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.     

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.     

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.     

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.     

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.     

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.     

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.     

T lymphocyte adhesion mechanisms within inflamed human kidney: studies with a Stamper-Woodruff assay

Renal inflammatory conditions are characterized by mononuclear cell recruitment to sites of inflammation. We have developed a modified Stamper-Woodruff assay system to analyze mechanisms of functional T cell adhesion to cryostat sections of renal biopsy material from patients with vasculitic glomerulonephritis (GN) and acute allograft rejection. Peripheral blood T cells adhered to intraglomerular, periglomerular, and tubulointerstitial regions of the cortex. Blocking monoclonal antibodies against tissue expressed ICAM-1, VCAM-1, and the CS-1 domain of fibronectin (CS-1Fn) differentially attenuated T cell adhesion. Glomerular adhesion in vasculitic GN and tubulointerstitial adhesion in acute rejection were particularly sensitive to both anti-ICAM-1 and anti-VCAM-1 antibodies, indicating a prominent role for ICAM-1 and VCAM-1 at glomerular sites in vasculitis and at tubulointerstitial sites in rejection. Furthermore, using KL/4 cells (LFA-1 expressing) and Jurkat cells (VLA-4 expressing), we demonstrated specific LFA-1/ICAM-1- and VLA-4/VCAM-1-mediated interactions within glomerular and tubulointerstitial compartments. Jurkat cells also adhered to VCAM-1-free sites, and binding was inhibitable by anti-CS-1Fn antibody, thereby demonstrating a role for VLA-4/fibronectin interactions especially at intraglomerular sites in acute rejection where VCAM-1 is notably absent. We therefore propose a prominent functional role for ICAM-1, VCAM-1, and CS-1 domain fibronectin in T cell recruitment to the inflamed kidney.     

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

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

Changes in the pattern of fibronectin mRNA alternative splicing in acute experimental mesangioproliferative nephritis

Fibronectins (FN) regulate cell migration, proliferation, and matrix formation during tissue injury. In humans, up to 20 different FN isoforms are generated by alternative splicing in three regions called EIIIA, EIIIB, and V, which have been implicated in the process undergoing wound healing and embryonic development. Specifically, EIIIA- and EIIIB-containing isoforms have been implicated in the regulation of cell proliferation and migration, whereas FN isoforms containing the full-length V region (named V120) are ligands to the VLA-4 integrin. To study the changes in the expression of FN isoforms in the anti-Thy-1 nephritis, an acute and self-resolutive model of mesangioproliferative nephritis, we analyzed the FN splicing patterns by means of ribonuclease protection assays. At Day 7 after anti-Thy-1 monoclonal injection, time of the maximal matrix expansion and glomerular hypercellularity, EIIIA+, EIIIB-, and V120 FN mRNA isoforms were increased. In accordance with the mRNA studies, FN proteins, including the EIIIA and V120 regions, increased in the mesangium of nephritic rats, as assayed by immunohistochemistry. Coinciding with the EIIIA and V120 isoforms up-regulation, there was an increase in mesangial cell proliferation and in the number of VLA-4+ infiltrating cells. At Day 14, in parallel with a remission of the above-mentioned changes, there was a decline in the mRNA and protein FN isoforms increased in the previous phase. The marked and reversible changes in the pattern of FN isoforms and their temporal association with other indicators of glomerular injury suggest that certain FN isotypes are important and coordinated components of the mechanisms attempting to reverse glomerular damage.     

Allicin inhibits SDF-1alpha-induced T cell interactions with fibronectin and endothelial cells by down-regulating cytoskeleton rearrangement, Pyk-2 phosphorylation and VLA-4 expression

Allicin, a major ingredient of fresh garlic extract that is produced during the crushing of garlic cloves, exerts various beneficial biological effects, including a broad spectrum of antimicrobial activity, antihyperlipidaemic and antihypertensive effects. However, how allicin affects the immune system is less well known, and its effect on human T cells has never been studied. Here, we examined the in-vitro effects of allicin on the functioning of T cells related to their entry to inflamed extravascular sites. We found that allicin (20-100 microm) inhibits the SDF-1alpha (CXCL12)-induced T cell migration through fibronectin (FN), and that this inhibition is mediated by the down-regulation of (i) the reorganization of cortical actin and the subsequent T cell polarization, and (ii) T cell adhesion to FN. Moreover, allicin also inhibited T cell adhesion to endothelial cells and transendothelial migration. The mechanisms underlying these inhibitory effects of allicin are associated with its ability to down-regulate the phosphorylation of Pyk2, an intracellular member of the focal adhesion kinases, and to reduce the expression of the VCAM-1- and FN-specific alpha4beta1-integrin (VLA-4). The ability of allicin to down-regulate these chemokine-induced and VLA-4-mediated T cell functions explains its beneficial biological effects in processes where T cells play an important role and suggests that allicin may be used therapeutically with chronic inflammatory diseases.     

The low molecular weight Dextran 40 inhibits the adhesion of T lymphocytes to endothelial cells

Dextrans are complex colloidal macromolecules widely used as haemorrheologic substances and anti-thrombotic agents. Here we describe a novel function of Dextran 40 by demonstrating an inhibition of T lymphocyte adhesion to endothelial cells (EC). We applied an established microassay in which constitutive and tumour necrosis factor-alpha (TNF-α)-induced binding of mouse T lymphoma cells (TK-1) to mouse endothelioma (eEND.2) cells is mediated by the interaction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on EC with their counter-receptors the LFA-1 heterodimer (CD11a/CD18) and VLA-4 on T cells. Dextran 40 in therapeutically achievable levels (2–32 mg/ml) reduced both constitutive and TNF-α-stimulated TK-1 adhesion to eEND.2. Selective preincubation of eEND.2 or TK-1 revealed that Dextran 40 acted exclusively on the T cells. To explore further the mechanisms by which Dextran 40 interfered with TK-1 adhesion, their LFA-1 and VLA-4 expression was analysed by FACS. The surface expression levels of neither receptor were affected by Dextran 40. However, confocal microscopy revealed that Dextran 40 interfered with the activation-dependent capping and clustering of LFA-1 and VLA-4 on the surface of TK-1. We conclude that Dextran 40 inhibits the capacity of TK-1 T cells to adhere to eEND.2 endothelial cells and thus may be useful for therapeutic intervention in diseases associated with enhanced T lymphocyte binding to microvascular endothelium.     

VLA-2 is the integrin used as a collagen receptor by leukocytes.

Cultured T cells and freshly isolated mononuclear leukocytes are able to bind collagen specifically. These leukocytes express equivalent levels of the integrins VLA-1, VLA-2 and VLA-3 which are collagen-binding receptors on other cells. However, only solubilized VLA-2 is able to bind collagen and only monoclonal antibodies specific for alpha 2 or beta 1 subunits are able to block the binding of intact cells to collagen. This restriction provides another example of the dependence of integrin specificity on the cell type on which it is expressed. It was also speculated that the inserted or I domain on the alpha subunits of VLA-1, VLA-2 and the beta 2 integrin family might have a role in collagen binding on the basis of its sequence homology to other types of collagen binding proteins. However, LFA-1, CR3 and p150,95 showed no collagen binding activity, suggesting that the I domain has another function.     

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

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

Differential regulation of VLA-2 expression on Th1 and Th2 cells: a novel marker for the classification of Th subsets

We found that T(h)1 cells derived from ovalbumin (OVA)-specific TCR transgenic (DO11.10) mice showed significantly higher levels of VLA-2 (CD49b/CD29) expression than T(h)2 cells. In the early days (until 6 days) during induction of T(h)1 or T(h)2 cells, the expression of VLA-2 was gradually increased on both T(h) subsets. Thereafter, VLA-2 expression was further up-regulated on T(h)1 cells until 13 days, while a significant decrease of VLA-2 was observed in T(h)2 cells, resulting in a marked difference of expression at day 13. Up-regulation of VLA-2 on T(h)1 cells was not impaired in IFN-gamma(-/-) T(h) cells nor blocked by anti-IL-12 mAb treatment on wild-type T(h) cells, suggesting that up-regulation of VLA-2 on T(h)1 cells occurs in an IFN-gamma- and IL-12-independent manner. In contrast, T(h) cells cultured under IL-4-depleted T(h)2 conditions abrogated the down-regulation of VLA-2 expression, suggesting that down-regulation of VLA-2 expression on T(h)2 cells was dependent on IL-4. The finding that STAT6(-/-) T(h)2 cells did not show any down-regulation of VLA-2 expression and expressed the same levels of VLA-2 as T(h)1 cells indicated a critical role for the IL-4 receptor/STAT6 signaling pathway in IL-4-dependent down-regulation of VLA-2 on T(h)2 cells. Stimulation of T(h)1 cells by VLA-2 ligands such as collagen type I or agonistic mAb provided co-stimulation for anti-CD3 mAb-induced IFN-gamma production. However, these ligations had little effect on the IL-4 production of T(h)2 cells. Together, these results indicate that VLA-2 is a novel functional marker that dissociates T(h)1 from T(h)2 cells, and thus might be useful for therapeutic monitoring of T(h)1-dependent immune diseases such as rheumatoid arthritis or Crohn's disease.     

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.     

T lymphocyte adhesion to the fibronectin and laminin components of the extracellular matrix is regulated by the CD4 molecule.

The adhesion of T cells to components of the extracellular matrix (ECM) is mediated by the beta 1 subfamily of integrin receptors, designated VLA. It has been recently demonstrated that the binding of VLA receptors to protein components of the ECM is rapidly augmented by the activation of the T cells without, however, any actual change in the level of expression of the VLA receptors for fibronectin (FN) or laminin (LN). Thus, it is likely that activation of existing VLA receptors is required for binding. The activation must be regulated by T cell surface molecules capable of transducing signals into the cell. We studied the role of the CD4 molecule in the binding of rat CD4+ T cells to the FN and LN components of the ECM. We now report that the CD4 molecule appears to play a major role in regulating T cell interactions with ECM. This conclusion is based on the following observations: (a) monoclonal antibodies directed against the CD4 molecule inhibited T cell adhesion to both FN and LN; (b) down-regulation of the CD4 molecule resulted in partial loss of the ability of CD4+ T cells to adhere to FN and LN; (c) a CD4+ T cell clone adhered to both FN and LN while a CD4-CD8- clone expressing an identical T cell receptor bound weakly to both proteins and (d) treatment of the CD4+ T cells with an inhibitor of the CD4-associated tyrosine protein kinase activity inhibited T cell adhesion to both ECM proteins.     

T lymphocyte adhesion to fibronectin (FN): a possible mechanism for T cell accumulation in the rheumatoid joint.

The accumulation of T cells within the joint is responsible for the perpetuation of synovitis. This process is partly regulated by selective binding to endothelium. However, adhesion to extra-cellular matrix proteins, like FN, may also be important. FN binding is mediated by certain members of the VLA (beta 1 integrin) family of proteins. To investigate the role of Tc-FN interactions in synovitis the binding of synovial fluid (SF) and peripheral blood (PB) T cells to FN-coated wells, and the expression of cell surface VLA molecules on these cells by double label immunofluorescence, were studied. SF T cells bound better to FN than PB T cells. VLA alpha 4 and VLA beta 1 but not VLA alpha 5 were up-regulated on SF compared with PB T cells. Anti-VLA alpha 4, VLA beta 1 and VLA alpha 5 MoAbs inhibited the binding of SF T cells to FN. The increased binding of SF T cells to FN could have been related to activation and/or to their predominantly memory phenotype. Purified resting memory or naive T cells bound poorly to FN. In contrast, compared with SF T cells, concanavalin A-activated T cells showed a very similar level of binding to FN, comparable expression of VLA molecules and the same pattern of inhibition of binding to FN by MoAbs. Thus, VLA molecules may play an important role in the retention of T cells in the joint and since T cells can be activated via VLA-FN interactions, this mechanism may perpetuate chronic inflammation.     

Role of cell-cell communication in inhibiting butyric acid-induced T-cell apoptosis

We have previously demonstrated that human gingival fibroblasts rescue butyric acid-induced T-cell apoptosis via proinflammatory cytokines such as interleukin 6 (IL-6) and IL-11, which are produced by fibroblasts stimulated with butyric acid. In this study, we determined if T-cell adhesion to human gingival fibroblasts influenced the susceptibility of T cells to butyric acid-induced apoptosis. We have shown that the number of Jurkat T cells adherent to gingival fibroblasts (Gin-1 cells) was significantly increased by the addition of butyric acid. All Jurkat cells that adhered to Gin-1 cells remained viable, while the nonadherent Jurkat cells dropped into apoptosis. The increase in T-cell adhesion to fibroblasts was also observed when Jurkat cells, but not Gin-1 cells, were pretreated with butyric acid. The expression levels of CD44, very late antigen 2 (VLA-2) and VLA-5 but not of leukocyte function-associated antigen 1 (LFA-1) and VLA-4 on Jurkat cells were increased following treatment with butyric acid. Furthermore, pretreatment of butyric acid-sensitized Jurkat cells with monoclonal antibodies against CD44, VLA-2, and VLA-5, but not LFA-1 and VLA-4, followed by coculture with Gin-1 cells inhibited T-cell adhesion to fibroblasts and increased apoptosis of nonadherent T cells after coculture of gingival fibroblasts and Jurkat cells. These results indicate that T-cell adherence to fibroblasts is enhanced by butyric acid and that butyric acid-induced T-cell apoptosis is down-regulated by T-cell adhesion to gingival fibroblasts through an interaction with the adhesion molecules CD44, VLA-2, and VLA-5 expressed on T cells stimulated with butyric acid.     

Expression and functions of very late antigen 1 in inflammatory joint diseases

In the human immune system, very late antigen 1 (VLA-1), a putative collagen receptor, is expressed on the surface of T lymphocytes that have undergone mitogenic or antigenic stimulation. A new VLA-1-specific monoclonal antibody, 1B3.1, was used to probe the expression and function of VLA-1 on T lymphocytes in patients with arthritis. Synovial mononuclear cells from the joints of patients with rheumatoid arthritis or other joint diseases contained 32.9±13.8% 1B3.1-positive cells (42.8±10.4% in patients with rheumatoid arthritis and 28 ± 12.6% in non rheumatoid patients). In the peripheral blood, patients with active rheumatoid arthritis expressed VLA-1 on 11.7±6.0% of their mononuclear cells, compared to 1.9±1.5% in controls (P<0.001). Using dual fluorescence analysis, virtually all the 1B3.1-positive synovial cells were CD3+ T lymphocytes and included both CD4+ and CD8+ T cells. When 1B3.1-expressing synovial mononuclear cells orin vitro activated T lymphocytes were triggered with anti-CD3 antibodies, marked augmentation of their proliferation occurred if they were simultaneously cross-linked with mab 1B3.1. Collagen type IV, a putative ligand of VLA-1, also augmented T-cell proliferation to anti-CD3. The data suggest that the VLA-1 molecule could play an important role in the pathophysiology of arthritis by modulating T-cell activation in these diseases.