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We have studied the transcriptional activation of the human TNF-alpha gene by the superantigen staphylococcal enterotoxin A (SEA) in the human premonocytic cell line THP-1. Nuclear proteins from SEA-stimulated THP-1 cells bound strongly to kappa 3, the most proximal of three putative NF-kappa B binding sites (kappa 1-kappa 3) found in the 5' regulatory region of the TNF-alpha gene, but only weakly to kappa 1, the most distal of the NF-kappa B binding sites, and showed no binding to kappa 2. The mobility of the kappa 3-nucleoprotein complex was identical to that of complexes formed between nuclear proteins and the consensus NF-kappa B seuqence. Moreover, both 5' and 3' mutants of kappa 3 were unable to displace kappa 3 binding, suggesting that the kappa 3 binding complex induced by SEA has the characteristics of NF-kappa B. Studies using Abs directed against the NF-kappa B subunits p50 and p65 suggested that both p50 and p65 bind to the kappa 3 sequence. Reporter gene assays showed that deletion of kappa 3 (-99 to -89 bp) and point mutation of the three 5' guanine bases in the kappa 3 sequence reduced the inducibility of the TNF-alpha promoter by SEA and LPS. These results indicate that superantigen induces NF-kappa B in human monocytic cells and suggest that binding of NF-kappa B to the kappa 3 site of the TNF-alpha promoter plays an important role in the transcriptional activation of the TNF-alpha gene by superantigen.

Staphylococcal exotoxins (SE) bind to MHC class II molecules and induce the production of IL-1 beta and TNF-alpha in human monocytic cells. Here we show that stimulation of peripheral blood monocytes with toxic shock syndrome toxin-1 (TSST-1) induced rapid increase in tyrosine phosphorylation of cytosolic protein substrates, accumulation of inositol phosphates, and de novo tyrosine phosphorylation of the PLC-gamma 1 isozyme. Accumulation of inositol phosphates was inhibited by preincubation of cells with inhibitors of protein tyrosine kinases (PTK). Stimulation of monocytes with TSST-1 furthermore led to activation of protein kinase C (PKC). PTK and PKC activation plays a role in the induction of monokine gene transcription by SE because inhibitors of PTK and PKC reduced TSST-1-stimulated IL-1 beta and TNF-alpha gene expression. We therefore propose a model in which the induction of monokine gene transcription by TSST-1 in monocytes necessitates activation of tyrosine kinase(s) and of PKC, the latter probably by way of PLC-gamma 1.

Microbial superantigens bind to major histocompatibility complex (MHC) class II molecules and activate gene transcription in monocytes. In search of transcription factors that potentially mediate the effects of superantigens at the nuclear level, we examined the capacity of staphylococcal superantigens to stimulate the activity of the transcriptional promoter factor activator protein-1 (AP-1). Electrophoretic mobility shift assays showed an increase in nuclear proteins that bound to the consensus AP-1 motif within 5 min following the stimulation of the monocytic cell line THP-1 with toxic shock syndrome toxin-1 (TSST-1) or staphylococcal endotoxin A. We show that mRNA levels for the subunits that compose AP-1, the protooncogenes c-fos and c-jun, are upregulated by stimulation of THP-1 cells with TSST-1. The activated AP-1 complexes were functional, as evidenced by the capacity of TSST-1 to stimulate the expression of an AP-1-driven reporter gene construct transfected into THP-1 cells. These results establish that the engagement of MHC class II molecules by superantigens increases the activity of functional AP-1 complexes and that this may proceed in part by transcriptional activation of c-fos and c-jun protooncogenes.

Staphylococcal superantigens bind to MHC class II molecules and induce transcriptional activation of IL-1 beta and TNF-alpha genes in human monocytic cells. The understanding of the mechanisms by which superantigens activate cytokine gene expression is incomplete. In this study, we demonstrate that toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A and B induce the activation of NF-kappa B, a transcriptional enhancer that binds to sequences found in both the IL-1 beta and TNF-alpha promoters. Electrophoretic mobility-shift assays showed a rapid induction of nuclear proteins that bound to the consensus kappa B motif. Furthermore, TSST-1 potently stimulated chloramphenicol acetyltransferase (CAT) expression by THP-1 cells transfected with a consensus NF-kappa B-promoter CAT construct, indicative of induction of NF-kappa B enhancer function. Induction of both NF-kappa B DNA-binding proteins and NF-kappa B enhancer function was down-regulated by inhibitors of protein kinase C and protein tyrosine kinase, indicating a role for these protein kinases in the induction of NF-kappa B by MHC class II ligands. Using neutralizing antibodies, we demonstrated that after the stimulation of cells with TSST-1, TNF-alpha, but not IL-1 beta, acted to up-regulate binding of NF-kappa B to DNA and the activation of the NF-kappa B-promoter CAT construct. These results indicate that induction of NF-kappa B by superantigens is up-regulated in part by an autocrine loop involving TNF-alpha.