Nitric Oxide and Prostaglandin E2 Regulation in Neonatal Porcine Cerebral Endothelial Cells Infected by Group B Streptococcus (GBS) † 1444

GBS-induced meningitis impairs cerebral blood flow and triggers inflammatory mediators in newborn piglets. We examined 1) the regulation at a molecular level of inflammatory biochemical mediators induced by GBS meningitis: Nitric Oxide (NO) and its synthases (bNOS, ecNOS and iNOS), Prostaglandin E2 (PGE2) and the cyclooxygenases (COX-1 and COX-2) and 2) the role of endothelial cells (EC) in the inflammatory cascade initiated by GBS, in the primary culture of piglet brain microvessel endothelial cells (EC). Treatment of EC (n=4) with vehicle (saline) and GBS (10⁵ & 10⁹ cfu) over a 24 hr time course showed a significant dose response relationship. GBS 10⁵ increased PGE2 levels (ELISA) compared to control by 20 fold (p=.0067 r=0.9324) and 10⁹ by 13 fold (p=0.0072 r=0.9299), at 4 hrs. No significant differences were noted in the control and 10⁹ cfu GBS treated groups however a significant difference was noted in 10⁵ cfu GBS treatment (p<0.0001 r=0.9579) (ANOVA). Gene regulation of COX1, COX2, ecNOS and bNOS by GBS was analyzed by determining the protein and mRNA levels of GBS treated EC. Western blot protein analysis showed that GBS 10⁵ cfu increased levels of COX1, COX2 and bNOS (2.5, 4.3 and 3.3 fold respectively) after 1-4 hrs but returned to basal level after 24 hrs. mRNA levels for COX1, COX2, bNOS and ecNOS were determined using a semi-quantitative rtPCR assay and Northern blot analysis. Induction of COX2, bNOS and ecNOS mRNA by GBS confirmed our protein data and suggest that GBS affected transcription of the major PG-synthase and NO-synthase genes. In addition, we have tested cytotoxicity, cell viability and the appearance of apoptosis (programmed cell death) in endothelial cells exposed to different doses of GBS (10⁵, 10⁷ and 10⁹ cfu). GBS doses greater than 10⁷ cfu were toxic to brain EC in culture and decreased cell viability by 50% after 72 hrs. GBS did not induce apoptosis in EC (analyzed by DNA fragmentation assay) suggesting low susceptibility of brain endothelial cells to GBS induced apoptosis. Our data suggest that GBS deregulates gene expression for the major and interacting inflammatory mediators in brain microvascular endothelial cells. Increased synthesis of these inflammatory mediators may result in brain and cellular injury during GBS infection in newborn babies.