REGULATION OF SUPEROXIDE ANION GENERATION IN BOVINE ALVEOLAR MACROPHAGES BY BACTERIAL LIPOPOLYSACCHARIDE, SERUM-PROTEINS, AND MODULATORS OFSIGNAL-TRANSDUCTION

The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbici dal and potentially damaging to host tissues. We investigated regulati on of the respiratory burst of alveolar macrophages in response to lip opolysaccharide (LPS) derived from gramnegative bacteria, serum protei ns, and several modulators of signal transduction. When employed as a single stimulus, LPS (E. coli 055:B5, 10 ng/ml-1 mu g/ml) was a weak s timulus for generation of superoxide anion (O-2(-)) 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 feta l bovine serum (FBS; 0.4-1.0% vol/vol, equivalent to 128-320 mu g prot ein/ml), O-2(-) generation was enhanced approximately two-fold over LP S alone. A chromatographically-derived bovine serum fraction which con tained bovine lipopolysaccharide-binding protein (bLBP; 0.25-1.0 mu g/ ml) was an effective substitute for FBS at a much lower protein concen tration than whole FBS, and a similar synergistic effect with LPS on O -2(-) generation was observed. Stimulation of macrophages for generati on of O-2(-) either with LPS alone or with LPS plus serum/serum fracti on was suppressed by the protein tyrosine kinase inhibitor herbimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12 mu M), but not by mo dulators of G-proteins, including pertussis toxin (10 ng/ml) and chole ra toxin (5 mu g/ml protein). Essentially complete inhibition of O-2(- ) synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1 mu g/ml protein), but only p artial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256 mu g/ml protein). These results indicate that when LPS is used as a sole stimulus it induces modest respirator y burst activity. However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amo unts of O-2(-). Cellular signaling pathways important in stimulation o f macrophages by LPS and serum components are protein tyrosine kinase- and Ca++-dependent, but do not relay on G-protein-mediated signaling.