocyte migration, as seen in the role in this process. current study, previous reports showed that LPS is able to augment The phosphorylation of GRK2 on Ser670 induced by LPS results

monocyte migration in response to MCP-1 through the induc- in a profound impact on CCR2 internalization and availability, as http://www.jimmunol.org/ tion of IL-8/CXCL8 expression (18) or through the activation well as subsequent effects on monocyte migration. As evidenced of MARCKS-related protein (25), both of which synergistically by the results from the current study, LPS-induced phosphoryla- amplify MCP-1–induced monocyte migration. However, the cur- tion of GRK2 on Ser670 inhibited GRK2 translocation to the cell rent study provides a new mechanism by which LPS directly membrane and subsequent CCR2 internalization, which was as- enhances CCR2 availability through the suppression of GRK2- sociated with enhanced monocyte migration. These effects of LPS induced CCR2 desensitization by internalization. The role of could be reversed by prevention of the phosphorylation of GRK2 TLR4 signaling in mediating the LPS-enhanced monocyte mi- on Ser670 by inhibiting p38. Furthermore, the negative regulatory gration was shown using TLR42/2 monocytes and in vivo studies effects of ERK were suppressed by ERK inhibitor PD98059, with using TLR42/2 mice. Genetic deletion of TLR4 completely blocked enhanced LPS-induced phosphorylation of GRK2 on Ser670, aug- by guest on September 29, 2021 the effect of LPS on augmenting monocyte migration both in vitro mented cell surface expression of CCR2, and increased cell mi- and in vivo. Of note, the current finding may represent a rapid gration (Fig. 6). regulation mechanism that may play a particularly important role It was observed in the study that knock down of GRK2 did not in the early stages of inflammation. Alternative regulatory mech- significantly enhance MCP-1–induced monocyte migration. This anisms may take place at later time points, such as LPS regulation observation may be explained by the following facts: first, it is of GRK mRNA (9) or CCR2 mRNA expression (34, 35), to further important to note that using an siRNA approach could not com- influence leukocyte migration behavior. pletely knock down GRK2 expression in RAW264.7 cells (Fig. For a large number of related GPCRs, rapid desensitization is 3A); thus, MCP-1 may not be able to induce enhanced cell mi- initiated through agonist-promoted receptor phosphorylation by gration in GRK2-knockdown cells to the same degree as that in- GRKs (36–38). There are seven members of the GRK family (39, duced by LPS+MCP-1 in WT cells. Second, the cell-migration 40). GRK2, the most widely studied member, is known to mod- data from the microchamber experiments were a net end result

FIGURE 7. Model of TLR4 and chemokine receptor cross-talk. (A) MCP-1 binding to CCR2 induces monocyte migration, as well as GRK2 translocation to the membrane, which results in CCR2 internalization and desensitization (blue arrow and dashed line), thereby negatively regulating monocyte migration. (B) LPS, acting through the TLR4-signaling and p38 MAPK pathway, induces the phosphorylation of GRK2 at Ser670 and inhibits the translocation of GRK2 to membrane, thereby suppressing the internalization and desensitization of CCR2, which leads to augmented monocyte migration (red arrows and dotted lines). TLR4 signaling–activated ERK plays a negative regulatory role in the phosphorylation of GRK2 at Ser670; this role can be observed while ERK is inhibited. 8 TLR4 ACTING THROUGH GRK2 AUGMENTS MONOCYTE CHEMOTAXIS at 6 h after cell stimulation, whereas the cell surface expression of (MRP) affects transmigration in activated RAW264.7 cells. Cell. Immunol. 256: 92–98. CCR2 detected by flow cytometry reflected the changes at 1 h 19. Ashida, N., H. Arai, M. Yamasaki, and T. Kita. 2001. Differential signaling for after cell stimulation. The expression level of CCR2 at this time MCP-1-dependent integrin activation and chemotaxis. Ann. N. Y. Acad. Sci. 947: point may not entirely match the alteration in cell migration. 387–389. 20. Liu, X., B. Ma, A. B. Malik, H. Tang, T. Yang, B. Sun, G. Wang, R. D. Minshall, Third, analysis of cell surface expression of CCR2 showed that, Y. Li, Y. Zhao, et al. 2012. Bidirectional regulation of neutrophil migration by in WT cells, LPS+MCP-1 decreased cell surface CCR2 by 6% mitogen-activated protein kinases. Nat. Immunol. 13: 457–464. compared with the control group, whereas MCP-1 in GRK2- 21. Zhang, J., N. Zhu, Q. Wang, J. Wang, Y. Ma, C. Qiao, Y. Li, X. Li, B. Su, and B. Shen. 2010. MEKK3 overexpression contributes to the hyperresponsiveness knockdown cells decreased cell surface CCR2 by 23%. This sig- of IL-12-overproducing cells and CD4+ T conventional cells in nonobese dia- nificant difference may also contribute to the difference in cell betic mice. J. Immunol. 185: 3554–3563. 22. Bu¨nger, S., U. J. Roblick, and J. K. Habermann. 2009. Comparison of five migration between these groups. Finally, although the data dem- commercial extraction kits for subsequent membrane protein profiling. Cyto- onstrate a predominant role for GRK2 in regulating monocyte technology 61: 153–159. migration, other GRKs may have an effect, which leads to a more 23. Yadav, A., V. Saini, and S. Arora. 2010. MCP-1: chemoattractant with a role beyond immunity: a review. Clin. Chim. Acta 411: 1570–1579. complicated outcome. 24. Ebihara, N., S. Yamagami, S. Yokoo, S. Amano, and A. Murakami. 2007. In- In summary, we identified a novel interaction between TLR4 and volvement of C-C chemokine ligand 2-CCR2 interaction in monocyte-lineage chemokine receptors that can control monocyte migration, an essen- cell recruitment of normal human corneal stroma. J. Immunol. 178: 3288–3292. 25. Gouwy, M., S. Struyf, H. Verbeke, W. Put, P. Proost, G. Opdenakker, and J. Van tial component of the innate immune response. The results support a Damme. 2009. CC chemokine ligand-2 synergizes with the nonchemokine G new model, as shown in Fig. 7, in which LPS–TLR4 signaling aug- protein-coupled receptor ligand fMLP in monocyte chemotaxis, and it coop- erates with the TLR ligand LPS via induction of CXCL8. J. Leukoc. Biol. 86: ments monocyte migration by modulating the cell surface expression 671–680. of chemokine receptors in a GRK2-dependent manner. Intervention 26. Moser, E., J. Kargl, J. L. Whistler, M. Waldhoer, and P. Tschische. 2010. G Downloaded from in the specific signaling pathway that involves the regulation of protein-coupled receptor-associated sorting protein 1 regulates the postendocytic sorting of seven-transmembrane-spanning G protein-coupled receptors. Phar- GRK2 subcellular translocation may represent a therapeutic strat- macology 86: 22–29. egy for controlling inflammation during bacterial infection. 27. Marchese, A., M. M. Paing, B. R. Temple, and J. Trejo. 2008. G protein-coupled receptor sorting to endosomes and lysosomes. Annu. Rev. Pharmacol. Toxicol. 48: 601–629. Disclosures 28. Mack, M., J. Cihak, C. Simonis, B. Luckow, A. E. Proudfoot, J. Plachy´,

The authors have no financial conflicts of interest. H. Bru¨hl, M. Frink, H. J. Anders, V. Vielhauer, et al. 2001. Expression and http://www.jimmunol.org/ characterization of the chemokine receptors CCR2 and CCR5 in mice. J. Immunol. 166: 4697–4704. 29. Otten, J. J., S. C. de Jager, A. Kavelaars, T. Seijkens, I. Bot, E. Wijnands, References L. Beckers, M. M. Westra, M. Bot, M. Busch, et al. 2013. Hematopoietic 1. Kamei, M., and C. V. Carman. 2010. New observations on the trafficking and G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion diapedesis of monocytes. Curr. Opin. Hematol. 17: 43–52. formation in LDL receptor-knockout mice. FASEB J. 27: 265–276. 2. Steevels, T. A., and L. Meyaard. 2011. Immune inhibitory receptors: essential 30. Elorza, A., S. Sarnago, and F. Mayor, Jr. 2000. Agonist-dependent modulation of regulators of phagocyte function. Eur. J. Immunol. 41: 575–587. G protein-coupled receptor kinase 2 by mitogen-activated protein kinases. Mol. 3. Weber, C., A. Schober, and A. Zernecke. 2004. Chemokines: key regulators of Pharmacol. 57: 778–783. mononuclear cell recruitment in atherosclerotic vascular disease. Arterioscler. 31. Penela, P., C. Ribas, and F. Mayor, Jr. 2003. Mechanisms of regulation of the Thromb. Vasc. Biol. 24: 1997–2008. expression and function of G protein-coupled receptor kinases. Cell. Signal. 15:

4. Cochran, B. H., A. C. Reffel, and C. D. Stiles. 1983. Molecular cloning of gene 973–981. by guest on September 29, 2021 sequences regulated by platelet-derived growth factor. Cell 33: 939–947. 32.Yeh,T.M.,S.H.Liu,K.C.Lin,C.Kuo,S.Y.Kuo,T.Y.Huang,Y.R.Yen, 5. Deshmane, S. L., S. Kremlev, S. Amini, and B. E. Sawaya. 2009. Monocyte R. K. Wen, L. C. Chen, and T. F. Fu. 2013. Dengue virus enhances thrombomo- chemoattractant protein-1 (MCP-1): an overview. J. Interferon Cytokine Res. 29: dulin and ICAM-1 expression through the macrophage migration inhibitory factor 313–326. induction of the MAPK and PI3K signaling pathways. PLoS ONE 8: e55018. 6. van Zoelen, M. A., M. I. Verstege, C. Draing, R. de Beer, C. van’t Veer, 33. Arefieva, T. I., N. B. Kukhtina, O. A. Antonova, and T. L. Krasnikova. 2005. S. Florquin, P. Bresser, J. S. van der Zee, A. A. te Velde, S. von Aulock, and MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent T. van der Poll. 2011. Endogenous MCP-1 promotes lung inflammation induced on activation of different signaling cascades. Cytokine 31: 439–446. by LPS and LTA. Mol. Immunol. 48: 1468–1476. 34. Sica, A., A. Saccani, A. Borsatti, C. A. Power, T. N. Wells, W. Luini, 7. Aragay, A. M., M. Mellado, J. M. Frade, A. M. Martin, M. C. Jimenez-Sainz, N. Polentarutti, S. Sozzani, and A. Mantovani. 1997. Bacterial lipopolysaccha- C. Martinez-A, and F. Mayor, Jr. 1998. Monocyte chemoattractant protein-1- ride rapidly inhibits expression of C-C chemokine receptors in human mono- induced CCR2B receptor desensitization mediated by the G protein-coupled cytes. J. Exp. Med. 185: 969–974. receptor kinase 2. Proc. Natl. Acad. Sci. USA 95: 2985–2990. 35. Zhou, Y., Y. Yang, G. Warr, and R. Bravo. 1999. LPS down-regulates the ex- 8. Lefkowitz, R. J. 1998. G protein-coupled receptors. III. New roles for receptor pression of chemokine receptor CCR2 in mice and abolishes macrophage in- kinases and beta-arrestins in receptor signaling and desensitization. J. Biol. filtration in acute inflammation. J. Leukoc. Biol. 65: 265–269. Chem. 273: 18677–18680. 36. Lefkowitz, R. J. 1993. G protein-coupled receptor kinases. Cell 74: 409–412. 9. Sun, L., and R. D. Ye. 2012. Role of G protein-coupled receptors in inflam- 37. Lohse, M. J., C. Krasel, R. Winstel, and F. Mayor, Jr. 1996. G-protein-coupled mation. Acta Pharmacol. Sin. 33: 342–350. receptor kinases. Kidney Int. 49: 1047–1052. 10. Gainetdinov, R. R., L. M. Bohn, J. K. Walker, S. A. Laporte, A. D. Macrae, 38. Premont, R. T., J. Inglese, and R. J. Lefkowitz. 1995. Protein kinases that M. G. Caron, R. J. Lefkowitz, and R. T. Premont. 1999. Muscarinic supersen- phosphorylate activated G protein-coupled receptors. FASEB J. 9: 175–182. sitivity and impaired receptor desensitization in G protein-coupled receptor ki- 39. Penela, P., C. Murga, C. Ribas, V. Lafarga, and F. Mayor, Jr. 2010. The complex nase 5-deficient mice. Neuron 24: 1029–1036. G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physio- 11. Benovic, J. L., A. DeBlasi, W. C. Stone, M. G. Caron, and R. J. Lefkowitz. 1989. pathological targets. Br. J. Pharmacol. 160: 821–832. Beta-adrenergic receptor kinase: primary structure delineates a multigene family. 40. Ribas, C., P. Penela, C. Murga, A. Salcedo, C. Garcıá-Hoz, M. Jurado-Pueyo, Science 246: 235–240. I. Aymerich, and F. Mayor, Jr. 2007. The G protein-coupled receptor kinase 12. Penn, R. B., A. N. Pronin, and J. L. Benovic. 2000. Regulation of G protein- (GRK) interactome: role of GRKs in GPCR regulation and signaling. Biochim. coupled receptor kinases. Trends Cardiovasc. Med. 10: 81–89. Biophys. Acta 1768: 913–922. 13. Penela, P., C. Ribas, I. Aymerich, and F. Mayor, Jr. 2009. New roles of G protein- 41. Evron, T., T. L. Daigle, and M. G. Caron. 2012. GRK2: multiple roles beyond G coupled receptor kinase 2 (GRK2) in cell migration. Cell Adhes. Migr. 3: 19–23. protein-coupled receptor desensitization. Trends Pharmacol. Sci. 33: 154–164. 14. Penn, R. B., and J. L. Benovic. 1994. Structure of the human gene encoding the 42. Penela, P., C. Murga, C. Ribas, A. Salcedo, M. Jurado-Pueyo, V. Rivas, beta-adrenergic receptor kinase. J. Biol. Chem. 269: 14924–14930. I. Aymerich, and F. Mayor, Jr. 2008. G protein-coupled receptor kinase 2 15. Fan, J., and A. B. Malik. 2003. Toll-like receptor-4 (TLR4) signaling augments (GRK2) in migration and inflammation. Arch. Physiol. Biochem. 114: 195–200. chemokine-induced neutrophil migration by modulating cell surface expression 43. Gurevich, E. V., J. J. Tesmer, A. Mushegian, and V. V. Gurevich. 2012. G of chemokine receptors. Nat. Med. 9: 315–321. protein-coupled receptor kinases: more than just kinases and not only for 16. Francke, A., J. Herold, S. Weinert, R. H. Strasser, and R. C. Braun-Dullaeus. GPCRs. Pharmacol. Ther. 133: 40–69. 2011. Generation of mature murine monocytes from heterogeneous bone marrow 44. Jimeńez-Sainz, M. C., B. Fast, F. Mayor, Jr., and A. M. Aragay. 2003. Signaling and description of their properties. J. Histochem. Cytochem. 59: 813–825. pathways for monocyte chemoattractant protein 1-mediated extracellular signal- 17. Hintzen, C., S. Quaiser, T. Pap, P. C. Heinrich, and H. M. Hermanns. 2009. regulated kinase activation. Mol. Pharmacol. 64: 773–782. Induction of CCL13 expression in synovial fibroblasts highlights a significant 45. Pitcher, J. A., J. J. Tesmer, J. L. Freeman, W. D. Capel, W. C. Stone, and role of oncostatin M in rheumatoid arthritis. Arthritis Rheum. 60: 1932–1943. R. J. Lefkowitz. 1999. Feedback inhibition of G protein-coupled receptor kinase 18. Chun, K. R., E. M. Bae, J. K. Kim, K. Suk, and W. H. Lee. 2009. Suppression 2 (GRK2) activity by extracellular signal-regulated kinases. J. Biol. Chem. 274: of the lipopolysaccharide-induced expression of MARCKS-related protein 34531–34534. The Journal of Immunology

Corrections

Liu, Z., Y. Jiang, Y. Li, J. Wang, L. Fan, M. J. Scott, G. Xiao, S. Li, T. R. Billiar, M. A. Wilson, and J. Fan. 2013. TLR4 signaling augments monocyte chemotaxis by regulating G protein–coupled receptor kinase 2 translocation. J. Immunol. 191: 857–864.

In the grant support footnote of the original publication, the number for National Key Basic Research (973) Program of China was incorrect. It should read “National Key Basic Research (973) Program of China No. 2010CB529704 (to Y.J.).” www.jimmunol.org/cgi/doi/10.4049/jimmunol.1390044