Rolling of Th1 Cells via P-Selectin Glycoprotein Ligand-1 Stimulates LFA-1-Mediated Cell Binding to ICAM-1
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免疫学杂志 2005年第3期
Abstract
Activated T cells migrate from the blood into nonlymphoid tissues through a multistep process that involves cell rolling, arrest, and transmigration. P-Selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed on subsets of activated T cells such as Th1 cells and mediates cell rolling on vascular endothelium. Rolling cells are arrested through a firm adhesion step mediated by integrins. Although chemokines presented on the endothelium trigger integrin activation, a second mechanism has been proposed where signaling via rolling receptors directly activates integrins. In this study, we show that Ab-mediated cross-linking of the PSGL-1 on Th1 cells enhances LFA-1-dependent cell binding to ICAM-1. PSGL-1 cross-linking did not enhance soluble ICAM-1 binding but induced clustering of LFA-1 on the cell surface, suggesting that an increase in LFA-1 avidity may account for the enhanced binding to ICAM-1. Combined stimulation by PSGL-1 cross-linking and the Th1-stimulating chemokine CXCL10 orCCL5 showed a more than additive effect on LFA-1-mediated Th1 cell adhesion as well as on LFA-1 redistribution on the cell surface. Moreover, PSGL-1-mediated rolling on P-selectin enhanced the Th1 cell accumulation on ICAM-1 under flow conditions. PSGL-1 cross-linking induced activation of protein kinase C isoforms, and the increased Th1 cell adhesion observed under flow and also static conditions was strongly inhibited by calphostin C, implicating protein kinase C in the intracellular signaling in PSGL-1-mediated LFA-1 activation. These results support the idea that PSGL-1-mediated rolling interactions induce intracellular signals leading to integrin activation, facilitating Th1 cell arrest and subsequent migration into target tissues.
Introduction
In response to inflammatory stimuli, leukocytes migrate from the blood into tissues through several sequential adhesive steps (1). Initially, leukocytes are captured from the bloodstream by adhesion receptors through rapid and transient interactions. These interactions are primarily mediated by the following selectins: L-selectin (CD62L), which is expressed on most leukocytes, and E-selectin (CD62E) and P-selectin (CD62P), which are expressed on activated endothelium. The relatively low-affinity interactions mediated by the selectins and their ligands allow leukocytes to roll along the vascular surface under flow conditions (2, 3, 4). To migrate into tissues, rolling leukocytes must stop through a firm adhesion step, which is mediated by integrins. LFA-1 (CD11a/CD18, L2) and Mac-1 (CD11b/CD18, M2) represent the major 2 integrins on leukocytes that mediate their binding to ICAMs, the Ig superfamily integrin ligands (1). Among the ICAMs, ICAM-1 (CD54) serves as the predominant ligand for LFA-1 and Mac-1 and is up-regulated on endothelial cells during inflammation.
2 integrins are constitutively expressed on leukocytes and require activation to initiate adhesion. Various chemokines presented on the endothelium, where leukocytes roll, trigger rapid integrin activation through Gi-linked receptors in a number of leukocyte types (5). A second mechanism has been proposed in which signaling mediated by rolling receptors directly activates integrins. For example, the cross-linking of L-selectin on neutrophils up-regulates the adhesive function of Mac-1 (6). Stimulation of L-selectin via glycosylation-dependent cell adhesion molecule-1, a soluble ligand for L-selectin, also stimulates the LFA-1-mediated binding of naive lymphocytes to ICAM-1 (7).
P-selectin glycoprotein ligand-1 (PSGL-13; CD162) is a major P-selectin ligand on myeloid cells and subsets of lymphoid cells and mediates the tethering and rolling of leukocytes on the vascular endothelium (8). The physiologic role of PSGL-1 as the predominant P-selectin ligand has been clearly demonstrated by the deficient P-selectin-mediated rolling in PSGL-1-deficient mice (9). PSGL-1 can also bind to E-selectin and L-selectin, and the physiologic roles of these interactions have been shown in vivo (10, 11, 12). To bind selectins, PSGL-1 requires specific core 2-type O-glycans containing the sialyl LewisX moiety (13). The expression of some glycosyltransferases involved in the synthesis of selectin-binding glycans, including core 2 -1,6-N-acetylglucosaminyltransferase and -1,3-fucosyltransferase, is dynamically regulated in lymphocytes during activation and differentiation. Thus, despite the fact that virtually all T cells express PSGL-1, only particular subsets of T cells express a form of PSGL-1 that can bind selectins. Notably, in vitro-differentiated Th1 cells express PSGL-1 in a selectin-binding form (10, 14).
In contrast to naive T cells, which traffic through secondary lymphoid organs, activated T cells can migrate into nonlymphoid tissues through a multistep process similar to that used by neutrophils and monocytes. The migration of Th1 cells into the inflamed skin in a contact hypersensitivity model is dependent on P-selectin and E-selectin (15), and PSGL-1 is the major ligand for P-selectin as well as one of the ligands for E-selectin in this process (10, 14). Th1 cells also exhibit a preferential expression of chemokine receptors, typically CXCR3 and CCR5, which may be responsible for the distinct migration properties of these cells (16, 17). A CXCR3 ligand, CXCL10, and a CCR5 ligand, CCL5, have been implicated in integrin-mediated T cell adhesion (18, 19, 20).
Although the role of PSGL-1 as a rolling receptor is well established, less is known about its role as a signal-transducing molecule. Previous studies have demonstrated signaling events that are induced by PSGL-1 ligation via P-selectin binding or Ab-mediated cross-linking. PSGL-1 ligation enhances tyrosine phosphorylation and activates MAPKs in human neutrophils as well as cytokine release by neutrophils and monocytes (21). Cross-linking of PSGL-1 on mouse neutrophils with an Ab-like chimeric protein of P-selectin increases their binding to ICAM-1-transfected Chinese hamster ovary cells through LFA-1 and Mac-1 (22). Signaling events induced by P-selectin binding in T cells have also been reported. P-Selectin in conjunction with an anti-TCR mAb augments the production of GM-CSF by Ag-primed human T cells (23). P-Selectin binding also induces the phosphorylation of several substrates, including focal adhesion kinase and Syk, in T cells (24, 25). Although Ab-mediated PSGL-1 cross-linking and P-selectin binding have been used to examine PSGL-1-induced cellular signaling, the effect of PSGL-1 ligation under physiological conditions, i.e., PSGL-1-mediated rolling, on intracellular signaling remains unknown.
In this study, we report that cross-linking of PSGL-1 on Th1 cells enhances their LFA-1-mediated binding to ICAM-1, which may be attributed in part to the clustering of LFA-1 on the cell surface. We show that PSGL-1 cross-linking combined with chemokine stimulation exerts a more than additive effect on LFA-1-mediated Th1 cell adhesion. In addition, we show that PSGL-1-mediated rolling on P-selectin dramatically enhances the Th1 cell accumulation on ICAM-1 under flow conditions. The enhanced cell accumulation is inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC), implicating PKC pathways in the intracellular signaling from PSGL-1-mediated rolling to LFA-1 activation. To our knowledge, this is the first demonstration of a role for PSGL-1-mediated rolling in triggering integrin activation.
Materials and Methods
Mice
C57BL/6J (B6) mice were purchased from CLEA Japan. PSGL-1-deficient mice on a B6 background (9, 10) were provided by Dr. B. Furie (Harvard Medical School, Boston, MA). All mice used were 6–12 wk old. The mice were housed at the Institute of Experimental Animal Sciences at Osaka University Medical School. All studies and procedures were approved by the Ethics Review Committee for Animal Experimentation of the Osaka University Graduate School of Medicine.
Chimeric proteins
The expression plasmid for mouse P-selectin-human IgM chimeric protein (P-selectin-IgM) was provided by Dr. J. Lowe (University of Michigan Medical School, Ann Arbor, MI). The plasmid was transfected into COS-7 cells using DEAE-dextran. Chimeric proteins were purified from culture supernatants using biotinylated anti-human IgM (American Qualex) bound to avidin-agarose (Pierce Biotechnology) and concentrated using Centriplus YM-30 (Millipore). Mouse P-selectin-human IgG chimeric protein (P-selectin-IgG) was purified using protein A-Sepharose (Amersham Biosciences) as described previously (10). The expression plasmid for rat ICAM-1-human IgG chimeric protein (ICAM-1-IgG) was provided by Dr. Y. Iigo (Daiichi Pharmaceutical, Tokyo, Japan). The plasmid was transfected into COS-7 cells, and the chimeric protein was purified as described previously (26).
Generation of Th1 and Th2 cells
Splenic CD4+ T cells were isolated by autoMACS (Miltenyi Biotec) using biotinylated anti-CD11b (M1/70; BD Biosciences), biotinylated anti-B220 (RA3-6B2; BD Biosciences), and biotinylated anti-CD8 (53-6.7; Southern Biotechnology Associates) followed by Streptavidin MicroBeads (Miltenyi Biotec). The enriched population was 88–95% positive for CD4 staining. Purified CD4+ T cells were cultured on 24-well tissue culture plates coated with 10 μg/ml anti-CD3 (145-2C11; BD Biosciences) and 10 μg/ml anti-CD28 (37.51; BD Biosciences) for 2 days in the presence of 4 ng/ml IL-2 (R&D Systems) and either 8 ng/ml IL-12 (R&D Systems) and 0.2 μg/ml anti-IL-4 (11B11; BD Biosciences) to promote Th1 differentiation, or 100 ng/ml IL-4 (Genzyme) to promote Th2 differentiation. The cells were then transferred to uncoated plates and cultured for an additional 3 days. Freshly activated cells were prepared by stimulating MACS-isolated CD4+ T cells with plate-bound anti-CD3 and anti-CD28 in the presence of IL-2 for 2 days.
Flow cytometry
All mAbs used for the flow cytometric analyses were purchased from BD Biosciences. They included anti-CD4-FITC (RM4-5), anti-CD11a-FITC (M17/4), anti-CD25-FITC (7D4), anti-CD44-FITC (IM7), anti-CD62L-FITC (MEL-14), and anti-CD162-PE (2PH1). Cells were stained with mAbs for 30 min on ice, washed, and analyzed on an EPICS XL flow cytometer (Beckman Coulter). To assess the ICAM-1-IgG binding by flow cytometry, cells were incubated with 40 μg/ml ICAM-1-IgG for 30 min on ice, washed, and then incubated with FITC-labeled anti-human IgG (Biomeda) for 30 min on ice. To assess the P-selectin-IgM binding, cells were incubated with a COS-7 supernatant containing P-selectin-IgM, washed, and then incubated with biotinylated anti-human IgM. The cells were then washed and stained with streptavidin-PE (BD Biosciences).
Cell adhesion assays under static conditions
ICAM-1-IgG or control human IgG (Sigma-Aldrich) was immobilized on Costar 3690 plates (5 μg/ml, 25 μl/well) at 4°C overnight, and the plates were blocked with FCS at 37°C for 2 h. The cells were labeled with 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes) at 37°C for 30 min, washed, and resuspended in RPMI 1640 with 10 mM HEPES (RPMI-HEPES). To cross-link PSGL-1 with Abs, the labeled cells were incubated with 5 μg/ml anti-PSGL-1 mAb 2PH1 or control rat IgG1 (both from BD Biosciences) at 37°C for 30 min, washed, and then incubated with 50 μg/ml goat F(ab')2 anti-rat IgG (Immunotech) at 37°C for 30 min. The cells were washed and resuspended at 4 x 106/ml in RPMI-HEPES and 10% FCS (RPMI-HEPES/FCS), added to the plate, and incubated for 20 min on ice. In some experiments, PSGL-1-cross-linked cells were preincubated with an anti-LFA-1 mAb KBA (27) or its isotype control rat IgG2a (Zymed) before addition to the plate. The plates were filled with RPMI-HEPES/FCS, inverted, and incubated at 37°C for 30 min. Supernatants were aspirated, and bound cells were lysed with 0.1% Igepal CA-630 (Sigma-Aldrich) in PBS. Fluorescence intensity was measured using a SpectraMax Gemini XS (Molecular Devices). To assess the effect of kinase inhibitors on cell adhesion, BCECF-labeled cells were treated with 0.5 μM staurosporine, 0.1 μM calphostin C, 50 μM LY294002, 50 μM PD098059, or 4–40 μM rottlerin (all from Sigma-Aldrich) at 37°C for 30 min before cross-linking PSGL-1.
For short-term cell adhesion assays, BCECF-labeled Th1 cells were incubated with 2PH1 (5 μg/ml) at 37°C for 30 min and resuspended at 4 x 106 cells/ml in RPMI-HEPES/FCS. Secondary Abs (20 μg/ml) and chemokines at several concentrations in RPMI-HEPES/FCS were added to plates (5 μl/well) coated with ICAM-IgG or human IgG. The cells were then added to the plates (25 μl/well), incubated for 18 min on ice, and then rapidly warmed in a 37°C water bath for 2 min. The plates were washed three times with RPMI-HEPES, and the bound cells were quantified as described above.
Cell adhesion assays under flow conditions
ICAM-1-IgG or control human IgG (100 μg/ml) was immobilized on the inside walls of glass capillaries (inner diameter, 0.69 mm; Drummond Scientific) at 4°C overnight. The capillaries were then blocked with FCS for 5 min at room temperature. PSGL-1 on Th1 cells was cross-linked with 2PH1 and a secondary Ab. The cells were resuspended at 1 x 106 cells/ml in RPMI-HEPES/FCS and infused into the capillaries mounted on the stage of an inverted microscope (Diaphot 300; Nikon) at a shear force of 1 dyn/cm2. The rate of flow was controlled by a PHD 2000 syringe pump (Harvard Apparatus). Four minutes after the start of infusion, cell images were recorded with a cell-viewing system (SRM-100; Nikon) and video recorder (BR-S600; Victor). For the detachment assays, cells were infused at 0.25 dyn/cm2 for 8 min, and the shear stress was increased stepwise every 20 s until it reached 20 dyn/cm2. At the end of each shear stress treatment, the number of cells that remained bound was determined.
To examine the effect of P-selectin-dependent rolling of Th1 cells on their adhesion to ICAM-1, P-selectin-IgG or control human IgG (5 μg/ml) was immobilized on the inside walls of capillaries at 4°C overnight. The capillaries were then blocked with FCS for 5 min at room temperature. A capillary coated with P-selectin-IgG or human IgG was connected in tandem with an ICAM-1-IgG-coated capillary and washed with 2 ml of RPMI-HEPES/FCS at 10 dyn/cm2. Th1 or Th2 cells were resuspended at 8 x 105 cells/ml in RPMI-HEPES/FCS, and 1 ml of cell suspension was infused into the capillaries at 1 dyn/cm2. In some experiments, the cells were preincubated with polyclonal Abs raised against the N-terminal region (QVVGDDDFEDPDYTY) or cytoplasmic region (EPSGDRDGDDLTLHSFLP) of PSGL-1. The cells were also preincubated with 0.1 μM calphostin C or vehicle. Four minutes after the start of the infusion, cell images were recorded for 10 min, and the bound cells were counted.
Immunocytochemical staining
Th1 cells were incubated with 5 μg/ml 2PH1 or 20 μg/ml P-selectin-IgM at 37°C for 30 min. The cells were then incubated at 37°C for 30 min with 20 μg/ml goat F(ab')2 anti-rat IgG or goat F(ab')2 anti-human IgM (OEM Concepts), both conjugated with Alexa Fluor 594 using the Alexa Fluor 594 mAb labeling kit (Molecular Probes). PSGL-1-cross-linked cells were fixed with 1% paraformaldehyde for 15 min at room temperature. To block the nonspecific binding of FITC-labeled Abs, cells were incubated with 200 μg/ml rat IgG for 30 min on ice. The cells were stained with 10 μg/ml FITC-labeled anti-CD11a or control FITC-labeled rat IgG2a for 30 min on ice. The cells were washed, resuspended in ProLong Antifade (Molecular Probes), and transferred onto slides. The cells were observed under a confocal microscope (LSM 510 UV/META; Carl Zeiss).
To assess changes in cell morphology and staining patterns, 100 cells in each sample were evaluated using differential interference contrast (DIC) and confocal images. Cells with >1.3 ellipticity (the ratio between the longest and shortest axes) were considered to exhibit the morphological change, and cells with <30% of their surface showing condensed staining were considered to show the redistribution of cell surface molecules.
Western blotting
Th1 cells were incubated with 5 μg/ml 2PH1 at 37°C for 30 min and then incubated with 50 μg/ml goat F(ab')2 anti-rat IgG at 37°C for the indicated times. The cells were lysed in a buffer containing 1% Triton X-100, 25 mM HEPES (pH 7.5), 150 mM NaCl, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF, 1 mM EDTA, and 1 mM Na3VO4 for 30 min, and then cleared by centrifugation. The lysates were resolved by SDS-PAGE under reducing conditions, and the proteins were transferred to an Immobilon-P membrane (Millipore). The membranes were probed with Abs against various phosphorylated PKC isoforms from phospho-PKC Ab sampler kit (Cell Signaling Technology).
Results
Cross-linking of PSGL-1 on Th1 cells stimulates cell binding to ICAM-1
To examine the effect on integrin activation of the ligation of the PSGL-1 on T cells, we first tested the adhesion of in vitro-generated Th1 cells to ICAM-1-IgG immobilized on 96-well microtiter plates after cross-linking PSGL-1. To cross-link PSGL-1, Th1 cells were incubated with an anti-PSGL-1 mAb, 2PH1, for 30 min at 37°C, washed, and then incubated for 30 min at 37°C with a secondary goat F(ab')2 anti-rat IgG. The cross-linking of PSGL-1 on Th1 cells using 2PH1 and a secondary Ab induced a 10-fold increase in the number of Th1 cells bound to ICAM-1-IgG compared with cells that were not incubated with any Abs (Fig. 1A). No significant increase was observed in the Th1 cells binding to control human IgG after cross-linking. Incubation of Th1 cells with 2PH1 alone, a secondary Ab alone, or an isotype-matched control rat IgG followed by a secondary Ab did not significantly affect the Th1 cell binding to ICAM-1-IgG (Fig. 1A).
FIGURE 1. PSGL-1 cross-linking enhances the binding of Th1 cells to ICAM-1. A, Incubation of Th1 cells with 2PH1 and a secondary Ab (PSGL-1 cross-linking) enhances the cell binding to ICAM-1. In vitro-generated Th1 cells were labeled with BCECF-AM. The labeled cells were incubated with or without 2PH1 or control rat IgG, washed, and then incubated with or without a secondary goat F(ab')2 anti-rat IgG. The cells were added to 96-well plates coated with ICAM-IgG or human IgG. The plates were incubated for 20 min, unbound cells were removed, and the fluorescence per well was determined. Bound cells are expressed as a percentage of the total cells added. B, Binding of PSGL-1-cross-linked Th1 cells treated with an anti-LFA-1 mAb to ICAM-1. Th1 cells that had been incubated with 2PH1 and a secondary Ab were treated with either the anti-LFA-1 mAb KBA or its isotype control for 30 min before addition to the wells. Cells bound to ICAM-IgG are expressed as a percentage of the untreated control. C, Binding of PSGL-1-deficient Th1 cells to ICAM-1. PSGL-1-deficient (KO) or wild-type (WT) Th1 cells were incubated with or without 2PH1 and a secondary Ab and added to plates coated with ICAM-IgG or human IgG. D, Binding of unstimulated CD4+ T, freshly activated CD4+ T, Th1, and Th2 cells to ICAM-1. Freshly isolated or freshly activated CD4+ T cells or in vitro-generated Th1 or Th2 cells were incubated with or without 2PH1 and a secondary Ab and added to plates coated with ICAM-IgG or human IgG. Each data point represents the mean ± SEM from triplicate wells. E, Expression of cell surface markers on unstimulated CD4+ T, freshly activated CD4+ T, Th1, and Th2 cells. Freshly isolated or freshly activated CD4+ T cells or in vitro-generated Th1 or Th2 cells were stained with the indicated mAbs (shaded histograms) or isotype-matched control IgGs (open histograms) and analyzed by flow cytometry. Mean fluorescence intensity values for PSGL-1 and LFA-1 are also indicated. F, P-selectin-IgM binding of Th1 and Th2 cells. Cells were incubated with P-selectin-IgM (shaded histograms) or control human IgM (open histograms). Bound P-selectin-IgM was detected using biotinylated anti-human IgM and streptavidin-PE.
The binding of cross-linked Th1 cells to ICAM-1-IgG was dependent on LFA-1, given that it was almost completely blocked by the incubation of the cross-linked cells with the LFA-1-blocking mAb KBA, but not with a control rat IgG (Fig. 1B). Incubation of Th1 cells from PSGL-1-deficient mice with 2PH1 and a secondary Ab did not induce an increase in the number of cells bound to ICAM-1-IgG, confirming that the effect induced by cross-linking was mediated by PSGL-1 (Fig. 1C). Cross-linking PSGL-1 on unstimulated CD4+ T cells by 2PH1 and a secondary Ab did not show an increase in the number of bound cells (Fig. 1D). These cells expressed both PSGL-1 and LFA-1, albeit at a lower level than did Th1 and Th2 cells (Fig. 1E). In contrast, cross-linking PSGL-1 on Th2 cells enhanced their binding, although the number of bound cells was 40% of that of Th1 cells (Fig. 1D). The level of expression of PSGL-1 and LFA-1 on the cell surface was only slightly different between Th1 and Th2 cells (Fig. 1E). We also confirmed that Th1 cells, but not Th2 cells, bound P-selectin, as detected by the binding of P-selectin-IgM chimeric proteins (Fig. 1F).
To examine whether the differences in the effect of PSGL-1 cross-linking on the cell binding to ICAM-1 between unstimulated and differentiated CD4+ T cells are due to activation-dependent alterations in the signaling pathway, we tested freshly activated CD4+ T cells for their binding to ICAM-1. These cells exhibited high forward scatter and were high in CD25 and CD44 expression, confirming an activated status (Fig. 1E). The basal binding of freshly activated CD4+ T cells to ICAM-1 was markedly higher than that of unstimulated cells and differentiated Th1 and Th2 cells. Cross-linking PSGL-1 on these activated cells did not significantly enhance cell adhesion (Fig. 1D), suggesting that T cell activation alone is not sufficient to induce PSGL-1-mediated LFA-1 activation.
Cross-linking the PSGL-1 on Th1 cells increases their resistance to detachment from ICAM-1 under flow conditions
We next examined whether stimulation through PSGL-1 cross-linking would affect cell binding to ICAM-1 under flow conditions. When Th1 cells were infused into a capillary tube coated with ICAM-1-IgG at 1 dyn/cm2, a small population of cells bound without rolling, and only minimal binding to human IgG was observed (Fig. 2A). PSGL-1 cross-linking enhanced the cell binding to ICAM-1-IgG, reaching a 3.5-fold increase in the number of bound cells compared with non-cross-linked cells at 8 min after the start of recording (Fig. 2A). The Th1 cell binding to ICAM-1-IgG under flow conditions was also dependent on LFA-1, given that it was almost completely blocked by incubating the cross-linked cells with KBA (data not shown). We also assessed the overall strength of the Th1 cell adhesion to ICAM-1-IgG by increasing the shear stress stepwise to 20 dyn/cm2. Whereas 67% of the non-cross-linked cells remained adherent on ICAM-1-IgG at 20 dyn/cm2, 84% of the cross-linked cells remained bound at this level of shear stress (Fig. 2B). Thus, cross-linking PSGL-1 not only increased the number of cells accumulated on ICAM-1 but also significantly increased the cell resistance to detachment from ICAM-1 compared with non-cross-linked cells.
FIGURE 2. Cross-linking of PSGL-1 enhances the accumulation of Th1 cells on ICAM-1 under flow conditions. A, Accumulation of Th1 cells on ICAM-1 under flow conditions. Th1 cells were incubated with or without 2PH1 and a secondary Ab and infused into capillaries coated with ICAM-1-IgG or human IgG at a shear stress of 1 dyn/cm2. The number of cells bound to the capillaries was determined. One of three similar independent experiments is shown. B, Resistance of Th1 cells to detachment from ICAM-1. Th1 cells were incubated with or without 2PH1 and a secondary Ab and infused into ICAM-1-IgG-coated capillaries. The cells were allowed to accumulate at a shear stress of 0.25 dyn/cm2 for 8 min. The shear stress was then increased every 20 s to 20 dyn/cm2. The number of cells remaining bound at the end of each interval was determined and expressed as a percentage of the cells accumulated before applying increasing shear stress. Each data point represents the mean ± SEM from three independent experiments.
PSGL-1 cross-linking and chemokine stimulation induce a combined effect on the binding of Th1 cells to ICAM-1
During leukocyte migration to inflammatory sites, chemokines presented on the endothelium trigger the activation of integrins on rolling leukocytes, leading to the firm adhesion and subsequent transmigration of the leukocytes into tissues. We therefore examined the effect of combining PSGL-1 cross-linking with the addition of the Th1-stimulating chemokine CXCL10 or CCL5. We confirmed that in vitro-generated Th1 cells express mRNA for both CXCR3 and CCR5 (data not shown). Because chemokines stimulate integrins with very fast kinetics (28), short-term adhesion assays were performed to assess the effect of simultaneous stimulation by chemokines and cross-linking. Th1 cells were first incubated with 2PH1 for 30 min at 37°C, washed, and added to ICAM-1-IgG-coated microtiter plates, where the cells were stimulated with a secondary Ab and chemokine for 2 min at 37°C. This short-term cross-linking of PSGL-1 induced a 70% increase in Th1 cell adhesion to ICAM-1-IgG, compared with non-cross-linked cells (Fig. 3A). Stimulation of Th1 cells with CXCL10 alone at 1 μM induced a 30% increase in the number of bound cells (Fig. 3A). When cells were stimulated with 1 μM CXCL10 combined with PSGL-1 cross-linking, the number of bound cells increased by 175% compared with non-cross-linked cells (Fig. 3A). Similarly, when Th1 cells were stimulated with CCL5 alone, only a 25% increase in cell adhesion was seen at 1 μM, but a 140% increase was observed when the cells were stimulated simultaneously with 1 μM CCL5 and PSGL-1 cross-linking (Fig. 3B). The increase in adhesion by PSGL-1 cross-linking and chemokine stimulation was more than additive compared with activation by a single stimulus alone. These results indicate that PSGL-1 cross-linking and chemokine stimulation exert a combined effect on the Th1 cell adhesion to ICAM-1.
FIGURE 3. PSGL-1 cross-linking and the addition of Th1-stimulating chemokine exert a combined effect on Th1 cell adhesion to ICAM-1. BCECF-labeled Th1 cells were incubated with or without 2PH1 and added to ICAM-1-IgG-coated plates containing a secondary Ab and either CXCL10 (A) or CCL5 (B). The plates were incubated for 18 min on ice and then warmed in a 37°C water bath for 2 min. The plates were washed, and the fluorescence was determined. Data are expressed as the percentage of increase in the number of bound cells relative to the number of unstimulated cells bound. Each data point represents the mean ± SEM from triplicate wells. Each figure represents one of three similar experiments.
Cross-linking of PSGL-1 does not significantly affect soluble ICAM-1-IgG binding
Two major modes of LFA-1 activation have been demonstrated. One is an increase in affinity by conformational changes of LFA-1, and the other is an increase in lateral mobility, leading to the formation of clusters and increased avidity (29, 30). To clarify the mechanisms involved in the increase in Th1 cell adhesion by PSGL-1 cross-linking, we first examined whether PSGL-1 cross-linking would affect LFA-1 affinity using soluble ICAM-1-IgG (31). As shown in Fig. 4A, no significant alteration in LFA-1 expression on the cell surface was observed after cross-linking PSGL-1 on Th1 cells with 2PH1 and a secondary Ab. Although PSGL-1 cross-linking increased the binding of Th1 cells to ICAM-1-IgG immobilized on a plate (Fig. 1A), this treatment failed to induce the binding of soluble ICAM-1-IgG under the conditions used (Fig. 4B). In contrast, Mn2+, which is known to activate integrins, induced soluble ICAM-1-IgG binding to both unstimulated and PSGL-1-cross-linked cells (Fig. 4B). These results suggest that cross-linking of PSGL-1 did not detectably increase the LFA-1 affinity.
FIGURE 4. PSGL-1 cross-linking does not increase soluble ICAM-1-IgG binding. A, Expression of LFA-1 on Th1 cells that were incubated with or without 2PH1 and a secondary Ab. Th1 cells were incubated with or without 2PH1 and a secondary Ab and stained with FITC-anti-CD11a (shaded histogram) or isotype control (open histogram). B, Soluble ICAM-1-IgG binding to Th1 cells incubated with or without 2PH1 and a secondary Ab. Th1 cells were incubated with or without 2PH1 and a secondary Ab and stained with ICAM-I-IgG (shaded histogram) or human IgG (open histogram) followed by FITC-labeled anti-human IgG. Mn2+ was used for the detection of positive ICAM-1-IgG binding.
Cross-linking of PSGL-1 induces clustering of PSGL-1 and LFA-1
We next examined the distribution of PSGL-1 and LFA-1 on the cell surface following PSGL-1 cross-linking. PSGL-1 on untreated Th1 cells was evenly distributed over the entire cell surface with only a few small clusters (Fig. 5A). LFA-1 was also localized over the entire cell surface but formed more discrete clusters than did PSGL-1. Cross-linking PSGL-1 with Abs resulted in the clustering of PSGL-1 on almost all the cells, which formed a cap-like structure (Fig. 5D). Clustering of LFA-1 was also significantly increased after PSGL-1 cross-linking, with 53% of cells observed to carry clustered LFA-1, whereas 29% of untreated cells exhibited LFA-1 clustering (Fig. 5D and Table I). LFA-1 clustering was further enhanced by the combined stimulation with PSGL-1 cross-linking and either CXCL10 or CCL5 (Fig. 5, E and F, and Table I). When P-selectin-IgM chimeric protein and a secondary anti-human IgM Ab were used to cross-link PSGL-1, a similar combined effect on LFA-1 redistribution was observed (Fig. 5, G–I). In addition, as shown in the DIC images of Fig. 5, a change in cell morphology from a spherical to an elongated shape (marked by asterisks) was also enhanced by the combined stimulation. These results indicate that ligation of PSGL-1 enhances the clustering of LFA-1, which is further enhanced by the addition of chemokines. The parallel effects on cell adhesion to ICAM-1 and clustering of LFA-1 may suggest that enhanced LFA-1 clustering may at least in part account for the increased binding ability mediated by LFA-1.
FIGURE 5. Redistribution of PSGL-1 and LFA-1 by PSGL-1 cross-linking and chemokine stimulation. Th1 cells were incubated without Abs (A–C), with 2PH1 and Alexa Fluor 594-labeled anti-rat IgG (D–F), or with P-selectin-IgM (P-IgM) and Alexa Fluor 594-labeled anti-human IgM (G–I) in the absence (A, D, and G) or presence of either 1 μM CXCL10 (B, E, and H) or CCL5 (C, F, and I). Cells were fixed and then stained with FITC-anti-CD11a. Non-cross-linked cells (A–C) were stained for PSGL-1 using 2PH1 and Alexa Fluor 594-labeled anti-rat IgG after fixing the cells. PSGL-1 and LFA-1 staining, the corresponding DIC images, and the merged images are shown for each stimulus. Clustering of LFA-1 is marked by arrows. Elongated cells are marked by asterisks in DIC images.
Table I. Cell ellipticity and clustering of PSGL-1 and LFA-1 by PSGL-1 cross-linking and chemokine stimulationa
Th1 cell rolling on P-selectin increases cell binding to ICAM-1 under flow conditions
To evaluate the effect of PSGL-1 ligation on integrin-mediated cell adhesion in a more physiological setting, we examined the effect of PSGL-1-mediated rolling on Th1 cell adhesion to ICAM-1 under flow conditions. We used the flow assay using two tandem-connected capillary tubes, one coated with P-selectin-IgG or control human IgG and the other with ICAM-1-IgG, through which cells were infused at 1 dyn/cm2 (Fig. 6A). This assay enabled us to evaluate the effect of the initial P-selectin-dependent rolling on the subsequent cell adhesion to ICAM-1. Th1 cells rolled on P-selectin-IgG but not on control human IgG. They rolled continuously on P-selectin-IgG for at least 1 min through the observed area (1 mm long). Although some of the cells that passed through the human IgG-coated tube were arrested on ICAM-1-IgG, the number of cells accumulated on ICAM-1-IgG was dramatically increased when the cells first passed though the P-selectin-IgG-coated tube, where the cells rolled (Fig. 6B). The speed of the cells entering the ICAM-1-IgG-coated tube was equivalent between the cells that had passed through the P-selectin-IgG-coated tube and the human IgG-coated tube. The increase in cell binding after rolling on the P-selectin-IgG was almost completely abrogated by preincubating the cells with a polyclonal Ab against the N-terminal domain of PSGL-1 (Fig. 6B). The Ab directed against this region was shown to inhibit the binding of PSGL-1 to P-selectin (10). In contrast, an anti-PSGL-1 Ab directed against the cytoplasmic domain of PSGL-1, which does not inhibit the PSGL-1-P-selectin interaction, did not show an inhibitory effect (data not shown). These results confirm that the effect of rolling on P-selectin-IgG on cell adhesion to ICAM-1-IgG is mediated by PSGL-1 interacting with P-selectin. In addition, Th2 cells, which did not detectably roll on P-selectin, did not show an increase in cell arrest on ICAM-1-IgG (Fig. 6C), further supporting the idea that rolling on P-selectin activates cell adhesion to ICAM-1.
FIGURE 6. PSGL-1-mediated rolling enhances the Th1 cell accumulation on ICAM-1 under flow conditions. A, Schematic representation of the flow adhesion assays using tandem-connected capillaries. B, Th1 cell rolling on P-selectin-IgG enhanced the cell accumulation on ICAM-1. Th1 cells were preincubated with or without a blocking anti-PSGL-1 Ab and infused into the capillaries described in A at a shear stress of 1 dyn/cm2. The number of cells bound to the ICAM-IgG-coated capillaries was determined. One of three similar independent experiments is shown. C, Comparison of Th1 and Th2 cells for their accumulation on ICAM-1. Th1 and Th2 cells were infused into the capillaries described in A at a shear stress of 1 dyn/cm2. The number of cells bound to the ICAM-IgG-coated capillaries was determined. One of three similar independent experiments is shown.
PSGL-1-mediated stimulation of Th1 cell binding to ICAM-1 involves PKC pathways
To clarify the PSGL-1-mediated signaling pathways that lead to the enhanced cell binding to ICAM-1, we first tested the effect of various kinase inhibitors on PSGL-1-mediated stimulation of Th1 cell adhesion. Pretreatment of Th1 cells with staurosporine, a broad inhibitor of both serine/threonine protein kinases and tyrosine kinases, inhibited PSGL-1-mediated stimulation of cell adhesion by 90% (Fig. 7A). Pretreatment of the cells with calphostin C, a specific inhibitor of PKC, also inhibited cell adhesion by 80% (Fig. 7A). These results suggest that PKC-dependent pathways play an important role in PSGL-1-mediated LFA activation. In contrast, neither PD098059, a MAPK cascade inhibitor, nor LY294002, a specific PI3K inhibitor, affected PSGL-1-stimulated cell adhesion. Combined treatment of the cells with PD098059 and LY294002 slightly inhibited cell adhesion. Rottlerin partly inhibited cell adhesion at 4 μM, a concentration specific for PKC inhibition over other PKC isoenzymes, but markedly inhibited cell adhesion at 40 μM, a concentration that can inhibit PKC, PKC, and PKC. These results suggest that PKC pathways play a dominant role in signaling from PSGL-1 stimulation to LFA-1 activation, whereas MAPK and PI3K pathways play a minor role.
FIGURE 7. PKC activation is involved in PSGL-1-mediated stimulation of Th1 cell adhesion to ICAM-1. A, Binding of PSGL-1-cross-linked Th1 cells preincubated with kinase inhibitors. Th1 cells were incubated with 0.5 μM staurosporine (Sta), 0.1 μM calphostin C (Cal), 50 μM PD098059 (PD), 50 μM LY294002 (LY), or 4–40 μM rottlerin (Rot) at 37°C for 30 min before cross-linking PSGL-1. Cells bound to ICAM-IgG are expressed as a percentage of the untreated control. B, PKC activity in PSGL-1-cross-linked Th1 cells. Th1 cells were cross-linked for the indicated times, and the lysates were examined for PKC activity using Abs against pan-phospho-PKC, phospho-PKC/II, phospho-PKC (T505), phospho-PKC (S643), phospho-PKC, and phospho-PKC/. C, Effect of calphostin C on Th1 cell accumulation on ICAM-1 induced by rolling on P-selectin-IgG. Th1 cells were preincubated with 0.1 μM calphostin C or vehicle and infused into the capillaries described in Fig. 6A at a shear stress of 1 dyn/cm2. The number of cells bound to the ICAM-IgG-coated capillaries was determined. One of three similar independent experiments is shown.
We next examined whether PSGL-1 cross-linking would induce PKC activation. Although PSGL-1 cross-linking did not detectably up-regulate the phosphorylation of PKC detected by the activation-specific Ab against most isoforms of PKC, it induced activation of a species recognized by the Ab directed against phospho-PKC/II at 5 min (Fig. 7B). Up-regulation of phosphorylation of PKC, PKC, and PKC/ was not observed after PSGL-1 cross-linking. These results suggest that activation of PKC or PKCII is involved in PSGL-1-mediated signaling.
The involvement of PKC pathways in Th1 cell adhesion induced by PSGL-1-mediated rolling was also tested. Pretreatment of Th1 cells with calphostin C reduced the cell adhesion to ICAM-1 induced by PSGL-1-mediated rolling (Fig. 7C). Calphostin C showed no effect on the number and speed of Th1 cells rolling on P-selectin-IgG (data not shown). These results suggest that PKC pathways are major components of the intracellular signals from PSGL-1-mediated rolling to LFA-1 activation in Th1 cells.
Discussion
In this study, we investigated the role of PSGL-1-mediated signaling in integrin activation in T cells. We demonstrated that PSGL-1 cross-linking on Th1 cells enhances LFA-1-mediated cell adhesion to ICAM-1. PSGL-1 cross-linking induced the clustering of LFA-1 on the cell surface, suggesting that the increase in LFA-1 avidity may account for the enhanced adhesion to ICAM-1. Our data also show that PSGL-1 cross-linking combined with chemokine stimulation exerted a more than additive effect on LFA-1-mediated Th1 cell adhesion. In addition, we showed that PSGL-1-mediated rolling on P-selectin enhances Th1 cell accumulation on ICAM-1 under flow conditions. To our knowledge, this is the first study demonstrating that PSGL-1-mediated rolling directly induces integrin activation.
Signaling induced by P-selectin binding has been studied in different leukocyte types, including neutrophils, monocytes, and lymphocytes. Earlier reports showed inhibitory effects of P-selectin binding in neutrophils, such as the inhibition of 2 integrin-mediated binding (32) and superoxide generation (33). More recently, stimulatory effects of P-selectin via PSGL-1 on tyrosine phosphorylation and the activation of MAPKs in human neutrophils (21) and on cytokine release by human neutrophils and monocytes (21, 34) have been shown. The effect of PSGL-1 cross-linking on integrin activation has also been reported for mouse neutrophils (22).
Integrins are normally expressed on leukocytes in a state with a low ability to bind to ligands and require activation to initiate adhesion. One mechanism for integrin activation is an up-regulation of integrin affinity by conformational changes (29, 30). The other mechanism is an increase in integrin avidity, which occurs through integrin clustering in discrete areas of the plasma membrane. Chemokines can trigger the integrin-dependent adhesion of a number of leukocyte subtypes (5). CCL19, CCL21, and CXCL12 enhance both the affinity and the clustering of LFA-1 in naive lymphocytes (35). In our experiments, an alteration in LFA-1 affinity as detected by soluble ICAM-1-IgG binding was not observed after PSGL-1 cross-linking. In contrast, PSGL-1 cross-linking with an anti-PSGL-1 mAb or P-selectin-IgM followed by their respective secondary Abs induced the clustering of LFA-1. These results suggest that the mechanisms for enhanced Th1 cell binding to ICAM-1 induced by PSGL-1 cross-linking involve at least LFA-1 clustering. Our results also showed that the cross-linking of PSGL-1 on activated T cells, including Th1 and Th2 cells, but not unstimulated T cells, enhanced the cell binding to ICAM-1. Because unstimulated T cells express LFA-1 at a lower level than do activated T cells, the enhanced adhesion induced by the clustering of LFA-1 may require a certain level of LFA-1 expression. Our results also showed that the cross-linking of PSGL-1 on Th1 cells enhanced the binding of more cells compared with Th2 cells. Th1 cells express PSGL-1 and LFA-1 at a slightly higher level than Th2 cells, which may account for the differences in adhesion induced by PSGL-1 cross-linking in these two cell types. Alternatively, T cells may acquire or activate the signaling machinery required for PSGL-1-mediated integrin activation during cell activation and differentiation.
Early responses in leukocyte activation during their migration into tissues include a transition from a spherical to a polarized morphologic conformation. Chemokines cause the polarization of lymphocytes and the redistribution of several adhesion molecules to the uropod, a cytoplasmic projection that forms at the rear end of a moving cell (36). Previous reports have shown that PSGL-1 is redistributed to the uropod upon stimulation by chemoattractants in leukocytes (37, 38). Similarly, stimulation of neutrophils with fMLP induces the redistribution of 2 integrin Mac-1 to the uropod (39). Our results showed that PSGL-1 cross-linking induced polarization of the cell shape and clustering of both PSGL-1 and LFA-1, which were often, but not always, colocalized in one pole of the cell. These results show that not only chemokines but also PSGL-1 cross-linking can induce cell polarization and adhesion molecule redistribution, both of which are thought to be important for cell migration. Although the relationship between PSGL-1 and LFA-1 clustering is not clearly addressed in this study, it is likely that PSGL-1 clustering is a prerequisite for LFA-1 clustering, because the increase in LFA-1-dependent adhesion was not induced by incubation of the cells with 2PH1 alone, but required the secondary Ab. In support of this idea, a recent report suggests that the clustering of rolling receptors such as L-selectin and PSGL-1 transduces signals leading to 2 integrin clustering in neutrophils (40).
Th1 cells preferentially express the chemokine receptors CXCR3 and CCR5. Both CXCL10 and CCL5 have been implicated in the activation of the integrin-dependent adhesion of T cells (18, 19, 20). As expected, the stimulation of Th1 cells with CXCL10 or CCL5 enhanced cell binding to ICAM-1, which was accompanied by increased cell polarization and the redistribution of both LFA-1 and PSGL-1. Costimulation with chemokines and PSGL-1 cross-linking further enhanced the cell binding as well as LFA-1 and PSGL-1 clustering. Synergy between L-selectin cross-linking and chemokine stimulation for integrin-mediated cell adhesion has been reported (41). In this regard, it is of note that L-selectin stimulation enhances cell responses to CXCL12 by regulating the functional expression of surface CXCR4 in lymphocytes (42). It should be clarified whether similar mechanisms are involved in the combined effect of PSGL-1 cross-linking and chemokine stimulation observed in this study.
Ab-mediated cross-linking of PSGL-1 or P-selectin binding has been used as a model system to ligate PSGL-1 in vitro. However, PSGL-1 engagement in vivo most likely occurs during cell rolling on activated endothelium expressing P-selectin. Thus, we used a flow assay involving two tandem-connected capillary tubes, one coated with P-selectin-IgG and the other with ICAM-1-IgG. Our data showed that the number of cells that bound to ICAM-1 was dramatically increased when they first rolled on P-selectin, suggesting that PSGL-1-mediated rolling directly induces the intracellular signaling that leads to integrin activation. In addition, we showed that PKC pathways were involved in integrin activation induced by PSGL-1-mediated rolling.
In conclusion, our data show that Ab-mediated cross-linking of PSGL-1 or PSGL-1-mediated rolling on P-selectin stimulates the LFA-1-mediated binding of Th1 cells to ICAM-1. We also show that PSGL-1-mediated signaling functions cooperatively with that induced by chemokines to induce LFA-1 activation and that the signaling pathways from PSGL-1-mediated rolling to integrin activation involve at least PKC activation. Clarification of the PSGL-1-mediated signaling pathways leading to LFA-1 activation in Th1 cells will help us to understand and control the migration of Th1 cells under various inflammatory conditions.
Acknowledgments
We thank Drs. Bruce Furie and Barbara Furie for PSGL-1-deficient mice, Dr. John Lowe for selectin-IgM constructs, Dr. Yutaka Iigo for the ICAM-1-IgG construct, Dr. Hideo Yagita for the KBA mAb, Dr. Peter Smith for the protocol for selectin-IgM purification, and Drs. Toshiyuki Tanaka, Toshiyuki Murai, and Haruko Hayasaka for valuable comments. We also thank Shinobu Yamashita and Miyuki Komine for secretarial assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant-in-aid for the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
2 Address correspondence and reprint requests to Dr. Takako Hirata, Laboratory of Molecular and Cellular Recognition, Osaka University Graduate School of Medicine C8, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail address: thirata@orgctl.med.osaka-u.ac.jp
3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; PKC, protein kinase C; BCECF, 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; DIC, differential interference contrast.
Received for publication July 12, 2004. Accepted for publication November 16, 2004.
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Activated T cells migrate from the blood into nonlymphoid tissues through a multistep process that involves cell rolling, arrest, and transmigration. P-Selectin glycoprotein ligand-1 (PSGL-1) is a major ligand for P-selectin expressed on subsets of activated T cells such as Th1 cells and mediates cell rolling on vascular endothelium. Rolling cells are arrested through a firm adhesion step mediated by integrins. Although chemokines presented on the endothelium trigger integrin activation, a second mechanism has been proposed where signaling via rolling receptors directly activates integrins. In this study, we show that Ab-mediated cross-linking of the PSGL-1 on Th1 cells enhances LFA-1-dependent cell binding to ICAM-1. PSGL-1 cross-linking did not enhance soluble ICAM-1 binding but induced clustering of LFA-1 on the cell surface, suggesting that an increase in LFA-1 avidity may account for the enhanced binding to ICAM-1. Combined stimulation by PSGL-1 cross-linking and the Th1-stimulating chemokine CXCL10 orCCL5 showed a more than additive effect on LFA-1-mediated Th1 cell adhesion as well as on LFA-1 redistribution on the cell surface. Moreover, PSGL-1-mediated rolling on P-selectin enhanced the Th1 cell accumulation on ICAM-1 under flow conditions. PSGL-1 cross-linking induced activation of protein kinase C isoforms, and the increased Th1 cell adhesion observed under flow and also static conditions was strongly inhibited by calphostin C, implicating protein kinase C in the intracellular signaling in PSGL-1-mediated LFA-1 activation. These results support the idea that PSGL-1-mediated rolling interactions induce intracellular signals leading to integrin activation, facilitating Th1 cell arrest and subsequent migration into target tissues.
Introduction
In response to inflammatory stimuli, leukocytes migrate from the blood into tissues through several sequential adhesive steps (1). Initially, leukocytes are captured from the bloodstream by adhesion receptors through rapid and transient interactions. These interactions are primarily mediated by the following selectins: L-selectin (CD62L), which is expressed on most leukocytes, and E-selectin (CD62E) and P-selectin (CD62P), which are expressed on activated endothelium. The relatively low-affinity interactions mediated by the selectins and their ligands allow leukocytes to roll along the vascular surface under flow conditions (2, 3, 4). To migrate into tissues, rolling leukocytes must stop through a firm adhesion step, which is mediated by integrins. LFA-1 (CD11a/CD18, L2) and Mac-1 (CD11b/CD18, M2) represent the major 2 integrins on leukocytes that mediate their binding to ICAMs, the Ig superfamily integrin ligands (1). Among the ICAMs, ICAM-1 (CD54) serves as the predominant ligand for LFA-1 and Mac-1 and is up-regulated on endothelial cells during inflammation.
2 integrins are constitutively expressed on leukocytes and require activation to initiate adhesion. Various chemokines presented on the endothelium, where leukocytes roll, trigger rapid integrin activation through Gi-linked receptors in a number of leukocyte types (5). A second mechanism has been proposed in which signaling mediated by rolling receptors directly activates integrins. For example, the cross-linking of L-selectin on neutrophils up-regulates the adhesive function of Mac-1 (6). Stimulation of L-selectin via glycosylation-dependent cell adhesion molecule-1, a soluble ligand for L-selectin, also stimulates the LFA-1-mediated binding of naive lymphocytes to ICAM-1 (7).
P-selectin glycoprotein ligand-1 (PSGL-13; CD162) is a major P-selectin ligand on myeloid cells and subsets of lymphoid cells and mediates the tethering and rolling of leukocytes on the vascular endothelium (8). The physiologic role of PSGL-1 as the predominant P-selectin ligand has been clearly demonstrated by the deficient P-selectin-mediated rolling in PSGL-1-deficient mice (9). PSGL-1 can also bind to E-selectin and L-selectin, and the physiologic roles of these interactions have been shown in vivo (10, 11, 12). To bind selectins, PSGL-1 requires specific core 2-type O-glycans containing the sialyl LewisX moiety (13). The expression of some glycosyltransferases involved in the synthesis of selectin-binding glycans, including core 2 -1,6-N-acetylglucosaminyltransferase and -1,3-fucosyltransferase, is dynamically regulated in lymphocytes during activation and differentiation. Thus, despite the fact that virtually all T cells express PSGL-1, only particular subsets of T cells express a form of PSGL-1 that can bind selectins. Notably, in vitro-differentiated Th1 cells express PSGL-1 in a selectin-binding form (10, 14).
In contrast to naive T cells, which traffic through secondary lymphoid organs, activated T cells can migrate into nonlymphoid tissues through a multistep process similar to that used by neutrophils and monocytes. The migration of Th1 cells into the inflamed skin in a contact hypersensitivity model is dependent on P-selectin and E-selectin (15), and PSGL-1 is the major ligand for P-selectin as well as one of the ligands for E-selectin in this process (10, 14). Th1 cells also exhibit a preferential expression of chemokine receptors, typically CXCR3 and CCR5, which may be responsible for the distinct migration properties of these cells (16, 17). A CXCR3 ligand, CXCL10, and a CCR5 ligand, CCL5, have been implicated in integrin-mediated T cell adhesion (18, 19, 20).
Although the role of PSGL-1 as a rolling receptor is well established, less is known about its role as a signal-transducing molecule. Previous studies have demonstrated signaling events that are induced by PSGL-1 ligation via P-selectin binding or Ab-mediated cross-linking. PSGL-1 ligation enhances tyrosine phosphorylation and activates MAPKs in human neutrophils as well as cytokine release by neutrophils and monocytes (21). Cross-linking of PSGL-1 on mouse neutrophils with an Ab-like chimeric protein of P-selectin increases their binding to ICAM-1-transfected Chinese hamster ovary cells through LFA-1 and Mac-1 (22). Signaling events induced by P-selectin binding in T cells have also been reported. P-Selectin in conjunction with an anti-TCR mAb augments the production of GM-CSF by Ag-primed human T cells (23). P-Selectin binding also induces the phosphorylation of several substrates, including focal adhesion kinase and Syk, in T cells (24, 25). Although Ab-mediated PSGL-1 cross-linking and P-selectin binding have been used to examine PSGL-1-induced cellular signaling, the effect of PSGL-1 ligation under physiological conditions, i.e., PSGL-1-mediated rolling, on intracellular signaling remains unknown.
In this study, we report that cross-linking of PSGL-1 on Th1 cells enhances their LFA-1-mediated binding to ICAM-1, which may be attributed in part to the clustering of LFA-1 on the cell surface. We show that PSGL-1 cross-linking combined with chemokine stimulation exerts a more than additive effect on LFA-1-mediated Th1 cell adhesion. In addition, we show that PSGL-1-mediated rolling on P-selectin dramatically enhances the Th1 cell accumulation on ICAM-1 under flow conditions. The enhanced cell accumulation is inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC), implicating PKC pathways in the intracellular signaling from PSGL-1-mediated rolling to LFA-1 activation. To our knowledge, this is the first demonstration of a role for PSGL-1-mediated rolling in triggering integrin activation.
Materials and Methods
Mice
C57BL/6J (B6) mice were purchased from CLEA Japan. PSGL-1-deficient mice on a B6 background (9, 10) were provided by Dr. B. Furie (Harvard Medical School, Boston, MA). All mice used were 6–12 wk old. The mice were housed at the Institute of Experimental Animal Sciences at Osaka University Medical School. All studies and procedures were approved by the Ethics Review Committee for Animal Experimentation of the Osaka University Graduate School of Medicine.
Chimeric proteins
The expression plasmid for mouse P-selectin-human IgM chimeric protein (P-selectin-IgM) was provided by Dr. J. Lowe (University of Michigan Medical School, Ann Arbor, MI). The plasmid was transfected into COS-7 cells using DEAE-dextran. Chimeric proteins were purified from culture supernatants using biotinylated anti-human IgM (American Qualex) bound to avidin-agarose (Pierce Biotechnology) and concentrated using Centriplus YM-30 (Millipore). Mouse P-selectin-human IgG chimeric protein (P-selectin-IgG) was purified using protein A-Sepharose (Amersham Biosciences) as described previously (10). The expression plasmid for rat ICAM-1-human IgG chimeric protein (ICAM-1-IgG) was provided by Dr. Y. Iigo (Daiichi Pharmaceutical, Tokyo, Japan). The plasmid was transfected into COS-7 cells, and the chimeric protein was purified as described previously (26).
Generation of Th1 and Th2 cells
Splenic CD4+ T cells were isolated by autoMACS (Miltenyi Biotec) using biotinylated anti-CD11b (M1/70; BD Biosciences), biotinylated anti-B220 (RA3-6B2; BD Biosciences), and biotinylated anti-CD8 (53-6.7; Southern Biotechnology Associates) followed by Streptavidin MicroBeads (Miltenyi Biotec). The enriched population was 88–95% positive for CD4 staining. Purified CD4+ T cells were cultured on 24-well tissue culture plates coated with 10 μg/ml anti-CD3 (145-2C11; BD Biosciences) and 10 μg/ml anti-CD28 (37.51; BD Biosciences) for 2 days in the presence of 4 ng/ml IL-2 (R&D Systems) and either 8 ng/ml IL-12 (R&D Systems) and 0.2 μg/ml anti-IL-4 (11B11; BD Biosciences) to promote Th1 differentiation, or 100 ng/ml IL-4 (Genzyme) to promote Th2 differentiation. The cells were then transferred to uncoated plates and cultured for an additional 3 days. Freshly activated cells were prepared by stimulating MACS-isolated CD4+ T cells with plate-bound anti-CD3 and anti-CD28 in the presence of IL-2 for 2 days.
Flow cytometry
All mAbs used for the flow cytometric analyses were purchased from BD Biosciences. They included anti-CD4-FITC (RM4-5), anti-CD11a-FITC (M17/4), anti-CD25-FITC (7D4), anti-CD44-FITC (IM7), anti-CD62L-FITC (MEL-14), and anti-CD162-PE (2PH1). Cells were stained with mAbs for 30 min on ice, washed, and analyzed on an EPICS XL flow cytometer (Beckman Coulter). To assess the ICAM-1-IgG binding by flow cytometry, cells were incubated with 40 μg/ml ICAM-1-IgG for 30 min on ice, washed, and then incubated with FITC-labeled anti-human IgG (Biomeda) for 30 min on ice. To assess the P-selectin-IgM binding, cells were incubated with a COS-7 supernatant containing P-selectin-IgM, washed, and then incubated with biotinylated anti-human IgM. The cells were then washed and stained with streptavidin-PE (BD Biosciences).
Cell adhesion assays under static conditions
ICAM-1-IgG or control human IgG (Sigma-Aldrich) was immobilized on Costar 3690 plates (5 μg/ml, 25 μl/well) at 4°C overnight, and the plates were blocked with FCS at 37°C for 2 h. The cells were labeled with 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes) at 37°C for 30 min, washed, and resuspended in RPMI 1640 with 10 mM HEPES (RPMI-HEPES). To cross-link PSGL-1 with Abs, the labeled cells were incubated with 5 μg/ml anti-PSGL-1 mAb 2PH1 or control rat IgG1 (both from BD Biosciences) at 37°C for 30 min, washed, and then incubated with 50 μg/ml goat F(ab')2 anti-rat IgG (Immunotech) at 37°C for 30 min. The cells were washed and resuspended at 4 x 106/ml in RPMI-HEPES and 10% FCS (RPMI-HEPES/FCS), added to the plate, and incubated for 20 min on ice. In some experiments, PSGL-1-cross-linked cells were preincubated with an anti-LFA-1 mAb KBA (27) or its isotype control rat IgG2a (Zymed) before addition to the plate. The plates were filled with RPMI-HEPES/FCS, inverted, and incubated at 37°C for 30 min. Supernatants were aspirated, and bound cells were lysed with 0.1% Igepal CA-630 (Sigma-Aldrich) in PBS. Fluorescence intensity was measured using a SpectraMax Gemini XS (Molecular Devices). To assess the effect of kinase inhibitors on cell adhesion, BCECF-labeled cells were treated with 0.5 μM staurosporine, 0.1 μM calphostin C, 50 μM LY294002, 50 μM PD098059, or 4–40 μM rottlerin (all from Sigma-Aldrich) at 37°C for 30 min before cross-linking PSGL-1.
For short-term cell adhesion assays, BCECF-labeled Th1 cells were incubated with 2PH1 (5 μg/ml) at 37°C for 30 min and resuspended at 4 x 106 cells/ml in RPMI-HEPES/FCS. Secondary Abs (20 μg/ml) and chemokines at several concentrations in RPMI-HEPES/FCS were added to plates (5 μl/well) coated with ICAM-IgG or human IgG. The cells were then added to the plates (25 μl/well), incubated for 18 min on ice, and then rapidly warmed in a 37°C water bath for 2 min. The plates were washed three times with RPMI-HEPES, and the bound cells were quantified as described above.
Cell adhesion assays under flow conditions
ICAM-1-IgG or control human IgG (100 μg/ml) was immobilized on the inside walls of glass capillaries (inner diameter, 0.69 mm; Drummond Scientific) at 4°C overnight. The capillaries were then blocked with FCS for 5 min at room temperature. PSGL-1 on Th1 cells was cross-linked with 2PH1 and a secondary Ab. The cells were resuspended at 1 x 106 cells/ml in RPMI-HEPES/FCS and infused into the capillaries mounted on the stage of an inverted microscope (Diaphot 300; Nikon) at a shear force of 1 dyn/cm2. The rate of flow was controlled by a PHD 2000 syringe pump (Harvard Apparatus). Four minutes after the start of infusion, cell images were recorded with a cell-viewing system (SRM-100; Nikon) and video recorder (BR-S600; Victor). For the detachment assays, cells were infused at 0.25 dyn/cm2 for 8 min, and the shear stress was increased stepwise every 20 s until it reached 20 dyn/cm2. At the end of each shear stress treatment, the number of cells that remained bound was determined.
To examine the effect of P-selectin-dependent rolling of Th1 cells on their adhesion to ICAM-1, P-selectin-IgG or control human IgG (5 μg/ml) was immobilized on the inside walls of capillaries at 4°C overnight. The capillaries were then blocked with FCS for 5 min at room temperature. A capillary coated with P-selectin-IgG or human IgG was connected in tandem with an ICAM-1-IgG-coated capillary and washed with 2 ml of RPMI-HEPES/FCS at 10 dyn/cm2. Th1 or Th2 cells were resuspended at 8 x 105 cells/ml in RPMI-HEPES/FCS, and 1 ml of cell suspension was infused into the capillaries at 1 dyn/cm2. In some experiments, the cells were preincubated with polyclonal Abs raised against the N-terminal region (QVVGDDDFEDPDYTY) or cytoplasmic region (EPSGDRDGDDLTLHSFLP) of PSGL-1. The cells were also preincubated with 0.1 μM calphostin C or vehicle. Four minutes after the start of the infusion, cell images were recorded for 10 min, and the bound cells were counted.
Immunocytochemical staining
Th1 cells were incubated with 5 μg/ml 2PH1 or 20 μg/ml P-selectin-IgM at 37°C for 30 min. The cells were then incubated at 37°C for 30 min with 20 μg/ml goat F(ab')2 anti-rat IgG or goat F(ab')2 anti-human IgM (OEM Concepts), both conjugated with Alexa Fluor 594 using the Alexa Fluor 594 mAb labeling kit (Molecular Probes). PSGL-1-cross-linked cells were fixed with 1% paraformaldehyde for 15 min at room temperature. To block the nonspecific binding of FITC-labeled Abs, cells were incubated with 200 μg/ml rat IgG for 30 min on ice. The cells were stained with 10 μg/ml FITC-labeled anti-CD11a or control FITC-labeled rat IgG2a for 30 min on ice. The cells were washed, resuspended in ProLong Antifade (Molecular Probes), and transferred onto slides. The cells were observed under a confocal microscope (LSM 510 UV/META; Carl Zeiss).
To assess changes in cell morphology and staining patterns, 100 cells in each sample were evaluated using differential interference contrast (DIC) and confocal images. Cells with >1.3 ellipticity (the ratio between the longest and shortest axes) were considered to exhibit the morphological change, and cells with <30% of their surface showing condensed staining were considered to show the redistribution of cell surface molecules.
Western blotting
Th1 cells were incubated with 5 μg/ml 2PH1 at 37°C for 30 min and then incubated with 50 μg/ml goat F(ab')2 anti-rat IgG at 37°C for the indicated times. The cells were lysed in a buffer containing 1% Triton X-100, 25 mM HEPES (pH 7.5), 150 mM NaCl, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF, 1 mM EDTA, and 1 mM Na3VO4 for 30 min, and then cleared by centrifugation. The lysates were resolved by SDS-PAGE under reducing conditions, and the proteins were transferred to an Immobilon-P membrane (Millipore). The membranes were probed with Abs against various phosphorylated PKC isoforms from phospho-PKC Ab sampler kit (Cell Signaling Technology).
Results
Cross-linking of PSGL-1 on Th1 cells stimulates cell binding to ICAM-1
To examine the effect on integrin activation of the ligation of the PSGL-1 on T cells, we first tested the adhesion of in vitro-generated Th1 cells to ICAM-1-IgG immobilized on 96-well microtiter plates after cross-linking PSGL-1. To cross-link PSGL-1, Th1 cells were incubated with an anti-PSGL-1 mAb, 2PH1, for 30 min at 37°C, washed, and then incubated for 30 min at 37°C with a secondary goat F(ab')2 anti-rat IgG. The cross-linking of PSGL-1 on Th1 cells using 2PH1 and a secondary Ab induced a 10-fold increase in the number of Th1 cells bound to ICAM-1-IgG compared with cells that were not incubated with any Abs (Fig. 1A). No significant increase was observed in the Th1 cells binding to control human IgG after cross-linking. Incubation of Th1 cells with 2PH1 alone, a secondary Ab alone, or an isotype-matched control rat IgG followed by a secondary Ab did not significantly affect the Th1 cell binding to ICAM-1-IgG (Fig. 1A).
FIGURE 1. PSGL-1 cross-linking enhances the binding of Th1 cells to ICAM-1. A, Incubation of Th1 cells with 2PH1 and a secondary Ab (PSGL-1 cross-linking) enhances the cell binding to ICAM-1. In vitro-generated Th1 cells were labeled with BCECF-AM. The labeled cells were incubated with or without 2PH1 or control rat IgG, washed, and then incubated with or without a secondary goat F(ab')2 anti-rat IgG. The cells were added to 96-well plates coated with ICAM-IgG or human IgG. The plates were incubated for 20 min, unbound cells were removed, and the fluorescence per well was determined. Bound cells are expressed as a percentage of the total cells added. B, Binding of PSGL-1-cross-linked Th1 cells treated with an anti-LFA-1 mAb to ICAM-1. Th1 cells that had been incubated with 2PH1 and a secondary Ab were treated with either the anti-LFA-1 mAb KBA or its isotype control for 30 min before addition to the wells. Cells bound to ICAM-IgG are expressed as a percentage of the untreated control. C, Binding of PSGL-1-deficient Th1 cells to ICAM-1. PSGL-1-deficient (KO) or wild-type (WT) Th1 cells were incubated with or without 2PH1 and a secondary Ab and added to plates coated with ICAM-IgG or human IgG. D, Binding of unstimulated CD4+ T, freshly activated CD4+ T, Th1, and Th2 cells to ICAM-1. Freshly isolated or freshly activated CD4+ T cells or in vitro-generated Th1 or Th2 cells were incubated with or without 2PH1 and a secondary Ab and added to plates coated with ICAM-IgG or human IgG. Each data point represents the mean ± SEM from triplicate wells. E, Expression of cell surface markers on unstimulated CD4+ T, freshly activated CD4+ T, Th1, and Th2 cells. Freshly isolated or freshly activated CD4+ T cells or in vitro-generated Th1 or Th2 cells were stained with the indicated mAbs (shaded histograms) or isotype-matched control IgGs (open histograms) and analyzed by flow cytometry. Mean fluorescence intensity values for PSGL-1 and LFA-1 are also indicated. F, P-selectin-IgM binding of Th1 and Th2 cells. Cells were incubated with P-selectin-IgM (shaded histograms) or control human IgM (open histograms). Bound P-selectin-IgM was detected using biotinylated anti-human IgM and streptavidin-PE.
The binding of cross-linked Th1 cells to ICAM-1-IgG was dependent on LFA-1, given that it was almost completely blocked by the incubation of the cross-linked cells with the LFA-1-blocking mAb KBA, but not with a control rat IgG (Fig. 1B). Incubation of Th1 cells from PSGL-1-deficient mice with 2PH1 and a secondary Ab did not induce an increase in the number of cells bound to ICAM-1-IgG, confirming that the effect induced by cross-linking was mediated by PSGL-1 (Fig. 1C). Cross-linking PSGL-1 on unstimulated CD4+ T cells by 2PH1 and a secondary Ab did not show an increase in the number of bound cells (Fig. 1D). These cells expressed both PSGL-1 and LFA-1, albeit at a lower level than did Th1 and Th2 cells (Fig. 1E). In contrast, cross-linking PSGL-1 on Th2 cells enhanced their binding, although the number of bound cells was 40% of that of Th1 cells (Fig. 1D). The level of expression of PSGL-1 and LFA-1 on the cell surface was only slightly different between Th1 and Th2 cells (Fig. 1E). We also confirmed that Th1 cells, but not Th2 cells, bound P-selectin, as detected by the binding of P-selectin-IgM chimeric proteins (Fig. 1F).
To examine whether the differences in the effect of PSGL-1 cross-linking on the cell binding to ICAM-1 between unstimulated and differentiated CD4+ T cells are due to activation-dependent alterations in the signaling pathway, we tested freshly activated CD4+ T cells for their binding to ICAM-1. These cells exhibited high forward scatter and were high in CD25 and CD44 expression, confirming an activated status (Fig. 1E). The basal binding of freshly activated CD4+ T cells to ICAM-1 was markedly higher than that of unstimulated cells and differentiated Th1 and Th2 cells. Cross-linking PSGL-1 on these activated cells did not significantly enhance cell adhesion (Fig. 1D), suggesting that T cell activation alone is not sufficient to induce PSGL-1-mediated LFA-1 activation.
Cross-linking the PSGL-1 on Th1 cells increases their resistance to detachment from ICAM-1 under flow conditions
We next examined whether stimulation through PSGL-1 cross-linking would affect cell binding to ICAM-1 under flow conditions. When Th1 cells were infused into a capillary tube coated with ICAM-1-IgG at 1 dyn/cm2, a small population of cells bound without rolling, and only minimal binding to human IgG was observed (Fig. 2A). PSGL-1 cross-linking enhanced the cell binding to ICAM-1-IgG, reaching a 3.5-fold increase in the number of bound cells compared with non-cross-linked cells at 8 min after the start of recording (Fig. 2A). The Th1 cell binding to ICAM-1-IgG under flow conditions was also dependent on LFA-1, given that it was almost completely blocked by incubating the cross-linked cells with KBA (data not shown). We also assessed the overall strength of the Th1 cell adhesion to ICAM-1-IgG by increasing the shear stress stepwise to 20 dyn/cm2. Whereas 67% of the non-cross-linked cells remained adherent on ICAM-1-IgG at 20 dyn/cm2, 84% of the cross-linked cells remained bound at this level of shear stress (Fig. 2B). Thus, cross-linking PSGL-1 not only increased the number of cells accumulated on ICAM-1 but also significantly increased the cell resistance to detachment from ICAM-1 compared with non-cross-linked cells.
FIGURE 2. Cross-linking of PSGL-1 enhances the accumulation of Th1 cells on ICAM-1 under flow conditions. A, Accumulation of Th1 cells on ICAM-1 under flow conditions. Th1 cells were incubated with or without 2PH1 and a secondary Ab and infused into capillaries coated with ICAM-1-IgG or human IgG at a shear stress of 1 dyn/cm2. The number of cells bound to the capillaries was determined. One of three similar independent experiments is shown. B, Resistance of Th1 cells to detachment from ICAM-1. Th1 cells were incubated with or without 2PH1 and a secondary Ab and infused into ICAM-1-IgG-coated capillaries. The cells were allowed to accumulate at a shear stress of 0.25 dyn/cm2 for 8 min. The shear stress was then increased every 20 s to 20 dyn/cm2. The number of cells remaining bound at the end of each interval was determined and expressed as a percentage of the cells accumulated before applying increasing shear stress. Each data point represents the mean ± SEM from three independent experiments.
PSGL-1 cross-linking and chemokine stimulation induce a combined effect on the binding of Th1 cells to ICAM-1
During leukocyte migration to inflammatory sites, chemokines presented on the endothelium trigger the activation of integrins on rolling leukocytes, leading to the firm adhesion and subsequent transmigration of the leukocytes into tissues. We therefore examined the effect of combining PSGL-1 cross-linking with the addition of the Th1-stimulating chemokine CXCL10 or CCL5. We confirmed that in vitro-generated Th1 cells express mRNA for both CXCR3 and CCR5 (data not shown). Because chemokines stimulate integrins with very fast kinetics (28), short-term adhesion assays were performed to assess the effect of simultaneous stimulation by chemokines and cross-linking. Th1 cells were first incubated with 2PH1 for 30 min at 37°C, washed, and added to ICAM-1-IgG-coated microtiter plates, where the cells were stimulated with a secondary Ab and chemokine for 2 min at 37°C. This short-term cross-linking of PSGL-1 induced a 70% increase in Th1 cell adhesion to ICAM-1-IgG, compared with non-cross-linked cells (Fig. 3A). Stimulation of Th1 cells with CXCL10 alone at 1 μM induced a 30% increase in the number of bound cells (Fig. 3A). When cells were stimulated with 1 μM CXCL10 combined with PSGL-1 cross-linking, the number of bound cells increased by 175% compared with non-cross-linked cells (Fig. 3A). Similarly, when Th1 cells were stimulated with CCL5 alone, only a 25% increase in cell adhesion was seen at 1 μM, but a 140% increase was observed when the cells were stimulated simultaneously with 1 μM CCL5 and PSGL-1 cross-linking (Fig. 3B). The increase in adhesion by PSGL-1 cross-linking and chemokine stimulation was more than additive compared with activation by a single stimulus alone. These results indicate that PSGL-1 cross-linking and chemokine stimulation exert a combined effect on the Th1 cell adhesion to ICAM-1.
FIGURE 3. PSGL-1 cross-linking and the addition of Th1-stimulating chemokine exert a combined effect on Th1 cell adhesion to ICAM-1. BCECF-labeled Th1 cells were incubated with or without 2PH1 and added to ICAM-1-IgG-coated plates containing a secondary Ab and either CXCL10 (A) or CCL5 (B). The plates were incubated for 18 min on ice and then warmed in a 37°C water bath for 2 min. The plates were washed, and the fluorescence was determined. Data are expressed as the percentage of increase in the number of bound cells relative to the number of unstimulated cells bound. Each data point represents the mean ± SEM from triplicate wells. Each figure represents one of three similar experiments.
Cross-linking of PSGL-1 does not significantly affect soluble ICAM-1-IgG binding
Two major modes of LFA-1 activation have been demonstrated. One is an increase in affinity by conformational changes of LFA-1, and the other is an increase in lateral mobility, leading to the formation of clusters and increased avidity (29, 30). To clarify the mechanisms involved in the increase in Th1 cell adhesion by PSGL-1 cross-linking, we first examined whether PSGL-1 cross-linking would affect LFA-1 affinity using soluble ICAM-1-IgG (31). As shown in Fig. 4A, no significant alteration in LFA-1 expression on the cell surface was observed after cross-linking PSGL-1 on Th1 cells with 2PH1 and a secondary Ab. Although PSGL-1 cross-linking increased the binding of Th1 cells to ICAM-1-IgG immobilized on a plate (Fig. 1A), this treatment failed to induce the binding of soluble ICAM-1-IgG under the conditions used (Fig. 4B). In contrast, Mn2+, which is known to activate integrins, induced soluble ICAM-1-IgG binding to both unstimulated and PSGL-1-cross-linked cells (Fig. 4B). These results suggest that cross-linking of PSGL-1 did not detectably increase the LFA-1 affinity.
FIGURE 4. PSGL-1 cross-linking does not increase soluble ICAM-1-IgG binding. A, Expression of LFA-1 on Th1 cells that were incubated with or without 2PH1 and a secondary Ab. Th1 cells were incubated with or without 2PH1 and a secondary Ab and stained with FITC-anti-CD11a (shaded histogram) or isotype control (open histogram). B, Soluble ICAM-1-IgG binding to Th1 cells incubated with or without 2PH1 and a secondary Ab. Th1 cells were incubated with or without 2PH1 and a secondary Ab and stained with ICAM-I-IgG (shaded histogram) or human IgG (open histogram) followed by FITC-labeled anti-human IgG. Mn2+ was used for the detection of positive ICAM-1-IgG binding.
Cross-linking of PSGL-1 induces clustering of PSGL-1 and LFA-1
We next examined the distribution of PSGL-1 and LFA-1 on the cell surface following PSGL-1 cross-linking. PSGL-1 on untreated Th1 cells was evenly distributed over the entire cell surface with only a few small clusters (Fig. 5A). LFA-1 was also localized over the entire cell surface but formed more discrete clusters than did PSGL-1. Cross-linking PSGL-1 with Abs resulted in the clustering of PSGL-1 on almost all the cells, which formed a cap-like structure (Fig. 5D). Clustering of LFA-1 was also significantly increased after PSGL-1 cross-linking, with 53% of cells observed to carry clustered LFA-1, whereas 29% of untreated cells exhibited LFA-1 clustering (Fig. 5D and Table I). LFA-1 clustering was further enhanced by the combined stimulation with PSGL-1 cross-linking and either CXCL10 or CCL5 (Fig. 5, E and F, and Table I). When P-selectin-IgM chimeric protein and a secondary anti-human IgM Ab were used to cross-link PSGL-1, a similar combined effect on LFA-1 redistribution was observed (Fig. 5, G–I). In addition, as shown in the DIC images of Fig. 5, a change in cell morphology from a spherical to an elongated shape (marked by asterisks) was also enhanced by the combined stimulation. These results indicate that ligation of PSGL-1 enhances the clustering of LFA-1, which is further enhanced by the addition of chemokines. The parallel effects on cell adhesion to ICAM-1 and clustering of LFA-1 may suggest that enhanced LFA-1 clustering may at least in part account for the increased binding ability mediated by LFA-1.
FIGURE 5. Redistribution of PSGL-1 and LFA-1 by PSGL-1 cross-linking and chemokine stimulation. Th1 cells were incubated without Abs (A–C), with 2PH1 and Alexa Fluor 594-labeled anti-rat IgG (D–F), or with P-selectin-IgM (P-IgM) and Alexa Fluor 594-labeled anti-human IgM (G–I) in the absence (A, D, and G) or presence of either 1 μM CXCL10 (B, E, and H) or CCL5 (C, F, and I). Cells were fixed and then stained with FITC-anti-CD11a. Non-cross-linked cells (A–C) were stained for PSGL-1 using 2PH1 and Alexa Fluor 594-labeled anti-rat IgG after fixing the cells. PSGL-1 and LFA-1 staining, the corresponding DIC images, and the merged images are shown for each stimulus. Clustering of LFA-1 is marked by arrows. Elongated cells are marked by asterisks in DIC images.
Table I. Cell ellipticity and clustering of PSGL-1 and LFA-1 by PSGL-1 cross-linking and chemokine stimulationa
Th1 cell rolling on P-selectin increases cell binding to ICAM-1 under flow conditions
To evaluate the effect of PSGL-1 ligation on integrin-mediated cell adhesion in a more physiological setting, we examined the effect of PSGL-1-mediated rolling on Th1 cell adhesion to ICAM-1 under flow conditions. We used the flow assay using two tandem-connected capillary tubes, one coated with P-selectin-IgG or control human IgG and the other with ICAM-1-IgG, through which cells were infused at 1 dyn/cm2 (Fig. 6A). This assay enabled us to evaluate the effect of the initial P-selectin-dependent rolling on the subsequent cell adhesion to ICAM-1. Th1 cells rolled on P-selectin-IgG but not on control human IgG. They rolled continuously on P-selectin-IgG for at least 1 min through the observed area (1 mm long). Although some of the cells that passed through the human IgG-coated tube were arrested on ICAM-1-IgG, the number of cells accumulated on ICAM-1-IgG was dramatically increased when the cells first passed though the P-selectin-IgG-coated tube, where the cells rolled (Fig. 6B). The speed of the cells entering the ICAM-1-IgG-coated tube was equivalent between the cells that had passed through the P-selectin-IgG-coated tube and the human IgG-coated tube. The increase in cell binding after rolling on the P-selectin-IgG was almost completely abrogated by preincubating the cells with a polyclonal Ab against the N-terminal domain of PSGL-1 (Fig. 6B). The Ab directed against this region was shown to inhibit the binding of PSGL-1 to P-selectin (10). In contrast, an anti-PSGL-1 Ab directed against the cytoplasmic domain of PSGL-1, which does not inhibit the PSGL-1-P-selectin interaction, did not show an inhibitory effect (data not shown). These results confirm that the effect of rolling on P-selectin-IgG on cell adhesion to ICAM-1-IgG is mediated by PSGL-1 interacting with P-selectin. In addition, Th2 cells, which did not detectably roll on P-selectin, did not show an increase in cell arrest on ICAM-1-IgG (Fig. 6C), further supporting the idea that rolling on P-selectin activates cell adhesion to ICAM-1.
FIGURE 6. PSGL-1-mediated rolling enhances the Th1 cell accumulation on ICAM-1 under flow conditions. A, Schematic representation of the flow adhesion assays using tandem-connected capillaries. B, Th1 cell rolling on P-selectin-IgG enhanced the cell accumulation on ICAM-1. Th1 cells were preincubated with or without a blocking anti-PSGL-1 Ab and infused into the capillaries described in A at a shear stress of 1 dyn/cm2. The number of cells bound to the ICAM-IgG-coated capillaries was determined. One of three similar independent experiments is shown. C, Comparison of Th1 and Th2 cells for their accumulation on ICAM-1. Th1 and Th2 cells were infused into the capillaries described in A at a shear stress of 1 dyn/cm2. The number of cells bound to the ICAM-IgG-coated capillaries was determined. One of three similar independent experiments is shown.
PSGL-1-mediated stimulation of Th1 cell binding to ICAM-1 involves PKC pathways
To clarify the PSGL-1-mediated signaling pathways that lead to the enhanced cell binding to ICAM-1, we first tested the effect of various kinase inhibitors on PSGL-1-mediated stimulation of Th1 cell adhesion. Pretreatment of Th1 cells with staurosporine, a broad inhibitor of both serine/threonine protein kinases and tyrosine kinases, inhibited PSGL-1-mediated stimulation of cell adhesion by 90% (Fig. 7A). Pretreatment of the cells with calphostin C, a specific inhibitor of PKC, also inhibited cell adhesion by 80% (Fig. 7A). These results suggest that PKC-dependent pathways play an important role in PSGL-1-mediated LFA activation. In contrast, neither PD098059, a MAPK cascade inhibitor, nor LY294002, a specific PI3K inhibitor, affected PSGL-1-stimulated cell adhesion. Combined treatment of the cells with PD098059 and LY294002 slightly inhibited cell adhesion. Rottlerin partly inhibited cell adhesion at 4 μM, a concentration specific for PKC inhibition over other PKC isoenzymes, but markedly inhibited cell adhesion at 40 μM, a concentration that can inhibit PKC, PKC, and PKC. These results suggest that PKC pathways play a dominant role in signaling from PSGL-1 stimulation to LFA-1 activation, whereas MAPK and PI3K pathways play a minor role.
FIGURE 7. PKC activation is involved in PSGL-1-mediated stimulation of Th1 cell adhesion to ICAM-1. A, Binding of PSGL-1-cross-linked Th1 cells preincubated with kinase inhibitors. Th1 cells were incubated with 0.5 μM staurosporine (Sta), 0.1 μM calphostin C (Cal), 50 μM PD098059 (PD), 50 μM LY294002 (LY), or 4–40 μM rottlerin (Rot) at 37°C for 30 min before cross-linking PSGL-1. Cells bound to ICAM-IgG are expressed as a percentage of the untreated control. B, PKC activity in PSGL-1-cross-linked Th1 cells. Th1 cells were cross-linked for the indicated times, and the lysates were examined for PKC activity using Abs against pan-phospho-PKC, phospho-PKC/II, phospho-PKC (T505), phospho-PKC (S643), phospho-PKC, and phospho-PKC/. C, Effect of calphostin C on Th1 cell accumulation on ICAM-1 induced by rolling on P-selectin-IgG. Th1 cells were preincubated with 0.1 μM calphostin C or vehicle and infused into the capillaries described in Fig. 6A at a shear stress of 1 dyn/cm2. The number of cells bound to the ICAM-IgG-coated capillaries was determined. One of three similar independent experiments is shown.
We next examined whether PSGL-1 cross-linking would induce PKC activation. Although PSGL-1 cross-linking did not detectably up-regulate the phosphorylation of PKC detected by the activation-specific Ab against most isoforms of PKC, it induced activation of a species recognized by the Ab directed against phospho-PKC/II at 5 min (Fig. 7B). Up-regulation of phosphorylation of PKC, PKC, and PKC/ was not observed after PSGL-1 cross-linking. These results suggest that activation of PKC or PKCII is involved in PSGL-1-mediated signaling.
The involvement of PKC pathways in Th1 cell adhesion induced by PSGL-1-mediated rolling was also tested. Pretreatment of Th1 cells with calphostin C reduced the cell adhesion to ICAM-1 induced by PSGL-1-mediated rolling (Fig. 7C). Calphostin C showed no effect on the number and speed of Th1 cells rolling on P-selectin-IgG (data not shown). These results suggest that PKC pathways are major components of the intracellular signals from PSGL-1-mediated rolling to LFA-1 activation in Th1 cells.
Discussion
In this study, we investigated the role of PSGL-1-mediated signaling in integrin activation in T cells. We demonstrated that PSGL-1 cross-linking on Th1 cells enhances LFA-1-mediated cell adhesion to ICAM-1. PSGL-1 cross-linking induced the clustering of LFA-1 on the cell surface, suggesting that the increase in LFA-1 avidity may account for the enhanced adhesion to ICAM-1. Our data also show that PSGL-1 cross-linking combined with chemokine stimulation exerted a more than additive effect on LFA-1-mediated Th1 cell adhesion. In addition, we showed that PSGL-1-mediated rolling on P-selectin enhances Th1 cell accumulation on ICAM-1 under flow conditions. To our knowledge, this is the first study demonstrating that PSGL-1-mediated rolling directly induces integrin activation.
Signaling induced by P-selectin binding has been studied in different leukocyte types, including neutrophils, monocytes, and lymphocytes. Earlier reports showed inhibitory effects of P-selectin binding in neutrophils, such as the inhibition of 2 integrin-mediated binding (32) and superoxide generation (33). More recently, stimulatory effects of P-selectin via PSGL-1 on tyrosine phosphorylation and the activation of MAPKs in human neutrophils (21) and on cytokine release by human neutrophils and monocytes (21, 34) have been shown. The effect of PSGL-1 cross-linking on integrin activation has also been reported for mouse neutrophils (22).
Integrins are normally expressed on leukocytes in a state with a low ability to bind to ligands and require activation to initiate adhesion. One mechanism for integrin activation is an up-regulation of integrin affinity by conformational changes (29, 30). The other mechanism is an increase in integrin avidity, which occurs through integrin clustering in discrete areas of the plasma membrane. Chemokines can trigger the integrin-dependent adhesion of a number of leukocyte subtypes (5). CCL19, CCL21, and CXCL12 enhance both the affinity and the clustering of LFA-1 in naive lymphocytes (35). In our experiments, an alteration in LFA-1 affinity as detected by soluble ICAM-1-IgG binding was not observed after PSGL-1 cross-linking. In contrast, PSGL-1 cross-linking with an anti-PSGL-1 mAb or P-selectin-IgM followed by their respective secondary Abs induced the clustering of LFA-1. These results suggest that the mechanisms for enhanced Th1 cell binding to ICAM-1 induced by PSGL-1 cross-linking involve at least LFA-1 clustering. Our results also showed that the cross-linking of PSGL-1 on activated T cells, including Th1 and Th2 cells, but not unstimulated T cells, enhanced the cell binding to ICAM-1. Because unstimulated T cells express LFA-1 at a lower level than do activated T cells, the enhanced adhesion induced by the clustering of LFA-1 may require a certain level of LFA-1 expression. Our results also showed that the cross-linking of PSGL-1 on Th1 cells enhanced the binding of more cells compared with Th2 cells. Th1 cells express PSGL-1 and LFA-1 at a slightly higher level than Th2 cells, which may account for the differences in adhesion induced by PSGL-1 cross-linking in these two cell types. Alternatively, T cells may acquire or activate the signaling machinery required for PSGL-1-mediated integrin activation during cell activation and differentiation.
Early responses in leukocyte activation during their migration into tissues include a transition from a spherical to a polarized morphologic conformation. Chemokines cause the polarization of lymphocytes and the redistribution of several adhesion molecules to the uropod, a cytoplasmic projection that forms at the rear end of a moving cell (36). Previous reports have shown that PSGL-1 is redistributed to the uropod upon stimulation by chemoattractants in leukocytes (37, 38). Similarly, stimulation of neutrophils with fMLP induces the redistribution of 2 integrin Mac-1 to the uropod (39). Our results showed that PSGL-1 cross-linking induced polarization of the cell shape and clustering of both PSGL-1 and LFA-1, which were often, but not always, colocalized in one pole of the cell. These results show that not only chemokines but also PSGL-1 cross-linking can induce cell polarization and adhesion molecule redistribution, both of which are thought to be important for cell migration. Although the relationship between PSGL-1 and LFA-1 clustering is not clearly addressed in this study, it is likely that PSGL-1 clustering is a prerequisite for LFA-1 clustering, because the increase in LFA-1-dependent adhesion was not induced by incubation of the cells with 2PH1 alone, but required the secondary Ab. In support of this idea, a recent report suggests that the clustering of rolling receptors such as L-selectin and PSGL-1 transduces signals leading to 2 integrin clustering in neutrophils (40).
Th1 cells preferentially express the chemokine receptors CXCR3 and CCR5. Both CXCL10 and CCL5 have been implicated in the activation of the integrin-dependent adhesion of T cells (18, 19, 20). As expected, the stimulation of Th1 cells with CXCL10 or CCL5 enhanced cell binding to ICAM-1, which was accompanied by increased cell polarization and the redistribution of both LFA-1 and PSGL-1. Costimulation with chemokines and PSGL-1 cross-linking further enhanced the cell binding as well as LFA-1 and PSGL-1 clustering. Synergy between L-selectin cross-linking and chemokine stimulation for integrin-mediated cell adhesion has been reported (41). In this regard, it is of note that L-selectin stimulation enhances cell responses to CXCL12 by regulating the functional expression of surface CXCR4 in lymphocytes (42). It should be clarified whether similar mechanisms are involved in the combined effect of PSGL-1 cross-linking and chemokine stimulation observed in this study.
Ab-mediated cross-linking of PSGL-1 or P-selectin binding has been used as a model system to ligate PSGL-1 in vitro. However, PSGL-1 engagement in vivo most likely occurs during cell rolling on activated endothelium expressing P-selectin. Thus, we used a flow assay involving two tandem-connected capillary tubes, one coated with P-selectin-IgG and the other with ICAM-1-IgG. Our data showed that the number of cells that bound to ICAM-1 was dramatically increased when they first rolled on P-selectin, suggesting that PSGL-1-mediated rolling directly induces the intracellular signaling that leads to integrin activation. In addition, we showed that PKC pathways were involved in integrin activation induced by PSGL-1-mediated rolling.
In conclusion, our data show that Ab-mediated cross-linking of PSGL-1 or PSGL-1-mediated rolling on P-selectin stimulates the LFA-1-mediated binding of Th1 cells to ICAM-1. We also show that PSGL-1-mediated signaling functions cooperatively with that induced by chemokines to induce LFA-1 activation and that the signaling pathways from PSGL-1-mediated rolling to integrin activation involve at least PKC activation. Clarification of the PSGL-1-mediated signaling pathways leading to LFA-1 activation in Th1 cells will help us to understand and control the migration of Th1 cells under various inflammatory conditions.
Acknowledgments
We thank Drs. Bruce Furie and Barbara Furie for PSGL-1-deficient mice, Dr. John Lowe for selectin-IgM constructs, Dr. Yutaka Iigo for the ICAM-1-IgG construct, Dr. Hideo Yagita for the KBA mAb, Dr. Peter Smith for the protocol for selectin-IgM purification, and Drs. Toshiyuki Tanaka, Toshiyuki Murai, and Haruko Hayasaka for valuable comments. We also thank Shinobu Yamashita and Miyuki Komine for secretarial assistance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant-in-aid for the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
2 Address correspondence and reprint requests to Dr. Takako Hirata, Laboratory of Molecular and Cellular Recognition, Osaka University Graduate School of Medicine C8, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail address: thirata@orgctl.med.osaka-u.ac.jp
3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; PKC, protein kinase C; BCECF, 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; DIC, differential interference contrast.
Received for publication July 12, 2004. Accepted for publication November 16, 2004.
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