Chemokine Stimulation of Lymphocyte α4Integrin Avidity but Not of Leukocyte Function-associated Antigen-1 Avidity to Endothelial Ligands under Shear Flow Requires Cholesterol Membrane Rafts*

VLA-4 and LFA-1 are the major vascular integrins expressed on circulating lymphocytes. Previous studies suggested that intact cholesterol rafts are required for integrin adhesiveness in different leukocytes. We found the α4 integrins VLA-4 and α4β7 as well as the LFA-1 integrin to be excluded from rafts of human peripheral blood lymphocytes. Disruption of cholesterol rafts with the chelator methyl-β-cyclodextrin did not affect the ability of these lymphocyte integrins to generate high avidity to their respective endothelial ligands and to promote lymphocyte rolling and arrest on inflamed endothelium under shear flow. In contrast, cholesterol extraction abrogated rapid chemokine triggering of α4-integrin-dependent peripheral blood lymphocytes adhesion, a process tightly regulated by Gi-protein activation of G protein-coupled chemokine receptors (GPCR). Strikingly, stimulation of LFA-1 avidity to intercellular adhesion molecule 1 (ICAM-1) by the same chemokines, although Gi-dependent, was insensitive to raft disruption. Our results suggest that α4 but not LFA-1 integrin avidity stimulation by chemokines involves rapid chemokine-induced GPCR rearrangement that takes place at cholesterol raft platforms upstream to Gi signaling. Our results provide the first evidence that a particular chemokine/GPCR pair can activate different integrins on the same cell using distinct Gi protein-associated machineries segregated within defined membrane compartments.

To extravasate the bloodstream at specific sites of inflammation or antigen presentation, circulating lymphocytes must rapidly develop firm adhesion to specific endothelial ligands (1,2). Lymphocyte arrest on vascular endothelia is nearly exclusively mediated by the integrins VLA-4 1 (␣ 4 ␤ 1 ), its related integrin, ␣ 4 ␤ 7 , and by LFA-1, counter-receptors for endothelial VCAM-1, MAdCAM-1, and ICAM-1, respectively. As these integrins occur in largely inactive states on circulating leukocytes, their in situ activation must take place within fractions of seconds at endothelial contact zones (3,4). Apically expressed endothelial chemokines confer these integrins with high avidity and shearresistant adhesion more efficiently than their soluble counterparts (4,5). This suggests that chemokine-occupied GPCRs may transmit local signals to neighboring integrins at the lymphocyte plasma membrane. Segregation of the GPCR machinery, its proximal effectors, and their target integrins within preformed supramolecular structures may facilitate chemokine triggering of integrin avidity under shear flow (6).
Candidate membrane microdomains for such segregation of GPCR complexes are lipid rafts, cholesterol-and glycosphingolipid-enriched lipid microdomains, which serve as key signaling platforms in many cell types (7). Because lymphocyte rafts contain several key potential modulators of integrin adhesion stimulation by chemokines, including GPCR subsets and their associated G-proteins (8 -10), PI3K and Src (11)(12)(13), we hypothesized that avidity stimulation of integrins by GPCRs should take place within or proximal to raft microdomains. Recently, thymocyte LFA-1 has also been shown to partition to rafts and undergo avidity modulation upon raft ligation (14). Lymphocyte rafts have been also implicated in ␤ 1 integrin adhesion modulation by the T-cell receptor machinery (15) and by other agonists, as measured in prolonged static contacts (16). However, very little is known about the involvement of rafts in rapid stimulation of leukocyte integrin avidity by chemokines at endothelial contacts. We report here that although spontaneous integrin-mediated adhesion of resting PBL to inflamed endothelium under shear flow does not require raft integrity, chemokine triggering of VLA-4 or ␣ 4 ␤ 7 avidity to their respective endothelial ligands requires the integrity of cholesterol rafts. Interestingly, chemokine-triggered avidity of LFA-1 to endothelial ICAM-1 is insensitive to raft disruption by cholesterol depletion. Our results suggest that triggering of ␣ 4 integrin avidity by major GPCRs on resting T cells occurs through a cholesterol-dependent mechanism, not utilized by the LFA-1 integrin.
Cells-Human PBL were isolated from citrate-anticoagulated whole blood as described (18) and consisted of Ͼ90% CD3ϩ T lymphocytes. Cells were cultured in lipopolysaccharide-free RPMI culture medium for 18 h before use. The pre-B cell line 300.19 (19) was maintained in RPMI 1640, supplemented with 10% fetal calf serum, 2 mM glutamine, 0.1 M 2-mercaptoethanol, and antibiotics. The human T lymphoblastoide Jurkat E6-1 and the human promonocytic U937 cell line, gifts of Dr. Y. Shimizu (Minnesota University, Minneapolis, MN) and Dr. S. Levy (Stanford University, Stanford, CA), respectively, were maintained in RPMI 1640, supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics. Chinese hamster ovary cells transfected with full-length human VCAM-1, a gift of Dr. R. Lobb (Biogen), were maintained in ␣-minimal essential medium, supplemented with 10% dialyzed fetal calf serum, 4 mM L-glutamine, and 200 nM methotrexate (Sigma). Primary cultures of human umbilical cord endothelial cells (HUVEC) were established as previously described (4) and passages 3-4 were taken for adhesion experiments. HUVEC were stimulated for 24 h with TNF-␣ (2 ng/ml, 200 units/ml, R&D Systems).
Immunofluorescence Confocal Microscopy-Human PBL were washed in PBS and attached to poly-L-lysine-coated glass slides at room temperature. Surface rafts were patched by incubating PBL with anti-CD59 mAb (MEM-43, Serotec Ltd., Oxford, United Kingdom) for 30 min at 4°C, followed by washing and 20 min incubation at 37°C with Alexa Fluor-546-conjugated anti-mouse Ab (Molecular Probes, Inc., Eugene, OR). Cells were washed and fixed in 3% paraformaldehyde in PBS (30 min, room temperature). Control cells were fixed immediately after attachment to poly-L-lysine and then incubated with the various Abs. Fluorescence microscopy was performed with a confocal microscope (Nicon eclipse TE300 with the Laser Scanning System 2000-BIO-RAD) with ϫ100 objective lens, using laser excitation at 488 and 543 nm.
Flow Cytometry-Cells were washed once with PBS, resuspended in FACS-wash buffer (PBS and 10% bovine serum), and incubated with primary antibody (10 g/ml) for 30 min at 4°C. The samples were then washed and incubated with secondary antibody for 30 min at 4°C. Cells were washed and analyzed immediately on a FACScan flow cytometer (BD Biosciences, Erembodegem, Belgium).
Isolation of Lipid Rafts-Detergent-insoluble glycosphinogolipid fractions were derived as described (11) with slight modifications. In brief, 1 ϫ 10 9 T cells were washed and lysed for 10 min in 1 ml of cold 25 mM MES buffer, 150 mM NaCl, pH 6.5, containing 1% Triton X-100 supplemented with a protease and phosphatase inhibitor mixture. Lipid fractions were separated by sucrose density gradient (30 to 5%), formed by 18 h centrifugation (250,000 ϫ g using a Beckman SW41 Ti rotor) at 4°C. Twelve 0.8-ml fractions were recovered and stored at Ϫ70°C until analysis.
Western Blot Analysis-LFA-1 and ␣ 4 integrins in lipid fractions were probed by Western blotting with polyclonal goat Ab directed against the ␣ L or ␣ 4 integrin subunits (Santa Cruz Biotechnology Inc., Santa Cruz, CA), respectively, and developed with peroxidase-conjugated IgG (Jackson, West Grove, PA). Lck was probed with monoclonal anti-Lck Ab (Santa Cruz). CXCR4 was probed with rabbit polyclonal anti-CXCR4 Ab (Proscience Inc., Poway, CA). Densitometric scanning analysis was performed using NIH Image 1.62 software. GPCRmediated chemokine activation of ERK1/2 in intact or M␤CD-treated PBL was quantified as previously described (20) by immunoblot analysis using an anti-phosphospecific ERK1/2 Ab, a kind gift from Dr. Rony Seger (Weizmann Institute).
Shear Flow Assays-The preparation of all chemokine-containing adhesive substrates and of the cells analyzed in the laminar flow adhesion assays were performed as previously described (4,5,20). ICAM-1-Ig or MAdCAM-1-Ig-coated substrates were prepared by pre-coating substrates with 1 g/ml protein A in coating medium (21), followed by co-immobilization of either inactivated or intact chemokines at 2 g/ml in the presence of 2 g/ml carrier human serum albumin. The protein A/chemokine substrates were overlaid overnight at 4°C with the indicated concentration of the ICAM-1-Ig or MAdCAM-1-Ig (in PBS supplemented with 20 mg/ml albumin). Cell monolayers and adhesion ligand/chemokine-coated substrates were assembled on the lower wall of the flow chamber (260-m gap) and extensively washed with binding medium (Hanks' balanced salt solution containing 2 mg/ml bovine serum albumin, 10 mM HEPES, pH 7.4, and Ca 2ϩ and Mg 2ϩ at 1 mM each). Chemokine pretreatment was performed by incubating cells for 5 min in binding medium at room temperature in the presence of 0.5 g/ml soluble chemokine followed by cell perfusion into the chamber. Disruption of lymphocyte rafts was performed by pretreating cells (10 min at room temperature) in cell binding medium with 15 mM M␤CD followed by washing and immediate introduction into the flow chamber. M␤CD did not affect lymphocyte viability within a 30min period after completion of cholesterol extraction. This treatment was found to deplete 60 -65% of the entire cholesterol content in cultured lymphocyte lines (Jurkat and the Pre B line, 300.19 data not shown). Cell treatment with 30 mM M␤CD did not further deplete cellular cholesterol, suggesting that the majority of surface extractable cholesterol was depleted by 15 mM M␤CD. Mild disruption of the actin cytoskeleton was induced by pretreating cells for 1 min at room temperature with 20 M Cyto D followed by immediate introduction into the flow chamber with binding medium containing 2 M Cyto D. For blocking G i protein ␣-subunit signaling, PBL were cultured for 15 h at 37°C in culture medium in the presence of 100 ng/ml pertusis toxin. All flow experiments were conducted at 37°C. The entire period of cell perfusion was recorded on a videotape with a long integration LIS-700 CCD video camera (Applitech, Holon, Israel) and a time lapse SVHS-Video recorder (AG-6730, Panasonic, Osaka, Japan). All cellular interactions with the adhesive substrates were determined by manually tracking the motions of individual cells along 0.9-mm paths for 1 min within different fields of view (each one 0.17 mm 2 of area). Three categories of cell tethers under flow were determined: tethers were defined as transient if cells attached briefly (Ͻ2 s) to the substrate; as rolling, if cells established rolling for at least 3 s; and as arrests if immediately arrested on the substrate as previously described (4). Frequencies of adhesive categories within differently pretreated cells were determined as a percentage of cells flowing immediately over the substrates, as previously described (4). To assess rapid development of integrin avidity to ligand at stationary contacts, cells were perfused into the flow chamber and allowed to settle onto the substrate for 1 min. Flow was then initiated at 0.5 dyn/cm 2 (unless mentioned otherwise) and increased stepwise every 5 s by a programmed set of rates generated by a syringe pump (4). At the indicated shear stresses, the number of cells that remained bound was expressed relative to the number of cells originally settled on the substrate.
To assess rapid rearrangement of lymphocyte-surface GPCRs at subsecond contacts, PBL were perfused over surface-bound anti-GPCR mAb (12G5) coated with inactivated or active chemokine, as previously described (4,22). Frequency and type of PBL tethers (transient or immediate arrests) served as a measure of spontaneous or chemokinetriggered GPCR avidity developed by the PBL interacting with the mAb-coated adhesive contacts under shear flow.

Cholesterol Extraction from PBL Does Not Abrogate Intrinsic
Integrin Adhesiveness-To test the involvement of cholesterol rafts in the regulation of selectin-and integrin-mediated adhesion of PBL to inflamed endothelium, freshly isolated PBL were perfused under physiological shear flow over a monolayer of TNF-␣-activated HUVECs, a well established inflamed endothelial model. PBL establish persistent rolling on the endothelial cell monolayer, which is mediated primarily by E-selectin and VCAM-1 induced on these endothelial cells (4). Interestingly, brief T cell pretreatment with 15 mM M␤CD, which depleted the entire extractable cholesterol from the surface of cultured lymphocyte lines (see "Experimental Procedures"), but retained lymphocyte viability and shape (data not shown), had no effect on PBL capture and rolling on either activated HUVEC or on isolated E-selectin (Fig. 1, A and B). Furthermore, M␤CD treatment also did not impair the spontaneous ability of VLA-4 to support capture, rolling, or immediate arrest on VCAM-1-bearing cell surfaces (Fig. 1C) or on purified VCAM-1 coated at different densities (Fig. 1D). Similar to VLA-4, ␣ 4 ␤ 7 integrin-mediated tethering and rolling of PBL on MAdCAM-1 were also resistant to M␤CD (Fig. 1E). Consistent with these results, ␣ 4 integrin expression was not affected by M␤CD treatment (Fig. 1F). Thus, intact rafts are not required for the inherent ability of ␣ 4 integrins on resting T cells to interact with their respective ligands under shear flow and to spontaneously develop medium or high avidity to these ligands at rapid adhesive contacts.
Cholesterol Extraction Abrogates Chemokine-stimulated ␣ 4 Integrin-mediated PBL Adhesion but Not Stimulation of LFA-1-mediated Adhesion-Immobilized chemokines bind lymphocyte GPCRs and trigger robust VLA-4 integrin-mediated adhesion to VCAM-1 at static contacts through activation of pertussis toxin-sensitive G i -protein machinery (4,5,23). We next asked how chemokine-stimulated adhesion of VLA-4 is affected by disruption of cholesterol rafts on PBL. The prototypic PBL chemokines, SDF-1␣ (CXCL12) and secondary lymphoid tissue chemokine (CCL21), co-immobilized with VCAM-1, trigger robust VLA-4-dependent adhesion of PBL at 1-min long static contacts ( Fig. 2A). VLA-4-dependent adhesion triggered by both chemokines as well as by Mig (CXCL9) was entirely abrogated by M␤CD pretreatment of PBL ( Fig. 2A and data not shown), as well as by pertussis toxin pretreatment of lymphocytes (Ref. 4, and data not shown). The inhibitory effect of M␤CD on chemokine-stimulated VLA-4 avidity was reversed in the presence of exogenous cholesterol ( Fig. 2A), consistent with cholesterol depletion accounting for the inhibitory effect of M␤CD. Similarly, M␤CD pretreatment of murine B lymphocytes abrogated their ability to develop high avidity VLA-4mediated adhesion to VCAM-1 in response to the B cell chemo-kine B cell attracting chemokine-1 (CXCL13, data not shown). Thus, although intact rafts are not required for spontaneous VLA-4 and ␣ 4 ␤ 7 adhesiveness developed at rapid adhesive contacts, they are necessary for chemokine-stimulated VLA-4 adhesiveness. These results could suggest that GPCR signaling triggered by SDF-1␣ and secondary lymphoid tissue chemokine is severely impaired by M␤CD treatment. Strikingly, however, both chemokines could normally trigger robust LFA-1-dependent PBL adhesion to purified ICAM-1, and such adhesion was insensitive to cholesterol extraction, yet sensitive to PTX pretreatment of lymphocytes (Fig. 2B, and data not shown). In support of these results, both the expression level and intrinsic signaling activity of the SDF-1␣ GPCR (CXCR4) to ERK was fully retained in M␤CD-treated PBL (Fig. 2, C and D). Spontaneous LFA-1-mediated PBL adhesiveness to ICAM-1 was also fully retained upon raft disruption (data not shown). Thus, G i protein-mediated enhancement of LFA-1-dependent adhesion by major chemokines is fully retained upon raft disruption in PBL, although G i stimulation of VLA-4 adhesiveness by identical chemokines in the same cells is totally abrogated.
Cholesterol Rafts Are Necessary for Subsecond G i -proteindependent Enhancement of VLA-4 Avidity to Ligand under Shear Flow-Chemokines also trigger VLA-4 avidity at subsecond contacts promoting lymphocyte capture to VCAM-1 under continuous shear flow (4). The involvement of rafts in these earliest signaling events of chemokines to target integrins on the lymphocyte surface was next investigated. In agreement with its suppressive effect on chemokine-stimulated VLA-4 adhesion at static adhesive contacts ( Fig. 2A), cholesterol extraction from PBL impaired rapid SDF-1␣ stimulation of VLA-4 avidity to VCAM-1 at subsecond contacts and abrogated chemokine-triggered capture and immediate lymphocyte arrest Frequency of intact and treated PBL tethers to TNF-␣-stimulated HUVEC cells (HUVEC) was measured at 1.5 dyn/cm 2 (A) or to E-selectin-Ig (at 1 g/ml) on protein A precoating determined at 1 dyn/cm 2 (B) and is depicted in the stacked bars. The different tether categories were expressed as described under "Experimental Procedures." C, frequency and category of tethers mediated by intact or M␤CD-treated PBL interacting with a VCAM-1 expressing Chinese hamster ovary cell monolayer at 1.5 dyn/cm 2 . D, frequency of intact or M␤CD-treated PBL interacting under shear stress of 1 dyn/cm 2 with high density sVCAM-1 (5 g/ml) or with medium density sVCAM-1 (2.5 g/ml). E, frequency of intact or M␤CD-treated PBL interacting under shear stress of 1 dyn/cm 2 with MAdCAM-1-Ig (overlaid at 10 ng/ml on protein A). Where indicated, PBL were pretreated with the anti-␣ 4 ␤ 7 blocking mAb (10 g/ml Act-1) to demonstrate specificity of the binding to the ␣ 4 ␤ 7 integrin. The results in A-E represent an average Ϯ range of two fields of view. Experiments are representative of two independent runs. F, flow cytometry of intact (black line) and M␤CD (gray line) pretreated PBL stained with antibody to ␣ 4 integrin (HP1/2 mAb) followed by fluorescein isothiocyanate-conjugated anti-mouse (Jackson).
on VCAM-1-bearing surfaces (cell-free or on the surface of a cell monolayer; Fig. 3, A and B, respectively). Cholesterol extraction also abrogated SDF-1␣-stimulated G i -dependent (PTXsensitive) transient tethers mediated by VLA-4 on low density VCAM-1 confirming that rafts are obligatory to chemokineenhanced avidity regardless of the level of avidity stimulated or duration of the adhesive contact (Fig. 3C). Similar to VLA-4, rapid chemokine-triggered ␣ 4 ␤ 7 avidity to MAdCAM-1 was also abrogated in M␤CD-treated lymphocytes (Fig. 3D), although spontaneous lymphocyte capture and rolling on MAdCAM-1 were insensitive to cholesterol extraction (Fig. 1E). However, subsecond chemokine stimulation of LFA-1 avidity to isolated ICAM-1, required to trigger capture and immediate arrest of lymphocytes on ICAM-1 at low shear flow (Fig. 3E), was entirely insensitive to identical cholesterol extraction and raft disruption. Furthermore, robust SDF-1␣-triggered integrindependent arrest of PBL captured on TNF-␣Ϫactivated HUVEC was also insensitive to M␤CD-mediated cholesterol depletion (Fig. 3F), in agreement with the major role of LFA-1 in rapid adhesion strengthening developed by lymphocytes on these HUVEC cells (24). Intact cholesterol-enriched rafts appear therefore obligatory for the ability of chemokine GPCRs to FIG. 2. Cholesterol depletion abolishes ␣ 4 integrin but not LFA-1 stimulation by chemokines without interfering with GPCR expression and signaling. PBL were pretreated with M␤CD as described in the legend to Fig. 1. A, adhesion of PBL to purified sVCAM (at 2 g/ml) co-immobilized with heat-inactivated chemokines (Ϫ) or functional SDF-1␣ or secondary lymphoid tissue chemokine-1 (each at 2 g/ml). Values depict the fraction of cells originally settled for 1 min that resisted detachment to a shear stress of 1 dyn/cm 2 for 5 s. Where indicated, PBL were pretreated with M␤CD in the presence of cholesterol (2 g/ml) for 10 min. B, adhesion of PBL to purified ICAM-1 (at 0.25 g/ml) co-immobilized with heat-inactivated (Ϫ) or intact chemokine (SDF-1␣ at 2 g/ml). Values depict the fraction of cells originally settled for 1 min on the substrates that resisted detachment by 1 dyn/cm 2 applied for 5 s. The results in A and B represent an average Ϯ range of 2-3 fields of view. The experiments are each representative of three using multiple PBL donors. C, immunofluorescnce flow cytometry of intact (black line) and M␤CD (gray line) pretreated PBL stained with antibody to CXCR4 (rabbit polyclonal CXCR4 antibody, 10 g/ml) followed by phycoerythrin-conjugated anti-rabbit (Jackson). D, effect of M␤CD on chemokine signaling. SDF-1␣ (10 nM, 30 s) stimulation at 37°C of ERK1/2 phosphorylation in PBL (intact or M␤CD pretreated). Immunoblotting with anti-phosphospecific ERK1/2 (upper panel) and anti-ERK (lower panel) is shown. Intact PBL were also stimulated with SDF-1␣ in the presence of the CXCR4 blocker, AMD3100 (5 M, 10 min) (43).
FIG. 3. Cholesterol depletion abolishes ␣ 4 integrin but not LFA-1 stimulation by chemokines at subsecond contacts. A, frequency of intact or M␤CD-treated PBL interacting under shear stress of 1 dyn/cm 2 with sVCAM-1 (2 g/ml) co-immobilized with heat-inactivated (Ϫ) or functional SDF-1␣ (2 g/ml). The different tether categories were expressed as described under "Experimental Procedures." The data represent mean values of determinations in two fields of view. One experiment representative of three is shown. B, frequency and category of tethers mediated by intact or M␤CD-treated PBL interacting with a VCAM-1 expressing Chinese hamster ovary cell monolayer at 1.5 dyn/ cm 2 . SDF-1␣ (100 ng/ml) was overlaid on the monolayer and washed extensively before PBL perfusion. C, frequency of transient tethers initiated by PBL on low density sVCAM (1 g/ml) co-immobilized with functional or heat-inactivated (Ϫ) chemokine (SDF-1␣ 2 g/ml) under shear stress (1 dyn/cm 2 ). Indicated PBL samples were pretreated with pertusis toxin (PTX) to block their G i signaling. D, frequency of intact or M␤CD-treated PBL interacting under shear stress of 1 dyn/cm 2 with MAdCAM-1-Ig (overlaid at 10 ng/ml) on protein A precoated with heatinactivated chemokine (Ϫ) or functional SDF-1␣ as described under "Experimental Procedures." E, frequency of intact or M␤CD-treated PBL interacting, under low shear stress of 0.5 dyn/cm 2 , with ICAM-Ig (overlaid at 0.5 g/ml on protein A) co-immobilized with heat-inactivated chemokine (Ϫ) or functional SDF-1␣ (2 g/ml). High avidity LFA-1 binding to ICAM-1 required for lymphocyte capture in the absence of selectins could be triggered only at this or lower shear stresses. The data shown in B-E each represent an average Ϯ range of measurements in two fields of view. Each experiment shown is representative of two. F, frequency of intact and M␤CD-treated PBL firmly arrested on TNF-␣-stimulated HUVEC, overlaid with SDF-1␣ (100 ng/ml) at 1.5 dyn/cm 2 . VLA-4 and LFA-1 integrins on intact PBL were preblocked with HP1/2 and TS 1/18 mAbs (20 g/ml), respectively. Values are the mean Ϯ range of two fields. A representative experiment of five is shown. trigger rapid avidity of distinct ␣ 4 integrins but not of LFA-1 integrin at subsecond endothelial contacts under shear flow.
Rafts Partitioning of Lymphocyte Integrins-This vastly different cholesterol dependence of ␣ 4 integrin avidity modulation could reflect preferential partition of these integrins together with their stimulatory GPCRs within cholesterol-containing raft domains. Strikingly, however, both ␣ 4 integrins and LFA-1 were largely excluded from detergent-resistant low density lipid fractions recovered by sucrose gradient highly enriched with the raft marker p56 Lck (Fig. 4A). Thus, raft partitioning of ␣ 4 integrin did not determine the cholesterol dependence of their GPCR-mediated avidity stimulation. Nevertheless, ␣ 4 integrins and LFA-1 appeared to be compartmentalized in distinct membranal domains of PBL as evident by their inability to co-localize (Fig. 4B). This dichotomy in M␤CD sensitivity of chemokine-stimulated ␣ 4 integrin and LFA-1 avidity was not restricted to PBL. U937 monocytes and Jurkat T lymphoblastoids express endogenous CXCR4 (4,25). Similar to PBL, SDF-1␣-triggered VLA-4 avidity to VCAM-1 in both types of leukocytes was highly sensitive to cholesterol extraction (data not shown). SDF-1␣-triggered LFA-1 avidity in the same cells was, however, insensitive to raft disruption (Fig. 5A, and data not shown). Notably, a major fraction of LFA-1 in Jurkat cells (40%) was found to partition within Lck-enriched low-density lipid fractions (Fig. 5B). This further suggests that raft enrichment of LFA-1 does not render its chemokine/GPCR-mediated avidity stimulation more susceptible to raft disruption. Thus, the M␤CD sensitivity of chemokine-stimulated ␣ 4 integrins, which is not shared by LFA-1, was not the result of a preferential partition of ␣ 4 integrins within rafts and did not occur even in cases where LFA-1 was present in rafts.

Raft Patching Is Insufficient to Trigger VLA-4 Avidity to VCAM-1 at Rapid
Adhesive Contacts-Clustering of thymocyte rafts has been reported to stimulate integrin adhesiveness through activating PI3K (14). We therefore next asked whether raft clustering could play any stimulatory role in rapid VLA-4 adhesion stimulation. Because the levels of the raft sphingolipid marker, GM1, on resting PBL were low (data not shown), we used CD59, an abundant glycosylphosphatidylinositollinked raft-enriched protein on resting PBL to induce raft patching (Fig. 6, top panel) and to test its effects on stimulation of VLA-4 avidity to VCAM-1 (Fig. 6, lower panel). Notably, under conditions where chemokines triggered robust VLA-4 avidity to VCAM-1, CD59 cross-linking had no proadhesive effect on VLA-4-dependent PBL adhesion to VCAM-1 (Fig. 6). Furthermore, murine pre-B 300.19 cells that express high levels of GM1 also failed to develop stronger VLA-4-mediated adhesion to VCAM-1 in response to GM1 patching by the GM1 receptor, cholera toxin B subunit, or to cross-linking of their glycosylphosphatidylinositol-linked protein, CD24 (data not shown). These results suggest that raft clustering on its own is insufficient to stimulate rapid ␣ 4 integrin avidity.
Cholesterol Rafts Are Implicated in GPCR Rearrangement by Immobilized Chemokine Obligatory for VLA-4 Avidity Stimulation-Because chemokine stimulation of ␣ 4 integrin avidity appears to involve both integrin and GPCR clustering events at subsecond adhesive contacts (4) and because soluble chemokines fail to trigger ␣ 4 avidity in resting PBL (4), we hypothesized that clustering of GPCRs is triggered within rafts by immobilized, but not soluble chemokines during ␣ 4 integrin avidity stimulation. We therefore tested whether GPCR clustering triggered on PBL by surface-bound chemokine is sensitive to disruption of cholesterol rafts. We chose to focus on the CXCR4/SDF-1␣ interaction as a model (22). Only a minority of CXCR4 on PBL was recovered in low-density fractions (Fig.  7A). Furthermore, CXCR4Ϫmediated PBL adhesion to anti-CXCR4 immobilized at low density under shear flow, a sensitive measure of the surface density, availability, and cytoskel- etal anchorage of the antigenic determinant (4,26,27) was largely resistant to raft disruption by M␤CD (Fig. 7B). Thus, CXCR4 surface levels and accessibility in PBL do not depend on integrity of cholesterol rafts. However, CXCR4-mediated PBL adhesion to high density anti-CXCR4 mAb under shear flow was substantially suppressed by cholesterol depletion by M␤CD (Fig. 7B). A similar requirement for intact cholesterol rafts in PBL was observed when the lymphocytes were allowed to adhere to low density anti-CXCR4 mAb co-immobilized with the native CXCR4 ligand, SDF-1␣ (Fig. 7C). In this assay (Fig.  7D), the frequency and stability of cellular tethers formed on the surface-bound mAb serves as a readout of CXCR4 microclusters, spontaneous or chemokine-triggered (22). Consistent with this notion, neither soluble SDF-1␣ nor immobilized chemokine to an irrelevant GPCR, CCR7 (data not shown), could augment PBL adhesion to the low density anti-CXCR4 mAb (Fig. 7C). Both transient and firm PBL tethers on the mAbcoated substrates were stabilized over 0.1-0.5 s in a PTXresistant manner (Fig. 7C, and data not shown). This indicated that CXCR4 reorganization by mAb and chemokine was completed within subsecond time frames of adhesive contact independent of G i -protein activation. Taken together these results suggest that M␤CD treatment of PBL abolishes SDF-1␣-augmented CXCR4 clustering at subsecond contacts, a process that requires intact rafts, but not G i -protein signaling.
Raft clustering and associated signaling depends on an intact actin cytoskeleton and involves enrichment of F-actin at sites of raft patches (28 -30). Thus, SDF-1␣-triggered PBL adhesion to anti-CXCR4 mAb may involve active clustering of rafts sensitive to disruption of the actin cytoskeleton. Indeed, short treatment of T lymphocytes with Cyto D abolished SDF-1␣-triggered PBL adhesion to low density anti-CXCR4 mAb under shear flow (Fig. 8A). VLA-4 avidity stimulation by SDF-1␣ was also completely disrupted by the Cyto D treat-ment, even though the short treatment did not affect spontaneous VLA-4 adhesiveness to VCAM-1 in treated PBL (Fig.  8B). Nevertheless, identical Cyto D treatment had no effect on LFA-1 avidity stimulation induced by SDF-1␣ (Fig. 8C). These results therefore suggest that SDF-1␣ stimulates VLA-4 avidity through an M␤CD-sensitive cytoskeletally regulated CXCR4 clustering upstream to G i signaling. This clustering of CXCR4 is not required, however, for SDF-1␣ to transduce G i signals and optimally trigger LFA-1 avidity to ICAM-1. DISCUSSION Chemokines, in addition to orchestrating cell motility and chemotaxis, are potent stimulants of integrin avidity and adhesive processes (31). Although cholesterol-and sphingolipid- Lower panel, adhesion of PBL (intact or CD59 ligated) to purified sVCAM, at 2 g/ml, analyzed in a flow chamber assay. CD59 ligation was performed by incubating cells with anti-CD59 mAb (15 g/ml for 5 min), washing, and cross-linking with secondary Ab (3 g/ml for 5 min). Ligated PBL were immediately settled on the sVCAM-1 substrate and extent of adhesion was determined as described in the legend to Fig. 2A. As a control, adhesion of intact PBL induced by immobilized SDF-1␣ to a similar sVCAM-1 substrate is shown. The results are an average Ϯ range of four fields of view and are representative of two experiments. FIG. 7. Cholesterol extraction impairs CXCR4 rearrangement implicated in SDF-1 stimulation of avidity to surface-bound anti-CXCR4 mAb. A, immunoblot of sucrose density gradient fractions of PBL, 1% Triton X-100 lysate, with anti-Lck or rabbit polyclonal CXCR4 Abs. B, tethering frequency, measured at 0.5 dyn/cm 2 , of intact or M␤CD-treated PBL interacting with surface-bound CXCR4 mAb (12G5), coated at low or high density (0.2 or 1 g/ml, respectively). Tether type was expressed as described under "Experimental Procedures." C, tethering frequency, measured at 0.75 dyn/cm 2 , of intact or M␤CD-treated PBL interacting with surfaces coated with the CXCR4 mAb 12G5 (0.2 g/ml), co-immobilized with either heat-inactivated (Ϫ) or functional SDF-1␣ (2 g/ml). Tether type was expressed as described under "Experimental Procedures." Where indicated, PBL were pretreated with soluble SDF-1␣ (0.5 g/ml) or with pertusis toxin as described under "Experimental Procedures." Values represent mean Ϯ range of measurement in two fields of view. Adhesion experiments are representative of three experiments. D, a schematic representation of spontaneous or chemokine-triggered CXCR4 avidity to mAb developed by PBL interacting with an anti-CXCR4 mAb-coated substrate under shear flow, as shown in B and C. Postulated CXCR4 organization on the lymphocyte surface prior to (1), or immediately after interacting with a surface coated with high density anti-CXCR4 mAb (2), or with low density anti-CXCR4 mAb co-immobilized with SDF-1␣ (3). Enhanced in situ CXCR4 clustering results in augmented binding avidity of the cluster to the mAb and increased number and strength of lymphocyte tethers to the surface. enriched lipid raft domains have been implicated in chemokinetriggered cell polarization and chemotaxis (32), the role of these signaling platforms in rapid chemokine signaling events has not been investigated to date. At endothelial contacts, leukocytes must respond within fractions of seconds to localized signals transmitted by endothelial displayed surface-bound chemokines to specific GPCRs on the leukocyte surface (3,4). This localized stimulation of integrins is designed to up-regulate integrin clustering and affinity to endothelial ligands within subsecond contacts under the continuous presence of disruptive shear forces (4,23,25). Our studies on chemokine triggering of VLA-4 avidity in lymphocytes as well as in hematopoietic progenitors have also shown that VLA-4 avidity to endothelial VCAM-1 involves rapid induction of integrin clus-tering triggered preferentially by immobilized chemokines juxtaposed to the integrin ligand. Rapid chemokine signaling to VLA-4 therefore appears to depend on specialized GPCR rearrangement events proximal to the regulated integrin target. The present study provides the first indication that although both are transduced through G i protein stimulation, VLA-4 and LFA-1 avidity stimulation by surface bound chemokines involves different GPCR rearrangement mechanisms. These rearrangements appear to operate upstream to the heterotrimeric G-protein activation step essential for the integrin avidity stimulation (Fig. 9) because they are insensitive to pertussis toxin blockage of G i signaling (Figs. 3C and 7C), but take place in the context of cholesterol rafts.
Subsets of CXCR4 have been reported to translocate into raft domains upon CXCR4 patching with human immunodeficiency virus envelope proteins (9,33). Our results on CXCR4 suggest that the vast majority of this prototypic leukocyte chemokine receptor is excluded from rafts in PBL. These findings are in agreement with a recent study demonstrating CXCR4 exclusion from detergent-resistant raft fractions in resting and SDF-1␣-treated T cells (34). Nevertheless, CXCR4, CCR7, and CXCR5, as well as other subsets of leukocyte GPCRs, are expected to operate in the vicinity of lipid rafts by way of association with their signaling units, the heterotrimeric Gproteins that partition into rafts through their G␣ subunits (10). Thus, our results are consistent with the notion that GPCRs, normally excluded from rafts, can utilize their raftassociated effectors to transmit subsecond signals from their cognate chemokines to target integrins on the cell surface. Notably, these target integrins, i.e. VLA-4 and ␣ 4 ␤ 7 , can also be largely excluded from rafts and still respond to chemokine signals in the context of raft-associated effectors. Furthermore, in cells like the T cell line, Jurkat, where LFA-1 was found enriched within detergent-insoluble raft fractions, the integrin response to chemokine stimulation was still independent of cholesterol raft integrity, whereas VLA-4 response to chemokine was still dependent of intact cholesterol rafts, as in PBL (data not shown). Thus, the ␣ 4 -integrin response to a GPCRmediated avidity stimulatory signal within a raft compartment Chemokinetriggered integrin avidity elevation involves two sequential steps: chemokine-induced rearrangement (clustering) of the GPCR (step 1) followed by activation of the associated G i protein (G i signal, step 2). GPCR subsets clustered in the vicinity of raft microdomains (which are M␤CD and Cyto D sensitive in the left part of the membrane) are required for successful transmission of the G i signal to ␣ 4 integrins but are not required for triggering of LFA-1 avidity. GPCRs that do not undergo clustering within raft domains can still transmit their G i signals to LFA-1 although they are incapable of stimulating ␣ 4 integrin avidity. For simplicity, integrins and ligands are not included in the scheme.
does not require this integrin to partition into this compartment. The GPCR signaling machinery utilized by an integrin, rather than the partition of the integrin in rafts, appears to dictate whether chemokine-mediated stimulation of this integrin is dependent on raft integrity. Considering that cholesterol rafts are extremely small lipid entities with radii ranging from 10 to 30 nm (35), it is possible that GPCRs can transmit local signals to proximal integrin targets near such rafts within fractions of seconds, even if both the GPCR and the integrin do not co-partition in these rafts. We cannot exclude, however, the possibility that GPCR signals triggered preferentially by immobilized chemokines can mobilize ␣ 4 integrins but not LFA-1 into cholesterol rafts where their clustering is in situ stimulated by chemokine-occupied GPCRs. This process is unlikely to involve, however, GPCR signaling linked to Src activation, because this pathway is not involved in subsecond induction of either ␣ 4 or LFA-1 integrin avidity by chemokines (20). 2 How then do cholesterol rafts serve as platforms of chemokine signaling to integrins and what is the basis for the raft segregation of chemokine signaling to ␣ 4 integrins as opposed to LFA-1? In PBL, VLA-4 and LFA-1 avidity regulation by chemokines at rapid contact sites appear to employ distinct G protein-transduced machineries, even when stimulated by a common chemokine GPCR (4,23). For instance, although both integrins do not require intact PI3K and classical protein kinase C machinery to undergo avidity up-regulation by chemokines, LFA-1 but not VLA-4 avidity stimulation may depend on GPCR-triggered Ca 2ϩ mobilization (4, 23). 2 Furthermore, LFA-1 and ␣ 4 integrins differ substantially with respect to both their surface distribution and cytoskeletal associations (36 -38). It is therefore possible that a specialized GPCR effector of ␣ 4 avidity, which operates independently of PI3K, protein kinase C, or intracellular free Ca 2ϩ , is regulated by G i signaling within or proximal to raft platforms. Notably, disruption of the actin cytoskeleton abolished both CXCR4-dependent adhesion and ␣ 4 integrin avidity stimulation by SDF-1␣ without affecting LFA-1 avidity stimulation (Fig. 8). These results, and the finding that VLA-4 avidity stimulation is restricted to immobilized chemokines (4) suggest that a critical cytoskeletal-mediated raft-dependent GPCR clustering step must be coupled to G i signaling to optimally trigger ␣ 4 integrin avidity at subsecond adhesive contacts (Fig. 9). In contrast to ␣ 4 integrin simulation, enhancement of LFA-1 avidity by chemokines is triggered in the absence of CXCR4 clustering, because interference with this step by disruption of cholesterol rafts (Fig. 7, B and C) or by mild disruption of the actin cytoskeleton (Fig. 8A) did not affect the ability of CXCR4 to mediate optimal SDF-1␣ triggering of LFA-1 avidity (Fig. 8C). Because SDF-1␣-triggered PBL adhesion to the anti-CXCR4 mAb as well as to the isolated integrin ligands takes place at 0.1-0.5 long contacts, de novo actin polymerization events triggered by CXCR4 at such contact sites are unlikely to underlie ␣ 4 avidity stimulation. These subsecond contacts are much more rapid than earliest actin polymerization processes reported to date (39).
In contrast to the role of rafts in GPCR-mediated chemokine signaling to ␣ 4 integrins, we did not detect any raft requirement for ␣ 4 or other integrins to spontaneously capture lymphocytes from the flow and to generate high avidity to ligand at short-lived adhesive contacts independent of chemokine activation (Fig. 1). M␤CD-treated lymphocytes could bind to VCAM-1, MadCAM-1, or ICAM-1 under shear flow at comparable efficiencies to intact lymphocytes (Fig. 1). ␣ 4 Integrin presentation and clustering on lymphocyte microvilli have been suggested to regulate lymphocyte capture on ligand under shear flow (26,36). Microvilli structures on PBL appeared to remain largely intact in M␤CD-treated cells, because both ␣ 4 integrin-mediated and L-selectin-mediated lymphocyte capture and rolling on respective ligands were not affected by cholesterol extraction (Fig. 1, B and D). 3 Our present data contrast recent reports suggesting that agonist-induced or constitutive LFA-1 adhesiveness in primary T cells, thymocytes, or in the Jurkat T cell line, is strongly suppressed by raft disruption (14,16). In our hands, however, both spontaneous, phorbol ester-stimulated or mAb-activated LFA-1-dependent adhesion to ICAM-1 were entirely insensitive to cholesterol depletion (data not shown). Furthermore, raft clustering was shown to stimulate PI3K activity and thereby trigger LFA-1-mediated thymocyte adhesion (14). Raft patching also induces Src activation and downstream actin polymerization processes in adherent lymphocytes (29). However, spontaneous VLA-4 and LFA-1 adhesiveness developed by T cells at rapid adhesive contacts are insensitive to PI3K inhibition. 4 Raft clustering alone is also insufficient to stimulate VLA-4 integrin adhesiveness at these short-lived contacts (Fig. 6). Thus, the raft clustering events that have been reported to promote T cell adhesion do so via late post-ligand occupancy events rather than by directly enhancing integrin avidity to immobilized ligand. Furthermore, raft ligation alone was recently shown to translocate both free and ligand-occupied LFA-1 and VLA-4 integrins into rafts (16,40) where these integrins and their cytoskeletal adaptors can be activated de novo or stabilized by raft-associated kinases (15,41). In contrast, spontaneous integrin-mediated adhesiveness at rapid contacts do not appear to employ these slow adhesion strengthening processes. Rapid integrin-mediated adhesive contacts may rely on integrin molecules with preformed potential to generate high avidity to endothelial ligands independent of raft-associated machineries (42).
In conclusion, our results provide a first indication that chemokine signaling through a common GPCR to different integrins coexpressed on a given leukocyte can take place at different lipid microdomains. PBL rafts may stabilize an, as yet unidentified, GPCR effector that affects ␣ 4 avidity through a specialized rearrangement process not required for LFA-1 avidity stimulation. Spontaneous avidity generation of either ␣ 4 integrins or LFA-1 do not require, on the other hand, intact cholesterol rafts, ruling out preferential partition of ␣ 4 integrins or of their specific avidity regulatory effectors within these rafts. Thus, it is the integrin activating machinery, rather than the integrin itself, that requires intact rafts to convert chemokine signaling through GPCRs into productive integrin avidity stimulation at rapid contacts. The notion that GPCR effectors need to undergo specialized rearrangements to translate chemokine signals into avidity enhancement of particular integrins opens up new opportunities to selectively disrupt functional stimulation of distinct integrins by a given chemokine.