Cutting Edge: Suppression of T Cell Chemotaxis by Sphingosine 1-Phosphate Markus Graeler, Geetha Shankar and Edward J. Goetzl This information is current as J Immunol 2002; 169:4084-4087; ; of September 28, 2021. doi: 10.4049/jimmunol.169.8.4084 http://www.jimmunol.org/content/169/8/4084 References This article cites 14 articles, 8 of which you can access for free at: Downloaded from http://www.jimmunol.org/content/169/8/4084.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on September 28, 2021 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology ● Cutting Edge: Suppression of T Cell Chemotaxis by Sphingosine 1-Phosphate1 Markus Graeler,* Geetha Shankar,† and Edward J. Goetzl2* Blood unstimulated CD4 T cells express high levels of LPA2 Murine CD4 and CD8 T cells express predominantly types 1 (Edg-4) receptors constitutively, whereas CD8 T cells from the and 4 sphingosine 1-phosphate (S1P) G protein-coupled recep- same source show only traces of LPA1 (Edg-2) (8). In studies of tors (designated S1P1 and S1P4 or previously endothelial dif- regulation of expression of LPA receptors, mitogen activation of ferentiation gene-encoded 1 and 6) for S1P, which has a nor- blood CD4 T cells down-regulated expression of LPA2 and up- mal plasma concentration of 0.1–1 ␮M. S1P now is shown to regulated that of LPA1, but such activation of CD8 T cells did not enhance chemotaxis of CD4 T cells to CCL-21 and CCL-5 by alter expression of any LPA receptor (9). IL-2 production by blood Downloaded from ␮ ␮ up to 2.5-fold at 10 nM to 0.1 M, whereas 0.3–3 M S1P CD4 T cells was suppressed through LPA2 and enhanced through inhibits this chemotaxis by up to 70%. Chemotaxis of S1P1, but LPA1. In contrast, LPA2 transduced LPA-evoked chemotaxis and not S1P4, transfectants to CXCL1 and CXCL4 was similarly chemokinesis of CD4 T cells, whereas LPA1 failed to signal mi- affected by S1P. Activation of CD4 T cells, which decreases gration directly but mediated LPA suppression of chemokine-elic- S1P receptor expression, suppressed effects of S1P on chemo- ited chemotaxis of CD4 T cells (10). Thus, the effects of activation taxis. Pretreatment of labeled CD4 T cells with S1P before of blood CD4 T cells on LPA receptors converted LPA from a http://www.jimmunol.org/ reintroduction into mice inhibited by a maximum of 75% their migration-enhancing and cytokine production-inhibiting factor to a migration into chemokine-challenged s.c. air pouches. The migration-inhibiting and cytokine production-enhancing factor for effector T cells. S1P-S1P1 receptor axis thus controls recruitment of naive T cells by maintaining their response threshold to diverse lym- In the course of studies of mouse splenic T cell LPA and S1P photactic factors. The Journal of Immunology, 2002, 169: receptors, S1P1 and S1P4 were found to be the vastly predominant S1P receptors of CD4 and CD8 T cells and LPA receptors were 4084–4087. detected at only very low levels (11). S1P was noted to elicit T cell chemotactic responses at concentrations well below those typical by guest on September 28, 2021 he lysophospholipid growth factors sphingosine 1-phos- of plasma (11). It was also discovered that the high level of ex- 3 phate (S1P) and lysophosphatidic acid (LPA) are gener- pression of S1P1 and S1P4 receptors by mouse splenic CD4 and T ated predominantly by stimulated platelets and leuko- CD8 T cells is suppressed nearly completely by TCR-dependent cytes, resulting in up to micromolar concentrations in normal activation (11). Chemotactic responses of both unstimulated CD4 plasma and some other extracellular fluids (1–3). S1P and LPA and CD8 T cells were elicited by 1 nM S1P, attained a maximum evoke cellular proliferation and diverse functional responses by at 0.1 ␮M S1P, and were reduced in parallel with suppression of binding to members of a family of homologous G protein-coupled the levels of S1P receptors by TCR-dependent activation (11). A receptors (GPCRs), which originally were designated endothelial pharmacological probe of S1P receptors (12, 13) also inhibited differentiation gene-encoded or Edg receptors (4–6). Recently, a chemotactic responses of T cells to S1P in vitro and in vivo (11). nomenclature subcommittee of the International Union of Pharma- It is now reported that the major effect of plasma concentrations cologists has renamed these receptors for their principal ligand and of S1P on T cells is suppression of their responses to other che- order of discovery, so that the LPA receptors are LPA1 (Edg-2), LPA2 motactic factors, such as chemokines, in a basement membrane (Edg-4), and LPA3 (Edg-7), and the S1P receptors are S1P1 (Edg-1), transmigration assay. TCR-dependent activation of the T cells, re- S1P2 (Edg-5), S1P3 (Edg-3), S1P4 (Edg-6), and S1P5 (Edg-8) (7). sulting in decreased expression of S1P receptors, reduced the ex- tent of suppression of chemotactic responses by S1P. S1P1 recep- tor is the major transducer of S1P effects based on findings that S1P suppressed chemokine-evoked chemotaxis of transfectants ex- *Departments of Medicine and Microbiology-Immunology, University of California † Medical Center, San Francisco, CA 94143; and Ceretek, LLC, Alameda, CA 94502 pressing only S1P1 receptors, but not that of transfectants express- Received for publication July 23, 2002. Accepted for publication August 22, 2002. ing only S1P4 receptors. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Isolation, activation, and fluorescent labeling of mouse spleen 1 The study was supported by Grant HL 31809. CD4 T cells 2 Address correspondence and reprint requests to Dr. Edward J. Goetzl, University of California, UB8B, UC Box 0711, 533 Parnassus at 4th, San Francisco, CA 94143- Mouse CD4 T cells were isolated from splenocytes of 6- to 8-wk-old 0711. E-mail address: [email protected] C57BL/6 female mice at a minimum purity of 97% using metallic mi- 3 Abbreviations used in this paper: S1P, sphingosine 1-phosphate; LPA, lysophos- crobeads bearing anti-CD4 mAbs and two cycles of magnetic retention phatidic acid; Edg, endothelial differentiation gene-encoded; FAF, fatty acid free; chromatography (Miltenyi Biotec, Auburn, CA), as described elsewhere (8, FM-DiI, methylbenzamido-1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindo-dicarbocya- 9). For some studies, 1-ml suspensions of purified T cells in RPMI 1640-50 nine; GPCR, G protein-coupled receptor. ␮g/ml fatty acid-free BSA (FAF-BSA) (Calbiochem, La Jolla, CA) with Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 ● The Journal of Immunology 4085 100 U/ml penicillin G and 50 ␮g/ml streptomycin (RPMI 1640-BSA) were activated by incubation for 24 h on 2 ␮g each of adherent anti-CD3 plus anti-CD28 mAbs (BD PharMingen, San Diego, CA) in six-well plates. Stock solutions of 50 ␮g of methylbenzamido-1,1Ј-dioctadecyl-3,3,3Ј,3Ј- tetramethylindo-dicarbocyanine (FM-DiI; Molecular Probes, Eugene, OR) in 50 ␮l of DMSO were prepared immediately before labeling each prep- aration of CD4 T cells. Suspensions of 2 ϫ 106 CD4 T cells from normal C57BL/6 mice/ml Ca2ϩ- and Mg2ϩ-free PBS were incubated with 2 ␮g/ml FM-DiI for 5 min at 37°C and 15 min at 4°C, washed three times in PBS, and resuspended in 1 ml of RPMI 1640-FAF-BSA for incubation with S1P for1hat37°C before i.p. injection into test mice. The fluorescent labeling did not affect T cell viability or chemotactic responses in vitro nor was any fluorescent label transferred to mouse adherent peritoneal macrophages by labeled T cells, at a cellular ratio of 1:5, after6hofincubation at 37°C. Generation of HTC4 cell-S1P receptor transfectants The cDNAs encoding human S1P1 and S1P4 were subcloned into pLXSN and introduced into the EcoHEK-293 packaging cell line (Clontech Lab- oratories, Palo Alto, CA). Viral particles secreted by this line were filtered, FIGURE 1. S1P regulation of CD4 T cell chemotactic responses to che- treated with polybrene, and substituted for culture medium over HTC4 rat mokines. Each bar depicts the mean Ϯ SD of the results of three studies hepatoma cells. HTC4 cells resistant to 400 ␮g/ml geneticin were sub- performed in duplicate. Mean control chemotactic responses (100%) were L cloned, washed, and resuspended in MEM with 10% FBS and 4 mM - 3.2, 4.1, and 4.2% of the CD4 T cells initially added to each chamber for Downloaded from glutamine, and then incubated for 1–4 h. The mRNAs encoding S1P1 and 50 nM CCL-21; 12.9, 9.2, and 16.5% for 200 nM CCL-21; and 5.4, 10.4, S1P4 extracted from the respective HTC4 cell lines were quantified by and 7.8% for 100 nM CCL-5.