Gram-Positive and Gram-Negative Bacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways

Abstract

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) andStaphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1␣ (IL-1␣), IL-1ra, and tumor necrosis factor alpha (TNF-␣) production, whereas blocking of the ERK1/2 pathways inhibited IL-1␣, IL-1␤, and IL-1ra but not TNF-␣ production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1␣ production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.Invasive infection by gram-positive and gram-negative bacteria in humans results in septic shock and death. The early cellular response to both groups of microorganisms is still unclear. Several investigators have proposed that lipopolysaccharides (LPS) from gram-negative bacteria, as well as several components from gram-positive bacteria, trigger monocytic cytokine production following interaction with membrane-bound CD14 (mCD14) and activation of Toll-like receptors (TLRs; the human homologues of Drosophila Toll) which are present on the cell membrane (23,30,43,61). Several lines of evidence suggest that after binding to mC14, LPS initiates intracellular signaling pathways activating a number of tyrosine kinases as an initial step. Various ␣ subunits of heterodimeric G proteins and Src kinases, physically associated with membrane-bound CD14 molecules in LPS-stimulated normal human monocytes, induce p38 mitogen-activated protein (MAP) kinase activation, which is involved in cytokine synthesis (44). Other proteins that are phosphorylated upon LPS stimulation include p42 and p44 MAP kinases, which are encoded by the erk2 and erk1 genes, respectively (10, 25). However, the upstream events that occur during ERK1, ERK2, and p38 kinase phosphorylation remain unclear. There is some evidence that LPS activates Ras and consequently Raf, resulting in MEK-1 and MAP kinase phosphorylation, but the precise pathways are not fully understood (8). In addition to MEK and MAP kinase activation, Syk molecules are phosphorylated upon LPS stimulation of macrophages. However, several studies have indicated that neither Syk, Src family tyrosine kinases, Hck, Lyn, nor Fgr is indispe...

Toll-Like Receptor (TLR)2 and TLR4 Agonists Regulate CCR Expression in Human Monocytic Cells

“…TLR2 and TLR4 agonists cause the differential expression of a growing number of genes including IFN-␤, IL-6, IL-12, IFN-␥, monocyte chemoattractant protein (MCP)-5, and macrophage-inflammatory protein (MIP)-2 in murine macrophages (22) and induce distinct patterns of cytokine production in RAW 264.7 macrophages (23)(24)(25), DCs (19), and mast cells (20). In keeping with these observations, stimulation with TLR2 and TLR4 agonists cause similar, but not identical, activation of MAPK (19,24,26), whereas treatment with specific kinase inhibitors indicates that p38 activation is involved in TLR4, but not TLR2, signaling (27).…”

“…Again, conflicting data have been published regarding the effects of MAPK inhibitors following TLR stimulation. For example, blocking the p38 pathway with SB203580 before TLR4 stimulation decreased cytokine production from human monocytes, but had no effect on cytokine production elicited by TLR2 activation, leading the authors to suggest that TLR2 signaling does not involve the p38 signaling pathway (27). However, a second group found that SB203580 partially inhibits TLR2-and TLR4-induced secretory IL-1R antagonist gene expression in RAW 264.7 cells (25).…”

Response of monocytes exposed to phagocytosable particles and discs of comparable surface roughness

“…LPS stimulation of THP-1 cells induced activated/differentiated cells, characterized by thin and elongated bodies that appeared to be attached to the surface of the culture wells (Figure 3e-h). Such morphological changes are indicative of monocyte activation via Toll-Like-Receptor by LPS [14], lending a visual analog to the cytokine expression data seen in Figures 5.…”

Role of the Phosphatidylinositol 3 Kinase-Akt Pathway in the Regulation of IL-10 and IL-12 byPorphyromonas gingivalisLipopolysaccharide

“…‫,ء‬ Significant differences (p Ͻ 0.05) compared with P. gingivalis LPS-stimulated cultures. mouse or human monocytes/macrophages (24,(67)(68)(69)(70)(71). To determine whether the ability of P. gingivalis LPS to activate the PI3K-Akt pathway played a functional role in the regulation of cytokine production, we next examined the role of PI3K in modulating P. gingivalis LPS-induced IL-12 p35, IL-12 p40, IL-12 p70, and IL-10 production in human monocytes.…”