Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms.

ABSTRACT

Local anesthetics have anti-inflammatory effects in vivo and inhibit neutrophil functions in vitro, but how these agents act on neutrophils remains unclear. Phagocytosis and bactericidal activity of neutrophils are enhanced by exposure to bacterial components such as lipopolysaccharide (LPS); this process is termed priming, which for enhanced release of superoxide (O2-) causes mobilization of intracellular granules that contain cytochrome b558, a component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We studied whether local anesthetics affected LPS priming for enhanced release of O2- in response to triggering by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), and we investigated which element in the LPS signaling pathway might be the target of local anesthetics. Neutrophils were incubated with 10 ng/ml LPS and 1% plasma+/-local anesthetics, washed, and triggered with fMLP. Local anesthetics all inhibited LPS priming, and 50% inhibition was at 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine. Local anesthetics inhibited LPS-induced mobilization of specific granules and secretory vesicles. Local anesthetics inhibited LPS-induced up-regulation of cytochrome b558 but not LPS-induced translocation of p47phox. Inhibition of priming by local anesthetics was reversed by washing and incubating for 5 min. Tetracaine alone, but not the other local anesthetics, inhibited LPS activation of p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (kinases in the LPS signaling pathway). The p38 MAPK inhibitors SB203580 and PD169316 also blocked LPS priming. Thus, tetracaine and the other local anesthetics inhibit by disparate mechanisms, but all the local anesthetics impaired up-regulation of cytochrome b558 and all impaired priming of NADPH oxidase by LPS.