Fibroblast Interferon Research Articles

Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli.

The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the lambda phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed. The cloned T4 pin gene does not seem to affect the turnover of normal E. coli proteins.

Chromosomal localization of human leukocyte, fibroblast, and immune interferon genes by means of in situ hybridization

Chromosomal localization of human leukocyte, fibroblast, and immune interferon genes by means of <i>in situ</i> hybridization

Potentiation of the cytolytic activity of peripheral blood monocytes by lymphokines and interferon.

Human peripheral blood monocytes obtained by EDTA-reversible adherence to plastic surfaces precoated with autologous serum can rapidly lyse a variety of tumor cells. That the effector cells in this system are indeed monocytes has been demonstrated (1). Using a short-term (3 to 4 hr) 51Cr-release assay and the single cell conjugate cytotoxic assay, we studied the effects of lymphokine-rich supernatants containing gamma-interferon and partially purified fibroblast interferon on the monocyte cytolytic activity. Overnight incubation of the monocytes in fetal bovine serum-containing medium resulted in a relatively small decrease in cytotoxic activity compared to the one obtained with monocytes incubated in autologous serum. The addition of lymphokines or interferon under both incubation conditions resulted in augmented activity as measured in the 51Cr-release assay. However, the proportions of binding and cytotoxic monocytes, determined in the single cell conjugate assay, did not increase. These results suggest that augmented activity is not due to recruitment of inactive cells. Kinetics studies of tumor cell lysis indicate the increase in killing efficiency is probably due to both an increase in the rate of killing and in the recycling ability of the cytotoxic cells. Using the conjugate/agarose technique, we also demonstrated that excess tumor cells could impair the lytic machinery of freshly isolated monocytes, whereas monocytes treated with lymphokines or interferon partially lost their sensitivity to this inhibitory effect. The ability of tumor cells to impair the lytic machinery of monocytes could be one of the mechanisms by which tumors escape immunosurveillance.

Human brain tumor--derived cell lines: growth rate reduced by human fibroblast interferon.

The biological response modifier human beta-interferon had pronounced antigrowth effects on various histologic types of human brain tumor cells but no effects on a nontransformed cell line, MRC-5. The cultures of brain tumor cells showed severe alterations indicative of cell injury and death after exposure to beta-interferon for 2 to 6 days. Similar results were obtained with cells freshly explanted from human brain tumors. The results indicate that it may be possible to use fresh, explanted tumor tissue to identify patients who might benefit from therapy with beta-interferon.

Effect of treatment with interferon and cyclophosphamide on the growth of a spontaneous liposarcoma in rats.

A spontaneous non-immunogenic transplantable liposarcoma in BN rats was found to be sensitive to the antitumor effects of rat fibroblast interferon (RIF) when it was administered from the day of tumor implantation onwards. Treatment with RIF starting at 7 days after implantation was not effective. Tumor growth was markedly inhibited by cyclophosphamide (Cyclo). At the time and dose schedules used, Cyclo was more effective than RIF. When the growth of the tumor was inhibited by Cyclo, subsequent treatment with RIF did not lead to an additional retardation of tumor growth. Administration of RIF interfered with the beneficial effect of Cyclo when both agents were given concomitantly. RIF and Cyclo gave similar results to those obtained with RIF alone and these were inferior to those obtained with Cyclo alone.

A unique set of polypeptides is induced by gamma interferon in addition to those induced in common with alpha and beta interferons.

Human immune interferon (IFN-gamma) differs from leukocyte interferon (IFN-alpha) and fibroblast interferon (IFN-beta) in cell origin, inducing agents, physical and biological properties and amino acid sequence. These differences have led to interest in possible differences in the biological properties of IFN-gamma compared with IFN-alpha and IFN-beta. IFN-gamma has the same broad range of biochemical and biological actions as do IFN-alpha and IFN-beta, although relative potencies vary depending on the cell type and function investigated. There has so far been no direct evidence that IFN-gamma alters normal cell functions differently from other interferons. We report here striking qualitative and quantitative differences in the intracellular response of human fibroblasts to IFN-gamma compared with IFN-alpha and IFN-beta. Two-dimensional gel electrophoresis demonstrates, in addition to the induction of a common group of polypeptides, the existence of a set of polypeptides whose synthesis is uniquely induced by IFN-gamma.

Deletion mapping of the inducible promoter of human IFN-beta gene.

Human fibroblast cells produce beta-type interferon only in response to viral infection or treatment with an inducer such as poly(rI) X poly(rC); this event is most probably controlled at the transcriptional level (for review see ref. 1). To study the induction process, we inserted the human fibroblast interferon (IFN-beta) gene, with or without its promoter region, into recombinant simian virus 40 (SV40) plasmid vectors which subsequently were transfected into monkey AP-8 cells. We report here that upon induction with poly(rI) X poly(rC) there was a 10-30-fold increase in IFN-beta synthesis. This inducer had no effect on interferon production when the coding region only was inserted into the vector plasmid, which indicates that the promoter region is required for inducibility of this gene. Deletion mapping implicates the region between nucleotides -144 and -186 from the mRNA initiation site in the specific regulation of the IFN-beta gene. This region contains a sequence that is remarkably homologous with a consensus sequence found in the 5' flanking region of steroid hormone responsive genes, which might be involved in binding the progesterone-receptor complex.

Antiviral effects of human fibroblast interferon from Escherichia coli against encephalomyocarditis virus infection of squirrel monkeys.

Recombinant DNA methodology has allowed the production of human fibroblast interferon (IFN-beta) from Escherichia coli and this material, in highly purified form, has been shown to reduce viraemia and mortality in encephalomyocarditis (EMC) virus-infected squirrel monkeys. These effects are dose related: six treatments over 4 days at 10(6) U/kg and 3 x 10(3) U/kg have comparable efficacy, whereas treatments at 10(3) U/kg are ineffective. The recombinant DNA-derived IFN-beta appears to be as effective as natural fibroblast cell-derived IFN-beta and both materials are effective by the intramuscular or intravenous routes. Thus, even though previous studies have shown that low circulating concentrations of IFN-beta are observed after intramuscular injections, the present data indicate that this slow release from the muscle can still confer protection.

Growth inhibitory activity of human lymphoblastoid and fibroblast interferons in vitro.

Growth inhibitory activities of both human fibroblast (HuIFN-beta) and lymphoblastoid (HuIFN-alpha) interferons against 17 human cultured cell lines derived from leukemias and lymphomas were measured quantitatively by regrowth assay. Daudi cells were the most sensitive to both interferons. Three B-cell lines, one T-cell line and one non-T, non-B cell line were moderately sensitive to both interferons. Although the levels of sensitivity of these cell lines to the interferons were different, cells could be killed by the interferons. Eleven other cultured cell lines including three B-cell lines, three T-cell lines, two non-T, non-B cell lines, two myeloid cell lines and one monocytoid cell line were not sensitive to these interferons. The results indicated that the growth inhibitory activity of interferons was not always cell lineage-specific and cell lines which were sensitive to the one interferon were always sensitive to the other interferon, although the level of sensitivity was different. Cytocidal action of interferons was analyzed by use of the most sensitive Daudi cells. The results indicated that both interferons had a time-dependent cytocidal action, but not a concentration-dependent one. Knowledge of the mode of the cytocidal action may be useful for the design of optional therapeutic schedules using the interferons. The prediction of clinical effectiveness of interferon against hematologic malignancies is discussed, based on the level of in vitro sensitivity of cultured leukemia and lymphoma cell lines.

Immune-enhancing effect of recombinant human interferon-beta produced in Escherichia coli on human peripheral blood mononuclear cells in vitro.

Highly purified recombinant fibroblast interferon produced by Escherichia coli (ReIFN-beta) was tested for its ability to stimulate natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity (ADCC) and monocyte-macrophage phagocytic activity in human peripheral blood mononuclear cells (PBM) in comparison with natural human fibroblast interferon (IFN-beta). The NK cell activity against target cells (K-562, Molt-3, CCRF-HSB-2, CCRF-CEM, Daudi, and HeLa S3) was enhanced by ReIFN-beta and IFN-beta. Augmentation of cytotoxicity of NK cells was observed at a concentration of ReIFN-beta as low as 1 U/ml and increased in a dose-dependent manner. The enhancing effect of ReIFN-beta was expressed sufficiently during a 1-hr incubation with effector cells, and IFN-beta showed kinetics and potency similar to those of ReIFN-beta. ADCC was measured with chicken red blood cells (CRBC) or CCRF-CEM coated with each antibody, as target cells. Pretreatment of PBM with ReIFN-beta caused a significant increase in ADCC activity against both targets, and the enhancing activity of ReIFN-beta was similar to that of IFN-beta. Phagocytic activity of adherent cells in PBM against CRBC was enhanced by ReIFN-beta and IFN-beta as determined by microscopical cytologic examination and by the 51Cr-labeled CRBC-uptake method. From these results, it can be concluded that recombinant human IFN-beta modulates NK cell activity, ADCC and phagocytic activity of human PBM as effectively as natural human IFN-beta.

Antiproliferative Activities of Bacterial-Derived Human Alpha and Beta Interferons on Human and Mouse Tumor Cells

We tested the antiproliferative properties of five bacterial derived leukocyte interferons (IFNs), IFN-αA and IFN-αD, and the hybrids IFN-αAD (Bg1), IFN-αAD (Pvu), and IFN-αDA (Bgl), bacterial derived human fibroblast (IFN-β1,) and buffy coat IFNs. McCoy cells were completely resistant to the effects of buffy coat and IFN-β1 IFNs while HeLa cells were very sensitive to both preparations. At a concentration of 104 U/ml IFN-β1 produced an 84% inhibition while the buffy coat IFN reduced the growth to 50% of the control cells. Both cell types proved to be quite insensitive to IFN-αA and IFN-αD, even at concentrations as high as 104 U/ml. IFN-αA had no effect on McCoy cells while approximately a 25% inhibition was observed with IFN-αA on HeLa cells and IFN-αD on both cell lines. The hybrids IFN-αAD (Pvu) and IFN-αAD (Bgl), on the other hand, effectively inhibited the proliferation of these cell lines. IFN-αAD (Pvu) and IFN-αAD (Bgl) at concentrations of 104 U/ml inhibited HeLa cell proliferation by 74% and 69%, respectively. In McCoy cells IFN-αAD (Bgl) exerted a slightly greater antiproliferative activity (49% inhibition) compared with IFN-αAD (Pvu) (40% inhibition). The third hybrid IFN tested, IFN-αDA (Bgl), had no effect on the growth of McCoy cells and only minimal activity on HeLa cells (17% inhibition at 104 U/ml). Combinations of IFNs could have a synergistic, additive, or antagonistic effect on their antiproliferative activities. To analyze these possibilities we assayed several mixtures of the cloned preparations. In all cases an additive effect was obtained either when the two parent IFNs or the hybrids were combined.

Cytostatic action of human fibroblast interferon in direct growth inhibition: effect on human malignant melanoma.

The mode of action of the direct antiproliferative effect of human fibroblast interferon (HuIFN-beta) on tumor cells was analyzed in vitro with a human malignant melanoma cell line, HMV-1. HuIFN-beta inhibited the growth of HMV-1 cells in a time-dependent fashion (50%-inhibition concentration: less than 50 IU/ml). Its action was cytostatic. DNA synthesis was inhibited before the antitumor effect appeared, and apparent dose-dependent inhibitions of RNA and protein synthesis were induced. An increase of cell protein content was seen with the occurrence of growth inhibition. Increase of cell volume and prolongation of doubling time were seen from the second day after the addition, which corresponded well with the effects of HuIFN-beta on the cell cycle (the accumulation of cells in the S phase). These results indicate that the direct antitumor effect of HuIFN-beta on HMV-1 cells may result from cell cycle retardation mediated through obstruction of DNA synthesis.

Biological properties of human interferon beta 1 synthesized in recombinant bacteria.

Human fibroblast interferon, designated IFN-beta 1, has been produced in E. coli by direct expression of the cloned cDNA coding for the mature polypeptide. Bacterial lysates from recombinant cultures contain a polypeptide with an apparent molecular weight of 17,500 that corresponds in size to the unglycosylated IFN-beta 1 molecule. The latter could be specifically immunoprecipitated by antibodies to purified natural IFN-beta and could inhibit the replication of Herpes simplex virus types 1 and 2 in many different cell lines. Like the natural fibroblast IFN-beta, the bacterial IFN-beta 1 was active in many human cell lines, less active in a monkey cell line and inactive in rabbit and mouse fibroblasts. The antibody titre required to neutralise the anti-herpes activity of both IFN preparations was similar suggesting that they have the same specific activities. Similarly, the bacterial IFN-beta 1 was equally active in inhibiting the proliferation of Daudi cells grown in culture. Bacterial IFN-beta 1 was also capable of enhancing natural killer cell activity and antibody-dependent cellular cytotoxicity in vitro. Thus, IFN-beta 1 produced in recombinant bacteria displays a large range of biological properties ascribed to the natural fibroblast IFN-beta molecule.

Effects of human fibroblast interferon on human gliomas transplanted into nude mice.

Daily intratumor injection of human fibroblast interferon (HuIFN-beta) resulted in significant growth inhibition of human gliomas transplanted into nude mice. Light and electron microscope examinations were conducted to estimate the efficacy of HuIFN-beta. A 29-day daily treatment with HuIFN-beta induced complete regression of oligodendroglioma KG-1--a slowly growing tumor line positive for S-100 protein and negative for glial fibrillary acidic protein (GFAP)--in 9 out of 10 mice receiving 6 X 10(5) IU and 6 out of 9 given 1 X 10(5) IU. In mice with glioblastoma multiforme TMIMS-583, which showed more rapid growth and was positive for GFAP, dose-dependent growth inhibition was observed during 23 days of HuIFN-beta administration. Pathological changes of TMIMS-583 induced by HuIFN-beta were characterized by a decrease of the metaphase tumor cells, by enhanced degeneration of tumor cells with stromal reaction and round cell infiltrates, and by prominence of multinuclear giant cells containing glial filaments. Tumor weights were well correlated with the number of tumor cells in the metaphase (r = 0.89).

Partial dissociation of antiviral and antimitogenic activities of murine interferon after its incorporation into liposomes.

Murine fibroblast interferon (MuIFN, 90% beta, 10% alpha) was associated with both positively and negatively charged liposomes formed by reverse-phase evaporation. This interferon-liposome association occurred predominantly in a manner that resulted in protection of a significant portion of the IFN's antiviral activity from trypsin digestion, yet also permitted biological expression of this activity without prior liposome disruption. A differential dissociation of antiviral and antimitogenic activities was observed with MuIFN associated with positively vs negatively charged liposomes, as reflected by differential sensitivities to trypsin inactivation. This may reflect either (1) differential associations of various molecular species of MuIFN with liposomes, or (2) that different portions of the IFN molecule are responsible for the antiviral and the antimitogenic activities.

Inhibition of human cytomegalovirus by trifluorothymidine.

The antiviral activity of trifluorothymidine (TFT) singly and in combination with other antiviral agents against human cytomegalovirus (HCMV) was evaluated by using an infectious center plaque reduction assay. The 50% inhibitory dose of TFT against six different patient HCMV strains was 0.57 (+/- 0.24, standard deviation) microM and ranged from 0.32 to 0.97 microM. The 50% inhibitory dose for the laboratory-adapted HCMV strain, AD-169, was 2.1 microM. When TFT (0.17 microM) was combined with human fibroblast interferon (25 U/ml), the combination was additive against all four HCMV isolates evaluated. Synergism was observed when TFT (0.17 microM) was combined with phosphonoformic acid (25 microM) for all strains studied or with acyclovir (20 microM) for three of the four clinical HCMV strains tested. Each of the three antiviral agents, when combined with TFT, exhibited additive effects against strain AD-169. TFT at concentrations of 0.5, 1.7, and 3.5 microM had an increasing inhibitory effect on uninfected human embryonic lung fibroblast (HEL) cell growth over 72 h, with 16% growth inhibition at 3.5 microM after 3 days. There was no increased toxicity to growing HEL cells when the paired antiviral agent combinations were evaluated. These findings suggest that TFT may be useful singly or in combination with other antiviral agents in treating HCMV infections.

Interferon as an in vitro modifier of immune responses induced by combined vaccine (DTP) in mice.

The ability of DTP (diphtheria, tetanus, pertussis) vaccine to stimulate IgG-plaque forming cells (PFC) in mice was markedly inhibited by human leukocyte and mouse fibroblast interferon (IF). This effect of IF was not dose-dependent. The maximum inhibitory effect of human IF was observed on Day 10 after the administration of the vaccine. The inhibition of IgM-PFC was more severe when human IF was used. Although IF abrogated the effect of DTP vaccine on IgG-PFC, at the same time it stimulated PFC in the control group of non-vaccinated animals. The administration of DTP vaccine decreased IgM-PFC on Day 5, the effect was enforced by the addition of both human and mouse IF. IF decreased IgM-PFC in the control group. Small and large doses of DTP suppressed the leukocyte adherance inhibition (LAI) on Day 5 after administration of the vaccine, the effect was not affected by addition of IF. The suppressive effect of DTP was lost on Day 10 when addition of IF increased the percent of LAI in all experimental groups. Thus IF appears to interfere with the effect of DTP and selectively modulate the different immune responses.

Human fibroblast interferon RNA transcripts of different sizes in poly(I).poly(C) induced cells.

Northern blot analysis reveals that total RNA from human fibroblastoid cells (MG 63) induced with poly(I).poly(C) under conditions of IFN-beta production, contains predominantly a +/- 1,200 nucleotide long poly (A) mRNA (mRNA.M) which hybridizes with a Hu IFN-beta cDNA specific probe. But hybridization with this probe also enabled the detection of a polyadenylated RNA (RNA.I) with a length of between 3.5 kb-3.8 kb, representing 0.6% of the total hybridizable cellular RNA in superinduced cells. Mapping shows that the RNA.I contains all the sequence information present in mRNA.M. Furthermore, it also hybridizes to sequences, located downstream from the IFN-beta gene up to 2.5 kb beyond its poly A attachment site, while no hybridization to fragments located upstream of the IFN-beta mRNA cap site was observable. Hence this RNA.I corresponds to a transcript that starts at the same position as the major mRNA.M but which extends up to 2.5 kb beyond the 3'-end of mRNA.M where another polyadenylation signal is located.

Inhibition of interferon or soluble immune response suppressor (SIRS) mediated suppression by levamisole

Soluble immune response suppressor (SIRS), a product of concanavalin A-activated murine T cells, nonspecifically suppresses antibody responses in vitro. SIRS acts via macrophages (Mø) which after exposure to SIRS, release a second factor, Mø-derived suppressor factor (Mø-SF), that is directly responsible for suppression. Mø-SF appears to be modified SIRS since hydrogen peroxide can convert SIRS to Mø-SF directly. Murine fibroblast interferon (IFNβ) also activates suppressor T cells which produce a factor(s) with properties identical to SIRS; thus accounting for at least some of the immunosupressive properties of INFβ. Levamisole (L (−) 2,3,5,6,-tetrahydro-6-phenyl-imidazo [2,1–6] thiazole enhances immune responses in vivo and in vitro and is of value in improving immune responses in clinically depressed hosts. The effects of levamisole on IFNβ- or SIRS-mediated suppression were examined to gain a better understanding of the mechanism of action of this agent. SIRS or IFNβ suppressed PFC responses to 10–20 % of control responses; under optimal conditions, these responses were increased to ∼ 80 % of control levels in the presence of levamisole. To block suppression, levamisole was required during the first 24 h of the 5 day culture. Levamisole did ot inactivate IFNβ, SIRS or Mφ-SF directly, but inhibited production of Mφ-SF by SIRS-treated Mφ and conversion of SIRS to Mφ-SF by peroxide. Kinetic analyses suggested that levamisole acted as a competitive inhibitor of the SIRS-H 2O 2 reaction. Collectively these data indicate that levamisole inhibits SIRS- or IFNβ-mediated suppression by preventing formation of Mφ-SF and suggest that some immunoenhancin properties of levamisole may be accounted for by interference with nonspecific suppressor T cell pathways.

Natural killer activity in (C57BL/6 X DBA/2)F1 hybrids undergoing acute and chronic graft-vs.-host reaction.

The present findings demonstrate that a total i.v. transfer of 100 X 10(6) C57BL/6 (B6) parental spleen cells into untreated (C57BL/6 X DBA/2)F1 hybrids (B6D2F1) resulted in acute runting, which was associated with a significantly elevated graft-vs.-host (GVH) index over a one-month period following GVH induction. Furthermore, this B6-induced acute GVH disease was associated with a marked depression of natural killer (NK) cell activity (spleen and peripheral blood) (with or without addition of mouse fibroblast interferon), which correlated with lymphoid cell hypocellularity, prominent splenic extramedullary hematopoiesis (EMH), and parallel depressions of both concanavalin A- and lipopolysaccharide-induced mitogenesis. Significantly increased killing by antibody-dependent cellular cytotoxicity of antibody-coated chicken red blood cells, as well as increased T cell killing of the NK-insensitive cell line P815 (as compared to the significantly decreased killing of the NK-sensitive cell line YAC-1) was also observed in the spleens of this 100 X 10(6) B6-injected F1 group. In marked contrast to this 100 X 10(6) B6-injected acute GVH group, untreated mice injected i.v. with the same or greater numbers of parental DBA/2 spleen cells (100 X 10(6)-150 X 10(6) DBA/2 spleen cells) exhibited a milder and more chronic form of GVH disease, which was not associated with a significant decrease of NK activity. It was of considerable interest that a total i.v. transfer of 50 X 10(6) B6 spleen cells (i.e. one-half of that required to produce acute GVH, markedly depressed NK, and prominent splenic EMH) into B6D2F1 hybrids also resulted in a more chronic form of GVH disease, but was associated with significantly increased levels of NK activity at two weeks post GVH induction.