Cytochrome b co-fractionates with gelatinase-containing granules in human neutrophils

Abstract

Subcellular fractionation studies in resting human neutrophils indicated a bimodal distribution for cytochrome b. A major peak of cytochrome b co-sedimented with gelatinase under different experimental conditions. This localization was partially overlapped with specific granules (using lysozyme and lactoferrin as specific granule markers), but clearly resolved from azurophilic granules, plasma membrane, mitochondria, as well as from a novel alkaline phosphatase-rich intracellular organelle. A minor localization of cytochrome b was found in fractions enriched in both the plasma membrane marker 5'-nucleotidase and alkaline phosphatase. A significant portion of ubiquinone cell content co-fractionated with the gelatinase-containing granules. After phorbol myristate acetate (PMA)-cell stimulation, cytochrome b was mobilized to fractions showing respiratory burst activity and enriched in 5'-nucleotidase activity. This mobilization paralleled secretion of gelatinase and lysozyme to the extracellular medium. Furthermore, neutrophil stimulation with fluoride in the absence of cytochalasin B induced release of gelatinase and generation of superoxide anion with only minimal release of lysozyme. Preincubation of cells with the anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) prevented lysozyme release, but had only a minor effect on the release of gelatinase and did not inhibit the superoxide anion generation elicited by N-formyl-methionyl-leucyl-phenylalanine or PMA. These results suggest a main location of cytochrome b in mobilizable gelatinase-containing granules, which can constitute a subpopulation of specific granules. Furthermore, these findings show that the gelatinase-containing granule is functionally involved in the respiratory burst in neutrophils and that membrane fusion between plasma membrane and the gelatinase-containing granule occurs during activation of cells.

Divergence of Mechanisms Regulating Respiratory Burst in Blood and Sputum Eosinophils and Neutrophils from Atopic Subjects

“…NADPH oxidase activation is therefore unlikely to involve crystalloid granules in eosinophils. This is in contrast to neutrophils, which express cytochrome b 558 in specific granules (48,49,53,59). The majority of NADPH oxidase activity in neutrophils undergoing arachidonate-or PMA-induced respiratory burst was found to localize to specific granules (58,60).…”

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

“…cytochrome b, required for superoxide anion generation, are mainly located in tertiary granules, with a secondary location in specific granules (6,7,9). Fusion of cytochrome b-rich granules with the plasma membrane is required for the production of reactive oxygen species (8,35).…”

“…Therefore, prevention of tertiary/specific granule fusion with phagosomes containing azurophilic granules may hamper the triggering of major anti-infection mechanisms. Likewise, lack of fusion of tertiary and specific granules, which harbor cytochrome b (6,8,9) responsible for the respiratory burst, with parasite-containing phagosome would prevent the triggering of the generation of oxygen metabolites highly toxic for the parasite. In fact, we found that L. major parasites did not trigger the respiratory burst in human neutrophils.…”

Role of Vesicle-Associated Membrane Protein-2, Through Q-SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor/R-SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Interaction, in the Exocytosis of Specific and Tertiary Granules of Human Neutrophils

“…To prepare the distinct subcellular fractions, freshly prepared neutrophils (ϳ3-5 ϫ 10 8 ) were gently disrupted, and the postnuclear fractions were fractionated in 15-40% (w/w) continuous sucrose gradients as described previously (5,34). Subcellular fractions were analyzed for marker proteins for each organelle, namely lactate dehydrogenase (cytosol), HLA (plasma membrane), latent alkaline phosphatase (secretory vesicles), gelatinase (tertiary granules), lactoferrin (specific granules), and peroxidase (azurophilic granules) (34 -37).…”