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 activatio n 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 stim ulation and by simultaneous assays for CD11b expression and morphology . The maximal CD11b expression was higher with PMA than with fMLP, sug gesting different activation pathways. Under these experimental condit ions, a morphological response to fMLP was not observed. However, sign ificant shape change was detected in PMA treated samples and was suppr essed by either the removal of extracellular calcium or staurosporine at the concentrations above 14.5 mu M. Calcium ionophore induced a sim ilar light scattering pattern to that by PMA and enhanced CD11b expres sion, both of which were not inhibitable by staurosporine. These obser vations, for the first time, indicated that Ca2+ was a mediator in act ivation processes and that the treatment of PMN with PMA resulted in C a2+ influx.