Bacterial cell envelopes (ghosts) and LPS but not bacterial S-layers induce synthesis of immune-mediators in mouse macrophages involving CD14

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Abstract
The synthesis of inflammatory mediators in human macrophages/monocytes seen after stimulation with lipopolysaccharide (LPS) involves the binding of CD14 to LPS complexed to lipopolysaccharide binding protein (LBP). The binding mechanisms of different LPS domains to LBP and CD14, as well as the interaction of the entire bacterial cell wall and its components with CD14 and LBP, are poorly understood. We, therefore, studied the effects of anti-mouse CD14 antibodies on the synthesis of TNFα and PGE2 in RAW 264.7 mouse macrophages stimulated by bacterial cell envelopes (ghosts) of Escherichia coli 026:B6 and Salmonella typhimurium C5, LPS, lipid A, and crystalline bacterial cell surface layer (S-layer) preparations. Ghosts and S-layers, with distinct activities on the immune-system, are presently under investigation for their use as vaccines. Whereas LPS and E. coli ghosts exhibited a strong endotoxic activity in the Limulus amoebocyte lysate assay, the endotoxic activity of S-layer preparations was several orders of magnitude lower. LPS, ghosts, and bacterial S-layers all induced TNFα and PGE2 synthesis as well as the accumulation of TNFα mRNA. Pre-incubation with anti-mouse CD14 antibodies resulted in a dose-dependent inhibition of TNFα and PGE 2 synthesis after stimulation by LPS, lipid A (30-50%) and ghosts (40-70%). The bacterial S-layer-induced mediator synthesis remained unchanged following the addition of anti-mouse CD14 antibodies. Reproducible differences could be observed for the inhibition of TNFα induced by LPS of different species by anti-CD14. Adding fetal calf serum (FCS) strongly enhanced the release of cell mediators stimulated by low doses of LPS and bacterial ghosts. These effects of the FCS may be due to the presence of LBP in the FCS. The results show that CD14 is highly relevant for the activation of mouse macrophages by bacterial cells, LPS, and lipid A. Specially defined bacterial cell wall constituents such as bacterial S-layers might act through other activation pathways.