Use of lipophilic probes of membrane potential to assess human neutrophil activation. Abnormality in chronic granulomatous disease

Abstract

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O2-) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H.M., and G. Weissmann. 1978. Proc. Natl. Acad. Sci. U.S.A. 75: 3818-3822). To further define the interelationship of these probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethyl-phosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O2- production (chronic granulomatous disease [GGD]), and from patients with defective degranulation and/or chemotaxis (Chediak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formly-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore r467w1.w2 A23187 and phorbol myristate actate (PMA). The results obtained with 3'3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with GGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells to f-Met-Leu-Phe and PMA could not be attributed to the absence of O2-, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.