Lipopolysaccharide priming of human neutrophils for an enhanced respiratory burst. Role of intracellular free calcium.
Lipopolysaccharide (LPS) pretreatment "primes" neutrophils to release increased amounts of superoxide anion (O2-) when stimulated. We investigated the molecular basis of this enhanced activity. Comparison of kinetic parameters of the respiratory burst NADPH oxidase in unstimulated LPS-primed and control neutrophils disclosed a similar Km for NADPH and no difference was seen in the content of cytochrome b. Pertussis toxin, which inhibits some G proteins, did not prevent priming. Change in membrane potential (delta psi) was five-fold greater in LPS-primed cells and paralleled the increased O2- release. Cytofluorographic analysis indicated that the increased change in delta psi was due to the creation of a new population of active cells. Changes in the concentration of intracellular free Ca2+ ([Ca2+]i) are believed to antecede changes in delta psi. There was a consistent increment (67 +/- 8%, n = 12) in resting [Ca2+]i in cells preincubated with LPS compared with control. When stimulated, the peak [Ca2+]i was significantly higher in LPS-primed cells. Ca2+-dependent protein kinase C activity was unaltered in resting and FMLP-stimulated neutrophils preexposed to LPS. Addition to cells of the intracellular Ca2+ chelator MAPTAM before preincubation with LPS blocked the changes in [Ca2+]i and the enhanced respiratory burst that characterize LPS priming. The increased resting [Ca2+]i in LPS-primed cells may enhance stimulus-induced cellular activity by modifying a Ca2+-dependent step in signal transduction.
Forehand
J R
JR
Department of Pediatrics, University of Pennsylvania School of Medicine, Children's Hospital of Philadelphia, Philadelphia 19104.
Pabst
M J
MJ
Phillips
W A
WA
Johnston
R B
RB
D002118:Calcium / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008564:Membrane Potentials; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects*; D010101:Oxygen Consumption / Q000187:drug effects*; D037342:Pertussis Toxin; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D003574:Cytochrome c Group; D008070:Lipopolysaccharides; D010566:Virulence Factors, Bordetella; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D037342:Pertussis Toxin; D002118:Calcium