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The Journal of Immunology, 2006, 176: 1474-1480.
Copyright © 2006 by The American Association of Immunologists

Enhanced FTY720-Mediated Lymphocyte Homing Requires G{alpha}i Signaling and Depends on beta2 and beta7 Integrin1

Oliver Pabst*, Heike Herbrand*, Stefanie Willenzon*, Tim Worbs*, Angela Schippers{dagger}, Werner Müller{dagger}, Günter Bernhardt* and Reinhold Förster2,*

* Institute of Immunology, Hannover Medical School, Hannover, Germany; and {dagger} Department of Experimental Immunology, Gesellschaft für Biologische Forschung, Braunschweig, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of beta2 and beta7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either beta2 or beta7 integrin but not of beta2-deficient lymphocytes, which in addition were blocked by anti-beta7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte G{alpha}i signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The efficient induction of adaptive immune responses relies on the continuous migration of naive lymphocytes through secondary lymphoid organs (SLO)3 thereby increasing the probability of lymphocytes to encounter their cognate Ags. The homeostatic migration of lymphocytes is controlled by a complex molecular network involving selectins, integrins, as well as signaling induced by chemokines and sphingosine-1-phosphate (S1P) (1). Interference with this network could provide promising therapeutic strategies that would act by altering migratory properties of cells without interfering with lymphocyte activation.

Regulation of lymphocyte homeostasis is controlled at several checkpoints including lymphocyte immigration to and egress from lymphoid organs. In contrast to memory T cells that enter lymph nodes (LN) via afferent lymphatics, naive T and B cells enter LN through specialized high endothelial venules (HEV) (2). A multistep model has been proposed for the immigration of lymphocytes across HEV (3, 4): interaction of L-selectin (CD62L) expressed on naive lymphocytes with sialyl-LewisX carbohydrate moieties on HEV enables the cells to roll along the endothelium. Chemokines present at the luminal site of the HEV activate their cognate receptors on contacting lymphocytes. Chemokine receptor signaling in turn induces a high affinity conformation of integrins that subsequently causes a stable arrest of lymphocytes on the apical surface of the endothelium. Firmly attached lymphocytes finally cross the endothelial cell (EC) layer to enter the LN (1, 3). The major contribution to the arrest of rolling lymphocytes in peripheral LN is achieved by the {alpha}Lbeta2 integrin (LFA-1, CD11a/CD18) (5), whereas {alpha}4beta7 integrin is more important in gut-associated lymphoid tissue (6). Although expressed on lymphocytes, beta1 integrin appears to be dispensable for their homing into SLO. Correspondingly, in peripheral LN the beta2 integrin ligand ICAM-1 is abundantly expressed on HEV whereas the beta7 integrin ligand MadCAM-1 is expressed on HEV in the gut-associated lymphoid tissue as well as in intestinal venules (7, 8).

In contrast to this thoroughly analyzed process of lymphocyte immigration, little is known regarding the molecular mechanisms that regulate lymphocyte egress from LN. Recently, the study of a novel immunomodulatory drug, FTY720, disclosed S1P receptor signaling to be essential for lymphocyte egress from LN and thymus (1, 9, 10, 11). In vivo FTY720 is rapidly converted into FTY720 phosphate (12), which has been suggested to represent the biologically active form of this drug via binding to four of five known S1P receptors (S1P1–5) (11, 13). Signaling via S1P receptors leads to the G{alpha}i-dependent activation of the small GTP proteins Rho, Rac, and Cdc42 that are crucially involved in modulating cell shape, motility, and adherence (14). In general, Rac/Cdc42 conducted activities mediated via S1P1 and S1P3 result in mobility, whereas Rho dominated signaling is coupled to S1P2 and in part S1P3 predisposing to a stationary phenotype (14, 15). Interference of FTY720 with S1P signaling hampers entry of lymphocytes into efferent lymphatics within LN, thereby delaying their subsequent return into the circulation. This retention of lymphocytes in the LN leads to blood lymphopenia and most likely accounts for the potency of FTY720 to suppress transplant rejection as well as autoimmune diseases including experimental autoimmune encephalitis (16, 17), arthritis (18, 19), systemic lupus erythematosus (20), and diabetes mellitus type 1 (21, 22).

Although inhibition of lymphocyte egress is believed to account for FTY720-mediated immunosuppression, the mode of action of this substance is still discussed controversially. It has been argued that FTY720 phosphate acts as an agonist on S1P receptors (11). In contrast, genetic analysis demonstrated that S1P1-deficient cells fail to egress from lymphoid organs in mice (9), thus mimicking the effect of FTY720 and potentially suggesting an antagonistic function for FTY720 with respect to signaling via this particular receptor. The supposed antagonistic functions of FTY720 were correlated with drug-induced S1P1 receptor internalization, thus desensitizing the cells for further S1P-mediated signaling (9, 23). Therefore it is still puzzling how the putative agonistic and/or antagonistic signaling events triggered by S1P and FTY720 phosphate combine to exert the observed phenomena and in particular how FTY720-modulated S1P receptor signaling affects lymphocyte migration and/or adhesion.

We have previously shown that FTY720 facilitates the otherwise inefficient homing of lymphocytes in the absence of chemokine receptor CCR7 signaling (24), indicating that FTY720 does not only affect egress of naive lymphocyte from SLO but also their homing into lymphoid organs. However, a potential role for FTY720 in the immigration of cells into SLO has barely been addressed so far. In this study we aim to further dissect the influence of FTY720 on the migration of lymphocytes in vivo in particular by analyzing the effect of FTY720 therapy on integrin function. We demonstrate that, in addition to its effect on lymphocyte egress, FTY720 reinforces lymphocyte-endothelium interactions in a G{alpha}i-dependent manner and rescues the immigration defects of beta2 or beta7 integrin single-deficient lymphocytes but not of beta2-deficient cells that were treated with inhibitory anti-beta7 integrin Abs. Our data demonstrate that FTY720 affects lymphocyte homeostasis not only at the level of LN egress but also at LN homing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and FTY720 treatment

Wild-type C57BL/6 mice were purchased from Charles River Breeding Laboratories. Mice deficient for the beta2 integrin on a C57BL/6 background were a gift from K. Scharffetter-Kochanek (University of Ulm, Ulm, Germany). beta7 integrin and VCAM-1-deficient mice on a C57BL/6 background have been described earlier (6, 25) and were bred at the Gesellschaft für Biotechnologische Forschung. MadCAM-1-deficient mice that have been generated in the laboratory of Dr. W. Müller (Gesellschaft für Biologische Forschung, Braunschweig, Germany) will be reported elsewhere. For FTY720 treatment, animals were fed with 1 mg/kg FTY720 in 100 µl of PBS and the drinking water supplemented with 2.5 µg of FTY720/ml. For adoptive transfer experiments, FTY720 treatment of recipient animals was started 48 h before adoptive cell transfer. All animal experiments have been performed in accordance with institutional guidelines and have been approved by the local committees.

Antibodies

The following Abs and conjugates were used in this study: anti-CD4 PerCp (BD Biosciences), anti-CD19-bio (Southern Biotechnology Associates), and anti-CD8beta (clone RM CD8) and anti-CD3 (clone 17A2) Abs provided by E. Kremmer (GSF-Forschungszentrum für Umwelt und Gesundheit, München, Germany). HEV were visualized using MECA79 Ab (BD Biosciences) followed by polyclonal mouse anti-rat biotin conjugate (The Jackson Laboratory) and detected using Streptavidin-Alexa Fluor 750 (Molecular Probes). For in vitro treatment of cells anti-{alpha}4beta7 integrin (clone DATK32) and anti-beta7 integrin (clone FIB27), both from BD Biosciences, were used at 1 µg/ml.

Adoptive transfers and flow cytometry

Adult mice were sacrificed by CO2 inhalation. To obtain single cell suspensions, peripheral and mesenteric LN were minced through a nylon mesh and washed with PBS supplemented with 3% FCS. Cells obtained from wild-type or integrin-deficient mice were labeled with CFSE or TAMRA. In brief, 5 x 106 cells/ml were preincubated for 30 min in RPMI 1640 containing 5% FCS at 37°C, subsequently incubated with 5 µM CFSE or TAMRA for 10 min and washed in ice-cold PBS with 3% FCS. Equal numbers of cells were mixed and i.v. transferred into 8- to 12-wk-old C57BL/6 wild-type recipients. The ratio of CFSE- and TAMRA-labeled cells was determined for CD4-, CD8-, and CD19-positive cells. Analysis was performed using FACSCalibur (BD Biosciences), and ratios obtained were corrected for the ratio present in the injected mixtures.

Immunofluorescence microscopy

Lymphoid organs were embedded in OCT on dry ice, and 6-µm cryosections were prepared. Sections were air dried, fixed for 10 min in ice-cold acetone, and subsequently stained with Abs against the indicated markers as previously described (26). Images were made using a motorized Axiovert 200M microscope (Carl Zeiss) and Axiovision software (Carl Zeiss).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
FTY720 induces lymphopenia in beta2 and beta7 integrin-deficient mice as well as ICAM-1-, VCAM-1-, and MadCAM-1-deficient mice

Earlier studies demonstrated that FTY720 rescues the homing defect of CCR7-deficient lymphocytes to SLO (24), suggesting that FTY720 treatment might allow the uncoupling of lymphocyte homing from the canonical multistep process (see opening discussion). Therefore, we analyzed the well-documented role of integrins and integrin ligands in the homeostasis of lymphocytes in FTY720-treated mice. beta2 and beta7 integrin-deficient, ICAM-1-, VCAM-1-, or MadCAM-1-deficient mice, and wild-type controls were fed with an initial dose of 1 mg/kg FTY720 and kept under constant FTY720 treatment by adding 2.5 µg/ml FTY720 to the drinking water. The total number of CD4+, CD8+, and CD19+ lymphocytes in the blood was determined before treatment and 24 h after FTY720 administration (Fig. 1A and data not shown). Interestingly, in all mutant mice analyzed, FTY720 treatment rapidly induced the disappearance of lymphocytes from the peripheral blood (Fig. 1A), demonstrating that the FTY720-mediated shift of lymphocyte homeostasis does not critically depend on any of the classical homing and adhesion molecules under investigation. Comparable results have been obtained with MadCAM-1/beta7 integrin and MadCAM-1/ICAM-1 double-deficient mice on a mixed genetic background (data not shown).


Figure 1
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  		FIGURE 1. FTY720 induces lymphopenia in integrin-deficient and integrin ligand-deficient mice. A, ICAM-1, VCAM-1, MadCAM-1, beta2 or beta7 integrin-deficient animals as well as wild-type controls were fed with an initial dose of 1 mg of FTY720/kg body weight and kept under constant treatment by adding 2.5 µg/ml FTY720 to the drinking water. The number of CD4+ and CD8+ lymphocytes was determined before ({square}) and 24 h after ({blacksquare}) the onset of treatment shown as mean + SD (n = 5–8 mice). B, Lymphopenia is induced in wild-type and beta2 integrin-deficient mice with similar kinetics. As described, the number of CD8+ lymphocytes in wild-type mice ({circ}) and beta2 integrin-deficient mice ({square}) was determined at various time points during the treatment with FTY720. Data obtained from individual animals of one of two independent experiments are shown and horizontal bars represent mean values.

 
In wild-type animals FTY720 treatment resulted in the removal of ~87% of CD4+ or CD8+ lymphocytes from the peripheral blood within 6 h of treatment (Fig. 1B and data not shown), whereas during the same period of time ~75% of CD4+ or CD8+ T cells disappeared from the blood in beta2 integrin-deficient mice. These data reveal that lymphopenia is induced with similar kinetics in wild-type and beta2-deficient mice (Fig. 1B) and suggests that FTY720 treatment results in increased homing of beta2 integrin-deficient lymphocytes.

FTY720 restores homing of beta2 or beta7 integrin-deficient cells but not beta2-deficient cells lacking functional beta7 integrin

To specifically address the effect of FTY720 on the homing of integrin-deficient lymphocytes, we performed adoptive transfer experiments. Cells were isolated from peripheral and mesenteric LN of wild-type mice or beta2 or beta7 integrin-deficient mice and labeled with green or red fluorescent dye. Equal numbers of wild-type and integrin-deficient cells were injected i.v. into wild-type recipients. The localization and frequency of transferred cells within SLO was analyzed 60 min after cell transfer. Importantly, immunohistology of sections from peripheral and mesenteric LN revealed that, within this short period of time, the majority of transferred wild-type cells were found in the proximity of HEV, whereas virtually no cells were identified close to or within the medullary sinus of the LN (data not shown). Therefore, the number of adoptively transferred cells present in SLO at that time almost exclusively reflects the efficiency of lymphocyte entry, whereas cell loss by lymphocyte egress is negligible. The ratio of transferred wild-type and mutant cells was analyzed by flow cytometry in peripheral and mesenteric LN, Peyer’s patches (PP), and spleen (Fig. 2). In line with published observations, beta2 integrin-deficient cells showed a 15-fold decreased migration into peripheral LN but efficiently entered mesenteric LN, PP, and spleen (Fig. 2). Conversely, deficiency of beta7 integrin selectively affected the migration into PP and mesenteric LN but not into peripheral LN and spleen (Fig. 2). Residual migration of beta2 integrin-deficient cells into peripheral LN could be completely abolished by in vitro treatment of beta2-deficient cells with neutralizing anti-{alpha}4beta7 Ab before adoptive transfer, suggesting that this integrin plays a major role in beta2 integrin-independent migration into peripheral LN (5) (Fig. 2). Similarly, the migration of lymphocytes into the mesenteric LN is also considerably impaired under these experimental conditions.


Figure 2
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  		FIGURE 2. beta7 integrin partially rescues the homing capacity of beta2 integrin-deficient cells. Lymphocytes were isolated from wild-type, beta2- or beta7-deficient donors, labeled with CFSE and mixed with equal numbers of TAMRA-labeled wild-type cells. At 60 min after transfer, the homing capacity of beta7 integrin, beta2 integrin, or beta2 integrin-deficient cells that were additionally treated with a neutralizing anti-{alpha}4beta7 mAb was analyzed and compared with wild-type cells. The homing efficiency is shown as the ratio of homed TAMRA/CFSE-labeled cells. pLN, peripheral LN; MLN, mesenteric LN; SPL, spleen. Results depicted are representative of three experiments with four recipients each.

 
Adoptive transfer experiments using CFSE- and TAMRA-labeled lymphocytes from wild-type and various gene-targeted mice, respectively, were conducted using untreated and FTY720-treated wild-type recipients. Interestingly, in FTY720-treated recipients beta2 integrin-deficient cells were significantly more effective in their homing to peripheral LN compared with untreated controls (Fig. 3A). Furthermore, the homing of beta7-deficient cells into mesenteric LN and PP could be restored by FTY720 treatment almost to the efficiency observed for wild-type cells (Fig. 3B). Further analysis showed that the homing of CD4+ and CD8+ beta2- or beta7-deficient T cells were affected equally by FTY720 treatment (data not shown). To test whether FTY720-mediated homing into LN is generally independent of integrin function, beta2 integrin-deficient cells were treated in vitro with a neutralizing anti-{alpha}4beta7 Ab before adoptive transfer. Interestingly, this blockade could not be overcome by FTY720 treatment of the recipient animals (Fig. 3C), suggesting that FTY720 mediated homing to LN relies on the function of beta2 and beta7 integrins.


Figure 3
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  		FIGURE 3. FTY720 treatment in recipients mediates homing into lymphoid organs. FTY720 rescues the homing defect of beta2 integrin (A) and beta7 integrin-deficient lymphocytes (B) but not of beta2-deficient cells treated with a blocking anti-{alpha}4beta7 mAb (C). The experimental setup is the same a described for Fig. 2. The homing efficiency is shown as ratio of homed wild-type cells to mutant cells 60 min after adoptive transfer into untreated ({square}) or FTY720-treated recipients ({blacksquare}). pLN, peripheral LN; MLN, mesenteric LN; SPL, spleen. Results depicted are representative of two (A) or three (B and C) experiments with four recipients each. **, p < 0.01; *, p < 0.05.

 
Short- vs long-time effects of FTY720 on the positioning of adoptively transferred lymphocytes in spleen and peripheral LN

We subsequently analyzed the tissue distribution pattern of lymphocytes immigrating into SLO in the presence or absence of FTY720 under short- and long-time homing conditions. To this end cells were labeled with TAMRA or CFSE and adoptively transferred into the same untreated or FTY720-treated wild-type recipients in two consecutive waves: 22.5 h after the transfer of TAMRA-labeled cells (long-time) the same recipients received CSFE-labeled cells (short-time) i.v. before they were sacrificed 90 min later. Tissue sections were stained for nuclei and T cells to visualize the compartments encompassing TAMRA- and CFSE-labeled cells. In this experimental setup CFSE-labeled cells were primarily restricted to the splenic red pulp irrespective of the presence of FTY720. TAMRA-labeled cells were present in both the white as well as the red pulp of untreated recipients representing lymphocyte recirculation in equilibrium. In contrast, in FTY720-treated recipients TAMRA-labeled cells (24 h presence) were largely absent from the splenic red pulp and appeared more frequently in the white pulp compared with untreated controls (Fig. 4), indicating interrupted lymphocyte homeostasis with no more cells entering the spleen whereas those in the white pulp accumulated without exiting. In peripheral LN no differences were observed regarding the distribution of TAMRA-labeled cells (long-time) between FTY720-treated and untreated recipients. Notably, we also did not observe any accumulation of these cells (long-time) in the subcapsular sinus or the medullary cords, the putative sites of lymphocyte exit from the peripheral LN. In marked contrast, CFSE-labeled cells, which had been in the recipients for 90 min, were strikingly more numerous in peripheral LN of FTY720-treated recipients compared with untreated controls and appeared to cluster around HEV (Fig. 4).


Figure 4
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  		FIGURE 4. FTY720 does not influence the positioning of cells within spleen and peripheral LN. A total of 107 TAMRA-labeled splenocytes (red) were adoptively transferred into FTY720-treated or untreated wild-type recipients. At 22.5 h later, CFSE-labeled splenocytes (green) were transferred into the same animals, and the positioning of TAMRA- and CFSE-labeled cells was analyzed 90 min later. Dashed lines demarcate the white pulp in the spleen; scale bars represent 400 µm.

 
FTY720 increases the adherence of lymphocytes on HEV

Increased numbers of freshly transferred cells in the proximity of HEV indicated that FTY720 might exert its effect on lymphocyte homing by modifying the immigration across the HEV endothelium. We therefore quantified the putative effect of FTY720 on lymphocyte entry by evaluating lymphocyte/HEV contacts. For this purpose the number of adoptively transferred cells that were found associated with HEV was quantitatively analyzed on sections of peripheral and mesenteric LN derived from FTY720-treated and untreated recipients. Interestingly, FTY720 treatment of the recipients significantly increased the number of wild-type cells associated with HEV nearly 3-fold (Fig. 5, A and B). Increased adherence to HEV could already be observed 30 min after transfer and did not depend on the size of HEV (data not shown). Notably, we did not observe increased adherence to either CD31+ blood vessels in liver and lung or to MadCAM-1+ venules of the intestine (data not shown), suggesting that the effect of FTY720 to lymphocyte endothelium interaction is selective for HEV.


Figure 5
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  		FIGURE 5. FTY720 enhances the interaction between lymphocytes and HEV. A, The number of adoptively transferred wild-type cells adhering to HEV in peripheral and mesenteric LN was determined for untreated ({square}) or FTY720-treated recipients ({blacksquare}). B, Microscopic images demonstrate adherence of wild-type cells (red) on HEV that were visualized with anti-MECA79 Ab (white). No differences were observed between HEV in mesenteric and peripheral LN for wild-type cells. C, The number of adoptively transferred beta7 integrin-deficient cells adhering to HEV in mesenteric LN was determined for untreated ({square}) or FTY720-treated recipients ({blacksquare}). D, Microscopic images demonstrate increased homing of beta7 integrin-deficient cells into MLN of FTY720-treated recipients and increased adherence of beta7 integrin-deficient cells (red) on HEV stained with MECA79 Ab (white). Bars represent the mean of two to three experiments with four to six recipients each. Scale bars represent 100 µm (B) and 400 µm (D). **, p < 0.01.

 
To determine whether increased adherence to HEV could also account for the improved immigration efficacy of integrin-deficient cells in LN of mice under FTY720 regimen, we subsequently analyzed the influence of FTY720 administration on the number of integrin-deficient lymphocytes associated with HEV. In analogy to the pattern observed for wild-type cells, we also noted an increased number of HEV-associated beta7 integrin-deficient cells in FTY720-treated recipients when compared with control mice. In the mesenteric LN of untreated recipients, only few beta7 integrin-deficient cells were occasionally observed on HEV (0.5 ± 0.5 cells/mm HEV). In marked contrast, FTY720 treatment significantly raised the number of HEV-associated cells (2.0 ± 0.4 cells/mm HEV), suggesting that in the presence of FTY720 beta7 integrin-deficient cells are also more effectively recruited to the HEV (Fig. 5, C and D). We thus conclude that the increased homing capacity of integrin-deficient cells in FTY720-treated recipients correlates well with their potential to adhere to HEV, which subsequently might induce transmigration.

FTY720-mediated adherence depends on G{alpha}i

Expression of S1P receptors has been reported on lymphocytes as well as on endothelia, suggesting that FTY720 could exert its effects on both cell types (11). Therefore, we tested whether pertussis toxin (PTX) blocking the signal transduction of G{alpha}i protein-coupled receptors would affect FTY720-mediated adherence to HEV. Lymphocytes were either incubated for 2 h in medium alone or in medium supplemented with 5 ng/ml PTX and subsequently labeled with CFSE or TAMRA, respectively. Immunofluorescence analysis revealed that PTX-treated cells could neither migrate into the white pulp of the spleen nor into peripheral LN irrespective of the presence of FTY720 in the recipients (Fig. 6A). In addition to this finding, PTX-treated cells failed to adhere to HEV of FTY720-treated recipients in vivo. Thus, signaling via G{alpha}i protein-coupled receptors in lymphocytes plays an essential role in the modulation of the increased adherence to HEV and lymphocyte homing in FTY720-treated animals.


Figure 6
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  		FIGURE 6. In vitro treatment of lymphocytes with PTX or FTY720. A, Wild-type lymphocytes were either mock-treated (green) or treated with 5 ng/ml PTX for 2 h in vitro (red), labeled with CFSE or TAMRA, respectively, and adoptively transferred into untreated or FTY720-treated recipients. Analysis was performed 90 min after transfer. Homing of PTX-treated cells into the splenic white pulp and into peripheral LN was abrogated in both untreated and FTY720-treated recipients. B, Lymphocytes isolated from peripheral LN of FTY720-treated (green) or untreated mice (red) were adoptively transferred into wild-type recipients. In vivo pretreatment with FTY720 did not affect the positioning of cells within the spleen or peripheral LN. Scale bars represent 400 µm.

 
FTY720 has been reported to mediate a rapid and long lasting internalization of S1P receptors on lymphocytes, decreasing their chemotactic activity toward S1P but not toward chemokines (23). We therefore analyzed the effect of FTY720 pretreatment of lymphocytes on their homing efficiency into SLO, their adherence to HEV, and their localization within SLO following adoptive transfer. To this end, splenocytes were either treated in vitro with FTY720 (100 ng/ml for 2 h or 16 h at 37°C) or mock-treated and then labeled with CFSE or TAMRA, respectively. A total of 107 TAMRA- and CSFE-labeled cells each were injected into naive or FTY720-treated wild-type recipients. After 90 min or 16 h, the ratio of CFSE to TAMRA fluorescent cells present in peripheral LN, mesenteric LN, spleen, and PP was determined by flow cytometry. We failed to observe any difference in the organs analyzed in regard to the ratio of both cell populations (data not shown). In addition, we performed similar experiments with cells isolated from peripheral LN or spleen of untreated mice or from mice that had received FTY720 treatment for 3 days. Again, this in vivo pretreatment with FTY720 did not affect the homing efficiency of lymphocytes that were adoptively transferred to untreated recipients (data not shown). Furthermore, we were not able to observe significant differences in the number of FTY720-experienced and control lymphocytes adhering to HEV (Fig. 6B) in this experimental setup. Thus, neither in vitro nor in vivo pretreatment of lymphocytes with FTY720 provokes conspicuous effects regarding the localization within lymphoid organs or the balance of lymphocyte homing to lymphocyte egress.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The immunomodulatory drug FTY720 essentially contributed to our understanding regarding the role of S1P receptors in the egress of lymphocytes from lymphoid organs. In this study we show that FTY720 also profoundly affects the homing of lymphocytes to LN. In the presence of this drug lymphocytes were more effectively recruited to the LN HEV irrespective of the presence or absence of beta2 or beta7 integrin. In conjunction with the observation that sustained adherence of lymphocytes driven by FTY720 correlates with their homing efficiency, our results indicate that FTY720 administration abrogates the biased use of beta2 or beta7 integrins in the homing of lymphocytes to particular SLO. However, adherence and homing were abolished once both the beta2 integrin and the beta7 integrin pathway are blocked. This demonstrates that FTY720 activates the known mechanisms for cell attachment operating via integrins instead of stimulating alternative pathways.

The observed activities of FTY720 were sensitive to PTX and thus depend on G{alpha}i signaling on lymphocytes most likely by blocking S1P receptor controlled signaling pathways. FTY720 has been suggested to act as a noncompetitive antagonist that induces the internalization of surface S1P receptors and therefore abolishes S1P signaling (9, 23). Ligand induced cyclical modulation of S1P1 surface expression has been proposed to be critically involved in determining lymphoid organ transit time (27). Yet lymphocytes pretreated in vitro or in vivo with FTY720 homed like control cells when adoptively transferred into recipient mice, and FTY720 failed to imprint an increased adherence capability to HEV. This result is in apparent conflict with reports showing that FTY720 mediated a long lasting S1P receptor internalization in vitro thereby rendering cells insensitive to signaling via these receptors (23). However, in vivo an enduring effect of FTY720 on S1P receptor surface expression might require a continuous presence of the drug. In addition this experimental setup neglects any potential contribution of an endothelia-derived FTY720 effect to the observed phenomena (see below).

A general obstacle in our understanding of S1P-driven biological mechanisms and their susceptibility to FTY720 diversion relates to the fact that S1P exerts pleiotropic effects. Virtually any cell type inside an organism expresses a characteristic pattern of S1P receptors, therefore possessing a specific S1P response signature. Although lymphocytes predominantly express S1P1 and S1P4, the EC repertoire includes S1P1, S1P2, and S1P3 yet varies depending on the endothelial subtype (28). The integrated signal triggered by the interaction of S1P with its diverse receptors present on the same cell will, for example, activate the small GTP proteins Rho, Rac, and Cdc42 to varying extents, wherefore the effect of S1P (or FTY720) may radically differ depending on the cell type or status (14). This effect is illustrated by the finding that immature dendritic cells (DC) possess a S1P receptor expression fingerprint different from that of mature DC. In immature DC, S1P-mediated signaling favors a Rho governed, sessile phenotype, whereas mature DC up-regulate S1P1 and S1P3 expression, shifting the S1P signaling toward Rac dominated cell motility, an effect that is in part reverted by FTY720 (29). In addition, processes like lymphocyte homing are composite events including the participation of different cell types (lymphocyte adhering to EC) and/or consist of several consecutive steps (lymphocyte immigration: adherence to HEV and diapedesis). Importantly we failed to observe any effect of FTY720 on the adherence of lymphocytes to ICAM-1, VCAM-1, or MadCAM-1 in in vitro adherence assays (data not shown), suggesting that FTY720 is not sufficient to increase the affinity of lymphocytes resident integrins to these ligands. A working hypothesis accounting for the effects of FTY720 on lymphocyte homeostasis should therefore take into consideration that upon administration FTY720 acts on both lymphocytes and EC. In lymphocytes, down-regulation of S1P1 may provoke a preponderance of Rho dominated activities by lowering the counterbalancing Rac influence coupled to S1P1 signaling. This result might enhance the propensity for attachment by decreasing a Rac driven disengagement tendency. An increased lymphocyte/endothelium interaction probability or time might provide an opportunistic setting now allowing several integrin-ligand interactions to operate in the establishment of lymphocyte contacts to HEV. This result may explain why FTY720 rescues attachment and homing defects of beta2- or beta7-deficient lymphocytes to the cognate SLOs. In contrast, FTY720 cannot prevent the collapse of homing when both beta2 and beta7 integrin are rendered nonfunctional. Therefore, FTY720 can only exploit the existing pool of integrins for its activities, whereas it might engage other than usual counter receptors on HEV for integrin-based binding of lymphocytes (see below).

On behalf of the endothelia, it seems necessary to discriminate HEV from afferent lymphatics and the sinus-lining EC inside the SLO. It was observed that FTY720 causes reinforcement of endothelial barrier function by promoting adherence junction assembly (30). This requires the redistribution of adhesive receptors on EC surfaces. Therefore, FTY720 might impede transgression of any cell at any EC layer, thereby contributing to the observed block of lymphocyte egress from SLO. However, FTY720 may also induce relocalization of adhesion molecules such as junctional adhesion molecule family members to the apical cell surface. Because these adhesion receptors participate in both adhesion and transmigration (31), they might assist bound lymphocytes in more than mere attachment, i.e., in diapedesis. If correct, this assumption would predict that such adhesion receptors are expressed in a HEV-specific fashion because increased adherence could not be observed on any other EC structure (see Results). Interestingly, FTY720 has been shown to act on endothelial HUVEC leading to the up-regulation of candidate genes that might facilitate interaction with lymphocytes in vivo such as ICAM-1 (32). In favor of transmigration HEV-based barriers tend to leak more compared with a regular EC layer due to the lesser extent of adherence junctions formed between the cells. Therefore, transmigration of stochastically attached lymphocytes across HEV might already be effective at a modest level of interaction between lymphocytes and the HEV.

All in all, FTY720 supposedly exerts in part opposing effects with respect to lymphocyte homeostasis by 1) lowering the threshold for lymphocyte adherence to the HEV and/or broadening the repertoire of lymphocyte expressed integrins competent to contribute to adhesion, 2) providing HEV-specific receptors that also support transmigration, and 3) raising the EC and HEV barrier function by reinforcing adherence junction formation. As a net result lymphocyte immigration might be slightly impaired or supported by FTY720 administration depending on the particular SLO analyzed (33). Regarding the dynamic nature of lymphocyte homeostasis, there is no need to radically change the parameters controlling this process. The S1P/S1P receptor system is exquisitely suitable for such fine-tuning due to the panel of S1P receptors expressed by each cell type in varying extent and composition. Thus already subtle changes such as the differing down-regulation kinetics of S1P1 triggered by S1P and FTY720, or the fact that FTY720 does not affect S1P2, may suffice to provoke such a striking phenotype like lymphopenia.

Although we provide strong in vivo evidence that FTY720 facilitates homing of lymphocytes to LN, it is currently unclear how this observation relates to the inhibition of cell egress from the same organs. Anatomical egress is accompanied by the entrance of lymphocytes into medullary cords that represent the exit point of LN and, like immigration, egress requires penetration of an EC barrier (34, 35). Injection of neutralizing anti-integrin Abs did not reveal any role for integrins in lymphocyte egress from SLO (27, 36), indicating a fundamental difference between lymphocyte immigration into and exit from a SLO. In contrast, our recent finding showing that egress of newly generated plasma cells from peripheral, but not mesenteric LN entirely depends on the expression of beta2 integrin, demonstrates that this integrin plays a substantial role in cell egress from distinct SLO (37). This finding suggests that in analogy to "postal codes" that determine LN homing, similar combinations of signals, at least consisting of S1P receptors and integrins, control the cell type-specific egress from lymphoid organs. Considering the profound biological differences between naive and Ag-activated lymphocytes regarding the presence of functional microenvironments, it might be even more important to control egress of activated lymphocytes from LN rather than selectively controlling the entry of naive cells.

In conclusion, our data establish that besides its well-renowned effect on lymphocyte egress, FTY720 also influences the process of lymphocyte immigration. We suggest that FTY720 treatment will not activate alternative homing mechanisms because homing under FTY720 remains obliged to those integrins well known to confer attachment of lymphocytes under standard conditions. By extending the retention time/probability of the lymphocytes on the HEV surfaces, FTY720 treatment rather seems to shift the mechanistic details within and also the balance between the stages comprising the canonical multistep model. Moreover, FTY720 pretreated lymphocytes fail to evoke any of the observed phenomena typical for this drug in adoptively transferred mice. This result suggests that FTY720 acts on both lymphocytes and endothelia simultaneously to be effective in vivo.


   Acknowledgments
 
We thank V. Brinkmann for providing FTY720, Karin Scharffetter-Kochanek for beta2 integrin-deficient mutants, and Niklas Czeloth and Lars Ohl for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Deutsche Forschungsgemeinschaft Grant SFB566-A14 (to O.P. and R.F.) and Forschergruppe Grant FOR 471/2 (to W.M.). Back

2 Address correspondence and reprint requests to Dr. Reinhold Förster, Institute of Immunology, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. E-mail address: foerster.reinhold{at}mh-hannover.de Back

3 Abbreviations used in this paper: SLO, secondary lymphoid organ; EC, endothelial cell; HEV, high endothelial venule; LN, lymph node; DC, dendritic cell; PP, Peyer’s patch; PTX, pertussis toxin; S1P, sphingosine-1-phosphate. Back

Received for publication July 19, 2005. Accepted for publication November 18, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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