Lipopolysaccharide-induced protein tyrosine phosphorylation in human macrophages is mediated by CD14.

Abstract

Induced protein tyrosine phosphorylation is an early intracellular event in LPS-stimulated murine macrophages that appears to play a role in signal transduction. We have now demonstrated that LPS also increases protein tyrosine phosphorylation in human monocyte-derived macrophages and in the human monocytic cell line, THP-1. This response was rapidly elicited by biologically active forms of LPS or lipid A, at concentrations of these bacterial components that stimulate anti-bacterial responses by human macrophages. Inhibition of the LPS-induced tyrosine phosphorylation response with the tyrosine kinase inhibitor, herbimycin A, was accompanied by the inhibition of the secretion of TNF-alpha by human macrophages. These results extend previous work with murine macrophages and provide further support for the hypothesis that induced protein tyrosine phosphorylation is an important signaling reaction in macrophages after LPS exposure. In addition, CD14, which is thought to be a receptor for LPS, appeared to mediate the induced phosphorylation response in human macrophages and THP-1 cells at low LPS concentrations. Two antibodies against CD14, 3C10 and 60b, which have been shown to prevent LPS binding to CD14, specifically inhibited the protein tyrosine phosphorylation induced by nanogram per milliliter concentrations of LPS in these cells. The antibody-mediated inhibition did not appear to involve engagement of surface FcR because a preparation of F(ab')2/Fab fragments of the 60b antibody also prevented LPS-induced tyrosine phosphorylation. At higher concentrations of LPS (> or = 10 ng/ml), however, anti-CD14 antibodies did not prevent the induction of protein tyrosine phosphorylation, suggesting that a lower affinity CD14-independent pathway also mediates the tyrosine phosphorylation response. Because CD14-dependent and CD14-independent recognition of LPS appear to lead to the same functional responses by macrophages, induced protein tyrosine phosphorylation may be part of a shared intracellular signaling pathway.