Priming of the oxidative burst in human neutrophils by physiological agonists or cytochalasin B results from the recruitment of previously non-responsive cells.
Using a sensitive flow cytometric assay, which measures the intracellular oxidation of 2'7' dichlorofluorescein (DCFH) by H2O2, we have assessed, at a single-cell level, the effects of a variety of physiological priming agonists and cytochalasin B (CB) on purified populations of neutrophils stimulated at different points along the signal response transduction pathway. Pretreatment of purified neutrophils with the physiological priming agonists monocyte interleukin-8 (IL-8), granulocyte-monocyte colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, and non-stimulatory doses of formyl-methionyl-leucyl-phenylalanine (FMLP), resulted in an increased percentage of cells generating an oxidative burst in response to subsequent receptor stimulation with FMLP. CB had a similar but much more pronounced effect on cellular recruitment to a receptor-mediated responsive state. Activation of protein kinase C (PKC) using the phorbol ester phorbol myristate acetate (PMA) resulted in a heterogeneous response, with all cells generating H2O2, but with two populations differing in their magnitude of response. Physiological priming agonists had no effect on the heterogeneity of the PMA response. However, pretreatment with CB dramatically altered the PMA response, producing a homogeneous population highly responsive to stimulation with PKC. In contrast, direct stimulation of G proteins with fluoride (A1F-4) was primed both by physiological priming agonists and by CB. These results demonstrate that priming of neutrophils by physiological agonists involves changes at the level of signal transduction which enable a previously non-responsive cell to respond to a secondary stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)
Daniels
R H
RH
Yamanouchi Research Institute (U.K.), Littlemore Hospital, Oxford.
Elmore
M A
MA
Hill
M E
ME
Shimizu
Y
Y
Lackie
J M
JM
Finnen
M J
MJ
D002478:Cells, Cultured; D003571:Cytochalasin B / Q000276:immunology*; D016207:Cytokines / Q000276:immunology*; D005459:Fluorides / Q000276:immunology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology; D006801:Humans; D007378:Interleukins / Q000276:immunology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000276:immunology; D009504:Neutrophils / Q000276:immunology*; D010972:Platelet Activating Factor / Q000276:immunology; D016897:Respiratory Burst / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016207:Cytokines; D007378:Interleukins; D010972:Platelet Activating Factor; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013755:Tetradecanoylphorbol Acetate; D005459:Fluorides