Regulation of superoxide anion generation in bovine alveolar macrophages by bacterial lipopolysaccharide, serum proteins, and modulators of signal transduction

Abstract

The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbicidal and potentially damaging to host tissues. We investigated regulation of the respiratory burst of alveolar macrophages in response to lipopolysaccharide (LPS) derived from gram-negative bacteria, serum proteins, and several modulators of signal transduction. When employed as a single stimulus, LPS (E. coli 055:B5, 10 ng/ml-1 microgram/ml) was a weak stimulus for generation of superoxide anion (O2-) as compared to the potent effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; 500 ng/ml). However, when LPS was combined with fetal bovine serum (FBS; 0.4-1.0% vol/vol, equivalent to 128-320 micrograms protein/ml), O2- generation was enhanced approximately two-fold over LPS alone. A chromatographically-derived bovine serum fraction which contained bovine lipopolysaccharide-binding protein (bLBP; 0.25-1.0 microgram/ml) was an effective substitute for FBS at a much lower protein concentration than whole FBS, and a similar synergistic effect with LPS on O2- generation was observed. Stimulation of macrophages for generation of O2- either with LPS alone or with LPS plus serum/serum fraction was suppressed by the protein tyrosine kinase inhibitor heribimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12 microM), but not by modulators of G-proteins, including pertussis toxin (10 ng/ml) and cholera toxin (5 micrograms/ml protein). Essentially complete inhibition of O2- synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1 microgram/ml protein), but only partial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256 micrograms/ml protein). These results indicate that when LPS is used as a sole stimulus it induces modest respiratory burst activity. However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amounts of O2-. Cellular signaling pathways important in stimulation of macrophages by LPS and serum components are protein tyrosine kinase- and Ca(++)-dependent, but do not relay on G-protein-mediated signaling.