FTY720-Enhanced Homing Is Dependent on CCR2, CCR5, CCR7, and CXCR4: Evidence for Distinct Compartments This information is current as of September 30, 2021. Adam C. Yopp, Shuang Fu, Shaun M. Honig, Gwendalyn J. Randolph, Yaozhong Ding, Nancy R. Krieger and Jonathan S. Bromberg J Immunol 2004; 173:855-865; ; doi: 10.4049/jimmunol.173.2.855 Downloaded from http://www.jimmunol.org/content/173/2/855

References This article cites 53 articles, 25 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/173/2/855.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021 • Fast Publication! 4 weeks from acceptance to publication

*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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

FTY720-Enhanced T Cell Homing Is Dependent on CCR2, CCR5, CCR7, and CXCR4: Evidence for Distinct Chemokine Compartments1

Adam C. Yopp,* Shuang Fu,* Shaun M. Honig,* Gwendalyn J. Randolph,* Yaozhong Ding,* Nancy R. Krieger,*† and Jonathan S. Bromberg2*†

FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G -coupled receptors for chemo- kines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell to CCR2-CCL2, but not to a variety of other , including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration Downloaded from of FTY720 induces chemokine-dependent migration of T cells in the , peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 adminis- tration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor- http://www.jimmunol.org/ stimulated T cell migration are dependent on the restricted usage of -ligand pairs within discrete anatomic compartments. The Journal of Immunology, 2004, 173: 855–865.

TY720 is a sphingosine analog immunomodulator derived transporters and sensitization of the CCR7 chemokine system, we from the Chinese herb, Iscaria sinclarii, that enhances previously showed that FTY720 sequentially stimulates the Abcb1 F solid organ allograft survival in humans and animal mod- and Abcc1 multidrug transporters, S1PR, and cysteinyl leukotriene els (1, 2). Originally thought to act via a ceramide-related apopto- (cysLT) receptors to sensitize the CCR7 chemokine receptor, tic mechanism, it has clearly been shown that FTY720 enhances thereby enhancing T cell migration to the CCR7 ligands, CCL19 allograft survival by promoting T cell egress from peripheral blood and CCL21 (8–10) (see Ref. 11 for review). Whether or how by guest on September 30, 2021 into secondary lymphoid tissues (3, 4). A single oral dose of FTY720 influences other chemokine receptor-ligand pairs remains FTY720 causes T cell egress from peripheral blood to lymph unclear. 3 nodes (LN) and Peyer’s patches, but does not affect T cell priming Henning et al. (12) reported that FTY720-induced T cell LN or activation, and does not interfere with the response to infectious migration occurs in both CCR7-dependent and independent fash- challenges (4, 5). ions, suggesting that other chemokine receptor-ligand pairs are in- The molecular mechanism of FTY720-enhanced T cell homing volved. Our previous results demonstrated that the CCR1, CCR3, to LN has only recently been elucidated. Mandala et al. and Brink- CCR5, CX3CR1, CXCR3, and CXCR5 chemokine receptors mann et al. (6, 7) have shown that both phosphorylated FTY720 (P-FTY720) and sphingosine 1-phosphate (S1P) are agonists for G failed to exhibit FTY720-driven T cell chemotaxis in both in vitro protein-coupled S1PR or endothelial differentiation receptors, and in vivo assays (10). Therefore, it is important to determine stimulation of which promotes leukocyte migration. Based on the whether other chemokine receptors are responsible for the CCR7- observation that migration to peripheral LN is de- independent action of FTY720-stimulated T cell migration, and pendent on activation of the Abcb1 and Abcc1 multidrug lipid whether they rely on the same mechanisms observed for CCR7- CCL19/21 interactions. In this study, we show that CCR2-CCL2- stimulated chemotaxis and migration are sensitive to FTY720. *Carl C. Icahn Center for Gene Therapy and Molecular Medicine, and †Recanti/ Similar to the CCR7 system, FTY720-stimulated CCL2-driven T Miller Transplantation Institute, Mount Sinai School of Medicine, New York, NY 10029 cell migration is dependent on activity of both the Abcb1 and the Received for publication January 6, 2004. Accepted for publication May 4, 2004. Abcc1 lipid transporters, as well as 5-lipoxygenase (5-LO) activ- The costs of publication of this article were defrayed in part by the payment of page ity. CCL2-driven T cell migration is more sensitive to both Abcc1 charges. This article must therefore be hereby marked advertisement in accordance and 5-LO activity than CCR7 responses. In vivo, the CCR2-CCL2 with 18 U.S.C. Section 1734 solely to indicate this fact. system is required for FTY720-driven T cell homing to LN. The 1 This work was supported by National Institutes of Health Grant R01 AI41428 (to results also demonstrate that S1PR-stimulated T cell migration oc- J.S.B.). curs by engaging different sets of chemokines and their receptors 2 Address correspondence and reprint requests to Dr. Jonathan S. Bromberg, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1104, New York, NY in the thymus and secondary lymphoid organs to determine the 10029-6574. E-mail address: [email protected] destination of the migrating T cell. Specifically, the results indicate 3 Abbreviations used in this paper: LN, lymph node; cysLT, cysteinyl leukotriene; that FTY720-stimulated T cell migration in vivo is dependent on DKO, double knockout; GPCR, -coupled receptor; 5-LO, 5-lipoxygenase; the anatomically restricted use of the CCR2-CCL2, CCR5-CCL5, LTD4,5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid; P-FTY720, phosphorylated FTY720; S1P, sphingosine 1-phosphate. CCR7-CCL19/21, and CXCR4-CXCL12 receptor-ligand pairs.

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 856 FTY720-ENHANCED T CELL HOMING

Materials and Methods mental group of mice; and thymus, peripheral blood, peripheral LN, and Mice spleen were harvested 18 h later. Cell counts were performed with a he- mocytometer, and cell subset was determined by flow cytometry. C57BL/6, FVB/NJ, C57BL/6 CCR5Ϫ/Ϫ (13), and B6/129 5-LOϪ/Ϫ (14) mice 8–10 wk of age were purchased from The Jackson Laboratory (Bar Ϫ Ϫ Ϫ Ϫ Statistics Harbor, ME). FVB/129 Abcb1 / and FVB/129 Abcc1 / mice were pur- chased from Taconic Farms (Germantown, NY). C57BL/6 plt (15), In vivo migration results represent samples from two to three mice per Ϫ/Ϫ Ϫ/Ϫ C57BL/6 CCR2 (16), C57BL/6 CCL2 (17), and C57BL/6 experiment. In vitro migration results represent mean values of triplicate Ϫ/Ϫ (CCR2 , plt) double knockout (DKO) mice were maintained in our fa- samples. All experiments were performed two to five times. SDs and p cility. All mice were housed in a specific pathogen-free facility in mi- values were calculated with Student’s t test using Microsoft Excel croisolator cages. All experiments were performed with age- and sex- software. matched mice in accordance with institutionally approved animal care criteria. Reagents Results CCR2-CCL2-driven migration is enhanced by S1PR activation PE- and FITC-conjugated rat anti-mouse CD4 mAb or CD8a mAb were purchased from BD Pharmingen (San Diego, CA). MK571, AA-861, 5(S)- We previously showed FTY720 increases T cell migration to the hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid CCR7-CCL19/21 chemokine receptor ligands (10). Whether other (LTD4), and S1P were purchased from BIOMOL (Plymouth Meeting, PA). chemokine receptor-ligand pairs similarly show FTY720-stimu- Fluo-3, DiOC , and CSFE were purchased from Molecular Probes (Eu- 2 lated T cell migration is unknown. In a series of in vitro migration gene, OR). Murine CCL2, human CCL7, human CCL8, human CCL13, murine CCL19, murine CCL1, murine CXCL12, murine CCL3, murine studies, we found that purified murine splenic T cells failed to Downloaded from CCL4, and murine CCL22 were purchased from R&D Systems (Minne- migrate to the CCR4-CCL22, CCR8-CCL1, CXCR3-CXCL9, apolis, MN). FTY720, phosphorylated FTY720, the biologically active R- CCR1/3/5-CCL5, CCR1/5-CCL3, and CCR5/8-CCL4 chemokine enantiomer AAL151, and the inactive L-enantiomer AAL149 were kind receptor-ligand pairs; and FTY720 also failed to stimulate migra- gifts from V. Brinkmann (Novartis Pharmaceuticals, Basel, Switzerland). AMD-3100 was obtained from the National Institutes of Health AIDS Re- tion to them (10) (data not shown). In vitro responses of purified search and Reference Reagant Program, Division of AIDS, National Insti- splenic T cells to the CXCR4-CXCL12 receptor-ligand pair tute of Allergy and Infectious Diseases, National Institutes of Health: bi- showed only a low level of T cell migration to CXCL12 that was cyclam JM-2987 (hydrobromide salt of AMD-3100). not increased by the addition of FTY720. Because a previous pre- http://www.jimmunol.org/ Cell preparations liminary report suggested that FTY720 stimulated T cell chemo- taxis to CCL2, we assessed in vitro migration of purified splenic T Mice (two to three per group) were sacrificed, and spleen, LN (cervical, cells to the CCR2 ligands CCL2, CCL7, CCL8, and CCL13 (18). periaortic, inguinal, and axillary), and thymus were removed and gently dissociated into single cell suspensions. Peripheral blood was collected by Dose-response experiments demonstrated effective migration of ␮ cardiac puncture. RBC were removed by Tris-NH4Cl lysis. If indicated, purified T cells to 1 g/ml CCL2, with 10–15% of total T cells cell suspensions were passed through T cell enrichment columns (R&D migrating in individual experiments. Approximately 5% of cells Systems); these cells were routinely 85–95% T cells. Cells were placed in migrated to 0.5 ␮g/ml CCL2, while lower doses caused only min- complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FCS, imal migration (Fig. 1A). Addition of the chemokine to the upper 1 mM sodium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin, 100 by guest on September 30, 2021 ␮g/ml streptomycin, 1ϫ nonessential amino acids, and 2 ϫ 10Ϫ5 M wells or both wells resulted in no migration (data not shown), 2-ME). indicating the migration is chemotactic, not chemokinetic. Various Flow cytometry concentrations and incubation times of FTY720 significantly in- creased chemotaxis to CCL2, with the optimal concentrations of Cell washes and Ab dilutions were performed in PBS plus 1% BSA at 4°C. FTY720 between 0.5 and 1.0 ␮g/ml, and the optimal incubation Flow cytometric analysis was performed on a FACScan flow cytometer (BD Biosciences, San Jose, CA). Forward and side scatter parameters were time 15 min at 37°C (Fig. 1A and data not shown). The remaining used to gate on live cells. Results are expressed as percentage of cells CCR2 ligands, CCL7, CCL8, and CCL13, induced only modest T staining above background. cell chemotaxis without demonstrating FTY720-stimulated migra- Migration assays tion (Fig. 1B). In vitro migration assays with cells from various knockout In vitro migration assays were performed, as previously described (10). A mouse strains were also performed to assess the role of CCL2 in ϫ 5 total of 5 10 splenic T cells was incubated with various doses of FTY720-driven T cell chemotaxis. Purified T cells from CCL2Ϫ/Ϫ FTY720, P-FTY720, or S1P for 15 min at 37°C. The cells were washed twice, resuspended in RPMI 1640 containing 0.5% BSA, and added in a mice demonstrated FTY720 increased T cell migration to CCL2 Ϫ Ϫ volume of 100 ␮l to the upper wells of a 24-well transwell plate with a (Fig. 2, top). However, CCR2 / T cells failed to migrate to 5-␮m insert (Corning Glass, Corning, NY). Lower wells contained various CCL2, with or without FTY720, as expected (Fig. 2, middle). Fur- ␮ Ϫ Ϫ doses of chemokines in 600 l of RPMI 1640/0.5% BSA. The number of thermore, FTY720 also increased CCR2 / T cell migration to T cells that migrated to the lower well following a 2-h incubation was counted in three high power fields using a hemocytometer. In vivo migra- CCL19, demonstrating that the absence of migration was not due tion assays were performed with mice given 0.3 mg/kg FTY720 via per os to another abnormality of the knockout strain, and that CCR7- gavage and/or 1 mg/kg AMD-3100 i.p., and sacrificed 18 h later. Thymus, CCL19-driven migration could take place independently of CCR2- peripheral blood, peripheral LN, and spleen were harvested and made into CCL2. We next evaluated whether or not T cell migration was single cell suspensions. Total cell numbers were counted using a hemocy- dependent on the biological specificity of FTY720 by performing tometer, and subset analysis was performed using fluorescent flow cytometry. in vitro migration assays with the active (AAL151) or inactive (AAL149) enantiomers of FTY720. T cell migration was observed Adoptive transfer assays with CSFE-labeled cells only with the active, but not the inactive enantiomer of FTY720 Single cell suspensions from the spleens of wild-type and various knockout (Fig. 2, bottom). strains were made, and erythrocytes were removed by lysis solutions. CFSE (5.0 ␮M; Molecular Probes) solution was added to a single cell suspension containing 2.0 ϫ 107 cells/ml and incubated at room temper- CCR2-CCL2-driven migration is dependent on multidrug ature for 5 min. The staining process was stopped by adding 20 ml of PBS transporter activity with 5% FCS and washed twice with PBS. A total of 2 ϫ 107 labeled splenocytes in 300 ␮l of PBS was injected into the tail veins of the recip- We previously demonstrated that FTY-stimulated T cell migration ient mice; 0.3 mg/kg FTY720 via per os gavage was given to the experi- to the CCR7-CCL19/CCL21 chemokine-ligand pair is dependent The Journal of Immunology 857 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021 FIGURE 1. FTY720 enhances T cell migration to CCL2, but not other CCR2 chemokine ligands. A, Dose response to various doses of CCL2 and FTY720. *, p Ͻ 0.05 vs no FTY720 control. B, In vitro T cell chemotactic responses to CCL2, CCL7, CCL8, and CCL13. In vitro migration results represent mean values Ϯ SE of triplicate samples. on sequential activation of the Abcb1 and Abcc1 multidrug trans- porters (10). To determine whether CCL2-driven and FTY720- stimulated T cell migration functioned through a similar mecha- nism, T cells from Abcb1- or Abcc1-deficient mice or wild-type T cells treated with the Abcb1 blocker, PSC833, or the Abcc1 blocker, MK571, were treated with or without FTY720, S1P, or P-FTY720 and migrated to CCL2. Cells pretreated with PSC833 or MK571, with or without FTY720, failed to migrate to CCL2 above background levels (Fig. 3A). Cells from Abcb1Ϫ/Ϫ mice showed only modest migration to CCL2 alone, and this was not enhanced by FTY720 (Fig. 3B2). Cells from Abcc1Ϫ/Ϫ mice failed to mi- grate to CCL2 alone, and did not show any increase with the ad- dition of FTY720 (Fig. 3B3). The results demonstrate that both the Abcb1 and Abcc1 multidrug transporters play an important role in both basal T cell migration to CCL2 and FTY720-stimulated T cell FIGURE 2. FTY720 enhances T cell migration to CCL2 and is depen- migration. dent on the biologically active FTY720 enantiomer, AA151. In vitro che- Ϫ/Ϫ Ϫ/Ϫ Exogenous P-FTY720 and S1P both directly activate cell sur- motactic responses to CCL2 for CCL2 and CCR2 T cells. Response of wild-type cells to the FTY720 enantiomers, AAL151 (active) and face S1P receptors and cause T cell migration, but do not require AAL149 (inactive), and CCL2. In vitro migration results represent mean Abcb1 efflux activity for these biological effects (10). We exam- values Ϯ SE of triplicate samples. ined in vitro chemotactic responses of wild-type and knockout strain cells to CCL2 along with P-FTY720 or S1P. Addition of either S1P or P-FTY720 increased wild-type T cell migration to gesting that efflux of FTY720 by Abcb1 is necessary for increased CCL2, similar to FTY720 (Fig. 4A). Unlike FTY720, P-FTY720 T cell migration to CCL2, and that exogenous phosphorylated also increased T cell migration of Abcb1Ϫ/Ϫ cells (Fig. 4B), sug- sphingosine ligand can bypass the need for Abcb1. Similar results 858 FTY720-ENHANCED T CELL HOMING Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 3. FTY720-enhanced T cell migration to CCL2 is dependent on the multidrug transporters, Abcb1 and Abcc1. In vitro chemotactic response of T cells from wild-type (A), FVB (B1), Abcb1Ϫ/Ϫ (B2), or Abcc1Ϫ/Ϫ (B3) mice to CCL2 or CCL19. MK571 (Abcc1 antagonist) or PSC833 (Abcb1 antagonist) was added, as indicated. In vitro migration results represent mean values Ϯ SE of triplicate samples.

were observed with wild-type cells in which Abcb1 was blocked CCR2-CCL2-driven migration is highly dependent on 5-LO and with PSC833; S1P and P-FTY720 caused migration, but FTY720 cysLTs did not (Fig. 4A). PSC833 can block Abcc1 (19), but at the doses used in this study it preferentially blocked only Abcb1, as shown We previously demonstrated that T cell migration to the CCR7- by the effectiveness of P-FTY720 to bypass the blockade and in- CCL19/21 receptor-ligand pairs is dependent on cysLT production crease migration. Conversely, S1P and P-FTY720 were unable to by 5-LO and its subsequent efflux through the Abcc1 transporter restore or stimulate migration of cells in which Abcc1 was blocked (8–10). To determine whether the CCR2-CCL2 receptor-ligand with MK571 (Fig. 4A). Taken together, these results show that pair functions with a similar mechanism, in vitro chemotaxis as- CCR2-CCL2 is dependent on the efflux activity of Abcb1 and says were performed with wild-type T cells treated with the 5-LO Abcc1, that Abcb1 is upstream of Abcc1, and that phosphorylated blocker, AA-861, and 5-LO knockout strain cells. With or without sphingosine ligands bypass the need for Abcb1 activity, all similar FTY720, AA-861-pretreated and 5-LOϪ/Ϫ T cells both migrated to the CCR7-CCL19/21 system (10). poorly or not at all to CCL2. In comparison, 5-LOϪ/Ϫ T cells The Journal of Immunology 859 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021 FIGURE 5. FTY720-enhanced T cell migration to CCL2 is dependent on 5-LO and cysLTs. A, In vitro chemotactic response of T cells from 5-LOϪ/Ϫ mice to CCL19 and CCL2. B, In vitro chemotactic response of T cells from wild-type mice treated with AA861 (5-LO antagonist) and/or

LTD4 to CCL19 or CCL2. In vitro migration results represent mean val- ues Ϯ SE of triplicate samples. All experiments were performed twice.

Compartmentalization of FTY720 enhanced T cell migration A single oral dose of FTY720 has been shown to induce T cell egress from the peripheral blood and spleen to the LN (4). Fur- thermore, reports demonstrated the CCR7-CCL19/CCL21 recep- tor-ligand pairs were important, but not exclusive, requirements for T cell sequestration in LN in response to FTY720 (10, 12). We FIGURE 4. PFTY720 and S1P cause Abcb1-independent and Abcc1- sought to determine whether these receptor-ligand pairs as well as dependent T cell migration to CCL2. In vitro chemotactic responses of T other chemokine receptor-ligand pairs affected in vivo T cell mi- cells from wild-type (A) or Abcb1Ϫ/Ϫ (B) mice to CCL2. MK571 (Abcc1 gration in the thymus, peripheral blood, peripheral LN, and spleen antagonist) or PSC833 (Abcb1 antagonist) was added, as indicated. In vitro following FTY720 administration. In vivo migration assays were migration results represent mean values Ϯ SE of triplicate samples. performed with various knockout mouse strains given a single 0.3 mg/kg oral dose of FTY720. The thymus, peripheral blood, spleen, and LN were harvested 18 h later, and cell numbers and subsets were quantified by cell counting and fluorescent flow cytometry. migrated to CCL19 (Fig. 5A). The addition of exogenous LTD4 In the thymus of wild-type, but not plt (mutants lacking CCL19 restored migration of the AA-861-treated T cells. Taken together and lymphoid CCL21), CCR2Ϫ/Ϫ, (CCR2Ϫ/Ϫ, plt) DKO, or with the results that leukotriene transporter Abcc1 knockout or CCR5Ϫ/Ϫ mice, there was a reduction in total T cell numbers, blocked T cells do not migrate to CCL2 (Fig. 3, A and B3), this CD4ϩ single-positive, CD8ϩ single-positive, and CD4ϩCD8ϩ shows that CCR2-CCL2 is very sensitive to 5-LO activity, cysLT double-positive cells following FTY720 administration (Figs. 6A production, and cysLT efflux by Abcc1. In contrast, CCR7-CCL19 and 8B). CCL2Ϫ/Ϫ mice had only a partial reduction of induces some T cell migration in the absence of Abcc1 or 5-LO cellularity in response to FTY720. These results suggest that the (Figs. 3B3 and 5A), although FTY720 still remains inactive on CCR2, CCR5, and CCR7 chemokine receptors and ligands are these cells (10). required for FTY720-stimulated T cell thymic egress. 860 FTY720-ENHANCED T CELL HOMING

FIGURE 6. FTY720 enhances T cell accumulation or egress from the thymus, peripheral blood, peripheral LN, and spleen based on chemokine and receptor dis- tribution. In vivo migration of T cells following FTY720 administration in the thymus (A), peripheral blood (B), LN (C), or spleen (D). Mouse strains are Downloaded from indicated, and results are expressed as percentage of control FTY720-untreated groups of the same strain. In vivo migration results represent mean values Ϯ SE of .p Ͻ 0.05 compared with control ,ء .n ϭ 3 mice http://www.jimmunol.org/ by guest on September 30, 2021

Peripheral blood T cells were all significantly reduced, not only estingly, the percentage of distribution of CD4 cells among naive in wild-type, but also in plt, CCL2Ϫ/Ϫ, (CCR2Ϫ/Ϫ, plt) DKO, and memory subsets, as determined by multicolor flow cytometry CCR2Ϫ/Ϫ, and CCR5Ϫ/Ϫ mice following FTY720 administration analysis for CD45RB and L-, showed no difference be- (Fig. 6B). Similarly, splenic T cells were reduced in all strains tween untreated and FTY720-treated LN (data not shown), sug- (Fig. 6D). Therefore, the absence of either the CCR5, CCR2, gesting that S1PR activation does not preferentially drive a par- and/or the CCR7 chemokine receptors and ligands does not affect ticular subset expressing a limited chemokine receptor repertoire T cell egress from the peripheral blood or spleen. These findings into the LN. suggest that none of these chemokine systems contributes to To further elucidate the role of the CCR2 and CCR7 chemokine FTY720-stimulated T cell egress from peripheral blood or the systems in FTY720-driven T cell migration, adoptive transfer stud- spleen. ies were performed with CSFE-labeled splenocytes from wild-type Sequestration of T cells in LN only occurred in the wild-type or CCR2Ϫ/Ϫ donors transferred into wild-type or plt recipients and CCR5Ϫ/Ϫ mice, with no significant increase observed in given FTY720. As shown in Fig. 7B, after adoptive transfer, CCR2Ϫ/Ϫ or (CCR2Ϫ/Ϫ, plt) DKO mice, compared with their re- CSFE-labeled T cells can be found in the peripheral blood, and spective controls (Fig. 6C). Slight peripheral LN T cell sequestra- they can be induced to exit from the peripheral blood following tion was observed in plt and CCL2Ϫ/Ϫ mice, but at a significantly FTY720 administration, regardless of the genotype of the trans- lower level compared with control mice. These findings demon- ferred cell or host. These results confirm the in vivo findings strate that the absence of CCL19 and lymphoid CCL21, or the above, showing that none of the chemokines examined contributes absence of the single CCR2 ligand, CCL2, partially prevents to T cell egress from the peripheral blood. However, the migration FTY720-stimulated T cell peripheral LN accumulation. The ab- patterns of these cells into LN were intrinsic to the genotype of sence of CCR2 prevents T cell accumulation to a much greater both the transferred cells and the recipient (Fig. 7B, right). We degree than the absence of CCL2. Similarly, the absence of CCR7 observed peripheral LN sequestration of CSFE-labeled wild-type has been demonstrated to more effectively inhibit LN accumula- T cells transferred into C57BL/6, but not plt mice, and no LN tion in response to FTY720 compared with plt mice (12). The roles sequestration with CCR2Ϫ/Ϫ cells transferred into either wild-type of these chemokines in FTY720 LN accumulation of T cells are or plt hosts. Furthermore, these adoptive transfer studies show that redundant or highly overlapping because (CCR2Ϫ/Ϫ, plt) DKO are the transferred cells migrate independently of surrounding host very similar in their LN response compared with CCR2Ϫ/Ϫ. Inter- cells. Thus, CCR2Ϫ/Ϫ adoptively transferred cells do not migrate The Journal of Immunology 861

showed that compared with AMD-3100 treatment alone, FTY720 was able to induce thymic egress in AMD-3100-treated mice; and compared with FTY720 treatment alone, AMD-3100 was able to induce thymic accumulation in FTY720-treated mice. This dem- onstrates that CXCR4 is mainly used for homeostatic, but not S1PR-driven thymic T cell egress. Splenic and peripheral blood total mononuclear cell and T cell counts were also assessed and revealed that T cell egress from spleen and blood occurred nor- mally following FTY720 administration, with or without AMD- 3100 (Fig. 8A). Overall, these findings reveal that CXCR4 is re- quired for FTY720-stimulated T cell LN accumulation, but not for splenic or peripheral blood egress, and that CXCR4 is mainly used for homeostatic, but not S1PR-stimulated thymic T cell egress.

Discussion Effect of FTY720 on CCR2 FTY720 stimulates T cell migration to peripheral LN by both

CCR7-dependent and independent mechanisms (12). FTY720 pro- Downloaded from motes CCL19/CCL21-dependent T cell LN sequestration through sequential activation of the Abcb1 lipid transporter, S1PR, 5-LO, Abcc1 multidrug transporter, and cysLT receptor, with subsequent sensitization of CCR7 and use of CD44- and VLA-4␣-dependent adhesion (10). In this study, we report that screening of the CCR1, CCR2, CCR3, CCR5, CCR8, CX3CR1, CXCR3, CXCR4, and http://www.jimmunol.org/ CXCR5 chemokine systems demonstrated that CCR2-CCL2 is the only other receptor-ligand pair for which in vitro T cell chemotaxis is increased by FTY720. Our results also demonstrate that T cell migration to CCR2-CCL2 is more sensitive to the production of cysLTs by 5-LO, and their subsequent transport through the Abcc1 transporter, than CCR7-CCL19/CCL21. Both rely similarly on transport by Abcb1 and on the activation of S1P receptors for FIGURE 7. FTY720-enhanced T cell migration to peripheral LN is de- increased T cell migration. We previously proposed a model (10) pendent on CCR2 and CCR7 and is intrinsic to cell genotype. In vivo in which the sequential engagement and activation of these two by guest on September 30, 2021 migration of A, recipient blood and LN T cells, and B, adoptively trans- multidrug transporters and three G protein-coupled receptor ferred donor CSFE-labeled T cells in blood and LN following FTY720 (GPCRs) (S1PRs, cysLTRs, and CCRs) occurred in an autocrine Ͻ ء administration. Results are expressed as actual cell counts. , p 0.05 or paracrine fashion. The precise biochemical events that sensitize compared with control. In vivo migration results represent mean values Ϯ cells to chemokine-driven migration are not known, but because SE of n ϭ 3 mice. cysLTs have a rapid effect on migration (9, 10), sensitization may relate to immediate second messengers that affect the cytoskeleton to LN, while host wild-type cells do (Fig. 7A, right). Conversely, and motility (23, 24). Differential sensitivity to cysLTs suggests wild-type adoptively transferred cells do not enhance migration of that CCR2 and CCR7 stimulate similar, but not identical molecular host plt cells, as expected, because T cells do not secrete CCL19 events related to migration, while other chemokine receptors may or CCL21. engage alternative migration mechanisms, because they are unaf- A previous report suggested a role for CXCR4-CXCL12 in ho- fected by S1PR stimulation and presumably cysLTs. In fact, CCR2 meostatic T cell LN migration that was apparent only when CCR7- and CCR7 differ in their engagement of DOCK2 and Jak2 tyrosine CCL19 was blocked (20). To determine whether the CXCR4- kinase (25, 26). With regard to the effects of FTY720 on different CXCL12 receptor-ligand pair influences FTY720-stimulated T chemokine-receptor pairs, it is important to note that the source of migration in vivo, even though, as noted above, CXCL12-driven in cells used for migration studies may influence in vitro results. For vitro migration is not stimulated by FTY720, a chemical com- example, preliminary experiments suggest that peripheral blood pound with unique antagonist specificity for CXCR4, AMD-3100 and splenic murine T cells respond to S1PR stimulation, while LN (21), was used. As shown in Fig. 8A, administration of AMD-3100 T cells do not (J.S.B., A.C.Y., unpublished observations), perhaps alone had no effect on peripheral LN accumulation in the absence areflection that LN-retained cells have down-modulated S1PR1 of FTY720. However, administration of AMD-3100 along with expression or activity (27–29). FTY720 in wild-type mice prevented T cell peripheral LN accu- mulation, compared with controls that received FTY720 alone. Effect of FTY720 on thymic egress Thus, CXCR4 blockade alone does not affect LN migration, sim- The results also demonstrate that in addition to CCR7-CCL19/ ilar to the previous report (20); but CXCR4 is required for S1PR- CCL21, CCR2-CCL2 also influences FTY720-driven T cell mi- driven migration initiated by FTY720 administration. A previous gration in vivo. Furthermore, the CCR5-CCL3/4/5 and CXCR4- report (22) suggested that CXCR4-CXCL12 contributes to normal CXCL12 chemokine systems influenced FTY720-driven T cell thymocyte emigration. We observed that administration of AMD- egress from the thymus and migration to the LN, but migration to 3100 alone prevented normal thymic T cell egress, as noted by an these chemokines was not directly affected by S1PR activation in acute increase in thymocyte numbers, affecting all subsets com- vitro. Rather, S1PR activation uncovered an increased role for pared with controls (Fig. 8, A, left; B, upper right). Further analysis CCR5 in thymic egress and CXCR4 in LN migration that was not 862 FTY720-ENHANCED T CELL HOMING Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 8. CXCR4 contributes to homeostatic thymic and S1PR-driven LN migration. A, In vivo migration of total T cells of mice treated with AMD-3100 or FTY720, as indicated. B, CD4 and CD8 plots of . In vivo migration results represent mean values Ϯ SE of triplicate samples. Flow cytometry plots are representative of three experiments; n ϭ 3 per group. apparent when S1PR were not activated. These in vivo results are presence or absence of S1PR stimulation further alters receptor summarized in Table I, which clearly shows that different chemo- usage. To understand the thymic effects, it is important to note that kine receptors are engaged in different compartments, and that the double-negative thymocytes express CXCR4 and migrate to

Table I. Summary of chemokine effects on T cell migration in lymphoid compartments in the presence and absence of FTY720

Peripheral LN Thymic Egress Blood Egress Splenic Egress Accumulation

Homeostatic state Dependent on CCR7 CCR2, CCR5, CCR7, and CCR7 affects Dependent on CCR7 (no FTY720) and CXCR4 (36, CXCR4 do not affect architecture and CXCR4 (20, 41) PBL counts (13, 15, (37) 48) 16, 20) SIPR stimulation Also dependent on Independent of CCR2, Independent of Also dependent on (ϩ FTY720) CCR2 and CCR5 CCR5, CCR7, and CCR2, CCR5, CCR2 and CXCR4 CCR7, and CXCR4 CXCR4 The Journal of Immunology 863

CXCL12 (30, 31); double-positive thymocytes express CXCR4 an inappropriate location to respond to FTY720-stimulated CCL2- and CCR9 and migrate to CXCL12 and CCL25 (30–33); transi- or CCL19-driven migration. This suggests a role for CCR5 in tional thymocytes between the double- and single-positive stages S1PR-driven, but not normal thymic emigration (Table II). express CCR4 and migrate to CCL22 (31); and single-positive Our results on the effects of FTY720 in the thymus are in con- thymocytes migrate to the CCR7 ligands, CCL19 and CCL21 (Ta- trast to a recent report suggesting that FTY720 inhibits thymic T ble II) (34, 35). Because adult CCR7Ϫ/Ϫ mice have increased thy- cell emigration in wild-type mice (42). However, in that report, mocyte numbers, but do not have reduced numbers of circulating thymic cellularity was only evaluated after 20 days of FTY720 T cells, this suggests that thymic emigration relies on both CCR7- administration, and not acutely as in our studies. Multiple other dependent and independent mechanisms (36, 37). FTY720-driven mechanisms could have influenced their results observed after T cell emigration is observed in wild-type controls, but is absent in long-term administration. It is unlikely that the decrease in thymic plt mice lacking thymic medullary CCL19. Taken together, these cellularity we observed was due to FTY720-driven apoptosis be- observations suggest that CCR7-CCL19 interactions are involved cause decreased thymocyte numbers were not observed in in both normal and FTY720-driven thymic emigration, but that CCR2Ϫ/Ϫ and plt mice, FTY720-driven apoptosis requires higher additional mechanisms may also drive T cells into the vessel lu- doses and is independent of chemokines (10), and flow cytometry men at the corticomedullary junction and out of the thymus. In did not reveal increased thymic cell death or apoptosis (data not accordance with this conclusion, we observed that FTY720-driven shown). Rosen et al. (43) demonstrated that acute administration of thymic emigration is also absent in CCR2Ϫ/Ϫ and partially absent the active enantiomer of FTY720 did not increase overall thymic in CCL2Ϫ/Ϫ mice. Because CCL2 is highly expressed by vascular cellularity, but accelerated CD69 loss, consistent with increased endothelial cells, but not within the thymus, CCR2-CCL2 interac- thymocyte maturation, which would be expected to enhance em- Downloaded from tions may drive T cells out of the thymus into the circulation (38). igration. They also suggested that S1PR activation prevents thymic Because CCR2Ϫ/Ϫ and CCL2Ϫ/Ϫ mice have normal thymic archi- egress because fewer thymic emigrants were found in the spleen; tecture, this suggests that CCR2-CCL2 is important for FTY720- however, they did not report whether these cells migrated to the stimulated, but not normal thymic egress (Table II). LN or other sites (43). Very recently, it has been demonstrated that Poznansky et al. (22) recently suggested that chemorepulsion or S1PR1 is required for thymic egress and that FTY720 can act first

fugetaxis away from the medullary chemokine CXCL12 may also as an agonist and then as an antagonist for S1PR1 with increasing http://www.jimmunol.org/ contribute to thymocyte emigration. The role of CXCR4-CXCL12 dose or duration of exposure (27–29). Our observation of de- in thymocyte emigration was examined in vivo by administration creased thymic cellularity measured 18 h after 6 ␮g of FTY720 of the CXCR4 antagonist AMD-3100 (Fig. 8). Blockade of was administered by oral gavage (Figs. 6 and 8) suggests that we CXCR4 alone prevented thymic T cell egress, which was partially have observed the agonistic effects of FTY720 on thymic egress. restored by the administration of FTY720. These results confirm Indeed, if 60 ␮g of FTY720 was administered, then thymic cellu- normal thymocyte egress through a CXCL12-dependent mecha- larity was increased at 18 h (our unpublished results). nism, and also suggest that S1PR-enhanced thymocyte egress is at least partially independent of CXCR4-CXCL12 (Table II). It is Effects of FTY720 on LN accumulation possible that CXCR4 blockade prevented normal intrathymic mi- Our results show that FTY720-driven T cell entry into LN follow- by guest on September 30, 2021 gration, so that the T cells were no longer in proximity to medul- ing FTY720 administration is dependent on CCR2, CCR7, and lary CCL19 or vascular CCL2, and thus could not completely re- CXCR4, but not CCR5 (Table I). There are several implications of spond to FTY720. This may also explain why FTY720 does not these observations. First, because blockade of any one of these influence CXCL12-driven migration in vitro, but is sensitive to three receptors prevents S1PR-stimulated LN accumulation, this CXCR4 blockade in vivo. suggests that all three are expressed on the same or highly over- In vitro T cell migration to CCR5 ligands is not enhanced by lapping T cell populations. CXCR4 and CCR7 are constitutively FTY720; CCR5Ϫ/Ϫ have normal thymic architecture; and expressed on naive peripheral T cells, and along with CCR2 are CCR5Ϫ/Ϫ display normal FTY720-driven T migration in blood, also expressed on memory/effector T cells (44–46); as noted ear- spleen, and LN. However, CCR5Ϫ/Ϫ display an abnormal thymic lier, FTY720 does not preferentially drive particular T cell subsets response to FTY720, demonstrating no S1PR-stimulated thymic into the LN. Second, there is a hierarchy of receptors. Okada et al. egress. CCR5 is expressed on CD4ϩCD8ϩ thymocytes and is nor- (20) demonstrated a role for CXCR4 in LN T cell migration only mally down-regulated on single-positive T cells (39–41). Thus, if CCR7-CCL19 was blocked. Similarly, we observed a role for there may be a failure of normal intrathymic migration of CXCR4 in FTY720-driven LN accumulation, an event that is CCR5Ϫ/Ϫ at the double-positive stage, and thymocytes may be in highly dependent on both CCR2 and CCR7. These data suggest a

Table II. Model for chemokine, chemokine receptor, and FTY720 effects on thymocyte migration

Role in Thymic Emigration

Thymic Location Cell Subset Chemokine Receptor Chemokine Ligand Ϫ FTY720 ϩ FTY720

Cortex DNa CXCR4 (30, 31) CXCL12 (30, 31) ϩϪ Cortex DPb CXCR4 (30, 31) CXCL12 ϩϪ CCR5 (39–41) CCL3/4/5 Ϫϩ CCR9 (32, 33) CCL25 ϩ ND Cortex Transitional CCR4 (31) CCL22 ϩ ND Medulla SPc CCR7 (34–37) CCL19 ϩϩ Corticomedullary junction vessels SP CCR2 (38) CCL2 Ϫϩ CCR7 (34–37) CCL19 ϩϩ

a DN, double negative. b DP, double positive. c SP, single positive. 864 FTY720-ENHANCED T CELL HOMING subordinate role for CXCR4 compared with CCR2 or CCR7 in Acknowledgments homeostatic LN accumulation of T cells, but a codominant role for We thank Volker Brinkmann at Novartis Pharmaceuticals for providing all three receptors in S1PR-driven accumulation. The mechanism valuable reagents. We acknowledge the technical contributions of Minwei of this is not certain, but suggests that CCR2- or CCR7-driven Mao and Dan Chen. events may place T cells in proximity to CXCL12, hence our ob- servation that FTY720 does not enhance in vitro migration to References CXCL12. Third, it is important to note that S1PR1 activity is also 1. Budde, K., R. L. Schmouder, R. Brunkhorst, B. Nashan, P. W. Lucker, T. Mayer, required for T cells to exit the LN (6, 12, 27–29, 47). Thus, che- S. Choudhury, A. Skerjanec, G. Kraus, and H. H. Neumayer. 2002. First human trial of FTY720, a novel immunomodulator, in stable renal transplant patients. mokines could influence not only entry into the LN, but also exit, J. Am. Soc. Nephrol. 13:1073. with LN accumulation being the balance between the two. 2. Brinkmann, V., S. Chen, L. Feng, D. Pinschewer, Z. Nikolova, and R. Hof. 2001. FTY720 alters homing and protects allografts without inducing gen- Effect of FTY720 on splenic and peripheral blood egress eral immunosuppression. Transplant. Proc. 33:530. 3. Nagahara, Y., M. Ikekita, and T. Shinomiya. 2002. T cell selective apoptosis by FTY720-driven T cell egress from the spleen was not inhibited in a novel immunosuppressant, FTY720, is closely regulated with Bcl-2. Ϫ/Ϫ Br. J. Pharmacol. 137:953. any of the strains tested in this study (Table I). Although CCR2 4. Yanagawa, Y., Y. Hoshino, H. Kataoka, T. Kawaguchi, M. Ohtsuki, K. Sugahara, Ϫ/Ϫ and CCR5 have normal splenic architecture, it might have been and K. Chiba. 1999. FTY720, a novel immunosuppressant, prolongs rat skin expected that at least the CCR2Ϫ/Ϫ would have abnormal splenic allograft survival by decreasing T-cell infiltration into grafts. Transplant. Proc. 31:1227. egress because there is a failure of LN sequestration, yet surpris- 5. Pinschewer, D. D., A. F. Ochsenbein, B. Odermatt, V. Brinkmann, ingly this was not observed. Although the splenic parenchyma H. Hengartner, and R. M. Zinkernagel. 2000. FTY720 immunosuppression im- lacks the high endothelial venules of the peripheral LN, CCL19 pairs effector T cell peripheral homing without affecting induction, expansion, Downloaded from and memory. J. Immunol. 164:5761. and CCL21 do play an important role in normal T cell migration 6. Mandala, S., R. Hajdu, J. Bergstrom, E. Quackenbush, J. Xie, J. Milligan, into the spleen, as plt and CCR7Ϫ/Ϫ mice have distorted splenic R. Thornton, G. J. Shei, D. Card, C. Keohane, et al. 2002. Alteration of lym- phocyte trafficking by sphingosine-1-phosphate receptor agonists. Science architecture, with T cells migrating to the red pulp instead of the 296:346. T cell-rich areas of the white pulp (48). Nonetheless, plt mice also 7. Brinkmann, V., M. D. Davis, C. E. Heise, R. Albert, S. Cottens, R. Hoff, exhibited normal splenic egress in response to FTY720, despite a C. Bruns, E. Prieschl, T. Baumruker, P. Hiestand, et al. 2002. The immune mod- ulator FTY720 targets sphingosine 1-phosphate receptors. J. Biol. Chem. lack of LN accumulation. The results suggest that other chemokine 277:21453. http://www.jimmunol.org/ systems or mechanisms must be involved in splenic egress. 8. Randolph, G. J., S. Beaulieu, M. Pope, I. Sugawara, L. Hoffman, R. M. Steinman, T cell egress from the peripheral blood relies on similar mech- and W. A. Muller. 1998. A physiologic function for P-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels. anisms as T cell homing to the high endothelial venules of periph- Proc. Natl. Acad. Sci. USA 95:6924. eral LN (49). Henning et al. (12) demonstrated that although 9. Robbiani, D. F., R. A. Finch, D. Jager, W. A. Muller, A. C. Sartorelli, and Ϫ Ϫ FTY720-driven T cell egress was observed in CCR7 / and con- G. J. Randolph. 2000. The leukotriene C4 transporter MRP1 regulates CCL19 (MIP-3␤, ELC)-dependent mobilization of dendritic cells to lymph nodes. Cell Ϫ/Ϫ trol mice, the CCR7 mice displayed delayed kinetics, suggest- 103:757. ing that other chemokine systems are involved in FTY720-en- 10. Honig, S. M., S. Fu, X. Mao, A. Yopp, M. D. Gunn, G. J. Randolph, and J. S. Bromberg. 2003. FTY720 stimulates multidrug transporter- and cysteinyl hanced peripheral blood T cell egress. Our results show that leukotriene-dependent T cell chemotaxis to lymph nodes. J. Clin. Invest. FTY720 and S1PR activation also affect CCR2. Because CCL2 is 111:627. by guest on September 30, 2021 localized to subendothelial and smooth muscle layers in blood ves- 11. Yopp, A. C., G. J. Randolph, and J. S. Bromberg. 2003. Leukotrienes, sphingo- lipids, and leukocyte trafficking. J. Immunol. 171:5. sels, we hypothesized that CCR2-CCL2 interactions would be re- 12. Henning, G., L. Ohl, T. Junt, P. Reiterer, V. Brinkmann, H. Nakano, sponsible for FTY720-driven peripheral blood egress of T cells; W. Hohenberger, M. Lipp, and R. Forster. 2001. CC chemokine receptor 7-de- however, this was not observed. Our results demonstrate FTY720- pendent and -independent pathways for lymphocyte homing: modulation by Ϫ/Ϫ FTY720. J. Exp. Med. 194:1875. stimulated T cell depletion of the peripheral blood in (CCR2 , 13. Kuziel, W. A., T. C. Dawson, M. Quinones, E. Garavito, G. Chenaux, plt) DKO-, CCL2Ϫ/Ϫ-, CCR2Ϫ/Ϫ-, CCR5Ϫ/Ϫ-, plt-, and AMD- S. S. Ahuja, R. L. Reddick, and N. Maeda. 2003. CCR5 deficiency is not pro- tective in the early stages of atherogenesis in apoE knockout mice. Atheroscle- 3100-treated mice that was equivalent to depletion in wild-type rosis 167:25. controls, suggesting that a yet undetermined factor is responsible 14. Chen, X. S., J. R. Sheller, E. N. Johnson, and C. D. Funk. 1994. Role of leuko- for FTY720-enhanced peripheral blood T cell egress. Previous re- trienes revealed by targeted disruption of the 5-lipoxygenase gene. Nature ␣ 372:179. ports show that L-selectin, CD44, VLA-4 , and fucosyltransferase 15. Luther, S. A., H. L. Tang, P. L. Hyman, A. G. Farr, and J. G. Cyster. 2000. VII do not affect T cell egress from the peripheral blood in re- Coexpression of the chemokines ELC and SLC by T zone stromal cells and sponse to S1PR activation (10, 50, 51). Because peripheral blood deletion of the ELC gene in the plt/plt mouse. Proc. Natl. Acad. Sci. USA 97:12694. egress is sensitive to pertussis toxin desensitization of GPCR (6), 16. Kuziel, W. A., S. J. Morgan, T. C. Dawson, S. Griffin, O. Smithies, K. Ley, and and because only a few of the known chemokine systems were N. Maeda. 1997. Severe reduction in leukocyte adhesion and extrav- examined in this study, it is possible that other chemokines and asation in mice deficient in CC chemokine receptor 2. Proc. Natl. Acad. Sci. USA 94:12053. their receptors are responsible for egress from peripheral blood. 17. Lu, B., B. J. Rutledge, L. Gu, J. Fiorillo, N. W. Lukacs, S. L. Kunkel, R. North, The disruption of thymic egress or LN accumulation by genetic C. Gerard, and B. J. Rolllins. 1998. Abnormalities in monocyte recruitment and expression in monocyte chemoattractant protein 1-deficient mice. or pharmacological means does not affect splenic or peripheral J. Exp. Med. 187:601. blood T cell egress following FTY720 administration (Table I). 18. Chen, S., K. B. Bacon, G. Garcia, R. Liao, Z. K. Pan, S. K. Sullivan, H. Nakano, This illustrates that distinct lymphoid compartments do not nec- A. Matsuzawa, V. Brinkmann, and L. Feng. 2001. FTY720, a novel transplan- tation drug, modulates lymphocyte migratory responses to chemokines. Trans- essarily directly interact with each other, so that entry, accumula- plant. Proc. 33:3057. tion, or egress from one compartment does not affect another com- 19. Aszalos, A., K. Thompson, J. J. Yin, and D. D. Ross. 1999. Combinations of partment. Indeed, given peripheral blood and splenic egress P-glycoprotein blockers, verapamil, PSC833, and cremophor act differently on the multidrug resistance asscociated protein (MRP) and on P-glycoprotein (Pgp). without LN accumulation, it is not clear to where Anticancer Res. 19:1053. have migrated in plt or CCR2Ϫ/Ϫ mice, and other tissues will need 20. Okada T., V. N. Ngo, E. H. Ekland, R. Forster, M. Lipp, D. R. Littman, and J. G Cyster. 2002. Chemokine requirements for entry to lymph nodes and to be examined in these animals. Although the focus in this study Peyer’s patches. J. Exp. Med. 196:65. is on chemokines, it is noteworthy that S1PR activation influences 21. Hatse, S., K. Princen, G. Bridger, E. De Clercq, and D. Schols. 2002. Chemokine endothelial cell barrier integrity (52, 53). FTY720 might engage receptor inhibition by AMD3100 is strictly confined to CXCR4. FEBS Lett. 527:255. this mechanism to influence migration, homing, or retention in a 22. Poznansky, M. C., I. T. Olszak, R. H. Evans, Z. Wang, R. B. Foxall, D. P Olson, chemokine-independent, but GPCR-dependent, fashion. K. Weibrecht, A. D. Luster, and D. T. Scadden. 2002. Thymocyte emigration is The Journal of Immunology 865

mediated by active movement away from stroma-derived factors. J. Clin. Invest. density lipoprotein induces monocyte chemotactic protein 1 in human endothelial 109:1101. cells and smooth muscle cells. Proc. Natl. Acad. Sci. USA 87:5134. 23. Capra, V., M. R. Accomazzo, S. Ravasi, M. Parenti, M. Macchia, S. Nicosia, and 39. Berkowitz, R. D., K. P. Beckerman, T. J. Schall, and J. M. McCune. 1998. G. E. Rovati. 2003. Involvement of prenylated in calcium signalling CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell induced by LTD4 in differentiated U937 cells. Prostaglandins Other Lipid Me- differentiation. J. Immunol. 161:3702. diat. 71:235. 40. Dairaghi, D. J., K. Franz-Bacon, E. Callas, J. Cupp, T. J. Schall, S. A. Tamraz, 24. Massoumi, R., C. K. Nielsen, D. Azemovic, and A. Sjolander. 2003. Leukotriene S. A. Boehme, N. Taylor, and K. B. Bacon. 1998. inflammatory D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and up- protein-1␤ induces migration and activation of human thymocytes. Blood ␣ ␤ regulation of 2 1-integrin. Exp. Cell Res. 289:342. 91:2905. 25. Fukui, Y., O. Hashimoto, T. Sanui., T. Oono, H. Koga, M. Abe, A. Inayoshi, 41. Zaitseva, M. B., S. Lee, R. L. Rabin, H. L. Tiffany, J. M. Farber, K. W. Peden, M. Noda, M. Oike, T. Shirai, and T. Sasazuki. 2001. Hematopoietic cell-specific P. M. Murphy, and H. Golding. 1998. CXCR4 and CCR5 on human thymocytes: CDM family protein DOCK2 is essential for lymphocyte migration. Nature biological function and role in HIV-1 infection. J. Immunol. 161:3103. 412:826. 42. Yagi, H., R. Kamba, K. Chiba, H. Soga, K. Yaguchi, M. Nakamura, and T. Itoh. 26. Stein, J. V., S. F. Soriano, C. M’rini, C. Nombela-Arrieta. G. G. de Buitrago, 2000. Immunosuppressant FTY720 inhibits thymocyte emigration. Eur. J. Im- J. M. Rodriguez-Frade, M. Mellado, J. P. Girard, and A. C. Martinez. 2002. munol. 30:1435. CCR7-mediated physiological lymphocyte homing involves activation of a ty- 43. Rosen, H., C. Alfonso, C. D. Surh, and M. G. McHeyzer-Williams. 2003. Rapid rosine kinase pathway. Blood 101:38. induction of medullary thymocyte phenotypic maturation and egress inhibition by 27. Allende, M. L., J. L. Dreir, S. Mandala, and R. L. Proia. 2004. Expression of the nanomolar sphingosine 1-phosphate receptor agonist. Proc. Natl. Acad. Sci. USA sphingosine 1-phosphate receptor, S1P1, on T-cells controls thymic emigration. 100:10907. J. Biol. Chem. 279:15396. 44. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two 28. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, subsets of memory T lymphocytes with distinct homing potentials and effector M. L. Allende, R. L. Proia, and J. G. Cyster. 2004. Lymphocyte egress from functions. Nature 401:708. thymus and peripheral lymph nodes is dependent on S1P receptor 1. Nature 45. Rabin, R. L., M. K. Park, F. Liao, R. Swofford, D. Stephany, and J. M. Farber. 427:355. 1999. Chemokine receptor responses on T cells are achieved through regulation 29. Graler, M. H., and E. J. Goetzl. 2004. The immunosuppressant FTY720 down-

of both receptor expression and signaling. J. Immunol. 162:3840. Downloaded from regulates sphingosine 1-phosphate G protein-coupled receptors. FASEB J. 46. Bonnecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, 18:551. A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, and R. Sinigaglia. 30. Kim, C. H., L. M. Pelus, J. R. White, and H. E. Broxmeyer. 1998. Differential 1998. Differential expression of chemokine receptors and chemotactic respon- chemotactic behavior of developing T cells in response to thymic chemokines. siveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129. Blood 91:4434. 31. Suzuki, G., Y. Nakata, Y. Dan, A. Uzawa, K. Nakagawa, T. Saito, K. Mita, and 47. Sanna, M. G., J. Liao, E. Jo, C. Alfonso, M. Y. Ahn, M. S. Peterson, B. Webb, T. Shirasawa. 1998. Loss of SDF-1 receptor expression during positive selection S. Lefebvre, J. Chun, N. Gray, and H. Rosen. 2004. Distinct S1P receptor sub- in the thymus. Int. Immunol. 10:1049. types S1P1 and S1P3 respectively regulate lymphocyte recirculation and heart rate. J. Biol. Chem. 279:13839.

32. Wurbel, M. A., J. M. Philippe, C. Nguyen, G. Victorero, T. Freeman, http://www.jimmunol.org/ P. Wooding, A. Miazek, M. G. Mattei, M. Malissen, B. R. Jordan, et al. 2000. The 48. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, and chemokine TECK is expressed by thymic and intestinal epithelial cells and at- H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ che- tracts double- and single-positive thymocytes expressing the TECK receptor mokine have defects in lymphocyte homing and dendritic cell localization. CCR9. Eur. J. Immunol. 30:262. J. Exp. Med. 189:451. 33. Norment, A. M., L. Y. Bogatzki, B. N. Gantner, and M. J. Bevan. 2000. Murine 49. Von Andrian, U. H., and C. R. Mackay. 2000. T-cell function and migration: two CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regu- sides of the same coin. N. Engl. J. Med. 343:1020. lated following pre-TCR signaling. J. Immunol. 164:639. 50. Yanagawa, Y., Y. Masubuchi, and K. Chiba. 1998. FTY720, a novel immuno- 34. Campbell, J. J., J. Pan, and E. C. Butcher. 1999. Cutting edge: developmental suppressant, induces sequestration of circulating mature lymphocytes by accel- switches in chemokine responses during T cell maturation. J. Immunol. 163:2353. eration of lymphocyte homing in rats. III. Increase in frequency of CD62L-pos- 35. Bleul, C. C., and T. Boehm T. 2000. Chemokines define distinct microenviron- itive T cells in Peyer’s patches by FTY720-induced lymphocyte homing. ments in the developing thymus. Eur. J. Immunol. 30:3371. Immunology 95:591.

36. Ueno, T., K. Hara, M. S. Willis, M. A. Malin, U. E. Hopken, D. H. Gray, 51. Bai, Y., J. Liu, Y. Wang, S. Honig, L. Qin, P. Boros, and J. S. Bromberg. 2002. by guest on September 30, 2021 K. Matsushima, M. Lipp, T. A. Springer, R. L. Boyd, et al. 2002. Role for CCR7 L-selectin-dependent lymphoid occupancy is required to induce alloantigen-spe- ligands in the emigration of newly generated T lymphocytes from the neonatal cific tolerance. J. Immunol. 168:1579. thymus. Immunity 6:205. 52. Lee, M. J., S. Thangada, K. P. Claffey, N. Ancellin, C. H. Liu, M. Kluk, M. Volpi, 37. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, and R. I. Sha’afi, and T. Hla. 1999. Vascular endothelial cell adherens junction as- M. Lipp. 1999. CCR7 coordinates the primary by establishing sembly and morphogenesis induced by sphingosine-1-phosphate. Cell 99:301. functional microenvironments in secondary lymphoid organs. Cell 99:23. 53. Schaphorst, K. L., E. Chiang, K. N. Jacobs, A. Zaiman, V. Natarajan, F. Wigley, 38. Cushing, S. D., J. A. Berliner, A. J. Valente, M. C. Territo, M. Navab, F. Parhami, and J. G. Garcia. 2003. Role of sphingosine-1 phosphate in the enhancement of R. Gerrity, C. J. Schwartz, and A. M. Fogelman. 1990. Minimally modified low endothelial barrier integrity by platelet-released products. Am. J. Physiol. 284L:1.