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.     

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.     

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.     

Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes.

The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.     

Adhesion and activation molecules expressed by human natural killer cells

Our study concerns the expression and the regulation of adhesion and activation receptors on human NK cells. In particular we provide evidence on: a) the expression on fresh human NK cells of VLA-4, VLA-5 and VLA-6, extracellular matrix (ECM) receptors of integrin family capable of mediating their adhesion to FN and LM; b) the role of PKC on the regulation of CD16, a differentiation antigen associated with FcR type III expressed by all NK cells, which mediate ADCC activity and trigger lymphokine production.     

Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules.

The capacity of purified fibronectin to costimulate human T cell DNA synthesis was examined. Low concentrations of immobilized fibronectin, but not soluble fibronectin, augmented anti-CD3-induced proliferation of highly purified human T cells. In the absence of anti-CD3 stimulation, immobilized fibronectin did not induce T cell proliferation alone or in the presence of IL-2 or phorbol dibutyrate. Although fibronectin is present in high concentrations in the serum, immobilized fibronectin was able to costimulate T cell proliferation when cells were cultured in serum-containing medium. Immobilized collagen type I did not enhance anti-CD3 stimulated T cell responses, whereas gelatin (denatured collagen) and laminin were able to enhance anti-CD3 stimulated T cell responses modestly. The effects of gelatin, however, appeared to be indirect, because it could not enhance responses in medium devoid of fibronectin. Immobilized fibronectin enhanced anti-CD3 induced proliferation of both CD45RA dim and CD45RA bright subsets within both the CD4+ and CD8+ subpopulations of T cells, although cells with the CD45RA dim phenotype were costimulated by lower concentrations of immobilized fibronectin. Enhancement of anti-CD3 induced proliferation by immobilized fibronectin was completely inhibited by a mAb to CD29, the integrin beta 1-chain (4B4) and not by a variety of other mAb. In contrast to its effects on proliferation, 4B4 only partially blocked T cell binding to anti-CD3 and fibronectin-coated macrowells. These findings suggested that the interaction between fibronectin and its receptor transduced a signal to the T cell and did not merely stabilize the interaction between anti-CD3 and the CD3 complex. Further experiments confirmed this observation. Thus fibronectin could enhance anti-CD3 responses when it was immobilized to a separate surface. The augmentation of anti-CD3 stimulated proliferation induced by immobilized fibronectin was also inhibited partially by mAb to either VLA-4 or VLA-5 and completely by a combination of the two mAb. The mAb to VLA-4 not only blocked the capacity of immobilized fibronectin to enhance anti-CD3-induced T cell proliferation but also directly costimulated T cell responses. Thus, at least two fibronectin receptors are involved in fibronectin-mediated costimulation of T cell proliferation. These studies indicate that signals are transduced through the fibronectin receptors, VLA-4 and VLA-5, that augment T cell responses and therefore implicate the extracellular matrix protein fibronectin as an important influence regulating T cell responsiveness in vivo.     

A monoclonal antibody to VLA-4 alpha-chain (CDw49d) induces homotypic lymphocyte aggregation

The complex processes of cellular adhesion involve a variety of receptor to ligand interactions that are extremely important during the development of immune function. Lymphocyte activation by Ag or mitogen, CTL- and NK-mediated cytolysis, homing to lymphoid-associated tissue, and the attachment of lymphocytes to extracellular matrix proteins are all governed, at least in part, by cell surface adhesion receptors. During the analysis of mAb for the ability to block human cytotoxic T lymphocyte-mediated killing an inhibitory mAb was noted that caused rapid and vigorous aggregation among the CTL. This antibody, mAb L25, also induced aggregation among human T and B tumor cell lines. mAb L25 binds to an epitope on the alpha 4 subunit of the integrin protein VLA-4 and induced an adhesion event requiring divalent cations, energy, a fluid plasma membrane, and an intact cytoskeleton. The Ag-independent homotypic adhesion induced by mAb L25 was not inhibited by mAb to the lymphocyte function associated Ag-1 (CD11a/CD18), CD2, CD4, and CD8, or to their ligands ICAM-1, LFA-3, MHC class I, or MHC class II. We believe that these experiments suggest a role for VLA-4 in a novel system of leukocyte adhesion.     

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.     

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.     

An alternative leukocyte homotypic adhesion mechanism, LFA-1/ICAM-1- independent, triggered through the human VLA-4 integrin

The VLA-4 (CD49d/CD29) integrin is the only member of the VLA family expressed by resting lymphoid cells that has been involved in cell-cell adhesive interactions. We here describe the triggering of homotypic cell aggregation of peripheral blood T lymphocytes and myelomonocytic cells by mAbs specific for certain epitopes of the human VLA alpha 4 subunit. This anti-VLA-4-induced cell adhesion is isotype and Fc independent. Similar to phorbol ester-induced homotypic adhesion, cell aggregation triggered through VLA-4 requires the presence of divalent cations, integrity of cytoskeleton and active metabolism. However, both adhesion phenomena differed at their kinetics and temperature requirements. Moreover, cell adhesion triggered through VLA-4 cannot be inhibited by cell preincubation with anti-LFA-1 alpha (CD11a), LFA-1 beta (CD18), or ICAM-1 (CD54) mAb as opposed to that mediated by phorbol esters, indicating that it is a LFA-1/ICAM-1 independent process. Antibodies specific for CD2 or LFA-3 (CD58) did not affect the VLA-4-mediated cell adhesion. The ability to inhibit this aggregation by other anti-VLA-4-specific antibodies recognizing epitopes on either the VLA alpha 4 (CD49d) or beta (CD29) chains suggests that VLA-4 is directly involved in the adhesion process. Furthermore, the simultaneous binding of a pair of aggregation-inducing mAbs specific for distinct antigenic sites on the alpha 4 chain resulted in the abrogation of cell aggregation. These results indicate that VLA-4-mediated aggregation may constitute a novel leukocyte adhesion pathway.     

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.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

Structure-function analysis of the human integrin VLA-4 (alpha 4/beta 1). Correlation of proteolytic alpha 4 peptides with alpha 4 epitopes and sites of ligand interaction.

The structure-function relationship of the human integrin VLA-4 (alpha 4/beta 1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the alpha 4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as alpha 4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa alpha 4 peptides. The trypsin-generated 80- and 65-kDa alpha 4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the beta 1 chain. Distinct anti-VLA-4 mAb against four different alpha 4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa alpha 4 chain both associated or non-associated with the beta 1 chain. The alpha 4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-alpha 4 mAb directed against the four different alpha 4 epitopes. On the other hand, the 55-kDa alpha 4 peptide was present in precipitates from anti-alpha 4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-alpha 4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-alpha 4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa alpha 4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.     

VLA-5-mediated Adhesion to Fibronectin Accelerates Hemin-stimulated Erythroid Differentiation of K562 Cells through Induction of VLA-4 Expression

Fibronectin plays important roles in erythropoiesis through the fibronectin receptors VLA-4 and VLA-5. However, the substantial role of these fibronectin receptors and their functional assignment in erythroid differentiation are not yet fully understood. Here, we investigated the effects of cell adhesion to fibronectin on erythroid differentiation using K562 human erythroid progenitor cells. Erythroid differentiation could be induced in K562 cells in suspension by stimulating with hemin. This hemin-stimulated erythroid differentiation was highly accelerated when cells were induced to adhere to fibronectin by treatment with TNIIIA2, a peptide derived from tenascin-C, which has recently been found to induce β1-integrin activation. Another integrin activator, Mn²⁺, also accelerated hemin-stimulated erythroid differentiation. Adhesive interaction with fibronectin via VLA-4 as well as VLA-5 was responsible for acceleration of the hemin-stimulated erythroid differentiation in response to TNIIIA2, although K562 cells should have been lacking in VLA-4. Adhesion to fibronectin forced by TNIIIA2 causally induced VLA-4 expression in K562 cells, and this was blocked by the RGD peptide, an antagonist for VLA-5. The resulting adhesive interaction with fibronectin via VLA-4 strongly enhanced the hemin-stimulated activation of p38 mitogen-activated protein kinase, which was shown to serve as a signaling molecule crucial for erythroid differentiation. Suppression of VLA-4 expression by RNA interference abrogated acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2. Thus, VLA-4 and VLA-5 may contribute to erythropoiesis at different stages of erythroid differentiation.Hematopoietic stem and progenitor cells proliferate and differentiate in the bone marrow and fetal liver (1–6). Stromal cells of the bone marrow and fetal liver form a hematopoietic microenvironment called a “niche.” This microenvironment niche plays a crucial role in the regulation of the proliferation and differentiation of hematopoietic stem and progenitor cells. Besides humoral factors that include hematopoietic growth factors, adhesive interaction of hematopoietic stem and progenitor cells with stromal cells and/or the extracellular matrix (ECM)² in the hematopoietic microenvironment is indispensable for hematopoietic development (1–6). The ECM in the hematopoietic microenvironment is composed of various macromolecules, such as fibronectin (FN), collagens, laminins, and proteoglycans. Among them, FN is one of the most important parts of the microenvironment niche (7–11). Also, in erythropoiesis, the importance of the adhesion of erythroid progenitors to FN via the FN receptors VLA-4 and VLA-5 has been reported (11–16). However, the substantial role of these FN receptors and their functional assignment in erythroid differentiation are not yet fully understood.We previously found that FN, which provides scaffolding for the adhesion of various cell types, has an alternative functional site opposing cell adhesion (17). A 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat of the FN molecule, termed FNIII14, strongly suppresses cell adhesion to FN by inhibiting the activation of β1-integrins including VLA-4 and VLA-5 (18, 19). Conversely, we have recently found that tenascin (TN)-C, which is an anti-adhesive ECM protein (20, 21), has a functional site for stimulating cell adhesion to FN (22). A 22-mer peptide derived from the FNIII repeat A2 in the TN-C molecule, termed TNIIIA2, can induce the conformational change necessary for functional activation of FN receptors through binding with syndecan-4 (22, 23). The active sites of FNIII14 and TNIIIA2 appear to be cryptic in the molecular structures of FN and TN-C but are exposed by conformational change through interaction with other ECM molecules or by processing with matrix metalloproteinase-2 (22, 24). Thus, these functional sites found in FN and TN-C molecules, which act in opposition to their parental ECM proteins, may act as a negative feedback loop for preventing excessive cellular responses to these ECM proteins in biological processes with ECM rearrangement. In any case, FNIII14 and TNIIIA2 enable us to control, either negatively or positively, the adhesion of various cell types to FN.Various hematopoietic progenitor cell lines have been used in in vitro studies of hematopoietic differentiation. However, most hematopoietic progenitor cell lines are nonadherent, because their cell surface β1-integrins, including FN receptors, have impaired ligand-binding activity (25, 26). Therefore, in order to investigate the role of cell adhesion to FN in hematopoietic differentiation, their FN receptors must be activated. Since TNIIIA2 can induce activation of FN receptors in various hematopoietic progenitor cell lines (22), this peptide factor may be useful for investigating the substantial role of cell adhesion to FN in hematopoietic differentiation. Here, we investigate the effects of cell adhesion to FN on erythroid differentiation using TNIIIA2 and Mn²⁺ as the integrin activator and the human erythroid progenitor cell line K562, which only expresses VLA-5, as the FN receptor (27). As a result, we show that hemin-stimulated erythroid differentiation of K562 cells is strongly enhanced when K562 cells are forced to adhere to FN. Sustained adhesion to FN via VLA-5, which is induced by TNIIIA2 or Mn²⁺, causes induction of VLA-4 expression. The resulting adhesive interaction with FN via newly expressed VLA-4 then generates a conspicuous increase in the hemin-stimulated phosphorylation/activation of p38 MAP kinase, which is shown to serve as a signaling molecule crucial for erythroid differentiation of K562 cells.     

Requirement of leukocytic cell adhesion molecules (CD11a-c/CD18) in the enhanced NK lysis of iC3b-opsonized targets

Treatment of Raji or Daudi cells with human serum under conditions which allow the alternative pathway of C activation results in their C3-opsonization and enhanced sensitivity to NK-mediated lysis. The effector lymphocytes have low buoyant density, carry CD16 and HNK1 markers as well as the CD11a-c/CD18 leukocytic cell adhesion molecules. One of these molecules, made up of CD11b-CD18 (alpha- and beta-chains), is also the receptor for iC3b. We studied the role of the cell adhesion molecules in the NK effect on targets with and without C3-fragments. We focused on the E/T interaction of opsonized cells in the presence of anti CD18 mAb. mAb directed to the CD11a molecule caused 0 to 30% inhibition of the lysis of both non-opsonized and opsonized cells whereas the mAb antibody directed to the CD11c molecule had no effect. Reagents reactive with the iC3b binding site of CD11b (alpha-chain of the CR3) molecule did not alter the lysis of non-opsonized targets whereas they abrogated the C3-mediated increment of the Nk effect on opsonized cells. Two mAb preparations, 60.3 and IB4, directed to the CD18 chain shared by the three cell adhesion molecules abrogated in a dose-dependent way the lysis of both non-opsonized and opsonized targets. The 60.3 mAb inhibited the iC3b binding site of CR3 (despite its localization on the alpha-chain) and in accordance it inhibited the binding of lymphocytes to the opsonized target also. The IB4 did not affect this site and in accordance it inhibited only partially the binding of effectors to the C3 fragment carrying Raji, nevertheless it inhibited their lysis. This result indicates that the iC3b-CR3 bridge is insufficient for triggering the lysis in absence of the contact through the adhesion molecules.     

CD11a-c/CD18 and GP84 (LB-2) adhesion molecules on human large granular lymphocytes and their participation in natural killing

Effect of mAb against the CD11a-c, CD18, and GP84 adhesion molecules on the binding and cytotoxicity of human NK cells was studied. The target cells were K562, MOLT-4, Raji, and fresh uncultured autologous endometrial carcinoma cells. Antibodies against adhesion relevant epitopes of CD11a(TA-1/LFA-1), CD11b(Mol/OKM1/Mac1), or CD11c (Leu-M5) did not inhibit NK function. The mAb 60.3 against CD18, the common beta-chain associated to CD11a-c, strongly inhibited both the binding and cytotoxicity of large granular lymphocytes (LGL) against all the target cells tested. Also the antibody LB-2 against the GP84 adhesion molecule inhibited NK function to some degree. 60.3 and LB-2 antibodies exerted an additive effect in the inhibition of both binding and cytotoxicity. However, even this antibody combination did not completely block NK activity, suggesting a heterogeneity of adhesion structures in the NK system. According to both FACS analyses and immunoprecipitation studies, all the tested antibodies recognized either a subpopulation or all of LGL. On the other hand, antibodies against CD11b, CD11c, and LB-2 showed only marginal reactivity with highly purified LGL-free T cells.     

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.     

ICAM-3 regulates lymphocyte morphology and integrin-mediated T cell interaction with endothelial cell and extracellular matrix ligands

Leukocyte activation is a complex process that involves multiple cross- regulated cell adhesion events. In this report, we investigated the role of intercellular adhesion molecule-3 (ICAM-3), the third identified ligand for the beta 2 integrin leukocyte function-associated antigen-1 (LFA-1), in the regulation of leukocyte adhesion to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the 38- and 80-kD fragments of fibronectin (FN40 and FN80). The activating anti-ICAM-3 HP2/19, but not other anti-ICAM-3 mAb, was able to enhance T lymphoblast adhesion to these proteins when combined with very low doses of anti-CD3 mAb, which were unable by themselves to induce this phenomenon. In contrast, anti-ICAM-1 mAb did not enhance T cell attachment to these substrata. T cell adhesion to ICAM-1, VCAM-1, FN40, and FN80 was specifically blocked by anti-LFA-1, anti-VLA alpha 4, and anti-VLA alpha 5 mAb, respectively. The activating anti-ICAM-3 HP2/19 was also able to specifically enhance the VLA-4- and VLA-5-mediated binding of leukemic T Jurkat cells to VCAM-1, FN40, and FN80, even in the absence of cooccupancy of the CD3-TcR complex. We also studied the localization of ICAM-3, LFA-1, and the VLA beta 1 integrin, by immunofluorescence microscopy, on cells interacting with ICAM-1, VCAM-1 and FN80. We found that the anti-ICAM-3 HP2/19 mAb specifically promoted a dramatic change on the morphology of T lymphoblasts when these cells were allowed to interact with those adhesion ligands. Under these conditions, it was observed that a large cell contact area from which an uropod-like structure (heading uropod) was projected toward the outer milieu. However, when T blasts were stimulated with other adhesion promoting agents as the activating anti-VLA beta 1 TS2/16 mAb or phorbol esters, this structure was not detected. The anti-ICAM-3 TP1/24 mAb was also unable to induce this phenomenon. Notably, a striking cell redistribution of ICAM-3 was induced specifically by the HP2/19 mAb, but not by the other anti-ICAM-3 mAb or the other adhesion promoting agents. Thus, ICAM-3 was almost exclusively concentrated in the most distal portion of the heading uropod whereas either LFA-1 or the VLA beta 1 integrin were uniformly distributed all over the large contact area. Moreover, this phenomenon was also observed when T cells were specifically stimulated with the HP2/19 mAb to interact with TNF alpha-activated endothelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.