?Fig

?Fig.5, treatment5, treatment of cells with PKC inhibitors resulted in a substantial reduction in TNF- secretion, and the inhibitory effect occurred in a dose-dependent manner. but not in monocytes. Pretreatment of cells with inhibitors of signal transduction pathways was employed to further define events in SEB-induced TNF- production. Neither protein kinase A inhibitors nor two protein tyrosine kinase inhibitors altered SEB-induced TNF- production. In contrast, SEB induced protein kinase C (PKC) BAF312 (Siponimod) translocation, and pretreatment of cultures with inhibitors of PKC blocked TNF- induction. Alteration of levels of diacylglycerol (DAG), an activator of PKC, by treatment with inhibitors of phospholipase C or DAG kinase also altered SEB-induced TNF- production. These data suggest that PKC activation plays a critical role in SEB-induced TNF- production in human T cells. Originally characterized for their ability to induce the emesis and diarrhea associated with food poisoning (5), staphylococcal enterotoxins (SEs) also exhibit biological activities that can lead to lethal shock (29, 39). SEs constitute a group of nine serologically distinct (types A to BAF312 (Siponimod) E and G to J) proteins that have sequence and structural homologies and are members of the functionally related family of pyrogenic exotoxins (8) that includes streptococcal pyrogenic exotoxin and toxic shock syndrome toxin 1 (TSST-1). These toxins function as superantigens (29), exhibiting the ability to activate large numbers of T cells. This property is a result of the toxin’s bifunctional conversation with both the major histocompatibility complex (MHC) class II receptors on antigen-presenting cells such as monocytes and the T-cell receptor of T lymphocytes expressing specific V chains to which an individual toxin binds (22). For several of the toxins, including staphylococcal enterotoxin B (SEB), the structural domains and amino Rabbit Polyclonal to PWWP2B acid residues participating in these receptor interactions have been identified and three-dimensional structural analyses of the binding of toxin to the MHC class II receptor and T-cell receptor have been described elsewhere (19, 23, 25). Binding of cell surface receptors leads BAF312 (Siponimod) to activation of gene expression through enlistment of signal transduction pathways. These pathways consist of a cascade of biochemical events that can include activation of a variety of kinases including protein tyrosine kinases (PTKs), protein kinase C (PKC), or protein kinase A (PKA). These kinases in turn modify other factors that control individual gene expression. One or more of these kinases may participate in controlling a gene’s expression. Ligand engagement of MHC class II receptors and T-cell receptors activates such signal transduction events (9, 18). The superantigen activity of SEs results in induction of T-cell proliferation and in synthesis of a variety of cytokines including interleukin-1 (IL-1), IL-2, IL-6, gamma interferon, and tumor necrosis factor alpha (TNF-) (24). It is the massive release of such cytokines that is thought to contribute to the immune dysfunction characteristic of superantigen toxicity including lethal shock (29). TNF- is an important cofactor in endotoxic shock (13). It mediates SEB-induced lethality in mouse models that involve both MHC class II and T-cell interactions (28, 33, 46). TNF- induced by superantigen can be produced by both monocytes and T cells (1, 15, 30). Previous studies have examined the induction of TNF- by SEA, SEB, or TSST-1 (15, 30, 38, 42, 43). In this study, we wished to characterize the induction of TNF- by SEB in mixed cultures of human monocytes in the presence of lymphocytes. We wanted to determine which cell types produce TNF- under these culture conditions and which signal transduction pathways are involved. In order to examine the induction of TNF- by SEB, we have employed receptor-binding mutants of SEB, immunodetection and FACScan analysis of TNF–producing cells, and inhibitors of signal transduction pathways. MATERIALS AND METHODS Reagents. SEB, lot 14-30, was BAF312 (Siponimod) obtained from the U.S. Army Research Institute of Infectious Diseases, Frederick, Md. SEB mutants F44R and N23F were constructed by site-directed mutagenesis and purified as described previously (35). The inhibitors genistein, H7, sphingosine, chelerythrine chloride, HA1004, H89, U73122, “type”:”entrez-nucleotide”,”attrs”:”text”:”R59949″,”term_id”:”830644″,”term_text”:”R59949″R59949, and tyrophostin 23 were purchased from Biomol (Plymouth Getting together with, Pa.). Phorbol 12-myristate 13-acetate (PMA) and the inhibitor tyrophostin AG1288 were obtained from Calbiochem (La Jolla, Calif.). Fluorescein isothiocyanate (FITC)-mouse immunoglobulin G1, CD3(Leu-4)-phycoerythrin (PE), and CD14(Leu-M3)-PE were from Becton Dickinson (Mansfield, Mass.). FITCCanti-human TNF- monoclonal antibody (MAb) and monensin were from PharMingen (San Diego, Calif.). Preparation of human monocytes and lymphocytes. Peripheral blood mononuclear cells were prepared from leukopacks from normal donors by centrifugation over lymphocyte separation medium as described previously (21). Monocytes and lymphocytes were then further purified from these preparations by counterflow centrifugation-elutriation with pyrogen-free Ca2+- and Mg2+-free phosphate-buffered saline as the eluant. This method resulted in cell preparations made up of 95 to 99% of the indicated cell type with viability greater than 95%. Unless indicated otherwise, monocytes and lymphocytes were mixed in a 1:4 ratio prior to use. That ratio had been found in.