Flow cytometric investigation of neutrophil activation pathways by n-formyl-Met-Leu-Phe and phorbol myristate acetate.

Recent evidence suggests that multiple pathways exist in PMN activation and that specific leukocyte response may be due to the activation of a particular signaling pathway. Using flow cytometry, PMN activation pathways were studied through the parallel comparison of n-formyl-Met-Leu-Phe (fMLP)- and phorbol-12-myristate-13-acetate (PMA)-induced stimulation and by simultaneous assays for CD11b expression and morphology. The maximal CD11b expression was higher with PMA than with fMLP, suggesting different activation pathways. Under these experimental conditions, a morphological response to fMLP was not observed. However, significant shape change was detected in PMA treated samples and was suppressed by either the removal of extracellular calcium or staurosporine at the concentrations above 14.5 microM. Calcium ionophore induced a similar light scattering pattern to that by PMA and enhanced CD11b expression, both of which were not inhibitable by staurosporine. These observations, for the first time, indicated that Ca2+ was a mediator in activation processes and that the treatment of PMN with PMA resulted in Ca2+ influx.