The Sphingosine‐1‐Phosphate Receptor Agonist FTY720...

来自 : 发布时间：2024-04-28

Free Access The Sphingosine-1-Phosphate Receptor Agonist FTY720 Modulates Dendritic Cell Trafficking In Vivo Yuk Yuen Lan, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorAn De Creus, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorBridget L. Colvin, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorMasanori Abe, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorVolker Brinkmann, Novartis Institutes for Biomedical Research, Basel, SwitzerlandSearch for more papers by this authorP. Toby H. Coates, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorAngus W. Thomson, Corresponding Author Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA*Corresponding author: Angus W. Thomson, thomsonaw@upmc.eduSearch for more papers by this author Yuk Yuen Lan, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorAn De Creus, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorBridget L. Colvin, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorMasanori Abe, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorVolker Brinkmann, Novartis Institutes for Biomedical Research, Basel, SwitzerlandSearch for more papers by this authorP. Toby H. Coates, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USASearch for more papers by this authorAngus W. Thomson, Corresponding Author Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA*Corresponding author: Angus W. Thomson, thomsonaw@upmc.eduSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat AbstractThe pro-drug FTY720 is undergoing phase III clinical trials for prevention of allograft rejection. After phosphorylation, FTY720 targets the G protein-coupled –sphingosine-1-phosphate receptor 1 (S1PR1) on lymphocytes, thereby inhibiting their egress from lymphoid organs and their recirculation to inflammatory sites. Potential effects on dendritic cell (DC) trafficking have not been evaluated. Here, we demonstrate the expression of all five S1PR subtypes (S1PR1–5) by murine DCs. Administration of FTY720 to C57BL/10 mice markedly reduced circulating T and B lymphocytes within 24 h, but not blood-borne DCs, which were enhanced significantly for up to 96 h, while DCs in lymph nodes and spleen were reduced. Numbers of adoptively transferred, fluorochrome-labeled syngeneic or allogeneic DCs in blood were increased significantly in FTY720-treated animals, while donor-derived DCs and allostimulatory activity for host naïve T cells within the spleen were reduced. Administration of the selective S1PR1 agonist SEW2871 significantly enhanced circulating DC numbers. Flow analysis revealed that CD11b, CD31/PECAM-1, CD54/ICAM-1 and CCR7 expression on blood-borne DCs was downregulated following FTY720 administration. Transendothelial migration of FTY720-P-treated immature DCs to the CCR7 ligand CCL19 was reduced. These novel data suggest that modulation of DC trafficking by FTY720 may contribute to its immunosuppressive effects.Introduction The novel immunomodulatory pro-drug FTY720 is a structural analogue of ISP-1 (myriocin), a fermentation product of the ascomycete Isaria sinclairii (1, 2). FTY720 = 2-amino-2-[2-(4-octylphenyl-ethyl)]-1,3-propane-diol hydrochloride (C19H33NO2:HCl) represents the first member of a new class of immunosuppressants. Unlike conventional immunosuppressive agents, it does not inhibit T or B cell activation or proliferation, or their effector function (3, 4). FTY720 causes rapid and dramatic depletion of circulating lymphocytes, but not granulocytes or monocytes, within 2–6 h of intake (5). Mature lymphocytes are sequestered in peripheral and mesenteric lymph nodes and intestinal Peyer\'s patches, leading to decreases also in lymphocytes in the thoracic duct lymph and spleen (6, 7). The sequestration is completely reversible and infused labeled lymphocytes reappear in the blood after drug withdrawal (4, 7, 8). FTY720 prolongs rodent heart and skin allograft survival, and acts synergistically with cyclosporine to suppress canine and nonhuman primate kidney graft rejection (9-12). It has proved a promising anti-rejection agent in clinical renal transplantation (13, 14) and is currently undergoing phase III clinical trials. The exact mechanism(s) underlying the influence of FTY720 on leukocyte trafficking and its immunosuppressive action are still poorly understood. A recent theory suggests that FTY720 activates G-protein-coupled sphingosine-1-phosphate receptors (S1PRs), known previously as endothelial differentiation gene (Edg) receptors, stimulation of which promotes leukocyte migration (1, 8, 15). Upon binding sphingosine-1-phosphate (S1P), these receptors act downstream on the small GTPases Cdc42, Rac and Rho, and evoke a S1PR-dependent activation of Cdc42, Rac and Rho that initiates cytoskeletal rearrangements that determine leukocyte morphology and motility (16-18). FTY720 is phosphorylated in vivo to its active metabolite FTY720-phosphate (FTY720-P), a structural homolog of S1P that binds to four of the five S1PRs (S1PR1, 3, 4, 5) in vitro (1, 8). In vivo, FTY720-P acts as a high-affinity agonist at S1PR1 on lymphocytes, thereby inducing aberrant internalization of the receptor, rendering the cells unresponsive to S1P, and depriving them of an obligatory signal to egress from lymphoid organs (19). Consequently, FTY720 inhibits egress of lymphocytes from secondary lymphoid organs (SLO) into efferent lymph and blood, thereby preventing recirculation of auto- and allo-aggressive effector T cells to peripheral inflammatory sites. To date, there have been few reports concerning potential effects of FTY720 on dendritic cells (DCs). DCs are uniquely well equipped bone marrow (BM)-derived migratory antigen-presenting cells that induce and regulate immune responses (20-24). There is recent evidence (25, 26) that human DCs express S1PR, and that S1P and FTY720 or FTY720-P induce changes in human DC migration, cytokine production profile, and T cell stimulatory function in vitro. In the present study, we have investigated the influence of FTY720 and the selective S1PR1 agonist SEW2871 on blood-borne and secondary lymphoid tissue DCs in mice. The data suggest that FTY720 administration can impair DC migration from blood and downregulate the expression of key intercellular adhesion molecules and the CC chemokine receptor CCR7 required for DC transendothelial migration and homing to SLO. These effects on DCs may contribute to the immunosuppressive action of FTY720. Male C57BL/10J (B10; H2b) and C3H/HeJ (C3H; H2k) mice, 8–12 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the specific pathogen-free Central Animal Facility of the University of Pittsburgh. Experiments were conducted in accordance with the National Institutes of Health Guide for use and care of laboratory animals and under an Institutional Animal Care and Use Committee–approved protocol. Mice were injected with a single i.p. dose of 3.5 mg/kg FTY720 (Novartis, Basel, Switzerland) or with 20 mg/kg SEW2871 = 5-(4-phenyl-5-trifluoromethylthiophen-2-yl)-3-(3-trifluoromethylphenyl)-(1, 2, 4)-oxadiazole (Calbiochem, La Jolla, CA). FTY720 was dissolved in distilled water and FTY720-P (Novartis) in DMSO/50 mM HCl, then diluted to the appropriate concentrations. The selective S1PR1 agonist SEW2871 was dissolved in DMSO before dilution to the appropriate concentration. The DC poietin recombinant (r) human fms-like tyrosine kinase 3 ligand (Flt3L) (chinese hamster ovary cell–derived), used to mobilize DCs, was a gift from Amgen (Seattle, WA). Recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 were gifts from Schering-Plough (Kenilworth, NJ). Complete medium (CM) comprised RPMI-1640 (BioWhittacker, Walkersville, MD) supplemented with 10% v/v fetal calf serum (FCS; Nalgene, Miami, FL), nonessential amino acids, l-glutamine, sodium pyruvate, penicillin-streptomycin and 2-mercaptoethanol (all Life Technologies, Gaithersburg, MD). BD PharM Lyse® NH4Cl lysing reagent, Annexin V-FITC apoptosis detection kit and all anti-mouse monoclonal antibodies (mAbs) except PE-anti-CCR7 (BioLegend, San Diego, CA) were obtained from BD PharMingen (San Diego, CA). Vybrant™ CFDA SE cell tracer kit was purchased from Molecular Probes (Eugene, OR). Lympholyte-M® was purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). Collagenase IV and Escherichia coli lipopolysaccharide (LPS; serotype 026:B6) were obtained from Sigma (St. Louis, MO). Magnetic microbeads and separation columns were from Miltenyi Biotech (Auburn, CA). Trizol reagent was purchased from R&D Systems (Minneapolis, MN) and the Advantage™ RT-for-PCR kit from Clontech (Palo Alto, CA). Mouse r CCL19 was purchased from R&D Systems. Heparinized blood collected from pairs of mice was pooled. Samples (200 μL) were then immunostained for T cells (FITC-anti-CD3), B cells (FITC-anti-CD19), NK cells (PE-anti-NK1.1) or DCs (FITC-anti-CD11c) simultaneously with red blood cell (RBC) lysis using BD PharM Lyse®, according to the manufacturer\'s instructions. Lymph node (submandibular, axillary and inguinal) and splenic mononuclear cells were isolated as described (27), then immunostained with PE-anti-CD11c, and FITC-anti-CD8α, FITC-anti-CD3 or FITC-anti-CD19 mAbs to determine DC subsets, T-cell and B-cell populations, respectively. Following flow analysis, absolute cell number was calculated by multiplying the total leukocyte number by the percentage (%) of each population of interest. Results were expressed as percent normal control value ±1 standard deviation (SD). DCs were propagated from normal B10 mouse BM in GM-CSF+ IL-4 (28) then positively selected using anti-CD11c magnetic microbeads, as described in (29). Their purity was consistently 95%. The purified CD11c+ cells were characterized by two-color flow cytometric analysis and were CD40lo, CD80lo, CD86lo and MHC class II (IAb)lo, as shown previously (30). Bone marrow dendritic cells (BMDCs) and blood-borne DCs were positively selected using anti-CD11c bead separation, as described (29). Spleen DCs were first enriched using a 16% v/v nycodenz gradient (500g; 4°C; 20 min) before anti-CD11c bead separation. DC purity was 95%. Total RNA was extracted from the purified DCs using the Trizol method (28). cDNA was synthesized from the RNA samples with Advantage™ RT-for-PCR kit. PCR primers were: S1PR1 (681 bp): S1PR1a (5′-GGG ACA CAA TTA GCA GCT AT-3′, sense) and S1PR1b (5′-GTA GAG GAT GGC GAT GGA AAG-3′, antisense); S1PR2 (461 bp): S1PR2a (5′-TTA ACT CCC GTG CAG TGG TTT GC-3′, sense) and S1PR2b (5′-ACG ATC GTC ACC GTC TTG AGC A-3′, antisense); S1PR3 (681 bp): S1PR3a (5′-CGC ATG TAC TTT TTC ATT GGC AA-3′, sense) and S1PR3c (5′-GGG TTC ATC GCG GAC TTC AG-3′, antisense); S1PR4 (445 bp): S1PR4a (5′-GAG TCA TAC CCA CAG TTG C-3′, sense) and S1PR4b (5′-CAG TGT GAT GTT CAG CAG G-3′, antisense); S1PR5 (571 bp): S1PR5e (5′-CTA CTG CTA CAG ACT GAC G`-3′, sense) and S1PR5f (5′-GCT CTG TTT CCT CTG TAG C-3′, antisense). The PCR mix for S1PR1–3 was run 35 cycles (94°C, 30 s; 54°C, 30 s; 72°C, 2 min), with a final extension step of 10 min at 72°C. The PCR mix for S1PR4, 5 was run 35 cycles (94°C, 30 s; 58°C, 30 s; 72°C, 30 s), with a final extension step of 7 min at 72°C. PCR samples were then analyzed on 1% w/v agarose gel stained with ethidium bromide. The active metabolite of FTY720, FTY720-P, used in in vitro experiments was diluted in CM from a stock solution of 10−3 M. BMDCs were cultured in the presence of a clinically relevant concentration of 10−6M FTY720-P for 24 h. DC apoptosis was monitored by staining of phosphatidylserine translocation with FITC-Annexin V in combination with propidium iodide (PI) according to the manufacturer\'s instructions. Cells were co-stained with CD11c to allow specific analysis of DCs by flow cytometry. CCR mRNA expression by highly purified DCs was determined by RNase protection assay (RPA) as described (31). Staining of DCs for cell surface CCR7 and subsequent flow cytometric analysis was performed using anti-CCR7 mAb. Migratory responses of DCs to S1P were quantified in Transwell® (Corning Inc., Corning, NY) chemotaxis assays, as described (31). For analyzing DC transendothelial migration in response to CCL19, resting murine endothelial cells (EC; MS1, a gift from Dr T. M. Carlos, Department of Medicine, University of Pittsburgh) were layered (7.5–8 × 104) on each Transwell® filter (5 μm), 24–48 h before the addition of DCs (105), and then the chambers were incubated for 4 h. Migrated DCs were enumerated using a Coulter counter. Control or FTY720-P-treated (10−6M; 24 h), bead-purified BMDCs were labeled with CMFDA (15 μM). Then 5 × 105 cells were added to an EC monolayer (2.105) in 1.5 mL complete medium for 1.5 h at 37°C. The wells were washed gently with warm PBS, and the number of labeled DCs adherent to ECs in 10 randomly chosen high-power (×400) fields counted using a fluorescence microscope. Blood DCs from control or FTY720-treated mice, or cultured BMDCs were double-stained with PE-anti-CD11c and either FITC-anti-CD11b, -anti-CD31/PECAM-1, -anti-CD54/ICAM-1 or anti-CD62L and analyzed by flow cytometry. Freshly isolated BM cells were incubated in pre-warmed PBS containing CFSE (5 μM) for 15 min at 37°C, and washed extensively before use. Fifty million labeled cells were injected intravenously into syngeneic recipients via the lateral tail vein, 2 h after FTY720 administration. Mice were killed 18 h later. Blood samples were processed as described above, then stained with PE-anti-CD11c mAb. Flow cytometry was used to analyze the double-positive, CFSE-labeled CD11c+ population. BM cells from Flt3L-mobilized B10 mice (10 μg i.p. for 10 days) comprising 15–30% CD11c+ DCs (32) were labeled with CFSE, as described above. Similar to the syngeneic DC tracking experiment, 50 × 106 labeled BM cells were injected intravenously into either untreated or FTY720-treated allogeneic (C3H) mice. Blood and spleen samples were collected and processed as in the syngeneic cell transfer experiment. The capacity of C3H host spleen cells, harvested 24 h after i.v. infusion of 50 × 106 Flt3L-mobilized allogeneic (B10) BM cells, to stimulate proliferative responses in freshly isolated, naïve C3H splenic T cells was determined in 72 h MLR, as described (30). Significances of differences between means were calculated using the Wilcoxon–Mann–Whitney test for small samples or Student\'s t-test as appropriate. Differences between groups were considered significant at p 0.05. In the first set of experiments, we employed RT-PCR to analyze the expression of S1PR1–5 (formerly Edg-1,-5,-3,-6 and -8, respectively) (33) by freshly-isolated (immature) B10 mouse blood and spleen DCs and by in vitro propagated, immature BM-derived myeloid DCs. As shown in Figure 1A, each DC population expressed all five S1PR. Expression of each S1PR was also evident following DC maturation induced by overnight culture in the presence of the Toll-like receptor (TLR) 4 ligand, LPS, with evidence of higher expression of S1PR1–3 on mature DCs (Figure 1B,C; data shown only for BMDCs). We also examined the in vitro migratory responses of immature versus LPS-matured BMDCs to S1P using Transwell® chemotactic assays, as described (31). As shown in Figure 1D, mature BMDCs exhibited modest but significant migratory responses to S1P, corresponding to the higher expression of S1PR1–3 on these DCs. C57BL10 (B10) mouse DCs express S1P receptors. (A) RT-PCR analysis of S1P receptor (S1PR1–5; S1P1–5 in this figure) expression was performed on highly purified, freshly isolated C57BL/10 (B10) blood and spleen DCs, and on immature, bone-marrow-derived DCs (BMDCs), as described in the Materials and Methods. Data are from one experiment representative of two performed. 1Kb markers are displayed on the left. (B) both immature (i) and LPS-matured (m) BMDCs express S1PR, with evidence (C) of higher expression of S1PR1–3 on mature DCs. (D) although iBMDCs did not migrate in vitro to the S1PR ligand S1P, mBMDC displayed a modest chemotactic response, corresponding to their higher expression of S1PR1–3. Values are means ±1SD. *p 0.05. Data are from one experiment representative of three (B, C) or two performed (D). FTY720-P does not interfere with DC differentiation or affect the apoptotic death of DCs To examine its influence on DC differentiation, FTY720-P (10−6 M) was added at the beginning of BMDC cultures and replenished every 2 days. No significant influence on the numbers of DCs recovered from the cultures at 7 days (control: 4.8 ± 0.8 × 106/well; FTY720-P:4.7 ± 0.7 × 106/well) or on the expression of cell surface MHC class II (IAb) or costimulatory molecules (CD40, CD80 and CD86) was observed (Figure 2A). To ascertain whether FTY720-P might affect the apoptotic death of DCs, BMDCs were exposed to 10−6 M FTY720-P in CM (10% FCS-supplemented) for 24 h. As shown in Figure 2B, no significant influence on the incidence of Annexin V+/PI− cells, determined by flow analysis was found. When serum was removed from the medium in similar experiments, to enhance apoptotic cell death over the ensuing 72-h period, and the influence of FTY720-P reassessed, no significant effect was again observed (Figure 2B). Thus, under either steady state or apoptosis-promoting conditions, FTY720-P did not affect the death of DCs. Exposure to FTY720-P does not affect DCs differentiation or apoptosis. (A) FTY720-P (10−6 M) was added at the start of 7-day B10 BMDC cultures and replenished every 2 days. Cell surface expression of MHC class II (IAb) or costimulatory molecules, detected by flow cytometry at day 7 were unaffected. Percentages of positive cells are shown. All events were gated on CD11c+ cells. (B) BMDCs exposed to FTY720-P (10−6M) in the presence or absence of serum for the indicated time periods, show no difference in the incidence of apoptotic cells compared with untreated controls. Apoptosis of CD11c+ cells was detected by staining with FITC-Annexin V in combination with PI, as described in the Materials and Methods. Data shown are from one experiment and are representative of two performed. FTY720 administration markedly depletes circulating T and B lymphocytes, but enhances blood DCs We next examined whether systemic administration of FTY720 might affect DC trafficking in vivo. Circulating lymphocytes (T, B and NK cells) and DCs (CD11c+ NK1.1−) were analyzed, 24 h after drug administration to B10 mice, by single or two-color flow cytometry, as described in the Materials and Methods. As shown in Figure 3, and as anticipated, FTY720 administration caused substantial reductions in the incidence of circulating CD3+ T cells and CD19+ B cells in blood. By contrast, an increased incidence of blood-borne CD11c+ (NK1.1−) DCs was observed. The incidence of NK cells that did not co-express the DC-associated marker CD11c (NK1.1+ CD11c−) was unaffected. FTY720 administration depletes blood T (CD3+) and B (CD19+) lymphocytes, but not circulating DCs (CD11c+ NK1.1−) or NK cells (NK 1.1+). Blood samples were obtained from groups of untreated control B10 mice, or from animals 24 h after FTY720 administration (3.5 mg/kg). Isolated leukocytes from pooled blood were immunostained with FITC-anti-CD3, FITC-anti-CD19 or PE-anti-NK1.1 and PE-anti-CD11c mAbs, then analyzed by flow cytometry, as described in the Materials and Methods. Data shown are from one experiment, and are representative of three performed. To ascertain its influence on DCs and lymphocyte populations in SLO, lymph node and spleen mononuclear cells were obtained 24 h to 7 days after systemic FTY720 administration. Concomitant with their approximate, 2.5-fold elevation in the circulation at 24 h, that was sustained at 48 and 96 h, but no longer evident at 7 days, DC numbers were reduced modestly but significantly in the spleen (Figure 4B). Similar reductions were evident in both classic myeloid (CD11c+ CD8α−) and ‘lymphoid-related’ (CD11c+ CD8α+) DC subsets (Figure 4A). FTY720 also decreased the numbers of DCs in lymph nodes at 48 and 96 h (Figure 4B). There were concomitant reductions in the incidences of T and B lymphocytes in the spleen, whereas some increase in B cells in lymph nodes was observed (Figure 4A). FTY720 administration depletes DCs in the spleen and lymph nodes. (A) Single cell suspensions from blood, lymph nodes or spleens of groups of control B10 mice or animals 24 h after FTY720 administration (3.5 mg/kg) were prepared as described in the Materials and Methods. The cells were then immunostained with FITC-anti-CD3, FITC-anti-CD19 or PE-anti-CD11c mAbs to assay T cell, B cell and DC populations, respectively, by flow cytometry. Data were calculated as absolute cell numbers and shown as percent control values (absolute number × 100/absolute cell number in controls). DCs in spleens decreased consistently, together with T and B cells, whereas DC numbers in lymph nodes appeared unaffected at 24 h. Both CD8α+ (‘lymphoid-related’) and classic CD8α− (myeloid) DC subsets were modestly reduced. Data are mean values ±1SD obtained from three experiments performed. (B) Reduced numbers of DCs were observed in both spleen and lymph nodes, 48 and 96 h after FTY720 administration, concomitant with the increased numbers of DCs in blood. Cell numbers were restored to normal by day 7. Data are mean values ±1SD obtained from three experiments performed. *p 0.01. Greater numbers of infused syngeneic DCs are retained in the circulation of FTY720-treated mice To evaluate further the influence of FTY720 administration on circulating DCs, we infused CFSE-labeled syngeneic B10 BM cells (50 × 106) intravenously into either untreated controls, or into mice 2 h after FTY720 administration, then monitored the fate of the labeled cells. As shown in Figure 5, a higher proportion (approx. 7-fold) and absolute number (50% increase) of CFSE-labeled DCs were retained in the circulation of FTY720-treated mice compared with normal controls when examined 18 h later. At the same time, splenic CFSE-labeled DCs that trafficked to the spleen were reduced modestly, but significantly. Enhanced numbers of donor DCs are retained in the circulation of FTY720-treated mice following syngeneic BM cell infusion. Fifty million CFSE-labeled B10 BM cells were injected intravenously into each normal syngeneic recipient via the lateral tail vein, 2 h after systemic FTY720 administration (3.5 mg/kg). Pooled blood samples from control or FTY720-treated mice were collected 18 h later. Cells were stained with PE-anti-CD11c mAb for subsequent flow cytometric analysis. Results were expressed as percent control (absolute cell number × 100/absolute cell number in controls). (A) A higher incidence and (B) an approximate, 2-fold higher absolute number of double-positive, CFSE-labeled DCs were observed in blood of animals treated with FTY720. A small but significant reduction in CFSE-labeled DCs was observed concomitantly in the spleen. Data are from one experiment representative of three performed (A) or means ±1SD obtained from three separate experiments (B). *p 0.05. Greater numbers of donor DCs are retained in the blood of FTY720-treated animals following allogeneic BM transplantation: association with reduced naive T cell allostimulatory activity in host spleens Following allogeneic BM or organ transplantation, donor-derived DCs migrate via the blood to SLO and therein instigate anti-donor T cell responses (32, 34). To determine its influence on the migration of fully MHC-mismatched DCs from the blood, 50 × 106 CFSE-labeled, Flt3L-mobilized B10 BM cells were injected intravenously into normal control or FTY720-treated C3H recipients. As shown in Figure 6 A,B, FTY720 administration enhanced retention of allogeneic DCs (CD11c+) in the blood 18 h later by approx. 2-3–fold, whereas CFSE-labeled CD11c+ cells in the spleen were reduced concomitantly. The reduction in donor-derived DCs within host spleens was associated with reduced allostimulatory activity of splenocytes for naïve host T cells (Figure 6C), indicating that reduced trafficking of donor-derived DCs to the spleens of FTY720-treated mice was associated with a reduced level of T cell priming activity. Enhanced numbers of donor DCs are retained in the blood of FTY720-treated mice after allogeneic BM transplantation: association with reduced donor DCs and naïve T cell allostimulatory activity within host spleens. Fifty million allogeneic (B10; H2b), Flt3L-mobilized CFSE-labeled BM cells were infused intravenously via the lateral tail vein of untreated control C3H (H2k) mice or mice given FTY720 (3.5 mg/kg) 2 h earlier. Blood and spleen were collected 18 h later, and mononuclear cells isolated and stained with anti-CD11c mAb. (A) Dot plots show an increased incidence of labeled DCs in the blood, but not in the spleen of treated mice. Percentages of CFSE-labeled CD11c+ cells are shown. Data are from one experiment representative of three performed. (B) The absolute number of labeled DCs increased 2–3 fold in the blood, but decreased concomitantly in the spleens of FTY720-treated mice compared with controls. Percent control = absolute cell number × 100/absolute cell number in controls. Data are means ±1SD obtained from three independent experiments. *p 0.01. (C) The decrease in number of allogeneic DCs reaching the spleen of FTY720-treated recipients was associated with reduced in vitro allostimulatory activity of host splenocytes for naïve C3H T cells, as determined by MLR. Data (means ± 1SD) are from one experiment representative of two performed. *p 0.05. To ascertain whether the influence of FTY720 on blood DCs could be reproduced by a selective S1PR1 agonist, we administered SEW2871 i.p. and determined blood and SLO DC levels 6 and 24 h later. As shown in Figure 7, there were significantly increased levels of DCs in the blood of SEW2871-treated animals compared with controls at 6 h. This increase was no longer apparent at 24 h. No significant changes in splenic or lymph node DC numbers were observed following SEW2871 administration. The selective S1PR1 agonist SEW2871 increases DC numbers in the blood. B10 mice were injected i.p. with SEW2871 (20 mg/kg), then 6 h and 24 h later, CD11c+ DCs were quantified in (A) blood and (B) secondary lymphoid organs. SEW2871 significantly increased DCs in blood at 6 h, but the effect was no longer apparent at 24 h. Percent control = absolute cell number × 100/absolute cell number in controls. Data are means ±1SD obtained from three experiments. *p 0.05. To ascertain whether FTY720 might affect DC adhesion molecule expression in vivo, we treated mice with FTY720, then 24 h later, examined the expression of various cell surface adhesion molecules on blood DCs. Immunostaining with PE-anti-CD11c and either FITC-anti-CD11b, -anti-CD31/PECAM-1, -anti-CD54/ICAM-1 or anti-CD62L was performed simultaneously. As shown in Figure 8, FTY720 administration down-modulated expression of CD11b, CD31 and CD54, but no consistent effect on surface CD62L expression was observed. When we examined directly, the influence of 24 h FTY720-P pre-treatment of immature BMDCs on their subsequent adhesion to resting mouse endothelium, no significant effect was found (data not shown). FTY720 administration downregulates adhesion molecule expression on circulating DCs. Pooled blood samples from normal control or FTY720-treated B10 mice were collected 24 h after FTY720 administration (3.5 mg/kg). Immunostaining of blood leukocytes with PE-anti-CD11c and either FITC-anti-CD11b, -anti-CD31/PECAM-1, -anti-CD54/ICAM-1 or -anti-CD62L was performed simultaneously with RBC lysis, as described in the ‘Materials and Methods’ section. The level of expression (mean fluorescence intensity; MFI) of CD11b, CD31 and CD54 was reduced significantly on DCs from FTY720-treated mice compared with those from control animals. Results are means ± 1SD. The experiment was repeated three times, with similar results. *p 0.05. FTY720-P or FTY720 administration downmodulates CCR7 expression on DCs: association with reduced transendothelial migration to CCL19 Modulation of chemokine receptor expression on DCs is important in regulating migratory responses of these cells to chemokines, expressed either constitutively or in sites of inflammation (35, 36). We considered that FTY720 might affect the expression of CC chemokine receptors that affect transendothelial migration by DCs and their homing to SLO. RPA of highly purified, unstimulated BMDCs following their in vitro exposure to FTY720-P or S1P for 8 or 24 h was performed as described (31). No significant influence of FTY720-P or S1P on CCR1, 5, 6 or 7 mRNA expression was detected (data not shown). We also examined cell surface expression of CCR7 on the FTY720-P-treated BMDCs and on blood DCs, 24 h after FTY720 injection. As shown in Figure 9A, FTY720-P reduced the incidence of CCR7+ immature BMDCs (by approx. 30%) and to a lesser extent (approx. 10–15%) the incidence of CCR7+ mature BMDCs. Moreover, the percentage of blood DCs expressing CCR7 was reduced (approx. 40%) in FTY720-treated animals. The decreased expression of CCR7 on BMDCs was accompanied by a reduction in in vitro migratory responses to the CCR7 ligand CCL19 (Figure 9B). FTY720/FTY720-P downmodulates CCR7 expression on DCs: association with reduced transendothelial migration in response to CCL19. (A) Immature or LPS-activated mature BMDCs were treated with FTY720-P (10−6M) for 24 h, then stained with FITC-anti-CD11c and PE-anti-CCR7 mAbs. Blood samples from FTY720-treated B10 mice were lysed with PharM Lyse® NH4Cl lysing reagent and stained in the same way. Controls were untreated. Flow cytometric analysis reveals reduced levels of CCR7 expression on both immature and mature BMDCs and on blood DCs, following FTY720-P or FTY720 treatment, respectively. Data are representative of three separate experiments; gated on CD11c+ cells. (B) Transendothelial migration of untreated or FTY720-P (10−6M)-treated immature BMDCs in Transwell® assays. Migrated DCs were enumerated 4 h after start of incubation using a Coulter counter. FTY720-P-treated BMDCs showed reduced migration to the CCR7 ligand CCL19 at 0.1 and 10 nM. The experiment was repeated three times, each with similar results. Here, we show for the first-time that in normal animals, not only circulating T and B lymphocytes, but also numbers of blood DCs are affected by systemic FTY720 administration. Freshly isolated blood or spleen DCs, or in vitro-propagated murine myeloid DC subsets, expressed all five S1PR subtypes. This finding is consistent with the recent demonstration of S1PR1–4 expression by human monocyte-derived DCs (25, 26). Treatment of mice with a single dose of either FTY720, or the selective S1PR1 agonist SEW2871 (37), increased the number of DCs in blood. By contrast, DC subsets were reduced significantly in the spleens and lymph nodes of FTY720-treated animals. Accordingly, systemic FTY720 administration resulted in enhanced retention of adoptively transferred, unstimulated syngeneic or allogeneic DCs in the blood. Increases in blood-borne DCs were associated with downmodulation of specific cell surface adhesion molecules (CD11b, CD31 and CD54/ICAM-1) and the CC chemokine receptor CCR7. Reduction in CCR7 expression may contribute to the altered homing of DCs to SLO. Moreover, lower numbers of donor-derived DCs within the spleens of FTY720-treated mice were associated with reduced stimulatory (priming) activity for host naïve T cells in MLR, verifying the impact of altered DC migration on induction of alloimmune reactivity. DC trafficking is determined by various factors, in particular cell maturation status, cognate G-protein-coupled receptor expression, chemokine signaling and expression of cell surface intercellular adhesion molecules (38-40). In the present study, exposure of immature BMDCs (equivalent to those that circulate in the blood) to physiologic concentrations of FTY720-P did not affect their chemokine receptor (CCR1, 5, 6 and 7) mRNA expression significantly. However, cell surface expression of CCR7 on FTY720-P-treated immature BMDCs and their migratory responses to the CCR7 ligand CCL19 were reduced compared with control cells. These latter observations correlate with the increased numbers of blood-borne DCs and with the significant reductions in DC traffickings from blood to SLO in FTY720-treated mice. Thus, we consider it unlikely that DC precursors/immature DCs in the BM undergo enhanced motility/migration into the blood in response to FTY720 administration. Indeed, FTY720-P-treated immature human DCs show decreased chemotactic responses to RANTES and stromal-derived factor-1α (26), associated with a reduction in actin polymerization, a pre-requisite for cell migration. DC migration from the blood is regulated by cell surface adhesion molecules on the DCs and their ligands on ECs. CD11b/CR3 is important for DC adhesion to endothelium (41). In addition, blocking mAb against CD31/PECAM-1 inhibits DC migration across activated endothelium (42), whereas CD54/ICAM-1 mediates LFA-1-dependent monocyte migration through human umbilical vein EC (43). In the present study, blood-borne DCs from FTY720-treated animals showed reduced surface expression of CD11b, CD31 and CD54, suggesting that modulation of intercellular adhesion molecule expression may underlie/contribute to the influence of FTY720 on the blood-tissue distribution of DCs. Although we found no significant effect of FTY720 administration on CD62L expression by blood DCs, the implications of this finding are uncertain. CD62L expression is usually associated with the migratory behavior of T cells, although CD62L has been implicated in regulation of human plasmacytoid DC migration from blood to SLO (44). Our failure to demonstrate a consistent effect of FTY720-P on adhesion molecule expression by, or adherence of BMDCs to vascular endothelial cells in vitro does not exclude the possibility that, in vivo, intercellular adhesion and transendothelial migration by DCs may be affected by FTY720 administration, particularly as in vivo, both DCs and ECs are exposed to the drug. Interestingly, while expression of these intercellular adhesion molecules on FTY720- or FTY720-P-treated human DCs was not examined in the recent study of Muller et al. (26), the authors did observe reduced expression of CD18 that forms heterodimers with CD11a, b and c, which could also interfere with the migratory activity of DCs. DCs homing from blood to SLO clearly involves trafficking across multiple barriers. When circulating DC precursors fail to transmigrate, the number of DCs reaching the lymph nodes and spleen decreases. Spleen DCs have a rapid turnover [half-life, 1.5–2.9 days (45)], do not recirculate, and remain in the lymphoid tissue until they die. Thus, reduction in migration of immature DCs from blood would quickly result in DC depletion in the spleen, while recovery (following drug withdrawal) would be expected to take several days, as observed in this study. Our in vitro data also reveal that FTY720-P does not induce DC apoptosis, either under steady-state conditions, or when cell death is enhanced in vitro by serum deprivation. This makes it unlikely that (as with lymphocytes) (4), this mechanism accounts for the observed DC depletion in the spleens of FTY720-treated mice. It is tempting to speculate that FTY720-P, the principal in vivo metabolite of FTY720, may downmodulate S1PR, particularly S1PR1, on DCs, thereby depriving them of signals provided by serum S1P that might be critical for the regulation of adhesion molecule expression. Agonistic interaction of FTY720-P with S1PR on DCs could also lead to receptor internalization, particularly of S1PR1. This, in turn, could deprive DCs of an S1P signal required for migration from blood into SLO. In line with this view, it has been suggested that DCs may upregulate S1PR1 upon maturation and Ag uptake, prior to migration into SLO, and that DCs downmodulate S1PR1 once in lymphoid tissue (46). Our data indeed suggest that maturation of DCs is associated with modest upregulation of S1PR1 expression at the mRNA level. While this finding contrasts with a recent report of similar amounts of mRNA for S1PR on immature and mature human DCs (25), the apparent inconsistency may relate to the different experimental conditions employed. The need to understand the contributions of specific S1PR to the mode of action of the relatively nonspecific S1PR agonist FTY720 has become more urgent as, in clinical trials, its immunosuppressive action is accompanied by transient bradycardia (14, 47, 48). The discovery of selective S1P agonists, as exemplified by SEW2871 (37), has led to the observation that agonism of S1PR1 alone is sufficient to control lymphocyte egress/recirculation. Use of selective agonism, together with S1PR3−/− mice, has revealed that S1PR1 and S1PR3, respectively, regulate lymphocyte circulation and heart rate (37). Our data obtained using SEW2871 suggest that agonism of S1PR1 alone is also sufficient to affect DC numbers in blood and SLO, and rapidly enhances circulating numbers of these important APC. We conclude that FTY720 promotes the retention of DCs in the circulation, associated with downregulation of several cell surface intercellular adhesion molecules believed to be important for endothelial adhesion and transmigration. In addition, cell surface expression of the SLO homing receptor CCR7 was also downregulated. We propose that FTY720 induces these effects as the result of FTY720-P binding to S1PR, in particular S1PR1, that are expressed by murine DCs. As with T-cells, FTY720 may functionally inactivate/internalize S1PR1 (46), thereby affecting the migration of DCs to SLO. 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