Simultaneous recording of calcium transients and reactive oxygen intermediates of human polymorphonuclear granulocytes in response to formyl-Met-Leu-Phe and the environmental agent sulfite

Background: Human polymorphonuclear granulocytes (PMN) are an essential com ponent in the immunological defense network against a variety of harmful pa thogens. We have studied the effects of the airborne pollutant sulfite on t he calcium metabolism and respiratory burst of these cells simultaneously. Methods: A flow cytometric method was developed using the fluochromes Indo- 1 and DHR-123. This method allowed us to investigate the real-time kinetics of intracellular free calcium and reactive oxygen intermediates in viable cells with a temporal resolution of 1 s over a time course of 17 min. An ad ditional feature was the possibility to discriminate between reacting and n onreacting cells after treatment with defined stimuli, thus gaining additio nal insight into the behavior of cell subpopulations. Results: We analyzed the effects of sulfite on PMN before and after stimula tion with formyl-Met-Leu-Phe (FMLP). Treatment with sulfite alone (0.001-1 mM) caused a small, nontransient increase in intracellular calcium. Preincu bation with sulfite reduced the maximal calcium response elicited by FMLP. A significant increase in steady-state calcium levels after stimulation wit h FMLP was observed after treatment with sulfite in concentrations of 10 an d 100 mM. Regarding the respiratory burst, treatment with sulfite alone in concentrations of 0.001-1 mM induced a significant increase in DHR-123-deri ved fluorescence, whereas concentrations of 5 and 10 mM caused a significan t depression of this fluorescence below baseline values. Sulfite caused a m aximal twofold increase of DHR-123-derived fluorescence compared with the F MLP response. Similar results were obtained after preincubation with sulfit e before treatment with FMLP, showing that the effect of sulfite on the res piratory burst was additive to the FMLP response. Regarding the fractions o f responding cells, treatment with sulfite up to 1 mM induced a concentrati on-dependent increase of burst-reactive PMN, whereas preincubation before s timulation with FMLP showed no correlation between sulfite concentration an d fraction of burst-reacting cells. Conclusions: By simultaneous registration of [Ca2+](i) and [H2O2](i) of PMN after treatment with FMLP and sulfite, the essential responses were alread y observed within a short time interval(15 min). Striking differences were found in the response of calcium as second messenger and respiratory burst in PMN treated with sulfite. Until a critical concentration (0.5-1 mM), sul fite caused a concentration-dependent increase of [H2O2](i), in addition to the FMTS-induced response. The [Ca2+](i) changes induced by sulfite alone, however, were found to be small and showed no correlation with the respira tory burst response. (C) 2000 Wiley-Liss, Inc.