Mouse Interferon Preparations Research Articles

The antitumor effects of interferon: A personal history

Early experiments showed that administration of mouse interferon preparations inhibited the development of viral-induced or spontaneous viral associated leukemias in mice. Interferon α/β was also shown to inhibit the growth of transplantable tumors of different origins in all strains of mice tested. The finding that interferon α/β inhibited the growth of sublines of tumors selected for resistance to interferon α/β indicated the role of interferon induced host mechanisms in the antitumor effects observed. The different host antitumor mechanisms and especially the interaction of interferon α/β with the immune system have been briefly discussed. Injection of mice with a neutralizing antibody to interferon α/β demonstrated the essential role of endogenous interferon α/β in the defense of the mouse against the development of syngeneic, allogeneic and xenogeneic tumors.

Interferon treatment markedly inhibits the development of tumor metastases in the liver and spleen and increases survival time of mice after intravenous inoculation of Friend erythroleukemia cells.

To investigate the effect of interferon treatment on the development of tumor metastases, DBA/2 mice were injected i.v. with 2 X 10(6) Friend erythroleukemia cells (FLC) (equivalent to about 5 X 10(5) LD50). FLC multiplied rapidly in the liver and spleen and all untreated or control treated mice died between 7 and 12 days. Daily treatment of mice with potent preparations of mouse interferon alpha/beta was initiated 3 to 72 hr after i.v. inoculation of tumor cells, at times when FLC were already present in the liver and spleen. Interferon treatment resulted in a 100 to 1,000-fold inhibition of the multiplication of FLC in the liver and spleen and a marked increase in mean survival time. Small numbers of tumor cells persisted in the liver and spleen in some interferon-treated mice and could be recovered by bioassay several weeks after tumor inoculation. Most interferon-treated mice died with tumor in the ensuing months. Three of 34 interferon-treated mice were considered cured as they were alive at 386, 325 and 284 days after tumor inoculation. Daily treatment of tumor-inoculated mice with human recombinant interferons alpha D and alpha BDDD, which had antiviral activity on mouse cells in culture, also increased the survival time of mice injected i.v. with FLC. The use of the interferon-resistant 3C18 line of FLC suggests that the marked inhibition of development of established liver and spleen metastases was not due to a direct effect of interferon on the tumor cells, but was host-mediated.

Interferon alpha/beta induces changes in the metabolism of polyenoic phospholipids and diacylglycerols in the livers of suckling mice.

Suckling mice were injected daily from birth for 10 days with potent preparations of mouse interferon alpha/beta. Interferon treatment resulted in a markedly lower concentration of polyunsaturated fatty acids (20:4 omega 6 and 22:6 omega 3) in the two principal liver phospholipids, phosphatidylcholine and phosphatidylethanolamine, than in livers of control-treated mice. This effect appeared to correlate with a low level of synthesis of polyunsaturated phospholipids in the livers of interferon-treated mice. Thus, in control mice, synthesis of species of polyunsaturated phospholipids increased markedly in the first 10 days of life, whereas in 10-day-old interferon-treated mice, the level of synthesis of species of polyunsaturated phospholipids was comparable to that in newborn mice. In parallel, a marked increase in the diacylglycerol content without change of its renewal was observed in the livers of interferon-treated mice. We suggest that interferon treatment results in an inhibition of one of the processes that leads to activation of the enzymatic systems responsible for the synthesis of species of polyunsaturated phosphatidylcholine and phosphatidylethanolamine in the liver of suckling mice. It seems likely that these results are related to the inhibition of liver cell maturation and the marked cell necrosis that are observed in interferon-treated suckling mice.

Interferon alters the composition and metabolism of lipids in the liver of suckling mice.

Daily injection of suckling Swiss, C3H, and C57BL/6 mice with potent preparations of mouse interferon (IFN) alpha/beta resulted in a decrease in phospholipids and a decrease in the phospholipid-to-protein ratio in the liver. This decrease in polar lipids was accompanied by a marked accumulation of triglycerides without a pronounced change in free cholesterol. In contrast, a similar IFN treatment of adult mice did not change the content of liver phospholipids, although there was an increase in liver triglycerides. In suckling mice, after 10 days of IFN administration there was a marked decrease in the incorporation of 1(3)-[3H]glycerol into liver phospholipids, which was not observed in IFN-treated adult mice. Our findings suggest that IFN treatment results in the inhibition of some component of the system of phospholipid biosynthesis during the maturation of hepatocytes in suckling mice.

Interferon-gamma inhibits growth of Coxiella burnetii in mouse fibroblasts.

We studied the effects of various mouse interferon preparations on the growth of Coxiella burnetii in mouse fibroblasts. The addition of both recombinant interferon-gamma and a crude lymphokine preparation that contained interferon-gamma to infected L929 cells inhibited the growth of C. burnetii, whereas the addition of a crude preparation of type I interferons did not. Cycloheximide suppressed the inhibitory effects of recombinant interferon-gamma and crude lymphokines.

Injection of mice with antibody to interferon enhances the growth of transplantable murine tumors.

Injection of DBA/2, C57Bl/6, or BALB/c mice with antibody to mouse interferon alpha/beta enhanced the i.p. transplantability of six different murine tumors, as manifested by an increase in the percentage of tumor-bearing mice and a decrease in survival time. The effect was observed in mice injected with antibody to interferon raised in three sheep, a goat, and a rabbit, but not with sheep antibody to "impurities" present in the mouse interferon preparations or with normal sheep or goat globulins. The enhancement in transplantability was most marked when tumor cells had been previously passaged in vitro and were of low tumorigenicity. Analysis of some of the experimental conditions using interferon-sensitive and interferon-resistant lines of Friend erythroleukemia cells (FLC) showed that the enhancing effect was observed over a wide range of tumor cell inocula, was directly related to the amount of antibody to interferon injected and was most pronounced when antibody was administered at the time of tumor cell injection. Enhancement was also observed when FLC were injected subcutaneously (s.c.). Antibody did not act directly on the tumor cells in vitro. Although we were unable to demonstrate any biologically active interferon in mice before or after tumor cell inoculation, the results suggest that endogenous interferon is present and plays a role in inhibiting the transplantability of some murine tumors in immunocompetent mice.

Failure to Modify Scrapie in Mice by Administration of Interferon or Anti-interferon Globulin

Administration of potent mouse interferon preparations or anti-mouse interferon globulin did not influence the evolution of scrapie in mice after intraperitoneal injection of the agent. We conclude that the interferon system is probably not involved in scrapie.

Homogeneous interferon from E. coli depresses hepatic cytochrome P-450 and drug biotransformation

A highly purified homogeneous human interferon produced from cloned genes depressed the levels of hepatic cytochrome P-450 and related xenobiotic metabolism. Using another cloned human interferon and several impure preparations of human and mouse interferon, it appears that only interferons with antiviral activity in the mouse depress cytochrome P-450 in that species. This is the first direct evidence that interferon decreases hepatic drug biotransformation and likely explains the depression of drug elimination which occurs during viral infections or following the administration of interferon inducers.

Interferon inhibits the suppressor T cell response of delayed-type hypersensitivity.

We have recently shown that pretreatment of BALB/c mice with Corynebacterium parvum preferentially inhibits suppressor T lymphocytes (Ts) which have been induced by an epicutaneous antigen overload1 or by intravenous (i.v.) injection of 2,4-dinitrobenzenesulphonic acid (DNBSO3)2. Serum obtained 24 h after C. parvum injection had a similar suppressive effect on Ts but no effect on T effector lymphocytes of delayed-type hypersensitivity (TDH)3 Inhibition of Ts by this serum could be neutralized by an anti-interferon globulin4. A crude mouse fibroblast interferon (IFN) preparation also inhibited the Ts response4, which suggested that IFN in C. parvum serum might be the Ts inhibitory factor. We have now studied the effect of an electrophoretically purified mouse interferon (IFN-α and β) preparation on the Ts response to dinitrofluorobenzene (DNFB) in BALB/c mice. Small amounts (1,000 U) of the preparation inhibited the generation of Ts by DNBSO3 in vivo, and the function of Ts both in the recipient animals when injected i.v. 2 h after transfer of the Ts and after incubation of the spleen ceUs from tolerant animals with interferon before transfer into recipients. The TDH response was not affected by this amount of interferon.

60] Differentiation of types of interferon by kinetics of development of the antiviral state

This chapter presents the different kinetics of activation of the antiviral state by immune and virus-induced interferons is shown with crude preparations of both human and mouse interferon, and it is quite reproducible provided that a rigorous control of the temperature at 37 ° is achieved throughout the cell activation phase. Another important variable that must be considered is the type of cell culture to be used in the test. All the primary or diploid cells tested may be activated by treatment for a few minutes with fibro blastor leukocyte interferons, whereas most continuous cell lines may require longer treatment, although some rapidly responding clones may be found among human and mouse cell lines. For instance, a clone of mouse L cells is indentified and one of human WISH cells with a rapid response similar to that shown by primary or diploid cell cultures. In general, however, kinetics of activation by immune interferon appears to be much slower even in cells that require a relatively long exposure to fibroblast and leukocyte interferon to develop a substantial level of antiviral resistance. It has to be pointed out, however, that the choice of rapidly responding cells is preferable for a more practical and accurate differentiation of the two types of kinetics. When these considerations are taken into account, comparative determination of the kinetics of activation of the antiviral state provides a practical, reliable, and accurate method to differentiate immune from fibroblast and leukocyte interferons.

Effect of virus-induced interferon on the antibody response of suckling and adult mice.

Continued administration of potent virally induced mouse interferon (IFN type I) preparations to suckling mice resulted in an inhibition of the primary antibody response to sheep erythrocytes (14-day-old mice). When slightly older suckling mice were immunized (17 days old), a delay (about 2 days) in the kinetics of the response was observed, but the amplitude of the response was similar in both control and IFN-treated animals. In adult mice, potent IFN preparations did not inhibit the antibody response under a variety of experimental conditions (different doses of IFN, schedules of treatment, routes of injection, times of assay). Although IFN does inhibit the in vitro antibody response, we conclude that under most experimental conditions, injection of IFN type I is not immunosuppressive in adult mice. When administered after immunization, IFN clearly enhanced the primary antibody response. It is of interest that IFN, a product of viral infection, enhances in vivo those components of the immune system that have been implicated in the host response to viral infections (i.e. delayed-type hypersensitivity, natural killer cell and macrophage activity, expression of lymphocyte membrane molecules), and, as is shown here, the primary antibody response.

Interferon-induced disease in mice and rats.

Treatment of newborn mice with potent mouse interferon preparations resulted in an acute "early" syndrome characterized by inhibition of growth, delay in maturation of several organs, diffuse liver cell necrosis and death. When interferon treatment was discontinued at 1 week of life, mice appeared to recover, but subsequently developed a progressive glomerulonephritis ("late syndrome"). Treatment of newborn rats with potent rat interferon preparations also resulted in inhibition of growth, delay in maturation, and the subsequent development of glomerulonephritis. After infection at birth with lymphocyte choriomeningitis (LCM) virus, most strains of mice developed a similar acute early syndrome and surviving mice subsequently developed glomerulonephritis. We postulated that the endogenous interferon induced by LCM virus early in life was partially responsible for these syndromes. Administration of a potent anti-mouse interferon serum to LCM virus-infected mice neutralized the circulating endogenous interferon and inhibited the development of both the early and late syndromes. Our results suggest that large amounts of exogenous or endogenous interferon at a crucial stage of rapid growth or development of mice and rats can induce lesions in several different organs. Some lesions (i.e. the kidney) only become apparent weeks or even months after exposure to interferon.

Characterization of a subspecies of mouse interferon cross-reactive on human cells and antigenically related to human leukocyte interferon

Mouse interferon preparations from various virus-induced cell cultures exhibited some antiviral activity (about 0.1% of homologous cell titers) on human cells. When analyzed by SDS-polyacrylamide gel electrophoresis the activity of mouse interferon on mouse cells migrated as two bands with activity peaks at 38,000 and 22,000 daltons, while the activity on human cells was detectable only in the faster migrating portion of the lower molecular weight band, at about 21,000 daltons. When the mouse interferon preparation was chromatographed on a column of immobilized anti-human leukocyte interferon antibodies, the specifically retained interferon was found to migrate in SDS-polyacrylamide gels as a 21,000-dalton species which exhibited similar degrees of activity on both mouse and human cells. Thus, mouse interferon preparations contain a low molecular weight interferon species which is both active on mouse cells and is responsible for all the heterologous antiviral activity on human cells and is antigenically related to human leukocyte interferon. These data suggest significant structural similarities between the active cores of certain interferons from phylogenetically diverse animal species.

Enhancement of the human mixed lymphocyte culture response by pretreating the stimulating cells with interferon.

The influence of interferon (IF) on the human mixed lymphocyte culture (MLC) reaction was examined. The presence of human IF in the MLCs but not pretreatment of the responding cell population with IF reduced the reaction. Pretreatment of the stimulating cells with human IF enhanced their MLC stimulatory capacities. This effect was observed with four different IF preparations of varying origin and purity, but not with a human mock or a mouse IF preparation.

Spontaneous loss and subsequent stimulation ofH-2 expression in clones of a heterozygous lymphoma cell line

The expression of H-2Kk antigens in a (C3H × DBA/2)F1 lymphoma cell line growing in vitro was investigated with monoclonal antibodies specific for a public antigen of theH-2K k region (H-2.m3) in fluorescence analysis and microcytotoxicity assays and in cell-mediated cytotoxicity with allogeneically stimulated effector cells. Estimates of relative levels of H-2Kk-antigen expression obtained by the different methods were highly correlated. The uncloned, unselected population gradually lost H-2Kk surface antigen expression under culture conditions. This was due to the appearance of H-2Kk negative variants. Fifteen cloned sublines of a population enriched for cells expressing antigen H-2.m3 in the fluorescence activated cell sorter contained either two distinct populations, one consisting of H-2.m3 negative and one of H-2.m3 positive cells, or consisted of H-2.m3 negative cells only. The expression of the H-2.m3 determinant of H-2Kk paralleled that of other serological H-2Kk determinants and of H-2Kk target determinants for cell-mediated cytotoxicity. In nearly all clones where two populations could be detected, the proportion of H-2.m3 negative cells increased with time in culture. The amounts of H-2Kk antigen expressed by the clones appeared not to be correlated to the amounts of H-2Dk antigens on the cell surface as judged by cell-mediated cytotoxicity. In at least one clone and in the uncloned population, H-2Kk-antigen expression detectable by fluorescence analysis could be stimulated by growing the cells in the peritoneal cavities of (C3H × DBA/2)F1 mice or by adding mouse interferon preparations to the cell cultures. The increase in susceptibility to cell-mediated lympholysis of cells grown in vivo paralleled the increase inH-2 expression detected by fluorescence. In contrast, cells growing in the presence of interferon in vitro showed reduced sensitivity to lysis by alloreactive lymphocytes, although H-2 antigens were strongly expressed as measured by fluorescence.

Impairment of reovirus mRNA ‘cap’ methylation in interferon-treated mouse L929 cells

Reovirus mRNAs synthesized in vitro by the virionassociated enzyme have a 5′ ‘cap 1’ structure (m 7G(5′)ppp(5′)GmpCp …). However, about one third to one half of the reovirus mRNAs formed in mouse L929 cells have a 5′ ‘cap 2’ structure (m 7G(5′)ppp(5′)GmpCmp …) and the rest have a 5′ ‘cap 1’ structure. The finding that virus mRNA ‘cap’ methylation is impaired in extracts of interferon-treated cells prompted us to study the effect of interferon on virus mRNA ‘cap’ methylation in vivo. Using labeling with [ 3H]-guanosine and dual labeling with [ 3H]methionine and [ 14C]uridine we compared the 5′ structures of reovirus mRNAs accumulating between 5 and 11 h after infection in: L929 cells treated with 390 to 2600 U/ml of a partially purified mouse interferon preparation and untreated L929 cells. The treatment resulted in a 70 to 98% decrease in the 24 h virus yield and in a 50 to 55% decrease in the label accumulated in virus mRNAs. The ‘capping’ of virus mRNAs and the methylation of their 5′ terminal and adjacent G residues were not diminished in interferon-treated cells. However, the percent of ‘cap 2’ termini was 36 to 47% lower in virus mRNAs from interferon-treated cells than in virus mRNAs from control cells. The interferon treatment did not result in the appearance of additional methylated nucleotides in the virus mRNAs.

The mode of production of endotoxin-induced interferon in rabbit tissue cells. I. Development of priming by pretreatment with interferon.

In vitro production of endotoxin-induced interferon in rabbit tissue cell cultures could be enhanced by pretreatment with interferon. The enhancible state developed from the first hr of incubation at 37 C and a maximal priming effect was attained at 6 hr of incubation. Yields of interferon from unprimed cultures were usually 20-200 units/ml. In contrast, the primed cultures constantly yielded 1,000-2,500 units/ml of interferon. The pretreatment with interferon seemed to cause an earlier appearance of detectable interferon and the primed cells became more sensitive to endotoxin. It turned out that 10--30 units/ml of rabbit interferon were enough to develop the maximal priming. Even when cells were pretreated with higher doses of rabbit interferon such as 1.0 x 10(4) - 1.0 x 10(5) units/ml, the same level of priming effect was always observed without diminution. Various types of homologous (rabbit) and heterologous (human and mouse) interferon preparations showed similar dose-dependent enhancement of interferon production in proportion to the antiviral titers of these preparations as tested with RK-13 cells of rabbit origin.

Inhibition by Interferon of Thymidine Uptake in Chemostat Cultures of L1210 Cells

Mouse interferon preparations inhibited the incorporation of [3H]-thymidine into acid-insoluble material of mouse leukemia L1210 cells cultivated under steady-state conditions in a chemostat. Interferon exerted only a transitory and less pronounced effect on [3H]-deoxyadenosine incorporation and had no effect on the content of total DNA per cell. Study of [3H]-thymidine uptake at 1 degree into acid-soluble cellular material showed that interferon reduced the uptake of this labeled nucleoside whereas the uptake of [3H]-deoxyadenosine and [3H]-deoxy-D-glucose was not inhibited. The effect of interferon on [3H]-thymidine uptake occurred prior to the inhibitory action on cell multiplication. It is suggested that the inhibitory of [3H]-thymdine uptake reflected specific changes in the cell membrane of interferon-treated cells which may be relevant to the understanding of the antiviral and other biological effects of interferon.

The immunosuppressive effect of type II mouse interferon preparations on antibody production

Preparations containing Type II (immune induced) interferon suppressed the immune response to sheep erythrocytes (SRBC) both in vitro and in vivo. Type II interferon preparations were 250 times more active in immunosuppression than Type I (L cell) interferon preparations in parallel experiments. The antiviral and immunosuppressive activities shared several unique physical-chemical activities including pH 2 lability, 56 °C stability, and resistance to inactivation by anti-L-cell interferon antibody. Both activities were denatured by boiling at 100 °C for 2.5 min, but were not renatured by boiling for 1 min with 1% SDS, 1% β-mercaptoethanol and 5 M urea. The bulk of both the immunosuppressive and antiviral activities were recovered from a 41–60% saturated ammonium sulfate precipitate of Type II interferon. Sephadex G-100 column chromatography of Type II interferon preparations yielded two major peaks of anti-viral activity of molecular weights of approximately 40,000 and 90,000, both of which together contained the total immunomodulating activity observed in the proteins of the Chromatographic effluent. The 90,000-dalton species was also detected by its anti-viral and immunosuppressive activity on polyacrylamide gel electrophoresis.

Interferon, double-stranded RNA and RNA degradation. Characteristics of an endonuclease activity.

Extracts from interferon‐treated Ehrlich ascites tumor cells differ in various biochemical characteristics from extracts from control cells. Thus, as reported earlier, double‐stranded RNA (dsRNA) promotes the phosphorylation by ATP of at least two proteins in extracts from interferon‐treated cells, but not, or to only a lesser extent, in extracts from control cells. Moreover reovirus mRNAs are degraded faster in reaction mixtures containing extracts from interferon‐treated cells than in those containing extracts from control cells but only if the reaction mixtures are supplemented with dsRNA and ATP. The faster RNA degradation in extracts from interferon‐treated cells is due to enhanced endonuclease action. We designated the agent(s) catalyzing this as endonucleaseINT. There is some enhancement of nuclease activity by dsRNA and ATP also in extracts from control cells. The extent of this enhancement is, however, much smaller than in extracts from interferon‐treated cells. We report now that the promotion of mRNA cleavage in extracts from interferon‐treated cells by dsRNA and ATP is apparently not a consequence of a possible impairment in the attachment of the mRNA to ribosomes since (a) the protein synthesis inhibitors (sparsomycin and edeine) do not affect the degradation and (b) dsRNA and ATP enhance RNA degradation also in the 200000 × g supernatant fraction from extracts of interferon‐treated cells and this fraction is essentially free of ribosomes. (The ribosome concentration in our 200000 × g supernatant fraction is less than 1% of that in the 30000 × g extract.) GTP or the ATP analogs AMP(CH2)PP or AMP‐P(CH2)P do not substitute for ATP and dsDNA or DNA · RNA hybrids do not substitute for dsRNA in activating endonucleaseINT. The optimal concentration of ATP in activating endonucleaseINT is 1 mM or higher. The addition of dsRNA and ATP to extracts from interferon‐treated cells affects the degradation of some RNAs much more than that of others. Thus it promotes the degradation of reovirus mRNA from the large size class strongly, the degradation of those from the medium size class to an intermediate extent and that of those from the small size class the least. Furthermore it enhances the degradation of mRNA from either control or interferon‐treated Ehrlich ascites cells more than that of Ehrlich ascites cell ribosomal RNA or mouse globin mRNA. Reovirus dsRNA is not cleaved by endonucleaseINT. The partially purified mouse interferon preparation (specific activity 8 × 108 NIH reference standard units/mg protein) used in several experiments was free of nuclease activity under our experimental conditions. Moreover the addition to extracts from control cells (or extracts from interferon‐treated cells) of this interferon preparation together with dsRNA and ATP did not enhance nuclease activity.