Convergence between CD98 and integrin-mediated T-lymphocyte co-stimulation.

CD98 is a widely expressed cell surface heterodimeric glycoprotein, which is rapidly up-regulated upon activation of T lymphocytes. Monoclonal antibody (mAb) 80A10 recognizes an epitope on CD98 and in combination with CD3 antibody causes proliferation of peripheral blood T lymphocytes. CD98 co-stimulatory activity, mediated by either mAb 80A10 or 4F2, a well-characterized CD98-specific mAb, is blocked in the presence of the soluble beta1 integrin antibody 18D3. Previously we have reported that co-stimulatory activity of antibodies to integrins alpha4beta1, alpha5beta1, alphaLbeta2 and alpha4beta7 is inhibited by 18D3, whereas co-stimulation mediated by non-integrins was unaffected. Thus the non-integrin CD98 is uniquely sensitive to the inhibitory effects of beta1 integrin-blocking antibodies, which may reflect convergent signalling mechanisms between integrins and CD98. This is consistent with recent reports suggesting that CD98 may regulate integrin-mediated adhesive events.

Fibronectin fragments modulate monocyte VLA-5 expression and monocyte migration.

To identify the mechanisms that cause monocyte localization in infarcted myocardium, we studied the impact of ischemia-reperfusion injury on the surface expression and function of the monocyte fibronectin (FN) receptor VLA-5 (alpha(5)beta(1) integrin, CD49e/CD29). Myocardial infarction was associated with the release of FN fragments into cardiac extracellular fluids. Incubating monocytes with postreperfusion cardiac lymph that contained these FN fragments selectively reduced expression of VLA-5, an effect suppressed by specific immunoadsorption of the fragments. Treating monocytes with purified, 120-kDa cell-binding FN fragments (FN120) likewise decreased VLA-5 expression, and did so by inducing a serine proteinase-dependent proteolysis of this beta(1) integrin. We postulated that changes in VLA-5 expression, which were induced by interactions with cell-binding FN fragments, may alter monocyte migration into tissue FN, a prominent component of the cardiac extracellular matrix. Support for this hypothesis came from experiments showing that FN120 treatment significantly reduced both spontaneous and MCP-1-induced monocyte migration on an FN-impregnated collagen matrix. In vivo, it is likely that contact with cell-binding FN fragments also modulates VLA-5/FN adhesive interactions, and this causes monocytes to accumulate at sites where the fragment concentration is sufficient to ensure proteolytic degradation of VLA-5.

Angiogenesis induced by tumor necrosis factor-agr; is mediated by alpha4 integrins.

Tumor necrosis factor-alpha (TNF-alpha) and fibroblast growth factor-2 (FGF-2 or bFGF) are potent stimulators of angiogenesis. TNF- alpha, but not FGF-2, can induce the expression of vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells. The soluble form of VCAM-1 has recently been demonstrated to function as an angiogenic mediator. Here we demonstrate that monoclonal antibodies directed against VCAM-1 or its alpha4 integrin counter- receptor inhibited TNF-alpha-induced endothelial cell migration in vitro. Angiogenesis induced in vivo in rat corneas by TNF-alpha was inhibited by a neutralizing antibody directed against the rat alpha4 integrin subunit. A peptide antagonist of the a4 integrins blocked TNF-alpha-induced endothelial cell migration in vitro and angiogenesis in rat corneas in vivo. No inhibition by the antibodies or peptide antagonist was observed either in vitro or in vivo when FGF-2 was used as the stimulus. The peptide antagonist did not inhibit TNF-a binding to its receptor nor did it block the function of alphavbeta3, an integrin previously implicated in TNF-a and FGF- 2 mediated angiogenesis. These results demonstrate that angiogenic processes induced by TNF-alpha are mediated in part by agr;4 integrins possibly by a mechanism involving the induction of soluble VCAM-1.

Liposomal muramyl tripeptide upregulates adhesion molecules on the surface of human monocytes.

We previously demonstrated that liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE), a biologic response modifier now undergoing phase III clinical trial in osteosarcoma, upregulated monocyte expression of several cytokines' mRNA and the subsequent production of these proteins. In the present work, we investigated whether L-MTP-PE upregulated adhesion molecules on the surface of normal human monocytes. Flow-cytometric analysis showed that several subunits of the integrins, including alpha L, alpha 5, and beta 1 subunits, and intercellular adhesion molecule-1 on the monocytes were upregulated following their stimulation with 2 micrograms/ml L-MTP-PE for 24 h. Anti-alpha L antibodies blocked monocyte-mediated tumor cell killing stimulated by L-MTP-PE. We conclude that L-MTP-PE also stimulates the increase of several molecules on the monocyte cell surface. These adhesion molecules may contribute to the increased activation of monocyte-mediated tumor cell killing seen following L-MTP-PE exposure.

Identification of a combinatorial epitope expressed by the integrin alpha 4 beta 1 heterodimer involved in the regulation of cell adhesion.

The alpha 4 integrin subunit can associate with either the beta 1- or beta 7-integrin subunit to form two unique adhesion receptors alpha 4 beta 1 and alpha 4 beta 7. We developed a monoclonal antibody (mAb 19H8) that immunoprecipitated alpha 4 beta 1, induced homotypic leukocyte aggregation, and blocked the binding of cells to a synthetic peptide corresponding to the CS-1 peptide region of fibronectin. Aggregation cross-blocking analysis suggested that mAb 19H8 belonged to the group of mAbs that react with the B2 epitope of the alpha 4 subunit (alpha 4.B2 epitope); however, unlike the alpha 4.B2-specific mAb L25, mAb 19H8 did not immunoprecipitate alpha 4 beta 7. In addition, mAb 19H8 did not bind to beta 1-positive cells unless transfected with alpha 4 cDNA. These results indicated that mAb 19H8 was not specific for an individual alpha 4, beta 1, or beta 7 subunit but reacted with an epitope formed from the association of alpha 4 with beta 1. Separating the alpha 4 from the beta 1 subunit, by removing divalent cations or by treatment with high pH, disrupted mAb 19H8 binding. In contrast, the alpha 4-specific mAb L25 and the beta 1-specific mAb 18D3 could react with their respective subunits without subunit association. Therefore, mAb 19H8 defined a novel regulatory epitope expressed by the integrin alpha 4 beta 1.

Homotypic leukocyte aggregation triggered by a monoclonal antibody specific for a novel epitope expressed by the integrin beta 1 subunit: conversion of nonresponsive cells by transfecting human integrin alpha 4 subunit cDNA.

The monoclonal antibody 33B6 was found to be specific for the beta 1 integrin subunit. Treatment of leukocytes with this antibody induced a vigorous homotypic aggregation that had similar physiologic conditions as aggregation induced by a monoclonal antibody specific for the alpha 4 subunit. Expression of a beta 1 subunit on the cell surface was not sufficient for mAb 33B6-mediated aggregation to occur, since cells of the K562 erythroleukemia line failed to respond even though they expressed the beta 1 subunit and the 33B6 epitope. However, after transfection with cDNA encoding the alpha 4 subunit, K562 cells acquired the ability to aggregate in response to mAb 33B6 binding. By contrast, mAb 33B6 blocked cell binding to the endothelial surface protein vascular cell adhesion molecule-1 and the extracellular matrix protein fibronectin. These results suggest that the beta 1 epitope defined by mAb 33B6 may play a novel role in regulating leukocyte adhesive interactions.

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.