Glucocorticoids Suppress CCR9-Mediated Chemotaxis, Calcium Flux, and Adhesion to MAdCAM-1 in Human T Cells This information is current as Emily Wendt, Gemma E. White, Helen Ferry, Michael of September 25, 2021. Huhn, David R. Greaves and Satish Keshav J Immunol published online 25 March 2016 http://www.jimmunol.org/content/early/2016/03/24/jimmun ol.1500619 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/03/24/jimmunol.150061 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 25, 2016, doi:10.4049/jimmunol.1500619 The Journal of Immunology Glucocorticoids Suppress CCR9-Mediated Chemotaxis, Calcium Flux, and Adhesion to MAdCAM-1 in Human T Cells Emily Wendt,* Gemma E. White,† Helen Ferry,‡ Michael Huhn,* David R. Greaves,† and Satish Keshav* CCR9 expressed on T lymphocytes mediates migration to the small intestine in response to a gradient of CCL25. CCL25-stimulated activation of a4b7 integrin promotes cell adherence to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed by vascular endothelial cells of the intestine, further mediating gut-specific homing. Inflammatory bowel disease is a chronic inflam- matory condition that primarily affects the gastrointestinal tract and is characterized by leukocyte infiltration. Glucocorticoids (GCs) are widely used to treat inflammatory bowel disease but their effect on intestinal leukocyte homing is not well understood. Downloaded from We investigated the effect of GCs on the gut-specific chemokine receptor pair, CCR9 and CCL25. Using human peripheral blood- derived T lymphocytes enriched for CCR9 by cell sorting or culturing with all-trans retinoic acid, we measured chemotaxis, intracellular calcium flux, and a4b7-mediated cell adhesion to plate-bound MAdCAM-1. Dexamethasone (DEX), a specific GC receptor agonist, significantly reduced CCR9-mediated chemotaxis and adhesion to MAdCAM-1 without affecting CCR9 surface expression. In contrast, in the same cells, DEX increased CXCR4 surface expression and CXCL12-mediated signaling and downstream functions. The effects of DEX on human primary T cells were reversed by the GC receptor antagonist mifepristone. http://www.jimmunol.org/ These results demonstrate that GCs suppress CCR9-mediated chemotaxis, intracellular calcium flux, and a4b7-mediated cell adhesion in vitro, and these effects could contribute to the efficacy of GCs in treating intestinal inflammation in vivo. The Journal of Immunology, 2016, 196: 000–000. he chemokine CCL25 is constitutively expressed in the peripheral blood, the frequency of circulating CCR9+ T cells is small intestine and interacts with a single signaling re- elevated during small intestinal Crohn’s disease, suggesting a role T ceptor, CCR9 (1, 2). In humans, CCR9 is expressed on of CCR9+ T cells in small intestinal inflammation (11). CCR9, ∼ most (58–97%) T cells in the small intestine, 25% in the colon, CCL25, a4b7, and MAdCAM-1 are attractive therapeutic targets and a small minority (3–5%) in peripheral blood (2, 3). CCR9+ in IBD because of their tissue and cell specificity, and clinical by guest on September 25, 2021 T cells coexpress integrin a4b7, which binds to mucosal addressin studies show positive effects on blocking these proteins in subsets cell adhesion molecule-1 (MAdCAM-1) (4). MAdCAM-1 is consti- of IBD patients (12–15). tutively expressed on vascular endothelial cells in the small and Glucocorticoids (GCs) have broad-ranging anti-inflammatory large intestine, and together CCR9 and a4b7 preferentially me- actions, are highly effective in many chronic inflammatory dis- diate the adherence and migration of effector T cells into the orders, and have been the mainstay of IBD treatment since the intestine (5, 6). 1950s (16, 17). The anti-inflammatory effects of GCs are mainly Inflammatory bowel disease (IBD) is characterized by leukocyte mediated through interaction with cytosolic GC receptors (GCRs) infiltration, increased levels of proinflammatory cytokines in the to modify the transcription of proinflammatory genes (18, 19). intestine, and increased expression of MAdCAM-1 (7-10). In the Previously characterized effects of GCs on T cells include inhibiting TCR signaling, suppressing cell proliferation, inducing *Translational Gastroenterology Unit, Nuffield Department of Medicine, John Radcliffe IkB synthesis, and subsequently reducing NF-kB–mediated tran- † Hospital, Oxford OX3 9DU, United Kingdom; Sir William Dunn School of Pathology, scription of proinflammatory cytokines (19–21). However, little is University of Oxford, Oxford OX1 3RE, United Kingdom; and ‡Experimental Medicine Division, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DU, known about the effects of GCs on intestine-specific cell recruit- United Kingdom ment. We investigated the effect of GCs on CCR9 expression and Received for publication April 1, 2015. Accepted for publication February 22, 2016. function, as well as downstream a4b7 activation in primary human This work was supported by the Oxford Biomedical Research Centre, funded by the T cells, in vitro. United Kingdom National Institutes for Health Research Grant A93081 (to S.K.), as In this study we demonstrate that CCR9-mediated chemotaxis, well as by an unrestricted grant from ChemoCentryx, Inc. (Mountain View, CA). The funders had no role in the study design, data collection and analysis, decision to intracellular calcium flux, and adhesion to MAdCAM-1 are sig- publish, or preparation of the manuscript. nificantly suppressed following corticosteroid treatment, and this is Address correspondence and reprint requests to Dr. Satish Keshav, Gastroenterology mediated through activation of GCRs. As a control, we compared Unit, Nuffield Department of Medicine, Level 5, John Radcliffe Hospital, Headley + Way, Oxford OX3 9DU, U.K. E-mail address: [email protected] the effects of GCs on CXCR4, which is coexpressed on CCR9 The online version of this article contains supplemental material. T cells. CXCR4 is stimulated by a single ligand, CXCL12, and consistent with previous reports, GCs increase CXCR4 expression Abbreviations used in this article: ATRA, all-trans retinoic acid; DEX, dexamethasone; DOC, deoxycorticosterone acetate; GC, glucocorticoid; GCR, glucocorticoid receptor; and enhance CXCL12-mediated functions (22, 23). These results IBD, inflammatory bowel disease; MAdCAM-1, mucosal addressin cell adhesion mole- identify novel effects of GCs on the function of primary human cule-1; MFI, mean fluorescence intensity; MIF, mifepristone; PRED, prednisolone. T cells that are likely to be physiologically and clinically relevant Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 in intestinal inflammation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500619 2 GLUCOCORTICOIDS SUPPRESS CCR9 FUNCTION Materials and Methods Calcium mobilization assay All-trans retinoic acid–cultured cells Vehicle (H2O) and DEX-treated cells were separately labeled with anti- PBMCs were depleted of monocytes by CD14+ bead selection (Miltenyi CCR9 (R&D Systems, clone 248621) followed by anti-mouse IgG con- Biotec, Surrey, U.K.). Remaining leukocytes were activated by culturing on jugated to allophycocyanin in PBS with 0.1% BSA. Vehicle-treated cells plate-bound anti-CD3 (3 mg/ml) (BioLegend, Cambridge, U.K., clone OKT3) were labeled with anti-CD3 Alexa Fluor 700 and DEX-treated cells with and anti-CD28 (3 mg/ml) (BioLegend, clone CD28.2) in complete culture anti-CD3 allophycocyanin–eFluor 780 (eBioscience, clone UCHT1). An medium (RPMI 1640 supplemented with 10% FCS, penicillin-streptomycin) equivalent number of vehicle- and DEX-treated cells were combined in with 100 U IL-2 (PeproTech, Rocky Hill, NJ) and 100 nM all-trans retinoic HBSS with 0.02% Pluronic F-127, 1 mg/ml Fura Red AM (Life Tech- acid (ATRA). After 72 h, cells were removed from CD3/CD28 stimulation and nologies), and incubated for 30 min at 37˚C. Cells were resuspended in resuspended in complete culture medium with IL-2 and ATRA. Cells were HBSS with 0.1% BSA, 1 mM CaCl2, 0.5 mM MgCl2, and 10 mM HEPES used for assays between days 6 and 9 from initial isolation. with Live/Dead cell marker, SYTOX Green (Life Technologies), and in- cubated at 37˚C until flow cytometry was performed. Data were acquired on an LSR II SORP and analysis was performed in FlowJo. The gating Steroid and steroid receptor antagonist treatment 2 strategy was as follows: live cells (SYTOX Green ), excluding cell dou- Cells were resuspended in RPMI 1640 supplemented with 10% FCS, blets (determined by forward light scatter area versus forward light scatter 50 U/ml penicillin, and 50 mg/ml streptomycin. Cells were