An antiserum against the PC12 cell line defines cell surface antigens specific for neurons and Schwann cells

Abstract

Antibodies raised in rabbits and in guinea pigs against nerve growth factor-treated PC12 cells were absorbed exhaustively with three non-neuronal cell lines. In immunofluorescent staining experiments, these absorbed sera, designated anti-PC12, labeled specifically several different types of cultured neurons, including cerebral, cerebellar, spinal, dorsal root, and superior cervical neurons. Non-neuronal cells in these cultures, such as astrocytes, oligodendrocytes, and fibroblasts, were not labeled. However, neural crest-derived adrenal chromafhn cells and Schwann cells were stained by anti-PC12. Further absorption with adrenal tissue rendered anti-PC12 specific for neurons and Schwann cells, while still further absorption with Schwann cells yielded an antiserum that was reactive only with dorsal root and superior cervical neurons. Absorption of anti-PC12 with adult brain resulted in the loss of activity against all cells except PC12 cells.These absorption experiments suggested that anti-PC12 recognized at least four distinct cell surface components. Some anti-PC12-reactive components were identified by using the anti-PC12 serum to prepare immune precipitates from detergent extracts of 12"1-labeled cells. The immune precipitates were analyzed by polyacrylamide gel electrophoresis. Anti-PC12 precipitated two components with apparent molecular weights of 190,000 and 140,000 that were common to Schwann cells, chromaffin cells, and all types of neurons. Smaller quantities of these two components could also be precipitated from cerebellar glial cells. In addition, anti-PC12 precipitated a glycoprotein of greater than 200,000 daltons from the cultured neurons, the neuronal cell lines, and the Schwann cells, but not from chromaffin cells or from cerebellar glia. Each neuronal cell type expressed a characteristic form of this large glycoprotein, as judged by electrophoretic mobility. For example, the molecules found on four cell lines, PC12, B35, N18, and /3HC, had apparent molecular weights of 235,000, 225,000, 220,000, and 215,000, respectively. The 235,000-dalton glycoprotein from PC12 cells is likely to be identical to the NILE glycoprotein described by McGuire et al. (McGuire, J., L. Greene, and A. Furano (1978) Cell 15: 357-365), while the 220,000-dalton component from N18 cells is probably similar to that described by Akeson and Hsu (Akeson, R., and W. Hsu (1978) Exp. Cell Res. 115: 367-377). Adrenal-absorbed anti-PC12 did not precipitate the 190,000-and 140,000-dalton components from any of the cell types but continued to precipitate the 215,000-to 235,000-dalton glycoproteins from the neurons and Schwann cells. These high molecular weight glycoproteins thus appear to be cell surface markers for neurons and Schwann cells.The PC12 cell line (Greene and Tischler, 1976) shares many properties with sympathetic neurons and has thus provided an extremely useful model system for studying several types of neuronal mechanisms, including the function of neuronal acetylcholine receptors (Patrick and

Protein complexity of central nervous system cell lines

“…(4) Finally, proteins shed from the cell surface are released as a singlemolecular-weight species and are not found in the culture medium as a series of lower molecular weight peptides. This is true for fibroblast proteins (see, for example, McCormick et al, 1979) as well as for surface proteins of nerve and glial cells (RichterLandsberg et al, 1984;Stallcup et al, 1983;Sweadner, 1983). The inclusion of the protease inhibitor Kallikrein in the culture medium had no effect upon the pattern of extracellular protein synthesis (unpublished observations).…”

“…The major criterion on which the classification of the nerve and glial cells was made was electrical excitability, but a large number of other markers, such as cell-surface antigens, are in agreement with the physiology. Nile and Nl are nerve-specific antigens (Richter-Landsberg et al, 1984;Stallcup, 198 1;Stallcup et al, 1983), and G2 is found only on some glial cells (Stallcup and Cohn, 1976). The clonal mesoderma1 cell lines were derived from both normal embryonic and neoplastic cells.…”

Involvement of the nerve growth factor-inducible large external glycoprotein (NILE) in neurite fasciculation in primary cultures of rat brain.

“…Binding of anti-NILE, anti-N-CAM, tetanus toxin, and antibody to glial fibrillary acidic protein to cells in primary culture was detected by immunofluorescence as described previously (5,13). For experiments in which neurite outgrowth or fasciculation was to be quantitated, control cultures or cultures that had been incubated in the presence of tetanus toxin, anti-NILE or anti-N-CAM were stained by using sequential 30-min incubations with tetanus toxin, rabbit antitoxin, and fluorescein-labeled goat antibody against rabbit immunoglobulin.…”

“…Closely related glycoproteins have now been found on the surfaces of all neuronal cell lines examined and all neurons in primary culture, including neurons of central, sensory, and sympathetic origin (4-7). Schwann cells also express a NILE-related component (5,6). Although they are immunologically cross-reactive, these neuronal glycoproteins are not identical.…”

“…Anti-NILE antibodies are useful not only for biochemical studies of this family of neuronal glycoproteins but also for immunohistochemical identification of neuronal cells in primary culture (5,6) and in tissue sections (8). In central nervous system tissue, astrocytes, oligodendrocytes, and fibroblasts do not express a NILE-related component, so that the anti-NILE antibody can be used with confidence to identify neurons.…”