Abstract. In this paper we report that the assembly of interendothelial junctions containing the cell typespecific vascular endothelial cadherin (VE-cadherin or cadherin-5) is a dynamic process which is affected by the functional state of the cells.Immunofluorescence double labeling of endothelial cells (EC) cultures indicated that VE-cadherin, a,-catenin, and fl-catenin colocalized in areas of cell to cell contact both in sparse and confluent EC monolayers. In contrast, plakoglobin became associated with cell-cell junctions only in tightly confluent cells concomitantly with an increase in its protein and mRNA levels. Furthermore, the amount of plakoglobin coimmunoprecipitated with VE-cadherin increased in closely packed monolayers.Artificial wounding of confluent EC monolayers resulted in a major reorganization of VE-cadherin, ot-catenin, ~-catenin, and plakoglobin. All these proteins decreased in intensity at the boundaries of EC migrating into the lesion. In contrast, EC located immediately behind the migrating front retained junctional VE-cadherin, ot-catenin, and/3-catenin while plakoglobin was absent from these sites. In line with this observation, the amount of plakoglobin coimmunoprecipitated with VE-cadherin decreased in migrating EC.These data suggest that VE-cadherin, ot-catenin, and /3-catenin are already associated with each other at early stages of intercellular adhesion and become readily organized at nascent cell contacts. Plakoglobin, on the other hand, associates with junctions only when cells approach confluence. When cells migrate, this order is reversed, namely, plakoglobin dissociates first and, then, VE-cadherin, a-catenin, and/3-catenin disassemble from the junctions. The late association of plakoglobin with junctions suggests that while VEcadherin/a-catenin//J-catenin complex can function as an early recognition mechanism between EC, the formation of mature, cytoskeleton-bound junctions requires plakoglobin synthesis and organization.

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Abstract. The integrity of the endothelial layer, which lines the entire cavity of the vascular system, depends on tight adhesion of the cells to the underlying basement membrane as well as to each other. It has been previously shown that such interactions occur via membrane receptors that determine the specificity, topology, and mechanical properties of the surface adhesion. Cell-cell junctions between endothelial cells, in culture and in situ, involve both Ca2+-dependent and -independent mechanisms that are mediated by distinct adhesion molecules. Ca2+-dependent cell-cell adhesion occurs mostly via members of the cadherin family, which locally anchor the microfilament system to the plasma membrane, in adherens junctions. Ca2+-independent adhesions were reported to mainly involve members of the Ig superfamily.In this study, we performed three-dimensional microscopic analysis of the relative subcellular distributions of these two endothelial intercellular adhesion systems. We show that cadherins are located at adjacent (usually more apical), yet clearly distinct domains of the lateral plasma membrane, compared to PECAM-1. Moreover, cadherins were first organized in adherens junctions within 2 h after seeding of endothelial cells, forming multiple lateral patches which developed into an extensive belt-like structure over a period of 24 h. PECAM-1 became associated with surface adhesions significantly later and became progressively associated with the cadherin-containing adhesions. Cadherins and PECAM-1 also differed in their detergent extractability, reflecting differences in their mode of association with the cytoskeleton. Moreover, the two adhesion systems could be differentially modulated since short treatment with the Ca 2+ chelator EGTA, disrupted the cadherin junctions leaving PECAM-1 apparently intact.These results confirm that endothelial cells possess distinct intercellular contact mechanisms that differ in their spatial and temporal organization as well as in their functional properties.

Although glutamic acid decarboxylase (GAD) has been implicated in IDDM, there is no direct evidence showing GAD-reactive T cells are diabetogenic in vivo. To address this issue, 3-wk-old NOD mice received two injections of purified rat brain GAD; one mouse rapidly developed diabetes 3 wk later. Splenocytes from this mouse showed a proliferative response to purified GAD, and were used to generate a CD4+ T cell line, designated 5A, that expresses TCRs encoding Vbeta2 and Vbeta12. 5A T cells exhibit a MHC restricted proliferative response to purified GAD, as well as GAD65 peptide 524-543. After antigen-specific stimulation, 5A T cells secrete IFNgamma and TNFalpha/beta, but not IL-4. They are also cytotoxic against NOD-derived hybridoma cells (expressing I-Ag7) that were transfected with rat GAD65, but not nontransfected hybridoma cells. Adoptive transfer of 5A cells into NOD/SCID mice produced insulitis in all mice. Diabetes occurred in 83% of the mice. We conclude that GAD injection in young NOD mice may, in some cases, provoke diabetes due to the activation of diabetogenic T cells reactive to GAD65 peptides. Our data provide direct evidence that GAD65 autoimmunity may be a critical event in the pathogenesis of IDDM.

Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses. In this study we demonstrate that engagement of the β2 integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF-α, GM-CSF, and IL-3 mRNA, as well as a chimeric β-globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element from the GM-CSF mRNA 3′-untranslated region. This integrin-enhanced mRNA stability leads to augmented protein production, as determined by TNF-α ELISPOT assays. Furthermore, T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic translocation of the mRNA-stabilizing protein HuR, which in turn is capable of binding an AU-rich element sequence in vitro. Abrogation of HuR function by use of inhibitory peptides, or marked reduction of HuR levels by RNA interference, prevents LFA-1 engagement-mediated stabilization of T cell TNF-α or IFN-γ transcripts, respectively. Thus, HuR-mediated mRNA stabilization, stimulated by integrin engagement and controlled at the level of HuR nuclear export, is critically involved in T cell activation.