Human neutrophil heterogeneity identified using flow microfluorometry to monitor membrane potential

Abstract

Previous studies of neutrophil nitroblue tetrazolium dye reduction in response to endotoxin and rosetting of IgG-coated erythrocytes have suggested functional heterogeneity of peripheral blood neutrophils. In the following study we utilized flow microfluorometry and the membrane potential-sensitive fluorescent dye 3-3'-dipentyloxacarbocyanine to assess the heterogeneity of neutrophils upon activation by a variety of stimuli. Unstimulated neutrophils from normal subjects exhibited a unimodal distribution of fluorescence, suggesting that all the cells possessed the same resting membrane potential. As neutrophils aged (>5 hr), some cells lost fluorescence producing a bimodal distribution. In studies with fresh cells, the secretagogue phorbol myristate acetate (20 ng/ml) stimulated a uniform loss of fluorescence (apparent depolarization). The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) (0.1 μM) caused the neutrophils to assume and maintain (for >30 min) a bimodal fluorescence distribution in which 65 ± 5% of the neutrophils first decreased and then increased fluorescence (apparent depolarization/partial repolarization), and 35 ± 5% of the cells exhibited either an increase in fluorescence (apparent hyperpolarization) or no change. Treatment of neutrophils with cytochalasin B before stimulation caused the cells to respond homogeneously to f-Met-Leu-Phe. Additional studies using neutrophils from patients with chronic granulomatous disease, which exhibit abnormal membrane potential responses, indicated that this defect affected all such neutrophils uniformly. These observations demonstrate the need to investigate the neutrophil function and indicate that the f-Met-Leu-Phe-induced changes in membrane potential observed in bulk population cell studies are the summation of two different responses.