A successful execution and balance of adaptive immune responses requires a controlled positioning and navigation of dendritic cells (DC) into and inside secondary lymphoid organs. Whereas mechanisms were identified governing the migration of DC from peripheral nonlymphoid organs into their draining lymph nodes, little is known about the molecular cues controlling the proper positioning of spleen or lymph node resident DC. In this study, we show that the sphingosine-1 phosphate (S1P) receptor 1 influences the positioning of immature DC inside the murine spleen. Following treatment with FTY720 or SEW2871, drugs known to interfere with S1P1-mediated signaling, the 33D1+ DC subpopulation homogeneously redistributes from the bridging channels to the marginal zone. In contrast, the CD205+ DC subset remains associated with the T cell zone. Upon in vivo LPS treatment, the maturing DC assemble in the T cell zone. The LPS-driven redistribution occurs in the absence of CCR7 and cannot be prevented by FTY720, indicating that guiding mechanisms differ between immature and mature DC. Along with the observed DC subtype-specific S1P receptor expression pattern as well as the profound up-regulation of S1P1 and S1P3 accompanying DC maturation, these results suggest a decisive contribution of S1P signaling to intrasplenic DC motility and migration.

Dendritic cells (DC)3 possess unique properties that enable them to sample, process, and present Ag and thus represent the immune system‘s most important APCs (1). DC are important in instructing adaptive immune responses (2) as well as maintaining tolerance to self (3, 4, 5). Immature DC reside in nearby any peripheral tissue, where they constantly sample and process Ag. Microbial stimulation and inflammatory conditions causes these cells to mature and to migrate to draining lymphoid organs, where they interact with and stimulate Ag-specific T cells (6). The process of maturation is accompanied by profound phenotypic and functional changes. In addition, the maturing DC substantially modify their chemokine receptor repertoire, enabling them to migrate toward chemokines expressed by lymphatic vessels and in the T cell areas of secondary lymphoid organs (7). CCR7 and its ligands CCL19 and CCL21 play an especially important role in directing DC toward secondary lymphoid organs (8, 9). Therefore, it is assumed that the same or similar chemokines account for the positioning and migration of spleen resident DC. Splenic DC appear phenotypically immature and are located in the T cell zone, the red pulp, the bridging channels, and in the marginal (MZ) surrounding the follicle (10, 11). The splenic DC can be divided into major subsets differing in function and anatomical localization. Both CD8α+ and CD8α DC are known to efficiently present extracellular Ag on MHC class II (MHCII) and induce CD4 T cell responses (12). In contrast, only CD8α+ DC have the unique capability to present exogenously acquired Ag in the context of MHC class I (cross-presentation). They are competent to cross-prime CD8 T cells and elicit antiviral responses (13). A large part of the splenic DC resides in the bridging channels near the T cell zone (14) and minor numbers can be found in the MZ or the red pulp. All of these DC are CD8α and coexpress the protein recognized by the mAb 33D1 (14). 33D1 was recently shown to recognize the C-type lectin DCIR2 (14) which mediates Ag uptake and presentation on MHCII. In contrast, CD8α+ DC exclusively reside in the T cell zone and coexpress the marker CD205 (DEC205). Once activated by microbial stimuli, all DC rapidly migrate into the T cell zone and concomitantly acquire a more mature phenotype (15).

Sphingosine-1 phosphate (S1P) is a recently identified lipid mediator that controls egress of T and B cells from secondary lymphoid organs (16, 17). Most of its effects are caused by binding to one of the five known S1P-specific receptors, named S1P1–5. The S1P receptors belong to the family of G protein-coupled receptors and exhibit different affinities to S1P as well as unique effector functions (18). Members of the S1P receptor family are expressed in virtually all tissues, yet many of their biological functions have remained enigmatic thus far. S1P1 is the best characterized receptor of this group. Deficiency (receptor knockout) for S1P1 leads to a strong lymphopenia by inhibiting the egress of lymphocytes from thymus and lymphoid organs (17). S1P receptor deficiency may also be mimicked by the administration of agents such as FTY720 and SEW2871, which specifically interfere with S1P receptor function. Available evidence suggests that FTY720 becomes phosphorylated in vivo and interrupts S1P signaling mediated by all S1P receptors except for S1P2. It is a matter of debate as to whether FTY720 acts as an agonist or antagonist on S1P receptors. Indeed, FTY720 might represent a superagonist eliciting a short burst of S1P receptor signaling but also causing a sustained receptor internalization, thereby generating an antagonistic phenotype in the long run. Under FTY720 treatment, the block of lymphocyte egress is accompanied by their enhanced integrin-dependent adhesion to high endothelial venules (19). This may support lymphopenia by guiding peripheral lymphocytes into secondary lymphoid organs (19). These characteristics qualified FTY720 as an immunosuppressive drug, greatly facilitating transplant tolerance by silencing alloreactivity to grafted tissue (20). SEW2871 differs from FTY720 by blocking only S1P1 activity, thus allowing us to address the function(s) of this receptor more precisely. Just like FTY720, SEW2871 binding to S1P1 in vivo resembles the phenotype of receptor deficiency with regard to lymphocyte trafficking.

S1P1 function has also been shown to be crucial for various other biological activities such as vascular development (21) and positioning of MZ B cells in the MZ (22). However, the role of S1P1 for the migration of DC has not been studied extensively so far. We have previously shown that murine DC generated in vitro acquire the ability to migrate to S1P upon maturation (23). The general importance of a physiologically active S1P/S1P receptor system was affirmed in vivo by the finding that the migration of maturing DC from skin or lung to their corresponding draining lymph node could be inhibited by FTY720 treatment. In this study, we show that the positioning of immature DC in the spleen is altered by a disrupted S1P1 activity caused by FTY720 or SEW2871. In contrast, the LPS-induced assembly of matured DC in the T cell areas cannot be influenced by FTY720 treatment. The FTY720 triggered redistribution of immature DC and the migration of mature DC inside the spleen was also observed in CCR7-deficient mice. These data suggest that intrasplenic positioning of immature DC requires active S1P signaling and may operate unrestricted in the absence of CCR7-based pathways.

FTY720 was a gift from V. Brinkmann (Novartis). SEW2871 was purchased from BIOMOL and LPS was purchased from Sigma-Aldrich.

Cells were analyzed on a FACSCalibur or LSR II (BD Biosciences) after staining with the following mAbs: allophycocyanin-labeled anti-CD11c, biotinylated anti-MHCII (1Ab), biotinylated anti-ICAM-1 (CD54), purified anti-MAdCAM-1 (clone Meca-367; BD Biosciences), PE-labeled anti-CD86, PE-labeled anti-CD40 (Immunotech), purified anti-CD11c (Invitrogen Life Technologies), purified/biotinylated anti-DC 33D1 (eBioscience), purified anti-DEC-205 (CD205, clone NLDC-145, produced in-house), FITC-labeled anti-αL integrin (CD11a), FITC-labeled anti-β1 integrin (CD29), FITC-labeled anti-β2 integrin (CD18) (BD Pharmingen and BD Biosciences), and PE-labeled anti-α4β7 integrin (Sigma-Aldrich). As secondary reagents, Cy5-labeled mouse anti-rat (Dianova), Cy3-labeled mouse anti rat, Cy3-labeled streptavidin (Jackson ImmunoResearch Laboratories), PerCP-labeled streptavidin (BD Pharmingen and BD Biosciences), Cy5-labeled streptavidin (Invitrogen Life Technologies), and Alexa Fluor 594-labeled goat anti-hamster IgG (Molecular Probes) were used. For some histological stainings, the tyramide amplification system was used (PerkinElmer). Cell sorting was performed on a FACSAria (BD Biosciences). Nuclei of cells were stained with 4′,6-diamidino-2- phenylindole. Composite histology micrographs were taken on an Axiovert200M (Zeiss) equipped with a motorized table and an Orca ER camera (Hamamatsu). Micrographs were taken using the Axiovision software (Zeiss) and subsequently brightness, contrast, and γ correction were adjusted using Adobe Photoshop. All composite micrographs were taken at room temperature with a ×10 magnification and a numerical aperture of 0.55. Histological samples were prepared on Histobond-coated microscopic slides from Marienfeld laboratory glassware (Lauda-Königshofen) and mounted with Mowiol (Polysciences).

Eight- to 12-wk-old C57/BL6 mice were purchased from Charles River Breeding Laboratories. CCR7 (9)-, ICAM-1-, and MAdCAM-1- deficient mice were backcrossed to a C57BL/6 background. MAdCAM-1-deficient mice were generated by W. Müller and will be described in detail elsewhere. Cryosections of spleens from wild-type and MAdCAM-1-deficient mice were stained with anti-MAdCAM-1 Ab, and the absence of such stain in the spleen of knockout animals confirmed their allele status (data not shown). ICAM-1/MAdCAM-1 double-deficient animals were on a mixed C57BL/6/SV129 background. Animals were kept under specified pathogen-free conditions. Animal care and experiments were done in compliance with institutional guidelines and the German law for Welfare of Laboratory Animals.

Mice were treated orally (by gavage) with 40 μg/mouse per day FTY720 in H2O or with 400 μg/mouse SEW2871 in PBS/10% Tween 20/10% DMSO/10% ethanol at the indicated time points. Drug effectiveness was confirmed by analyzing lymphopenia induction in treated animals. For experiments applying SEW2871, control mice were treated with diluent alone. However, an effect of diluent application on splenic architecture or DC positioning was never observed (data not shown). For integrin α4 blocking studies, 500 μg of anti-α4 integrin Ab (clone PS/2) was injected i.v. 6 h before FTY720 gavage. To induce in vivo maturation of DC, mice were injected i.p. with 50 μg of LPS (serotype E26:B6) in PBS.

Total RNA was prepared using the Absolutely RNA Microprep Kit (Stratagene) according to the manufacturer’s protocol. RNA was reverse transcribed (Superscript II reverse transcriptase; Invitrogen Life Technologies) using random hexamer primers. The expression of GAPDH, S1P1, S1P2, S1P3, S1P4, and S1P5 was analyzed using a Lightcycler 2.0 (Roche) and the Fast Start DNA Master plus SYBR Green Kit (Roche) or the Sybr Premix Ex Taq Kit (Takara). The following primer pairs were used: S1P1-S: 5′-tct ctga cta tgg gaa cta tg-3′; S1P1-AS: 5′-cca gga tga ggg aga tga c-3′; S1P2-S: 5′-cct taa ctc act gct caa tcc-3′; S1P2-AS: 5′-gct gga aga tag gac aga cag-3′; S1P3-S: 5′-aca agg tcc ggg tgc tga g-3′; S1P3-AS: 5′-gta atg ttc ccg gag agt gtc-3′; S1P4-S: 5′-gct atg ccc att gtc cag tag-3′; and S1P4-AS: 5′-gga cca ggt act gat gtt cat g-3′. Standardization and absolute relative quantification of expression levels was previously described (23). Additionally, the Mm_Edg8_1_SG QuantiTect Primer Assay (Qiagen) was used to determine expression levels of S1P5.

We have previously demonstrated that DC generated in vitro from bone marrow express a distinct pattern of S1P receptors depending on their state of activation. Immature DC were found to express S1P2–4 but little to none S1P1. Upon stimulation, these immature DC were induced to mature and concomitantly up-regulated expression of S1P1 and S1P3, whereas the levels of S1P2 and S1P4 remained unchanged (23). Remarkably, this switch in S1P receptor repertoire correlated with the capacity of the matured cells to migrate in vitro in response to S1P, whereas immature cells lack such activity. Moreover, immature/mature skin-derived DC exert the same characteristic switch in their S1P receptor expression profile. Once more the up-regulation of S1P1 and S1P3 is associated with the migration of the maturing DC (23) into the draining lymph node.

In continuation of our earlier work, we were interested in studying the impact of S1P receptors on DC biology in vivo. We focused on DC residing in secondary lymphoid tissue. Following FTY720 application, we monitored the localization of DC inside peripheral lymph nodes (pLN) and the spleen by microscopic inspection of cryosections stained with CD11c, CD3, and MHCII (Fig. 1, A and B, and data not shown). No apparent changes in the DC distribution pattern in pLN were evident in mice treated with FTY720 even though subtle rearrangements of the DC might have escaped detection. In contrast, we noticed a conspicuous redistribution of CD11c+ cells inside the spleen of animals fed with FTY720 (Fig. 1,A). The FTY720 borne redistribution comes into effect rather quickly because the ring-shaped arrangement of CD11c+ cells was detectable 12 h after initial application of the drug. Moreover, this effect was reversible upon withdrawal of the drug (data not shown). The rather rapid and reversible kinetics favor the idea that this intrasplenic relocalization of DC was caused by cells already residing in the spleen and not by newly immigrating cells differentiating into DC. Indeed, FTY720 treatment exerted no influence on the percentage of DC found in the spleen (Fig. 1,C). It is remarkable that a pronounced effect of FTY720 was observed in spleen and not in pLN, considering the fact that pLN contain a more diverse array of DC subpopulations compared with spleen. Spleen diverges from pLN by possessing a distinct anatomical feature, the MZ (24). The MZ represents an area surrounding the white pulp and is known to be the home of MZ B cells, a subpopulation of B cells largely found only in spleen (22). Moreover, the MZ overlaps in an asymmetric fashion with the so-called bridging channels, a structure believed to allow the passage of lymphocytes coming from the red pulp on their travel to the white pulp (25). By microscopic inspection it was already evident that a large part of the splenic DC aggregates in the bridging channels and relocates uniformly throughout the MZ under the FTY720 regimen, thereby surrounding the follicle (Fig. 1A). The colocalization of these DC with ICAM-1 confirms their distribution to the MZ overlapping with the outer border of the MAdCAM-1- positive marginal sinus (Fig. 1, B and D). At the same time, the clustering of the DC in the regions of the bridging channels disappeared under FTY720 influence.

FIGURE 1.

Displacement of splenic DC into the MZ after treatment with FTY720. Mice were treated or left untreated for 3 days with 40 μg/day FTY720. A and B, Redistribution of DC in the spleen following FTY720 treatment. A, Composite micrographs of splenic cryosections from untreated (untreated) or FTY720-treated (FTY720) mice were prepared and stained with anti-CD3 (blue) and anti-CD11c (green). Bar, 200 μm. B, Cryosection of spleens stained with anti-CD11c (green), anti-ICAM-1 (red), and anti-MHCII (blue) as indicated. C, FTY720 does not alter the number of DC within the spleen nor the percentage of CD8α+ DC. Spleens from FTY720-treated and untreated animals were analyzed by flow cytometry for the percentage of CD11c+MHCII+ DC (left), and the percentage of CD8α+ cells within the CD11c+ MHCII+ population has been determined (right; pooled data from three independent experiments). D, Cryosection from spleens of untreated or FTY720-treated mice were stained with anti-MAdCAM (red) and anti-CD11c (green). Note the close proximity of marginal sinus lining endothelial cells (MAdCAM-1+) and CD11c+ DC in the case of FTY720 treatment.

FIGURE 1.

Displacement of splenic DC into the MZ after treatment with FTY720. Mice were treated or left untreated for 3 days with 40 μg/day FTY720. A and B, Redistribution of DC in the spleen following FTY720 treatment. A, Composite micrographs of splenic cryosections from untreated (untreated) or FTY720-treated (FTY720) mice were prepared and stained with anti-CD3 (blue) and anti-CD11c (green). Bar, 200 μm. B, Cryosection of spleens stained with anti-CD11c (green), anti-ICAM-1 (red), and anti-MHCII (blue) as indicated. C, FTY720 does not alter the number of DC within the spleen nor the percentage of CD8α+ DC. Spleens from FTY720-treated and untreated animals were analyzed by flow cytometry for the percentage of CD11c+MHCII+ DC (left), and the percentage of CD8α+ cells within the CD11c+ MHCII+ population has been determined (right; pooled data from three independent experiments). D, Cryosection from spleens of untreated or FTY720-treated mice were stained with anti-MAdCAM (red) and anti-CD11c (green). Note the close proximity of marginal sinus lining endothelial cells (MAdCAM-1+) and CD11c+ DC in the case of FTY720 treatment.

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Because FTY720 specifically affects S1P signaling (26), we subsequently analyzed the pattern of S1P receptors expressed by splenic CD11c+MHCII+ DC (Fig. 2). Similar to the results obtained for skin DC, we found a generally low level of expression in real-time PCR analyses. However, among all S1P receptors, S1P1,4,5 are expressed most prominently, whereas S1P2- and S1P3-specific signals were rather low yet significant. Although splenic DC express more than one S1P receptor and FTY720 is known to inhibit the activity of all S1P receptors except that of S1P2 (26), we made use of the drug SEW2871, recently shown to interfere exclusively with S1P1-mediated cellular activities (27). We therefore repeated the experiments described above and analyzed the DC localization pattern in spleens of mice treated with SEW2871. The observed effect of SEW2871 on DC localization was identical to that triggered by FTY720 (Fig. 3). Furthermore, FTY720 was not found to interfere with the maturation stage of the DC (based on flow cytometric analysis of the activation markers CD80, CD86, CD40; data not shown). This indicates that the mis-localization of the DC into the MZ is caused by interrupted S1P1 signaling and not by epiphenomena like the onset of maturation (or altered integrin expression; see later) possibly prompted by the application of the drug.

FIGURE 2.

Regulation of S1P receptors on DC in the spleen under inflammatory conditions and influence of the S1P receptor-specific drug FTY720 on maturation-induced migration of splenic DC. A, Immature DC (MHCII+CD11chigh) and in vivo-matured DC (MHC IIhighCD11chigh) were sorted from spleen of untreated animals (immature) or animals that had been injected with LPS (mature) 6 h before sorting. B, RNA was prepared from sorted cells shown in A and analyzed for the expression of S1P receptor mRNA by quanitative RT-PCR. (mean +SD; n = 3 independent experiments). C, Mice were left untreated or treated for 3 days with FTY720 to induce redistribution of DC into the MZ. Subsequently, the mice were injected with LPS for 6 h. Cryosections were stained with anti-CD11c (green) and anti-ICAM-1 (red). Note that FTY720 treatment had no effect on the positioning of in vivo-matured spleen DC.

FIGURE 2.

Regulation of S1P receptors on DC in the spleen under inflammatory conditions and influence of the S1P receptor-specific drug FTY720 on maturation-induced migration of splenic DC. A, Immature DC (MHCII+CD11chigh) and in vivo-matured DC (MHC IIhighCD11chigh) were sorted from spleen of untreated animals (immature) or animals that had been injected with LPS (mature) 6 h before sorting. B, RNA was prepared from sorted cells shown in A and analyzed for the expression of S1P receptor mRNA by quanitative RT-PCR. (mean +SD; n = 3 independent experiments). C, Mice were left untreated or treated for 3 days with FTY720 to induce redistribution of DC into the MZ. Subsequently, the mice were injected with LPS for 6 h. Cryosections were stained with anti-CD11c (green) and anti-ICAM-1 (red). Note that FTY720 treatment had no effect on the positioning of in vivo-matured spleen DC.

Close modal
FIGURE 3.

Localization of DC within the bridging channels depends on S1P1. Mice were left untreated or where gavaged with the S1P1-specific drug SEW2871 (SEW). After 6 h cryosections of spleens were stained with anti-CD11c (green) and anti-CD3 (blue; bars, 200 μm)

FIGURE 3.

Localization of DC within the bridging channels depends on S1P1. Mice were left untreated or where gavaged with the S1P1-specific drug SEW2871 (SEW). After 6 h cryosections of spleens were stained with anti-CD11c (green) and anti-CD3 (blue; bars, 200 μm)

Close modal

Since the vast majority of the spleen resident DC are rather immature, we made use of LPS to induce their maturation. To this end, animals were injected with a low dose of LPS and their spleens were analyzed 6 h later. As expected, LPS induced migration of the CD11c+ DC to the T cell zone of the white pulp in wild-type animals, indicating DC maturation (15). Concomitantly, CD11c+MHCIIhigh DC were sorted and subjected to S1P receptor analysis by real-time PCR (Fig. 2,A). The LPS-matured DC showed a strong up-regulation of S1P1 as well as of S1P3, with the S1P1 signal predominating. In contrast, S1P2,4,5 levels remained unchanged (Fig. 2,B). This parallels the results obtained earlier with skin-derived DC (23). The LPS stimulation in vivo was also performed with mice pretreated with FTY720. Upon staining splenic sections from treated and untreated animals, it was evident that in both cases the DC had relocalized to the T cell zone in an indistinguishable fashion (Fig. 2 C). This suggests that LPS- driven maturation overrides the FTY720-triggered MZ localization. However, it also overrules the original positioning of the DC in the bridging channels, indicating that S1P signaling has a direct impact on positioning of most of the immature DC but not mature DC. Despite a considerable up-regulation of S1P1,3 expression, apparently other guiding mechanisms dominate DC positioning following LPS application.

Upon closer inspection of the DC stained in the splenic sections, it was apparent that not all DC redistributed to the MZ following FTY720 or SEW2871 treatment. A smaller proportion of the cells settled in the T cell zone already before FTY720 application and a similarly intense CD11c stain remained detectable there following FTY720 exposure (Fig. 1,A). The splenic DC subpopulation residing in the T cell follicle was identified earlier to represent CD8+CD205+ DC, whereas those lodging in/near the bridging channels belong to the CD8CD4+33D1+ subtype (14). We consequently stained sections of FTY720-treated mice using the two discriminatory markers 33D1 and CD205 (Fig. 4, A and B). When compared with untreated spleen, we found that exclusively the 33D1+ DC but not the CD205+ DC moved into the MZ upon FTY720 influence. The latter remained in the T cell zone even if a subtle T zone-internal movement may have occurred. Thus, inactivation of S1P1 signaling triggered a DC subtype- specific mobilization into the MZ. We were therefore interested in exploring the S1P receptor signature characteristic for the two subtypes under scrutiny. Splenic DC were sorted according to Fig. 4 C and analyzed by real-time PCR. Interestingly, only receptors S1P1 and S1P2 were found to be expressed differentially by the two DC subtypes, whereas S1P3,4,5 levels were identical. Because FTY720 treatment remained ineffective regarding a displacement of CD205+ DC, their even higher S1P1 level appears irrelevant for their retention in the T cell zone. FTY720 does not inhibit S1P2-mediated signaling (26). Therefore, it is possible that this particular S1P receptor, whose expression is elevated in these cells compared with 33D1+ DC, may contribute to the unaltered positioning of CD205+ DC inside the T area. However, because 33D1+ DC also express S1P2, this idea is difficult to reconcile with the observation that 33D1+ DC chose the MZ as destination instead of the T cell zone under an otherwise identical FTY720 treatment. Since a long-term treatment with FTY720 ranging over several days did not change the distribution pattern compared with that found after 12 h either, it is unlikely that the CD205+ DC received insufficiently high amounts of the drug to elicit an effect (data not shown). These observations would suggest that CD205+ DC obey another guiding/adhesion system keeping them in the T cell area and thus rendering them unresponsive to any effects on their S1P signaling with regard to cellular localization.

FIGURE 4.

Differential effect of FTY720 on different splenic DC subpopulation and their expression of S1P receptors. A and B, Spleen sections from untreated and FTY720-treated animals were stained with anti-B220 (blue) and Abs to CD205 (A) or 33D1 (B). Note that FTY720 treatment affects the positioning of 33D1+ but not CD205+ DC. C, Sorting strategy for 33D1+ and CD205+ DC from spleen. D, RNA was prepared from sorted cells and mRNA levels of S1P receptors were analyzed by real-time PCR. mRNA levels are depicted as fold GAPDH. Each data point represents the value of individual mRNAs of three independent experiments.

FIGURE 4.

Differential effect of FTY720 on different splenic DC subpopulation and their expression of S1P receptors. A and B, Spleen sections from untreated and FTY720-treated animals were stained with anti-B220 (blue) and Abs to CD205 (A) or 33D1 (B). Note that FTY720 treatment affects the positioning of 33D1+ but not CD205+ DC. C, Sorting strategy for 33D1+ and CD205+ DC from spleen. D, RNA was prepared from sorted cells and mRNA levels of S1P receptors were analyzed by real-time PCR. mRNA levels are depicted as fold GAPDH. Each data point represents the value of individual mRNAs of three independent experiments.

Close modal

The primordial importance of the integrin system in migration and localization of immune cells is well documented. For instance, it has been previously reported that ICAM-1 in combination with VCAM-1 is required for keeping MZ B cells within the MZ, whereas a dependency on either integrin ligand was not found (28). To check for the expression of integrins on DC and integrin ligands on splenic stromal cells, we analyzed the respective molecules histologically and by flow cytometry. ICAM-1 was strongly expressed throughout the MZ and the T cell zones of the spleen, whereas MAdCAM-1 was present along the sinus lining endothelial cells in the marginal sinus (Fig. 5,A). Overlapping expression of MAdCAM-1 and ICAM-1 can be found in the marginal sinus (Fig. 5,A, inset). DC in the spleen express integrins that mediate adhesion to ICAM-1 (integrin αLβ2) and MAdCAM-1 (integrin α4β7 or α4β1). These integrins are present in high levels on the surface of the DC and their expression remains unaltered under FTY720 treatment (Fig. 5,B). To test whether the observed delocalization phenomenon of 33D1+ DC depends on distinct integrin ligands, we treated ICAM-1-deficient and MAdCAM-1- deficient animals with FTY720. Neither deficiency influenced the redistribution of the 33D1+ DC subset in the corresponding mice treated with FTY720 (Fig. 6, A and B). However, in mice double deficient for ICAM-1 and MAdCAM-1, the DC were no longer equally distributed throughout the MZ even if the FTY720 effect could not be reverted entirely in the double-deficient mice. Accumulations of DC in the bridging channels (Fig. 6,C, white arrowheads) were still present, confirming incomplete mobilization of DC by FTY720 treatment. The double deficiency for ICAM-1 and MAdCAM-1 may have unexpected side effects such as an altered architecture of the splenic MZ. Therefore, we made use of an Ab blocking integrin α4, thus integrin α4 interactions with MAdCAM-1 and VCAM-1 were suppressed. However, when investigating the positioning of splenic DC in ICAM-1- deficient mice treated with this Ab along with FTY720, we observed a phenotype identical to that detected in the ICAM-1/MAdCAM-1-deficient animals (Fig. 6 D). Taken together, these data point to a role for ICAM-1 and MAdCAM-1 driving the DC toward the MZ. Probably because of the known redundancies in the integrin/cell adhesion molecule (CAM)-adhesion system, we failed to observe complete suppression of the FTY720 effect on DC even in ICAM-1/MAdCAM-1 double-deficient mice. Other ligands such as VCAM-1 may therefore also play a role in these processes.

FIGURE 5.

Integrin ligands are expressed in the spleen marginal sinus while the corresponding integrins are expressed on splenic DC. A, Spleen sections were stained with anti-ICAM-1 (green) and anti-MAdCAM-1 (red). Insets show magnification of single integrin ligand expression and merge of ICAM-1 and MAdCAM-1 expression. Bar, 200 μm. B, Integrin expression on CD11chighMHCII+ DC from spleens of untreated and FTY720-treated animals (shown are representative results of at least two animals per group analyzed).

FIGURE 5.

Integrin ligands are expressed in the spleen marginal sinus while the corresponding integrins are expressed on splenic DC. A, Spleen sections were stained with anti-ICAM-1 (green) and anti-MAdCAM-1 (red). Insets show magnification of single integrin ligand expression and merge of ICAM-1 and MAdCAM-1 expression. Bar, 200 μm. B, Integrin expression on CD11chighMHCII+ DC from spleens of untreated and FTY720-treated animals (shown are representative results of at least two animals per group analyzed).

Close modal
FIGURE 6.

FTY720-induced distribution of DC in the spleen depends on the presence of integrin ligand. Mice deficient for ICAM-1 (A), MAdCAM-1 (B), or double deficient for ICAM-1 and MAdCAM-1 (C) were left untreated or treated orally with FTY720 for 2–4 days. Spleen sections were stained with anti-CD11c (green) and anti-CD3 (blue). Note that in FTY720-treated mice double-deficient for ICAM-1 and MAdCAM-1 the distribution of DC to the MZ is strongly reduced with DC still residing in the bridging channels (arrowheads in C; representative micrographs from three (A and B) or at least nine animals (C) per group). D, Additionally ICAM-1-deficient mice were treated with blocking anti-α4-integrin Abs 6 h before FTY720 gavage. Spleen sections of untreated (top), FTY720-treated (middle), and Ab plus FTY720-treated mice (bottom) are shown. B cells (B220) are shown in red and DC (CD11c) in green. Blocking of MAdCAM-1 interactions in ICAM-1−/− mice with the anti-α4 integrin Abs resembles the phenotype observed in ICAM-1/MAdCAM-1 double-deficient animals (bar, 200 μm, representative of at least three mice per group are shown).

FIGURE 6.

FTY720-induced distribution of DC in the spleen depends on the presence of integrin ligand. Mice deficient for ICAM-1 (A), MAdCAM-1 (B), or double deficient for ICAM-1 and MAdCAM-1 (C) were left untreated or treated orally with FTY720 for 2–4 days. Spleen sections were stained with anti-CD11c (green) and anti-CD3 (blue). Note that in FTY720-treated mice double-deficient for ICAM-1 and MAdCAM-1 the distribution of DC to the MZ is strongly reduced with DC still residing in the bridging channels (arrowheads in C; representative micrographs from three (A and B) or at least nine animals (C) per group). D, Additionally ICAM-1-deficient mice were treated with blocking anti-α4-integrin Abs 6 h before FTY720 gavage. Spleen sections of untreated (top), FTY720-treated (middle), and Ab plus FTY720-treated mice (bottom) are shown. B cells (B220) are shown in red and DC (CD11c) in green. Blocking of MAdCAM-1 interactions in ICAM-1−/− mice with the anti-α4 integrin Abs resembles the phenotype observed in ICAM-1/MAdCAM-1 double-deficient animals (bar, 200 μm, representative of at least three mice per group are shown).

Close modal

The outcome of the S1P receptor signaling with respect to cellular localization may be modulated by the chemokine/receptor system. Indeed, MZ B cells losing their S1P1-mediated signaling were displaced from the MZ to the B cell zone via CXCR5 activity (22). Accordingly, this redistribution was found to be absent in cells deficient in CXCR5 (data not shown) or its ligand (22). CCR7 and its ligands CCL19/CCL21 are part of the most important chemokine receptor/ligand system for the maintenance of structures in lymph nodes and spleen (9). CCL21 is expressed in the T cell zones of the spleen (data not shown) and, among the defects observed, CCR7 deficiency or a lack in the ligands (plt/plt) causes a less well-organized T cell area in the spleen (Fig. 7). However, domains distinguished by accumulations of T cells still exist along with the follicular areas. The distribution pattern of the DC also appears much less structured when compared with wild-type spleen. Although many DC locate to the T domains, others are found to be scattered throughout the nonfollicular areas. To test its effect on DC localization, we treated CCR7−/− and plt/plt mice with FTY720. The bulk of the CD11c+ DC distributed homogeneously throughout the ICAM-1-expressing MZ surrounding the follicle (Fig. 7 and data not shown), whereas a minor fraction remained associated with the T cell domains, a pattern highly reminiscent of that observed in wild-type mice upon FTY720 application. Therefore, we conclude that the FTY720-driven MZ allocation of 33D1+ DC as well as the retention of DEC205+ DC in the T cell domains is independent of CCR7.

FIGURE 7.

Mobilization of DC into and out of the MZ are independent of CCR7. Spleen sections of CCR7-deficient mice were stained with anti-CD3 (red), anti-CD11c (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue). Mice were either untreated (A), treated with FTY720 for 3 days (B), or treated for 3 days with FTY720 and subsequently injected with LPS for 6 h (C). Representative results from at least four mice per group are shown.

FIGURE 7.

Mobilization of DC into and out of the MZ are independent of CCR7. Spleen sections of CCR7-deficient mice were stained with anti-CD3 (red), anti-CD11c (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue). Mice were either untreated (A), treated with FTY720 for 3 days (B), or treated for 3 days with FTY720 and subsequently injected with LPS for 6 h (C). Representative results from at least four mice per group are shown.

Close modal

It was hypothesized that CCR7 signals guide DC into the T cell zone following microbial stimulation. Therefore, CCR7−/− mice were first treated with FTY720 and subsequently with LPS. Untreated CCR7−/− mice that were injected with LPS do not show gross alterations in DC distribution within the spleen (data not shown). This observation may be explained by the fact that due to the less organized splenic architecture in CCR7-deficient mice, larger aggregations of DC serving as landmarks are missing. Interestingly, when the mice were treated with FTY720 before LPS injection, nearly all DC were found associated with the T cell domains, whereas the MZ was devoid of any CD11c stain (Fig. 7). Thus, as in wild-type animals, the FTY720 effect is overruled by the LPS-induced maturation and migration. More importantly, because already CD205+ DC reside in T domains and also the mature 33D1+ DC find their way to the T cell domains in the absence of CCR7, it is assumed that hitherto unresolved chemoattractant mechanisms control the intrasplenic migration and positioning of immature and mature DC.

The importance of S1P signaling for the migration and positioning of DC has not been studied intensively so far. Previous work suggested an important role for S1P in the mobilization of DC from skin (23) and lung (29) to draining lymph nodes. We detected that the DC maturation program in vitro as well as in vivo encompassed up-regulation of S1P1 and S1P3 and assumed that the concomitant activation of the small G proteins Rac/Cdc42/Rho provided the stimulated cell with sufficient motility to abandon their sessile stage (23). In the present study, we provided evidence that a subpopulation of splenic immature DC, the 33D1+ DC, require S1P-mediated signaling via S1P1 to maintain their subanatomical location in/near the bridging channels. Disruption of S1P1 signaling due to FTY720 or SEW2871 treatment causes the immature 33D1+ DC to leave their position in the bridging channels and to distribute equally into the MZ. We are not aware of any other report describing a similar finding for other immature DC populations residing either in the periphery or inside secondary lymphoid tissue. Remarkably, immature CD205+ as well as 33D1+ DC express a similar pattern of S1P receptors, yet only the positioning of the latter DC subtype seems to depend on S1P receptor activity. We hypothesize that the withdrawal of incoming S1P signals predominantly via S1P1 loosened their anchoring to the adhesive support provided by the bridging channels even though the nature of the adhesive system accomplishing this remains to be determined. The cells start to drift and subsequently halt at cells expressing ICAM-1/MAdCAM-1 and additional adhesion receptors, thus causing the observed redistribution of 33D1+ cells to the MZ. A similar scenario may apply for the CD205+ DC. Although they apparently fail to respond to FTY720- triggered S1P receptor inactivation, they already reside in an area of detectable expression of ICAM-1 (and probably others). The juxtaposed CAM ligand expression in the T cell zone may thus provoke the impression of unresponsiveness.

It is considered unlikely that a chemoattractive system guides the 33D1+ DC to the MZ. Although it was shown that 33D1+ DC express chemokine receptors, these cells failed to migrate in vitro to all chemokines tested (30) as well as to S1P (data not shown). This applies also to the CD205+ DC and indicates that the immature splenic DC are ignorant to migratory stimuli, probably because the apparatus needed for migration is discontinued. This is in line with the concept that immature DC should stay where they are and move on only when Ag uptake and transport are required. But this also raises the question how these cells arrive at all at their home base in the bridging channels or T cell zones. The turnover rate of intrasplenic DC is rather high and the existing DC are replaced by new ones (10, 31). Most likely, the DC precursors are recruited from the blood or an intrasplenic pool of precursors (32), migrate to the corresponding locations, and differentiate into immature DC on site, acquiring a nonmigratory phenotype. Only upon further differentiation into mature DC do the cells regain migratory potential. As a first step, motility is enhanced by up-regulating S1P1 and perhaps S1P3. Complementary to this, the cells gain responsiveness to chemokines or other attractants navigating their active migration. The rather low expression level of S1P receptors observed in immature DC and their subsequent up-regulation in maturing DC as a feature shared by many DC subtypes (at least all studied thus far) would lend further support to this idea.

The pronounced reallocation of the DC involving the MZ is reminiscent of the published effect of FTY720 on MZ B cells. However, whereas DC settle the MZ, MZ B cells leave that zone and migrate into the B cell follicle when animals receive FTY720. The FTY720 effects under discussion demonstrate that S1P is a critical factor in keeping these cells at their original subanatomical locations. Indeed, MZ B cells express S1P1 and S1P3 apart from marginal amounts of S1P4 and it was shown that S1P1 is critically responsible for the retention of MZ B cells in their perifollicular localization inside the MZ (22). With their rather high levels of S1P1 and S1P3, MZ B cells resemble the mature DC that just up-regulated the levels of these receptors. In addition, MZ B cells are able to respond to chemokine signals just as mature DC can. This illustrates that the context of the S1P receptor-mediated effects matters: cell type, original location, adhesion receptor repertoire, maturation stage, general responsiveness to chemokines, and more. All of these factors combine to create an array of different responses and may help to explain why one cell type leaves the MZ whereas the other settles the same zone under otherwise identical external conditions. In this context, it is also of importance to consider that several cell types inside the spleen express S1P receptors. Apart from DC and MZ B cells, endothelial cells express S1P1, rendering them susceptible to FTY720. Thus, a combined effect of several S1P receptor-positive cell types may drive the observed DC relocalization. However, it would be conceivable that this preferentially affects the destination of the shifting DC rather than the fact that they start moving after all. The latter is caused most likely by DC intrinsic effects triggered by functional loss of S1P1.

Remarkably, MZ B cell migration to S1P in vitro is mediated via S1P3 signaling, whereas their localization in vivo depends on S1P1 activity (22). Similarly, it was recently reported that migration of mature DC generated in vitro also depends on a functional S1P3 receptor (33), whereas the results presented here provide evidence that the positioning of 33D1+ DC in the bridging channels requires S1P1 activity. Thus, in both cases, cell retention may rely on S1P1, whereas migration is triggered via S1P3. Because the physical elements overlap considerably, it is difficult to assess each receptor‘s contributions to either positioning or migration. Positioning is predominantly dependent on adhesive/sessile features, whereas migration requires a mix of reiterating steps where adhesive/sessile attributes alternate with motile elements. Since it is known that S1P receptors differentially activate small G proteins responsible in large parts for these activities (34, 35, 36, 37, 38, 39), it is conceivable that combined signaling from two or more S1P receptors contributes to migration as well as positioning. Withdrawal of either receptor activity may then cause an imbalance in the activities of Rac/Cdc42/Rho (35, 40). Moreover, the net result “migration” is subject to many contributions because CAMs as well as chemokine receptor signaling is known to influence components required for and contributing to migration (41, 42, 43, 44, 45, 46, 47, 48).

It was surprising to observe that the maturation-induced migration of intrasplenic DC was accomplished in the absence of CCR7 (Fig. 7). This contrasts with the absolute necessity of functional CCR7 for the immigration of maturing DC from the periphery into their draining lymph nodes (8). Currently, the driving force underlying the intrasplenic migration of mature DC into T cell domains in the CCR7-deficient mice is unknown. Although it is widely accepted that CCR7 contributes substantially to this process in the wild-type scenario (49), our results suggest that alternative routes exist which can fully compensate the CCR7 deficiency at least in the migratory aspects under discussion. Whether the responsible factors attracting mature DC are produced by T cells, the splenic stroma cells, or both remains to be determined. S1P seems not to be involved, since FTY720 treatment could not prevent maturation-induced DC redistribution.

We are indebted to V. Brinkmann for providing FTY720.

The authors have no financial conflict of interest.

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 has been funded by a Deutsche Forschungsgemeinschaft Grant (SFB566-A14) to R.F. and a Deutsche Forschungsgemeinschaft Grant (Forschergruppe Grant f 471/2) to W.M.

3

Abbreviations used in this paper: DC, dendritic cell; S1P, sphingosine-1 phosphate; MZ, marginal zone; CAM, cell adhesion molecule; pLN, peripheral lymph node.

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