Characteristics of isolated erythrocyte complement receptor type one (CR1, C4b-C3b receptor) and CR1-specific antibodies.

Abstract

Abstract Complement receptor type one (CR1) was isolated from detergent-solubilized human erythrocyte (E) membranes by modification of a previously described technique. The isolated CR1 was labeled with 125I and was shown to be bound by either C4b- or C3b-coated sheep E (EAC14b or EC3b), but not by sheep E coated with either C3bi or C3d (EC3bi or EC3d). When analyzed by sodium dodecyl sulfate Polyacrylamide gel electrophoresis, the 125l-la-beled CR1 that was bound to EAC14b or EC3b consisted of a single protein of 195,000 daltons, with or without prior reduction of disulfide bonds. Rabbit antibody prepared by immunization with purified CR1 totally inhibited the CR1-specific EAC14b and EC3b rosetting activity of human E, peripheral blood and tonsil B lymphocytes, B lymphoblastoid cells, monocytes, and neutrophils. By contrast, saturation of membrane CR1 with monovalent Fab’-anti-CR1 did not inhibit lymphocyte CR2-dependent or monocyte-neutrophil CR3-dependent rosette formation with EC3d and EC3bi, respectively, confirming the independence and separation of CR1 from both CR2 and CR3 on the membrane surface. Direct immunofluorescence assays with anti-CR1 confirmed previous rosette assay data that most peripheral blood and tonsil B lymphocytes that expressed CR1 also bore surface immunoglobulins (Ig). Peripheral blood monocytes and neutrophils demonstrated especially intense CR1-specific fluorescence staining, whereas human E, Raji and Daudi lymphoblastoid cells, and T lymphocytes were negative for CR1 immunofluorescence. The negative staining of human E was probably due to the low number of CR1 receptors per cell, since a radioimmune assay with 125l-labeled Fab’-anti-CR1 demonstrated less than 2000 CR1 antigenic sites per human E cell. With all of the various lymphoblastoid cell lines examined, CR1-specific staining correlated with the ability of the cells to form rosettes with EAC14b rather than EC3b, since some cell lines lacking CR1 converted EC3b to EC3bi that then bound to CR2. A comparison of C-receptor-dependent rosetting with the direct immunofluorescence assay for the enumeration of CR1-bearing cells indicated that the two methods were of approximately comparable sensitivity with most cell types tested with the exception of both human E and the lg−CR1+ peripheral blood lymphocyte subpopulation that apparently express very few CR1 per cell.