Granulocyte-macrophage colony-stimulating factor from cultured normal rat kidney cell line

Abstract

Serum-free medium conditioned by the normal rat kidney (NRK) cell line contains colony-stimulating factors (CSF) that stimulate the in vitro formation of granulocyte and macrophage colonies from rat, mouse, and human marrow. There are two types of CSF: NRK-CSF I stimulates rat and mouse marrow, while NRK-CSF II stimulates the human marrow. The NRK-CSF I has been partially purified to a specific activity of 5 X 10(7) units/mg by employing methods such as preparative isoelectrofocusing, gel filtration chromatography, and preparative gel electrophoresis. It has an isoelectric point of 5.1 and an apparent molecular weight of 35,000 daltons as estimated by gel filtration. It is stable at 50 degrees C for 30 min and relatively resistant to papain, but highly sensitive to trypsin, chymotrypsin, and subtilisin. The secretion of CSF by NRK cells is inhibited by actinomycin D (0.5 micrograms/ml) and cycloheximide (0.5 micrograms/ml), but not by cytosine arabinoside (5 micrograms/ml). Although the CSF activity is inactivated by periodate oxidation (5 mM), thus suggesting its glycoprotein nature, treatment of the CSF with neuraminidase and other glycosidases has no effect on its activity. Anti-NRK-CSF I antibody inhibits the rat/mouse-active CSF from all rat sources, but has no effect on the human-active CSF. Comparative studies with CSF from media conditioned by the transformed cell line (442) and from rat lung and spleen have shown that CSF I from different rat sources share similar isoelectric points, molecular weights, and immunological properties.