LPS directly induces oxygen radical production in human monocytes via LPS binding protein and CD14

In human monocytes, superoxide (O 2 ‐ ) generation accompanies phagocytosis and is important for bactericidal activity. It also contributes to tissue damage in inflammation. In the present study we investigated, whether lipopolysaccharide (LPS) directly stimulates monocyte O 2 ‐ production with kinetics known for other LPS effects and, if so, by which mechanism. LPS caused a time‐ and dose‐dependent O 2 ‐ release in nonadherent purified monocytes. The effect appeared after 5 min, peaked at 30 min, and disappeared after 2 h. It was maximal with 10 ng/ml lipid A (+148 ± 22%, P < .001), 1 ng/ml LPS Escherichia coli Re (+226 ± 68%, P < .001), and 100 ng/ml LPS Salmonella abortus equi sm (+272 ± 52%, P < .001), respectively. The effect was not observed in buffer, even when using 10 ü g/ml LPS. It was dependent on the presence of heat‐inactivated AB serum, with a maximal effect at ≥;0.5%. Serum could be replaced by LPS‐binding protein (LBP). Polymyxin B and anti‐LBP antiserum, respectively, blocked the LPS effect. LPS‐induced O 2 ‐ generation was also completely blocked by anti‐CD14 antibodies (3C10 and 63D3) and by their corresponding F(ab') 2 fragments. Monocytes treated with phosphoinositol‐specific phospholipase C and monocytes from patients with paroxysmal nocturnal hemoglobinuria, lacking the phosphatidylinositol‐anchored CD14, did not respond to LPS stimulation with O 2 ‐ production. Similarly to LPS, E. coli caused stronger O 2 ‐ production with heat‐inactivated serum than without, and this effect was blocked by anti‐CD14 antibodies. In conclusion, these data indicate that LPS directly stimulates O 2 ‐ production in human monocytes via CD14 depending on LBP. J. Leukoc. Biol . 57: 440–449; 1995.