Autologous immunoglobulin-G binding to erythrocytes subjected to cyclical deoxygenation in vitro

Abstract

This study demonstrates that low-density metabolically replete HbSS erythrocytes suspended in heat-inactivated autologous plasma and subjected to 15 hr of cyclical deoxygenation (under nitrogen) bind significantly increased quantities of autologous IgG as compared with oxygenated paired samples. IgG binding to the erythrocyte surface was quantified by a nonequilibrium 125-iodinated protein A binding assay and by flow cytometry. Sickle cells deoxygenated 15 hr (37 degrees C) in the presence of 2 mM calcium bound 2.2 +/- 0.2 (mean +/- SD)-fold more IgG (p less than 0.01) than oxygenated paired samples. Sickle erythrocytes deoxygenated in 0.4 mM EDTA bound 1.7 +/- 0.3 (mean +/- SD)-fold more autologous IgG than oxygenated controls (p less than 0.05). Indirect immunofluorescence assays also demonstrated that the relative levels of autologous IgG bound to sickle cells after 15 hr cyclical deoxygenation in the presence or absence of calcium was increased as compared with IgG binding by oxygenated paired samples. After 3 hr of cyclical deoxygenation in the presence of 2 mM calcium sickle erythrocytes exhibited a 40-60% increase in IgG binding, as compared with 10-20% increased IgG binding by paired samples treated in EDTA. These findings demonstrate that repeated morphologic sickling will increase the IgG binding capacity of low-density sickle cells, and suggest that sickling-associated alterations of the cell surface will produce new binding sites recognized by autologous IgG. These studies also show that the sickling-induced increase in IgG binding may be slightly enhanced by the presence of extracellular calcium.