Surface events in polymorphonuclear leucocyte activation: insights from a hydrophobic membrane antigen that triggers the respiratory burst.
The rat monoclonal antibodies (Mab) 1A10.4 and IG4 were raised to a solubilized NADPH-oxidase preparation from guinea-pig polymorphonuclear neutrophils (PMN). They bind to a surface antigen restricted to guinea-pig myelomonocytic cells and on binding to the PMN surface can stimulate the respiratory burst (RB) and degranulation. This response was specific to these Mab and was not found with several other Mab restricted to myelomonocytic cells but with other antigenic epitopes. In order to understand the role of this molecule in stimulating the RB, we have characterized the antigen (Ag) by a variety of techniques. It is a hydrophobic membrane molecule with an apparent molecular weight of 8-10(x10(3] and a pI of 6.2. The Ag has been partially purified by extraction in organic solvents and high-pressure liquid chromatography on silica and is probably a proteolipid. Studies on Mab-dependent triggering of PMN secretion indicated that cross-linking of the cell surface was critical. We therefore used direct immunofluorescence under triggering and non-triggering conditions to show that stimulation of the RB by Mab correlated with redistribution of the surface Ag into patches. This patching was associated with aggregation of surface Ag and transfer of Ag from a Triton X-100-extractable to a Triton X-100-inextractable membrane domain. These movements of surface Ag, which included both patching and capping resulting from aggregation of a hydrophobic membrane component, occurred at 4 degrees C and were insensitive to inhibition by cytoskeletal inhibitors. These specific probes that control triggering of the RB have permitted the dissection of PMN stimulation into discrete membrane events by correlating the biochemical and morphological characteristics of a new PMN surface Ag as it stimulates exocytosis.
Rosen
H
H
Sir William Dunn School of Pathology, University of Oxford, UK.
Gordon
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology* / Q000302:isolation & purification; D002462:Cell Membrane / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005260:Female; D006168:Guinea Pigs; D007111:Immunity, Cellular; D066298:In Vitro Techniques; D008297:Male; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000276:immunology*; D012997:Solvents
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000939:Epitopes; D012997:Solvents