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.     

Chemokines stimulate human T lymphocyte transendothelial migration to utilize VLA-4 in addition to LFA-1

Lymphocyte infiltration in inflammation is induced by the dual actions of chemokines and cell adhesion molecules. The role of LFA-1 and VLA-4 in chemokine-induced T cell transendothelial migration (TEM) across cytokine-activated endothelium has not been examined. LFA-1, but not VLA-4, mediated blood T cell TEM to RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and stromal cell-derived factor-1 (SDF-1), and across tumor necrosis factor alpha (TNF-alpha) or interferon-gamma (IFN-gamma) -stimulated endothelial cells (EC). Chemokine stimulation in combination with TNF-alpha activation of EC induced TEM, which was partially mediated by VLA-4. SDF-1 increased a beta1-integrin activation epitope on T cells and enhanced VLA-4-mediated adhesion. Thus, LFA-1 mediates TEM under most conditions, but VLA-4 can also mediate TEM, although, in contrast to LFA-1, this requires exogenous chemokines and EC activation. In addition, an LFA-1- and VLA-4-independent pathway of lymphocyte TEM can also be induced by SDF-1.     

Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF.     

Expression and functional activity of the very late activation antigen-4 molecule on human natural killer cells in different states of activation

In the present study we describe the expression and functional activity of the alpha4beta1 heterodimer molecule on human natural killer (NK) cells. Flow cytometric analyses showed that fresh and activated NK cells expressed high levels of very late activation antigen-4 (VLA-4) molecules. These cells bound to fibronectin (FN) and to its 38 000-MW proteolytic fragment through the VLA-4 integrin that was blocked with HP2/1 anti-alpha4 monoclonal antibodies (mAbs) and with the FN peptide fragment CS1. No inhibitory effects were observed in the presence of anti-alpha5 mAb, FN peptide fragment CS2 or other irrelevant mAb. Fresh NK cells were unable to aggregate, despite their expression of VLA-4, and only activated (cultured and lymphocyte-activated killer cells) NK cells showed homotypic aggregation with HP1/7 and HP2/4 anti-alpha4 mAb related to cellular activation. These results underline new evidence of how NK cells in different states of activation maintain different constitutive levels of alpha4beta1 integrin activity, and highlight the possibility of a different functional regulation by the cells bearing VLA-4, in the expression of these epitopes and their ability to interact with their ligands.     

Heparin-disaccharide affects T cells: inhibition of NF-kappaB activation, cell migration, and modulation of intracellular signaling

We previously reported that disaccharides (DS), generated by enzymatic degradation of heparin or heparan sulfate, inhibit T cell-mediated immune reactions in rodents and regulate cytokine [tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and IL-1beta] secretion by T cells, macrophages, or intestinal epithelial cells. Here, we investigated the effects of a trisulfated heparin DS (3S-DS) on two aspects of T cell function: secretion of proinflammatory cytokines and migration to an inflamed site. 3S-DS down-regulated nuclear factor-kappaB activity and reduced the secretion of TNF-alpha and interferon-gamma (IFN-gamma) by anti-CD3-activated T cells. In addition, 3S-DS inhibited CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor-1alpha)-dependent migration in vitro and in vivo and decreased CXCL12-induced T cell adhesion to the extracellular matrix glycoprotein, fibronectin (FN). This inhibition was accompanied by attenuation of CXCL12-induced Pyk2 phosphorylation but did not involve internalization of the CXCL12 receptor, CXCR4, or phosphorylation of extracellular-regulated kinase. Despite inhibiting CXCL12-induced adhesion, 3S-DS, on its own, induced T cell adhesion to FN, which was accompanied by phosphorylation of Pyk2. A monosulfated DS showed no effect. Taken together, these data provide evidence that 3S-DS can regulate inflammation by inducing and modulating T cell-signaling events, desensitizing CXCR4, and modulating T cell receptor-induced responses.     

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.     

Induction of mast cell interactions with blood vessel wall components by direct contact with intact T cells or T cell membranes in vitro

BACKGROUND: Mast cells exert profound pleiotropic effects on immune cell reactions at inflammatory sites, where they are most likely influenced not only by the extracellular matrix (ECM) and inflammatory mediators but also by the proximity of activated T lymphocytes. We recently reported that activated T cells induce mast cell degranulation with the release of TNF-alpha, and that this activation pathway is mediated by lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) binding. OBJECTIVE: To determine how this contact between the two cell types can modulate mast cell behaviour in an inflammatory milieu by examining the adhesion of mast cells to endothelial cells and ECM ligands in an integrin-dependent manner. METHODS: Human mast cells (HMC-1) were co-cultured with resting or activated T cells followed by testing their adhesion to endothelial cell and ECM ligands, stromal derived factor-1alpha (SDF-1alpha)-induced migration, and western blotting. RESULTS: Co-culturing HMC-1 with activated, but not with resting T cells resulted in marked stimulation of mast cell adhesion to vascular cell adhesion molecule-1 and ICAM-1 in a very late antigen-4- and LFA-1-dependent fashion. In addition, activated T cells or T cell membranes promoted HMC-1 adhesion to fibronectin (FN) and laminin. This effect was accompanied by the phosphorylation of extracellular regulated kinase and p38, but not of c-Jun N-terminal kinase. Importantly, the adhesive property of mast cells depended exclusively on the direct contact between the two cell types, since neither supernatants from activated T cells nor separation of the two cell populations with a porous membrane affected mast cell adhesion to FN. Furthermore, similar results were obtained when mast cells were incubated with purified membranes from activated T cells. These results suggest that, in addition to stimulating mast cell degranulation, the proximity of activated T lymphocytes to mast cells can mediate the adhesion of mast cell precursors to the endothelial ligands and ECM. Activated T cells also stimulated SDF-1alpha-induced mast cell migration. CONCLUSION: This symbiotic relationship between the two types of immune cells may serve to direct mast cells to specific sites of inflammation where their effector functions are required.     

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.     

Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin alpha4beta1

The alpha4beta1 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that alpha4beta1 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4(+)CD8(+) and CD4(+)CD8(-) adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9(+)/alpha4beta1(+) human T cell line Molt-4. To study the role on CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP-interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM), we knocked down their expression and tested transfectant attachment to alpha4beta1 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial alpha4beta1-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by alpha4beta1 could contribute to control their trafficking in the thymus during maturation, and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside-out signaling, leading to stimulated adhesion.     

VLA-4-dependent adhesion activities of U937 cells and guinea pig bronchoalveolar lavage leukocytes

VLA-4-dependent binding to fibronectin (FN) and to a human vascular cell adhesion molecule (hVCAM-1)-transfected murine cell line was measured using U937 cells and guinea pig (GP) bronchoalveolar lavage (BAL) cells. A species cross-reactive, blocking monoclonal antibody directed against human VLA-4 (TY 21.6) inhibited U937/FN binding by 71±7%. The presence of TY 21.6 inhibited the stimulated binding of U937 cells to hVCAM-1 by 84%. However, TY 21.6 was unable to inhibit the BAL/FN binding. With the addition of TY 21.6, the binding of PMA-stimulated BAL cells to hVCAM-1 was inhibited by 57±5%. In summary, human and guinea-pig leukocytes express binding activity to both FN and hVCAM-1. A specific VLA-4 blocking monoclonal antibody, TY 21.6, inhibited U937 and BAL cell binding to hVCAM-1, but only inhibited FN binding with U937 cells.In memory of Robert Sturm, January 1993.     

Effects of the very late adhesion molecule 4 antagonist WAY103 on human peripheral blood eosinophil vascular cell adhesion molecule 1-dependent functions

BACKGROUND: Eosinophil infiltration to the lung in allergic inflammation can be initiated by the tethering of circulating cells through very late adhesion molecule 4 (VLA-4; alpha4beta1, CD49d/CD29) to vascular cell adhesion molecule 1 (VCAM-1) expressed on pulmonary vascular endothelium. Small-molecule VLA-4 antagonists have been proposed as a therapeutic mechanism to prevent eosinophil infiltration in asthma; however, they might affect other eosinophil functions. OBJECTIVE: The small-molecule VLA-4 antagonist (2S)-3-(4-Dimethylcarbamoyloxyphenyl)-2-{[(4R)-5,5-dimethyl-3-(1-methyl-1H-pyrazole-4 sulfonyl)thiazolidine-4-carbonyl]amino}propionic acid (WAY103) was assessed for its effects on eosinophil VLA-4-dependent functions, including adhesion, migration, respiratory burst, and degranulation. METHODS: Human peripheral blood eosinophils were preincubated with WAY103, anti-alpha4, and/or anti-beta2 integrin mAbs and then assessed for adhesion to recombinant VCAM-1, intercellular adhesion molecule 1, and endothelial cell monolayers. Transmigration was measured by using human pulmonary microvascular endothelial cell monolayers and Transwell filters. Superoxide anion generation was determined by means of cytochrome C reduction and degranulation by means of eosinophil-derived neurotoxin release. RESULTS: WAY103 inhibition of eosinophil adhesion to recombinant VCAM-1 was dose dependent (63% inhibition with 100 nM WAY103, P < .04) and comparable with inhibition caused by anti-alpha4 mAb (60.1% inhibition). Although pretreatment with WAY103 also decreased eosinophil adhesion to TNF-alpha plus IL-4-activated human pulmonary microvascular endothelial cell monolayers, it did not prevent eosinophil transendothelial migration in response to RANTES. Finally, WAY103 inhibited VCAM-1-stimulated superoxide generation but enhanced cytokine-activated eosinophil-derived neurotoxin degranulation. CONCLUSION: Although small-molecule VLA-4 antagonists, such as WAY103, might reduce eosinophil adhesion, this approach might not be sufficient to eliminate this cell from in vivo allergic airway inflammatory participation and could even promote specific cell activation.     

The role of very late antigen-1 in immune-mediated inflammation

The alpha1beta1 integrin, also known as "very late antigen" (VLA)-1, is normally expressed on mesenchymal cells, some epithelial cells, activated T cells, and macrophages, and interacts, via the I-domain of the extracellular domain of the alpha1 subunit, with collagen molecules in the extracellular matrix (ECM). By "outside-in" transmembranal signaling to the interior of the cell, it mediates adhesion, migration, proliferation, remodeling of the ECM, and cytokine secretion by endothelial cells, mesangial cells, fibroblasts, and immunocytes. Importantly, its expressions and functions are enhanced by inflammatory cytokines including interferon (IFN)gamma and tumor necrosis factor (TNF)alpha, thus augmenting angiogenesis and fibrosis linked, in particular, to inflammation. Moreover, within the immune system, VLA-1 marks effector memory CD4+ and CD8+ T cells that are retained in extralymphatic tissues by interactions of the integrin with collagen and produce high levels of IFNgamma. Thus, immune-mediated inflammation in vivo is inhibited by blockade of the VLA-1-collagen interaction in experimental animal models of arthritis, colitis, nephritis, and graft versus host disease (GVHD), suggesting that inhibiting the interaction of the alpha1 I-domain with its ligands or modulating "outside-in" signaling by VLA-1 would be a useful approach in the human diseases simulated by these experimental models.     

Dual inhibition of VLA-4 and LFA-1 maximally inhibits cutaneous delayed-type hypersensitivity-induced inflammation.

Lymphocytes express surface receptors that mediate adhesion to endothelial cells and control T cell migration into inflammatory sites. Lymphocyte VLA-4 and LFA-1 mediate adhesion to cytokine-activated endothelium, but the contribution of these molecules to in vivo migration and lymphocyte mediated inflammation is not clear. Here we show that both VLA-4 and LFA-1 contribute to not only lymphocyte adhesion but to in vivo lymphocyte migration in the rat and that nearly complete inhibition of lymphocyte accumulation is observed when both integrins are blocked. Furthermore, inhibition of delayed-type hypersensitivity-induced inflammation, as quantified by skin induration and fibrin deposition, is observed with either anti-VLA-4 or anti-LFA-1, but much stronger inhibition is observed with a blockade of both integrins. Thus, dual inhibition of the VLA-4 and LFA-1 pathways is required for a maximal anti-inflammatory effect in some types of T cell-mediated inflammation.     

Modulation of integrin-mediated intercellular adhesion during the interaction of thymocytes with stromal cells expressing VLA-4 and LFA-1 ligands

Mature peripheral T cells closely regulate their intercellular interactions by modulating integrin adhesion functions. The ability of members of the integrin family to mediate intercellular adhesion is dependent on signals from within the cells (inside-out signaling) that increase the avidity of integrins for their ligands. These changes in avidity are independent of the quantitative changes on the number of receptors, and there is evidence to suggest that phosphorylation events play a predominant role in the regulation of the avidity state of the integrins. Whether such regulatory mechanisms are operative during T cell development had hitherto been an opened question. In the present work, we have used an in vitro adhesion assay between thymocytes and target cells expressing VLA-4 and LFA-1 counter ligands to determine how thymocytes can discriminate between integrin-specific signals during T cell development. Our findings are that VLA-4, but not LFA-1, is constitutively expressed in its high-avidity state during the early stages of T cell development, and that the high-avidity state of thymocytes for VCAM-1-expressing cells is closely regulated by signaling through protein kinase C and protein tyrosine kinase pathways. At later stages of development, mature thymocytes prior to leaving the thymus turn off both VLA-4 and LFA-1 adhesion functions. Our results show that the low-affinity state of integrins on peripheral mature T cells is established before mature thymocytes leave the thymus. Only when mature T cells recognize antigenic peptides in the context of major histocompatibility complex in the periphery will they turn on the adhesion function of VLA-4 and/or LFA-1 integrins.     

Activation-dependent changes in soluble fibronectin binding and expression of beta1 integrin activation epitopes in T cells: relationship to T cell adhesion and migration

The relationship between activation-dependent changes in beta1 integrin conformation, T cell adhesion to immobilized fibronectin, and T cell migration in vitro was analyzed in this study. Stimulation of Jurkat T cells and peripheral T cells with Mn(2+), the activating beta1 integrin-specific monoclonal antibody (mAb) TS2 /16, CD2, or CD28 stimulation led to increased adhesion, soluble fibronectin (FN) binding and expression of the activation epitope defined by the beta1 integrin mAb HUTS-21. Phorbol 12-myristate 13-acetate treatment increased adhesion, but not soluble FN binding or HUTS-21 epitope expression. In peripheral T cells, CD3 or CD7 stimulation also led to increased adhesion, soluble FN binding and HUTS-21 epitope expression. Soluble FN blocked peripheral T cell adhesion induced by Mn(2+) or TS2/16, but had no effect on adhesion induced by the other integrin-activating signals. In contrast, migration induced by TS2/16, CD2, CD3, CD7 or CD28 stimulation was blocked by excess soluble FN. Phosphoinositide 3-OH kinase (PI 3-K) inhibitors blocked receptor-mediated increases in cell adhesion, but not soluble FN binding or HUTS-21 expression. Migration was similarly unaffected by PI 3-K inhibitors, with the exception of CD7- and CD28-induced migration, which was specifically blocked by LY294,002. These results suggest that activation-dependent changes in beta1 integrin conformation are PI 3-K-independent and are involved in T cell migration but not adhesion.     

Defective up-regulation of CD49d in final maturation of NOD mouse macrophages

Macrophages are potent regulators of both innate and adaptive immunity. They play a central role in the development of autoimmune diabetes and are among the first cells to appear in peri-islet infiltrates of NOD mice that spontaneously develop diabetes. Since efficient adhesion and migration are crucial for proper macrophage trafficking, we examined the migration and fibronectin (FN) adhesion capacity of NOD macrophages, as well as the regulation and expression of the FN receptors alpha4beta1 and alpha5beta1. When compared to macrophages from control strains, resident NOD macrophages showed a reduced ability to adhere to and migrate on FN, a delayed clearance following peritoneal inflammation, and substantially lower expression levels of the alpha4beta1 integrin alpha chain, CD49d. NOD bone marrow-derived macrophages were specifically defective in the LPS-induced increase in CD49d expression. Moreover, the mitogen-activated protein kinase extracellular signal-regulated kinase-1/2 negatively regulated macrophage CD49d expression and strongly suppressed its expression in NOD macrophages. The data presented herein indicate that the LPS-activated signaling cascade plays a critical role in CD49d expression of macrophages. Mature NOD macrophages are characterized by decreased CD49d expression and show defective CD49d-mediated adhesion to FN.     

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.     

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.     

整合素β1及纤连蛋白、层粘连蛋白对胶质瘤细胞浸润性的影响

目的 探讨整合素β1和纤连蛋白（FN）、层粘连蛋白（LN）对人胶质瘤浸润性的影响和作用机制。方法 以U251人胶质母细胞瘤（U251MG）细胞为研究对象,通过细胞黏附实验、迁移实验和体外侵袭实验,检测整合素β1及LN、FN对人恶性胶质瘤细胞黏附、迁移和转移能力的影响。通过荧光染色结合激光扫描共聚焦显微镜观察和扫描电镜方法,观察细胞微丝数量、分布和细胞表面伪足情况,比较整合素β1及FN、LN对微丝骨架的影响。结果 （1）FN对U251MG细胞黏附能力无明显影响,但抗整合素β1抗体可减少U251MG细胞的黏附数量（P〈0．01）;LN增加U251MG细胞黏附能力（P〈0．01）,抗整合素β1抗体对此作用影响较小。（2）抗整合素β1抗体减弱U251MG细胞在FN的运动、迁移能力（P〈0．05）。（3）U251MG细胞内可见清晰的微丝结构,FN、LN使细胞内纤维型肌动蛋白（F-actin）形成束状纤维,粗壮而密集;抗整合素B1抗体处理的细胞内,难以见到清晰的细胞微丝骨架,并常见大量絮团状的F-actin。（4）扫描电镜观察显示,FN、LN使细胞表面的伪足数量明显增加,而抗整合素β1抗体使细胞伪足数量明显减少,甚至消失。（5）FN和抗整合素β1抗体对U251MG细胞的体外侵袭能力无明显影响;LN可促进U251MG细胞的体外侵袭能力,抗整合素β1抗体可抑制这种作用（P〈0．01）。结论 （1）U251MG细胞通过整合素β1和FN相互作用,改变细胞微丝骨架、伪足结构和数量而促进U251MG细胞的运动、迁移能力。（2）整合素β1参与了LN介导的U251MG细胞体外侵袭作用。     

LPAM-1 (integrin α4β7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen,a fibronectin-like polymer and RGD-like cyclic peptides

The α4 integrin LPAM-1 (α4β7) mediates lymphocyte attachment within the extracellular matrix (ECM) by adhering to the connecting segment (CS)-1 site of fibronectin (FN). Here we reveal that very late antigen (VLA)-4⁻ LPAM-1⁺ T cell lymphoma TK-1 cells bind via LPAM-1 to multiple copies of the RGD sequence engineered within an FN-like polymer. Further, the small conformationally restrained RGD-like cyclic peptides 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys and Arg-Cys-Asp-thioproline-Cys inhibit the adhesion of TK-1 cells to immobilized CS-1 peptide, and to endothelial counterreceptors for LPAM-1, namely mucosal addressin cell adhesion molecule (MAdCAM)-1 and vascular cell adhesion molecule (VCAM)-1. Spontaneous adhesion of the VLA-4⁻ LPAM-1⁺ B lymphoma cell line RPMI 8866 to CS-1 was likewise inhibited, confirming a previously undocumented ability of LPAM-1 to recognize the RGD tripeptide. The RGD-binding site in LPAM-1 either overlaps or is identical to sites required for interaction with MAdCAM-1, VCAM-1, and the CS-1. The binding of LPAM-1 and VLA-4 to RGD-containing ligands may have relevance in vivo given that fibrinogen at physiological concentrations is able to partially block the binding of TK-1 cells to MAdCAM-1. Hence fibrinogen and other vascular RGD-containing proteins may have mild anti-inflammatory activity required for maintaining effective homeostasis, analogous to the anti-thrombogenic activity of the vascular endothelium.