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 mu g/ml LPS. It was dependent on the presence of heat-inactivated AB serum, with a maximal effect at greater than or equal to 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.