VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.

Cytokine-activated human endothelial cells express vascular cell adhesion molecule-1 (VCAM-1), which binds lymphocytes. We now identify the integrin VLA-4 as a receptor for VCAM-1 because VLA-4 surface expression on K-562 cells (following transfection of the VLA alpha 4 subunit cDNA) resulted in specific cell adhesion to VCAM-1, and anti-VLA-4 antibodies completely inhibited VCAM-1-dependent cell-cell attachment. In addition, VLA-4 expression allowed K-562 cells to attach to the heparin II binding region (FN-40) of fibronectin. However, VLA-4/VCAM-1 and VLA-4/FN-40 interactions are readily distinguishable: only the former was inhibited by the anti-VLA-4 monoclonal antibody HP1/3, and only the latter was inhibited by soluble FN-40. The VCAM-1/VLA-4 ligand-receptor pair may play a major role in the recruitment of mononuclear leukocytes to inflammatory sites in vivo.     

Immobilized stromal cell-derived factor-1alpha triggers rapid VLA-4 affinity increases to stabilize lymphocyte tethers on VCAM-1 and subsequently initiate firm adhesion

The integrin VLA-4 (alpha(4)beta(1)) mediates tethering and rolling events as well as firm adhesion of leukocytes to VCAM-1. Unlike selectins, VLA-4 integrin-mediated lymphocyte adhesiveness can be modulated by chemokines through intracellular signaling pathways. To investigate the effects of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) on VLA-4-mediated lymphocyte adhesion, human PBL were flowed over VCAM-1 substrates in a parallel plate flow chamber with surface-immobilized SDF-1alpha, a potent activator of firm adhesion. The initial tethering interactions had a median lifetime of 200 ms, consistent with the half-life of low-affinity VLA-4-VCAM-1 bonds. Immobilized SDF-1alpha acted within the lifetime of a primary tether to stabilize initial tethering interactions, increasing the likelihood a PBL would remain interacting with the surface. As expected, the immobilized SDF-1alpha also increased the ratio of PBL firm adhesion to rolling. An LDV peptide-based small molecule that preferentially binds high-affinity VLA-4 reduced PBL firm adhesion to VCAM-1 by 90%. The reduction in firm adhesion due to blockage of high-affinity VLA-4 was paralleled by a 4-fold increase in the fraction of rolling PBL. Chemokine activation of PBL firm adhesion on VCAM-1 depended on induction of high-affinity VLA-4 rather than recruitment of a pre-existing pool of high-affinity VLA-4 as previously thought.     

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.     

The VLA-4/VCAM-1 molecules participate in gamma delta cell interaction with endothelial cells.

The accumulation of T lymphocytes at the site of chronic inflammation depends on a number of factors including adherence of T cells to vascular endothelial cells (EC) and endothelial permeability. We examined the effects of human gamma delta + T lymphocytes on the permeability of EC to macromolecules and characterized the cell surface molecules that are involved in these interactions. In this model, the flux of [125I]albumin was measured across the EC monolayer after a short-term culture with cloned gamma delta cells. Our results show that coculture of activated, but not resting, gamma delta cells with EC enhances endothelial permeability by a cytolytic process. Pretreating gamma delta cells with monoclonal antibodies directed at either LFA-1 or VLA-4 molecules or pretreating EC with monoclonal antibodies directed against either ICAM-1 or VCAM-1 molecules significantly inhibited gamma delta cell-mediated enhancement in endothelial permeability. This indicated that VLA-4/VCAM-1 and LFA-1/ICAM-1 adhesion pathways participate in gamma delta cell-EC interaction.     

Alpha 4/beta 1 integrin (VLA-4) ligands in arthritis. Vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes.

Expression of vascular cell adhesion molecule-1 (VCAM-1) in synovial tissue was determined using the immunoperoxidase technique. Normal, rheumatoid arthritis (RA), and osteoarthritis (OA) synovia bound VCAM-1 antibodies in the intimal lining as well as blood vessels. The amount of VCAM-1 was significantly greater in the synovial lining of RA and OA tissues compared with normal synovium (p less than 0.002). There was also a trend toward greater levels of VCAM-1 staining in blood vessels of arthritic tissue (RA greater than OA greater than normal). Because VCAM-1 staining was especially intense in the synovial lining, VCAM-1 expression and regulation was studied on cultured fibroblast-like synoviocytes (FLS) derived from this region. Both VCAM-1 and intercellular adhesion molecule 1 were constitutively expressed on FLS. VCAM-1 expression was further increased by exposure to IL-1 beta, TNF-alpha, IL-4, and IFN-gamma. These cytokines (except for IL-4) also induced intercellular adhesion molecule 1 expression on FLS. ELAM was not detected on resting or cytokine-stimulated FLS. The specificity of VCAM-1 for FLS was demonstrated by the fact that only trace amounts were detected on normal and RA dermal fibroblasts. Cytokines induced intercellular adhesion molecule 1 display on dermal fibroblasts but had minimal effect on VCAM-1 expression. Finally, in adherence assays, Jurkat cell binding to resting FLS monolayers was inhibited by antibody to alpha 4/beta 1 integrin (VLA-4), CS-1 peptide from alternatively spliced fibronectin (which is another VLA-4 ligand), and, to a lesser extent, anti-VCAM-1 antibody. After cytokine stimulation of FLS, Jurkat-binding significantly increased, and this increase was blocked by anti-VCAM-1 antibody. Therefore, both CS-1 and VCAM-1 participate in VLA-4-mediated adherence to resting FLS in vitro, and VCAM-1 is responsible for the increase in Jurkat binding mediated by cytokines.     

The roles of Akt and NOSs in regulation of VLA-4-mediated melanoma cell adhesion to endothelial VCAM-1 after UVB-irradiation

UVB-reduced avidity between M624 melanoma and HUVEC cells is dependent on the interaction of VLA-4 with its endothelial ligand VCAM-1. Our previous studies suggested that a spatial organization of α4 integrin, one of the two subunits of VLA-4, on the melanoma cell surface contributed to the changes in avidity for VCAM-1 upon UVB-irradiation. In this study, we demonstrate that Akt plays an important role in regulation of the expression and surface level of α4 integrin on melanoma cells upon UVB-irradiation. While the cell surface level of α4 integrin is not significantly affected by UVB-irradiation or Akt inhibitor alone, it is dynamically altered after UVB-irradiation when Akt is inhibited. Inhibition of Akt also reverses the reduction of avidity of cells after the irradiation. Our data also shows that UVB reduces the level of Akt. The inhibition of Akt activity correlates with a reduced amount of coupled cNOS and reduced amount of iNOS after UVB-irradiation. However, the effect of NOSs on melanoma cell adhesion appears due to their roles in regulation of apoptosis after UVB-irradiation. Base on these results, we propose that the UVB-induced reduction of avidity of melanoma cells is coordinatively regulated by NOSs and Akt through two differential mechanisms.     

The integrin VLA-4 supports tethering and rolling in flow on VCAM-1

Selectins have previously been shown to tether a flowing leukocyte to a vessel wall and mediate rolling. Here, we report that an intergrin, VLA- 4, can also support tethering and rolling. Blood T lymphocytes and alpha 4 integrin-transfected cells can tether in shear flow, and then roll, through binding of the intergrin VLA-4 to purified VCAM-1 on the wall of a flow chamber. VLA-4 transfectants showed similar tethering and rolling on TNF-stimulated endothelium. Tethering efficiency, rolling velocity, and resistance to detachment are related to VCAM-1 density. Tethering and rolling did not occur on ICAM-1, fibronectin, or fibronectin fragments, and tethering did not require integrin activation or the presence of an alpha 4 cytoplasmic domain. Arrest of rolling cells on VCAM-1 occurred spontaneously, and/or was triggered by integrin activating agents Mn2+, phorbol ester, and mAb TS2/16. These agents, and the alpha 4 cytoplasmic domain, promoted increased resistance to detachment. Together the results show that VLA-4 is a versatile integrin that can mediate tethering, rolling, and firm arrest on VCAM-1.     

Difference in Th1 and Th17 lymphocyte adhesion to endothelium

T cell subset-specific migration to inflammatory sites is tightly regulated and involves interaction of the T cells with the endothelium. Th17 cells often appear at different inflammatory sites than Th1 cells, or both subsets appear at the same sites but at different times. Differences in T cell subset adhesion to endothelium may contribute to subset-specific migratory behavior, but this possibility has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo and we characterized their migratory phenotype by flow cytometry and quantitative RT-PCR. More Th17 than Th1 cells interacted with E-selectin. Fewer Th17 than Th1 cells bound to TNF-α-activated E-selectin-deficient endothelial cells, and intravital microscopy studies demonstrated that Th17 cells engage in more rolling interactions with TNF-α-treated microvessels than Th1 cells in wild-type mice but not in E-selectin-deficient mice. Th17 adhesion to ICAM-1 was dependent on integrin activation by CCL20, the ligand for CCR6, which is highly expressed by Th17 cells. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. These data provide evidence that E-selectin- and ICAM-1-dependent adhesion of Th17 and Th1 cells with endothelium are quantitatively different.     

IL-4 acts synergistically with IL-1 beta to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1.

Adhesion of lymphocytes to endothelial cells (EC) is the requisite first element in the multistep process of transmigration from blood across the postcapillary venules. Selective expression of cell adhesion molecules (CM) by microvascular EC in lymphoid organs (e.g., lymph nodes) and during tissue inflammation modulates this traffic in a site-directed manner. CAM synthesis by EC is regulated in turn by cytokines released in the local microenvironment. Studies done largely with human umbilical vein EC have implicated IL-1, IFN-gamma, and TNF-alpha as cytokines which promote leukocyte adhesion to EC. In the work reported here, the responses of cultured microvascular EC derived from macaque lymph nodes to IL-1beta, IL-2, IFN-gamma, and IL-4 were examined. Increases in lymphocyte adhesion after preculture of microvascular EC in IL-1beta or IFN-gamma were typically 2-to 4-fold above controls and comparable to those reported for human umbilical vein EC. IL-2 had no effect. In contrast, IL-4 markedly enhanced adhesion to microvascular EC. IL-4-induced adhesion was observed as early as 4 h after induction, plateaued by 24 h, was stable through 72 h of culture, but decayed to basal levels within 72 h after removal of IL-4 from the cultures. IL-1beta, but not IL-2 or IFN-gamma, synergistically enhanced the action of IL-4 on cultured microvascular EC to promote lymphocyte binding. Adhesion triggered in this manner required de novo protein synthesis. However, the avidity of IL-4-activated microvascular EC for lymphocytes, and analyses of kinetics, cation and temperature dependence, and/or lack of blockade with mAb to endothelial leukocyte adhesion molecule-1, intra-cellular adhesion molecule-1, and MECA-79 indicated that these CAM were not central to the phenomenon. To aid identification of the relevant CAM, mAb specific to IL-4-induced microvascular EC were produced. One of these, 6G10, blocked up to 90% of lymphocyte adhesion to IL-4-induced microvascular EC, immunoprecipitated an IL-4-induced cell-surface molecule of 110-kDa molecular mass, and reacted specifically with Chinese hamster ovary cells transfected with human vascular cell adhesion molecule-1. Our results suggest that IL-4 may have potent effects on lymphocyte recirculation in vivo.     

Factors involved in cell adhesion to vascular endothelium

The adhesion of blood cells to endothelium can be studied in vitro using human endothelial cells in culture. This experimental model and radiometric techniques provide us with a simple system to quantify the adhesion of blood cells to endothelium. Normal human granulocytes isolated by density gradient adhere to normal endothelial cells in a proportion of 25%. Human promyelocytic cells (HL 60) induced by retinoic acid into mature cells adhere as well as normal granulocytes while the noninduced adhere poorly to endothelium. A small percentage of normal red cells attach to endothelial cells while red cells from patients with sickle cell anemia or diabetes mellitus have a significantly increased adhesion to endothelial cells (P greater than 0.001). This adhesion is statistically correlated with the extent and severity of vascular complications in diabetes mellitus (P less than 0.05). The addition of fibrinogen significantly increased (P less than 0.01) the adhesion of normal red cells, red cells from patients with sickle cell anemia or diabetes mellitus while gamma-globulins did not modify adhesion. Fibronectin potentiated the adhesion of normal red cells.     

B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1

VCAM-1 is one of the main ligands of VLA-4, an integrin that is highly expressed on the surface of mature B cells. Here we find that coexpression of VCAM-1 on an antigen-bearing membrane facilitates B-cell activation. Firstly, this is achieved by mediating B-cell tethering, which in turn increases the likelihood of a B cell to be activated. Secondly, VLA-4 synergizes with the B-cell receptor (BCR), providing B cells with tight adhesion and enhanced signalling. This dual role of VCAM-1 in promoting B-cell activation is predominantly effective when the affinity of the BCR for the antigen is low. In addition, we show that the VCAM-1 ectodomain alone is sufficient to carry out this function. However, it requires the transmembrane domain to segregate properly into a docking structure characteristic of the B-cell immunological synapse (IS). These results show that the VLA-4/VCAM-1 interaction during membrane antigen recognition enhances B-cell activation and this function appears to be independent of its final peripheral localization at the IS.     

Integrin VLA-4 enhances sialyl-Lewisx/a-negative melanoma adhesion to and extravasation through the endothelium under low flow conditions

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.     

Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis.

Mammalian myogenesis is biphasic: primary myoblasts fuse to form primary myotubes, then secondary myoblasts align along the primary myotubes and form secondary myotubes, which comprise most of adult muscle. We provide evidence that an integrin (VLA-4) and its counter receptor (VCAM-1) have a role in secondary myogenesis. Both receptors are synthesized by cultured muscle cells: VLA-4 is induced as myotubes form, whereas VCAM-1 is present on myoblasts and myotubes. In vivo, both molecules are expressed at sites of secondary myogenesis, VLA-4 on primary and secondary myotubes, and VCAM-1 on secondary myoblasts and on regions of secondary myotubes apposed to primary myotubes. These patterns suggest that VLA-4-VCAM-1 interactions influence alignment of secondary myoblasts along primary myotubes and/or the fusion of secondary myoblasts. In support of the latter possibility, antibodies to VLA-4 or VCAM-1 inhibit myotube formation in culture.     

Expression and pathogenesis of VCAM-1 and VLA-4 cytokines in multiple myeloma

ObjectiveThe objective of this study is to investigate the expression of Vascular cell adhesion molecule-1 (VCAM-1) and very late appearing antigen-4 (VLA-4) cytokines in MM (multiple Myeloma).MethodForty patients with MM are selected as the experimental group and 30 healthy persons as the control group. Flow cytometry is used to detect the expression of VCAM-1 (CD106), VLA-4 (CD49d), CD38 and CD138 antigens in experimental group and control group. ELISA (Enzyme Linked Immunosorbent Assay) is used to detect the concentration of VCAM-1 in serum of experimental group and control group. RT-PCR is used to detect the expression of VCAM-1.ResultsThe positive rate and antigen expression rate of VACM-1 antigen in the experimental group were significantly higher than those in the control group (P < 0.05). There were statistical differences of VLA-4 and VCAM-1 antigens between the initial diagnosis group and the relapse/refractory group, and between the relapse/refractory group and the platform stage group (P < 0.05). There were significant differences between VLA-4 antigen and VACM-1 antigen, phase I and phase II, and between phase I and phase III (P < 0.05). The concentration of VCAM-1 and the expression of VCAM-1 mRNA in the experimental group were significantly higher than (P < 0.01). In the different stages of ISS (International Staging System) and different disease groups in the experimental group, the concentration of VCAM-1 and the expression level of VCAM-1 mRNA are significantly different among the three groups of stage I, II and III (P < 0.01). There is a significant difference between the initial diagnosis group, the relapse/refractory group and the platform group (P < 0.05).ConclusionThere are abnormal expressions of adhesion molecules VCAM-1 and VLA-4 in multiple myeloma patients, which are related to ISS staging.     

Influence of beta 1 integrin intracytoplasmic domains in the regulation of VLA-4-mediated adhesion of human T cells to VCAM-1 under flow conditions

The VLA-4 integrin supports static cell-cell, cell-matrix adhesion, and dynamic interactions with VCAM-1. Although functions for well-conserved beta(1) integrin cytoplasmic domains in regulating static cell adhesion has been established, the molecular basis for beta(1) integrin-dependent arrest on VCAM-1 under flow conditions remains poorly understood. We have transfected the beta(1) integrin-deficient A1 Jurkat T cell line with beta(1) cDNA constructs with deletions of the NPXY motifs and specific mutations of tyrosine residues. Deletion of either NPXY motif impaired static adhesion induced by CD2 or CD47 triggering or direct beta(1) integrin stimulation. In contrast, PMA-induced adhesion to VCAM-1 was unaffected by deletion of the NPIY motif and only slightly impaired by deletion of NPKY. Moreover, deletion of the NPIY motif resulted in enhanced rolling and reduced arrest on VCAM-1 under shear flow conditions. In contrast, deletion of the NPKY motif did not alter arrest under flow. Although tyrosine to phenylalanine substitutions within two NPXY motifs did not alter static adhesion to VCAM-1, these mutations enhanced arrest on VCAM-1 under flow conditions. Furthermore, although deletion of the C'-terminal 5 AA of the beta(1) cytoplasmic domain dramatically impaired activation-dependent static adhesion, it did not impair arrest on VCAM-1 under flow conditions. Thus, our results demonstrate distinct structural requirements for VLA-4 function under static and shear flow conditions. This may be relevant for VLA-4 activity regulation in different anatomic compartments, such as when circulating cells arrest on inflamed endothelium under shear flow and when resident cells in bone marrow interact with VCAM-1- positive stromal cells.     

CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM- 1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.     

The L1 adhesion molecule is a cellular ligand for VLA-5

The L1 adhesion molecule is a member of the immunoglobulin superfamily shared by neural and immune cells. In the nervous system L1 can mediate cell binding by a homophilic mechanism. To analyze its function on leukocytes we studied whether L1 could interact with integrins. Here we demonstrate that VLA-5, an RGD-specific fibronectin receptor on a wide variety of cell types, can bind to murine L1. Mouse ESb-MP cells expressing VLA-5 and L1 could be induced to aggregate in the presence of specific mAbs to CD24 (heat-stable antigen), a highly and heterogeneously glycosylated glycophosphatidylinositol-linked differentiation antigen of hematopoietic and neural cells. The aggregation was blocked by both mAbs to L1 and VLA-5, respectively. Aggregation was blocked also by a synthetic RGD-containing peptide derived from the Ig-domain VI of the L1 protein. ESb-MP subclones with low L1 expression could not aggregate. In heterotypic binding assays mouse bone marrow cells could adhere in an L1-dependent fashion to platelets that expressed VLA-5. Also purified L1 coated to polystyrene beads could bind to platelets. The binding of L1-beads was again inhibited by mAbs to L1 and VLA-5, by soluble L1 and the L1-RGD peptide in a dose-dependent manner. Thymocytes or human Nalm-6 tumor cells expressing VLA-5 could adhere to affinity-purified L1 and to the L1- derived RGD-containing peptide coated to glass slides. The adhesion was strongly enhanced in the presence of Mn(2+)-ions and blocked by mAbs to VLA-5. We also demonstrate a direct L1-VLA-5 protein interaction. Our results suggest a novel binding pathway, in which the VLA-5 integrin binds to L1 on adjacent cells. Given its rapid downregulation on lymphocytes after induction of cell proliferation, L1 may be important in integrin-mediated and activation-regulated cell-cell interactions.     

The peripheral lymph node homing receptor, LECAM-1, is involved in CD18-independent adhesion of human neutrophils to endothelium.

The binding of polymorphonuclear granulocytes (PMN) to activated vascular endothelium is a crucial step in the recruitment of PMN to an inflammatory site. Studies employing cytokine-activated endothelium in culture have shown that PMN binding involves the CD18 family of leukocyte integrins, but also CD18-independent adhesion mechanism(s) on PMN that have not been defined. We unify here two previously disparate approaches to study cell adhesion events between endothelial cells and leukocytes. We show that antibodies to human LECAM-1, the peripheral lymph node homing receptor that is also expressed on PMN, partially inhibit the adhesion of human PMN not only to HEV in frozen sections of lymph node tissue, but also to cytokine-activated human umbilical vein endothelium in vitro. Inhibition with anti-LECAM-1 antibodies and anti-CD18 antibodies is additive. Furthermore, the anti-LECAM-1 antibodies inhibit the adhesion of CD18-deficient PMN to cytokine activated human endothelial cells. These findings indicate that LECAM-1 and CD18-mediated binding mechanisms are independent, and act coordinately or sequentially to mediate PMN attachment to cytokine activated endothelium.     

Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin

To investigate the functional significance of putative integrin divalent cation binding sites, several mutated alpha 4 subunit cDNAs were constructed. Mutants contained the conservative substitution of Glu for Asp or Asn at the third position in each of three putative divalent cation sites. Transfection of wild-type or mutated alpha 4 into K562 cells yielded comparable expression levels and immunoprecipitation profiles. However, for all three alpha 4 mutants, adhesion to CS1/fibronectin was greatly diminished in either the presence or absence of the stimulatory anti-beta 1 mAb TS2/16. Constitutive adhesion to vascular cell adhesion molecule (VCAM) 1 was also diminished but, unlike CS1 adhesion, was restored upon TS2/16 stimulation. In contrast, adhesion to the bacterial protein invasin was minimally affected by any of the three mutations. For each of the mutants, the order of preference for divalent cations was unchanged compared to wild-type alpha 4, on CS1/fibronectin (Mn2+ > Mg2+ > Ca2+), on VCAM-1 (Mn2+ > Mg2+ = Ca2+) and on invasin (Mg2+ = Ca2+). However for the three mutants, the efficiency of divalent cation utilization was decreased. On VCAM-1, 68-108 microM Mn2+ was required to support half-maximal adhesion for the mutants compared with 14-18 microM for wild-type alpha 4. These results indicate (a) that three different ligands for VLA-4 show widely differing sensitivities to mutations within putative divalent cation sites, and (b) each of the three putative divalent cation sites in alpha 4 have comparable functional importance with respect to both divalent cation usage and cell adhesion.