Characterization of antisera raised against cultured rat sympathetic neurons

Abstract

Antisera were prepared against monolayer cultures of newborn rat sympathetic neurons and characterized by 51Cr release microcomplement fixation, cytotoxicity and indirect immunofluorescence assays. After absorption with rat liver, kidney, spleen and thymus to remove activity against common rat antigens, the antisera retained activity against cultured sympathetic neurons, brain and adrenals. Indirect immunofluorescence showed that the antigens recognized were widely distributed in the brain, while in the adrenal gland they were limited to the medullary cells. Absorption with the brain completely removed the activity of the antisera against sympathetic neurons; in contrast, partial activity was retained after repetitive absorption with adrenals. The antisera were also characterized with respect to recognition of the PC12 clonal line of rat pheochromocytoma cells. Under normal growth conditions, PC12 cells resemble their normal counterparts, adrenal chromaffin cells, while after treatment with nerve growth factor, they acquire the differentiated properties of sympathetic neurons. Both untreated and treated cells were recognized by the antisera and indirect histofluorescence on living cells indicated that at least some of the antigens on these cells (as well as on sympathetic neurons) are exposed on the cell surface. However, differences were noted in recognition of nerve growth factor treated and untreated PC12 cells. For example, the former, but not the latter were recognized in cytotoxicity assays. Also, absorption of the antisera with treated cells removed all activity against rat sympathetic neurons, whereas repetitive absorption with untreated PC12 cells removed only part of the activity. Such findings indicate that PC12 cells treated with nerve growth factor and sympathetic neurons exhibit antigenic similarities and that our antisera can be used to identify, isolate, characterize and localize neural-specific surface antigens.