Markedly Enhanced Production of Gamma Interferon in Murine T Lymphocytes Treated with Lentil Lectin and the Diterpene Ester,Mezerein

Abstract

Gamma interferon (IFN-γ) was induced in murine splenocytes first stimulated to grow by concanavalin A (Con A) and subsequently treated for 3 h with the diterpene ester, mezerein (MZN) and then with lectin from Lens culinaris for 24 h. Yields as high as 60,000 u/ml were obtained in cells from either male or female, random-bred, white Swiss mice or inbred C67B1/6 mice. Antibody to Thy 1.2 surface antigen completely obliterated the mouse gamma interferon (MuIFN-γ) response, whereas anti-Lyt 1.2 and anti-Lyt 2.2 each destroyed a portion of the lymphocyte population responsible for MuIFN-γ production. Kinetic analysis of production and release showed that IFN was detectable in culture fluids within 4 h after treatment with very little IFN remaining cell-associated (less than 10%). A simple, rapid, and economical two-step purification procedure involving ammonium sulfate fractionation and yeast RNA affinity chromatography resulted in as much as 770-fold purification to achieve specific activities >10⁷ u/mg protein. The purified MuIFN-γ was shown to be predominantly acid-labile, inactivated by sodium dodecyl sulfate (SDS), and neutralized by antiserum to MuIFN-γ. Approximately 10% of the MuIFN-γ was acid-stable and SDS-resistant, but was still neutralized by anti-MuIFN-γ serum. Two molecular weight peaks of about 40 and 20 kD were demonstrated by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing in polyacrylamide slab gels gave a relatively heterogeneous band of activity between pH 5.5 and 6.5. The mechanism by which the combination treatment described enhances MuIFN-γ production so markedly remains unknown, but the degree of enhancement is greater than additive.