Effects of anti-human neutrophil antibodies in vitro. Quantitative studies

Opsonic, antiphagocytic, cytotoxic, and metabolic effects of homologous and heterologous antibodies against human neutrophils were analyzed by means of quantitative assays to facilitate detection of antibody activity, and to probe membrane function of these cells. Normal human neutrophils were purified by gradient centrifugation, sensitized with heat-inactivated antineutrophil antisera, and incubated with rabbit alveolar macrophages in balanced salt solution containing nitroblue tetrazolium. The macrophages engulfed sensitized neutrophils and reduced nitroblue tetrazolium to formazan in phagocytic vacuoles. The initial rate of nitroblue tetrazolium reduction by macrophages ingesting the neutrophils was measured spectrophotometrically. Neutrophils treated with rabbit anti-human leukocyte antiserum or IgG, with sera from mothers of infants with neonatal isoimmune neutropenia, and with 27% of sera from frequently transfused patients promoted rapid rates of nitroblue tetrazolium reduction by alveolar macrophages. This indicates that antineutrophil antibodies without added complement opsonized neutrophils for ingestion by the macrophages. Some sera from frequently transfused patients with opsonic activity for certain donors' neutrophils did not agglutinate these neutrophils (44%), did not lyse them in the presence of fresh plasma (47%), and did not inhibit phagocytosis of particles by the neutrophils (26%). The reverse was not observed. The opsonic activity of antineutrophil antiserum appears to be the most sensitive and a quantitative means of detecting antibody activity in vitro.Low concentrations of rabbit anti-human leukocyte antisera or IgG stimulated the ingestion rate of unopsonized or opsonized particles by human neutrophils, and, as previously reported by others, enhanced rates of oxidation of [1-(14)C]glucose by the cells. High concentrations of the antisera or IgG inhibited ingestion. All concentrations of homologous antineutrophil antisera tested only inhibited ingestion of particles by neutrophils and none altered rates of resting glucose oxidation by the cells. The findings suggest that heterologous antibodies disturb membrane antigens that trigger oxidative metabolism and enhance as well as inhibit ingestion, and that these antigens are common to all human neutrophils. In contrast to other studies with antimacrophage antibodies, antineutrophil antibodies altered phagocytic rates of both unopsonized and opsonized particles although there were differences in dose-response curves depending on the type of particle tested. Thus, antineutrophil antibodies do not merely cover selected receptor sites.