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Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

ABSTRACT: An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.

SUBMITTER: Kjeldsen L

PROVIDER: S-EPMC1133208 | biostudies-other | 1992 Oct

REPOSITORIES: biostudies-other

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Project description:The expression of cell-surface adhesion proteins and the release of extracellular-matrix degradative enzymes constitute crucial processes for the attachment of neutrophils to the endothelium and for the subsequent extravasation of these cells through the endothelial layer. We have analysed in resting human neutrophils the subcellular localization of heparanase, a heparan-sulphate-degrading endoglycosidase that can degrade basement-membrane components, thereby facilitating neutrophil passage into the tissue during an inflammatory reaction. By subcellular fractionation of postnuclear supernatants from resting human neutrophils on continuous sucrose gradients, we have found that heparanase activity was mainly located in gelatinase-containing tertiary granules. Using a specific antibody, the 96-kDa heparanase protein was further located in the gelatinase-rich subcellular fractions. Following immunoblotting and immunoprecipitation analysis in the distinct subcellular fractions, we also found co-localization of heparanase and Mo1 (CD11b/CD18), a leucocyte integrin involved in the attachment of neutrophils to the endothelium, in the fractions enriched in gelatinase-containing tertiary granules. Treatment of human neutrophils with tumour necrosis factor or granulocyte/macrophage colony-stimulating factor induced an increase in the CD11b/CD18 cell-surface expression, as well as the release of both gelatinase (matrix metalloproteinase-9) and heparanase, but not of other granule markers, indicating a major co-localization of gelatinase, heparanase and CD11b/CD18 in the same organelle. Furthermore, confocal laser scanning microscopy using specific antibodies against gelatinase and heparanase revealed a major co-localization of both enzymes in intracellular cytoplasmic granules. The major localization of heparanase and CD11b/CD18 in the gelatinase-containing tertiary granule supports the notion that mobilization of this organelle can regulate extravasation of human neutrophils.

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Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

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