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Treatment of leukaemic mouse L 1210 S cells with beta or gamma interferon results in the development of antiviral and anticellular effects, both of which are also induced by interferon in L 1210 R cells selected for their resistance to gamma-interferon. Here we show that treatment of L 1210 S cells with beta or gamma interferon results in the induction of two double-stranded (ds) RNA dependent enzymes which have been described previously in beta and gamma-interferon treated mouse L-929 cells: pppA (2'p5'A)n synthetase and protein kinase(s). Treatment of L 1210 R cells with beta-interferon does not result in the induction of any detectable enhanced pppA (2'p5'A)n synthetase and protein kinase activities whereas gamma-interferon is capable of inducing both of these enzymes. These results indicate that although beta and gamma-interferon induce similar intracellular events, they may have different mechanism of interaction with their target cells. In addition, here we show that similar to the action of beta-interferon, treatment of mouse embryonal carcinoma cells with gamma-interferon results in the induction of pppA (2'p5'A)n synthetase in the absence of any detectable protein kinase activity or any apparent development of antiviral and anti-cellular effects. The significance of this in relation to the mechanism of action of interferon is discussed.

Mouse gamma antigen-induced interferon (IFN) was produced in vivo by injecting PPD into BCG-sensitized mice. The IFN activity present in the serum was partially purified by adsorption on silicic acid and elution with buffer containing ethylene glycol, followed by chromatography of the eluate on Blue-Trisacryl. This method yielded IFN preparations with a specific activity of 1.5 x 10(5) mu/mg protein (10,000-fold purification). BCG IFN showed high affinity for polynucleotides and apparent hydrophobicity, two characteristics which allowed it to bind to Poly I agarose and Phenyl Sepharose, respectively. The chromatographic behavior of BCG IFN on Con A Sepharose indicates that BCG IFN is glycosylated. However, binding of the antiviral activity to the lectin column was only possible when purified IFN was used for chromatography. Although the purification method described here allowed purification of the antiviral activity as a major fraction, molecular heterogeneity was detected under certain chromatographic conditions. Two fractions with different molecular weights of 27,000 and 56,000, respectively, were always obtained by sieving purified BCG IFN on Sephacryl S 200.

Gamma interferon (gamma IFN) was produced in human lymphocyte cultures stimulated by PHA. Titers were in the range of 10,000-30,000 U/ml. Crude gamma IFN was adsorbed on silicic acid, from which the antiviral activity was eluted by a buffer containing a high salt concentration and ethylene glycol. This treatment allowed quantitative recovery of gamma IFN with a specific activity of 5 X 10(5)-1 X 10(6) U/mg of proteins. IL2 and B-cell helper activities were adsorbed and eluted from silicic acid together with the antiviral activity. This finding might be of practical interest for the purification of these lymphokines, particularly IL2. Gamma interferon was further purified on Blue Sepharose to a specific activity of 2 X 10(7) U/mg. The resulting preparations still contained IL2 and B cell helper activities. However, taking advantage of the differences in apparent hydrophobicity and in isoelectric point, we were able to dissociate antiviral activity from lymphokines. Such dissociation should facilitate the study of the biological properties of human natural gamma IFN.

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.

In a previous report, we presented preliminary data on interferon-alpha A (IFN-alpha A) pharmacokinetics through the simultaneous study of distribution of the radiolabeled protein by numerical dynamic scintigraphy and the trichloroacetic acid (TCA)-precipitable radioactivity in whole blood and serum, as well as the radioactivity linked to blood mononuclear cells. The interferon preparation then used was contaminated with free iodine which gave parasite images and counts. Here we present further analysis of those data, enlightened by additional information collected in new studies with interferon preparations without free iodine. Interferon uptake by the kidneys, liver, and heart was a consistent finding. On the contrary, images in stomach and duodenum seem mostly related to their well-known iodine uptake. At present, whether interferon is present in saliva and nasal secretion is unclear. The extent to which blood radioactivity corresponded to blood cells or plasma was analyzed. TCA-precipitable radioactivity measured after administration of filtered radiolabeled interferon was mainly confined to serum. The marginal amount bound to cells seemed to be in leukocytes. Current research points to compare the pharmacokinetic profiles of IFN-alpha and IFN-gamma.