Toll-Like Receptor Signaling Alters the Expression of Regulator of G Protein Signaling Proteins in Dendritic Cells: Implications for G Protein-Coupled Receptor This information is current as Signaling of September 29, 2021. Geng-Xian Shi, Kathleen Harrison, Sang-Bae Han, Chantal Moratz and John H. Kehrl J Immunol 2004; 172:5175-5184; ; doi: 10.4049/jimmunol.172.9.5175 Downloaded from http://www.jimmunol.org/content/172/9/5175 References This article cites 49 articles, 26 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/172/9/5175.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Toll-Like Receptor Signaling Alters the Expression of Regulator of G Protein Signaling Proteins in Dendritic Cells: Implications for G Protein-Coupled Receptor Signaling Geng-Xian Shi,1 Kathleen Harrison,1 Sang-Bae Han,1 Chantal Moratz, and John H. Kehrl2 Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein- coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of Downloaded from TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down- regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expres- ␣ ␣ sion are likely important for DC function, because both proteins inhibit G i- and G q-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, ؊/؊ bone marrow-derived DCs from Rgs1 mice have a heightened migratory response to both CXCL12 and CCL19 when com- http://www.jimmunol.org/ pared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines. The Journal of Immunology, 2004, 172: 5175– 5184. endritic cells (DC)3 function as the sentinels of the im- main. TLR signaling leads to NF-␬B activation, a requirement for mune system (reviewed in Refs. 1 and 2). Immature DCs the differentiation of iDC to mDC (5). D (iDC) traffic from the blood to inflamed tissues where iDCs express the chemokine receptors CCR1, CCR2, CCR5, they capture Ag, and differentiate into mature DC (mDC). Subse- and CXCR1, and respond to their respective ligands, chemokines quently, they move to the draining lymphoid nodes to prime naive often expressed in inflamed tissues (6, 7). In addition, iDCs mi- by guest on September 29, 2021 T cells. iDC are highly endocytic and well adapted for the capture grate in response to other inflammatory mediators that couple to G of Ag, but they function poorly as APCs. In contrast, mDC are protein-coupled receptors (GPCRs) including histamine (8), sphin- efficient APCs and important modulators of T cell function. Many gosine-1-phosphate (S-1P) (9), lysophosphatidic acid (LPA) (10), pathogen-derived substances are efficient inducers of iDC matura- and ATP (11). Maturing DCs lose their migratory response to tion, and do so predominantly by the engagement of Toll-like re- many of these inflammatory chemoattractants by either receptor ceptors (TLRs) (reviewed in Refs. 3 and 4). In humans, 10 TLR down-regulation or receptor desensitization, and acquire respon- homologs have been identified, the majority displayed by DCs. siveness to CC chemokine ligand (CCL)19 and CCL21 via the TLR contain two major domains, an extracellular domain charac- acquisition of high levels of CCR7 (6, 7). CCL19 and CCL21 have terized by leucine-rich repeats and an intracellular Toll-like do- significant roles in the accumulation of Ag-loaded DCs in T cell- rich areas of draining lymph nodes. Exposure of maturing DCs to B Cell Molecular Immunology Section, Laboratory of Immunoregulation, National histamine, S-1P, LPA, or ATP no longer induces a chemotactic Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, response, but rather down-regulates IL-12 and enhances IL-10 pro- MD 20892 duction (8–11). A number of prior studies have demonstrated that Received for publication September 29, 2003. Accepted for publication February signaling via chemokine and other GPCRs can modulate DC IL-12 13, 2004. production (reviewed in Ref. 12). For example, the production of The costs of publication of this article were defrayed in part by the payment of page ϩ charges. This article must therefore be hereby marked advertisement in accordance IL-12 by CD8␣ murine DCs can be triggered by CCR5 with 18 U.S.C. Section 1734 solely to indicate this fact. signaling (13). 1 G.-X.S., K.H., and S.-B.H. contributed equally to the completion of this study. Ligand-activated GPCRs such as chemokine receptors act as a 2 Address correspondence and reprint requests to Dr. John H. Kehrl, B Cell Molecular guanine nucleotide exchange factor for G␣ subunit of the hetero- Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy ␣ and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. E-mail trimeric G protein (reviewed in Refs. 14 and 15). Once the G address: [email protected] subunit exchanges GDP for GTP, it dissociates from the G␤␥ het- 3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, erodimer, thereby allowing both G␣ and G␤␥ to activate down- mature DC; TLR, Toll-like receptor; GPCR, G protein-coupled receptor; S-1P, sphin- stream effectors. However, G␣ subunits have an intrinsic GTPase gosine-1-phosphate; LPA, lysophosphatidic acid; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand; GAP, GTPase-activating protein; RGS, regulator of activity that limit the duration that they remain GTP bound and G protein signaling; BM, bone marrow; ERK, extracellular signal-regulated kinase; thus able to signal. In addition, GTPase-activating proteins (GAPs) med, medium; M1, muscarinic type 1; SRE, serum response element; TTBS, Tween for G␣ subunits termed regulator of G protein signaling (RGS) 20 plus TBS; MAPK, mitogen-activated protein kinase; PTX, pertussis toxin; IP3, inositol 1,4,5-trisphosphate; GFP, green fluorescent protein. proteins can further accelerate the intrinsic GTPase activity of G␣ Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 5176 TLR SIGNALING AND RGS PROTEINS subunits (reviewed in Ref. 16). Genetic studies in yeast, Aspergil- Generation of human monocyte-derived DCs lus nidulans, and Caenorhabditis elegans initially identified such PBMC were obtained from heparinized blood of healthy donors by Ficoll proteins (17–19). Providing evidence that they function by inter- density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, ␣ ␣ acting with G subunits, a yeast two-hybrid screen with G i3 iden- Sweden). The isolated PBMC were cultured in RPMI 1640 at 37°Cin tified a mammalian RGS protein originally termed GAIP and now 100-mm plate (Falcon, Franklin Lakes, NJ) for 3 h, and the nonadherent RGS19 (20). Cementing the functional relationship between the cells were discarded, and the adherent cells were washed with PBS for three times. After this procedure, the resulting cell population was repre- yeast and mammalian proteins, several human RGS proteins sub- sented by Ͼ98% CD14ϩ monocytes, as assessed by flow cytometry using stituted for Sst2p, a protein involved in the desensitizing of pher- FITC-CD14 Ab. Alternatively, elutriated monocytes prepared from leuco- omone signaling, a G protein-coupled signaling pathway in yeast paks were used as the starting population. The monocytes were maintained (21). Rapidly thereafter, RGS proteins were shown to possess GAP in RPMI 1640 medium supplemented with 10% FCS in the presence of GM-CSF (100 ng/ml) and IL-4 (50 ng/ml). After 4–6 days of culture, activity for Gi and Gq subfamily members (22–24). Coding regions nonadherent and loosely adherent cells were collected and used for sub- for ϳ25 human RGS proteins have now been identified. Two Rho sequent experiments. The purity of the recovered DCs exceeded 95% as guanine exchange factors, which possess divergent RGS domains, assessed by flow cytometry using PE-CD11c Ab. ␣ ␣ selectively act as GAPs for G 12 and G 13 (25, 26). Experimen- Isolation of mouse BM-derived DCs tally, the introduction of expression vectors for RGS1, RGS3, and RGS4 into B lymphocyte cell lines dramatically impairs chemo- DCs were generated from BM cells from 8- to 10-wk-old C57BL/6 female mice (30).