Separation and function of neutrophil karyogranuloplasts and comparison with cytoplasts and intact cells

Abstract

Since neutrophil cytoplasts lacking nucleus and granules were first prepared by centrifuging neutrophils over a discontinuous Ficoll gradient containing cytochalasin B, several functional deficits have been reported in these cytoplasts. Although these functional deficits have been considered to originate from the absence of organelles, cell damage during preparation could not be excluded. Therefore, in the following experiments the Ficoll gradient was modified to isolate both cytoplasts and karyogranuloplasts, which have a nucleus and granules and represent the cell after loss of the cytoplast. Electron microscopy and analysis of marker proteins and cell volume showed that karyogranuloplasts were distinct from neutrophils. The phorbol myristate acetate (PMA) or N-formylmethionylleucylphenylalanine (FMLP)-induced O2- release, corrected by surface area, was in the following order: neutrophils greater than cytoplasts greater than karyogranuloplasts. Both aggregation and membrane potential depolarization were maximal in neutrophils, intermediate in karyogranuloplasts, and lowest in cytoplasts when either PMA or FMLP was used as a stimulant. Extracellular release of the granule contents (degranulation) was triggered by FMLP in both neutrophils and karyogranuloplasts. Cytochalasin B pretreatment greatly enhanced FMLP-induced O2- release, degranulation, aggregation, and depolarization of membrane potential in neutrophils and karyogranuloplasts, but not in cytoplasts. The ability of cytochalasin B to potentiate FMLP-triggered cell function probably depends on granules or cell organelles which are depleted in cytoplasts. Chemokinesis and chemotaxis were impaired in both karyogranuloplasts and cytoplasts. Specific FML[3H]P binding was greater in karyogranuloplasts than in cytoplasts. Cellular actin content, measured by the DNase I inhibition assay, was abundant in cytoplasts and was extremely low in karyogranuloplasts. Karyogranuloplasts retain various neutrophil functions, except for chemotaxis, and provide an important control when studying the role of cell organelles in cytoplast function.