Involvement of G(i) proteins and Src tyrosine kinase in TNF alpha production induced by lipopolysaccharide, group B Streptococci and Staphylococcus aureus

Previous studies have suggested that heterotrimeric Gi proteins, Src tyrosine kinase and phosphatidylinositol-3 kinase (PI3 Kinase) are involved in signaling events induced by lipopolysaccharide (LPS) leading to pro-inflammatory cytokines gene expression. To investigate the involvement of these mediators in Gram-positive bacteria induced pro-inflammatory cytokine expression, LPS (10 ng/ml), heat killed group B Streptococci (GBS 1 lg/ml) and Staphylococcus aureus (SA 10 lg/ml) were used to induce TNFa production in the murine J774A.1 macrophage (MØ) cell line and human promonocytic THP-1 cell line. Pertussis toxin (PTx, 1 lg/ml), an inhibitor of Gi protein; pyrazolopyrimidine-2 (PP2, 1 or 25 lM), a Src tyrosine kinase inhibitor; and LY294002 (100 nM), an inhibitor of PI3 Kinase were used to examine the involvement of Gi, Src tyrosine kinase and PI3 Kinase, respectively, in TNFa production. In J774A.1 cells, pretreatment with PTx and PP2 attenuated TNFa production induced by LPS (60 9% and 81 11% inhibition, n 1⁄4 3, p < 0:05, respectively), GBS (95 1% and 80 6% inhibition, n 1⁄4 3, p < 0:05, respectively) and SA (51 18% and 68 16% inhibition, n 1⁄4 4, p < 0:05, respectively). However, pretreatment with LY 294002 inhibited LPS induced TNFa production (82 13% inhibition, n 1⁄4 3, p < 0:05), but did not inhibit GBS or SA induced TNFa production. In THP-1 cells, pretreatment with PTx, PP2 and LY 294002 inhibited TNFa production induced by LPS (84 3%, 59 12% and 84 4% inhibition, n 1⁄4 3, p < 0:05, respectively) and SA (56 7%, 87 1% and 35 6% inhibition, n 1⁄4 3, p < 0:05, respectively). These data support our hypothesis that Gi-coupled and Src tyrosine kinase-coupled signaling pathways are involved in both Gram-negative and Gram-positive bacteria induced pro-inflammatory cytokine expression. However, unlike LPS, involvement of PI3 Kinase in Gram-positive bacteria induced signaling pathways are species dependent. 2003 Elsevier Ltd. All rights reserved.