Cutting Edge: GPR35/CXCR8 Is the Receptor of the Mucosal CXCL17

This information is current as José L. Maravillas-Montero, Amanda M. Burkhardt, Peter A. of September 25, 2021. Hevezi, Christina D. Carnevale, Martine J. Smit and Albert Zlotnik J Immunol 2015; 194:29-33; Prepublished online 19 November 2014;

doi: 10.4049/jimmunol.1401704 Downloaded from http://www.jimmunol.org/content/194/1/29

Supplementary http://www.jimmunol.org/content/suppl/2014/11/19/jimmunol.140170 Material 4.DCSupplemental http://www.jimmunol.org/ References This article cites 33 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/194/1/29.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: GPR35/CXCR8 Is the Receptor of the Mucosal Chemokine CXCL17 Jose´ L. Maravillas-Montero,*,†,1,2 Amanda M. Burkhardt,*,†,1 Peter A. Hevezi,*,† Christina D. Carnevale,*,† Martine J. Smit,†,‡ and Albert Zlotnik*,† are chemotactic that direct the traf- In this article, we show that CXCL17 signals through the fic of leukocytes and other cells in the body. Chemokines orphan GPCR GPR35. This receptor is not known to bind bind to G protein–coupled receptors expressed on target chemokines (8); however, like CXCL17, it exhibits a mucosal cells to initiate signaling cascades and induce chemotaxis. expression pattern (9). Partly because of this, it has attracted Although the cognate receptors of most chemokines have attention as a potential therapeutic target (9). Because our been identified, the receptor for the mucosal chemokine findings indicate that it represents a novel , CXCL17 is undefined. In this article, we show that we suggest that it should be named CXCR8. Downloaded from GPR35 is the receptor of CXCL17. GPR35 is expressed in mucosal tissues, in CXCL17-responsive monocytes, Materials and Methods and in the THP-1 monocytoid cell line. Transfection Cells and reagents of GPR35 into Ba/F3 cells rendered them responsive to THP-1 leukemia cells and the pro-B cell line Ba/F3 were maintained in RPMI 1640. Abs used include rabbit IgG (Jackson ImmunoResearch, West Grove, CXCL17, as measured by calcium-mobilization assays. PA) and polyclonal rabbit anti-human GPR35 (Cayman Chemicals, Ann http://www.jimmunol.org/ Furthermore, GPR35 expression is downregulated in 2 2 Arbor, MI). A clone encoding human GPR35 was obtained from The the lungs of Cxcl17 / mice, which exhibit defects in Missouri S&T cDNA Resource Center, under GenBank accession number macrophage recruitment to the lungs. We conclude that AY275467 (http://www.ncbi.nlm.nih.gov/nuccore/AY275467). GPR35 is a novel chemokine receptor and suggest that it Body Index of Gene Expression database should be named CXCR8. The Journal of Immunology, The Body Index of Gene Expression is a comprehensive database of human 2015, 194: 29–33. gene expression (5, 10). Data from a probeset (210264_at) corresponding to GPR35 were used to determine its expression in the database. by guest on September 25, 2021 hemokines (chemotactic cytokines) are small secreted Quantitative real-time PCR analysis proteins that direct the migration of various cell types (1). They have been classified as homeostatic or in- Quantitative real-time PCR (qRT-PCR) data were generated with a Light- C Cycler 480 (Roche). cDNA was obtained from total RNA extracted from flammatory, depending on the stimuli that regulate their pro- THP-1 cells using QIAGEN kits. Gene-specific primers and corresponding duction (2). Chemokines bind receptors that belong to the class universal probes were used to quantify GPR35 or control gene transcripts. A G protein–coupled receptor (GPCR) superfamily. Binding these receptors triggers signaling cascades that promote multi- Chemotaxis assays ple cellular functions (3) through the coupling of G proteins. Chemotaxis assays were performed for 18–20 h using 5.0-mm, 24-well The human chemokine superfamily includes 48 ligands and 19 Transwell migration plates (Corning, Manassas, VA), with 200 ng/ml che- mokine (R&D Systems) in 600 ml incomplete RPMI added to the bottom receptors. The receptors for most of the ligands have been chambers (0.5–1.0 3 106 cells/well). Where noted, cells were pretreated with m identified (2), and only two chemokine ligands remain orphan 200 ng/ml Bordetella pertussis toxin (PTX) or 10 M PGE2 (both from (i.e., their receptors have not been identified: CXCL14 and Sigma-Aldrich, St. Louis, MO) for 24 h. CXCL17). CXCL17 was the last chemokine described (4), and Quantitation of chemotaxis by flow cytometry its expression pattern is closely associated with mucosal tissues (5, 6). Few reports exist on CXCL17, but it is known to This protocol was adapted from Proudfoot et. al (11). Briefly, the chemotaxed cells were resuspended in 200 ml13 PBS. Standards were generated through chemoattract macrophages both in vitro (4, 6), and in vivo (7). 10-fold dilutions ranging from 106 to 102 cells/200 ml. The cell counts were CXCL17 is also known to promote angiogenesis (6). the number of events in 30 s recorded in a FACSCalibur (Becton Dickinson).

*Department of Physiology and Biophysics, University of California, Irvine, Irvine, a University of California Institute for Mexico and the United States Postdoctoral CA 92697; †Institute for Immunology, University of California, Irvine, Irvine, CA Fellowship. 92697; and ‡Division of Medicinal Chemistry, Free University Amsterdam, 1081 HV Address correspondence and reprint requests to Dr. Albert Zlotnik, Department Amsterdam, the Netherlands of Physiology and Biophysics and Institute for Immunology, University of Cal- 1J.L.M.-M. and A.M.B. contributed equally to this work. ifornia, Irvine, 3034 Hewitt Hall, Irvine, CA 92697. E-mail address: azlotnik@uci. edu. 2Current address: Research Support Network, National Institute of Medical Sciences and Nutrition Salvador Zubiran–National Autonomous University of Mexico, Tlalpan, The online version of this article contains supplemental material. Mexico City, Mexico. Abbreviations used in this article: GPCR, G protein–coupled receptor; PTX, Bordetella Received for publication July 16, 2014. Accepted for publication October 27, 2014. pertussis toxin; qRT-PCR, quantitative real-time PCR; WT, wild-type.

This work was supported by National Institute of Allergic and Infectious Disease/ Ó National Institutes of Health Grant R01AI093548. J.L.M.-M. was supported by Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401704 30 CUTTING EDGE: GPR35/CXCR8 IS THE RECEPTOR OF CXCL17

GPR35-transfection assays A total of 2 3 107 cells/ml Ba/F3 cells was resuspended in 500 ml cytomix (12) and transferred to a 0.4-cm electroporation cuvette (USA Scientific). Then, 20 mg pcDNA3.1+/GPR35 DNA was added prior to electroporation using a Bio-Rad system (300 V, 960 mF). Cells were cultured in RPMI 1640 at 37˚C for 48 h before performing assays.

Calcium-mobilization assays A total of 5 3 107 THP-1 or Ba/F3 cells/ml was loaded with Calcium Green-1, AM and Fura Red, AM (Life Technologies, Carlsbad, CA) at 10 mmol/l for 30 min at 37˚C. After 30 s, cells were stimulated with human CXCL17 (R&D Systems) or 100 mM ionomycin (Sigma-Aldrich; positive control). The Calcium Green/Fura Red fluorescence ratio was measured in a FACS- Calibur before and after the addition of activators and was analyzed with FlowJo software.

2 2 Wild-type and Cxcl17 / mice 2 2 Lung tissue was collected from wild-type (WT) C57BL/6 or Cxcl17 / mice, which were obtained as described (13). All mouse studies were performed under a protocol approved by the Institutional Animal Care and Use Com- mittee of the University of California Irvine. Downloaded from Results and Discussion We report a novel chemokine receptor, GPR35, which we identified as the receptor of CXCL17, a chemokine first re- ported in 2006 (4). CXCL17 is a mucosal chemokine that has been associated with human diseases, including idiopathic http://www.jimmunol.org/ pulmonary fibrosis (5) and cancer (14, 15). Recently, we re- 2 2 ported that Cxcl17 / mice exhibit a paucity of macrophages in the lungs (7), whereas, conversely, injection of Cxcl17 i.p. induces strong macrophage recruitment in vivo (7). In vitro, macrophages (including THP-1 cells) chemotax to CXCL17, as well as to CCL2 (data not shown), a well-known macro- phage chemoattractant (16). These data indicate that CXCL17 is a major macrophage chemoattractant. by guest on September 25, 2021 CXCL17 induces chemotactic responses in the THP-1 hu- man monocyte cell line (Fig. 1A), indicating that THP-1 expresses the CXCL17 receptor. The chemotactic response of THP-1 to CXCL17 was amplified by pretreatment with PGE2 (Fig. 1A), as was shown for other chemokines (17). These responses are also sensitive to PTX (Fig. 1A), which inhibits a G i/o protein–signaling pathways, as observed for other che- mokine receptors (18). The binding of chemokines to their receptors induces an increase in cytosolic calcium (18). As shown in Fig. 1B, +2 CXCL17 induces a Ca flux in both resting and PGE2- treated cells; however, as observed with chemotactic responses (Fig. 1A), the Ca+2 fluxes observed in PGE -treated THP-1 2 FIGURE 1. THP-1 cells are responsive to CXCL17. (A) THP-1 cells were cells in response to CXCL17 were stronger (Fig. 1B). Both tested in CXCL17-directed chemotaxis Transwell assays, both under control CCL2 and CXCL17 induced calcium fluxes in THP-1 cells. or PGE2-pretreated conditions. These cells also were tested after pretreatment Chemokine receptors can be desensitized for a certain pe- with PTX (200 ng/ml). The bars show the total number of recovered cells riod of time following activation (19). As shown in Fig. 1C, (chemotaxed) in the lower chamber. (B) Representative calcium flux response CXCL17 desensitizes itself in THP-1 cells, but it does not of THP-1 cells (loaded with Ca+2-sensitive dyes), either resting or pretreated C desensitize the Ca+2 flux induced by CCL2 (a chemokine that with PGE2, when stimulated with 100 nM of CXCL17 (n = 2). ( ) De- signals through CCR2) (Fig. 1C) (20). Conversely, CCL2 did sensitization of the CXCL17 receptor expressed by THP-1 cells; 100 nM of CXCL17 or CCL2 was added at the indicated time points to induce THP-1 not desensitize CXCL17-mediated responses, indicating that calcium flux responses. Representative graphs are shown (n = 3). these two chemokines signal through different receptors (19). Before searching for the CXCL17 receptor, we tested whether CXCL17 bound or activated other known chemokine We screened the Body Index of Gene Expression database of receptors. We confirmed that CXCL17 does not bind or signal human gene expression (10) to identify GPCRs expressed by to CCR2, CCR5, CXCR2, CXCR3, CXCR4, or CXCR7 monocytes. We narrowed the list of possible candidates by (Supplemental Tables IA, IB). Therefore, we decided to selecting orphan GPCRs that exhibit structural characteristics search for orphan GPCRs expressed in macrophages as po- of chemokine receptors and a tissue-expression pattern similar tential candidates to be the CXCL17 receptor. to CXCL17. The orphan receptor GPR35 is known to be The Journal of Immunology 31 expressed in mucosal tissues, including the gastrointestinal Xaa-Pro motif, and a Cys-Trp-Xaa-Pro motif (27). These tract (21) and adult lung (22), and this expression profile regions are highly conserved in chemokine receptors (28, 29). correlates strongly with the expression of CXCL17. Impor- Additionally, some specific residues, known as “micro- tantly, it is also expressed by monocytes (23). The ImmGen switches,” which are also highly conserved in chemokine database (www.immgen.org) indicates that GPR35 is expressed receptors, are present in GPR35 (27). Taken together, these by dendritic cells, macrophages, and granulocytes, all of which structural characteristics are consistent with the conclusion chemotax to CXCL17 (4, 24). that GPCR35 is a signaling receptor for CXCL17. To assess the ability of CXCL17 to signal through GPR35, Kynurenic acid, 2-Acyl lysophosphatidic acid, and Zaprinast we sought to induce a CXCL17-mediated calcium flux in were reported to be GPR35 agonists (30); however, the previously nonresponsive cells by transfecting this receptor into responses observed with these ligands are in the micromolar a GPR35-null cell line. We transfected the mouse pro-B cell range. In contrast, CXCL17 induces calcium fluxes in the line Ba/F3, which does not express GPR35 (data not shown) nanomolar range (Fig. 2B), within physiologic concentrations (25). CXCL17 induced a robust calcium flux in GPR35- of this chemokine in vivo (31). transfected BA/F3 cells (Fig. 2A), and it exhibited a dose- We were able to increase the responsiveness of THP-1 cells response pattern with increasing CXCL17 concentrations to CXCL17 through pretreatment with PGE2 (Fig. 1A, 1B), (Fig. 2B). Another orphan chemokine, CXCL14, did not suggesting that PGE2 induces the expression of the CXCL17 induce calcium fluxes in GPR35-transfected BA/F3 cells (data receptor in THP-1 cells. Using qRT-PCR, we confirmed that not shown). Furthermore, transfection of HEK293 cells also GPR35 expression is significantly elevated in PGE2-treated Downloaded from 2 2 induced a CXCL17-mediated calcium flux (Fig. 2C). GPR35 THP-1 cells (Fig. 3A). We recently reported that a Cxcl17 / is located in human chromosome 2 near CXCR7, and mouse exhibits a paucity of macrophages in the lungs (7), and a phylogenetic analysis of chemokine receptors indicates that Cxcl17 chemoattracts macrophages from lungs of WT, but 2 2 it is closest to CXCR7, suggesting that these genes may share not Cxcl17 / , mice (7). These observations indicate that a common ancestor (Supplemental Fig. 1). GPR35 exhibits CXCL17 is a physiologic signal that recruits certain macro- a DRY box (Fig. 2D), a critical motif shared by all signaling phage subsets to the lungs that should express the CXCL17 http://www.jimmunol.org/ chemokine receptors that is required for G protein coupling receptor. This model predicts that cells expressing the (26). GPR35 also exhibits a conserved Asp residue, a Thr- CXCL17 receptor would not be recruited into the lungs of by guest on September 25, 2021 FIGURE 2. GPR35 is the CXCL17 chemokine receptor. (A) CXCL17 (100 nM) induces a calcium flux in GPR35- transfected BA/F3 cells loaded with Ca2+-sensitive dyes. Representative ex- periment is shown (n =6).(B)Dose response of CXCL17-induced calcium flux in GPR35-transfected cells. Repre- sentative experiment is shown (n =2).(C) CXCL17 (100 nM) induces a calcium flux in GPR35-transfected HEK293 cells. Representative experiment is shown (n =2).(D) GPR35 exhibits structural characteristics typical of chemokine re- ceptors. Alignment of protein sequences of CCR1, CCR2, CCR5, CXCR4, CXCR7, and GPR35. Conserved residues are highlighted in dark gray. The seven transmembrane (TM) do- mains are shown. The boxes show the DRY box, the Thr-Xaa-Pro (TxP) motif, and the Cys-Trp-Xaa-Pro (CWxP) motif. The arrowheads in- dicate the conserved aspartic acid at the second TM region and the con- served leucine/phenylalanine at the fifth TM region. The stars indicate the position of the conserved micro- switches. 32 CUTTING EDGE: GPR35/CXCR8 IS THE RECEPTOR OF CXCL17

Disclosures The authors have no financial conflicts of interest.

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SUPPLEMENTARY FIGURE 1

Sup. Fig. 1. GPR35 and chemokine receptor genes. A, localization of the GPR35 gene in the distal region of the long arm of the human chromosome 2; as depicted, it is possible to see the neighboring CXCR7 gene in the proximity. B, phylogenetic analysis of the protein sequences of the known chemokine receptors showing that the most closely related member to GPR35 is CXCR7..

SUPPLEMENTARY TABLES.

A. Radioligand displacement studies chemokine receptors.

Receptor Displacer chemokine pKi CXCR3 125I-CXCL10 CXCL11 10.4 ± 0.1 CXCL17 n.d CXCR4 125I-CXCL12 CXCL12 9.5 ± 0.1 CXCL17 n.d. CXCR7 125I-CXCL12 CXCL12 9.6 ± 0.1 CXCL17 n.d CCR2 125I-CCL2 CCL2 9.6 ± 0.2 CXCL17 n.d n.d = no displacement

Radioligand binding assay – Membranes from HEK293T cells transiently expressing the respective chemokine receptors were incubated with ~ 50 pmol 125I-chemokine and increasing concentrations of chemokine (control) or CXCL17. Cells were incubated for 3 h at 4°C and washed twice with binding buffer containing 0.5 M NaCl. After harvesting the samples with lysis buffer, the remaining cell-bound radioactivity was counted.

B. Chemokine-induced β-arrestin recruitmentva

Receptor Agonist pEC50 CXCR2 CXCL8 8.5 ± 0.3 CXCL17 no effect CCR5 CCL5 10.6 ± 0.1 CXCL17 no effect

! β -arrestin recruitment assay – PathHunter™ CCR5 or CXCR2 expressing β-arrestin cells (DiscoveRx (Fremont, CA)) were used to monitor chemokine-induced β-arrestin recruitment based on enzyme complementation.