DOSE EFFECTS OF LPS ON NEUTROPHILS IN A WHOLE-BLOOD FLOW CYTOMETRIC ASSAY OF PHAGOCYTOSIS AND OXIDATIVE BURST

Whole blood phagocytosis (P) and oxidative burst (OB), a rapid and sensitive flow cytometric method for quantifying neutrophil activation, was modified for single laser systems by using propidium iodide labeled Staphylococcus aureus and 2',7' dichlorofluorescein diacetate. The purpose of the present study was to characterize this assay with respect to the stimulatory activity of bacterial lipopolysaccharide (LPS) on phagocytosis. Blood from healthy donors was preincubated with log doses of bacterial LPS B (0.1-1,000 ng/ml) or sterile pyrogen-free saline at 37-degrees-C from 0-120 minutes. LPS increased both P and OB in a dose-dependent manner (up to 62 and 121%, respectively) at all time Points tested, and this effect on P and OB could be detected even with no preincubation. This LPS-induced phagocytic activity could be blocked by the addition of polymyxin B (10-mu-g/ml) during preincubation. The priming effect of LPS was maximal at 45 min. P and OB were inhibited by preincubation with EDTA at doses > 2 mM (60 and 80% inhibition, respectively). These observations are consistent with the exquisite sensitivity of the neutrophil to endotoxin. This method can evaluate neutrophil response to immunomodulatory and chemotherapeutic agents in a physiological milieu. These findings re-emphasize the necessity of using pyrogen-free reagents in any study of neutrophil function.