Synergistic Effect Of IFN-gamma Research Articles

Interferon-gamma regulates apoptosis by releasing soluble tumor necrosis factor receptors in a gastric epithelial cell line.

Interferon (IFN)-gamma and tumor necrosis factor (TNF) are predominant cytokines produced in the gastric mucosa of patients with Helicobacter pylori-infected gastritis. Several studies reported that IFN-gamma and TNF induced the synergistic effect on many cell lines. We attempted to clarify the apoptotic activity and the synergistic effect of IFN-gamma and TNF on the gastric epithelial cell, and whether IFN-gamma relates to soluble TNF receptors (sTNF-R) release from the gastric epithelial cell. On the gastric epithelial cell line MKN45, cytotoxic and apoptotic effects of IFN-gamma and TNF were examined. Next, sTNF-R released in response to IFN-gamma and the protective effect of sTNF-R against the cytotoxic activity of TNF and IFN-gamma were examined by blocking the release of sTNF-R with a serine protease inhibitor such as phenylmethylsulfonyl fluoride. Interferon-gamma significantly decreased cell viability, but TNF decreased it only slightly. Interferon-gamma and TNF did not make a synergistic effect on cell viability and apoptosis. Interferon-gamma and TNF induced sTNF-R release from gastric epithelial cells. Phenylmethylsulfonyl fluoride significantly inhibited shedding of sTNF-R and a synergistic effect of TNF and IFN-gamma on apoptosis was observed. These results suggest that sTNF-R released by IFN-gamma regulate the injury on the gastric epithelial cell line induced by TNF.

Inhibition by antioxidants of nitric oxide synthase expression in murine macrophages: role of nuclear factor kB and interferon regulatory factor 1

1. In view of the potential deleterious effects of high amounts of nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS) in inflammation, the prevention of the expression of this enzyme represents an important therapeutic goal. In cytokine-stimulated cells, activation of nuclear factor kappa B (NF-kappa B) is crucial for the increase in iNOS gene expression. Since NF-kappa B activation appears to involve a redox-sensitive step, we have investigated whether three structurally unrelated antioxidants, 5,7-dihydroxyflavone (chrysin), 3,4-dichloroisocoumarin (DCI) and N-acetyl 5-hydroxytryptamine (N-acetylserotonin, NAS), affect iNOS expression in cultured RAW 264.7 monocyte/macrophages stimulated with bacterial lipopolysaccharide (LPS, 140 ng ml-1) and interferon-gamma (IFN gamma, 5 u ml-1). 2. During a 6 h incubation period neither LPS nor IFN gamma alone exerted a significant effect but when combined, caused a prominent increase in nitrite formation, iNOS mRNA and protein abundance. Co-incubation with chrysin (50 microM), DCI (50 microM) or NAS (1 mM) markedly attenuated this increase in iNOS gene expression. 3. DCI, but not chrysin or NAS, prevented the activation of NF-kappa B in cells exposed to LPS plus IFN gamma for 30 min. In contrast, all three antioxidants significantly blunted the DNA-binding activity of interferon regulatory factor 1 (IRF-1), which mediates the synergistic effect of IFN gamma on iNOS gene expression in cells treated for 2 h with LPS plus IFN gamma. 4. DCI thus appears to inhibit iNOS gene expression at the transcriptional level by preventing the activation of both NF-kappa B and IRF-1. The inhibitory effect of DCI on NF-kappa B activation, however, does not seem to be related to its antioxidative properties, since DCI, unlike chrysin or NAS, is a potent serine protease inhibitor which stabilizes the inactive NF-kappa B complex by protecting the inhibitory I kappa B-alpha subunit from proteolytic degradation. 5. The virtually identical inhibitory effect of chrysin, DCI and NAS on the activation of IRF-1 points to a redox-sensitive step in the activation of this transcription factor, which in contrast to NF-kappa B requires de novo protein synthesis. 6. Since iNOS gene expression in human cells and tissues usually requires the combination of several cytokines, antioxidants such as chrysin and NAS which do not interfere with the activation of NF-kappa B may be of therapeutic value for selectively inhibiting the enhanced expression of this enzyme in inflammation.

Tumor necrosis factor-alpha and interferon-gamma modulate gene expression of type I 5'-deiodinase, thyroid peroxidase, and thyroglobulin in FRTL-5 rat thyroid cells.

Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) have many effects on a number of cell types, including thyrotrophs. In the present study, we used FRTL5 cells, a cultured rat thyroid follicular cell line, to examine the effects of IFN-gamma and TNF-alpha on type I 5'-deiodinase (5'D-I) activity and 5'D-I, thyroid peroxidase (TPO) and thyroglobulin (Tg) gene expression. Incubation of FRTL5 cells with the highest concentrations of TNF-alpha and IFN-gamma tested (1000 ng/ml or 1000 U/ml, respectively) for 72 h in the presence and absence of TSH had no effect on cell viability as assessed by trypan blue exclusion. In TSH-deprived FRTL-5 cells, TNF-alpha and IFN-gamma resulted in a small but dose-dependent decrease in 5'D-I activity. TNF-alpha or IFN-gamma blocked the TSH- or cAMP-induced rise in 5'D-I activity. 100 ng/ml TNF-alpha and 100 U/ml IFN-gamma completely blocked the TSH- or cAMP-induced rise in 5' D-I activity. However, when cells were incubated with TNF-alpha and IFN-gamma, in combination, there was a marked decrease in 5'D-I activity, with TNF-alpha (25 ng/ml) plus IFN-gamma (25 U/ml) completely blocking the TSH-induced rise in 5'D-I activity. Northern blot analyses were performed to examine the effect of TNF-alpha and IFN-gamma on 5'D-I gene expression. TNF-alpha had little effect on 5'D-I messenger RNA (mRNA) levels, while IFN-gamma resulted in a modest decrease in 5'D-I mRNA levels in TSH-deprived cells, and in TSH-stimulated FRTL-5 cells. However, when TNF-alpha and IFN-gamma were added in combination there was a marked decrease in 5'D-I gene expression with TNF-alpha (50 ng/ml) plus IFN-gamma (50 U/ml) decreasing 5'D-I mRNA levels by 89 percent in TSH-deprived cells. In TSH-stimulated cells incubated with 500 ng/ml TNF-alpha plus 500 U/ml IFN-gamma, 5'D-I mRNA levels were almost undetectable. We also examined the effect of IFN-gamma and TNF-alpha on TPO and Tg gene expression. As observed with 5'D-I mRNA levels, there was a synergistic effect of IFN-gamma and TNF-alpha on the inhibition of basal and TSH-stimulated TPO and Tg gene expression. These findings indicate that TNF-alpha and IFN-gamma in combination have a marked inhibitory effect on thyroid function, which is consistent with a decrease in thyroid hormone synthesis and metabolism.

Tumor necrosis factor-alpha and interferon-gamma modulate gene expression of type I 5'-deiodinase, thyroid peroxidase, and thyroglobulin in FRTL-5 rat thyroid cells

Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) have many effects on a number of cell types, including thyrotrophs. In the present study, we used FRTL5 cells, a cultured rat thyroid follicular cell line, to examine the effects of IFN-gamma and TNF-alpha on type I 5'-deiodinase (5'D-I) activity and 5'D-I, thyroid peroxidase (TPO) and thyroglobulin (Tg) gene expression. Incubation of FRTL5 cells with the highest concentrations of TNF-alpha and IFN-gamma tested (1000 ng/ml or 1000 U/ml, respectively) for 72 h in the presence and absence of TSH had no effect on cell viability as assessed by trypan blue exclusion. In TSH-deprived FRTL-5 cells, TNF-alpha and IFN-gamma resulted in a small but dose-dependent decrease in 5'D-I activity. TNF-alpha or IFN-gamma blocked the TSH- or cAMP-induced rise in 5'D-I activity. 100 ng/ml TNF-alpha and 100 U/ml IFN-gamma completely blocked the TSH- or cAMP-induced rise in 5' D-I activity. However, when cells were incubated with TNF-alpha and IFN-gamma, in combination, there was a marked decrease in 5'D-I activity, with TNF-alpha (25 ng/ml) plus IFN-gamma (25 U/ml) completely blocking the TSH-induced rise in 5'D-I activity. Northern blot analyses were performed to examine the effect of TNF-alpha and IFN-gamma on 5'D-I gene expression. TNF-alpha had little effect on 5'D-I messenger RNA (mRNA) levels, while IFN-gamma resulted in a modest decrease in 5'D-I mRNA levels in TSH-deprived cells, and in TSH-stimulated FRTL-5 cells. However, when TNF-alpha and IFN-gamma were added in combination there was a marked decrease in 5'D-I gene expression with TNF-alpha (50 ng/ml) plus IFN-gamma (50 U/ml) decreasing 5'D-I mRNA levels by 89 percent in TSH-deprived cells. In TSH-stimulated cells incubated with 500 ng/ml TNF-alpha plus 500 U/ml IFN-gamma, 5'D-I mRNA levels were almost undetectable. We also examined the effect of IFN-gamma and TNF-alpha on TPO and Tg gene expression. As observed with 5'D-I mRNA levels, there was a synergistic effect of IFN-gamma and TNF-alpha on the inhibition of basal and TSH-stimulated TPO and Tg gene expression. These findings indicate that TNF-alpha and IFN-gamma in combination have a marked inhibitory effect on thyroid function, which is consistent with a decrease in thyroid hormone synthesis and metabolism.

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium

To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. Treatment of HSE with interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-gamma (IFN gamma) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFN gamma and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN gamma also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNF beta/lymphotoxin. Immunoprecipitation of TNF alpha + IFN gamma-stimulated, 125I-labeled HSE cells with anti-ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFN gamma alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. These studies indicate that IFN gamma plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

Mitogenic activity of interferon gamma on growth-arrested human vascular smooth muscle cells.

Interferon gamma (IFN-gamma) is a multifunctional lymphokine secreted by activated T lymphocytes, which are found in atherosclerotic lesions. IFN-gamma has been reported to suppress the proliferation of vascular smooth muscle cells (SMCs). However, as we report in this paper, IFN-gamma is mitogenic for vascular SMCs under certain circumstances. Recombinant human IFN-gamma (1-100 units/ml), in a dose-dependent fashion, stimulated cell multiplication and [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation into DNA by cultured arterial SMCs that had been growth arrested by culturing in 1% plasma-derived serum for 5 days. IFN-gamma also accentuated the mitogenic activity of platelet-derived growth factor (PDGF)-BB. A time-course study revealed that there was a time lag of 4-6 hours between the G1-->S transition of quiescent SMCs stimulated by IFN-gamma and that of SMCs stimulated by PDGF-BB. A synergistic effect of IFN-gamma on the mitogenicity of PDGF became apparent after a similar time lag, suggesting that the IFN-gamma-related mitogenicity is mediated by a substance(s) secreted by IFN-gamma-treated SMCs. In fact, conditioned medium of IFN-gamma-treated SMCs was mitogenic for SMCs. Mitogenic activity in the conditioned medium was also detected by an assay using Swiss 3T3 cells, which originate from mice and, therefore, are not responsive to human IFN-gamma. The production of the mitogenic factor was blocked by anti-IFN-gamma antibody. Mitogenicity of the conditioned medium was not eliminated by addition of neutralizing antibody against PDGF, indicating that any autocrine growth factor(s) secreted by IFN-gamma-treated SMCs was not PDGF.(ABSTRACT TRUNCATED AT 250 WORDS)

55-kd Tumor Necrosis Factor Receptor Is Expressed by Human Keratinocytes and Plays a Pivotal Role in Regulation of Human Keratinocyte ICAM-1 Expression

Tumor necrosis factor alpha (TNF alpha) is a potent modulator of human keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression. TNF alpha is known to exert its biologic effects by binding to specific cell-surface receptors. Two distinct TNF binding molecules, the 55-kd and the 75-kd TNF receptor (TNFR) recently have been found to be expressed by human cells. These two receptor types are independently regulated and differ markedly in their intracellular regions, indicating functional dichotomy. In order to gain further insight into the mechanisms underlying ICAM-1 regulation in human keratinocytes, in the present study, the receptor molecules mediating TNF alpha induced ICAM-1 upregulation in human keratinocytes was defined. Human keratinocyte TNFR expression was assessed using monoclonal antibodies that specifically recognize the 55-kd or the 75-kd TNFR. Using FACS analysis, normal (HNK) as well as transformed (KB) human keratinocytes were found to react with anti-55-kd TNFR, but not anti-75-kd TNFR antibodies. These immunofluorescence data were confirmed by Northern blot analysis revealing clearly detectable amounts of mRNA specific for the 55-kd TNFR in KB cells. Incubation of human keratinocytes with anti-55-kd TNFR antibodies at 37 degrees C for 24 h increased ICAM-1 expression in a TNF alpha-like fashion. Moreover, the well known synergistic effect of IFN gamma plus TNF alpha on keratinocyte ICAM-1 induction could be mimicked by stimulation of cells with IFN gamma plus anti-55-kd TNFR antibodies. Synergistic ICAM-1 induction was not associated with increased expression of the 55-kd TNFR in IFN gamma-stimulated human keratinocytes. These studies indicate that human keratinocytes express the 55-kd TNF receptor and that this surface molecule may play an important role in regulation of human keratinocyte ICAM-1 expression.

Cytokine interactions in experimental cutaneous leishmaniasis. II. Endogenous tumor necrosis factor‐α production by macrophages is induced by the synergistic action of interferon (IFN)‐γ and interleukin (IL) 4 and accounts for the antiparasitic effect mediated by IFN‐γ and IL 4

Tumor necrosis factor-alpha (TNF-alpha) strongly activates murine peritoneal macrophages (M phi) for killing of amastigotes from Leishmania major in the presence of low amounts of interferon-gamma (IFN-gamma). Recently, we found that IFN-gamma and interleukin 4 (IL 4) also synergistically enhance the antileishmanial potential of M phi. In this report, evidence is provided that the synergism of IFN-gamma and IL 4 is based on the ability of the lymphokines to induce the endogenous production of TNF-alpha. First, both IFN-gamma and IL 4 as single agents and in combination were potent inducers of TNF-alpha production by M phi infected with L. major amastigotes. Second, the synergistic effect of IFN-gamma and IL 4 on parasite killing by M phi strongly correlated with their synergistic effect on the release of TNF-alpha. Third, the IFN-gamma/IL 4-mediated parasite elimination was completely abrogated not only in the presence of antibodies to IFN-gamma and IL 4, but also with an antibody specific for TNF-alpha. Consistent with the conclusion that endogenously produced TNF-alpha accounts for the synergism of IL 4 with IFN-gamma is the finding that N omega-monomethyl-L-arginine, an inhibitor of the L-arginine-dependent generation of microbicidal nitrogen intermediates, totally blocked the M phi activation induced by IFN-gamma combined with IL 4 as well as by IFN-gamma combined with TNF-alpha. These results underline the complex interplay of cytokines derived from lymphocytes and M phi and the role of TNF-alpha as pivotal factor for the induction of antileishmanial effector functions.

Synergistic in vitro and in vivo anti-tumor effect of daunomycin-anti-96-kDa melanoma-associated antigen monoclonal antibody CL 207 conjugate and recombinant IFN-gamma.

The mAb CL 207 recognizes a 96-kDa melanoma-associated Ag. The latter is not modulated by antibody but is highly susceptible to induction by immune IFN-gamma on human carcinoma and melanoma cells. The mAb CL 207 does not mediate C- and cell-dependent lysis of human carcinoma and melanoma cells. Conjugation of daunomycin with mAb CL 207 causes a slight reduction of its immunoreactivity and affinity but does not affect its serologic specificity or the toxicity of daunomycin. In combination with IFN-gamma, the daunomycin-mAb CL 207 conjugate displays a selective in vitro and in vivo toxic effect on tumor cells that express the 96-kDa MAA. The synergistic effect of IFN-gamma and daunomycin-mAb CL 207 conjugate is specific. The in vivo toxicity is influenced by the interval between injection of human tumor cells into nude mice and that of IFN-gamma and daunomycin-mAb CL 207 conjugate. The present results suggest that the 96-kDa melanoma-associated Ag may be a useful model to investigate the combined use of IFN-gamma and mAb for the selective destruction of tumor cells.

The synergistic influence of human interferon-gamma and interferon-alpha on suppression of hematopoietic progenitor cells is additive with the enhanced sensitivity of these cells to inhibition by interferons at low oxygen tension in vitro.

The influences of human interferons--natural gamma (2 X 10(7) NIH reference U/mg), recombinant gamma (approximately 5 X 10(6) U/mg), natural alpha (1.4 X 10(8) international reference U/mg), and natural beta (10(6) international reference U/mg)--were evaluated alone or in combination for their effects in vitro on colony formation by low density human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in normal incubator (approximately 20%) O2 tension or low (5%) O2 tension. Alone, these interferons demonstrated the same dose response inhibitory curves, as we reported previously, when cells were grown at 20% O2. Recombinant IFN-gamma gave the same dose response curve as natural IFN-gamma. Natural or recombinant interferon synergized with IFN-alpha to suppress colony formation at concentrations that were approximately 2 log units lower than that required by either interferon alone. Equal concentrations of these interferons were not needed for the synergistic effect and were still apparent when one was present at concentrations of 2 log units less than the other. IFN-gamma synergized to a lesser extent with IFN-beta, but IFN-alpha did not synergize with IFN-beta. Cells grown at 5% O2 were more sensitive to inhibition by 2 log units less IFN-gamma or IFN-alpha, and this effect was additive with the synergistic effects of IFN-gamma and IFN-alpha together. These results may have physiological, pathological, and/or clinical relevance.

Role of immune interferon in the monocytic differentiation of human promyelocytic cell lines induced by leukocyte conditioned medium

Conditioned medium (CM) from lectin-stimulated human leukocytes contains factors that induce human promyelocytic cell lines to differentiate along the monocytic pathway. In this report, we show that human promyelocytic cell lines are also induced to differentiate along this pathway by immune interferon (IFN gamma). Various preparations of IFN alpha tested did not induce this differentiation. In cultures containing IFN gamma, the cells are induced to coordinately express monocyte markers and functions such as monocyte-specific surface antigens, HLA-DR antigens, nonspecific esterase, receptors for the Fc fragment of IgG, and the ability to mediate antibody-dependent cell- mediated cytotoxicity. Our data indicate that differentiation induced by IFN gamma is not secondary to an arrest of growth of promyelocytic cell lines, but rather that a proportion of cells is induced along a programmed pathway of terminal differentiation similar to that of normal monocytes. CM contains IFN gamma, but its ability to induce differentiation is greater than expected on the basis of its content of IFN gamma. Treatments at 56 degrees C or at pH 2.0, which abolish IFN gamma activity, abrogate the differentiation ability of CM. The antiviral activity and the differentiation activity contained in the CM are coeluted from gel filtration and reverse-phase columns. Monoclonal antibodies anti-IFN gamma, which completely abrogate the differentiation ability of IFN gamma and the antiviral activity in the CM, completely suppress the induction of some monocyte markers by CM, but only reduce the expression of others. When IFN gamma is added to CM, promyelocytic cell lines are induced to differentiate to a much greater extent than that induced by either IFN gamma or IFN gamma- depleted CM alone. These results show that the differentiation activity of leukocyte CM is due to the synergistic effect of IFN gamma and other factors not yet identified.