High affinity binding of 125I-Labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties

Direct ligand-binding studies with highly purified ¹²⁵I-labeled virus-induced mouse interferon on mouse lymphoma L 1210 cells revealed a direct correlation of specific high-affinity binding with the biologic response to interferon. Neutralization of the antiviral effect by anti-interferon gamma globulin occurred at the same antibody concentration as the inhibition of specific binding. These results suggest that specific high-affinity binding of ¹²⁵I-interferon occurred at a biologically functional interferon receptor. Competitive inhibition experiments using ¹²⁵I- and ¹²⁵I-labeled interferon provided strong evidence that the fraction of ¹²⁵I-interferon inactivated upon labeling did not bind specifically. Scatchard analysis of the binding data yielded linear plots and thus suggested that interferon binds to homogeneous noncooperative receptor sites. In contrast to a characteristic property of several peptide hormone systems, binding of ¹²⁵I-interferon to its specific receptor did not induce subsequent ligand degradation. At 37° bound interferon was rapidly released in a biologically active form without evidence for molecular degradation. The expression of interferon receptors was not modified by treatment with interferon. Trypsin treatment of target cells and inhibition of protein synthesis abolished the specific binding of ¹²⁵I-interferon. Three major molecular weight species of Newcastle disease virus-induced mouse C 243 cell interferon were isolated separately and identified as mouse α and β interferons. These interferons were shown to inhibit competitively the specific binding of the highly purified labeled starting material which contained all three size species, thus providing evidence for a common receptor site for mouse α and β interferon.     

Specific high-affinity binding of 125I-labeled mouse interferon to interfevon resistant embryonal carcinoma cells in vitro

Multipotential mouse embryonal carcinoma cells are resistant to several biologic effects of mouse interferon: inhibition of viral multiplication and inhibition of cell division. Nevertheless using ¹²⁵I-labeled highly purified mouse interferon we have shown that these embryonal carcinoma cells express specific interferon receptors in similar number and of the same affinity as interferon sensitive differentiated embryonal carcinoma cells. Thus, mechanisms of interferon resistance are probably multiple: some cells are resistant because they lack specific binding sites for interferon (interferon resistant mouse L1210 cells) and other cells, as shown herein, are resistant to some of the effects of interferon despite binding of interferon to specific receptor sites. Furthermore, these binding sites may be considered functional, since interferon does induce an increase in 2-5A synthetase in these cells.     

Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin.

The receptors for cholera toxin and Escherichia coli heat-labile toxin (LT) in rabbit small intestinal epithelium were characterized and compared. (i) In vivo studies in ligated intestinal loops showed that whereas LT B subunits could block the fluid secretogenic action of purified LT as well as cholera toxin, cholera toxin B subunits did not inhibit the LT response even when tested in a concentration 100-fold higher than one which gave complete blocking of cholera toxin action. (ii) In vitro studies indicated that isolated intestinal epithelial cells or brush-border membranes could bind about 10-fold more of E. coli LT than of cholera toxin. (iii) All binding sites for cholera toxin in duodenal, jejunal, or ileal mucosal cells or brush-border membranes were extracted by chloroform-methanol-water (4:8:3), which removed lipids quantitatively but did not extract glycoproteins. The extracted cholera toxin binding sites were to greater than 95% recovered in a monosialoganglioside fraction; quantitatively these sites closely corresponded to the concentration of chromatographically identified mucosal GM1 ganglioside (1 nmol of cholera toxin was bound per 1 to 2 nmol of GM1). In contrast, a substantial fraction of mucosal binding sites for E. coli LT remained in the delipidized tissue residue, and these sites had properties consistent with a glycoprotein nature. Thus, whereas cholera toxin appeared to bind highly selectively to GM1 ganglioside receptor sites of rabbit small intestine, E. coli LT bound both to GM1 ganglioside and to a main glycoprotein receptor for which cholera toxin lacks affinity.     

Effect of cholera toxin on the antiviral and anticellular activities of human leukocyte interferon.

Cholera toxin added into cell cultures together with human leukocyte interferon inhibited the establishment of the antiviral state by interferon but not the anticellular activity of interferon in human cells. Sensitivities of various human cell lines to anticellular activities of interferon and cholera toxin were compared, but no direct correlation between both activities were demonstrated. These results suggest that antiviral and anticellular activities of interferon are due to different mechanism of actions, and cholera toxin does not act directly on the receptor site for interferon.     

Enhancement of cellular protein synthesis sensitivity to diphtheria toxin by interferon.

In attempts to determine whether, by analogy to cholera and tetanus toxins, diphtheria toxin (DT) can also relieve the antiviral effect of interferon (IF), we found that it rather enhanced the inhibitory effect of IF on the replication of murine leukemia virus in chronically infected NIH/3T3 cells. This enhancement was found to be a consequence of an increased sensitivity to DT of cellular protein synthesis in IF-treated cells. IF stimulated the anti-protein synthesis activity of DT in both mouse cells that are known to be highly resistant to this toxin and in human HeLa cells that are highly sensitive to this toxin. This stimulation was dependent on IF dose. The effect of IF on DT action was strictly species specific, indicating that it was not a consequence of the mere binding of IF to the cell membrane, but rather reflected the cellular changes that followed this initial binding. IF was found to be capable of potentiating intact DT, but could not potentiate its fragments in any combination. IF did not have any effect on the in vitro nicotinamide adenine dinucleotide glycohydrolase activity of DT, suggesting that the effect of IF is not due to molecular modification of the toxin.     

Ultrastructural distribution of interferon receptor sites on mouse L fibroblasts grown in suspension: ganglioside blockade of ligand binding.

Murine beta-interferon (IFN) receptors on L929 cells grown in suspension culture were visualized by indirect immunoferritin electron microscopy. Ferritin label on these cells was associated primarily with the coated areas and coated pits of the membrane, in contrast to previous observations with L929 cells grown in a monolayer, which did not reveal such coated areas or pits but showed ferritin label distributed randomly on the cell membrane (Kushnaryov et al., Infect. Immun. 36:811-821, 1982). On about 15% of the cell sections from suspension-grown cells, the ferritin label was found outside coated membrane areas. These findings suggest that different cell populations exist with respect to the localization and possibly the affinity of IFN receptors. In the same experiment, exogenously added gangliosides blocked the binding to cell surfaces not only of 125I-labeled IFN but also of unlabeled IFN as revealed by an immunospecific ferritin labeling technique, providing direct evidence that gangliosides interfere with the binding of IFN to specific receptor sites on the surface of mouse L929 cells. These studies establish that the binding of IFN to cell membranes, depending on cell growth conditions, can involve coated areas and coated pits, to which certain hormones and toxins have been shown to bind.     

Effect of serum on the antiviral and anticellular activities of mouse interferon

Summary Fetal bovine serum markedly decreased the ability of mouse L-929 interferon preparations to inhibit the formation of L-929 clones, but did not affect their ability to inhibit vesicular stomatitis virus (VSV) plaque formation in these cells. This dissociation of effects by interferon preparations indicates that: 1. the mechanism of action of interferon for its anticlonal and antiviral activities is different; or 2. the molecule responsible for the anticlonal activity is a separate growth inhibitory factor.With 2 Figures     

Rapid increase in guanosine 3',5'-cyclic-monophosphate interferon treated mouse leukemia L1210 cells: relationship to the development of the antiviral state and inhibition of cell multiplication

Treatment of mouse leukemia L1210 cells with electrophoretically pure mouse interferon caused a rapid (1 to 5 min) and transitory (duration 5 to 10 min) increase in the intracellular concentration of guanosine 3′,5′-cyclic monophosphate (cyclic GMP). No significant increase in the intracellular concentration of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) was observed until 2 to 3 hr after the addition of interferon. Electrophoretically pure mouse interferon caused a dose-dependent increase in the intracellular concentration of cyclic GMP at concentrations ranging from 1.0 to 10⁴ unite/ml. The interferon-induced increase in cyclic GMP was abrogated by specific antibody to mouse interferon. Furthermore, interferon had no effect on the intracellular concentration of cyclic GMP in a line of L1210 cells resistant to the action of interferon. The interferon-induced increase in cyclic GMP was calcium dependent and was inhibited by indomethacin and aspirin at concentrations of 10^?7 to 10^?6 M. Depletion of intracellular calcium prior to the addition of interferon abrogated the interferon-induced increase in cyclic GMP without preventing either the development of the antiviral state or the inhibition of cell multiplication in interferon-treated L1210 cells. These results suggest that cyclic GMP may not mediate these two biologic effects of interferon but may rather reflect rapid changes in interferon-treated cells.     

Purification of interferon by adsorption chromatography on controlled pore glass.

Human fibroblast and mouse L929 cell interferons can be purified by adsoprtion to and subsequent elution from Controlled Pore Glass. Purification of 40 to 90-fold to specific activities of 1 to 5 times 10(6) units/mg of protein can be achieved in a single step, with good recovery of activity. Human leukocyte interferon does not bind to the glass and cannot be purified in this way.     

Induction of delta 3(4) ketosteroid synthesis by interferon in mouse adrenal tumour cell cultures

Interferon, like cholera toxin, induces in mouse adrenal YI cells the production of delta 3(4) ketosteroids. In parallel, cyclic AMP (cAMP) is activated. At high concentrations, mouse but not human interferon induces a cell rounding effect, similar to that obtained with cholera toxin. In YI cells transformed by simian adenovirus 7, production of cAMP is still increased by that of steroids is blocked. The role if interferon in these cellular events is discussed.     

Effect of different interferon preparations on the synthesis of cellular DNA

The relationship between the age of interferon producers and the capacity of interferon to inhibit DNA synthesis in L929 cells was demonstrated on interferon models produced by fibroblast cultures from newborn and adult mice in the presence of blood sera of hemologous ages. Inhibition of DNA synthesis by both types of interferon partially purified with the use of porous glass and having similar antiviral activity depended both on the dose of the preparation and on the time of cell incubation with it. Under equal conditions, the interferon produced by newborn mouse cells inhibited DNA synthesis 1.9-fold less effectively than interferon produced by adult mouse cells.     

Inhibition of murine L cell interferon action by heparin

Summary Heparin added together with murine L cell interferon inhibited the development of antiviral activity in mouse L cells. When added after interferon treatment, heparin had no effect on antiviral activity. There was also no inhibition of interferon action in L cells treated with heparin before addition of interferon. On the other hand, heparin did not inhibit antiviral activities of human interferon and . Since murine L cell interferon, but not human interferon and , binds to a heparin affinity column and can be eluted with a solution of high salt, it is presumed that murine L cell interferon and heparin must interact with each other. The apparent interaction of heparin with murine L cell interferon was prevented by protamine, a drug that neutralizes heparin. Dextran sulfate inhibited murine L cell interferon action, but dextran and chondroitin sulfate A did not. These results suggests that heparin inhibited murine L cell interferon action by the binding via sulfate groups on its molecules. Heparin also inhibited antiviral activity of murine L cell interferon in mice infected with herpes simplex virus (+GC Miyama strain).With 6 Figures     

Chronic production of interferon in carrier mice congenitally infected with lymphocytic choriomeningitis virus

Congenital infection of mice with lymphocytic choriomeningitis virus leads to a lifelong virus carrier state. Here we provide evidence for the presence and action of interferon in such mice. The level of circulating interferon in adult carrier mice is 8–16 NIH units/ml of plasma. This interferon is acid stable and is capable of inducing 2–5A synthetase in mouse L 929 cells but not in human HeLa cells. Control, noinfected mice show less than 1 NIH unit/ml of plasma. In accord with these results, adult carrier mice have higher levels of the interferon-mediated pppA(2′p5′A)_n synthetase (2–5A synthetase) in their liver and spleen than normal mice. Congenitally infected newborn mice also have higher levels of 2–5A synthetase in their liver in contrast to newborn control mice. These results in congenitally infected newborn and adult mice suggest that interferon may play a role, at least in part, in the pathogenesis of infection.     

On the inhibition of interferon action by inhibitors of fatty acid cyclooxygenase

Experiments were carried out to investigate the proposed involvement of the prostaglandin (PG) biosynthesis pathway in interferon (IFN) action. Several known inhibitors of fatty acid cyclooxygenase (indomethacin, ibuprofen, oxyphenylbutazone) were found to inhibit the antiviral action of IFN in mouse L929 and human FS-4 cells. However, the concentrations of these inhibitors required for this effect were much higher than those used to inhibit PG biosynthesis. Furthermore, we found that the inhibitors tested also blocked RNA and protein synthesis, and that the concentration dependence for these effects of the inhibitors and for the inhibition of IFN action were similar. This could account for the inhibitory effect of these compounds on IFN action without invoking an involvement of the cyclooxygenase pathway.     

Production,purification and characterization of rat interferon

Summary Priming with heterologous mouse interferon, increased production of an antiviral substance induced in rat diploid fibroblasts by Newcastle disease virus. This substance was characterized as an acid stable interferon. This rat interferon exhibited marked cross-species antiviral activity when tested in mouse L929B cells, guinea pig embryo fibroblasts, human fibroblasts and bovine cells but was not active on chick embryo cells.     

Isolation of Rickettsia prowazekii with reduced sensitivity to gamma interferon.

The growth of Rickettsia prowazekii Madrid E was monitored in mouse L929 cells subcultured for several weeks in the presence of gamma interferon (IFN-gamma) to determine whether the rickettsiae would be eliminated from or would persist in these cultures. R. prowazekii exhibited two distinct patterns in these IFN-gamma-treated cultures. In some cases, IFN-gamma-induced inhibition of rickettsial growth led to elimination of the rickettsiae from the L929 cell cultures; in other cases, the initial inhibition of rickettsial growth was followed by establishment of a persistent rickettsial infection in the IFN-gamma-treated L929 cells. During the first 3 days after infection, the growth rate of the L929 cells was significantly lower and higher percentages of the cells were killed in the IFN-gamma-treated, R. prowazekii-infected cultures than in the untreated, R. prowazekii-infected cultures or the mock-infected cultures, whether treated or untreated. This suppression of cell growth occurred in the infected, IFN-gamma-treated cultures that eventually exhibited the elimination pattern as well as the IFN-gamma-treated cultures that became persistently infected. It was not possible to predict the outcome of a particular infection from the early growth pattern of the culture. It was determined that the L929 cells in the persistently infected, IFN-gamma-treated cultures had not lost the ability to respond to IFN-gamma. These cells, after treatment with an antibiotic to eliminate the persistent rickettsiae, retained the ability to inhibit both the replication of vesicular stomatitis virus and the growth of R. prowazekii Madrid E after treatment with IFN-gamma. In contrast, rickettsiae isolated from two persistently infected, IFN-gamma-treated cultures were less sensitive than R. prowazekii Madrid E to the antirickettsial effects of IFN-gamma in standard L929 cells. The maintenance of the phenotype of these altered rickettsiae during plaque purification and passage in the absence of IFN-gamma suggests an alteration at the genetic level rather than phenotypic adaptation.     

Inosiplex-dependent interferon production in vivo and in vitro

Inosiplex, an immunopotentiator, was shown to be capable of inducing interferon synthesis in vivo and in cell culture. Its concentrations of the order of 25 mg/mouse were to be most effective. The time course of production showed a late peak at 72 hours. The resulting interferon was not neutralized by antiserum to alpha- and beta-interferons. L929 cells of peritoneal exudate and splenocytes could synthesize interferon after treatment with inosiplex. The use of mixed cultures of these cells increased the effectiveness of interferon induction.     

Mechanism of immune interferon production in vitro: interaction between immune interferon-producing cells and antigenic cells.

Various processes of in vitro immune interferon production by sensitized spleen cells stimulated with allogeneic cells were investigated. When L cells, an interferon-inducing antigen, were fixed with methyl alcohol or paraformaldehyde, the ability to induce immune interferon disappeared. In this immune interferon production system, the majority of sensitized spleen cells adhered to target cells within 1 h of cocultivation. Adherence of immune interferon-producing cells to target cells was observed only when L cell-sensitized spleen cells were cocultured with L cells or with mouse embryo cells derived from C3H mice. Fixation of antigenic cells with methyl alcohol or paraformaldehyde significantly reduced cell adherence. When L cells alone or sensitized spleen cells alone were pretreated separately with cytochalasin D, neither cell type could bind to partner cells. Specific adherence did not take place at 4 degrees C, nor in the presence of dinitrophenol or sodium azide. Continuous protein synthesis in both cells was not required for immune cell adherence. Divalent cations, Ca2+ or Mg2+, were required for this immune specific adherence to take place. However, once stable adherence was established, treatment with cytochalasin D, ethylenediaminetetraacetic acid, or sodium azide, or simple reduction of temperature, did not disrupt the binding. Interaction between immune interferon-producing cells and antigenic cells can be subdivided into two phases according to the requirement for divalent cations: (i) lymphocytes and antigenic cells interact transiently, and divalent cations are required to maintain the binding; (ii) lymphocytes and antigenic cells form a stable interaction, and deprivation of divalent cations does not disrupt the binding. Colchicine showed an inhibitory effect in the period after cell-to-cell adherence. Colchicine did not inhibit the release of interferon. On the other hand, vinblastine, another antimicrotubule agent inhibited the secretion of immune interferon. Since interferon synthesis was not stopped immediately after addition of cycloheximide, continued protein synthesis of sensitized spleen cells was not required for interferon secretion. The present study showed that adherence of immune interferon-producing cells to antigenic cells was a complex phenomenon involving a series of successive events.     

Inhibition by interferon of Sendai virus cytolysis.

Treatment of HeLa cells with human interferons inhibited 51Cr release from cells induced by ultraviolet-inactivated Sendai virus. The inhibitory effect became apparent about 6 h after interferon treatment and persisted for 24 to 48 h. In the interferon-treated cells, the cytolysis was inhibited within 10 min after adding virus and the inhibitory action was suppressed by the treatment of the cells with cycloheximide. Mock interferon and mouse interferon did not inhibit the cytolysis and antiinterferon serum neutralized the effect of interferon. All these findings indicate that Sendai virus-induced cytolysis is inhibited by interferon per se. However, interferon did not have any influence on Sendai virus hemolysis.     

Role of the nitric oxide synthase pathway in inhibition of growth of interferon-sensitive and interferon-resistant Rickettsia prowazekii strains in L929 cells treated with tumor necrosis factor alpha and gamma interferon.

The ability of tumor necrosis factor alpha (TNF-alpha) alone and in combination with gamma interferon (IFN-gamma) to inhibit the growth of interferon-sensitive and -resistant Rickettsia prowazekii strains in mouse L929 cells was examined, and the possible role of the nitric oxide synthase pathway in the suppression of rickettsial growth induced by TNF-alpha, IFN-gamma, or both cytokines was evaluated. TNF-alpha inhibited the growth of strains Madrid E (IFN-gamma sensitive and alpha/beta interferon [IFN-alpha/beta] sensitive) and Breinl (IFN-gamma sensitive and IFN-alpha/beta resistant), but not that of strain 83-2P (IFN-gamma resistant and IFN-alpha/beta resistant), in L929 cells. Inhibition of the growth of the Madrid E strain in L929 cells treated with TNF-alpha and IFN-gamma in combination was greater than that observed with either TNF-alpha or IFN-gamma alone. Similarly, inhibition of the growth of the Breinl strain in L929 cells treated with both cytokines was greater than that observed with TNF-alpha alone; however, it did not differ significantly from the inhibition observed with IFN-gamma alone. Although strain 83-2P was resistant to TNF-alpha or IFN-gamma alone, its growth was inhibited in L929 cells treated with TNF-alpha and IFN-gamma in combination. Nitrite production was measured in mock-infected and infected L929 cell cultures, and the nitric oxide synthase inhibitors NG-methyl-L-arginine (NGMA) and aminoguanidine were used to evaluate the role of the nitric oxide synthase pathway in cytokine-induced inhibition of rickettsial growth. Nitrite production was induced in mock-infected or R. prowazekii-infected L929 cell cultures treated with IFN-gamma plus TNF-alpha, but not in mock-infected cultures that were untreated or treated with IFN-gamma or TNF-alpha alone. Nitrite production was also not induced in untreated, R. prowazekii-infected cultures; however, in some instances, it was induced in infected cultures treated with IFN-gamma or TNF-alpha alone. Nitrite production was blocked by NGMA or aminoguanidine, and these compounds markedly relieved the synergistic inhibitory effect of IFN-gamma plus TNF-alpha on the growth of strain 83-2P in L929 cells. In contrast, NGMA did not alleviate the inhibition of the growth of the Madrid E strain in L929 cells treated with IFN-gamma or TNF-alpha alone; however, it slightly and variably relieved the inhibition of the growth of the Madrid E strain in L929 cells treated with IFN-gamma and TNF-alpha in combination.(ABSTRACT TRUNCATED AT 400 WORDS)