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.     

Post-receptor occupancy events in leukocytes during β1 integrin-ligand interactions

Leukocyte adhesion to and subsequent spreading on the endothelium are the initial steps in the migration of these cells to the surrounding tissues. We have investigated the participation of different VLA heterodimers in cell spreading by using the anti-β1 TS2/16 monoclonal antibody (mAb) which induces a conformational change of different VLA integrin receptors, enabling a high-affinity interaction with their ligands. Both VLA-4 and VLA-5 fibronectin (FN), as well as VLA-2 collagen (COL) receptors mediated cell spreading and morphological changes. The spreading of U-937 and α4-transfected K-562 cells was induced in both FN and VCAM-1, suggesting that the morphological changes may be induced by cell-cell as well as cell-extracellular matrix (ECM) interactions. Furthermore, the β-regulated cell spreading on VCAM-1 and COL took place independently of the VLA-5 FN receptor function. The enhancing effect on cell attachment induced by anti-β1 TS2/16 mAb was observed in the presence of different doses of cytochalasin D, whereas cell spreading was abolished. Signal transduction during β1-stimulated integrin-ligand interaction was also investigated. We have found the co-localization of β1 integrins and tyrosine-phosphorylated proteins during the spreading of U-937 and α2- and α4-transfected K-562 cells on both ECM (FN and COL) and cellular (VCAM-1) ligands. Kinetic studies showed that tyrosine phosphorylation was almost coincident with cellular spreading. The tyrosine phosphorylation of polypeptides of 130 kDa and 77 kDa was triggered in U-937 cells by the interaction of FN with the VLA-5 receptor in a high-affinity conformation. However, no signaling was observed by inducing the high-affinity state of receptor in the absence of appropriate ligand. These data suggest that tyrosine kinase activation is a post-receptor occupancy event that might be critical in regulating the adhesive properties of integrins.     

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.     

Functional relevance during lymphocyte migration and cellular localization of activated β1 integrins

The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS-21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)-1 and an 80-kDa tryptic fragment of fibronectin (FN80) to the β1 integrins very late activation antigen (VLA)-4 and VLA-5, and that this effect is dependent on ligand concentration and is specific for β1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS-21 epitope localized exclusively to sites of integrin binding to fibronectin. These results indicate that mAb HUTS-21 recognizes a ligand-induced binding site (LIBS) on the common β1 subunit of VLA proteins. Engagement of β1 integrins through this LIBS epitope inhibited T lym-phoblast movement on fibronectin, as determined by quantitative time-lapse video microscopy studies. Furthermore, the HUTS-21 mAb also prevented T lymphoblast-directed migration through gradients of substratum-immobilized β1 integrin ligands such as fibronectin or VCAM-1, whereas it did not affect migration on intercellular adhesion molecule (ICAM)-1. This anti-LIBS mAb stimulated cell adhesion through postreceptor events, without affecting receptor affinity for ligand, and appears to interfere with cell migration by a mechanism distinct from that of other anti-β1 activating antibodies.     

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.     

Fibronectin at the Lymphocyte Surface. Evidence for Activation-Dependent Binding to VLA4 and VLA5 Integrins

The surface of in vitro cultured fixed and viable human T lymphocytes and certain T-cell lines was found to react with different monoclonal anti-fibronection (FN) antibodies as revealed by ELISA, immunocyto chemistry and FACS analysis. SDS-PAGE showed that anti-FN antibodies defined a high molecular weight lymphocyte component which could be iodinated using the lactoperoxidase method and which had gelatin binding capacity. FACS analysis showed that the reactivity of anti-FN antibodies with lymphocytes was most pronounced in activated cells and increased during the culture period. By contrast, FACS analysis revealed equal high expression of the VLA4 and VLA5 integrins on freshly purified as well as on mixed lymphocyte culture (MLC) activated cells, Freshly purified lymphocytes and lymphocytes cultured in vitro overnight did not bind ³H-labelled FN in solution whereas MLC-activated cells were capabie of 'spontaneous'binding of such [³H]-FN. However, brief 12- o -tetradecanoylphorbol-13-acetate (TPA) exposure rendered freshly purified lymphocytes capable of binding soluble FN. These interactions of the lymphocytes with ³H-labelled FN in solution could be almost completely blocked by monoclonal anti-VLA4 and VLA5 antibodies. These results indicate that activated T cells express fibronectin at their surface under'normal'culture conditions. Althotigh both freshly purified and MLC-activated lymphocytes have equal expression of the integrins VLA4 and VLA5, only activated cells are capable of 'spontaneous'binding FN in solution via an integrin-mediated process, probably via an increase in the affinity of these receptors for FN.     

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.     

VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization

The family of human T cell activation-associated proteins, named VLA complex, is formed by the molecular association of cell surface glycoproteins of 210, 165, 130 and 80 kDa. In this report, we describe eight different monoclonal antibodies (HP mAb) specific for the 80-kDa polypeptide which preferentially associates with the 165-kDa member. Comparative immunoprecipitation and cell-binding studies demonstrated that the HP mAb recognize an epitope(s) on the 165/80 kDa subset different from those recognized by other anti-VLA mAb previously described. Furthermore, cellular and tissue distribution studies by flow cytometry and peroxidase staining showed that the HP reactivity pattern is different from other VLA specificities. The 165/80-kDa complex defined by HP mAb is present on human thymocytes, peripheral blood lymphocytes as well as on T, B and myelomonocytic cell lines. However, only the 80-kDa subunit was precipitated by HP mAb from activated T lymphocytes. These results suggest that the association between the 165- and 80-kDa subunits diminishes during the activation process, and that the epitopes recognized by the HP mAb are located on the 80-kDa protein. The novel polypeptide association of 165/80 kDa has been termed VLA-3.     

CD9 antigen is an accessory subunit of the VLA integrin complexes

The CD9 antigen is a cell surface glycoprotein of unknown function which belongs to the tetraspans family. We demonstrate here, by precipitation, Western blotting and co-capping experiments, that this molecule is associated with a large fraction of β1 integrins in two cell lines, the pre-B cell line NALM-6 and the megakaryocytic cell line HEL. In HEL cells, CD9 antigen is only associated with VLA-4. In contrast, in NALM-6 cells, CD9 antigen is associated with both VLA-4 and VLA-5. On the other hand, only the β1 chain is co-precipitated with the CD9 antigen in transfected L cells. These data show that the CD9 antigen is associated with the β1 chain rather than with a particular integrin. CD9 monoclonal antibodies (mAb) did not modify the binding of HEL and NALM-6 cells to fibronectin, laminin or collagen. The association of CD9 antigen to VLA integrins is strengthened by the fact that both CD9 and anti-VLA mAb induce aggregation of the two cell lines and inhibit their migration in Transwell chambers. Because the aggregating effect, but not the inhibition of migration, is observed in CEM or CD9-transfected CEM cells, these two effects are likely to be mediated by different mechanisms.     

Expression and functional significance of an activation-dependent epitope of the β1 integrins in chronic inflammatory diseases

The avidity of VLA integrins for their ligands can be increased by their transition to an active conformational state. This conformational change can be detected with a novel monoclonal antibody (mAb), termed 15/7, that recognizes an activation-dependent conformational epitope on the common β1 polypeptide of different VLA αβ1 integrins. In an attempt to understand the possible role of the active conformational state of β1 integrins in vivo, we first investigated the expression of 15/7 epitope on T lymphocytes from patients with chronic inflammatory joint diseases. An enhanced expression of the 15/7 epitope was found in the synovial fluid (SF) T lymphocytes from these patients as compared to their peripheral blood (PB) T cells. The effect of different cytokines on the appearance of the 15/7 activation epitope in PB T lymphocytes was subsequently analyzed; interferon-γ, interleukin-2 and, to a lower extent, tumor necrosis factor-α were able to induce an increased expression of the 15/7 epitope. This enhanced 15/7 expression correlated with a higher binding ability to fibronectin of cytokine-activated T cells. The presence of this activation epitope was detected in a small proportion of T lymphocytes scattered within inflammatory foci of synovial membrane from rheumatoid arthritis and thyroid glands from Hashimoto's chronic thyroiditis. We then analyzed the possible role of 15/7 epitope expression on cell adhesion in vitro. Immunofluorescence studies showed that the 15/7 epitope displayed a spot-like distribution, selectively decorating adhesive contacts of U-937 myelomonocytic cells attached to the 80 kDa proteolytic fragment of fibronectin (FN80). Furthermore, the anti-β1 15/7 mAb was able to induce both T lymphocyte, Jurkat and U-937 cellular binding and spreading on FN80. Altogether these results indicate that an activated conformation of β1 integrins is detected in vivo in lymphocyte infiltrates from chronic inflammatory conditions. The active conformations of β1 integrins are regulated by physiologic mediators such as cytokines, play an important role in cellular attachment and spreading, and appear to be involved in the development of inflammatory processes.     

Beta 2 (CD18) and beta 1 (CD29) integrin mechanisms in migration of human polymorphonuclear leucocytes and monocytes through lung fibroblast barriers: shared and distinct mechanisms.

Accumulation of leucocytes in inflamed lung tissue and alveolar space involves their migration through vascular endothelium and then lung connective tissue. As a model of this process, we investigated human polymorphonuclear leucocyte (PMNL) and monocyte migration through a biological barrier of human lung fibroblasts (HLF) grown on polycarbonate filters. Very few PMNL (1-2%) or monocytes (3-8%) migrated through the HLF barriers spontaneously. Migration increased to 48-53% of added PMNL and 17-24% of added monocytes, when a C5a chemotactic gradient was present. The monocyte migration induced by C5a was not inhibited by monoclonal antibodies (mAb) to CD18 (beta 2 integrins). This CD18-independent migration was partially inhibited (35%) by mAb to gamma 5 of VLA-5 and completely inhibited by the combination of mAb to gamma 4 of VLA-4 with mAb to VLA-5, in the presence of mAb to CD18. In contrast, PMNL migration across HLF induced by C5a was partially inhibited by mAb to CD18 alone, but even with the addition of mAb to VLA-4, VLA-5 beta 1 and VLA-6, the greatest degree of inhibition was only 60%. Blocking the function of CD18 was not required to observe the inhibition by mAb to VLA-4, although the inhibitory effect of mAb to VLA-5 and VLA-6 alone or in combination was only observed when CD18 mechanisms were also blocked with anti-CD18 mAb. These results demonstrate that (a) both monocytes and PMNL can use either CD11/CD18 (beta 2 integrin) or beta 1 (CD49/CD29) integrins to migrate through HLF barriers; (b) in the case of monocytes, the VLA-4 and VLA-5 integrins account for essentially all the CD11/CD18-independent migration mechanisms; and (c) in contrast to monocytes, PMNL CD18-independent migration is mediated not only by VLA-4 and VLA-5, but also by VLA-6, and up to 40% of the migration appears to be via yet to be defined PMNL surface molecules.     

Involvement of β1 integrins in the binding and entry of Trypanosoma cruzi into human macrophages

We have investigated the role of integrin molecules in the binding and entry of Trypanosoma cruzi into human macrophages, with the help of monoclonal antibodies (mAb). Addition of Lia 1/2 mAb and in lesser extent of Lia 1/5 mAb, which are both specific for the β₁ subunit of the VLA integrin family, to human macrophages blocked T. cruzi uptake and subsequent replication inside the macrophages. This inhibition correlated with their respective ability to block Fibronectin (Fn) binding to macrophages. Furthermore, another anti-β₁ mAb, Alex 1/4, which binds to a different epitope on the β₁ molecule and was unable to block Fn binding, did not affect T. cruzi invasion. The inhibition by Lia 1/2 and Lia 1/5 was dose dependent and clearly observable with doses as low as 1 μg/ml. Moreover, this inhibition was T. cruzi specific since the Lia 1/2 and Lia 1/5 were unable to block uptake of Leishmania pifanoi or Escherichia coli by human macrophages. In contrast, the TS 1/18 mAb, which blocks ligand binding to β₂ integrin, inhibited entry of L. pifanoi but not of T. cruzi. Finally, mAb specific for the a₄ and a₅ subunits of the two major Fn binding molecules of macrophages (VLA-4 and VLA-5, respectively), either alone or in combination, were poor inhibitors of T. cruzi uptake, suggesting that several members of the VLA family, including VLA-4 and VLA-5, are involved in binding and entry of T. cruzi into macrophages.     

alpha9beta1 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers

Accumulation of leukocytes in inflamed tissue involves their migration through vascular endothelium and then in the connective tissue. Recently we utilized a barrier of human synovial, dermal, and lung fibroblasts (HSF, HDF, and HLF) grown on polycarbonate filters as a model of human polymorphonuclear leukocyte (PMN) migration through connective tissue. The beta2 integrins (CD 11/ CD18) and alpha4, alpha5, and alpha6beta1 (VLA-4, -5, and -6) integrins each contributed to this PMN migration. Here we report that on human blood leukocytes, alpha9beta1 (VLA-9) is expressed only on PMNs and that it is up-regulated after PMN activation. Based on monoclonal antibody (mAb) blocking studies, alpha9beta1 integrin contributed to C5a-induced PMN migration through fibroblast (HLF and HSF) barriers. This role was apparent only when alternate mechanisms such as CD18, alpha4, alpha5, and alpha6beta1 integrins were blocked and then mAb to alpha9beta1 integrin inhibited the residual PMN migration (by 40-50%) through the HLF or HSF barrier, resulting in > or = 75% inhibition overall. In contrast, PMN migration across interleukin-1-activated endothelium (HUVEC) in response to a C5a gradient, which is partly (30-40%) via CD11/CD18-independent mechanisms, was not inhibited by adhesion blocking by mAbs to alpha4, alpha5, alpha6, and alpha9beta1 even in combination. These results indicate that alpha9beta1 integrin on PMN may have a special role, in conjunction with other beta1 integrins, in mediating PMN migration in the extravascular space, and may contribute to differential neutrophil function within tissues.     

Human platelet alloantigens Br/Br are expressed on the very late activation antigen 2 (VLA-2) of T lymphocytes

We report the molecular localization of the human platelet alloantigens Br^a/Br^b on activated T lymphocytes. By radioimmunoprecipitation anti-Br^a precipitated two proteins from T lymphocytes of Br(a+) donors after long-term activation (24 days), but not from resting or short-term-activated T lymphocytes (4 days). No bands could be precipitated with anti-Br^a from T lymphocytes of Br(a−) donors, but the same two bands were seen with anti-Br^b. The apparent molecular weights of unreduced molecules were 155 and 130 kDa, respectively. Monoclonal antibody Gi14 directed against an epitope on the platelet membrane glycoprotein Ia/IIa complex precipitated the same two bands with long-term-activated T lymphocytes, whereas MoAb TS2/7 directed against very late antigen (VLA)-1 molecules precipitated two bands with M_r 180 and 130 kDa (₁β heterodimer). The physicochemical properties of these two bands precipitated by anti-Br^a, anti-Br^b or monoclonal antibody Gi14 with molecular weights of 155 and 130 kDa, respectively, correspond to VLA ₂ and β chains and fit defined criteria for the VLA-2 antigen. These results were corroborated by an assay employing monoclonal antibody for antigen immobilization showing that Br^a and Br^b antigens were present only on long-term activated T lymphocytes. Our results provide evidence for the expression of the Br^a/Br^b platelet alloantigen system on VLA-2 antigens of activated T lymphocytes and demonstrate a genetic polymorphism of VLA-2 molecules on both cell lines.     

Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of LFA-1, ICAM-1, VLA-4 and VCAM-1. off.

Lymphocyte adhesion to and migration across endothelial cell (EC) monolayers, derived from the rat blood-retinal barrier (BRB), were measured in vitro. The binding of concanavalin A (Con A)-activated peripheral lymph node lymphocytes and the migration of CD4+ T-cell lines could be significantly increased by treating the EC with interleukin-1 beta (IL-1 beta). To determine the role of various adhesion molecules during the processes of lymphocyte binding and transmonolayer migration (diapedesis), lymphocytes were treated with monoclonal antibody (mAb) specific for CD11a (alpha L subunit of leucocyte functional antigen-1; LFA-1), CD18 (beta 2 subunit of leucam family) and CD49d (alpha 4 subunit of very late activation antigen-4; VLA-4) and EC with mAb specific for CD54 (intercellular adhesion molecule-1; ICAM-1) and CD106 (vascular cell adhesion molecule-1; VCAM-1). Binding of the highly adhesive but non-migratory Con A-activated lymphocytes was inhibited by mAb to CD11a (reduced to 73% and 65% of control lymphocyte adhesion) and CD18 (42% and 54%) on non-activated and IL-1 beta-treated EC, respectively, but not by mAb to ICAM-1 or VCAM-1. Diapedesis of the highly migratory T-cell line lymphocytes was also blocked by antibodies to CD11a (reduced to 11% and 10% of control T-cell migration), CD18 (29% and 43%) but in addition was also inhibited by anti-ICAM-1 (17% and 53%) on non-activated and IL-1 beta treated EC, respectively. Both anti-VLA-4 and anti-VCAM-1 were also effective in producing a smaller reduction in migration, but only on IL-1 beta activated EC (66% and 58% of control migration, respectively). These studies indicate that lymphocyte adhesion to central nervous system (CNS) vascular EC is largely dependent on LFA-1 but not through its interaction with ICAM-1. In contrast, lymphocyte diapedesis is mostly supported through the LFA-1/ICAM-1 pairing, with a small proportion being mediated by VLA-4/VCAM-1 on IL-1 beta-activated EC. This latter pathway, however, also appears to be dependent on LFA-1 interacting with the EC.     

Identification and functional characterization of mouse CD29 with a mAb

The ß1 integrin subfamily, alternatively called verylate activation antigen (VLA), has been implicated in variouscellular functions. In this study, we generated a mAb againstthe mouse ß1 subunit (CD29) to examine the functionalproperty of mouse VLA proteins. After immunization with affinity-purifiedmouse VLA-4 (4ß1), a hamster mAb, HMß1-1,was established by screening mAb that reacted with 4-negatlveneuroblastoma C1300. The antigen defined by HMß1-1was widely distributed in various mouse cell lines and HMß1-1immunoprecipitated a 110-120 kDa protein common to VLA-1 andVLA-6, indicating that HMß1-1 recognizes the ß1subunit of mouse integrins. We then examined the inhibitoryeffect of HMß1-1 on VLA-dependent cell adhesion andactivation. HMß1-1 blocked the adhesion of mouse tumorcell lines to extracellular matrix proteins including collagen,laminin and fibronectin. Moreover, splenic T cell proliferationinduced by anti-CD3 mAb and allogeneic mixed lymphocyte responsewere strongly inhibited by HMß1-1 in combination withan anti-LFA-1 mAb. We conclude that HMß1-1 reactivewith mouse CD29 can inhibit VLA-dependent cellular functionsand, thus, would be useful for studying the physiological roleof ß1 integrins in vivo.     

Role of Kaposi's sarcoma cells in recruitment of circulating leukocytes: implications in pathogenesis

OBJECTIVE: We sought to identify and characterize mechanisms of interaction between Kaposi's sarcoma cells and circulating leukocytes leading to leukocyte migration into the lesion. STUDY DESIGN/METHODS: By using static and dynamic adhesion models, we measured the ability of late-stage KSY1 cells to support adhesion and transmigration of peripheral blood lymphocytes (PBL). RESULTS: We showed that resting as well as TNF-alpha- or PMA-activated KSY1 cells supported adhesion and transmigration of PBL with a higher efficiency compared with normal endothelial cells. The LFA1/ICAM1 pathway was totally involved in PBL adhesion to resting or TNF-alpha-activated KSY1 cells and partially responsible for adhesion to PMA-activated KSY1 cells. No inhibition of adhesion was observed by blockage of the VLA4 pathway. Under flow conditions, PBL/KSY1 cell interaction was totally dependent on L-selectin. CONCLUSION: Our data indicate that KS cells mimic an endothelium-like structure by regulating extravasation of lymphocytes into lesions.     

Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization

Ligation of very late antigen (VLA)-4 (α₄β₁ integrin) with a cross-linked anti-α₄ subunit monoclonal antibody (mAb) triggered a biphasic Ca²⁺ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca²⁺ response. Tumor necrosis factor-α-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca²⁺ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca²⁺ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca²⁺ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (～ 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca²⁺ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca²⁺ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca²⁺.     

Expression and function of homing-essential molecules and enhanced in vivo homing ability of human peripheral blood-derived hematopoietic progenitor cells after stimulation with stem cell factor

Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the alpha4beta1 and alpha5beta1 integrins very-late antigen (VLA)-4 and VLA-5 on peripheral blood-derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA-4 and VLA-5 on CD34+/c-kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient-derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long-term culture-initiating cell. The improvement of the homing abilities of SCF-stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic-scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.     

Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and −2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers

Recently we reported that monocyte migration through a barrier of human synovial fibroblasts (HSF) is mediated by the CD11/CD18 (β₂) integrins, and the β₁ integrins VLA-4 and VLA-5 on monocytes. Here we investigated in parallel the role of β₂ integrin family members, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on monocytes, and the immunoglobulin supergene family members, ICAM-1 and ICAM-2 on HSF and on human umbilical vein endothelial cells (HUVEC), in monocyte migration through HSF and HUVEC monolayers. Using function blocking monoclonal antibodies (mAb), when both VLA-4 and VLA-5 on monocytes were blocked, treatment of monocytes with mAb to both LFA-1 and to Mac-1 completely inhibited monocyte migration across HSF barriers, although blocking either of these β₂ integrins alone had no effect on migration, even when VLA-4 and VLA-5 were blocked. This indicates that optimal β₂ integrin-dependent monocyte migration in synovial connective tissue may be mediated by either LFA-1 or Mac-1. Both ICAM-1 and ICAM-2 were constitutively expressed on HSF and on HUVEC, although ICAM-2 was only minimally expressed on HSF. Based on results of mAb blockade, ICAM-1 appeared to be the major ligand for LFA-1-dependent migration through the HSF. In contrast, both ICAM-1 and ICAM-2 mediated LFA-1-dependent monocyte migration through HUVEC. However, neither ICAM-1 nor ICAM-2 was required for Mac-1-dependent monocyte migration through either cell barrier, indicating that Mac-1 can utilize ligands distinct from ICAM-1 and ICAM-2 on HSF and on HUVEC during monocyte transmigration.