Recombinant human tumor necrosis factor (TNF) was found to enhance the adherence of human peripheral blood neutrophils to human umbilical vein endothelial (HUVE) cell monolayers in vitro. The enhancement was due to the effects both on neutrophils and HUVE cells. The effect on neutrophils was maximally induced within 5 min and did not require protein or RNA synthesis. By contrast, maximal effects on HUVE cells took 4 hr to develop and required de novo protein and RNA synthesis; however, exposure of HUVE cells to TNF for as little as 5 min was sufficient to initiate changes leading to maximal adherence of neutrophils at 4 hr. Both the effect on neutrophils and that on HUVE cells were blocked by a monoclonal antibody against TNF. TNF also rapidly induced an increased surface expression of neutrophil antigens recognized by monoclonal antibodies directed against epitopes of a glycoprotein required for optimum adherence and for complement component C3bi receptor (CR3) function. Thus, the mechanism of action of TNF may involve the regulation of expression of cell surface molecules. Our observations show that TNF induces a process central to the development of all inflammatory reactions and that both blood neutrophils and endothelial cells are targets of TNF action. The regulation of inflammatory reactions by TNF or antagonists of TNF has wide-ranging clinical implications.The human cytokines tumor necrosis factor (TNF) and lymphotoxin have been identified by their tumoricidal and tumorostatic properties in vivo and in vitro (1-4). The cloning of these cytokines has allowed confirmation of their effects on malignant cells (5, 6) and the demonstration of their biological effects on the neutrophil, in which they enhanced phagocytic and antibody-dependent cytotoxic activities (7).Since neutrophils play a central role in inflammatory reactions, which may be involved in tumor rejections, we hypothesized that TNF may alter one of the essential components of the development of these reactions: namely, adherence of neutrophils to endothelial cells. In this communication we demonstrate that TNF is a powerful stimulator of neutrophil adherence to human umbilical vein endothelium (HUVE) Schwartz (9).Preparation and Radiolabeling of Neutrophils. Whole blood from normal healthy donors was drawn into syringes containing 0.2% ethylenediaminetetraacetic acid (EDTA). Purified neutrophils were isolated by Ficoll/Hypaque (Pharmacia) gradient centrifugation and dextran sedimentation with hypotonic lysis of contaminating erythrocytes (10).Radiolabeled neutrophils were prepared according to the method of Gallin et al. (11). Purified neutrophils (average purity >98%) were suspended (24 x 106 per ml) in HBSS with 1 mM Ca2+, 2 mM Mg2+, and 0.1% gelatin and incubated with 51Cr (24 ,uCi/ml, as sodium chromate, 200-500 Ci/g; New England Nuclear; 1 Ci = 37 GBq) at 37°C for 1 hr with periodic gentle agitation. After incubation, free 5tCr was removed in two washes with HBSS. Labeled neutrophils were resuspended in RPMI 1640 medium (GIBCO) ...

A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced Nformylmethionylleucylphenylalanine(FMLP)-stimulated degranulation ofCytochalasin B pretreated neutrophils and FMLPstimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mol antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocytemacrophage colony stimulating factor can selectively stimulate mature granulocyte function.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) are pleiotropic hemopoietic growth factors whose genes are closely linked and induced in T lymphocytes in a cyclosporin A (CsA)-sensitive fashion. Since we found that the human GM-CSF and IL-3 proximal promoters were not sufficient to account for the observed regulation of these genes, we mapped DNase I hypersensitive sites across the GM-CSF/IL-3 locus in the Jurkat human T-celi line to identify additional regulatory elements.We located an inducible DNase I hypersensitive site, 3 kb upstream of the GM-CSF gene, that functioned as a strong CsA-sensitive enhancer of both the GM-CSF and IL-3 promoters. Binding studies employing Jurkat cell nuclear extracts indicated that four sites within the enhancer associate with the inducible transcription factor AP1. Three of these AP1 elements lie within sequences that also associate with factors resembling the CsA-sensitive, T cell-specific transcription factor NFAT. We provide additional evidence suggesting that an APl-like factor represents one of the components of NFAT. We propose that the intergenic enhancer described here is required for the correctly regulated activation of both GM-CSF and IL-3 gene expression in T cells and that it mediates the CsA sensitivity of the GM-CSF/IL-3 locus.

Sphingosine-1-phosphate (S1P), the bioactive product of sphingosine kinase (SK) activation, is a survival factor for endothelial cells. The mechanism of SK-mediated survival was investigated in endothelial cells with moderately raised intracellular SK activity. Overexpression of SK mediated survival primarily through the activation of the phosphatidyl inositol 3-kinase (PI-3K)/protein kinase B (Akt/PKB) pathway and an associated up-regulation of the antiapoptotic protein B cell lymphoma gene 2 (Bcl-2) and down-regulation of the proapoptotic protein bisindolylmaleimide (Bcl-2 interacting mediator of cell death; Bim). In addition there was an up-regulation and dephosphorylation of the junctional molecule platelet endothelial cell adhesion molecule-1 (PECAM-1), which was obligatory for activation of the PI-3K/Akt pathway, for SK-induced cell survival, and for the changes in the apoptosis-related proteins. Thus, raised intracellular SK activity induced a molecule involved in cell-cell interactions to augment cell survival through a PI-3K/Akt-dependent pathway. This is distinct from the activation of both PI-3K/Akt and mitogen-activated pro- IntroductionEndothelial cell (EC) survival is regulated by cell-matrix and cell-cell interactions and by growth factors. Cell-matrix attachments are mediated by the integrins, and integrin ligation triggers downstream signaling events that inhibit apoptosis or anoikis. 1,2 Two cell-cell adhesion molecules are critical in the control of endothelial cell survival. The junctional molecule platelet endothelial cell adhesion molecule-1 (PECAM-1; CD31), a 130-kDa member of the immunoglobulin-immunoreceptor tyrosine-based inhibitory motif (Ig-ITIM) family of inhibitory receptors, possesses a functional cytoplasmic ITIM domain, is constitutively and abundantly expressed on normal endothelium 3 regulating leukocyte transmigration 4 and acts as a structural protein in cell-cell attachments. PECAM-1 is also able to suppress programmed cell death 5-9 by virtue of homophilic interactions. VE-cadherin, an endothelial-specific major structural protein involved in adherens junctions, is also involved in mediating the antiapoptotic effects of vascular endothelial cell growth factor 10 (VEGF). VEGF and basic fibroblast growth factor (bFGF) prevent EC apoptosis through activation of the phosphatidyl inositol 3-kinase (PI-3K)/protein kinase B (Akt/PKB) pathway and the Raf/MEK/MAPK (relative activity factor/mitogen-induced extracellular kinase/mitogenactivated protein kinase) pathway, both of which are critical pathways promoting cell survival. [11][12][13][14][15][16] Serum is also a recognized trophic factor for ECs since they undergo apoptosis within 24 hours of serum deprivation. 16 The protective factors in serum have been attributed to lysophosphatidic acid, sphingosine-1-phosphate (S1P), [17][18][19] and high-density lipoproteins. 20 The lipid mediator S1P is formed by the phosphorylation of membrane-associated sphingosine by sphingosine kinase (SK). S1P is stored in high concentrations in th...

The human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter binds a sequence-specific single-strand DNA binding protein termed NF-GMb. We previously demonstrated that the NF-GMb binding sites were required for repression of tumor necrosis factor-alpha (TNF-alpha) induction of the proximal GM-CSF promoter sequences in fibroblasts. We now describe the isolation of two different cDNA clones that encode cold shock domain (CSD) proteins with NF-GMb binding characteristics. One is identical to the previously reported CSD protein dbpB and the other is a previously unreported variant of the dbpA CSD factor. This is the first report of CSD factors binding to a cytokine gene. Nuclear NF-GMb and expressed CSD proteins have the same binding specificity for the GM-CSF promoter and other CSD binding sites. We present evidence that CSD factors are components of the nuclear NF-GMb complex. We also demonstrate that overexpression of the CSD proteins leads to complete repression of the proximal GM-CSF promoter containing the NF-GMb/CSD binding sites. Surprisingly, we show that CSD overexpression can also directly repress a region of the promoter which apparently lacks NF-GMb/CSD binding sites. NF-GMb/CSD factors may hence be acting by two different mechanisms. We discuss the potential importance of CSD factors in maintaining strict regulation of the GM-CSF gene.

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified ␤-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of ␤-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including ␤-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.

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Adhesion of blood cells to endothelial cells is an essential component of all inflammatory responses. The capacity of the endothelium to support adhesion of neutrophils is increased by cytokines such as tumor necrosis factor-alpha, interleukin-1, and endotoxin. Another cytokine, transforming growth factor-beta (TGF-beta), was a strong inhibitor of basalneutrophil adhesion and also decreased the adhesive response of endothelial cells to tumor necrosis factor-alpha (TNF-alpha). The ability of cells to respond to TGF-beta was related to the duration of culture of endothelial cells after explantation from umbilical veins. TGF-beta is likely to serve an anti-inflammatory role at sites of blood vessel injury undergoing active endothelial regeneration.