Endothelial cells play a central role in the inflammatory process. Tumor necrosis factor-␣ (TNF) is a multifunctional cytokine which elicits many of the inflammatory responses of endothelial cells. While TNF directly causes apoptosis of tumor cells and virally infected cells, normal cells are generally resistant. However, most resistant cells, including human endothelial cells, can be rendered susceptible to TNF by inhibiting RNA or protein synthesis. This finding suggests that TNF provides a cell survival signal in addition to a death signal. We have previously cloned a human Bcl-2 homologue, A1, and shown that it is specifically induced by proinflammatory cytokines but not by endothelial growth factors. In this study, we show that retroviral-mediated transfer of the A1 cDNA to a human microvascular endothelial cell line provides protection against cell death initiated by TNF in the presence of actinomycin D. The induction of A1 by TNF in this system is mediated via a protein kinase C pathway. Since TNF signaling has also been shown to proceed via ceramides, we tested whether exogenous ceramides could induce A1. Our findings indicate that ceramides do not induce A1 but do up-regulate c-jun and induce endothelial death. Ceramide-activated endothelial death is also inhibited by A1, suggesting that TNF may initiate divergent survival and death pathways via separate lipid second messengers. TNF1 is an inflammatory cytokine originally defined by its tumoricidal activity (1, 2). Subsequently, it has been shown to evoke multiple biological responses affecting virtually every cell type (3). Considerable attention has recently been paid to the apoptotic pathway elicited by TNF. A series of elegant experiments have defined a death pathway emanating from the TNF receptor 1. Engagement of the TNF receptor results in cell death by recruitment of a complex of proteins to the cell membrane including TRADD, FADD/Mort1, and MACH/FLICE which culminates in the activation of cysteine proteases (4 -10). TNF stimulation also results in signal transduction pathways mediated by two lipid second messengers, diacylglycerol and ceramide (3). While ceramide is a transducer of TNF-induced apoptosis (11, 12), diglyceride or phorbol ester pretreatment can protect against ceramide-induced cell death (11, 13). Ceramides appear to act downstream of FADD/Mort1 since a FADD dominant-negative can block TNF-induced apoptosis but not death mediated by ceramide (7).Although tumor cells and virally infected cells are susceptible to TNF-induced death, many normal cells are not (1, 2). In this respect, human endothelial cells which play a pivotal role in modulating the inflammatory response are not directly killed by TNF (14). However, it has previously been shown that in the presence of RNA or protein synthesis inhibitors, endothelial cells as well as other cells can be rendered sensitive to TNF (14 -16). Conversely, it has also been demonstrated that sensitive cells can be made resistant to TNF challenge by prior sublethal exposure to TNF (15, 16). Th...

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF- activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.

Abstract. The leukocyte ß1 integrin receptor very late activation antigen-4 (VLA-4) (cY4ßi, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium . A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered T HE emigration of lymphocytes to extravascular sites ofinflammation or immune reaction is mediated by the interaction ofadhesive surface proteins or lymphocytes with ligands on endothelial cells and matrix components . The binding of lymphocyte function-associated antigen-1 (LFA-1) , to intercellular adhesion molecule-1 (ICAM-1) and very late activation antigen-4 (VLA-4) with vascular cell adhesion molecule-1 (VCAM-1, inducible cell adhesion molecule-110 [INCAM-110]) accounts for a major component of lymphocyte binding to cytokine-activated cultured endothelium (7,16,32,34). LFA-1 (CDlla/CD18) is an integrin receptor of the 01 subfamily that interacts with the endothelial cell ligands ICAM-1(CD54) or ICAM-2, closely related members of the immunoglobulin superfamily (25,40,43,44) . VLA-4 (CD49d/CD29) is an integrin receptor of the ß, subfamily that has recently been shown to bind to both the CS-1 fragment of fibronectin (18, 50) andto vascular cell adhesion molecule-1 (VCAM-1) (12), a cytokine-induced endothelial protein of the immunoglobulin superfamily (28). by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes ß1 -subunit (CD29) of integrin receptors . In contrast to mAbs directed to VLA-4 alpha-subunit (tx4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL . Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin . This binding was blocked by mAbs to the VLA alpha-subunts a6 (CD49f), or «5 (CD49e) and a4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte ß, integrins, and should prove useful in studying the regulation of ß, integrin function.Migration of peripheral blood lymphocyte (PBL) through the subendothelial matrix is dependent at least in part upon other VLA antigens including VLA-5 (CD49e/CD29) and VLA-4 binding to fibronectin and VLA-6 (CD49f/CD29) to laminin (reviewed in 37) .The adhesive interactions of these receptors and ligands can be regulated at the level of the leukocyte or the endothelial cell. Expression of endothelial cell ICAM-1 is increased by interferon-gamma, lipopolysaccharide, interleukin-1, tumor necrosis factor (TNF) (11, 30), or phorbol esters (23), while VCAM-1 is induc...

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SummaryTumor necrosis factor (TNF) released by lipopolysaccharide (LPS)-stimulated mononuclear phagocytes is a critical mediator of sepsis. We examined the capacities of rough mutant Salmonella typhimurium LPS (Rc) and LPS partial structures lipid A, monophosphoryl lipid A (MPLA), lipid IVY, and lipid X to induce production of TNF in whole blood. Rc LPS (0.0001-10 ng/ml) produced a dose-dependent release ofTNF as determined by cytotoxicity ofactinomycin D-sensitized L929 murine fibroblasts . Lipid A, MPLA, lipid IVe, and lipid X exhibited decreasing capacities to stimulate production of TNF in whole blood, respectively. Fractional dearylation of LPS by incubation with acyloxyacyl hydrolase isolated from human leukocytes produced a reduction in the capacity ofLPS to induce TNF release in whole blood. Maximal enzymatic dearylation reduced activity of LPS by >100-fold .Coincubation with lipid IVY inhibited TNF release induced by Rc LPS or lipid A, but not by phorbol ester. In contrast, MPLA, lipid X, and deacylated LPS failed to inhibit LPS-stimulated release of TNF. Corresponding to the inhibition of the release of TNF protein, lipid IVn also inhibited the accumulation of TNF mRNA in LPS-stimulated mononuclear cells. These results suggest that lipid IVY may act as a competitive antagonist of LPS, perhaps at the receptor level .Gram-negativebacterial infection induces a complex array of inflammatory responses including fever, leu ocytosis, and activation of the coagulation, complement, and kinin systems . These manifestations ofGram-negative infection are primarily induced by heat-stable endotm ins released from the outer membrane ofGram-negative bacteria. LPS are the principal biologically active component of endotoxins that have a common macromolecular architecture. Three major domains comprise LPS structure : an 0-antigen side chain composed of repeating oligosaccharides, a core polysaccharide, and lipid A.Chemical degradation studies and bacterial mutants with defective synthesis of LPS have provided insights into biosynthesis and structure-function relationships of LPS and its components. Lipid A is the smallest substructure of LPS exhibiting all biological activities (1) . Lipid A from common pathogens exhibit the same general features, a 0-1,6 D-glucosamine disaccharide backbone, with four mole equivalents of 3-hydroxymyristate or 3-hydroxylaurate in amide and ester linkage, and two monophosphate groups at 1 and 4: Aryl residues are located at the 2, 2, 3, and 3' positions on the disaccharides, and the R-3 hydroxyl substitutes of the aryl 77 residues are further esterified with laurate and myristate, forming acyloxyacyl groups. Escherichia coli K12 lipid A has 2' and 3' acyloxyacyl groups, compared with Salmonella minnesota, which may have acyloxyacyl moieties at 2, 2, and 3:The biosynthesis of lipid A involves the formation of acylated monosaccharide precursors that are generated from UDP N-acetylglucosamine (2) . Two early intermediates are UDP-2,3-diacylglucosamine and 2,3-diacylglucosamine 1-phosphate (l...