Phosphatidylinositol mannoside from Mycobacterium tuberculosis binds alpha5beta1 integrin (VLA-5) on CD4+ T cells and induces adhesion to fibronectin

The pathological hallmark of the host response to Mycobacterium tuberculosis is the granuloma where T cells and macrophages interact with the extracellular matrix (ECM) to control the infection. Recruitment and retention of T cells within inflamed tissues depend on adhesion to the ECM. T cells use integrins to adhere to the ECM, and fibronectin (FN) is one of its major components. We have found that the major M. tuberculosis cell wall glycolipid, phosphatidylinositol mannoside (PIM), induces homotypic adhesion of human CD4+ T cells and T cell adhesion to immobilized FN. Treatment with EDTA and cytochalasin D prevented PIM-induced T cell adhesion. PIM-induced T cell adhesion to FN was blocked with mAbs against alpha5 integrin chain and with RGD-containing peptides. Alpha5beta1 (VLA-5) is one of two major FN receptors on T cells. PIM was found to bind directly to purified human VLA-5. Thus, PIM interacts directly with VLA-5 on CD4+ T lymphocytes, inducing activation of the integrin, and promoting adhesion to the ECM glycoprotein, FN. This is the first report of direct binding of a M. tuberculosis molecule to a receptor on human T cells resulting in a change in CD4+ T cell function.     

The neural cell adhesion molecule N-CAM enhances L1-dependent cell-cell interactions

On neural cells, the cell adhesion molecule L1 is generally found coexpressed with N-CAM. The two molecules have been suggested, but not directly shown, to affect each other's function. To investigate the possible functional relationship between the two molecules, we have characterized the adhesive interactions between the purified molecules and between cultured cells expressing them. Latex beads were coated with purified L1 and found to aggregate slowly. N-CAM-coated beads did not aggregate, but did so after addition of heparin. Beads coated with both L1 and N-CAM aggregated better than L1-coated beads. Strongest aggregation was achieved when L1-coated beads were incubated together with beads carrying both L1 and N-CAM. In a binding assay, the complex of L1 and N-CAM bound strongly to immobilized L1, but not to the cell adhesion molecules J1 or myelin-associated glycoprotein. N-CAM alone did not bind to these glycoproteins. Cerebellar neurones adhered to and sent out processes on L1 immobilized on nitrocellulose. N-CAM was less effective as substrate. Neurones interacted most efficiently with the immobilized complex of L1 and N-CAM. They adhered to this complex even when its concentration was at least 10 times lower than the lowest concentration of L1 found to promote adhesion. The complex became adhesive for cells only when the two glycoproteins were preincubated together for approximately 30 min before their immobilization on nitrocellulose. The adhesive properties between cells that express L1 only or both L1 and N-CAM were also studied. ESb-MP cells, which are L1-positive, but N-CAM negative, aggregated slowly under low Ca2+. Their aggregation could be completely inhibited by antibodies to L1 and enhanced by addition of soluble N-CAM to the cells before aggregation. N2A cells, which are L1 and N-CAM positive aggregated well under low Ca2+. Their aggregation was partially inhibited by either L1 or N-CAM antibodies and almost completely by the combination of both antibodies. N2A and ESb-MP cells coaggregated rapidly and their interaction was similarly inhibited by L1 and N-CAM antibodies. These results indicate that L1 is involved in two types of binding mechanisms. In one type, L1 serves as its own receptor with slow binding kinetics. In the other, L1 is modulated in the presence of N-CAM on one cell (cis-binding) to form a more potent receptor complex for L1 on another cell (trans-binding).     

Human natural killer cells express VLA-4 and VLA-5, which mediate their adhesion to fibronectin.

Very late Ag (VLA)-3, VLA-4, and VLA-5, belonging to the beta-1 subfamily of integrins, have been recently identified as receptors for different binding regions of fibronectin (FN). We have detected VLA-4 and VLA-5, but not VLA-3, on fresh CD3-, CD16+, CD56+ human NK cells by flow cytometry and immunochemical analyses using mAb directed against beta-1, alpha-3, alpha-4, and alpha-5 subunits. Binding assays, performed on FN-coated plates, showed that NK cells specifically adhere to FN and their binding capacity is increased by MgCl2 but not by CaCl2. Using as inhibitory probes a polyclonal antibody against the beta-1 chain of the human FN receptor, the synthetic peptide GRGDSP, which is able to inhibit cellular adhesion mediated by VLA-5, the CS1 fragment, which contains the principal adhesion site in the IIICS domain recognized by VLA-4, and functional mAb directed against alpha-4 or alpha-5 subunits, we show that both VLA-4 and VLA-5 mediate the adhesion of human NK cells to FN. The expression of these integrin receptors may be relevant for NK interaction with extracellular matrix components and other cell types.     

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.     

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18)

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac- 1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity- purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1- dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.     

Receptor functions for the integrin VLA-3: fibronectin, collagen, and laminin binding are differentially influenced by Arg-Gly-Asp peptide and by divalent cations

The capability of the integrin VLA-3 to function as a receptor for collagen (Coll), laminin (Lm), and fibronectin (Fn) was addressed using both whole cell adhesion assays and ligand affinity columns. Analysis of VLA-3-mediated cell adhesion was facilitated by the use of a small cell lung carcinoma line (NCI-H69), which expresses VLA-3 but few other integrins. While VLA-3 interaction with Fn was often low or undetectable in cells having both VLA-3 and VLA-5, NCI-H69 cells readily attached to Fn in a VLA-3-dependent manner. Both Arg-Gly-Asp (RGD) peptide inhibition studies, and Fn fragment affinity columns suggested that VLA-3, like VLA-5, may bind to the RGD site in human Fn. However, unlike Fn, both Coll and Lm supported VLA-3-mediated adhesion that was not inhibited by RGD peptide, and was totally unaffected by the presence of VLA-5. In addition, VLA-3-mediated binding to Fn was low in the presence of Ca++, but was increased 6.6-fold with Mg++, and 30-fold in the presence of Mn++. In contrast, binding to Coll was increased only 1.2-fold with Mg++, and 1.7-fold in Mn++, as compared to the level seen with Ca++. Together, these experiments indicate that VLA-3 can bind Coll, Lm, and Fn, and also show that (a) VLA-3 can recognize both RGD-dependent and RGD-independent ligands, and (b) different VLA-3 ligands have distinctly dissimilar divalent cation sensitivities.     

Divalent cation substitution reveals CD18- and very late antigen-dependent pathways that mediate human neutrophil adherence to fibronectin.

We investigated the mechanisms by which Mn2+ alters human neutrophil (PMN) adherence to various connective tissue proteins. Substitution of Mn2+ for Ca2+ and Mg2+ significantly increased adhesion of human PMN to plastic well coated with fibronectin, fibrinogen, and laminin but not gelatin. Anti-CD18 mAb almost completely blocked adherence to laminin, partly blocked adherence to fibrinogen, but did not inhibit adhesion to fibronectin at all. In contrast, anti-very late antigen (VLA)-5 mAb antibodies significantly reduced Mn(2+)-mediated PMN adherence to fibronectin, but not to laminin or fibrinogen, demonstrating that VLA-5-mediated PMN adherence to fibronectin, but not to fibrinogen or laminin. This was supported by experiments in which synthetic GRGDSP peptide significantly inhibited Mn(2+)-mediated adherence to fibronectin, but not to laminin or fibrinogen. Activation of PMN with phorbol ester or C5a stimulated VLA-5-mediated adhesion to fibronectin, but the contribution of VLA-5 to the forces mediating adherence could only be detected when CD18 function was either blocked with mAb, or when CD18 was congenitally absent. VLA-5 mediated adhesion was also more transient than CD18-dependent adhesion. These data further confirm the presence of PMN VLA integrins and demonstrate that PMN VLA-5 contributes to stimulated PMN adherence to fibronectin.     

Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath stromal cells in culture

Physical interaction between human lymphomas and murine bone marrow derived stromal cells were studied. Nalm-6 pre-B cells adhered to BMS2 stromal cells and subsequently migrated beneath them, while Ramos Burkitt lymphoma cells, adhered but did not migrate. Four mAbs were established against Nalm-6 cells, which were able to block initial adhesion of Nalm-6 cells. Two of them were directed against the alpha 4 chain of VLA-4, and other two recognized the beta 1 chain of VLA integrins. Therefore, the initial adhesion of Ramos and Nalm-6 cells to BMS2 was largely mediated by the VLA-4 integrin expressed on lymphocytes. The corresponding ligand on stromal cells appears to be VCAM-1, because antibodies against murine VCAM-1 blocked the adhesion. However, antibodies against the alpha chain of VLA-4 were not capable of blocking subsequent migration beneath stromal cells. In contrast, antibodies against the beta chain of VLA integrins blocked the migration beneath stromal cells as well as the initial adhesion. Because a common beta chain can be shared among integrins, the role of other VLA integrins in Nalm-6 cells migration was investigated. VLA-5 and VLA-6 as well as VLA-4 were expressed on Nalm-6 cells, but not on Ramos cells. Additional blocking experiments revealed that VLA-4 and VLA-5 are likely to work in concert to mediate the migration of Nalm-6 cells beneath stromal cells. Thus, particular VLA integrins appear to be responsible not only for lymphocyte adhesion but also for migration with respect to stromal cells. These findings may have implications for cell-cell interactions and directed migration of lymphocytes in bone marrow and other tissues.     

The Mac-1 and p150,95 beta 2 integrins bind denatured proteins to mediate leukocyte cell-substrate adhesion.

Activated monocytic cells and neutrophils adhere to substrates coated with a wide variety of proteins including albumins, catalase, casein, and various extracellular matrix proteins. This adhesion can be specifically inhibited by antibodies directed to the beta 2 integrin subunit. This adhesion to protein substrates shares some similarities with two known protein-protein recognition systems with little apparent binding specificity, namely, the interactions of heat shock proteins and histocompatibility antigens with denatured proteins or peptides. Cell adhesion and affinity chromatography experiments were performed to test the hypothesis that monocytes and neutrophils adhere to and migrate on protein substrates due to the presence of cell surface receptors that recognize common protein structures such as denatured protein epitopes. Adhesion experiments revealed that activated monocytic cells adhere more rapidly and extensively on substrates coated with denatured protein versus native protein. Both adhesion and migration on such substrates in vitro was dependent on beta 2 integrins since blocking antibodies completely interfered with these cellular responses. Affinity chromatography experiments revealed that the Mac-1 and p150,95 integrins could be isolated from monocyte-differentiated HL-60 cells or neutrophils on a denatured protein-Sepharose column. Much greater yields of the receptors were obtained on a denatured versus native protein Sepharose column. The binding of these receptors was specific in that the LFA-1 beta 2 integrin did not bind to the denatured protein column. These data provide evidence that the adhesion of activated monocytes and neutrophils to many protein substrates in vitro is due to the ability of Mac-1 and p150,95 to directly bind to denatured proteins. A model of leukocyte adhesion and invasion whereby activated leukocytes denature extracellular proteins during diapedesis, making them suitable for recognition by beta 2 integrins, is proposed.     

Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3

To analyze the binding requirements of LFA-1 for its two most homologous ligands, ICAM-1 and ICAM-3, we compared the effects of various LFA-1 activation regimes and a panel of anti-LFA-1 mAbs in T cell binding assays to ICAM-1 or ICAM-3 coated on plastic. These studies demonstrated that T cell binding to ICAM-3 was inducible both from the exterior of the cell by Mn2+ and from the interior by an agonist of the "inside-out" signaling pathway. T cells bound both ICAM ligands with comparable avidity. A screen of 29 anti-LFA-1 mAbs led to the identification of two mAbs specific for the alpha subunit of LFA-1 which selectively blocked adhesion of T cells to ICAM-3 but not ICAM-1. These two mAbs, YTH81.5 and 122.2A5, exhibited identical blocking properties in a more defined adhesion assay using LFA-1 transfected COS cells binding to immobilized ligand. Blocking was not due to a steric interference between anti-LFA-1 mAbs and N-linked carbohydrate residues present on ICAM-3 but not ICAM-1. The epitopes of mAbs YTH81.5 and 122.2A5 were shown to map to the I domain of the LFA-1 alpha subunit. A third I domain mAb, MEM-83, has been previously reported to uniquely activate LFA-1 to bind ICAM-1 (Landis, R. C., R. I. Bennett, and N. Hogg. 1993. J. Cell Biol. 120:1519-1527). We now show that mAb MEM-83 is not able to stimulate binding of T cells to ICAM-3 over a wide concentration range. Failure to induce ICAM-3 binding by mAb MEM-83 was not due to a blockade of the ICAM-3 binding site on LFA-1. This study has demonstrated that two sets of functionally distinct mAbs recognizing epitopes in the I domain of LFA-1 are able to exert differential effects on the binding of LFA-1 to its ligands ICAM-1, and ICAM-3. These results suggest for the first time that LFA-1 is capable of binding these two highly homologous ligands in a selective manner and that the I domain plays a role in this process.     

VLA-4-dependent and -independent pathways in cell contact-induced proinflammatory cytokine production by synovial nurse-like cells from rheumatoid arthritis patients

Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.     

Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes.

The Arg-Gly-Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell-to-cell and cell-to-matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD-containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona-free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD-containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000-coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 micrograms/ml, GRGDTP 150 micrograms/ml, laminin 80 micrograms/ml, and fibronectin 60 micrograms/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lovastatin and phospholipase Cgamma regulate constitutive and protein kinase C dependent integrin mediated interactions of human T-cells with collagen

We previously reported that human interleukin (IL)-2 dependent T cell lines derived from very late antigen (VLA)-1(+) CD45RO(+) peripheral blood (PB) T-cells adhere constitutively to collagen type IV, whereas lines from VLA-1(-) PB lymphocytes (L) adhere weakly. Here we report that the latter are induced to adhere by phorbol 12-myristate 13-acetate (PMA). Both PMA dependent and constitutive adhesion, including that of a Herpes Virus Saimiri (HVS) infected CD4(+)VLA-1(+) clone (HVST) were inhibited by anti-VLA-1 monoclonal antibodies (mAb), by inhibitors of phospholipase C (PLC)gamma and by lovastatin but not by a MEK1 inhibitor, whereas only PMA induced adhesion was blocked by inhibition of protein-kinase (PK) C. Furthermore, lovastatin enhanced PLCgamma and anti VLA-1 mAb blockade, and its effect was not reversed by mevalonic acid (MVA). Lovastatin also inhibited interferon (IFN)gamma secretion by T cells triggered with anti-CD3 and in cells detaching from collagen IV. These results suggest new ways for functional modulation of activated T-cells interacting with collagen.     

Expression of VLA-4 on thymocytes. Maturation stage-associated transition and its correlation with their capacity to adhere to thymic stromal cells.

The present study investigates the expression of VLA-4 on thymocytes at various stages of maturation and their capacity to adhere to thymic stromal cells. Whole thymocytes were stained with anti-CD4 and anti-CD8, as well as anti-VLA-4 antibodies. Flow microfluorometric analyses revealed that a) most of CD4-8- (double negative DN) and CD4-8intermediate thymocyte populations expressed large amounts of VLA-4, b) the levels of VLA-4 were considerably and markedly reduced on CD4+8+ (double positive DP) and single positive (SP) (CD4+8- or CD4-8+) populations, respectively. This contrasted with an increase in the levels of LFA-1 along with thymocyte maturation. DN, DP, and SP subsets were isolated and examined for their capacity to express VLA-4 and to adhere to fibronectin (FN) molecules as well as thymic stromal cells expressing FN. DN, DP, and SP subsets were confirmed to express the respective high, low, and very low levels of VLA-4, respectively. Approximately 70% of DN thymocytes became bound to FN-precoated culture plates, whereas 30 to 40% of DP and only 10 to 20% of SP cells adhered to FN. Similar patterns of adhesion were observed between these thymocyte subsets and thymic stromal monolayers. The binding of the DN subset to FN-plates or thymic stromal monolayers was inhibited only marginally by the RGDS peptide, but was efficiently inhibited by V10 peptide (cell-binding sequence that is located in the V region on FN and reacts with the VLA-4 integrin) or anti-VLA-4 antibody. Anti-VLA-4 antibody plus RGDS peptide strongly inhibited DN cell binding to FN-coated plates and thymic stromal monolayers. These results indicate that i) VLA-4 expressed on DN thymocytes functions as an important integrin for interacting with thymic stromal cells; ii) the expression level of this integrin decreases with the progress of thymocyte maturation, and iii) most of the mature thymocytes (SP) are rendered less adhesive to thymic stromal cells by reducing the level of VLA-4 expression.     

IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1.

The present studies were performed to explore potentially selective mechanisms of leukocyte adhesion in an attempt to understand how preferential recruitment of eosinophils and basophils might occur during allergic and other inflammatory reactions. Stimulation of human vascular endothelial cells for 24 h with IL-4 (30 to 1,000 U/ml) induced adhesion for eosinophils (up to approximately four-fold of control) and basophils (up to approximately twofold of control) but not neutrophils (less than 125% of control). Analysis of endothelial expression of adhesion molecules by flow cytometry revealed that IL-4 treatment induced vascular cell adhesion molecule-1 (VCAM-1) expression without significantly affecting the expression of other adhesion molecules, namely endothelial-leukocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1). The concentration-response curve for IL-4-induced VCAM-1 expression paralleled that for adhesion. Endothelial cells stimulated with IL-4 expressed adhesive properties for eosinophils by 3 h; the response increased steadily during a 24-h time course study. Eosinophils and basophils adhered to plates coated with a recombinant form of VCAM-1. This adhesion was blocked with antibodies to VCAM-1 but not ELAM-1. mAb directed against either VCAM-1 or VLA-4 inhibited (by approximately 75%) the binding of eosinophils and basophils to IL-4-stimulated endothelial cells. Because VLA-4 and VCAM-1 have been demonstrated to bind to each other in other adhesion systems, these results suggest that IL-4 stimulates eosinophil and basophil adhesion by inducing endothelial cell expression of VCAM-1 which binds to eosinophil and basophil VLA-4. The lack of expression of VLA-4 on neutrophils and the failure of IL-4 to stimulate neutrophil adherence support this conclusion. It is proposed that local release of IL-4 in vivo in allergic diseases or after experimental allergen challenge may partly explain the enrichment of eosinophils and basophils (vs neutrophils) observed in these situations.     

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.     

Dissection of murine lymphocyte-endothelial cell interaction mechanisms by SV-40-transformed mouse endothelial cell lines: novel mechanisms mediating basal binding, and alpha 4-integrin-dependent cytokine-induced adhesion.

Lymph node-derived endothelial cells were immortalized by infection with SV40 virus and subclones expressing the marker MECA 325 specific for high-endothelial venules (HEV) were selected. These transformed mouse endothelial (TME-) cell lines grow permanently without requirement for special growth factors. Staining of the selected clones with endothelium-specific antibodies and with anti-von Willebrand factor antiserum and uptake of acetylated low-density lipoprotein provide evidence for their endothelial origin. The vascular addressins identified by mAbs MECA 79 and MECA 367 on HEV are not detectable, indicating that the phenotype of the cells differs from that of HEV-type endothelium. The TME cells display a constitutive capacity to bind lymphocytes. An additional binding component is induced by treatment of the TME cells with TNF alpha. Antibodies against the homing receptor LECAM-1 (lectin-related leucocyte-endothelial cell adhesion molecule 1), alpha 4-integrins, vascular addressins, LFA-1, or ICAM-1 known to block lymphocyte interaction with particular types of HEV were unable to inhibit the basal adhesion to TME cells, indicating that a further binding mechanism in mice is displayed by this cell type. The adhesion component induced by TNF alpha is mediated by alpha 4-integrins since enhanced binding could be blocked by an antibody against mouse alpha 4 (lymphocyte-Peyer's patch adhesion molecule 1/2). TME cell lines therefore seem to be a useful model for the dissection and analysis of hitherto poorly characterized murine lymphocyte/endothelial cell interaction mechanisms.     

Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin

Given prior evidence that adhesion molecules play critical roles in T cell recognition, it is important to identify new adhesion pathways and explore their role in T cell activation. Our studies of T cell proliferation complement concurrent studies of T cell adhesion; both demonstrate that resting CD4+ human T lymphocytes express the VLA integrins VLA-4, VLA-5, and VLA-6, and can use these receptors to interact with the extracellular matrix (ECM) proteins fibronectin (VLA-4 and VLA-5) and laminin (VLA-6). VLA-dependent interaction of resting human CD4+ T cells with fibronectin (FN) and laminin (LN) facilitates CD3-mediated T cell proliferation. Specifically, T cells do not proliferate in response to a wide range of concentrations of a CD3 mAb, OKT3, immobilized on plastic. However, coimmobilization with the CD3 mAb of FN or LN, but not other ECM proteins such as fibrinogen and collagen, consistently results in strong T cell proliferation. mAb blocking studies demonstrate that three VLA integrin receptor/ligand interactions mediate costimulation: VLA-4/FN, VLA-5/FN, and VLA-6/LN. VLA-5-dependent binding to FN but not costimulation by FN can be specifically blocked with peptides containing the RGD (arg-gly-asp) tripeptide sequence whereas VLA-4-dependent binding and costimulation can both be efficiently inhibited by a 12 amino acid peptide, LHGPEILDVPST (leu-his-gly-pro-glu-iso-leu-asp-val-pro-ser-thr), derived from the alternatively spliced IIICS region of FN. The costimulation provided by FN and LN in this system is stronger than and distinct from costimulatory signals provided by cytokines, such as IL-1 beta, IL-6,, and IL-7. These results suggest that, such as other adhesion molecules, T cell VLA integrins may also function in a dual capacity as adhesion and signalling molecules. In addition, they suggest that the interaction of T cells in vivo with ECM via VLA integrins plays a role not only in T cell migratory processes but may also influence Ag-specific T cell recognition.     

Thrombospondin modulates alpha v beta 3 function through integrin- associated protein

Integrin-associated protein (IAP) is a receptor for the carboxyl- terminal "cell-binding domain" (CBD) of thrombospondin 1 (TS1). IAP associates with alpha v beta 3 integrin and mAbs against IAP inhibit certain integrin functions. Here we examine the effects of the TS1 CBD and 4N1K (KRFYVVMWKK), a cell-binding peptide derived from it, on the adhesion and spreading on vitronectin (VN) of C32 human melanoma cells which express IAP, alpha v beta 3, and alpha v beta 5. Cells adhere to VN at low surface densities via alpha v beta 5 and spread very slowly while adhesion to higher density VN involves both alpha v beta 5 and alpha v beta 3 and results in rapid spreading. Spreading of the cells, but not adhesion, on sparse VN coatings is markedly enhanced by the presence of soluble TS1, the recombinant CBD and 4N1K, but not the "mutant" peptide 4NGG, KRFYGGMWKK, which fails to bind IAP. This enhanced spreading is completely blocked by mAb LM609 against alpha v beta 3 and the anti-IAP mAb B6H12. Correlated with this enhanced spreading is increased tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and a protein of ca. 90 kD. The enhanced spreading induced by TS1 and 4N1K and the constitutive spreading on higher density VN are both blocked by calphostin C (100 nM), wortmannin (10 nM), and tyrosine kinase inhibitors. In contrast, pertussis toxin specifically blocks only the TS1 stimulated spreading on low density VN, indicating that IAP exerts its effects on signal transduction via a heterotrimeric Gi protein acting upstream of a common cell spreading pathway which includes PI-3 kinase, PKC, and tyrosine kinases.     

CHO cell aggregation induced by fibronectin-coated beads. Differences between wild-type and adhesion-variant cells

ADvF11 (F11), a Chinese Hamster Ovary (CHO) cell variant, is defective in its ability to adhere to fibronectin (Fn)-coated substrata but will adhere to substrata coated with poly-L-lysine, conA or extracellular matrix (ECM) [1]. We have observed that both F11 and CHO wild-type (WT) cells were able to bind 3H-Fn beads in a similar manner; however, only WT cells and not F11 cells aggregate in the presence of Fn beads. Both cell types aggregated similarly in the presence of lectins. Fn-bead-mediated aggregation was blocked by low temperature and aggregation did not occur when formaldehyde-fixed WT cells were used. Colchicine, tetracaine and cytochalasin B were not effective in blocking aggregation induced by Fn beads. These results suggest that: 1. Both WT and F11 cells have surface membrane-binding sites for Fn. 2. The aggregation defect in F11 cells is distal to the initial interaction between the cell surface and Fn, but proximal to the cytoskeletal rearrangements required for cell adhesion.