Translation of mRNA from phytohemagglutinin-stimulated human lymphocytes: characterization of interferon mRNAs

Abstract

Human lymphocytes obtained by cytapheresis were stimulated in spinner culture conditions by nonpurified PHA in order to study the production of gamma interferon, and the characterization of IFN-gamma mRNA. Titers of interferon prepared in 0.6 to 4 1 batches, varied in 20 preparations from 8,000 to 32,000 units/ml. This interferon was unstable at pH 2: the residual antiviral activity after 20 h treatment was less than 3%. Antibodies raised against gamma interferon from Con A and SEA-stimulated lymphocytes neutralized the interferon induced by PHA, indicating that all three preparations are antigenically related. Poly(A)RNA from control, noncultivated lymphocytes and from lymphocytes stimulated by PHA for 18 h were translated in reticulocyte lysates and analysed by polyacrylamide gel electrophoresis. The pattern of synthesized polypeptides was different suggesting modifications in the population of mRNA. When total poly(A)RNA was inoculated into Xenopus Laevis oocytes, interferon activity was found with both, control and stimulated mRNAs although only at low levels in the control. After sucrose gradient fractionation of poly(A)RNA, each fraction was inoculated into oocytes and interferon activity measured in the oocyte bathing medium. A low level was synthesized by the RNA fractions around 28 S from control as well as from stimulated lymphocytes. These interferons were not neutralized by anti-IFN-alpha or anti-IFN-gamma sera but they were neutralized by anti-IFN-beta serum. Only the 16 S RNA fraction from PHA-stimulated lymphocytes induced high levels of interferon in oocytes. This interferon has been characterized as gamma interferon. Each fraction obtained from sucrose gradients on poly(A)RNA from control and PHA-stimulated lymphocytes was translated in reticulocyte lysate. Gel analysis of the products showed striking differences when the same fraction of both RNAs were compared. Concerning particularly the 16 S RNA from PHA-stimulated lymphocytes, where gamma interferon mRNA was present, polypeptides ranged from 15 to 55 K with a bulk around 45 K, indicating heterogeneous RNA molecules.