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The Journal of Immunology, 2004, 173: 855-865.
Copyright © 2004 by The American Association of Immunologists

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*,{dagger} and Jonathan S. Bromberg2,*,{dagger}

* Carl C. Icahn Center for Gene Therapy and Molecular Medicine, and {dagger} Recanti/Miller Transplantation Institute, Mount Sinai School of Medicine, New York, NY 10029


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, 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 of FTY720 induces chemokine-dependent migration of T cells in the thymus, 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 administration. 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-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FTY720 is a sphingosine analog immunomodulator derived from the Chinese herb, Iscaria sinclarii, that enhances solid organ allograft survival in humans and animal models (1, 2). Originally thought to act via a ceramide-related apoptotic mechanism, it has clearly been shown that FTY720 enhances allograft survival by promoting T cell egress from peripheral blood into secondary lymphoid tissues (3, 4). A single oral dose of FTY720 causes T cell egress from peripheral blood to lymph nodes (LN)3 and Peyer’s patches, but does not affect T cell priming or activation, and does not interfere with the response to infectious challenges (4, 5).

The molecular mechanism of FTY720-enhanced T cell homing to LN has only recently been elucidated. Mandala et al. and Brinkmann et al. (6, 7) have shown that both phosphorylated FTY720 (P-FTY720) and sphingosine 1-phosphate (S1P) are agonists for G protein-coupled S1PR or endothelial differentiation gene receptors, stimulation of which promotes leukocyte migration. Based on the observation that dendritic cell migration to peripheral LN is dependent on activation of the Abcb1 and Abcc1 multidrug lipid transporters and sensitization of the CCR7 chemokine system, we previously showed that FTY720 sequentially stimulates the Abcb1 and Abcc1 multidrug transporters, S1PR, and cysteinyl leukotriene (cysLT) receptors to sensitize the CCR7 chemokine receptor, thereby enhancing T cell migration to the CCR7 ligands, CCL19 and CCL21 (8, 9, 10) (see Ref.11 for review). Whether or how FTY720 influences other chemokine receptor-ligand pairs remains unclear.

Henning et al. (12) reported that FTY720-induced T cell LN migration occurs in both CCR7-dependent and independent fashions, suggesting that other chemokine receptor-ligand pairs are involved. Our previous results demonstrated that the CCR1, CCR3, CCR5, CX3CR1, CXCR3, and CXCR5 chemokine receptors failed to exhibit FTY720-driven T cell chemotaxis in both in vitro and in vivo assays (10). Therefore, it is important to determine whether other chemokine receptors are responsible for the CCR7-independent action of FTY720-stimulated T cell migration, and 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. Similar to the CCR7 system, FTY720-stimulated CCL2-driven T cell migration is dependent on activity of both the Abcb1 and the Abcc1 lipid transporters, as well as 5-lipoxygenase (5-LO) activity. CCL2-driven T cell migration is more sensitive to both Abcc1 and 5-LO activity than CCR7 responses. In vivo, the CCR2-CCL2 system is required for FTY720-driven T cell homing to LN. The results also demonstrate that S1PR-stimulated T cell migration occurs by engaging different sets of chemokines and their receptors in the thymus and secondary lymphoid organs to determine the destination of the migrating T cell. Specifically, the results indicate that FTY720-stimulated T cell migration in vivo is dependent on the anatomically restricted use of the CCR2-CCL2, CCR5-CCL5, CCR7-CCL19/21, and CXCR4-CXCL12 receptor-ligand pairs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6, FVB/NJ, C57BL/6 CCR5–/– (13), and B6/129 5-LO–/– (14) mice 8–10 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME). FVB/129 Abcb1–/– and FVB/129 Abcc1–/– mice were purchased from Taconic Farms (Germantown, NY). C57BL/6 plt (15), C57BL/6 CCR2–/– (16), C57BL/6 CCL2–/– (17), and C57BL/6 (CCR2–/–, plt) double knockout (DKO) mice were maintained in our facility. All mice were housed in a specific pathogen-free facility in microisolator cages. All experiments were performed with age- and sex-matched mice in accordance with institutionally approved animal care criteria.

Reagents

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)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD4), and S1P were purchased from BIOMOL (Plymouth Meeting, PA). Fluo-3, DiOC2, and CSFE were purchased from Molecular Probes (Eugene, OR). Murine CCL2, human CCL7, human CCL8, human CCL13, murine CCL19, murine CCL1, murine CXCL12, murine CCL3, murine CCL4, and murine CCL22 were purchased from R&D Systems (Minneapolis, MN). FTY720, phosphorylated FTY720, the biologically active R-enantiomer AAL151, and the inactive L-enantiomer AAL149 were kind gifts from V. Brinkmann (Novartis Pharmaceuticals, Basel, Switzerland). AMD-3100 was obtained from the National Institutes of Health AIDS Research and Reference Reagant Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: bicyclam JM-2987 (hydrobromide salt of AMD-3100).

Cell preparations

Mice (two to three per group) were sacrificed, and spleen, LN (cervical, periaortic, inguinal, and axillary), and thymus were removed and gently dissociated into single cell suspensions. Peripheral blood was collected by cardiac puncture. RBC were removed by Tris-NH4Cl lysis. If indicated, cell suspensions were passed through T cell enrichment columns (R&D Systems); these cells were routinely 85–95% T cells. Cells were placed in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1x nonessential amino acids, and 2 x 10–5 M 2-ME).

Flow cytometry

Cell washes and Ab dilutions were performed in PBS plus 1% BSA at 4°C. Flow cytometric analysis was performed on a FACScan flow cytometer (BD Biosciences, San Jose, CA). Forward and side scatter parameters were used to gate on live cells. Results are expressed as percentage of cells staining above background.

Migration assays

In vitro migration assays were performed, as previously described (10). A total of 5 x 105 splenic T cells was incubated with various doses of 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 volume of 100 µl to the upper wells of a 24-well transwell plate with a 5-µm insert (Corning Glass, Corning, NY). Lower wells contained various doses of chemokines in 600 µl of RPMI 1640/0.5% BSA. The number of 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 migration assays were performed with mice given 0.3 mg/kg FTY720 via per os gavage and/or 1 mg/kg AMD-3100 i.p., and sacrificed 18 h later. Thymus, peripheral blood, peripheral LN, and spleen were harvested and made into single cell suspensions. Total cell numbers were counted using a hemocytometer, and subset analysis was performed using fluorescent flow cytometry.

Adoptive transfer assays with CSFE-labeled cells

Single cell suspensions from the spleens of wild-type and various knockout strains were made, and erythrocytes were removed by lysis solutions. CFSE (5.0 µM; Molecular Probes) solution was added to a single cell suspension containing 2.0 x 107 cells/ml and incubated at room temperature for 5 min. The staining process was stopped by adding 20 ml of PBS with 5% FCS and washed twice with PBS. A total of 2 x 107 labeled splenocytes in 300 µl of PBS was injected into the tail veins of the recipient mice; 0.3 mg/kg FTY720 via per os gavage was given to the experimental group of mice; and thymus, peripheral blood, peripheral LN, and spleen were harvested 18 h later. Cell counts were performed with a hemocytometer, and cell subset was determined by flow cytometry.

Statistics

In vivo migration results represent samples from two to three mice per experiment. In vitro migration results represent mean values of triplicate samples. All experiments were performed two to five times. SDs and p values were calculated with Student’s t test using Microsoft Excel software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CCR2-CCL2-driven migration is enhanced by S1PR activation

We previously showed FTY720 increases T cell migration to the CCR7-CCL19/21 chemokine receptor ligands (10). Whether other chemokine receptor-ligand pairs similarly show FTY720-stimulated T cell migration is unknown. In a series of in vitro migration studies, we found that purified murine splenic T cells failed to migrate to the CCR4-CCL22, CCR8-CCL1, CXCR3-CXCL9, CCR1/3/5-CCL5, CCR1/5-CCL3, and CCR5/8-CCL4 chemokine receptor-ligand pairs; and FTY720 also failed to stimulate migration to them (10) (data not shown). In vitro responses of purified splenic T cells to the CXCR4-CXCL12 receptor-ligand pair showed only a low level of T cell migration to CXCL12 that was not increased by the addition of FTY720. Because a previous preliminary report suggested that FTY720 stimulated T cell chemotaxis to CCL2, we assessed in vitro migration of purified splenic T cells to the CCR2 ligands CCL2, CCL7, CCL8, and CCL13 (18). Dose-response experiments demonstrated effective migration of purified T cells to 1 µg/ml CCL2, with 10–15% of total T cells migrating in individual experiments. Approximately 5% of cells migrated to 0.5 µg/ml CCL2, while lower doses caused only minimal migration (Fig. 1A). Addition of the chemokine to the upper wells or both wells resulted in no migration (data not shown), indicating the migration is chemotactic, not chemokinetic. Various concentrations and incubation times of FTY720 significantly increased chemotaxis to CCL2, with the optimal concentrations of FTY720 between 0.5 and 1.0 µg/ml, and the optimal incubation time 15 min at 37°C (Fig. 1A and data not shown). The remaining CCR2 ligands, CCL7, CCL8, and CCL13, induced only modest T cell chemotaxis without demonstrating FTY720-stimulated migration (Fig. 1B).



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  		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 < 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 ± SE of triplicate samples.

 
In vitro migration assays with cells from various knockout mouse strains were also performed to assess the role of CCL2 in FTY720-driven T cell chemotaxis. Purified T cells from CCL2–/– mice demonstrated FTY720 increased T cell migration to CCL2 (Fig. 2, top). However, CCR2–/– T cells failed to migrate to CCL2, with or without FTY720, as expected (Fig. 2, middle). Furthermore, FTY720 also increased CCR2–/– T cell migration to CCL19, demonstrating that the absence of migration was not due to another abnormality of the knockout strain, and that CCR7-CCL19-driven migration could take place independently of CCR2-CCL2. We next evaluated whether or not T cell migration was dependent on the biological specificity of FTY720 by performing in vitro migration assays with the active (AAL151) or inactive (AAL149) enantiomers of FTY720. T cell migration was observed only with the active, but not the inactive enantiomer of FTY720 (Fig. 2, bottom).



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  		FIGURE 2. FTY720 enhances T cell migration to CCL2 and is dependent on the biologically active FTY720 enantiomer, AA151. In vitro chemotactic responses to CCL2 for CCL2–/– and CCR2–/– T cells. Response of wild-type cells to the FTY720 enantiomers, AAL151 (active) and AAL149 (inactive), and CCL2. In vitro migration results represent mean values ± SE of triplicate samples.

 
CCR2-CCL2-driven migration is dependent on multidrug transporter activity

We previously demonstrated that FTY-stimulated T cell migration to the CCR7-CCL19/CCL21 chemokine-ligand pair is dependent on sequential activation of the Abcb1 and Abcc1 multidrug transporters (10). To determine whether CCL2-driven and FTY720-stimulated T cell migration functioned through a similar mechanism, 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 Abcb1–/– mice showed only modest migration to CCL2 alone, and this was not enhanced by FTY720 (Fig. 3B2). Cells from Abcc1–/– mice failed to migrate to CCL2 alone, and did not show any increase with the addition 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 migration.



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  		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), Abcb1–/– (B2), or Abcc1–/– (B3) mice to CCL2 or CCL19. MK571 (Abcc1 antagonist) or PSC833 (Abcb1 antagonist) was added, as indicated. In vitro migration results represent mean values ± SE of triplicate samples.

 
Exogenous P-FTY720 and S1P both directly activate cell surface S1P receptors and cause T cell migration, but do not require Abcb1 efflux activity for these biological effects (10). We examined 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 CCL2, similar to FTY720 (Fig. 4A). Unlike FTY720, P-FTY720 also increased T cell migration of Abcb1–/– cells (Fig. 4B), suggesting that efflux of FTY720 by Abcb1 is necessary for increased T cell migration to CCL2, and that exogenous phosphorylated sphingosine ligand can bypass the need for Abcb1. Similar results were observed with wild-type cells in which Abcb1 was blocked with PSC833; S1P and P-FTY720 caused migration, but FTY720 did not (Fig. 4A). PSC833 can block Abcc1 (19), but at the doses used in this study it preferentially blocked only Abcb1, as shown by the effectiveness of P-FTY720 to bypass the blockade and increase migration. Conversely, S1P and P-FTY720 were unable to restore or stimulate migration of cells in which Abcc1 was blocked with MK571 (Fig. 4A). Taken together, these results show that CCR2-CCL2 is dependent on the efflux activity of Abcb1 and Abcc1, that Abcb1 is upstream of Abcc1, and that phosphorylated sphingosine ligands bypass the need for Abcb1 activity, all similar to the CCR7-CCL19/21 system (10).



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  		FIGURE 4. PFTY720 and S1P cause Abcb1-independent and Abcc1-dependent T cell migration to CCL2. In vitro chemotactic responses of T cells from wild-type (A) or Abcb1–/– (B) mice to CCL2. MK571 (Abcc1 antagonist) or PSC833 (Abcb1 antagonist) was added, as indicated. In vitro migration results represent mean values ± SE of triplicate samples.

 
CCR2-CCL2-driven migration is highly dependent on 5-LO and cysLTs

We previously demonstrated that T cell migration to the CCR7-CCL19/21 receptor-ligand pairs is dependent on cysLT production by 5-LO and its subsequent efflux through the Abcc1 transporter (8, 9, 10). To determine whether the CCR2-CCL2 receptor-ligand pair functions with a similar mechanism, in vitro chemotaxis assays were performed with wild-type T cells treated with the 5-LO blocker, AA-861, and 5-LO knockout strain cells. With or without FTY720, AA-861-pretreated and 5-LO–/– T cells both migrated poorly or not at all to CCL2. In comparison, 5-LO–/– T cells migrated to CCL19 (Fig. 5A). The addition of exogenous LTD4 restored migration of the AA-861-treated T cells. Taken together with the results that leukotriene transporter Abcc1 knockout or blocked T cells do not migrate to CCL2 (Fig. 3, A and B3), this shows that CCR2-CCL2 is very sensitive to 5-LO activity, cysLT production, and cysLT efflux by Abcc1. In contrast, CCR7-CCL19 induces some T cell migration in the absence of Abcc1 or 5-LO (Figs. 3B3 and 5A), although FTY720 still remains inactive on these cells (10).



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  		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-LO–/– 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 values ± 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). Furthermore, reports demonstrated the CCR7-CCL19/CCL21 receptor-ligand pairs were important, but not exclusive, requirements for T cell sequestration in LN in response to FTY720 (10, 12). We sought to determine whether these receptor-ligand pairs as well as other chemokine receptor-ligand pairs affected in vivo T cell migration in the thymus, peripheral blood, peripheral LN, and spleen following FTY720 administration. In vivo migration assays were 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.

In the thymus of wild-type, but not plt (mutants lacking CCL19 and lymphoid CCL21), CCR2–/–, (CCR2–/–, plt) DKO, or CCR5–/– mice, there was a reduction in total T cell numbers, CD4+ single-positive, CD8+ single-positive, and CD4+CD8+ double-positive cells following FTY720 administration (Figs. 6A and 8B). CCL2–/– mice had only a partial reduction of thymocyte cellularity in response to FTY720. These results suggest that the CCR2, CCR5, and CCR7 chemokine receptors and ligands are required for FTY720-stimulated T cell thymic egress.



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  		FIGURE 6. FTY720 enhances T cell accumulation or egress from the thymus, peripheral blood, peripheral LN, and spleen based on chemokine and receptor distribution. In vivo migration of T cells following FTY720 administration in the thymus (A), peripheral blood (B), LN (C), or spleen (D). Mouse strains are indicated, and results are expressed as percentage of control FTY720-untreated groups of the same strain. In vivo migration results represent mean values ± SE of n = 3 mice. *, p < 0.05 compared with control.

 


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  		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 thymocytes. In vivo migration results represent mean values ± SE of triplicate samples. Flow cytometry plots are representative of three experiments; n = 3 per group.

 
Peripheral blood T cells were all significantly reduced, not only in wild-type, but also in plt, CCL2–/–, (CCR2–/–, plt) DKO, CCR2–/–, and CCR5–/– mice following FTY720 administration (Fig. 6B). Similarly, splenic T cells were reduced in all strains (Fig. 6D). Therefore, the absence of either the CCR5, CCR2, and/or the CCR7 chemokine receptors and ligands does not affect T cell egress from the peripheral blood or spleen. These findings suggest that none of these chemokine systems contributes to FTY720-stimulated T cell egress from peripheral blood or the spleen.

Sequestration of T cells in LN only occurred in the wild-type and CCR5–/– mice, with no significant increase observed in CCR2–/– or (CCR2–/–, plt) DKO mice, compared with their respective controls (Fig. 6C). Slight peripheral LN T cell sequestration was observed in plt and CCL2–/– mice, but at a significantly lower level compared with control mice. These findings demonstrate that the absence of CCL19 and lymphoid CCL21, or the absence of the single CCR2 ligand, CCL2, partially prevents FTY720-stimulated T cell peripheral LN accumulation. The absence of CCR2 prevents T cell accumulation to a much greater degree than the absence of CCL2. Similarly, the absence of CCR7 has been demonstrated to more effectively inhibit LN accumulation in response to FTY720 compared with plt mice (12). The roles of these chemokines in FTY720 LN accumulation of T cells are redundant or highly overlapping because (CCR2–/–, plt) DKO are very similar in their LN response compared with CCR2–/–. Interestingly, the percentage of distribution of CD4 cells among naive and memory subsets, as determined by multicolor flow cytometry analysis for CD45RB and L-selectin, showed no difference between untreated and FTY720-treated LN (data not shown), suggesting that S1PR activation does not preferentially drive a particular subset expressing a limited chemokine receptor repertoire into the LN.

To further elucidate the role of the CCR2 and CCR7 chemokine systems in FTY720-driven T cell migration, adoptive transfer studies were performed with CSFE-labeled splenocytes from wild-type or CCR2–/– donors transferred into wild-type or plt recipients given FTY720. As shown in Fig. 7B, after adoptive transfer, CSFE-labeled T cells can be found in the peripheral blood, and they can be induced to exit from the peripheral blood following FTY720 administration, regardless of the genotype of the transferred cell or host. These results confirm the in vivo findings above, showing that none of the chemokines examined contributes to T cell egress from the peripheral blood. However, the migration patterns of these cells into LN were intrinsic to the genotype of both the transferred cells and the recipient (Fig. 7B, right). We observed peripheral LN sequestration of CSFE-labeled wild-type T cells transferred into C57BL/6, but not plt mice, and no LN sequestration with CCR2–/– cells transferred into either wild-type or plt hosts. Furthermore, these adoptive transfer studies show that the transferred cells migrate independently of surrounding host cells. Thus, CCR2–/– adoptively transferred cells do not migrate to LN, while host wild-type cells do (Fig. 7A, right). Conversely, wild-type adoptively transferred cells do not enhance migration of host plt cells, as expected, because T cells do not secrete CCL19 or CCL21.



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  		FIGURE 7. FTY720-enhanced T cell migration to peripheral LN is dependent on CCR2 and CCR7 and is intrinsic to cell genotype. In vivo migration of A, recipient blood and LN T cells, and B, adoptively transferred donor CSFE-labeled T cells in blood and LN following FTY720 administration. Results are expressed as actual cell counts. *, p < 0.05 compared with control. In vivo migration results represent mean values ± SE of n = 3 mice.

 
A previous report suggested a role for CXCR4-CXCL12 in homeostatic T cell LN migration that was apparent only when CCR7-CCL19 was blocked (20). To determine whether the CXCR4-CXCL12 receptor-ligand pair influences FTY720-stimulated T migration in vivo, even though, as noted above, CXCL12-driven in vitro migration is not stimulated by FTY720, a chemical compound with unique antagonist specificity for CXCR4, AMD-3100 (21), was used. As shown in Fig. 8A, administration of AMD-3100 alone had no effect on peripheral LN accumulation in the absence of FTY720. However, administration of AMD-3100 along with FTY720 in wild-type mice prevented T cell peripheral LN accumulation, compared with controls that received FTY720 alone. Thus, CXCR4 blockade alone does not affect LN migration, similar to the previous report (20); but CXCR4 is required for S1PR-driven migration initiated by FTY720 administration. A previous report (22) suggested that CXCR4-CXCL12 contributes to normal thymocyte emigration. We observed that administration of AMD-3100 alone prevented normal thymic T cell egress, as noted by an acute increase in thymocyte numbers, affecting all subsets compared with controls (Fig. 8, A, left; B, upper right). Further analysis 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 demonstrates 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 normally following FTY720 administration, with or without AMD-3100 (Fig. 8A). Overall, these findings reveal that CXCR4 is required 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Effect of FTY720 on CCR2

FTY720 stimulates T cell migration to peripheral LN by both CCR7-dependent and independent mechanisms (12). FTY720 promotes 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{alpha}-dependent adhesion (10). In this study, we report that screening of the CCR1, CCR2, CCR3, CCR5, CCR8, CX3CR1, CXCR3, CXCR4, and 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 increased T cell migration. We previously proposed a model (10) in which the sequential engagement and activation of these two multidrug transporters and three G protein-coupled receptor (GPCRs) (S1PRs, cysLTRs, and CCRs) occurred in an autocrine or paracrine fashion. The precise biochemical events that sensitize cells to chemokine-driven migration are not known, but because cysLTs have a rapid effect on migration (9, 10), sensitization may relate to immediate second messengers that affect the cytoskeleton and motility (23, 24). Differential sensitivity to cysLTs suggests that CCR2 and CCR7 stimulate similar, but not identical molecular events related to migration, while other chemokine receptors may engage alternative migration mechanisms, because they are unaffected by S1PR stimulation and presumably cysLTs. In fact, CCR2 and CCR7 differ in their engagement of DOCK2 and Jak2 tyrosine kinase (25, 26). With regard to the effects of FTY720 on different chemokine-receptor pairs, it is important to note that the source of cells used for migration studies may influence in vitro results. For example, preliminary experiments suggest that peripheral blood and splenic murine T cells respond to S1PR stimulation, while LN T cells do not (J.S.B., A.C.Y., unpublished observations), perhaps a reflection that LN-retained cells have down-modulated S1PR1 expression or activity (27, 28, 29).

Effect of FTY720 on thymic egress

The results also demonstrate that in addition to CCR7-CCL19/CCL21, CCR2-CCL2 also influences FTY720-driven T cell migration in vivo. Furthermore, the CCR5-CCL3/4/5 and CXCR4-CXCL12 chemokine systems influenced FTY720-driven T cell egress from the thymus and migration to the LN, but migration to these chemokines was not directly affected by S1PR activation in vitro. Rather, S1PR activation uncovered an increased role for CCR5 in thymic egress and CXCR4 in LN migration that was not apparent when S1PR were not activated. These in vivo results are summarized in Table I, which clearly shows that different chemokine receptors are engaged in different compartments, and that the presence or absence of S1PR stimulation further alters receptor usage. To understand the thymic effects, it is important to note that double-negative thymocytes express CXCR4 and migrate to CXCL12 (30, 31); double-positive thymocytes express CXCR4 and CCR9 and migrate to CXCL12 and CCL25 (30, 31, 32, 33); transitional thymocytes between the double- and single-positive stages express CCR4 and migrate to CCL22 (31); and single-positive thymocytes migrate to the CCR7 ligands, CCL19 and CCL21 (Table II) (34, 35). Because adult CCR7–/– mice have increased thymocyte numbers, but do not have reduced numbers of circulating T cells, this suggests that thymic emigration relies on both CCR7-dependent and independent mechanisms (36, 37). FTY720-driven T cell emigration is observed in wild-type controls, but is absent in plt mice lacking thymic medullary CCL19. Taken together, these observations suggest that CCR7-CCL19 interactions are involved in both normal and FTY720-driven thymic emigration, but that additional mechanisms may also drive T cells into the vessel lumen at the corticomedullary junction and out of the thymus. In accordance with this conclusion, we observed that FTY720-driven thymic emigration is also absent in CCR2–/– and partially absent in CCL2–/– mice. Because CCL2 is highly expressed by vascular endothelial cells, but not within the thymus, CCR2-CCL2 interactions may drive T cells out of the thymus into the circulation (38). Because CCR2–/– and CCL2–/– mice have normal thymic architecture, this suggests that CCR2-CCL2 is important for FTY720-stimulated, but not normal thymic egress (Table II).


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

 

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

 
Poznansky et al. (22) recently suggested that chemorepulsion or fugetaxis away from the medullary chemokine CXCL12 may also contribute to thymocyte emigration. The role of CXCR4-CXCL12 in thymocyte emigration was examined in vivo by administration of the CXCR4 antagonist AMD-3100 (Fig. 8). Blockade of CXCR4 alone prevented thymic T cell egress, which was partially restored by the administration of FTY720. These results confirm normal thymocyte egress through a CXCL12-dependent mechanism, and also suggest that S1PR-enhanced thymocyte egress is at least partially independent of CXCR4-CXCL12 (Table II). It is possible that CXCR4 blockade prevented normal intrathymic migration, so that the T cells were no longer in proximity to medullary CCL19 or vascular CCL2, and thus could not completely respond to FTY720. This may also explain why FTY720 does not influence CXCL12-driven migration in vitro, but is sensitive to CXCR4 blockade in vivo.

In vitro T cell migration to CCR5 ligands is not enhanced by FTY720; CCR5–/– have normal thymic architecture; and CCR5–/– display normal FTY720-driven T migration in blood, spleen, and LN. However, CCR5–/– display an abnormal thymic response to FTY720, demonstrating no S1PR-stimulated thymic egress. CCR5 is expressed on CD4+CD8+ thymocytes and is normally down-regulated on single-positive T cells (39, 40, 41). Thus, there may be a failure of normal intrathymic migration of CCR5–/– at the double-positive stage, and thymocytes may be in an inappropriate location to respond to FTY720-stimulated CCL2- or CCL19-driven migration. This suggests a role for CCR5 in S1PR-driven, but not normal thymic emigration (Table II).

Our results on the effects of FTY720 in the thymus are in contrast to a recent report suggesting that FTY720 inhibits thymic T cell emigration in wild-type mice (42). However, in that report, thymic cellularity was only evaluated after 20 days of FTY720 administration, and not acutely as in our studies. Multiple other mechanisms could have influenced their results observed after long-term administration. It is unlikely that the decrease in thymic cellularity we observed was due to FTY720-driven apoptosis because decreased thymocyte numbers were not observed in CCR2–/– and plt mice, FTY720-driven apoptosis requires higher doses and is independent of chemokines (10), and flow cytometry did not reveal increased thymic cell death or apoptosis (data not shown). Rosen et al. (43) demonstrated that acute administration of the active enantiomer of FTY720 did not increase overall thymic cellularity, but accelerated CD69 loss, consistent with increased thymocyte maturation, which would be expected to enhance emigration. They also suggested that S1PR activation prevents thymic egress because fewer thymic emigrants were found in the spleen; however, they did not report whether these cells migrated to the LN or other sites (43). Very recently, it has been demonstrated that S1PR1 is required for thymic egress and that FTY720 can act first as an agonist and then as an antagonist for S1PR1 with increasing dose or duration of exposure (27, 28, 29). Our observation of decreased thymic cellularity measured 18 h after 6 µg of FTY720 was administered by oral gavage (Figs. 6 and 8) suggests that we have observed the agonistic effects of FTY720 on thymic egress. Indeed, if 60 µg of FTY720 was administered, then thymic cellularity was increased at 18 h (our unpublished results).

Effects of FTY720 on LN accumulation

Our results show that FTY720-driven T cell entry into LN following FTY720 administration is dependent on CCR2, CCR7, and CXCR4, but not CCR5 (Table I). There are several implications of these observations. First, because blockade of any one of these three receptors prevents S1PR-stimulated LN accumulation, this suggests that all three are expressed on the same or highly overlapping T cell populations. CXCR4 and CCR7 are constitutively expressed on naive peripheral T cells, and along with CCR2 are also expressed on memory/effector T cells (44, 45, 46); as noted earlier, FTY720 does not preferentially drive particular T cell subsets into the LN. Second, there is a hierarchy of receptors. Okada et al. (20) demonstrated a role for CXCR4 in LN T cell migration only if CCR7-CCL19 was blocked. Similarly, we observed a role for CXCR4 in FTY720-driven LN accumulation, an event that is highly dependent on both CCR2 and CCR7. These data suggest a subordinate role for CXCR4 compared with CCR2 or CCR7 in homeostatic LN accumulation of T cells, but a codominant role for all three receptors in S1PR-driven accumulation. The mechanism of this is not certain, but suggests that CCR2- or CCR7-driven events may place T cells in proximity to CXCL12, hence our observation that FTY720 does not enhance in vitro migration to CXCL12. Third, it is important to note that S1PR1 activity is also required for T cells to exit the LN (6, 12, 27, 28, 29, 47). Thus, chemokines could influence not only entry into the LN, but also exit, with LN accumulation being the balance between the two.

Effect of FTY720 on splenic and peripheral blood egress

FTY720-driven T cell egress from the spleen was not inhibited in any of the strains tested in this study (Table I). Although CCR2–/– and CCR5–/– have normal splenic architecture, it might have been expected that at least the CCR2–/– would have abnormal splenic egress because there is a failure of LN sequestration, yet surprisingly this was not observed. Although the splenic parenchyma lacks the high endothelial venules of the peripheral LN, CCL19 and CCL21 do play an important role in normal T cell migration into the spleen, as plt and CCR7–/– mice have distorted splenic architecture, with T cells migrating to the red pulp instead of the T cell-rich areas of the white pulp (48). Nonetheless, plt mice also exhibited normal splenic egress in response to FTY720, despite a lack of LN accumulation. The results suggest that other chemokine systems or mechanisms must be involved in splenic egress.

T cell egress from the peripheral blood relies on similar mechanisms as T cell homing to the high endothelial venules of peripheral LN (49). Henning et al. (12) demonstrated that although FTY720-driven T cell egress was observed in CCR7–/– and control mice, the CCR7–/– mice displayed delayed kinetics, suggesting that other chemokine systems are involved in FTY720-enhanced peripheral blood T cell egress. Our results show that FTY720 and S1PR activation also affect CCR2. Because CCL2 is localized to subendothelial and smooth muscle layers in blood vessels, we hypothesized that CCR2-CCL2 interactions would be responsible for FTY720-driven peripheral blood egress of T cells; however, this was not observed. Our results demonstrate FTY720-stimulated T cell depletion of the peripheral blood in (CCR2–/–, plt) DKO-, CCL2–/–-, CCR2–/–-, CCR5–/–-, plt-, and AMD-3100-treated mice that was equivalent to depletion in wild-type controls, suggesting that a yet undetermined factor is responsible for FTY720-enhanced peripheral blood T cell egress. Previous reports show that L-selectin, CD44, VLA-4{alpha}, and fucosyltransferase VII do not affect T cell egress from the peripheral blood in response to S1PR activation (10, 50, 51). Because peripheral blood egress is sensitive to pertussis toxin desensitization of GPCR (6), and because only a few of the known chemokine systems were examined in this study, it is possible that other chemokines and their receptors are responsible for egress from peripheral blood.

The disruption of thymic egress or LN accumulation by genetic or pharmacological means does not affect splenic or peripheral blood T cell egress following FTY720 administration (Table I). This illustrates that distinct lymphoid compartments do not necessarily directly interact with each other, so that entry, accumulation, or egress from one compartment does not affect another compartment. Indeed, given peripheral blood and splenic egress without LN accumulation, it is not clear to where lymphocytes have migrated in plt or CCR2–/– mice, and other tissues will need to be examined in these animals. Although the focus in this study is on chemokines, it is noteworthy that S1PR activation influences endothelial cell barrier integrity (52, 53). FTY720 might engage this mechanism to influence migration, homing, or retention in a chemokine-independent, but GPCR-dependent, fashion.


   Acknowledgments
 
We thank Volker Brinkmann at Novartis Pharmaceuticals for providing valuable reagents. We acknowledge the technical contributions of Minwei Mao and Dan Chen.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01 AI41428 (to J.S.B.). Back

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 10029-6574. E-mail address: jon.bromberg{at}mountsinai.org Back

3 Abbreviations used in this paper: LN, lymph node; cysLT, cysteinyl leukotriene; DKO, double knockout; GPCR, G protein-coupled receptor; 5-LO, 5-lipoxygenase; LTD4, 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid; P-FTY720, phosphorylated FTY720; S1P, sphingosine 1-phosphate. Back

Received for publication January 6, 2004. Accepted for publication May 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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