It is believed that tumor necrosis factor-alpha (TNF-α) plays an important role in the evolution of neonatal sepsis caused by group B Streptococcus (GBS). We previously have shown that stimulation of umbilical cord blood monocytes (C-MC) by GBS or its cell components induces TNF-α secretion via a CD14-independent pathway. However, the cell signaling pathways responsible for GBS-induced TNF-α secretion are not known. Since both GBS and LPS elicited quantitatively similar TNF-α patterns in C-MC, we investigated the effect of GBS on the activation of nuclear factor (NF)-κB and the transcriptional activation of the TNF-α gene upon infection of C-MC. Cord blood was collected immediately on delivery of the placenta during uncomplicated scheduled C-section. C-MC(107/ml) were stimulated with serotype III GBS (108 cfu/ml) for various time periods. Total RNA was isolated after stimulation (0, 30, 60, 240 min) and TNF-α mRNA was analyzed by Northern blot. Nuclear extracts (0, 30, and 60 min) were prepared and analyzed by electromobility shift assay. We observed upregulation of TNF-α mRNA and TNF-α release within 1hr after infection of C-MC with GBS. Furthermore, GBS induced a NF-κB DNA-protein complex in a time-dependent manner with enhanced binding of nuclear proteins observed in C-MC within 30 min of infection. The protein-DNA complexes were specifically competed by excess nonlabeled self-oligonucleotide confirming the specificity of the reactions. In preliminary studies, GBS peptidoglycan induced NF-κB activation in a dose-dependent manner. Inactivation of NF-κB by the protease inhibitor N-tosyl-1-phenylalanine chloromethyl ketone dramatically inhibited GBS-induced TNF-α mRNA expression. Thus, the activation of the immediate early factor NF-κB is sufficient for the transcriptional activation of the TNF-α gene in response to infection with GBS. We conclude that although GBS and LPS initiate monocyte activation via different cell surface events, the signaling pathways may ultimately converge to produce similar cellular responses. A better understanding of the molecular mechanisms involved in GBS- and other gram positive organism-induced cytokine production may lead to novel and perhaps more effective therapies for neonatal sepsis.
