Expression and function of alpha 4/beta 7 integrin on human natural killer cells.

In this report we have analysed the expression and function of the alpha 4/beta 7 heterodimer in human natural killer (NK) cells. The expression of alpha 4 beta 7 is induced in NK cells upon activation as the anti alpha 4 beta 2 ACT-1 monoclonal antibody (mAb) family stained a minority of peripheral blood NK cells, whereas it strongly reacted with in vitro long-term interleukin-2 (IL-2)-activated NK cells. Incubation with ACT-1 on its F(ab) fragments induced a strong homotypic adhesion of NK cells, comparable to than stimulated by the anti-alpha 4 HPI 7 mAb. Cell cell interaction induced by the ACT-1 mAb was only prevented by another anti-alpha 4 mAb (HP2.1) that recognizes a different epitope. In alpha 4 beta 7-mediated cell aggregation the alpha 4 beta 7 heterodimer was redistributed to intercellular contact sites thus, suggesting a direct involvement of this integrin in the formation of cell clusters. In NK cells attached to Fibronectin (FN38) or vascular cell adhesion molecule-1 (VCAM-1), both alpha 4 beta 7 and alpha 4 beta 7 integrins were redistributed at the ventral cellular membrane forming discrete contact sites. The ACT-1 mAb only partially blocked NK cell binding to FN38, but in combination with the anti-beta 7 mAb LIAI 2, NK cell binding to FN38 was completely inhibited. In contrast. ACT-1 did not modify NK cell adhesion to VCAM-1 thus supporting the theory that the alpha 4 beta 7 binding sites for both ligands appear to be different. Our results indicate that upon IL-2-activation, expression of functional alpha 4 beta-integrin is induced on NK cells potentially participating in their interaction with both extracellular matrix and endothelial cells.     

Differential expression of VLA-4 integrin by resident and peripheral blood B lymphocytes. Acquisition of functionally active α4β1-fibronectin receptors upon B cell activation

Very-late antigen (VLA)-4 (CD49d/CD29) constitutes the only member of the β1 integrin family that plays a role in the interaction of lymphoid cells with both extracellular matrix and endothelial cells through two identified ligands: fibronectin (FN) and VCAM-1, respectively. The expression and functional activity of VLA-4 has been studied in different maturation and activation stages of B cells from several cellular compartments. Resident B lymphocytes of different lymphoid organs were almost negative for VLA-4 as detected by both immunoperoxidase staining and flow cytometry analysis. However, a high expression of both chains of this heterodimer was observed when tonsillar B cells were activated in vitro with different stimuli, such as phorbol esters or Staphylococcus aureus Cowan I (SAC). Both nonactivated and in vitro activated B cells from peripheral blood constitutively expressed high levels of this surface antigen. The induced expression of VLA-4 after activation of tonsillar B lymphocytes was accompanied by the acquisition of the capacity to bind to a 38-kDa proteolytic fragment, containing the connecting segment I domain, of FN. Interestingly, nonactivated peripheral blood B cells were unable to attach to this FN fragment, in spite of their constitutive expression of VLA-4, and only acquired this functional capacity after cell activation with phorbol esters and SAC. This FN-binding acquisition was not affected by preincubation with inhibitors of protein and RNA synthesis. These results underline that the FN-binding activity of VLA-4 is dependent on processes affecting cellular activation as described for other members of the integrin family. By contrast, VLA-4-mediated homotypic aggregation of peripheral blood B cells could be triggered by anti-α4 monoclonal antibodies independently of the cell activation state.     

Anti-Gal IgG potentiates natural killer cell migration across porcine endothelium via endothelial cell activation and increased natural killer cell motility triggered by CD16 cross-linking

Xenoreactive antibodies (Ab) are important for the development of acute vascular rejection (AVR) of xenografts characterized by monocytes, natural killer (NK) cells and neutrophils infiltrating the graft. The mechanisms by which anti-galactose alpha 1,3galactose (alpha-Gal) IgG influence NK cell migration across porcine aortic endothelium (PAEC) were investigated. NK cell migration across PAEC increased in the presence of anti-alpha-Gal IgG. Anti-alpha-Gal IgG exposure activated PAEC as shown by an increased expression of CD62E and CD106. NK cells adhered, spread and showed motile forms on plastic surfaces coated with human IgG, IgG Fc and on mAb against CD16, but not on mouse IgG or BSA, suggesting that CD16 cross-linking can mediate increased adhesiveness. Increased NK cell motility was observed on Boyden filters coated with human IgG, IgG Fc, and mAb against CD16 and the alpha 4, alpha 5, alpha L, beta 1 and beta 2 integrin chains. No motile response was seen on mouse IgGor CD7, CD56 and alpha 6 integrin mAb. NK cell migration on human IgG and anti-CD16 Ab was blocked by anti-CD16 or anti-beta 2, but not anti-beta 1 Ab, implying that the motile response triggered by CD16 cross-linking is mediated via beta 2 integrins. Preformed or induced anti-alpha-Gal IgG may therefore contribute to AVR by stimulating innate immune cell infiltration of the graft.     

Beta1 integrin activation on human neutrophils promotes beta2 integrin-mediated adhesion to fibronectin

Although the importance of beta1 integrin-mediated binding to adhesion molecules and extracellular matrix (ECM) molecules is well established for most types of leukocytes, the expression patterns and functional importance of beta1 integrins on neutrophils have remained controversial. Using flow cytometry, we found that human neutrophils express the alpha4, alpha5, alpha9 and beta1 integrin subunits. To examine whether the integrins VLA-4 (alpha4/beta1) and VLA-5 (alpha5/beta1) have a functional role on neutrophils, we studied adhesion to their ligand fibronectin. Treatment of neutrophils with antibody 8A2, which specifically binds and activates beta1 integrins, resulted in increased binding to fibronectin. However, addition of blocking mAb revealed that 8A2-induced adhesion did not depend on beta1 integrins, but on the beta2 integrin CD11b/CD18. Similarly, activation of beta1 integrins by 8A2 resulted in CD11b-dependent binding of neutrophils to fibrinogen. 8A2 treatment increased expression of an activation epitope of CD11b/CD18, which depended on phosphoinositide 3-OH kinase activity and an adequate concentration of intracellular free Ca2+. These data suggest that engagement of beta1 integrins on neutrophils results in a cross-talk signal that leads to activation of the beta2 integrin CD11b/CD18, followed by CD11b-mediated adhesion. As transmigrated neutrophils are surrounded by both beta1 and beta2 ligands in the ECM, this integrin cross-talk could play a role in modifying migration and cellular activation in inflamed tissues.     

Functional role of alpha 2/beta 1 and alpha 4/beta 1 integrins in leukocyte intercellular adhesion induced through the common beta 1 subunit.

Whereas all of the integrins in the VLA protein subfamily are involved in cell-extracellular matrix interactions, only VLA-4 (through the alpha 4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein beta 1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti-beta 1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common beta 1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti-beta 1 mAb. Induction of cell aggregation by anti-beta 1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to alpha 4-mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA alpha 4/beta 1 heterodimer, intercellular adhesion induced through the VLA-beta 1 chain could be selectively inhibited by other anti-beta 1 mAb as well as by anti-alpha 4 mAb. Interestingly, anti-beta 1 mAb which induced strong aggregation of VLA-alpha 2- or VLA-alpha 4-transfected K562 cells, had minimal effect on the alpha 2- alpha 4- alpha 5+ K562 cell line. Furthermore, the beta 1-mediated induction of cell aggregation on alpha 2-K562- and alpha 4-K562-transfected cells was blocked by preincubation with either anti-alpha 2 or anti-alpha 4 mAb, respectively, as well as by other anti-beta 1 mAb. Interestingly, parental K562 cells were able to interact with both alpha 2- and alpha 4-transfected K562 cells, thus suggesting that counter-receptors for both integrins (VLA-2 and VLA-4) might exist on these cells. Together these results provide strong evidence supporting the involvement of alpha 2/beta 1 and alpha 4/beta 1 heterodimers in intercellular interactions and underline the pivotal role of the common beta 1 chain of VLA proteins in the integrin-mediated induction of cell aggregation.     

Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen-4 integrins suppresses neutrophil and T-lymphocyte migration to the joints and improves clinical disease.

The migration of leucocytes from blood into the joint is a key feature of human and experimental arthritis. Adhesion molecules on leucocytes and vascular endothelium are important in this process and may be therapeutic targets for intervention in arthritis. We investigated whether monoclonal antibody treatment to block the alpha 4 integrin, very late activation antigen-4 (VLA-4), and beta 2 integrins (CD11/CD18) administered to rats during the preclinical (day 5) or clinical phase (day 10+) would modify disease. When treatment was initiated 5 days after induction of disease, development of arthritis was significantly reduced by either anti-alpha 4 (TA-2) or anti-beta 2 (WT.3) monoclonal antibodies (mAb) and the combination of both mAb was even more effective (clinical scores: control 11.4; anti-alpha 4 6.6; anti-beta 2 6.8; anti-alpha 4 + anti-beta 2 3.9). When treatment was delayed until arthritis was apparent (day 10), the anti-alpha 4 + anti-beta 2 mAb combination still significantly diminished the arthritis score on day 14 (control 13; anti-alpha 4 + anti-beta 2 7.9). Treatment with anti-alpha 4 + anti-beta 2 mAb decreased the migration to the joints of blood polymorphonuclear leucocytes (PMNL) by 66-79% and of spleen T lymphocytes by 56-75%, depending on the joint. In contrast, PMNL migration was abolished (> 98%) and T-cell migration markedly (87%) inhibited to dermal inflammatory reactions in the same animals. These findings demonstrate: that blocking mAb to alpha 4 and beta 2 integrins can reduce the severity of adjuvant arthritis, even after joint inflammation has developed; that this treatment can markedly inhibit PMNL and T-lymphocyte migration to the joints; and that yet to be defined mechanisms distinct from alpha 4 (CD49d) and beta 2 (CD11/CD18) integrins, also contribute to leucocyte migration to inflamed joints. Identifying these additional adhesion mechanisms may be required to control joint inflammation further.     

Regulation of T-cell interaction with fibronectin by transforming growth factor-β is associated with altered Pyk2 phosphorylation

Although the involvement of transforming growth factor-beta (TGF-beta) in inflammatory reactions has been extensively studied, its mode of action in the context of the extracellular matrix (ECM) is still not fully understood. We undertook this study in an attempt to reveal the putative roles of TGF-beta in T-cell adhesion and migration. We found that a 60-min treatment of T cells with TGF-beta regulates T-cell adhesion to fibronectin (FN), a prototype cell adhesion protein of the ECM, depending on the presence of other activators. At 5 pg/ml to 1 ng/ml, TGF-beta alone induced T-cell adhesion to FN in an integrin alpha4/beta1- and integrin alpha5/beta1-dependent manner. TGF-beta also attenuated T-cell migration on the stromal cell-derived factor (SDF)-1alpha gradients. These effects of TGF-beta were not accompanied by alteration in the expression of very-late activation antigen type 4 (VLA-4) and VLA-5, nor were they mediated by the cyclo-oxygenase pathway. The cellular mechanism underlying the adhesion-regulating activities of TGF-beta involves adhesion-associated cytoskeletal elements. TGF-beta induced the phosphorylation of focal adhesion kinase Pyk2, but not extracellular signal-regulated kinase (ERK), and this effect was markedly increased in the presence of immobilized FN, suggesting a collaborative role for FN-specific integrins. Indeed, TGF-beta-induced Pyk2 phosphorylation was inhibited by monoclonal antibodies against VLA-4, VLA-5 and CD29. Thus, TGF-beta, which may appear at extravascular sites during inflammation, affects the adhesion of T cells to ECM glycoproteins and their migration by its ability to differentially induce or inhibit the phosphorylation of Pyk2.     

The beta 1 integrin, very late activation antigen-4 on human neutrophils can contribute to neutrophil migration through connective tissue fibroblast barriers.

Polymorphonuclear leucocyte (PMNL) accumulation in extravascular tissues and inflammatory exudates is dependent on their migration through blood vessel endothelium and then through connective tissue. Previously we utilized a barrier of human synovial and dermal fibroblasts (HSF or HDF) grown on microporous filters, as a model of PMNL migration through connective tissue. Those studies showed that beta 2 (CD18) and the beta 1 integrins, very late activation antigen-5 (VLA-5) and VLA-6, in part mediate this PMNL migration. Here we report that VLA-4, which can also be expressed at low levels on activated PMNL, is also involved in PMNL migration induced by C5a through fibroblast (HSF and HDF) barriers, because monoclonal antibody (mAb) to VLA-4 significantly inhibited (by 20-30%) PMNL migration. Blocking the function of CD18, VLA-5 or VLA-6 was not required for detection of the VLA-4-mediated migration. Combination treatment with mAb to VLA-4 and with mAb to VLA-5 or to VLA-6 further inhibited PMNL migration, irrespective of whether CD11/CD18 mechanisms were blocked with anti-CD18 mAb or not. Treatment of PMNL with a peptide based on the VLA-4-binding domain in the CS-1 fragment of fibronectin, but not a control peptide, inhibited PMNL migration to a comparable extent to treatment with mAb to VLA-4. A low level of VLA-4 was expressed on C5a-activated PMNL, detected by immunofluorescence flow cytometry. These results suggest that VLA-4 can be mobilized by human peripheral blood PMNL and can, in addition to VLA-5, VLA-6 and CD11/CD18 integrins, mediate PMNL migration through connective tissue. This is in marked contrast to PMNL transendothelial migration, where beta 1 integrins appear to play no significant role.     

VLA family in rheumatoid arthritis: evidence for in vivo regulated adhesion of synovial fluid T cells to fibronectin through VLA-5 integrin.

Adhesion of T cells to extracellular matrix (ECM) proteins through VLA integrin receptors is crucial for lymphocyte trafficking, tissue localization and inflammatory function. We have investigated the expression of different VLA integrins (VLA-1-5) on peripheral blood (PB) and synovial fluid (SF) T lymphocytes from patients with rheumatoid arthritis (RA). Their expression on different cell types from synovial membrane (SM) is also reported. The role of VLA-4 fibronectin (FN) receptors in the interaction of activated SF T cells from RA patients with a 38-kD fragment of FN has been previously demonstrated. Here we have focused functional studies on VLA-5 as an alternative FN receptor for RA T cells. A significant higher proportion of SF T cells were able to bind to an 80-kD fragment of FN, containing the Arg-Gly-Asp (RGD) cell binding site, compared with PB T cells. This attachment was almost completely inhibited by anti-VLA-5 MoAbs as well as by RGD peptides. This enhanced capability by SF T cells appears to be independent of the level of the surface expression of the receptor and correlates better with their activation state as determined by the expression of the activation molecule AIM (CD69). The evidence for the expression of VLA heterodimers on both SF and SM cells from RA patients suggests the possible implication of ECM proteins in mediating and perpetuating inflammation in vivo.     

Signaling through the LFA-1 leucocyte integrin actively regulates intercellular adhesion and tumor necrosis factor-α production in natural killer cells

The LFA-1 leucocyte integrin is known to participate in natural killer (NK) cytolytic activity, mediating effector target interactions. The possibility that LFA-1 may also play an active regulatory role in NK cells has been explored. To this end, we have employed a monoclonal antibody (HP1N) raised against recombinant interleukin-2 (rIL-2)-activated NK cells, which recognizes the α chain of the LFA-1 heterodimer (CD11a). In contrast to other anti-CD11a mAb the HP1N and its F(ab)₂ fragment did not affect NK cell-mediated cytotoxicity and triggered a strong homotypic adhesion of NK cells and other LFA-1⁺ cells. Cellular aggregation was inhibited by anti-CD18 mAb, anti-ICAM-1 mAb, and other anti-CD11a mAb. Remarkably, the HP1N mAb was also shown to induce tumor necrosis factor-α (TNF-α) production from NK cells upon costimulation with anti-CD16 mAb. Such an effect appeared to be independent from homotypic adhesion since it took place in Mg²⁺-free medium, where NK cell aggregation was inhibited. Moreover, incubation with the HP1N mAb triggered a Ca²⁺ influx into the cytosol; this effect was clearly observed upon cross-linking of cell bound HP1N and was also substantiated with other anti LFA-1 (CD11a and CD18) mAb. Taken together these results indicate that the LFA-1 molecule is capable of transducing signals in NK cells, which regulate the intercellular interaction with its ligand, and enhance the activation via Fey receptor type III.     

Adhesion to fibronectin primes eosinophils via alpha 4 beta 1 (VLA-4).

Human peripheral blood eosinophils adhered specifically to microtitre plates coated with plasma fibronectin (Fn) in a dose- and time-dependent fashion. Adhesion was optimal at 60 min at a concentration of 100 micrograms/ml. Adherence to Fn was up-regulated by platelet-activating factor (PAF; optimum concentration of 10(-6) M) and was significantly inhibited by a polyclonal anti-Fn antibody (P < 0.05). The following evidence suggested that eosinophil adhesion to Fn was mediated by alpha 4 beta 1: (1) eosinophil adherence to Fn was not inhibited by an Arg-Gly-Asp-Ser (RGDS) synthetic peptide; (2) there was a dose-dependent adherence of eosinophils to microtitre plates coated with the 40,000 MW proteolytic fragment of Fn that contains the CS-1 alpha 4 beta 1 binding region, whereas adherence to the 120,000 MW chymotryptic fragment of Fn, which contains the RGD-dependent binding site, was weak and only observed at high concentrations (> 250 micrograms/ml); (3) significant inhibition of eosinophil adherence to Fn was achieved by monoclonal antibodies (mAb) against the alpha chain of VLA-4 but not by a mAb against CD45 or a mouse myeloma antibody as negative controls. After adhesion to Fn, eosinophils were investigated for their capacity to release leukotriene C4 in response to stimulation with a suboptimal concentration of calcium ionophore (2 x 10(-6) M). Significant enhancement of release was detected with Fn-coated plates but not with the control bovine serum albumin (BSA) (P < 0.01). Furthermore, this enhancement was significantly inhibited by the alpha 4 beta 1 mAb HP2/1 (P < 0.05) but not by an anti-CD45 mAb. From these studies we conclude that (1) alpha 4 beta 1 (VLA-4) integrin is a major receptor for Fn on human eosinophils and (2) adhesion to Fn may prime eosinophils for mediator release during allergic inflammation.     

Enhancement of activation-induced cell death by fibronectin in murine CD4+ CD8+ thymocytes.

Development of T cells in the thymus is achieved through the interactions of thymocytes with their microenvironments. This study focused on the function of fibronectin (FN), a major extracellular matrix molecule in the thymus, in the cell death induced by activation via the T-cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti-CD3 monoclonal antibody (mAb), especially when a high concentration of anti-CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence-activated cell sorter (FACS) analysis revealed that the activation-induced cell death (AICD) caused by anti-CD3 mAb alone, or by a combination of anti-CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)-4 and VLA-5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA-4 was predominantly expressed at the CD4- CD8- stage, and thereafter the expression was reduced, whereas VLA-5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide but not in the presence of the connecting segment-1 (CS-1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA-5.     

Role of ligand-specific integrins in endothelial cell alignment and elongation induced by cyclic strain.

Ligand-specific integrins are thought to play a critical role in regulating multiple biological processes. However, the mechanisms by which ligand-specific integrins mediate external stimuli and activate intracellular signaling pathways remain to be elucidated. The aim of this study was to clarify the role of ligand-specific integrins in the morphological changes induced by cyclic strain (CS) via the p38 mitogen-activated protein kinase (p38 MAPK) pathway. Endothelial cells (ECs) were cultured on collagen (a ligand for integrin alpha 2 beta1, but not for alpha 5 and beta 4)-coated flexible plates and incubated for 24 h with or without anti-alpha2 integrin antibody (anti-alpha2), anti-alpha5, anti-beta1, or anti-beta4. ECs were then subjected to 15.6% average CS at 60 cycles/min up to 24 h. After exposure to CS, the cell shape index (defined as (4pi x cell area)/(cell perimeter)(2)), the cell orientation angle, and activation of p38 MAPK were assessed. ECs in the absence of integrin-blocking antibodies were elongated and aligned in response to CS. Anti-alpha 2 and anti-beta1 abolished both morphological changes of ECs as well as the activation of p38 MAPK. In contrast, anti-alpha 5 and anti-beta 4 inhibited neither morphological changes of ECs nor the activation of p38 MAPK. Our results indicate that ligand-specific integrins play a crucial role in the morphological changes of ECs induced by CS via the p38 MAPK pathway.     

Fibronectin synthesis by activated T lymphocytes: up-regulation of a surface-associated isoform with signalling function

Fibronectin (FN) is a major constituent of the extracellular matrix. We now provide evidence for a surface-associated isoform of FN that is synthesized by T cells upon activation. The T-cell-derived FN has an unusual splice pattern: an additional domain, EDB, is produced whereas sequences within another domain, IIICS, are spliced out. CS1, the binding domain for very late antigen-4 (VLA-4), however, is still generated. To study the potential function of surface-associated FN its synthesis was down-regulated by an antisense oligonucleotide, then proliferation of T cells was induced by cross-linked anti-CD3. Proliferation was reduced as was expression of CD25. Moreover, when T cells were cultured in high density, the synthetic peptide QILDVPST, corresponding to CS1, inhibited proliferation, as did antibodies to VLA-4. We propose that surface-associated FN is a ligand for VLA-4, which by binding to VLA-4 on an adjacent cell, provides a costimulatory signal, thus sustaining T-cell proliferation.     

Integrin alpha2beta1 on rat myeloma cells modulates interaction of alpha4beta1 integrin with vascular cell adhesion molecule-1 but not fibronectin

It is well established that alpha2beta1 integrin functions as a receptor for collagen and laminin; whereas alpha4beta1 integrin binds fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In the present study, we showed that rat myeloma YB2/0 cells constitutively expressed alpha4beta1 but not alpha2beta1 integrin. Transfection of cDNA of mouse a2 integrin subunit resulted in the expression of heterologous alpha2beta1 integrin on YB2/0 cells (YBmalpha2). The expression of alpha2beta1 conferred YBmalpha2 cells the ability to interact with collagen and laminin. In comparison with mock transfected YB2/0 cells (YBpF), YBmalpha2 cells exhibited increases in the binding and migration on VCAM-1; in contrast, both YBpF and YBmalpha2 were similar in their interactions with fibronectin or fibronectin fragment FN-40 that contains the binding site for alpha4beta1 integrin. The interaction of alpha4beta1 with VCAM-1 was further stimulated upon ligation with alpha2beta1-specific mAb. The use of specific inhibitory mAb demonstrated the role of alpha4beta1 in mediating the observed interactions with fibronectin and VCAM-1. Therefore, results show that expression of alpha2beta1 differentially regulated alpha4alpha1 integrin function by stimulating its interactions with VCAM-1 but not fibronectin. The in vivo significance of alpha2beta1 integrin expression was demonstrated by intravital videomicroscopy showing that ligation of alpha2beta1 enhanced alpha4beta1-mediated extravasation of YBmalpha2 cells in the liver.     

Human Natural Killer Cells Express Different Integrins and Spread on Fibronectin

Human natural killer (NK) cells adhered and most of them also actively spread on cellular fibronectin (cFn), plasma Fn (pFn) and its Mr 120,000-140,000 or Mr 105,000 cell-binding proteolytic Fn-fragments as well as on heparin-binding Fn-fragments containing the alternative cell binding site. The cells did not spread on vitronectin, laminin or collagens. Adhesion on Mr 105,000 Fn fragment containing the cell binding site, could be prevented by the synthetic peptide GRGDS but not by an inactive peptide, whereas adhesion on heparin-binding Fn fragments was unaffected by the peptide. Spreading of the NK cells led to a distinct reorganization of F-actin. Immunoprecipitation with monoclonal antibodies (MoAb) against the beta 1 integrin subunit of radioactively surface-labelled cells revealed a broad polypeptide band of Mr 140,000 under reducing conditions and a polypeptide doublet of Mr 160,000 and Mr 110,000 under non-reducing conditions. Identical polypeptides, corresponding to the alpha- and beta-subunits of the Fn-receptor complex, were bound to the Mr 105,000 chymotryptic Fn-fragment coupled to Sepharose. Similar experiments with small lymphocytes did not reveal any polypeptides. Immunofluorescence results with McAbs suggested that among the alpha-subunits of integrins, the alpha 3, alpha 4, and alpha 5 subunits are expressed in NK cells. The present results suggest that non-activated NK cells, but not small lymphocytes, express beta 1-integrins, and that at least the Fn-receptors alpha 4 beta 1 and alpha 5 beta 1 may function in the adhesion and migration of NK cells.     

Regulation of T cell proliferation by anti-CD49d and anti-CD29 monoclonal antibodies.

The beta 1 integrin VLA-4 (alpha 4 beta 1, CD49d/CD29), which is expressed on a large subpopulation of peripheral blood T lymphocytes, functions as a receptor for the endothelial adhesion protein VCAM-1 and the extracellular matrix protein fibronectin. Previous studies showed that immobilized fibronectin enhanced anti-CD3 monoclonal antibody (mAb)-induced T cell proliferation through binding to the integrins VLA-4 and VLA-5 (alpha 5 beta 1, CD49e/CD29). We studied the ability of the anti-CD49d mAb L25 to potentiate proliferation. T cell proliferation was induced by subthreshold concentrations of anti-CD3 mAb (mAb OKT3) coimmobilized with mAb L25 but not with coimmobilized anti-CD29 (beta 1) mAb. Soluble anti-CD29 mAb inhibited the proliferation induced by coimmobilized mAb OKT3 and L25 but not proliferation induced by mAb OKT3 with PMA or coimmobilized anti-CD26 mAb.     

Specific cleavage of the alpha 4 integrin associated with activation of peripheral T lymphocytes.

The heterodimeric VLA-4 integrin has been implicated in lymphocyte migration to inflamed peripheral tissues, lympho-haemopoiesis and autoimmune disease. To determine the structure and function of VLA-4 in physiological processes, molecular forms of the VLA-4 alpha-chain were analysed during T-cell activation. The results showed that prolonged activation of human peripheral T cells was associated with increased cleavage of the 150,000 MW alpha 4 chain into 80,000 and 66,000 MW fragments. Similar-sized alpha 4 fragments could also be generated from 150,000 MW alpha 4 on intact resting T cells by brief trypsinization, whereas trypsin treatment of isolated 150,000 MW alpha 4 resulted in nearly complete protein degradation. Native 80,000 and 66,000 MW alpha 4 chains on activated T cells could not be digested further by trypsin. These results indicated that specific cleavage of 150,000 MW alpha 4 was largely dependent on the tertiary structure of native alpha 4 chains. To determine the specific cleavage site in alpha 4 on peripheral T cells, VLA-4 was isolated and purified from in vitro-activated T cells and the N-terminus of the 66,000 MW fragment was partially sequenced. The sequence SKR/STE was identified as the specific alpha 4 cleavage site on T cells. These results indicate that T cells, upon activation, acquire an enhanced dipeptidase processing activity, which cleaves alpha 4 at a specific site.     

Signaling by vascular cell adhesion molecule-1 (VCAM-1) through VLA-4 promotes CD3-dependent T cell proliferation

Vascular cell adhesion molecule, VCAM-1, is an adhesion molecule expressed on activated endothelium thought to play a role in leukocyte migration to sites of inflammation. VCAM-1 adheres to leukocytes through the VLA-4 integrin. Recombinant soluble VCAM-1 (rsVCAM) and anti-CD3 mAb OKT3 were utilized to address the role of the VCAM-1/VLA-4 pathway in antigen-dependent T cell activation. Monocyte-depleted T cells proliferated upon exposure to co-immobilized OKT3 and rsVCAM but to neither alone. In contrast, an anti-VLA-4 mAb HP1/2 failed to co-activate with OKT3, despite the fact that both rsVCAM and HP1/2 support T cell adhesion comparably. These data indicate that adhesive function is not sufficient for co-stimulatory activity. They also reveal that VCAM-1 may play a role in regulating T cell immune responses as well as migration in vivo.