Integrin leukocyte function-associated antigen-1-mediated cell binding can be activated by clustering of membrane rafts

The leukocyte function-associated antigen-1 (LFA-1) integrin (CD11a/CD18) is an important adhesion molecule for lymphocyte migration and the initiation of an immune response. At the cell surface, LFA-1 activity can be regulated by divalent cations that enhance receptor affinity but also by membrane clustering induced by treatment of cells with substances such as phorbol esters. Membrane clustering leads to increased LFA-1 avidity. We report here that LFA-1-mediated binding of mouse thymocytes or activated T lymphocytes to intercellular adhesion molecule 1 can be rapidly induced by clustering of membrane rafts using antibodies to the glycosylphophatidylinositol-anchored molecule CD24 or cholera toxin (CTx). CD24 and CD18 were found to co-localize in rafts and cross-linking with CTx lead to enhanced LFA-1 clustering. We observed that disruption of raft integrity by lowering the membrane cholesterol content abolished the CTx and the phorbol 12-myristate 13-acetate-induced LFA-1 binding but left the ability to activate LFA-1 with Mg(2+)/EGTA unimpaired. In contrast to activation with Mg(2+)/EGTA, activation via raft clustering was dependent on PI3-kinase, required cytoskeletal mobility, and was accompanied by Tyr phosphorylation of a 18-kDa protein. Our results support the notion that rafts as preformed adhesion platforms could be important for the rapid regulation of lymphocyte adhesion.     

I-domain of lymphocyte function-associated antigen-1 mediates rolling of polystyrene particles on ICAM-1 under flow

In their active state, beta(2)-integrins, such as LFA-1, mediate the firm arrest of leukocytes by binding intercellular adhesion molecules (ICAMs) expressed on endothelium. Although the primary function of LFA-1 is assumed to be the ability to mediate firm adhesion, recent work has shown that LFA-1 can contribute to cell tethering and rolling under hydrodynamic flow, a role previously largely attributed to the selectins. The inserted (I) domain of LFA-1 has recently been crystallized in the wild-type (wt) and locked-open conformations and has been shown to, respectively, support rolling and firm adhesion under flow when expressed in alpha(L)beta(2) heterodimers or as isolated domains on cells. Here, we report results from cell-free adhesion assays where wt I-domain-coated polystyrene particles were allowed to interact with ICAM-1-coated surfaces in shear flow. We show that wt I-domain can independently mediate the capture of particles from flow and support their rolling on ICAM-1 surfaces in a manner similar to how carbohydrate-selectin interactions mediate rolling. Adhesion is specific and blocked by appropriate antibodies. We also show that the rolling velocity of I-domain-coated particles depends on the wall shear stress in flow chamber, I-domain site density on microsphere surfaces, and ICAM-1 site density on substrate surfaces. Furthermore, we show that rolling is less sensitive to wall shear stress and ICAM-1 substrate density at high density of I-domain on the microsphere surface. Computer simulations using adhesive dynamics can recreate bead rolling dynamics and show that the mechanochemical properties of ICAM-1-I-domain interactions are similar to those of carbohydrate-selectin interactions. Understanding the biophysics of adhesion mediated by the I-domain of LFA-1 can elucidate the complex roles this integrin plays in leukocyte adhesion in inflammation.     

Real-time analysis of the inside-out regulation of lymphocyte function-associated antigen-1 revealed similarities to and differences from very late antigen-4

Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by Gα(i)-coupled G protein-coupled receptors (GPCRs) and deactivated by Gα(s)-coupled GPCRs. The effects of Gα(s)-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific α I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step.     

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH(2) terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 mug/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.     

Specific activation of leukocyte beta2 integrins lymphocyte function-associated antigen-1 and Mac-1 by chemokines mediated by distinct pathways via the alpha subunit cytoplasmic domains

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (alphaMbeta2) but not lymphocyte function-associated antigen-1 (LFA-1; alphaLbeta2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1alpha in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1alpha were confirmed by expression of alphaM or alphaL in alphaL-deficient Jurkat cells. Moreover, expression of chimeras containing alphaL and alphaM cytoplasmic domain exchanges indicated that alpha cytoplasmic tails conferred the specific mode of regulation. Coexpressing alphaM or chimeras in mutant Jurkat cells with a "gain of function" phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the alphaL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of beta2 integrins. Our data suggest that a specific regulation of beta2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the alpha subunit cytoplasmic domains.     

The top of the inserted-like domain of the integrin lymphocyte function-associated antigen-1 beta subunit contacts the alpha subunit beta -propeller domain near beta-sheet 3

We find that monoclonal antibody YTA-1 recognizes an epitope formed by a combination of the integrin alpha(L) and beta(2) subunits of LFA-1. Using human/mouse chimeras of the alpha(L) and beta(2) subunits, we determined that YTA-1 binds to the predicted inserted (I)-like domain of the beta(2) subunit and the predicted beta-propeller domain of the alpha(L) subunit. Substitution into mouse LFA-1 of human residues Ser(302) and Arg(303) of the beta(2) subunit and Pro(78), Thr(79), Asp(80), Ile(365), and Asn(367) of the alpha(L) subunit is sufficient to completely reconstitute YTA-1 reactivity. Antibodies that bind to epitopes that are nearby in models of the I-like and beta-propeller domains compete with YTA-1 monoclonal antibody for binding. The predicted beta-propeller domain of integrin alpha subunits contains seven beta-sheets arranged like blades of a propeller around a pseudosymmetry axis. The antigenic residues cluster on the bottom of this domain in the 1-2 loop of blade 2, and on the side of the domain in beta-strand 4 of blade 3. The I domain is inserted between these blades on the top of the beta-propeller domain. The antigenic residues in the beta subunit localize to the top of the I-like domain near the putative Mg(2+) ion binding site. Thus, the I-like domain contacts the bottom or side of the beta-propeller domain near beta-sheets 2 and 3. YTA-1 preferentially reacts with activated LFA-1 and is a function-blocking antibody, suggesting that conformational movements occur near the interface it defines between the LFA-1 alpha and beta subunits.     

Transition from rolling to firm adhesion is regulated by the conformation of the I domain of the integrin lymphocyte function-associated antigen-1

The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation. We have measured for the first time the effect of conformational change on adhesive behavior in shear flow. We show that wild type and locked open I domains, expressed in alpha(L)beta(2) heterodimers or as isolated domains on the cell surface, mediate rolling adhesion and firm adhesion, respectively. alpha(L)beta(2) is thus poised for the conversion of rolling to firm adhesion upon integrin activation in vivo. Isolated I domains are surprisingly more effective than alpha(L)beta(2) in interactions in shear flow, which may in part be a consequence of the presence of alpha(L)beta(2) in a bent conformation. Furthermore, the force exerted on the C-terminal alpha-helix appears to stabilize the open conformation of the wild type isolated I domain and contribute to its robustness in supporting rolling. An allosteric small molecule antagonist of alpha(L)beta(2) inhibits both rolling adhesion and firm adhesion, which has important implications for its mode of action in vivo.     

DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.     

Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation,but is independent of post-translational modifications of osteopontin

Osteopontin (OPN) is a ligand for the α4ß1 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of post-translational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines and compared OPN interaction with α4 integrin to that of VCAM and fibronectin. Jurkat cells, whose α4 integrins are inherently activated, adhered to different preparations of OPN in the presence of Mn^2 +: the EC50 of adhesion was not affected by phosphorylation or glycosylation status. Thrombin cleavage of OPN at the C-terminus of the α4 integrin-binding site also did not affect binding affinity. THP-1 cells express a low-affinity conformation of the integrin and adhered to OPN only in the presence of Mn^2 + plus PMA or an activating antibody. This was in contrast to VCAM and fibronectin: THP-1 cells adhered to these ligands without integrin activation. Studies with ligand-induced binding site antibodies demonstrated that the SVVYGLR peptide of OPN bound to the α4 integrin with a similar affinity as the LDV peptide of fibronectin, suggesting that a high off-rate is responsible for the reduced binding of OPN to the low-affinity forms of this integrin. Together, the results suggest OPN has very low affinity for the α4 integrin on human leukocytes under physiological conditions.     

alpha4 beta1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation

alpha(4)beta(1)-Integrin plays a pivotal role in cell migration in vivo. This integrin has been shown to regulate the front-back polarity of migrating cells via localized inhibition of alpha(4)-integrin/paxillin binding by phosphorylation at the alpha(4)-integrin cytoplasmic tail. Here, we demonstrate that alpha(4)beta(1)-integrin regulates directionally persistent cell migration via a more complex mechanism in which alpha(4)-integrin phosphorylation and paxillin binding act via both cooperative and independent pathways. We show that, in response to shear flow, alpha(4)beta(1)-integrin binding to the CS-1 region of fibronectin was necessary and sufficient to promote directionally persistent cell migration when this integrin was ectopically expressed in CHO cells. Under shear flow, the alpha(4)beta(1)-integrin-expressing cells formed a fan shape with broad lamellipodia at the front and retracted trailing edges at the back. This "fanning" activity was enhanced by disrupting paxillin binding alone and inhibited by disrupting phosphorylation alone or together with disrupting paxillin binding. Notably, the phosphorylation-disrupting mutation and the double mutation resulted in the formation of long trailing tails, suggesting that alpha(4)-integrin phosphorylation is required for trailing edge retraction/detachment independent of paxillin binding. Furthermore, the stable polarity and directional persistence of shear flow-stimulated cells were perturbed by the double mutation but not the single mutations alone, indicating that paxillin binding and alpha(4)-integrin phosphorylation can facilitate directionally persistent cell migration in an independent and compensatory manner. These findings provide a new insight into the mechanism by which integrins regulate directionally persistent cell migration.     

Human eosinophil, but not neutrophil, adherence to IL-1-stimulated human umbilical vascular endothelial cells is alpha 4 beta 1 (very late antigen-4) dependent.

Eosinophils, through their ability to generate an array of potent mediators, are thought to be the major effector cells in a number of conditions, including parasitic infection, asthma, and other allergic diseases. The mechanism(s) by which eosinophils, as opposed to neutrophils, accumulate at inflammatory sites is unknown. One possible mechanism would be an eosinophil-specific pathway of adhesion to vascular endothelium. In this study we have demonstrated that human eosinophils, but not neutrophils, constitutively express alpha 4 beta 1 (CD49d/CD29). Expression was not increased on low density eosinophils or normal density cells stimulated with platelet-activating factor. Eosinophils, but not neutrophils, specifically adhered to COS cells transfected with vascular adhesion molecule-1 in a alpha 4 beta 1-dependent manner. Eosinophil, but not neutrophil, adhesion to IL-1 stimulated human umbilical vascular endothelial cells was significantly inhibited by alpha 4 beta 1 mAb at both 5 h (p less than 0.05) and 20 h (p less than 0.001). Inhibition of both resting and platelet-activating factor-(10(-7) M) stimulated eosinophil adhesion was observed. We conclude that the alpha 4 beta 1/vascular adhesion molecule-1 adhesion pathway may be involved in specific eosinophil, as opposed to neutrophil, migration into sites of eosinophilic inflammation.     

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.     

Homing of in vitro-generated donor antigen-reactive CD4+ T lymphocytes to renal allografts is alpha 4 beta 1 but not alpha L beta 2 integrin dependent

The extravasation and sequestration of Ag-reactive T lymphocytes into vascularized organ allografts depend on a cascade of complex interactions among circulating lymphocytes, endothelial cells, and extracellular matrix proteins. Ag-activated donor-specific CD4 T cells are major initiators and effectors in the allograft rejection response. Interfering with the intragraft homing of activated CD4 T cells may represent a novel therapeutic approach in transplant recipients. We have developed a FACS-based short-term homing assay that allows tracing in vitro-generated Ag-reactive CD4 T cells after adoptive transfer in test rat recipients. Allospecific cell lines were preincubated with anti-alpha(4)beta(1) or anti-alpha(L)beta(2) mAb, because of enhanced expression of both integrin receptors after alloactivation. The pretreated Lewis(BN) lymphocytes were carboxyfluorescein diacetate succinimidyl ester labeled and adoptively transferred into Lewis rat recipients of Brown Norway kidney allografts. The injection of equal numbers of PKH-26-labeled untreated cells allowed quantitative comparison of both populations in the same animal. Ex vivo treatment with anti-alpha(4)beta(1) mAb diminished intragraft infiltration of adoptively transferred T cells by 85% in a donor-specific fashion. In contrast, treatment with anti-alpha(L)beta(2) mAb did not affect intragraft cell sequestration. Hence, blocking alpha(4)beta(1) integrin interactions represents a novel strategy in preventing local intragraft recruitment of Ag-reactive CD4 T cells in transplant recipients.     

Clustering induces a lateral redistribution of alpha 2 beta 1 integrin from membrane rafts to caveolae and subsequent protein kinase C-dependent internalization

Integrin α2β1 mediates the binding of several epithelial and mesenchymal cell types to collagen. The composition of the surrounding plasma membrane, especially caveolin-1- and cholesterol-containing membrane structures called caveolae, may be important to integrin signaling. On cell surface α2β1 integrin was located in the raft like membrane domain, rich in GPI-anchored proteins, rather than in caveolae. However, when antibodies were used to generate clusters of α2β1 integrin, they started to move laterally on cell surface along actin filaments. During the lateral movement small clusters fused together. Finally α2β1 integrin was found inside caveolae and subsequently internalized into caveosome-like perinuclear structures. The internalization process, unlike cluster formation or lateral redistribution, was dependent on protein kinase Cα activity. Caveolae are known to be highly immobile structures and α2β1 integrin clusters represent a previously unknown mechanism to activate endocytic trafficking via caveolae. The process was specific to α2β1 integrin, because the antibody-mediated formation of αV integrin clusters activated their internalization in coated vesicles and early endosomes. In addition to natural ligands human echovirus-1 (EV1) gains entry into the cell by binding to α2β1 and taking advantage of α2β1 internalization via caveolae.     

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist.     

Regulation of macrophage activation by IL-3. I. IL-3 functions as a macrophage-activating factor with unique properties, inducing Ia and lymphocyte function-associated antigen-1 but not cytotoxicity

Here we report that IL-3 (also referred to as multi-CSF because of its colony-stimulating activity on a variety of hemopoietic cell lineages) can function as a macrophage-activating factor (MAF). IL-3 was able to regulate the expression of class II MHC Ag and the cellular interaction molecule lymphocyte function-associated Ag-1 on the surface of murine peritoneal exudate cells. The kinetics of IL-3-induced Ia expression appeared to be distinct from that induced by either IFN-gamma, IL-4, or granulocyte-macrophage-CSF. IL-3 was also distinguished from these factors by the finding that it did not induce macrophage tumoricidal activity. In addition to its inherent MAF activities, IL-3 also showed a marked synergy with low doses of LPS (0.05 to 0.5 ng/ml) as well as IFN-gamma in Ia induction. When lymphocyte function-associated Ag-1 expression was evaluated, the effects of these stimuli appeared to be only additive. Although LPS has been shown to inhibit IFN-gamma-induced Ia expression, in our experiments this property of LPS is manifest only when present at doses greater than or equal to 50 ng/ml. At lower concentrations, LPS potentiated both IL-3- and IFN-gamma-induced class II MHC Ag expression. Data presented here also suggest that the synergistic interactions between low doses of LPS and IL-3 are not mediated by known LPS-inducible cytokines of macrophage origin, because rIL-1, TNF-alpha, or IL-6 did not enhance the response to IL-3. Because IL-3 can also participate in the regulation of IL-1 expression, it appears that IL-3 can function as a MAF which selectively regulates the accessory cell characteristics required for Ag presentation, as opposed to the cytolytic functions of the macrophage.     

CCL25 and CCL28 promote alpha4 beta7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow

Inflammatory bowel disease is characterized by the recruitment of lymphocytes to the gut via mucosal vessels. Chemokines are believed to trigger alpha(4)beta(1)- and alpha(4)beta(7)-integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on mucosal vessels, although the contribution of each pathway and the chemokines involved are not well characterized. These interactions occur under conditions of hemodynamic shear, which is critical in determining how lymphocytes integrate chemokine signals to promote transmigration. To define the role of specific chemokines in mediating lymphocyte adhesion to VCAM-1 and MAdCAM-1, we studied the ability of immobilized chemokines to activate adhesion of human lymphocytes in a flow-based adhesion assay. Adhesion to immobilized MAdCAM-1 was alpha(4)beta(7) dependent, with no contribution from alpha(4)beta(1), whereas alpha(4)beta(1) mediated rolling and static adhesion on VCAM-1. Immobilized CC-chemokine ligand (CCL) 25 and CCL28 were both able to trigger alpha(4)beta(7)-dependent lymphocyte arrest on MAdCAM-1 under shear, highlighting a potential role for these chemokines in the arrest of lymphocytes on postcapillary venules in the gut. Neither had any effect on adhesion to VCAM-1, suggesting that they selectively trigger alpha(4)beta(7)-mediated adhesion. Immobilized CCL21, CCL25, CCL28, and CXC-chemokine ligand (CXCL) 12 all converted rolling adhesion to static arrest on MAdCAM-1 by activating lymphocyte integrins, but only CCL21 and CXCL12 also triggered a motile phenotype characterized by lamelipodia and uropod formation. Thus alpha(4)beta(1)/VCAM-1 and alpha(4)beta(7)/MAdCAM-1 operate independently to support lymphocyte adhesion from flow, and chemokines may act in concert with one chemokine triggering integrin-mediated arrest and a second chemokine promoting motility and transendothelial migration.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

The CD81 tetraspanin facilitates instantaneous leukocyte VLA-4 adhesion strengthening to vascular cell adhesion molecule 1 (VCAM-1) under shear flow

Leukocyte integrins must rapidly strengthen their binding to target endothelial sites to arrest rolling adhesions under physiological shear flow. We demonstrate that the integrin-associated tetraspanin, CD81, regulates VLA-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells. CD81 strengthens multivalent VLA-4 contacts within subsecond integrin occupancy without altering intrinsic adhesive properties to low density ligand. CD81 facilitates both VLA-4-mediated leukocyte rolling and arrest on VCAM-1 under shear flow as well as VLA-5-dependent adhesion to fibronectin during short stationary contacts. CD81 also augments VLA-4 avidity enhancement induced by either chemokine-stimulated Gi proteins or by protein kinase C activation, although it is not required for Gi protein or protein kinase C signaling activities. In contrast to other proadhesive integrin-associated proteins, CD81-promoted integrin adhesiveness does not require its own ligand occupancy or ligation. These results provide the first demonstration of an integrin-associated transmembranal protein that facilitates instantaneous multivalent integrin occupancy events that promote leukocyte adhesion to an endothelial ligand under shear flow.     

A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation.