TYROSINE PHOSPHORYLATION AND INTRACELLULAR ALKALINIZATION ARE EARLY EVENTS IN HUMAN NEUTROPHILS STIMULATED BY TUMOR-NECROSIS-FACTOR, GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR AND GRANULOCYTE COLONY-STIMULATING FACTOR

Tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte CSF (G-CSF) primed human neutrophils for enhanced release of superoxide in time- and dose-dependent manners. The priming effects of these cytokines were detected at 3 min and maximal at 10 min of preincubation. The potency of the maximal effect was TNF > GM-CSF > G-CSF. Exposure of human neutrophils to TNF, GM CSF and G-CSF resulted in tyrosine phosphorylation of a 42-kDa protein and intracellular alkalinization in a dose-dependent manner. The dose-response curves for triggering of tyrosine phosphorylation and intracellular alkalinization by each cytokine were similar to those for priming the cells. The potency of the maximal effect on tyrosine phosphorylation was TNF > GM-CSF > G-CSF, whereas that on intracellular alkalinization was GM-CSF > TNF > G-CSF. Tyrosine phosphorylation was detected at 3 min and maximal at 5-10 min after stimulation with each cytokine. Tyrosine phosphorylation induced by TNF declined at 20-40 min, whereas that induced by GM-CSF or G-CSF was maintained for at least 40 min. Intracellular alkalinization induced by each cytokine required a lag time of 3-5 min and was sustained for at least 40 min. Tyrosine phosphorylation preceded or occurred concomitantly with intracellular alkalinization and priming of the cells. These findings indicate that tyrosine phosphorylation and intracellular alkalinization are early events in human neutrophils stimulated by TNF, GM-CSF and G-CSF, and that these early events may, at least in part, mediate activation or priming of human neutrophils by these cytokines.