Granulocytes without degranulation: neutrophil function in granule-depleted cytoplasts.

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Abstract
Neutrophils [human] respond to a variety of stimuli by generating superoxide anion, degranulating and aggregating. Because it has been suggested that fusion of granules with the plasmalemma (degranulation) is necessary for aggregation and superoxide anion generation, whether these responses can be demonstrated in neutrophilic cytoplasts (granule-free vesicles of cytoplasm enclosed by plasmalemma) was tested. When examined by EM, cytoplasts were .apprx. 4 .mu.m in diameter and essentially granule free. Cytoplasts exposed to fMet-Leu-Phe (0.1 .mu.M) generated superoxide anion after a lag of 16 s but released no detectable .beta.-glucuronidase, lysozyme or elastase. Aggregation of cytoplasts, as measured by changes in light transmission, was also activated by fMet-Leu-Phe; no lag period was observed. EM of the aggregates demonstrated clusters of cytoplasts with a scalloped appearance. Superoxide anion generation and aggregation of cytoplasts were also activated by phorbol 12-myristate 13-acetate, concanavalin A and leukotriene B4. Exposure of cytoplasts to the dye 3,3''-dihexyloxacarbocyanine iodide [DiOC6(3)] led to dye uptake and enhancement of fluorescence, implying that the vesicles were sealed and maintained a membrane potential across the plasmalemma. Exposure of DioC6(3)-loaded cytoplasts to fMet-Leu-Phe and PMA [phorbol-12-myristate 13-acetate] caused a rapid loss of dye fluorescence that was not inhibited by CN-, compatible with their lack of mitochondria. Exposure of dye-loaded cytoplasts to concanavalin A or leukotriene B4 caused an increase in fluorescence, i.e., a hyperpolarization. Degranulation is not a prerequisite for aggregation or superoxide anion generation. The retention of ionic gradients and changes in membrane potential, as measured by DiOC6(3) fluorescence changes, suggest a fundamental role for ionic movements in activating superoxide anion generation and aggregation.