Initial kinetics of lysosomal enzyme secretion and superoxide anion generation by human polymorphonuclear leukocytes

Abstract

Human polymorphonuclear leukocytes (PMN) exposed to particulate and soluble stimuli secrete lysosomal enzymes. These stimuli cause prompt (< 10 sec) changes in membrane potential followed 30–45 sec later by superoxide anion (O ₂ ⁻ ) production. We describe a new technique utilizing flow dialysis apparatus which monitors the first stages of lysosomal enzyme release with a resolution of approximately 6 sec. Secretion ofβ-glucuronidase from cytochalasin B-treated PMN could be detected 19±5 sec after exposure to the chemotactic peptideN-formylmethionylleucylphenylalanine (FMLP). The “lag” times for release of this enzyme were different for other stimuli: 35±8 sec (BSA/anti-BSA immune complex); 48±8 sec (serum-treated zymosan, “STZ”); 60±25 sec (calcium ionophore A23187). The lag times for lysozyme release were less dependent upon the stimulus presented (28±16 sec for FMLP, 28±8 sec for BSA/antiBSA, 32±10 sec for STZ, and 38±8 seconds for Con A); only A23187 had a long lag period: 74±27 sec. Lag periods for the onset of O ₂ ⁻ production (measured by the same mathematical criteria) were comparable to those forβ-glucuronidase release: 21±4 sec for FMLP, 43±14 sec for BSA/anti-BSA, 61±7 sec for Con A, and 50±13 sec for A23187. Changes in FMLP dose up to 100-fold affected the magnitudes of O ₂ ⁻ generation andβ-glucuronidase release, but did not alter the time required for the onset of these processes. A variety of agents, such as corticosteroids, colchicine, 2-deoxyglucose, andN-ethyl maleimide, also affected the magnitudes of the responses, but not the lag periods when FMLP was used as the stimulus. When BSA/anti-BSA immune complex was used as the stimulus, 2-deoxyglucose andN-ethyl maleimide increased the lag period for superoxide anion generation, but not for lysosomal enzyme release. This new flow dialysis technique has permitted us to demonstrate that O ₂ ^- production and lysosomal enzyme secretion are concurrent but dissociable processes which are subsequent to earlier responses of the granulocyte to ligandreceptor interactions as reflected by changes in membrane potential.