Abstract
Mo1 (CD11b), a glycoprotein heterodimer that is involved in cellular adhesion processes and functions as the C3bi receptor of human myeloid cells, and T200 (CD45), a panleukocyte glycoprotein family whose function is still not well understood, increased their expression in the plasma membrane of human neutrophils after exposure to various stimuli which induce degranulation, such as formylmethionylleucylphenylalanine or calcium ionophore A23187. This increment in the expression of both molecules shows a good correlation with the release to the extracellular environment of gelatinase, a marker for an intracellular organelle named "tertiary granule" (Mollinedo, F., and Schneider, D. L. (1984) J. Biol. Chem. 259, 7143-7150). Flow cytometry studies indicate that at least 50% of the total Mo1 and T200 molecules are located in intracellular organelles. Furthermore, the subcellular distribution of Mo1 and T200 glycoproteins in resting human neutrophils was investigated by immunoprecipitation of the radiolabeled membrane proteins obtained from the distinct subcellular fractions. Both Mo1 and T200 were mainly localized in tertiary or specific intracellular granules, which were resolved from the azurophilic granules as well as from the cell membrane fraction. These findings suggest that the mobilization of intracellular Mo1 and T200 to the plasma membrane may regulate early events occurring upon neutrophil activation.
Highlights
From the 4Unidad de Biomembranas, Centro de InvestigacionesBioldgicas, Consejo Superior de InvestigacionesCientificas, Velcizquez 144 and the Wervicio de Inmunologia, Hospital de La Princesa, Diego de Leon 62, 28006 Madrid, Spain
By subcellular fractionation and immunoprecipitation studies, we have found that Mol and T200 molecules are constituents of neutrophil plasma membranes, and of intracellular granules, which contain a high proportion of these glycoproteins
There was a good correlation between Mol and T200 expression and gelatinase release, suggesting that the additional Mol and T200 expression at the cell surface was probably due to the discharged tertiary granules, whose membranes were incorporated into the plasma membrane as a result of the membrane fusion event, which is integral to the degranulation process
Summary
Subcellular Fractionation of Neutrophils-Neutrophils were frac- of both Mol and T200 cell-surface expression. The pellets, representing the membranous fractions, were resuspended in 50 mM Tris-HC1,pH 7.5, increase in Mol and T200 surface expression. FMLP or ionophore A23187, at the concentrations indicated in the (200 pg/ml) did not alter the above-described up-regulation of Mol andT200 cell-surface expression (data notshown). Gelatinase was significantly secreted upon cell preincubation in the presence of 5 pg/ml cytochalasin B, but in the absence of any stimulus, were run in parallel and processed as described above. Were incubated with 100 p1 of monoclonal antibody for 30 min at 4 "C, washed twice with phosphate-buffered saline, and suspended. 1. Effect of FMLP on surface antigen expression of ously described [39], and samples were subjected to sodium dodecyl human neutrophils. Chilled at 4 "C, labeled with the corresponding monoclonal antibodies and FITC fluorescein isothiocyanate-conjugated anti-
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