Marzenna Wiranowska-Stewart

Production, partial purification and characterization of human and murine interferons—Type II

Abstract Human and murine type II interferons were produced from mitogen stimulated lymphocyte suspensions. Priming of human lymphocytes with type I human interferon prior to induction slightly increased type II interferon production, but priming of mouse spleen lymphocytes with type I mouse interferon had no apparent effect on type II interferon production. Incorporation of β-mercaptoethanol into culture medium enhanced production of mouse type II interferon but had no effect on human type II interferon yields. Both human and murine type II interferons could be purified from crude culture medium by chromatography on polyribouridylic acid (poly U)-agarose by a method identical to that used for purification of type I interferons, to specific activities of about 104.5 U/mg protein. Both interferons could also be purified from culture medium by chromatography on controlled-pore glass by modifications of methods used for purification of type I interferons, giving specific activities of about 104.0 U/mg protein. When type II interferons were purified by sequential chromatography on controlled-pore glass followed by poly U-agarose, specific activities of approximately 106 units of type II interferon/mg protein were obtained. These partially purified type II human and murine interferons were compared to type I human and murine interferons purified by similar methods to similar specific activities, and each could be distinguished by their antigenicities, acid labilities and host cell specificities.

Pharmacology of interferons I. pharmacologic distinctions between human leukocyte and fibroblast interferons

Abstract Human leukocyte and fibroblast interferons were inoculated into rabbits and mice by various routes to determine their survival in circulation and their distributions in body tissues. Human leukocyte interferon inoculated intraperitoneally, intramuscularly, subcutaneously or intravenously was found to enter the circulation efficiently and was widely distributed in the tissues for several hours. However, human fibroblast interferon entered the blood inefficiently; human fibroblast interferon was only transiently detectable in the circulation and at significantly lower levels than the leukocyte interferon and could never be detected in any of the solid tissues. When these interferons were incubated with various body fluids from mice, rabbits or humans, both interferons had similar stabilities in blood and in cerebrospinal fluid, while fibroblast interferon was less stable than leukocyte interferon in urine. A marked distinction was found between the stabilities of the two human interferons in muscle tissue: human leukocyte interferon was completely stable in tissue homogenates prepared from mouse, rabbit or human muscles, whereas fibroblast interferon was rapidly inactivated by such suspensions. Human fibroblast interferon was also rapidly destroyed by a cell-free extract prepared from either human, mouse or rabbit muscle tissues. These data demonstrate that human leukocyte interferon has pharmacologically advantageous properties which allow it to enter efficiently into the circulation following intramuscular injection and to distribute to the body tissues, whereas human fibroblast interferon is unable to do so because the latter is inactivated by the tissues at the site of inoculation. Thus, for therapeutic exploitation, human fibroblast interferon may be limited to topical administration only, whereas human leukocyte interferon would be much preferred for systemic use.

Characterization of the heterogeneous molecules of human interferons: differences in the cross-species antiviral activities of various molecular populations in human leukocyte interferons.

Summary Human leukocyte interferon (HuLeIF) preparations were separated into populations of molecules with different sizes, by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and with different charges, by isoelectric focusing. These populations with different sizes and charges were analysed for their antiviral activity on homologous cells and on heterologous (bovine) cells. The distribution of interferon activity into two broad peaks by SDS-PAGE was similar whether assayed on human or bovine cells. However, within these peaks, the relative ratio of the activity in human cells and bovine cells varied significantly: while most of the size components had similar human/bovine cell activities, the fastest migrating component (apparent mol. wt. ∼ 13500) was more than 100 times more active on bovine cells than on human cells. The peaks of activity in isoelectric focusing were distributed from pH 5.5 to 7.0. There was generally correspondence between human and bovine cell activities, but while the more neutral pH range peaks were consistently slightly more active on human cells than on bovine cells, the more acid range peaks were always slightly more active on bovine cells than on human cells. However, with the most acidic peak, there was more than 100 times greater activity on bovine cells than on human cells. These data show that the heterogeneity of HuLeIFs is greater than merely two size populations, and data confirm that different forms of human leukocyte interferon can vary markedly in biological activity.

Characterization of Two Distinct Molecular Populations of Type I Mouse Interferons

The molecular heterogeneity of acid-stable (Type I) mouse interferons induced in C243 cells by Newcastle disease virus was analysed by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions, and the profiles of antiviral activities obtained were characterized biologicaly in mouse cells and in heterologous (guinea-pig) cells. Two bands of activity, A and B, were consistently present in all interferon preparations tested: under reducing conditions, the activity in all fractions of band A (with a peak of activity at about 38000 daltons) was uniformly increased, while that of band B (with a peak at about 22000 daltons) was uniformly diminished. All the active fraction in band A had only slight activity (less than 10% of homologous titres) on guinea-pig cells, whereas all those in band B were significantly more active an guinea-pig cells than on homologous L cells. Thus, mouse type I interferon preparations contain two molecular populations of interferons that can be distinguished physically (by size), biochemically (by the effect of reduction on reactivation from SDS) and biologically (by activity in heterologous cells).

Effect of glycosylation inhibitors on the production and properties of human leukocyte interferon

Abstract Human leukocyte “buffy coat” suspensions were induced to produce interferon by stimulation with Sendai virus and were incubated during the interferon production period in medium containing various concentrations of glycosylation inhibitors, either d -glucosamine or 2-deoxy-D-glucose. At 80 mM d -glucosamine or at 8 mM 2-deoxy- d -glucose, interferon production was inhibited by approximately 95%; however, at 20 and 2 mM, respectively, these inhibitors allowed interferon production at about 50% of levels produced by leukocytes not treated with the inhibitors. The residual interferons produced in the presence of glycosylation inhibitors were antigenically and biologically indistinguishable from native human leukocyte interferons in terms of neutralization by antisera to native interferons and antiviral activities on cells of heterologous species. However, when analyzed by electrophoresis in SDS-polyacrylamide gels, the interferons produced in the inhibitors exhibited markedly less size heterogeneity than found with native human leukocyte interferons, shifting toward the smaller size forms. Treatment of the native interferon with 0.01 M sodium periodate buffer, pH 4.5 at 4° reduced the size heterogeneity to the lower molecular weight form; such treatment had no detectable effect on the interferons produced in the presence of glycosylation inhibitors.

Repeated 'superinduction' of interferon in human diploid fibroblast cultures.

Human diploid fibroblast cultures induced to make interferon by the combination of polyriboinosinic acid-polyribocytidylic adic, cycloheximide and actinomycin D degenerate thereafter, owing to the irreversible nature of the inhibition induced by actinomycin D. However, cultures superinduced with the DNA-dependent RNA synthesis inhibitor 5,6-dichloro-I-beta-D-ribofuranosylbenzimadazole (DRB) survive, owing to the reversible nature of the inhibition induced by DRB, and can again be superinduced on several occasions.

The Pharmacologic Properties of Various Forms of Interferon

Several forms of human interferons were compared for their abilities to distribute systemically following various routes of inoculation in animals and for their survival in the circulatory system. Human leukocyte-derived interferons (HuIFN-α) were much more efficient at entering the blood than were fibroblast-derived interferons (HuIFN-β); the latter were unstable in tissue suspensions which were without effect on the former. While all forms of HuIFN-α isolated as distinct molecular species were similarly efficient at entering the blood following intramuscular injection, these HuIFN-α forms differed in the clearance rates from the blood. Thus, HuIFN-α (21K) was cleared much more rapidly than HuIFN-α (18K) or Namalva lymphoblastoid cell-derived HuIFN-α, which were in turn less stable in the bloodstream that the HuIFN-α (15K). Therefore, while HuIFN-α appears to have pharmacologically advantageous properties in comparison to HuIFN-β, each distinct molecular form of HuIFN-α can have different pharmacokinetic properties. Such evaluations will be important for each of the HuIFN-α forms produced by transformed E. coli clones.

Characterization of the size and charge heterogeneities of human leukocyte interferon populations

SummaryHuman leukocyte interferons are separable into two size components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and are separable into two charge-components by DEAE-BioGel A chromatography. However, each of the charge-components resolved by ion-exchange chromatography contained both size-components, when analysed by SDS-PAGE. Thus, there are more than two distinct molecular populations of human leukocyte interferons.