Interferon production by individual cells

A feline interferon production and assay system has been defined which allows measurement of the interferon produced by a single cell. It was found that 2–3 induced cells were required to reduce by 0.5 log¹⁰ the yield of vesicular stomatitis virus (VSV) from approximately 2500 target cells. The antiviral effect was characterized as interferon by several criteria. The presence of trypsin or pretreatment of the induced cells with actinomycin D eliminated the effect. The antiviral effect was species specific, and cell-free supernatants from target cell monolayers which had been exposed to induced cells could be used to protect other target cell monolayers. It was found that one interferon-producing cell provided significant (p < 0.05) 0.1 log₁₀ reduction of VSV yield from 2500 target cells. Analysis of the data obtained when 2 or 3 induced cells were used to provide antiviral protection indicated that approximately $\text{1}\text{3}$ of the cell population failed to respond measurably to the inducer. The number of cells required to protect target cells was found to be inversely proportional to the amount of interferon produced in monolayer culture.     

Immunochemical characterization of mouse brain-associated theta (Thy-1) antigen.

The Thy-1 antigens or rat brain and thymus have been isolated and chemically characterized, but those of mice have not been identified. Moreover, it is uncertain whether the antigens are glycolipids or glycoproteins. This study with highly purified preparations of gangliosides GM₁, ¹GD_1a, GD_1b and GT_1b from bovine brain and several ganglioside fractions from mouse brain showed that Thy-1 activity does not reside in gangliosides, but rather in the chloroform-methanol-insoluble residue of brain remaining after extraction of gangliosides. The antigen could be solubilized from this residue with a non-ionic detergent. The antigenic activity of the solubilized preparation was heat-labile but resistant to periodate. The chemical properties of the Thy-1 antigen of mouse brain are discussed.     

Plasma gangliosides in multiple sclerosis

The total concentration and the relative distribution of gangliosides isolated from plasma of patients with multiple sclerosis (MS) and controls were determined. The fatty acid and hexose constituents of GM₃ were also determined.GM₃ was found to be the major plasma ganglioside in both the MS and control groups. The total plasma ganglioside concentration, expressed as N-acetylneuraminic acid (sialic acid), was significantly lower in the MS group than in the control group. No difference was observed in the various ganglioside fractions between the two groups. Analyses by gas-liquid chromatography of the relative per cent composition of the fatty acids of the GM₃-gangliosides revealed no significant differences between the two groups. Analyses of the hexose moieties of the GM₃-gangliosides of the MS group revealed the presence of only glucose and galactose in a 1:1 ratio.     

Interferon induces an antiviral state in ganglioside-deficient transformed mouse fibroblasts

The antiviral activity of mouse fibroblast interferon against vesicular stomatitis virus was investigated in L-929 mouse fibroblasts and the ganglioside-deficient L-929 mutant cells (ATCC clone NCTC 2071). Although it has been widely reported that gangliosides serve as primary receptors for interferon at the cellular membrane, only a small difference in interferon sensitivity was observed between the wild-type L-929 and the ganglioside-deficient NCTC 2071 cells. It was not possible, however, to overcome this difference by administration of exogenous gangliosides.     

Interferon inhibits induction of endogenous murine leukemia virus production.

In 5-iododeoxyuridine (IUDR) induction of murine leukemia virus (MLV) in Balb cells transformed by SV40 (Balb SV40) or AKR cells, interferon inhibited virus production. This could have been due to inhibition of the X- and N-tropic virus induced or to inhibition of subsequent reinfection of the mouse cells by the N tropic virus. Cycloheximide, however, activates $\text{KBalb}\text{3T3}$ cells ($\text{Balb}\text{3T3}$ cells transformed by Kirsten sarcoma virus) to produce only an X-tropic virus which could not reinfect mouse cells. In this system, interferon treatment also decreases virus yields and, therefore, an event in the virus induction process.     

Interferon-induced alterations in sialic acid and glycoconjugates of L-929 cells

Changes in Sialic acid and glycosphingolipid (GSL) metabolism were demonstrated in interferon (IFN)-treated L-929 cells. IFN induced an increase in total cell Sialic acid, sialoglycoproteins, and gangliosides, as shown by calorimetric and radiolabeling techniques. Expression of cell surface (neuraminidase-releasable) Sialic acid on IFN-treated cells was markedly elevated, particularly in the GSL fractions. The incorporation of [¹⁴C]galactose into glycoproteins, most neutral GSL homologs, and most ganglioside homologs also was elevated, with the more striking effects (two- to threefold) in the lipid fractions. An increase in the concentration of a ganglioside with the migration of GM₂, as measured by chemical staining of chromatograms, was also shown. The observed effects were IFN dose dependent at ranges from 10 to 10,000 U/ml. As shown previously, IFN-treated L-929 cells became resistant to lysis by virus-induced IFN-activated natural killer cells. Correlations between high levels of surface Sialic acid, resistance to NK cell-mediated lysis, and tumor invasiveness have been shown in other systems.     

Interferon-induced antiviral actions in Friend leukemia cells: role of membrane gangliosides

The interactions between interferon (IFN) and membrane gangliosides have been analyzed in Friend leukemia cells (FLC). The G_M1-cholera toxin (CT) model has been selected as a reference system for a high-affinity ganglioside-ligand binding. Two gangliosides have been detected in FLC extracts: a disialoganglioside (～ 95%) and a monosialoganglioside (～ 5%) which migrate respectively as G_D1a and G_M1. Unilammellar liposomes containing either mixed commercial gangliosides or G_D1a or G_M1 bind three to eight fold more IFN (both partially purified and pure) than gangliosides-free liposomes. Only 3–4% of the input IFN was bound, whereas G_M1-containing liposomes bound almost 50% of [³H]acetyl-CT. Neuraminidase treatment of FLC, which converts G_D1a-like into the G_M1-like ganglioside, does not significantly modify the IFN-induced antiviral effects. CT binding to FLC as well as CT-induced elevation of endocellular cyclic AMP levels in FLC were instead markedly increased by the same neuraminidase treatment. Further, pretreatment of FLC with either G_D1a or G_M1 or gangliosides extracted from FLC did not change the IFN-induced establishment of the antiviral state. These data provide convincing evidence that FLC gangliosides do not represent the IFN high-affinity receptors nor specifically mediate IFN effects, even though they bind IFN to some extent.     

Glycosphingolipids as Novel Targets for T-Cell Suppression by the B Subunit of Recombinant Heat-Labile Enterotoxin

Heat-labile enterotoxin subunit B (LTB) is a noncatalytic protein derived from Escherichia coli that binds to ganglioside GM₁, a glycosphingolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitro. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB. CD8⁺ T cells bound twice as much as CD4⁺ T cells and B cells. Exposure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8⁺ T cells were more susceptible to rLTB than either CD4⁺ T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This was attributed to qualitative and quantitative differences in the CD4⁺ T cells. rLTB induced apoptosis in both T-cell subsets, but the level was significantly higher in CD8⁺ T cells. Apoptosis peaked at around 8 h after exposure to rLTB and incubation at 37°C. Binding to ganglioside GM₁ was essential for suppression, since rLTB/G33D, a mutant which does not bind GM₁, failed to inhibit proliferation or induce apoptosis. Naive T cells, which were acutely sensitive to rLTB, became more resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A 1-h pulse with rLTB was sufficient to inhibit T-cell proliferation and cytotoxic-T-lymphocyte generation in primary mixed lymphocyte reaction cultures. CD8⁺ T cells were preferentially depleted in these cultures. rLTB also induced functional modifications in T cells as indicated by inhibition of gamma interferon secretion after polyclonal activation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.     

Mechanism of interferon action. Species specificity of interferon and of the interferon-mediated inhibitor of translation from mouse, monkey, and human cells.

The species specificity of interferon and of the interferon-mediated ribosome-associated inhibitor of translation from mouse fibroblast, African green monkey kidney, and human amnion cells was investigated. The following results were obtained: (1) The antiviral activity of mouse fibroblast, monkey kidney, and human leukocyte interferons was maximal on the homologous species of cells. Mouse interferon was not active on heterologous monkey cells, and monkey interferon was not active on mouse cells. Monkey and human interferons were almost equally active on heterologous human and monkey cells, respectively; mouse and human interferons were about 10 and 125 times more active, respectively, on their homologous cell species than on heterologous human and mouse cells. (2) Ribosomal salt-wash fractions prepared from interferon-treated mouse fibroblast, monkey kidney, and human amnion cells inhibited the translation of reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. The corresponding fractions prepared from untreated mouse and human cells either did not affect or stimulated the translation of reovirus mRNA. Ribosomal washes from untreated monkey kidney cells were inhibitory, but not as inhibitory as washes from interferon-treated cells. (3) The concentration of KCl required to release the interferon-mediated ribosome-associated inhibitor from ribosomes of mouse fibroblast cells was higher than that required for ribosomes from human amnion and monkey kidney cells. (4) The translation of both methylated and unmethylated reovirus mRNA catalyzed by the mouse ascites cell-free system was inhibited by the mouse fibroblast ribosome-associated interferon-mediated factor. These results suggest that the interferon-mediated ribosome-associated antiviral factor(s) is not species specific, even though certain interferons are relatively species specific.     

Lipopolysaccharides from Campylobacter jejuni O:41 Strains Associated with Guillain-Barré Syndrome Exhibit Mimicry of GM1 Ganglioside

Three Campylobacter jejuni, biotype 2, serotype O:41 strains that were isolated from patients who developed Guillain-Barré syndrome (GBS) and one C. jejuni isolate from a patient who developed enteritis only were examined. The aim of the study was to determine the structure of the core oligosaccharide (OS) of the lipopolysaccharide (LPS) of C. jejuni serotype O:41, a serotype rarely associated with the development of GBS, and to determine if the LPS shares similar epitopes with any of the major human gangliosides. Electrophoretic analysis with silver staining or immunoblotting demonstrated that the strains had LPS profiles characteristic of low-molecular-weight LPS. Colorimetric analysis detected N-acetylneuraminic (sialic) acid in the core OSs of all the strains. Thin-layer chromatography with immunostaining showed that antisera raised against the GBS strains reacted with the GM₁ ganglioside, suggesting that C. jejuni serotype O:41 LPSs and the GM₁ ganglioside have similar epitopes. Furthermore, polyclonal anti-GM₁ and anti-asialoGM₁ antibodies cross-reacted with each C. jejuni O:41 LPS tested, suggesting that the serotype O:41 core OS has a GM₁- and asialoGM₁-like structure. LPSs extracted from C. jejuni serostrains O:2, O:3, and O:19 were also used in the study. Cholera toxin (a GM₁ ligand) and peanut agglutinin (a Galβ1–3GalNAc ligand) recognized all serotype O:41 LPSs and the serostrain O:2 LPS. Immunoadsorption results confirmed GM₁ relatedness. Moreover, the core OS was isolated from a GBS-associated C. jejuni O:41 LPS by gel permeation chromatography. An analysis by gas-liquid chromatography (GLC), GLC-mass spectrometry, and nuclear magnetic resonance showed the core OS of one of the C. jejuni O:41 GBS isolates to have a tetrasaccharide structure consistent with GM₁ mimicry.     

Interferon externalization by producing cell before induction of antiviral state.

The mechanism by which poly(I · C) induces the antiviral state in human fibroblast cells was studied using antisera which were selectively reactive with the inducer [poly(I · C)], the product (human fibroblast interferon), and the fibroblast cell surface. Anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Furthermore, when added to the medium of the cells in which interferon synthesis had already been induced by poly(I · C), interferon antiserum prevented the intracellular antiviral state from developing. Antibodies specific to double stranded RNA inhibited the antiviral activity of poly(I · C) whether it was in solution or bound to the cells, but the same antibodies were without effect when added after the induction of the antiviral state was initiated. Treatment with serum directed against the cell surface of human fibroblasts failed to inhibit the antiviral activity of poly (I · C) or of human interferon in these cells. These results indicate that the antiviral effect of poly(I · C) is interferon mediated, and that for the development of the antiviral state, interferon has to interact with the external part of the cellular membrane of the producing cell.     

Composition of membranes of cells transformed by tumorigenic DNA and RNA viruses.

Continuous cell lines obtained from mouse and hamster embryonic tissue and human lung contain a homologous series of acidic glycolipids known as gangliosides. These complex lipids are highly concentrated in plasma cell membranes. The ganglioside pattern becomes greatly simplified when cells are transformed by tumorigenic DNA and RNA viruses. Non-transforming viruses and lytic infection of cells by tumorigenic viruses without neoplastic tranformation do not cause changes in gangliosides. The alterations of ganglioside composition are the result of specific blocks in the biosynthesis of these lipids. The significance of these impairments of membrane glycolipid formation and the nature of the regulatory site(s) involved are considered with regard to the altered social behavior of transformed cells.     

Correlation of physicohemical and antigenic properties of human leukocyte interferon subspecies

Summary Human leukocyte interferon produced in primary cultures of buffy coat cells and human fibroblast interferon from cultures of the FS-4 foreskin cell strain were subjected to isoelectric focusing in polyacrylamide gels. Leukocyte interferon could be resolved into three major components (pI 5.5, 6.2 and 6.6, respectively) and one minor component (pI 7.0). Fibroblast interferon activity focused in a broad pH range of 6.8–7.8.The isoelectrically distinct subspecies of human leukocyte interferon were isolated and compared as to their antigenic nature, heterospecific antiviral activity in cultures of bovine cells, and apparent molecular weights upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The three major subspecies (pI 5.5, 6.2 and 6.6) were similar in their neutralization by antiserum against whole leukocyte interferon and in their relative heterospecific activities on bovine cells. When analyzed on SDS-PAGE, the component focusing at pH 5.5 migrated to a position corresponding to a molecular weight of 17,500 (Le _f), the component with the pI of 6.6 had its major peak corresponding to a molecular weight of 23,000 (Le _s), while the pI 6.2 component contained a mixture of the two molecular weight species. The minor isoelectric component focusing at pI 7.0 contained interferon with the antigenic specificity of fibroblast (F) interferon. It is concluded that the two major antigenic species of human interferon (Le andF) and two known subspecies of human leukocyte interferon (Le _s andLe _f) can be resolved by isoelectric focusing.With 3 Figures     

The facilitating effect of gangliosides on the electrogenic (Na+/K+) pump and on the resistance of the membrane potential to hypoxia in neuromuscular preparation

The effects have been investigated of a mixture of gangliosides from beef brain cortex (GM₁, GD_1a, GD_1b and GT₁) either added to the bathing medium or injected intraperitoneally on muscle fibres and nerve terminals in mouse diaphragm. The electrogenic (Na⁺/K⁺) pump activity of muscle fibres enriched with sodium was increased by 38% after 2-h pretreatment with gangliosides (5×10^–8 mol ·l^–1). Muscles from animals treated with gangliosides did not show the substantial depolarization of the resting membrane potential (RMP) in K⁺-free solution (6 h) shown by control muscles. Further, treatment with gangliosides slowed the changes in muscle fibre RMP and frequency of the miniature end-plate potentials in oxygen deprived muscles.     

Antagonism in action between mouse or human interferon and platelet growth factor

Summary Human leukocyte and mouse fibroblast interferons abolished the mitogenic effect of human or bovine platelet growth factor (PGF) on human or mouse cells. Conversely, the antiviral and anticellular activity of both forms of interferon was inhibited by PGF in the homologous and in certain heterologous cell systems.     

The synthesis and actions of mouse and human interferons in mouse-human hybrid cells.

Lines of mouse-human hybrid cells segregating either mouse or human chromosomes were used for the analysis of various aspects of the production and actions of mouse and human interferons. In one of the hybrid cell lines capable of producing both mouse and human interferons, the proportion of the two interferon activities produced varied greatly under different inducing conditions, suggesting that there are differences in the triggering mechanisms of the two interferons. Generally both mouse and human interferon production could be enhanced (“superinduced”) by sequential treatment with cycloheximide and actinomycin D; however, in one of the lines producing both mouse and human interferon, only the latter could be superinduced. There was no correlation between the capacity of the lines to produce mouse or human interferons and the sensitivity to their action. However, there was good correlation between the sensitivity to the antiviral action and the priming action (i.e., enhancement of subsequent interferon production) by the two interferons. Thus, line 55-91F2 produced both mouse and human interferons, but was sensitive to the antiviral and priming actions of human interferon only. Line GM52 × BalbVC15 produced only mouse interferon but was sensitive to the antiviral and priming actions of both interferons.     

Sensitivität verschiedener Primatenzellen und animaler Viren gegenüber der antiviralen Aktivität von menschlichem Leukocyteninterferon

U cells (a permanent, human amnion cell line) were protected against infection with Semliki Forest Virus (SFV) by human interferon (HIF) from peripheral leukocytes. Despite the usual genus-specific action of interferons, mouse L929 cultures (a permanent mouse fibroblast cell line) were also protected by HIF. The antiviral action of HIF in six other primate cell cultures was also examined. It is of interest that two lymphoblastoid cell lines, RPMI1788 and Kaplan, were insensitive to HIF. The sensitivity of ten different viruses against HIF in primary African green monkey kidney cell cultures was compared. Among the viruses tested SFV was the most sensitive whereas two strains of vaccinia virus were barely inhibited. In contrast, type 1 herpes simplex virus was relatively sensitive to the action of HIF.     

Type I and type II interferons: differential antiviral actions in transformed cells

In transformed mouse embryo cells, type II interferon had much less antiviral activity than type I interferon. In non-transformed cells, the two interferons had similar high activity, Reversal of the phenotype of Moloney sarcoma virus (MSV) transformed cells by sodium butyrate restored their sensitivity to the antiviral action of type II interferon. Additional evidence for a role of the cytoskeletal network in the action of type II interferon is that its antiviral effect is reduced by cytochalasin B, colchicine or vinblastine. MSV-transformed cells, selected for their resistance to the antiviral action of type I interferon, were sensitive to type II interferon. These differences in the effects of type I and II interferon on transformed cells are at present unexplained, but suggest that they have at least partially separate mechanisms of action.