Our studies suggest that the vascular endothelium is activated in patients with sickle cell anemia, regardless of the patients' clinical status. Adhesion proteins on activated endothelial cells may have a role in the vascular pathology of sickle cell disease.

have identified a new fibronectin receptor that is identical to the integrin receptor a4B1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the tx4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IrlCS for ot4B1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to or4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-glyasp) adhesion sequence, mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, ot5B1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both ot4/~l and ot5/31 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts.In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) ~x5~l is the receptor for the RGD containing cell adhesion domain, and (b) ot4~l is the receptor for a carboxyterminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that o~4~1 is the receptor for this adhesion site.

Abstract. Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and iaminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed a and 13, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed a and 13, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the ct subunits. Pulsechase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the a and 13 subunits of the class I and II antigens by peptide mapping indicated that the 13 subunits were identical while the ct subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody.The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I13 and 1113 subunits were structurally related to the 13 subunit of the fibronectin receptor described by others. However, none of these receptors shared the same ct subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.These results suggest that the class I and II receptors are two new members of a family of cell surface receptors for the extracellular matrix involved in mediating cell adhesion and shall be referred to as ECMRI and II. Each member of this family possesses a common 13 subunit and a unique a subunit. The class II receptor appears to be a primary mediator of specific cell adhesion to collagen. Th...

Abstract. Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin tx3B1, in focal adhesions (FAs), of cultured human keratinocytes (HFKs) as a mediator of HFK adhesion to secreted BMZ-like extracellular matrix (ECM; Carter, W. G., E. A. Wayner, T. S. Bouchard, and P. Kaur. 1990. J. Cell Biol. 110: 1387-1404. Here, we have examined the relation of integrins ct6/~4 and tx3B1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that ot6B4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with tx3B1-FAs to mediate adhesion to ECM, based on the following. (a) Comparison of secreted ECM, with exogenous laminin, fibronectin and collagen identified ECM as the preferred ligand for HFK adhesion and spreading and for formation of both tx6B4-SACs and ~x3B1-FAs. (b) Inhibition of HFK adhesion with combined anti-or3/31 (P1B5) and anti-c~6B4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while oe3B1 played a dominant role in spreading. (c) tx6fl4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both tx6~4-SACs and ot3fll-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to ot3~l-FAs, ot6~4-SACs were present only in nonmotile cells, not associated with stress fibers, and were relatively stable to detergents and urea, suggesting a nonmotile, or anchoring function for SACs and motility functions for tx3B1-FAs. (e) ct6B4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with ot6f14. (f) We suggest that ot6B4/BPA-SACs in culture restrict migration of HFKs on ECM while tx3fll-FAs form dynamic adhesions in spreading and migrating cells, a6fl4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.

Abstract. We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique ¢t subunit, recognized by the antibodies, and a common [~ subunit, each of ~140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, H, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains.ECENTLY, we identified a family of cell surface glycoproteins that mediate fibroblast adhesion to specific components of the extracellular matrix (ECM) ~ and are referred to as extracellular matrix receptors (ECMRs) I and II (Wayner and Carter, 1987). Monoclonal antibody to ECMR II (P1H5) specifically inhibited fibroblast (Wayner and Carter, 1987) and nonactivated platelet adhesion to collagen, while monoclonal antibody against ECMR I (P1B5) inhibited fibroblast adhesion to fibronectin (FN), collagen, and laminin (Wayner and Carter, T. J. Kunicki is an established investigator (83-186) of the American Heart Association.

Abstract. We investigated the role of the integrins c~vB3 and c~v/~5 in mediating vitronectin adhesion of three phenotypically distinct cell types. M21 human melanoma cells and H2981 lung carcinoma cells use both otv-containing integrins in adhering to vitronectin while UCLA-P3 lung carcinoma cells adhere exclusively with otv/~5. Specifically, monoclonal antibodies directed to functional epitopes on both receptors were required to block adhesion of M21 or H2981 cells while adhesion of UCLA-P3 cells to vitronectin could be blocked with a monoclonal antibody to t~v/~5. Although both receptors are involved in M21 and H2981 cell adhesion to vitronectin, only tav/33 can be detected in focal contacts, colocalizing with vinculin, talin, and the ends of actin filaments, while otv/~5 shows a distinct, nonfocal contact, distribution on the cell surface. These results provide the first evidence that two homologous integrins that recognize the same ligand distribute differentially on the cell surface.

The leukocyte receptor CD62, which is expressed on activated platelets and endothelial cells, is shown to mediate cell adhesion by binding a sialylated carbohydrate structure, sialyl-Lewis x, found on neutrophils, monocytes, and tumor cells. This structure has previously been identified as the ligand for another member of the LEC-CAM family of cell adhesion molecules, endothelial cell-eukocyte adhesion molecule 1, which also binds neutrophils and monocytes. The results demonstrate that although the two LEC-CAMs differ in their biological activities by their distribution and mode of expression, they are capable of mediating cell adhesion by recognition of the same carbohydrate ligand.Leukocyte trafficking and recruitment to sites of inflammation are mediated by three adhesion receptor families, the integrin and immunoglobulin superfamilies and the recently described LEC-CAM, or selectin family (1)(2)(3)(4) (100 nm) (13). Neutrophils were isolated from fresh human blood by centrifugation at room temperature through mono-poly resolving medium (Flow Laboratories) followed by three washes in ice-cold Hanks' balanced salt solution (GIBCO) containing 20 mM Hepes (GIBCO) and 0.2% glucose (Fisher). Human umbilical vein endothelial cells were obtained as described (6). HL-60 cells were cultured in RPMI 1640 containing 10%6 fetal calf serum. Chinese hamster ovary (CHO-Ki) cells, glycosylation mutants , and the carcinoma line were cultured in the a modification of Eagle's minimal essential medium (a-MEM) containing ribonucleosides, deoxyribonucleosides, and 10%1o fetal calf serum.Assays for CD62-Mediated Adhesion to Activated Platelets. Two assays were used, a fluid phase assay and a plate assay. The fluid phase assay (see Figs. 2-4) was performed essentially as described (10) for PADGEM (CD62). Platelets at 2 x 108 per ml were activated with thrombin at 0.25 units/ml for 20 min at room temperature without stirring. Twenty microliters of the activated platelet suspension was then mixed with an equal volume ofa suspension ofthe cells (2 x 106 cells per ml) to be assessed for adhesion. Adhesion was evaluated microscopically and was scored as the percent ofthe test cells that had bound two or more platelets. The plate assay used was a modification of the endothelial cell-neutrophil adherence assay previously described (20). A suspension of platelets (300 ,l) at 108 per ml was applied to each well of a 48-well plate that had previously been coated with 0.1% gelatin. The Gal431-4 GlcNAc / FUCCY1 3 Le' Abbreviations: LEC-CAM, a family of cell adhesion molecules, each of which contains an N-terminal lectin domain followed by an epidermal growth factor-like domain and a series of consensus repeats similar to those found in complement regulatory proteins; ELAM-1, endothelial cell-leukocyte adhesion molecule

Abstract. Human umbilical vein endothelial cells attach and spread on laminin-coated substrates. Affinity chromatography was used to identify the attachment receptor. Fractionation of extracts from surfaceiodinated endothelial cells on human lamininSepharose yielded a heterodimeric complex, the subunits of which migrated with molecular sizes corresponding to 160/120 kD and 160/140 kD under nonreducing and reducing conditions, respectively. The purified receptor bound to laminin and slightly less to fibronectin and type IV collagen in a radioreceptor assay. This endothelial cell laminin receptor was classified as an t~zB, integrin because monoclonal and polyclonal antibodies directed against the ct2 and/3, subunits immunoprecipitated the receptor. Cytofluorometric analysis arid immunoprecipitation showed that the ix2 subunit is an abundant integrin ~t subunit in the endothelial cells and that the ot subunits associated with laminin binding in other types of cells are expressed in these cells only at low levels. The ,v~, integrin appears to be a major receptor for laminin in the endothelial cells, because an anti-ix2 monoclonal antibody inhibited the attachment of the endothelial cells to human laminin. These results define a new role for the o~2 subunit in laminin binding and suggest that the ligand specificity of the tx2/3t integrin, which is known as a collagen receptor in other types of cells, can be modulated by cell type-specific factors to include laminin binding.