Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion...

Human neutrophils contain a rapidly mobilizable pool of so-called secretory vesicles distinct from the azurophil granules and specific granules. Using human albumin as a marker for these intracellular vesicles in immuno-electron microscopy, we found that part of the cytochrome b558 in non-purified whole blood neutrophils colocalized in these vesicles. This was detected with monoclonal antibody (MoAb) CLB-48, binding to the high molecular weight subunit of cytochrome b558. Approximately 65% of the albumin-containing vesicles showed MoAb CLB-48 labeling. This was also found in eosinophilic granulocytes and in monocytes. Cytofluorimetric determination of cytochrome b558 expression on the plasma membrane of intact, nonpurified granulocytes (and monocytes) with MoAb 7D5, which is directed against an extracellular epitope of cytochrome b558, did not show any binding. However, granulocytes (and monocytes) significantly bound 7D5 after density centrifugation. The positive binding of 7D5 to purified neutrophilic granulocytes correlated with a strongly reduced labeling of cytochrome b558 in the albumin-positive vesicles. Binding of CD11b MoAb CLB-B2.12 to the alpha subunit of the complement receptor type 3 (CR3) on the surface of intact, nonpurified neutrophils was detected to a limited extent in whole blood samples, but was strongly increased upon density gradient centrifugation of the cells, as we have described before. Investigation at the ultrastructural level showed that the CD11b antigen codistributed with albumin in vesicular structures in nonpurified phagocytes, especially in neutrophils and eosinophils. Together, these data substantiate the idea of an intracellular store that can be easily mobilized (even under the simple stress condition of density gradient centrifugation). Such mobilization may result in the expression of cytochrome b558 on the plasma membrane, as was indicated in this study. Apart from cytochrome b558, several other surface membrane molecules, as we show here for the integrin CD11b/CD18 (CR3), are probably also located in these rapidly mobilizable intracellular vesicles.