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* Immunology and Rheumatology,
Pharmacology, and
Cardiovascular Disease, Merck and Co., Inc., Merck Research Laboratories, Rahway, NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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-catenin, and zona occludens 1 in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S1P1, whereas S1P1 was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S1P1 agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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In our initial studies on the mechanism of FTY720, no changes in lymphocyte homing rates were detected, but lymphocytes were found accumulated around the abluminal side of sinus-lining ECs in mesenteric lymph nodes (MLNs) and appeared more clustered within the parenchyma (9). Increased clustering and adhesiveness may diminish the ability of lymphocytes to move within the node and may also limit diapedesis into efferent lymphatic vessels. Moreover, we discovered that FTY720 was a pro-drug that was phosphorylated in vivo to become a potent ligand (FTY-P) for G protein-coupled sphingosine-1-phosphate (S1P) receptors, S1P1, 3, 4, 5. More recent studies using conditional knockout mice and more S1P1-selective compounds have provided evidence that the immunosuppressive and lymphocyte sequestration effects of FTY720 are mediated via S1P1 (10, 11, 12, 13). FTY-P is a full agonist on S1P1 and induces GTP
S binding to the receptor, Ca+2 flux, and chemotaxis in human ECs and cells transfected with the receptor (1, 9, 14). However, genetic deletion of S1P1 from lymphocytes induces peripheral blood lymphopenia and recapitulates many of the defects in lymphocyte migration from thymus and SLO, suggesting that FTY-P may behave as a functional antagonist (12, 13).
Many G protein-coupled receptors undergo ligand-dependent receptor internalization, and protein kinase C-dependent and S1P-dependent phosphorylation sites on the C terminus of S1P1 have been shown to regulate phosphorylation and internalization (15). Recently, it has been shown that FTY720 binding to S1P1 transfected into CD4+ T cells causes internalization and degradation of the receptor through a protein kinase C
-dependent mechanism (16). Thus, sustained desensitization and down-regulation of S1P1 on lymphocytes is proposed to be the mechanism by which FTY720 and other potent agonists alter lymphocyte recirculation. The molecular components downstream of S1P1 that are involved in lymphocyte retention have not yet been characterized, and plausible mechanisms include decreased motility, increased adhesion, or failure to interact with a putative ligand on sinus-lining endothelium required for egress.
Lymphocyte sequestration alone is unlikely to account for the ability of FTY720 to prevent graft rejection, since rejection of allogeneic skin in mouse models can occur with as few as 1 x 103 donor-specific CD8+ T cells (17), and circulating lymphocytes are not fully depleted by the drug. The function and identity of the residual lymphocytes that escape depletion is unknown, but they may be long-lived effector memory T cells. These cells lack surface receptors needed for constitutive homing to SLOs and, thus, they should not be sequestered by S1P receptor agonists. Additionally, FTY-P has been shown to cause potent and rapid improvement in disease symptoms in rodent models of autoimmune encephalitis that occur before depletion of PBLs (18). Thus, other cells may contribute to the immunosuppressive activities of FTY720. ECs are known to express high levels of S1P1, and important functions for the receptor on ECs have been well characterized, including enhanced chemotaxis, survival, and barrier function.
The formation of specialized junctional complexes at endothelial cell-cell contacts, termed tight junctions and adherens junctions (AJs), is thought to be critical to endothelial barrier regulation (19). CD31 (PECAM), cadherins, -catenin, and zona occludin-1 (ZO-1) are membrane-associated adhesion molecules that are located in junctional complexes formed between ECs (19, 20, 21). Junctional complexes also function as signaling structures that regulate cell position and growth. Phosphorylated-FTY720 and S1P promote adherens junction assembly by inducing the translocation of vascular endothelial-cadherin (VE-cadherin) to the contact sites between ECs (22, 23). These compounds also promote the translocation of -catenin to focal contact sites, where it associates with the cytoskeletal elements to form cortical actin rings that help stabilize the endothelium (24). In addition, S1P-mediated phosphorylation of N-cadherin and subsequent formation of cadherin-catenin-actin complexes strengthens nascent endothelial cell adhesion to surrounding mural cells, a process known as vascular stabilization (25).
Maintaining the integrity of the barrier prevents vascular leak, which is an important component of inflammatory states such as adult respiratory distress syndrome and ischemia-reperfusion injury (24). FTY720 and S1P protected mice from vascular leak and inflammation in a murine model of acute lung injury (26). The barrier enhancing effect of FTY-P on endothelium in vivo was demonstrated by studies showing that vascular endothelial growth factor-induced permeability of murine ear vessels could be blocked by oral administration of FTY720 (23). Likewise, S1P was shown to rapidly increase the electrical resistance across monolayers of human and bovine ECs, as well as block vascular leakage induced by thrombin (27). The observation that treatment of endothelium by either FTY-P or S1P leads to a similar enhancement in barrier integrity suggests that desensitization through S1P1 internalization does not occur with endothelium, as it does with lymphocytes.
To further elucidate the structural changes occurring within lymph nodes upon FTY720 treatment and to characterize the cellular location of S1P1, we have undertaken immunofluorescent and immunoelectron microscopic studies of MLNs. In this study, we show that three cell types important for immune function display dramatically different levels of S1P1 receptor expression and respond differentially to FTY-P treatment. S1P1 expression on lymphocytes is nearly undetectable and almost exclusively intracellular, even in the absence of agonist treatment. Lymphocyte clustering seen at the light microscopy level in FTY-P-treated MLNs is due to increased contact between intranodal cells, including homotypic (lymphocyte:lymphocyte) and heterotypic (lymphocyte:macrophage or lymphocyte:endothelium) interactions within the parenchyma. HEVs and lymphatic endothelium were intensely labeled by S1P1 Abs, and cell surface expression remained high even after FTY-P treatment. At the same time, there was a marked increase in coexpression of CD31, -catenin, and ZO-1 in ECs by FTY-P. Macrophage functions have not previously been found to be altered by FTY720. We report here the novel observation that the subcapsular sinuses appeared to be expanded in size and contained increased numbers of S1P1-positive, CD68+ macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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C57BL/6 female mice (8–10 wk of age) were purchased from Taconic Farms and housed in a specific pathogen-free animal facility. Experiments were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee.
Immunofluorescent staining (IFS) and quantitation of MLN cells
C57BL/6 female mice were administered FTY-P (3 mg/kg/day; i.p.) or an equivalent volume of vehicle, and MLNs were harvested 18 h later. In some studies, FTY-P or vehicle was given daily for 14 days. Animals were euthanized by CO2 inhalation.
Single-cell suspensions of pooled MLNs were prepared by passage through a 40-µm sieve, following 1 h of collagenase treatment (30 U/ml; Sigma-Aldrich) at 37°C. Cells were washed in PBS containing 0.01% sodium azide and 0.1% BSA (FACS buffer). All samples were adjusted to 1 ml with FACS buffer and 100 µl was removed for quantitation of unstained cells. The rest of the sample was used for IFS. One hundred microliters of 4% paraformaldehyde was added to the 100-µl aliquot of unstained cells, and each sample was counted for 30 s on a FACScan (BD Biosciences). The total number of cells in each sample was determined using CellQuest software (BD Biosciences), based upon forward and side scatter characteristics. Blank particles (Spherotech; ACBP-100-10) were used as an internal standard to control for flow differences from sample to sample. For IFS, cells were incubated for 30 min on ice with 10 µg/ml anti-mouse CD16/CD32 (anti-FcIII/II receptor; BD Biosciences) and subsequently with 10 µg/ml FITC-conjugated rat anti-murine F4/80 (clone MCA497FB; Serotec), FITC-conjugated rat anti-murine CD3 (clone 17A2; BD Biosciences), or PE-conjugated rat anti-murine B220/CD45R (clone RA3-6B2; BD Biosciences). Cells were washed three times in FACS buffer, resuspended in 4% paraformaldehyde, and analyzed on a FACScan. Permeabilization was done before IFS with rabbit anti-mouse S1P1n Ab (20 µg/ml; 30 min), using a BD Cytofix/Cytoperm kit, following the manufacturer’s instructions (BD Biosciences). FITC-conjugated goat anti-rabbit IgG (10 µg/ml) was used as the secondary Ab (Santa Cruz Biotechnology).
Data were calculated as (average cell number per node) by dividing the total number of viable cells by the number of MLNs collected. The absolute number of each cell subset analyzed was determined by multiplying the (average cell number per node) by the percentage of the subset, determined by IFS on the pooled MLN samples. The sequestration index was calculated as the ratio between the compound-treated vs vehicle-treated animals.
Immunofluorescence microscopy (IFM)
MLNs were dissected from control and compound-treated mice immediately following euthanasia. MLNs were fixed in 4% formaldehyde (generated from paraformaldehyde; Fisher Scientific) in 0.1 M phosphate buffer (pH 7.4) at 4°C for a total of 2 h. Fixed MLNs were infused stepwise to final concentrations of 20% sucrose plus 5% glycerol in phosphate buffer (pH 7.2) overnight. MLNs were then placed in cryomolds filled with OCT compound, frozen in liquid nitrogen, and stored at –80°C. The nodes were cut into 5-µm frozen sections using a Hacker-Bright Cryostat (Hacker Instruments) fitted with an Instrumedix Sectioning Aid (Instrumedix). To obtain higher resolution immunofluorescent images, 0.5-µm cryosections were cut from blocks of MLNs prepared for immunoelectron microscopy (IEM) (see below) using glass knives and a Reichert UCT ultracryotome with a FCS cryoattachment (Reichert Scientific Instruments). To block nonspecific labeling, sections were treated with 5% donkey serum in PBS for 20 min, then with a clarified solution of 5% nonfat dry milk for 30 min, and finally with Fc blocker (Accurate Chemical) for 20 min. Sections were labeled overnight with the following primary Abs (5 µg to 10 µg/ml): rat anti-mouse CD45R/B220 (B cell) monoclonal IgG (BD Biosciences), rat anti-mouse CD68 (macrophage) monoclonal IgG (Serotec), affinity-purified rabbit anti-human S1P1c IgG (which also binds to rodent S1P1c) (10), affinity-purified rabbit anti-mouse S1P1n IgG (10), rat anti-mouse CD31 (PECAM) monoclonal IgG (BD Biosciences), FITC-conjugated anti- catenin monoclonal IgG (BD Biosciences), rat anti-mouse ZO-1 monoclonal IgG (Biogenesis), and affinity-purified rat anti-mouse reticular fibroblast protein monoclonal IgG (Cedarlane Laboratories). Control nonimmune IgGs were purchased from Jackson ImmunoResearch Laboratories. Slides were washed with PBS and incubated with affinity-purified F(ab')2 donkey anti-rabbit or rat Cy2 (green fluorescence)- or Cy3 (red fluorescence)-conjugated secondary Abs (5 µg/ml, 30 min) from Jackson ImmunoResearch Laboratories.
For double-labeling studies, combinations of primary Abs from different species, or corresponding species-specific fluorescent secondary Abs, were mixed together and incubated simultaneously on each slide. Slides were then fixed in 4% formaldehyde and coverslips were mounted on the slides with Vectashield plus 4',6'-diamidino-2-phenylindole (DAPI) nuclear stain (Vector Laboratories). Sections were photographed with an Everest imaging system (Intelligent Imaging Innovations) equipped with an Axioplan 2 microscope (Zeiss).
Immunoelectron microscopy
Fixation was performed on MLNs with a solution of 3.5% paraformaldehyde, 0.05% glutaraldehyde, and 0.1 M sucrose in PBS (pH 7.4) for 1 min at 4°C, followed by postfixation in Nakane’s solution (a mixture of paraformaldehyde, lysine, and periodate) (28) for 2 h at 4°C. Blocks of MLNs were infiltrated in 2% low-gelling temperature agarose, cryoprotected by overnight infiltration with 2.3 M sucrose and 50% polyvinylpyrrolidone in phosphate buffer (pH 7.2) (29), frozen by injection into liquid propane at –185°C in a KF-80 apparatus (Reichert Scientific Instruments), and stored under liquid nitrogen. To permit access of Abs to all sectioned subcellular compartments, ultrathin (
80-nm) frozen sections were cut with glass knives at –105°C (30) on a Reichert UCT ultramicrotome equipped with an FCS cryoattachment, and transferred to formvar-coated 200 mesh hexagonal nickel grids.
Sections were treated with a clarified solution of 5% nonfat dry milk and BSA buffer (1% BSA in PBS (pH 7.8) containing 0.1% sodium azide) for 30 min to block nonspecific binding, followed by overnight incubation in 5–10 µg/ml solutions of various IgGs (see above) in BSA buffer at 4°C. Controls consisted of 10 µg/ml solutions of the respective preimmune rabbit IgGs or isotype-matched nonimmune mAbs in BSA buffer. After extensive washing in PBS, the bound primary rabbit IgGs were detected with a donkey anti-rabbit 5-nm colloidal gold conjugate (GARG 5; Jackson ImmunoResearch Laboratories), and rat IgGs were labeled with donkey anti-rat 10-nm colloidal gold conjugate (GAMG10; Jackson ImmunoResearch Laboratories), either alone or as mixtures in BSA buffer for double labeling. The grids were then washed in PBS and fixed in 2% glutaraldehyde in PBS for 15 min, postfixed in 2% OsO4 in H2O for 20 min, stained with 2% uranyl acetate in H2O for 30 min, and embedded in 2% polyvinyl alcohol (30). Grids were examined and electron micrographs taken in a JEOL 200CX electron microscope at 80 kV at initial magnifications of x20,000–35,000.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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Sequestration of lymphocytes and macrophages. FTY720 and its phosphorylated active metabolite FTY-P have been shown to induce lymphopenia by inhibiting the egress of lymphocytes from the lymph node parenchyma into the sinuses, thereby blocking lymphocyte recirculation (9). We have now extended these observations by conducting IFM experiments on MLNs of mice treated with FTY-P, FTY720 or vehicle. Blood obtained by cardiac puncture was routinely checked for complete blood counts, and the expected degree of lymphopenia (80% depleted) was found in all animals dosed with FTY-P (data not shown). B cells were cleared from the subcapsular sinuses (SCS) and appeared to be highly aggregated within the MLN parenchyma following FTY-P administration (Fig. 1, A and B). Surprisingly, FTY-P treatment was also accompanied by numerous CD68+ macrophages concentrated in the SCS (Fig. 1, C and D). This observation was confirmed by quantitative immunofluorescent cell staining experiments demonstrating a 4.6-fold increase, on average, in macrophage accumulation in MLNs of FTY-P treated mice (Fig. 2A). Lymphocytes have previously been shown to accumulate in lymph nodes treated with FTY720 (3, 7) and, in our experiments, CD3+ and B220+ cells were also increased in MLNs after FTY-P treatment by 2.0- and 2.7-fold, respectively (Fig. 2A). Furthermore, the intracellular expression of S1P1 was unchanged following FTY-P treatment (Fig. 2B), strongly suggesting that this compound does not cause S1P1 degradation or up-regulation.
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Other cell types in control MLNs showed low to moderate amounts of S1P1 labeling, while increased S1P1 expression was seen in the SCS following FTY-P treatment (Fig. 1, E and F). To obtain higher resolution immunofluorescent images, semithin sections (0.5-µm thickness) of MLNs fixed and processed for IEM were analyzed (Fig. 3). The SCS of vehicle-treated MLNs contained moderate numbers of elongated cells and fibers that were strongly S1P1n+ (Fig. 3A). Following treatment with FTY720, the SCS appeared to be greatly enlarged and was filled with numerous S1P1n+ cells and cellular processes (Fig. 3B). In double-labeling experiments with B220 and S1P1n Abs, B lymphocytes were readily detected among the S1P1n+ cells of the control SCS, but were conspicuously depleted from the expanded S1P1n-positive SCS of FTY-P-treated MLNs (Fig. 3, C and D). S1P1n was also heavily expressed in macrophages distributed among B lymphocytes in the parenchyma and within the SCS (Fig. 3, E and F). However, no S1P1n labeling was detected on B220+ lymphocytes in the SCS or the parenchyma (Fig. 3, C–E). S1P1 immunolabeling was shown to be specific by the following criteria: 1) closely similar staining patterns were observed with both C-terminal and N-terminal peptide IgGs (see Figs. 1, 3, and 8), 2) the labeling was blocked by preincubation with relevant but not irrelevant peptides (data not shown) (10), 3) use of nonimmune IgGs abolished the staining (Fig. 1E, inset), 4) both S1P1 Abs labeled the proper proteins in immunoblots (data not shown) (10), and 5) S1P1–/– embryos did not label with these S1P1 IgGs (31).
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Effects of an S1P agonist on lymphatic endothelium |
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TopAbstractIntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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-catenin, is an important structural component of endothelial junctional complexes (19, 20), we studied the effects of S1P agonists on the localization of CD31 and -catenin in the MLN endothelium (Fig. 4). The SCS lymphatic endothelium of control MLNs exhibited a very delicate pattern of CD31 labeling that was coincident with a similar level of -catenin expression (arrowheads in Fig. 4, A–C). The parenchyma showed very intense CD31 and -catenin staining codistributed in HEVs (arrows in Fig. 4, A–C). In contrast, there was a marked increase in the coexpression of both CD31 and -catenin in the SCS lymphatic endothelium following FTY-P treatment (matching arrows in Fig. 4, D–F). Interestingly, this increase was accompanied by the localization of prominent -catenin labeling in the nuclei of many SCS cells (green arrowheads in Fig. 4E and corresponding arrowheads in Fig. 4, D and F).
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-catenin and ZO-1 Abs (Fig. 5). The SCS of vehicle-treated control MLNs displayed a delicate punctuate -catenin pattern that was colocalized with ZO-1 in the lymphatic endothelium, whereas HEVs showed much higher coexpression of both markers (Fig. 5, A–C). Following FTY-P treatment, there was a substantial increase in -catenin expression in the SCS lymphatic endothelium that was colocalized with enhanced ZO-1 labeling (Fig. 5, D–F).
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Transmission electron microscopy (TEM) of S1P agonist-treated MLNs |
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TopAbstractIntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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The SCS of control MLNs contained many lymphocytes and a few macrophages (Fig. 6A). After FTY-P treatment, most of the lymphocytes had disappeared from the SCS, revealing numerous macrophages and ECs within, while lymphocytes in the parenchyma appeared to be more clustered (Fig. 6, B and C). Lymphocytes within the control MLN parenchyma normally exhibited many cell surface microvilli (Fig. 7A). Following FTY-P administration, the surface microvilli disappeared, the lymphocyte pericellular space narrowed (Fig. 7B), and numerous close contacts developed between neighboring lymphocytes, as well as between lymphocytes and ECs (Fig. 7, C–E). Comparison of the number of close contacts between lymphocytes in the parenchyma of control vs FTY-P-treated MLNs revealed a 67.5% increase (Table I). After prolonged FTY-P treatment, lymphocytes in the parenchyma exhibited broad regions of cell surface membrane apposition (Fig. 7F).
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Macrophages in the parenchyma of control MLNs exhibited considerable S1P1n immunogold labeling on their cell surface membranes, while adjacent lymphocytes lacked S1P1n expression on their surfaces, and showed relatively minor S1P1n labeling in small cytoplasmic vesicles (Fig. 8A). Similarly, robust S1P1 immunostaining was present on both cell surface and cytoplasmic membranes of EC processes in the SCS of FTY-P-treated and control MLNs (Fig. 8, B and C). Administration of FTY-P or FTY720 did not substantially alter the subcellular distribution of S1P1n in parenchymal macrophages, and treatment with these compounds did not enhance the S1P1n staining of lymphocytes in the MLN parenchyma (Fig. 8, D and E). Significantly, regions of close contact between the cell surface membranes of lymphocytes and macrophages did not exhibit S1P1n labeling (arrows in Fig. 8, D and E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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When FTY-P was administered overnight, B220+ B cells in MLNs were increased (Fig. 1B), as was the total number of nodal cells (on average by 2.3-fold; Fig. 2). This compares favorably with previous studies (3) that reported a 1.8-fold rise in B220+ cells in the MLNs of rats treated for 24 h with FTY720. T cells also become sequestered in SLOs following FTY720 treatment (3, 7). This accumulation may be species, tissue, or cell type specific, since S1P1-deficient lymphocytes used in short-term homing experiments had a reduced ability to undergo integrin-mediated firm arrest in HEVs of peripheral lymph nodes (35). By contrast, S1P1-deficient T cells, but not S1P1-deficient B cells, appeared to home normally to Peyer’s patches (PPs). FTY720 treatment enhanced integrin-mediated arrest of both S1P1-expressing and S1P1-deficient T cells in HEVs in PPs, suggesting that some of FTY720’s effects, at least on PP-homing T cells, are independent of S1P1.
Lymphocytes showed a loss of microvilli after FTY-P administration, strikingly similar to the significant decrease in microvilli observed in FTY720-treated breast cancer cells. This change was accompanied by reduced metastases (36). We also observed statistically significant increases in the number of contacts between lymphocytes (Table I). TEM imaging of contacts showed that they contain electron-dense material (Fig. 7E) and more surface area than contacts between lymphocytes of controls. After 14 days of treatment, some lymphocytes transformed into epithelial-like sheets (Fig. 7F). In other cell types, S1P also induces changes in cytoskeletal proteins, surface adhesion molecules, and extracellular matrix components (22, 24, 37, 38, 39). Unfortunately, the epitopes of many adhesion molecules were inactivated by fixation, precluding their IEM identification at contact sites.
Recent data using receptor-selective agonists and mice with lymphocyte-specific deletions revealed that S1P1 is the target receptor for lymphocyte trafficking defects and immunosuppression. Despite the abundant levels of S1P1 mRNA previously measured (16), we observed few S1P1 receptors in intranodal lymphocytes. S1P1 staining was weakest in the lymphocyte-rich regions of MLNs, and the few receptors detected were almost exclusively intracellular (Figs. 1, E and F, 3, and 8, A, D, and E). This suggests that relatively small numbers of S1P1 receptors regulate lymphocyte egress, making the recirculation pathway exquisitely sensitive to modulation by agonists that sustain receptor down-modulation. However, we cannot rule out that S1P1 expression in intranodal lymphocytes may vary between cortical and medullary regions, where migratory behavior and stromal cell populations may differ significantly.
In contrast, ECs within MLNs expressed abundant levels of cell surface and intracellular S1P1 receptors (Figs. 1, 3, 5, and 8). We did not observe gross changes in S1P1 distribution in HEVs following FTY-P treatment. This is consistent with published reports that S1P and FTY-P act as agonists of endothelial barrier function (23, 24). In MLNs, many of the sinus resident cells had an endothelial morphology and were organized into capillaries; they expressed the endothelial cell Ag CD31 (PECAM; Fig. 4) and are presumably lymphatic ECs (20). These SCS ECs were still S1P1 positive after FTY-P treatment (Fig. 5F, inset) and showed no evidence of S1P1 down-regulation (Fig. 8, B and C).
Similar to ECs, S1P1 was found at the cell surface and in the cytoplasm of macrophages, and treatment with FTY-P did not result in significant redistribution of this receptor (Fig. 8). S1P1 desensitization has been studied in transfected cells, but relatively little is known about regulation of endogenous receptors. Recent studies indicate that S1P1 internalization, recovery, or down-modulation are regulated by distinct posttranslational modification pathways that may be differentially expressed in various cells (15, 40, 41, 42).
S1P1, originally cloned from ECs (43), plays a major role in regulating EC survival, migration, and differentiation. Using a transgenic mouse designed to express the -galactosidase reporter gene product from the S1P1 locus, the receptor was found to be expressed constitutively in selected vascular beds, e.g., in lung, heart, and liver ECs (44). It is also expressed strongly in newly formed vessels, e.g., in tumors (45). S1P promotes the assembly of -catenin- and VE-cadherin-containing AJs (22) and increases endothelial: mural cell adhesion, which is important for vascular maturation (25). -catenin in AJs promotes junctional stabilization by anchoring cadherins and CD31 to actin (21). The association of -catenin with VE-cadherin may prevent its translocation to the nucleus, where -catenin acts as a transcription factor for cyclin D1, leading to contact inhibition of growth (19, 46). ZO-1 is localized in tight junctions, which are frequently intermingled with AJs in EC junctions (19). Endothelial barrier function is enhanced by S1P-mediated cytoskeletal changes (24), thus we examined the effects of FTY-P on endothelial junctional proteins. We observed that CD31, -catenin, and ZO-1 were up-regulated and colocalized in junctions between SCS ECs in FTY-P-treated MLNs (Figs. 4 and 5). The enhanced expression of these molecules may increase the integrity of the SCS endothelial barrier.
FTY-P increased the area and intensity of SCS staining with S1P1-specific Abs, in part due to morphological changes in lymphatic ECs or to changes in other stromal cells (Figs. 1, 2, and 5). Some of the intensified S1P1 staining may be attributable to an increase in fibroblastic reticular cells (FRCs), which form a network of collagen-containing reticular fibers wrapped by FRC processes (47). FTY-P may alter the architecture of the network, since S1P can stimulate primary fibroblast proliferation and fibronectin matrix assembly (48, 49). These cells secrete reticular fibroblast protein (RFP; Fig. 8B), but a lineage-specific marker that would allow us to determine whether FRCs specifically express S1P1 is not available.
We observed a major increase in S1P1+ macrophages in the SCS (Figs. 1, 3, and 8). FTY720 does not deplete circulating murine monocytes, and altered macrophage functions have not been reported for this compound. Macrophages naturally reside in the SCS in resting nodes (50, 51), and it is incompletely understood how they enter MLNs. Some monocytes may enter SLOs via HEVs using MCP-1 and integrin-mediated arrest (52). CD68+ cells did not surround HEVs in our sections of treated nodes, but were found in the SCS; therefore, it is unlikely that FTY-P enhanced their diapedesis through HEVs. S1P prevented apoptosis of U937 cells, which occurred when their adherence to substrate was inhibited (53), and also prevented apoptosis of murine bone marrow-derived macrophages (54). Thus, enhanced adhesion and survival of macrophages may contribute to the increased number of CD68+ cells in FTY-P-treated nodes.
Nonactivated resident macrophages and monocyte-derived activated dendritic cells enter SLOs via draining afferent lymphatics (55, 56). Similarly, fluorescently labeled macrophages migrate to the SCS and medullary cords of lymph nodes following adoptive transfer (57). Lipid mediators, such as platelet-activating factor, inhibit the migration of macrophages and dendritic cells from peripheral sites to SLOs (56). FTY-P may accelerate this migration, and macrophages may become trapped within SCS afferent lymphatics due to the increased integrity of EC junctional complexes in treated MLNs.
In summary, our data show that S1P1 expression and distribution varies in the different MLN cell types. FTY-P evokes differential responses, including increased focal contacts in lymphocytes, enhanced expression, and colocalization of CD31, S1P1, -catenin, and ZO-1 in ECs and accumulation of macrophages in SCS. These findings strongly support the concept that multiple cell types are profoundly affected by treatment with S1P agonists and may contribute to the immunosuppressive effects of FTY720.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsEffects of an S1P...Transmission electron microscopy...DiscussionDisclosuresReferences |
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Footnotes |
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1 Address correspondence and reprint requests to Dr. Irwin I. Singer, Immunology and Rheumatology, Merck and Co., Inc., Merck Research Laboratories, Rahway, NJ 07065 or Dr. Elizabeth J. Quackenbush, Pharmacology, Merck and Co., Inc., Merck Research Laboratories, Rahway, NJ 07065. E-mail address: Irwin_Singer{at}Merck.com or Betsy_Quackenbush{at}Merck.com 
2 Abbreviations used in this paper: SLO, secondary lymphoid organ; MLN, mesenteric lymph node; FTY-P, phosphorylated FTY720; HEV, high endothelial venule; EC, endothelial cell; S1P, sphingosine-1-phosphate; AJ, adherens junction; ZO-1, zona occludens 1; VE, vascular endothelial; IFS, immunofluorescent staining; IFM, immunofluorescence microscopy; SCS, subcapsular sinus; PP, Peyer’s patches; FRC, fibroblastic reticular cell; DAPI, 4',6'-diamidino-2-phenylindole; RFP, reticular fibroblast protein; IEM, immunoelectron microscopy.
Received for publication March 15, 2005. Accepted for publication September 19, 2005.
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 M. L. Oo, S. Thangada, M.-T. Wu, C. H. Liu, T. L. Macdonald, K. R. Lynch, C.-Y. Lin, and T. Hla Immunosuppressive and Anti-angiogenic Sphingosine 1-Phosphate Receptor-1 Agonists Induce Ubiquitinylation and Proteasomal Degradation of the Receptor J. Biol. Chem., March 23, 2007; 282(12): 9082 - 9089. [Abstract] [Full Text] [PDF]
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 C. Nombela-Arrieta, T. R. Mempel, S. F. Soriano, I. Mazo, M. P. Wymann, E. Hirsch, C. Martinez-A., Y. Fukui, U. H. von Andrian, and J. V. Stein A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress J. Exp. Med., March 19, 2007; 204(3): 497 - 510. [Abstract] [Full Text] [PDF]
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P. J. Gonzalez-Cabrera, T. Hla, and H. Rosen Mapping Pathways Downstream of Sphingosine 1-Phosphate Subtype 1 by Differential Chemical Perturbation and Proteomics J. Biol. Chem., March 9, 2007; 282(10): 7254 - 7264. [Abstract] [Full Text] [PDF]
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P. Keul, M. Tolle, S. Lucke, K. von Wnuck Lipinski, G. Heusch, M. Schuchardt, M. van der Giet, and B. Levkau The Sphingosine-1-Phosphate Analogue FTY720 Reduces Atherosclerosis in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., March 1, 2007; 27(3): 607 - 613. [Abstract] [Full Text] [PDF]
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D. H. Walter, U. Rochwalsky, J. Reinhold, F. Seeger, A. Aicher, C. Urbich, I. Spyridopoulos, J. Chun, V. Brinkmann, P. Keul, et al. Sphingosine-1-Phosphate Stimulates the Functional Capacity of Progenitor Cells by Activation of the CXCR4-Dependent Signaling Pathway via the S1P3 Receptor Arterioscler. Thromb. Vasc. Biol., February 1, 2007; 27(2): 275 - 282. [Abstract] [Full Text] [PDF]
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) lacks S1P1 labeling; V indicates a small S1P1+ cytoplasmic vesicle. B, SCS of an FTY-P-treated MLN double labeled with anti- S1P1n (5 nm gold) and RFP (10 nm gold) Abs. The process of an EC exhibits intense expression of S1P1 on both cytoplasmic and cell surface membranes (arrowheads), while the pericellular connective tissue is positive for RFP (arrow). C, SCS of a vehicle-treated MLN immunostained with anti- S1P1c (5 nm gold). S1P1c is localized on cell surface (arrowheads) and on cytoplasmic membranes of an EC. D, Parenchyma of an FTY-P-treated MLN labeled with S1P1n (5 nm gold). The macrophage exhibits S1P1n expression on the cell membrane (arrowheads) and in cytoplasmic vesicles (V), while the contacting lymphocyte is unlabeled (arrows depict intercellular contact sites). E, Ultrathin cryosection of an FTY720-treated MLN labeled with anti-S1P1n (5 nm gold). The macrophage process shows both cell surface (arrowheads) and cytoplasmic S1P1n expression, whereas the attached lymphocyte is unlabeled; arrow delineates a large cell surface contact. M
