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Originally published online as doi:10.1189/jlb.0904487 on May 13, 2005

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(Journal of Leukocyte Biology. 2005;78:471-480.)
© 2005 by Society for Leukocyte Biology

Sphingosine 1-phosphate receptor agonist FTY720-phosphate causes marginal zone B cell displacement

Kalpit A. Vora*,1, Elizabeth Nichols*, Gene Porter*, Yan Cui*, Carol Ann Keohane*, Richard Hajdu*, Jeffery Hale{dagger}, William Neway{dagger}, Dennis Zaller* and Suzanne Mandala*

* Departments of Immunology and
{dagger} Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey

1 Correspondence: Department of Immunology, Merck Research Laboratories, 126 East Lincoln Avenue, P.O. Box 2000, Rahway, NJ 07065. E-mail address: Kalpit_vora{at}merck.com


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ABSTRACT
 
FTY720 is an immunosuppressive agent that modulates lymphocyte trafficking. It is phosphorylated in vivo to FTY720-phosphate (FTY-P) and binds to a family of G protein-coupled receptors recognizing sphingosine 1-phosphate (S1P) as the natural ligand. It has previously been reported that FTY-P blocks egress of lymphocytes from the thymus and lymph nodes, resulting in peripheral blood lymphopenia. We now report that FTY-P also causes displacement of marginal zone (MZ) B cells to the splenic follicles, an effect that is similar to that observed after in vivo administration of lipopolysaccharide. This effect is specific to B cells in the MZ, as treatment with FTY-P does not cause redistribution of the resident macrophage population. A small but statistically significant decrease in the expression of ß1 integrin on MZ B cells was observed with FTY-P treatment. The redistribution of MZ B cells from the MZ sinuses does not abolish the ability of these cells to respond to the T-independent antigen, trinitrophenol-Ficoll. It has been proposed that the displacement of MZ B cells to the follicles is an indication of cell activation. Consistent with this, FTY-P caused an increase in percentage of MZ B cells expressing activation markers CD9, CD1d, and CD24. These results suggest that S1P receptors on MZ B cells are responsible for their mobilization to follicles.

Key Words: lipopolysaccharide • rodents • marginal zone • spleen


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INTRODUCTION
 
Marginal zone (MZ) B cells are a unique subset of B cells intimately associated with MZ sinuses that are poised to respond to antigens entering the systemic circulation. These cells provide an early defense against blood-borne pathogens by mounting a rapid response that may be considered a hallmark of innate immunity [1 ]. Phenotypically, MZ B cells express soluble immunoglobulin M (IgM), lack surface IgD and CD23, and express high levels of CD21, CD1d, and CD9. Functionally, this subset of B cells produces antibodies in response to polysaccharide antigens without a requirement for T cell help [2 ]. MZ B cell populations are absent in mice with targeted deletions of Pyk2, nuclear factor-{kappa}B, Aiolos, DOCK2, or Lsc [3 4 5 6 7 ]. Studies in these knockout animals have demonstrated the importance of MZ B cells in T-independent (TI) responses. For example, Pyk2 and Lsc null animals are unable to mount an immune response to TI-II antigens.

The precursors of MZ B cells are derived from the bone marrow. Baff and Baff-R null mice have a block in B cell development at the transitional B cell stage and lack the MZ and follicular (FO) B cell population [8 ]. However, the exact molecular events involved in commitment to the MZ B cell pool versus the other types of B1 or B2 B cell populations remain to be elucidated. Once MZ B cells reach the marginal sinuses, their movement toward the B cell follicle-homing chemokine, CXC chemokine receptor 5 (CXCR5), is arrested. Studies by Lu and Cyster [9 ] demonstrated that the retention of MZ B cells was mediated by expression of high levels of {alpha}Lß2 and {alpha}4ß1 integrins on MZ B cells, and the ability of these cells to migrate toward a CXCR5 gradient was unaltered. Inactivating antibodies to {alpha}Lß2 and {alpha}4ß1 caused a rapid release of B cells from the MZ and spleen. Stimulation with lipopolysaccharide (LPS) or bacterial antigen induces a redistribution of MZ B cells via the bridging channels to the edges of periarteriolar lymphatic sheath at the boundary of red pulp and follicles but does not lead to release of MZ B cells into the circulation [10 ].

Intracellular signals that guide the retention or displacement of MZ B cells are not well understood, but several regulatory components of the actin cytoskeleton have been implicated. MZ B cells are absent in mice with a targeted deletion in downstream of CRK-180 homology-2 (DOCK-2), a CED-5, DOCK 180, myoblast city (CDM) family member that functions upstream of Rac [6 ]. MZ B cells are also absent in mice deficient in murine homolog of human p115 Rho GEF (Lsc), a Rho-specific guanosine 5'-triphosphate exchange factor that down-modulates G{alpha}12 and G{alpha}13 [4 ]. Ligands of G protein-coupled receptors that are known to activate Rac and Rho and thereby regulate cell adhesion and motility include lysolipids such as sphingosine 1-phosphate (S1P).

A critical role for S1P receptors in regulating lymphocyte migration has recently been revealed by studies designed to elucidate the mechanism of FTY720 [11 , 12 ], an immunosuppressive agent that promotes allograft survival and is efficacious in animal models of autoimmune disease [11 ]. There are five known G protein-coupled receptors that bind S1P [13 ]. We found that FTY720 is phosphorylated in vivo to become a high-affinity ligand for S1P1, 3, 4, 5 but not S1P2 [12 ]. FTY720-phosphate (FTY-P) blocks emigration of thymocytes and lymphocytes from lymph nodes, resulting in peripheral blood lymphopenia and causes redistribution of lymphocytes from spleen to secondary lymph nodes [14 15 16 ]. These effects are thought to contribute to the immunosuppressive properties of FTY720. We identified a series of nonhydrolyzable phosphonate analogs and structurally divergent S1P receptor agonists and found that the ability of these compounds to cause redistribution of lymphocytes correlated with their potency on the S1P1 receptor [12 , 17 ]. Deletion of S1P1 in lymphocytes by reconstitution of mice with fetal liver-derived S1P1 null hematopoietic progenitors or T cell-specific, conditional deletion of S1P1 inhibits thymic emigration and lymphocyte recirculation, similar to agonist treatment [18 , 19 ]. The similar outcome achieved by agonist treatment and genetic deletion can be reconciled by the sustained receptor down-regulation, which is observed upon treatment with the synthetic agonists that results in functional antagonism ([20 ] and S. Mandala, unpublished results). While this manuscript was under preparation, Cinamon et al. [21 ] published elegant studies, demonstrating that S1P1 expression on MZ B cells was necessary for their localization and retention in the splenic MZ.

To characterize the immunosuppressive effects of FTY720, most studies to date have focused on T cells, although B cells are also depleted from peripheral blood, albeit to a lesser extent than T cells. In this manuscript, we describe the effects of FTY-P on splenic B cell populations. We find that administration of FTY-P leads to dramatic changes in the distribution of MZ B cells in agreement with the findings of Cinamon et al. [21 ]. We further demonstrate that FTY-P mediates displacement of MZ B cells, an effect that is similar to that caused by LPS. Moreover, the displacement of MZ B cells to the follicles is reminiscent of antigen activation and does not alter their ability to respond to TI antigen.


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MATERIALS AND METHODS
 
Immunizations and dosing
Female C57Bl/6J mice, 8 weeks old at the time of study, were obtained from The Jackson Laboratory (Bar Harbor, ME) and were housed in a specific, pathogen-free rodent facility. Mice were immunized with 50 µg antigen trinitrophenol conjugated to Ficoll (TNP-Ficoll) intraperitoneally (i.p.; Biosearch Technologies, Novato, CA) in phosphate-buffered saline, as described earlier [22 ]. The Merck Institutional Animal Care and Use Committees (Rahway, NJ) approved all animal protocols. FTY-P was synthesized at Merck and dosed at 2.4 mg/kg i.p. every alternate day, starting at day 1 until the end of the experiment on day 21. Mice were bled via the retro-orbital sinus at the indicated time, and the levels of anti-TNP antibodies and their isotypes were determined by enzyme-linked immunosorbent assay (ELISA) with a protocol described previously [23 ]. In all other experiments, FTY-P was dosed i.p. at 2.4 mg/kg, 24 h prior to analysis of spleen, unless otherwise stated. Vehicle constituted 2% hydroxypropyl-ß-cyclodextrin in water. In vivo LPS (Escherichia coli strain O26:B6, Sigma Chemical Co., St. Louis, MO) stimulation was done with i.p. injections of 50 µg/mouse. In vitro splenocytes were stimulated with 25 µg/ml LPS overnight followed by fluorescein-activated cell sorter (FACS) analysis.

Determination of peripheral blood lymphocytes (PBL)
Mice (n=3) were dosed i.p. with 2.4 mg/kg FTY-P and 50 µg/mouse LPS, and PBL were determined after 24 h as described earlier [17 ]. Mice were euthanized via CO2 inhalation. 0.5 ml blood was withdrawn by cardiac puncture into EDTA, and hematology was evaluated using a clinical hematology AutoAnalyzer calibrated for performing murine differential counts (H2000, Careside, Culver City, CA).

Immunohistochemistry
Preparation of spleens for immunohistochemistry has been described previously [24 ]. Briefly, spleens were harvested at the indicated time and were snap-frozen in optical cutting temperature compound (Fisher Scientific, Bridgewater, NJ) in a 2-methylbutane bath cooled with dry ice. Sections (5-µm thick) were stained as described earlier [24 ]. The following reagents were used to stain frozen sections: Biotinylated anti-mouse mucosal addressin cell adhesion molecule-1 (MadCAM-1; MECA-367) was purchased from BD PharMingen (San Diego, CA). Rat anti-mouse metallomacrophages (MOMA-1), rat anti-mouse CD169 (3d6.112), and rat anti-mouse macrophage receptor with collagenous structure (MARCO; ED31) were obtained from Serotec Inc. (Raleigh, NC). Rat anti-mouse ER-TR9 was purchased from Accurate Chemicals and Scientific (Westbury, NY). Mouse S1P1 was detected by affinity-purified, N-terminal, peptide-specific rabbit antiserum [17 ]. The specificity of the reaction was determined by competition with the immunizing peptide. Alkaline phosphatase (AP)-conjugated mouse anti-rat IgG, biotinylated donkey anti-rabbit (H+L), biotinylated donkey anti-rabbit (H+L) F(ab')2, and horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgM were obtained from Jackson ImmmunoResearch Laboratories (West Grove, PA). Biotinylated primary antibodies were developed with StreptABComplex/AP (Dakocytomation, Carpinteria, CA). Bound AP and HRP activities were visualized by AP substrate kit III and NovaRED substrate kit for peroxidase staining (Vector Laboratories, Burlingame, CA).

FACS analysis
Flow cytometric analysis of spleen cells was performed on the Caliber II instrument as described previously [25 ]. The splenocytes were stained with fluorescein isothiocyanate-labeled anti-CD21, phycoerythrin-labeled anti-CD23, allophycocyanin-labeled anti-CD45 (B220), biotin-labeled anti-CD9, -CD1d, -CD24, -CD80, and -CD86, and anti-ß1 integrin (clone Ha2/5; BD PharMingen). Cells were permeablized for staining the S1P1 with N- and C-terminal rabbit antiserum. The specificity of S1P1 FACS staining was ascertained by competition with the cognate immunizing peptide and using unimmunized rabbit serum at similar concentrations as that of the immune sera. The secondary donkey anti-rabbit antibody used was adsorbed against mouse serum protein. Substituting the secondary with donkey anti-rabbit F(ab')2 did not alter the staining. Relevant isotype antibodies were used as controls for all other FACS staining. All antibodies were used as specified by the manufacturer. FACS data were analyzed with CellQuestTM (BD Immunochemistry Systems, San Jose, CA) software. Absolute cell numbers were determined by multiplying percentages of FO and MZ cells by total splenic counts.


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RESULTS
 
In vivo administration of FTY720 causes rapid lymphopenia as a result of inhibition of the egress of naïve lymphocytes from secondary lymphoid organs. Lymphocytes accumulate in mesenteric and peripheral lymph nodes but are partially depleted from spleen [16 ]. A closer histological examination of spleens revealed the loss of MZ B cells from the edges of the follicles 24 h following FTY-P treatment (Fig. 1A ). The dose of FTY-P, which caused this loss of MZ B cells, was sufficient to maintain lymphopenia for 24 h (data not presented). Despite the disappearance of MZ B cells from the MZ, FACS analysis revealed that the MZ B cells were not lost from the spleen (Fig. 1B) . The identity of MZ B cells was further ascertained by their higher levels of CD9 and CD1d expression, as compared with FO B cells (Fig. 1B) . The absolute numbers of MZ B cells in the FTY-P-treated spleens were similar to the control groups. Furthermore, the FO B cell numbers were also unaltered (Fig. 1C) . These results demonstrate that a single dose of FTY-P displaces MZ B cells from their natural niche surrounding the follicles around the marginal sinus. Immunohistochemical analysis revealed clusters of brightly staining IgM+ cells appearing around the bridging channels and red pulp. These clusters did not express CD1d on their surface. The frequencies of these clusters were higher upon FTY-P treatment as compared with the vehicle-treated animals. This pattern is reminiscent of what has been observed after activation of MZ B cells with blood-borne pathogens, polysaccharide antigens, or LPS [1 , 26 27 28 29 30 ].



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  		Figure 1. FTY-P causes displacement of MZ B cells without their loss from spleen. Immunohistochemical (A) and FACS (B) analysis of spleen from C57BL/6J mice dosed 1 day earlier with 2.4 mg/kg FTY-P. Animals were killed and spleen processed for histology as described in Materials and Methods. The MadCAM-1 (blue)-positive cells are present in the MZ, defining the boundaries of follicles in the vehicle and FTY-P-treated animals. The IgM+ (red) MZ B cells are clearly visible outside this region in the vehicle-treated mice. These cells are displaced in the FTY-P-treated mice. FACS analysis of splenocytes revealed that the B220+ CD21hi CD23lo/– MZ B cells (R3 gate) are not lost from the spleen of FTY-P-treated mice (B). The MZ B cell population (R3 gate) expresses higher levels of CD9 and CD1d as compared with FO B cells [R4 gate, (B)]. The absolute numbers of marginal and FO B cells were unaltered with FTY-P treatment (C). These data are representative of three animals analyzed. Original magnification of images was 40x.

It has been reported earlier by Karlsson et al. [31 ] that macrophages control the retention and trafficking of B-lymphocytes in the splenic MZ. Disruption of Src homology 2 containing inositol-5-phosphatase-1 resulted in the loss of MZ B cells that was attributed to migration of MARCO+ MZ macrophages to the red pulp. A similar pattern was seen after infection with Staphylococcus aureus, which led to the redistribution of MZ macrophages to the red pulp and induced migration of MZ B cells into the follicle. These studies prompted us to examine the integrity of the MZ macrophage population in spleens of animals treated with FTY-P. MZ macrophage populations are comprised of diverse subsets of macrophages identified by distinct surface marker expression [32 ]. MOMA-1 staining identifies metallophilic tissue-resident macrophages that are located in the marginal sinuses surrounding the follicle at the inner side of the MZ [33 ]. ER-TR9 staining defines a subset of macrophages present in the MZ, which expresses the murine analog of the human dendritic cell-specific ICAM-3 grabbing non-integrin (SIGN-R) receptor and is implicated in the uptake of polysaccharide antigens [34 35 36 ]. MARCO staining identifies MZ resident macrophages, which express bacterial scavenger receptors and are involved in binding to several bacterial antigens [37 ]. CD169 staining identifies stromal MZ macrophages expressing sialoadhesions [38 ]. Spleen sections from mice dosed 24 h prior with FTY-P or vehicle were stained for different macrophage populations and IgM+-bearing B cells. MOMA-1 (Fig. 2 ) and MadCAM-1 (Fig. 1) stain the inner sinus of MZ at the border on the FO side and hence, are the most useful in delineating the MZ-FO boundaries. The results demonstrated that all subsets of macrophages were unaltered by treatment with FTY-P at a dose that was sufficient to cause the displacement of MZ B cells (Fig. 2) . This implies that migration of macrophages was not associated with FTY-P-induced mobilization of MZ B cells



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  		Figure 2. Macrophage subsets in MZ of spleen are not displaced with FTY-P treatment. Parallel splenic sections were obtained from spleens described in Figure 1 . MZ macrophages (blue), defined by MARCO and ER-TR9 staining, MOMA-1 metallophilic macrophages, and CD169+ stromal macrophages were not displaced by FTY-P treatment. Sections were also stained for IgM+ B cells (red). Original magnification of images, 10x. These data are representative of more than three individual animals.

FTY-P is a nonselective agonist with activity on S1P1, 3, 4, and 5. However, recent evidence using more selective agonists and studies using receptor knockout mice have implicated S1P1 as the receptor driving lymphopenia [17 , 18 , 39 ]. To determine whether FTY-P was capable of direct activation of MZ B cells, we looked for S1P1 expression on splenocytes. Spleen sections were stained with a rabbit antiserum raised against the C-terminal peptide of S1P1. As shown in Figure 3B , positive staining was seen in the FO areas along with intense staining of the MZ. Most of the S1P1 staining was confined to the lymphocyte-rich follicles with little or no staining in the red pulp of spleen. This staining was specific, as it could be competed away with cognate S1P1-immunizing peptide (Fig. 3A) . It is interesting that the loss of MZ B cells after treatment with FTY-P was associated with a partial loss of S1P1-specific staining in the MZ. S1P1 expression on MZ B cells was quantified by FACS analysis. C57Bl/6 splenocytes were stained for B220, CD21, and CD23. The cells were then permeablized and stained with N- and C-terminal S1P1-specific rabbit antiserum (Fig. 3B) . Permeabilization is required for this antiserum to stain cells. Cells gated for MZ phenotype (B220+ CD21hi CD23lo/–) revealed positive staining with S1P1-specific N- and C-terminal rabbit antiserum. This staining was reduced to background levels, similar to that obtained with nonspecific rabbit sera, upon competition with cognate peptide (Fig. 3C) . The MZ B cells expressed higher levels of S1P1 than the FO B cells (B220+ CD21+ CD23hi). Thus, MZ B cells express high levels of S1P1, as indicated by immunohistochemistry and FACS analysis. Moreover, FTY-P treatment did not alter the intracellular S1P1 staining on MZ B cells (Fig. 3D) .



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  		Figure 3. MZ B cells express higher levels of S1P1 than FO B cells. (A) C-terminal anti-S1P1 antisera stain (blue) splenic MZ B cells. This staining is not a result of binding of the rabbit antisera to splenic Fc receptors, as the specific staining is not lost with the use of 10 µg/section Fc block. The specific staining is lost upon competition with a 100x excess cognate peptide. (B) Spleen sections from vehicle- and FTY-P (as described in Fig. 1 )-treated C57BL/6J mice were stained with rabbit antiserum to the C-terminal end of S1P1 (blue). The MZ sinuses along with MZ and FO B cells demonstrated positive staining in vehicle-treated spleens. Original magnification, 40x. (C) Spleen cells from naïve C57BL/6J mice were stained for MZ B cells (B220+, CD23lo/–, CD21hi) and FO B cells (B220+, CD23hi, CD21+) for FACS analysis. The cells were then permeabilized and stained with the N- or C-terminal S1P1-specific rabbit antiserum with and without 100x excess cognate peptide. Normal rabbit serum replaced the S1P1 antiserum in control samples. It is important to note that the MZ B cells have higher levels of S1P1 expression compared with the FO B cells. (D) S1P1 staining of permeabilized MZ B cells of spleens from FTY-P- and vehicle-treated (Veh) animals. Rb sera, Rabbit sera.

Another mechanism for localization of MZ B cells as proposed by Lu and Cyster [9 ] involves integrin-mediated retention around marginal sinuses. MZ B cells express higher levels of ß1, equivalent amounts of {alpha}4, and lower amounts of ß7 integrins compared with FO B cells. Combined antibody treatment with anti-{alpha}4 and anti-{alpha}L resulted in the loss of MZ B cells from spleens and release into blood. These observations prompted us to look for alteration of expression of {alpha}4ß1 on MZ B cells upon FTY-P treatment by FACS analysis (Fig. 4 ). MZ B cells expressed higher levels of ß1 on their surfaces compared with FO B cells, confirming earlier findings. Furthermore, a small (15–20%) but statistically significant decrease in the expression of ß1 was observed with FTY-P treatment. It is interesting to note that FO B cells from the same splenic cohorts did not alter their ß1 integrin levels.



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  		Figure 4. ß1 integrin levels are decreased on MZ B cells with FTY-P treatment. FACS analysis of mice injected with 2.4 mg/kg FTY-P and stained as described in Figure 1 . Levels of ß1 integrins were determined on MZ and FO B cell populations (n=6 animals). The levels are expressed as mean florescence intensity (MFI). P values were determined by standard Student’s t-test.

Pyk-2-deficient mice demonstrate a cell-autonomous defect in the production of MZ B cells and are unable to mount a humoral response to repetitive polysaccharide antigens of the TI-II [5 ]. We used TNP-Ficoll as the prototypic TI-II antigen to ascertain the functionality of displaced MZ B cells after FTY-P treatment. Mice received FTY-P 1 day prior to antigen stimulation, and the drug treatment was continued for 3 weeks (Fig. 5 ). Levels of anti-TNP total (IgG+IgM), IgM, and IgG3 produced in control and drug-treated animals were measured weekly. The drug-treated group showed a slight trend toward lower TNP-specific antibody titers, but these differences did not achieve statistical significance. Hence, the FTY-P-induced displacement of MZ B cells did not abolish the ability of the mice to mount a TI-II humoral response.



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  		Figure 5. Displaced MZ B cells are functionally active in mounting a response to TI antigen TNP-Ficoll. C57Bl/6 animals (n=6) were immunized with TNP-Ficoll and dosed with FTY-P as described in Materials and Methods. Mice were bled at indicated times, and levels of TNP-specific total, IgM, and IgG3 isotype-bearing antibodies were determined by ELISA. The points at which the optical density (OD) curves were 50% maximal were then used to calculate the relative dilution factor, giving an equivalent OD for each serum samples.

LPS caused lymphopenia in blood similar quantitatively to that caused by FTY-P (Fig. 6 ). The levels of peripheral blood monocytes and neutrophils were unaltered with LPS or FTY-P; however, the numbers of lymphocytes (84% and 82%, respectively) were reduced (data not shown). Displacement of MZ B cells to the bridging channels and red pulp boundary with LPS or antigen stimulation is considered an indication of cellular activation [1 , 27 , 29 ]. Kinetics of this response after immunization with TI-II antigen revealed that most of the responding MZ B cells were found at the T–B cells border within 4 h, followed by migration to red pulp through the bridging channels [26 ]. CD9, a surface glycoprotein belonging to the tetraspanin family and associated with plasmablast formation, is up-regulated on MZ B cells upon activation with LPS [28 ]. The population of CD9hiB220+CD21hi MZ B cells increased significantly after in vivo treatment with LPS (14.8%) and FTY-P (16%) compared with vehicle-treated mice (9%; Fig. 7 ). FTY-P did not change the expression of activation markers CD80, CD86, major histocompatibility complex (MHC) II, and CD69 on MZ B cells. Furthermore, percentages of MZ B cells expressing CD9, CD1d, and CD24 increased upon FTY-P treatment. It is interesting to note that B cells activated by LPS up-regulate CD24 expression. This up-regulation is not observed on B cells upon CD40 stimulation [40 ]. One significant difference between LPS and FTY-P was that in vitro treatment of splenocytes with LPS increased the population of CD9hi MZ B cells (23%), but FTY-P was ineffective when tested directly on isolated splenocytes (data not shown).



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  		Figure 6. LPS caused lymphopenia similar to that seen with FTY-P treatment. C57BL/6J mice (three per group) were dosed i.p. with vehicle, 2.4 mg/kg FTY-P, and 50 µg/animal LPS. PBL were determined the next day.



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  		Figure 7. FTY-P-activated MZ B cells similar to that observed with LPS. C57BL/6J mice were treated in vivo with vehicle, FTY-P, or LPS. MZ B cells were analyzed for the presence of the activation marker (R2 gate; A) CD9 (B), CD1d (C), and CD24 (D). Four to six individual animals for in vivo vehicle- and FTY-P-treated animals are shown. The data were analyzed via a standard linear model and ANOVA. P values less than 0.05 for all tests and comparisons were deemed significant unless otherwise indicated.


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DISCUSSION
 
Chiba and colleagues [16 ] made the observation that FTY720 depletes CD4+, CD8+, and B-lymphocytes from peripheral blood and causes them to sequester in secondary lymphoid organs, but not in the spleen. Within the lymph node, antigen presentation, activation, and proliferation of T cells appear to be normal, but effector cells are unable to egress to mount a peripheral immune response [14 , 41 ]. The results presented here demonstrate that FTY-P treatment induces splenic MZ B cell displacement in a manner that is reminiscent of that seen with LPS and TI-II polysaccharide antigens. MZ are a unique, anatomical niche in spleen, where blood-borne pathogens and antigens are sampled as a result of their close proximity to MZ sinuses, the entry point for blood flowing into spleen. MZ B cells are known to mount a rapid antibody response to polysaccharides of blood-borne bacteria. Activation of MZ B cells correlates with their displacement from the MZ to the B–T cell boundary and subsequent MZ B cell plasmablast formation at the edges of bridging channels and in the red pulp [27 , 29 ]. FTY-P-mediated displacement of MZ B cells did not disrupt the integrity of MZ macrophage populations, which as cells identified by MOMA-1+, ER-TR9+, CD169+, and MACRO+ surface markers, were present and unaltered around the MZ sinuses. Recently, it has been shown that that loss of MadCAM-1+ endothelial cell integrity in MZ led to migration of MZ B cells into the follicles [42 ]. In contrast to this observation, the integrity of MadCAM-1+ endothelial cell was intact after a single dose of FTY-P, and this did not prevent the displacement of MZ B cells.

Lu and Cyster [9 ] have demonstrated that retention of MZ B cells is mediated by the integrins lymphocyte function-associated antigen-1 and very late antigen-4, which are expressed on lymphocytes and interact with ICAM-1 and vascular cell adhesion molecule-1 expressing stromal and endothelial cells. Blockade of both integrins with anti-{alpha}4 and anti-{alpha}L resulted in loss of MZ B cells from spleens and release into blood [9 ]. It is interesting that they did not observe any substantial changes in integrin surface expression on MZ B cells after FTY treatment [21 ]. In contrast to these observations, we found that the displacement of MZ B cells after FTY-P treatment was accompanied by a modest down-regulation of ß1 integrin levels on MZ B cells. Although the decrease was small (15–20%), it was significant in comparison with FO B cells, which did not show altered integrin levels.

FTY-P is a nonselective agonist on a family of G protein-coupled receptors with activity on S1P1, 3, 4, 5. Recent data from gene knockout experiments and more selective compounds have identified S1P1 as the receptor involved in regulating lymphocyte trafficking [17 , 18 , 21 ]. Consistent with these results, we observed that MZ B cells express higher levels of S1P1 protein than FO B cells. A more selective S1P1 agonist, >100-fold selective over S1P3, also caused MZ B cell displacement (data not shown), which correlated with the ability of the compound to mediate peripheral blood lymphopenia [17 ]. Based on these observations and finding that MZ B cells express higher amounts of S1P1, we conclude that displacement of MZ B cells is likely to be a direct consequence of FTY-P binding to S1P1 on the B cell. It is interesting to note that the intracellular levels of S1P1 were unaltered upon FTY-P treatment. As our rabbit antisera could not detect S1P1 expression on the cell surface, it is difficult to make arguments for receptor down-modulation from surface upon ligand engagement.

The displaced MZ B cells were functionally active, as they retained the ability to mount a comparable response to a TI-II-antigenic challenge. The effect of FTY720 on in vivo MZ B cell dose responses to physiological relevant antigens requires further study. Consistent with the results reported here, functional TI responses under the cover of FTY720 treatment have been reported recently [43 ]. It is interesting to note that the ability of FO B cells to form germinal centers after one injection of FTY-P remained unaltered (data not shown). In addition, the activation-like displacement of MZ B cells upon FTY-P treatment argues against functional impairment of these cells.

Normally, the process of plasmablast formation takes 3–4 days after bacterial antigen stimulation [27 ], whereas LPS-mediated mobilization of MZ B cells and up-regulation of the early plasmablast marker CD9 occur with more rapid kinetics [28 ]. We found that FTY-P and LPS behaved similarly in causing displacement of MZ B cells and increase in percentages of CD9-expressing cells within 24 h of treatment. It has been reported previously that B cells activated by LPS, but not by CD40, up-regulate CD24 expression [40 ]. CD1d- and CD24-expressing MZ B cells increased in response to FTY-P, but the expression of other activation markers, such as CD80, CD86, MHC II, and CD69, on MZ B cells was not altered. Earlier studies documenting in vivo effects of LPS on MZ B cells had demonstrated their migration into splenic follicles and further noted the formation of IgD plasmablasts [30 ]. In contrast, the IgM+ plasmablasts that we observed at the edges of bridging channels after FTY-P treatment lacked surface expression of IgD and CD1d (data not shown). Another significant difference between FTY-P and LPS is the failure of FTY-P to activate MZ B cells when treated ex vivo. Stromal components may be required for FTY-P to exert its full effects for in vitro activation. LPS may affect S1P1 receptor expression by transcriptional down-regulation [21 ] or by down-modulation of S1P receptor signaling [44 ]. Alternatively, LPS may alter levels of the endogenous ligand by cytokine-induced activation of sphingosine kinase, similarly to that reported in endothelial cells [45 ]. An LPS-induced increase in proinflammatory cytokines may lead to local synthesis of S1P in lymphatic cells and tissues and activation and desensitization of S1P1, which in turn would result in MZ B displacement.

LPS-induced lymphopenia has been reported earlier [46 , 47 ]. We found that LPS induced lymphopenia similar to that of FTY-P at 24 h post-dose. The depletion of PBL from circulation by LPS and FTY-P was similar at earlier time-points (data not shown). Thus, LPS-triggered signaling pathways may also inhibit S1P1 signaling with a consequent alteration in PBL trafficking. Further studies are necessary to delineate the relationship amongst LPS-mediated Toll-like receptor 4 signaling, S1P release, and S1P1 receptor activation and down-modulation.


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ACKNOWLEDGEMENTS
 
We thank Drs. Edward O’Neill and Stephen O’Keefe for critical comments and Dr. Philip Davies for valuable support and guidance. We are grateful to Richard Wnek for FACS analysis.

Received September 2, 2004; revised April 7, 2005; accepted April 8, 2005.


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