Low Concentrations Of IFN-gamma Research Articles

Low-dose interferon-gamma-producing human neuroblastoma cells show reduced proliferation and delayed tumorigenicity.

Interferon-γ (IFN-γ) directs T helper-1 cell differentiation and mediates antitumour effects in preclinical models. However, high-dose IFN-γ is toxic in vivo, and IFN-γ-transfected neuroblastoma (NB) cells secreting high amounts of the cytokine may be lost due to cell apoptosis or differentiation. Two human NB cell lines (ACN and SK-N-BE2(c)) differing as to genetic and phenotypic features were transfected with the human IFN-γ gene and selected on the grounds of the low concentrations of IFN-γ produced. In both IFN-γ-transfected cell lines, autocrine and paracrine activation of IFN-γ-mediated pathways occurred, leading to markedly reduced proliferation rate, to increased expression of surface HLA and CD40 molecules and of functional TNF binding sites. ACN/IFN-γ cells showed a significantly delayed tumorigenicity in nude mice as compared to parental cells. ACN/IFN-γ tumours were smaller, with extensive necrotic area as a result of a damaged and defective microvascular network. In addition, a significant reduction in the proliferation index was observed. This is the first demonstration that IFN-γ inhibits in vivo proliferation of NB cell by acting on the tumour cell itself. This effect adds to the immunoregulatory and antiangiogenic activities operated by IFN-γ in syngeneic tumour-bearing hosts.

Interleukin-4 and transforming growth factor beta have opposing regulatory effects on gamma interferon-mediated inhibition of Cryptosporidium parvum reproduction.

It was shown previously that enterocytes activated by gamma interferon (IFN-gamma) are efficient effector cells in the killing of Cryptosporidium parvum. How this function is regulated is not clearly understood, but transforming growth factor beta (TGF-beta) and the Th2 regulatory cytokines may play a role. Using an in vitro cell culture system, we investigated how the key regulatory cytokines interleukin-4 (IL-4), IL-10, IL-13, and TGF-beta might modulate the effect of IFN-gamma in inducing resistance to infection in enterocyte cell lines. The results showed that TGF-beta can abolish the inhibitory effect on C. parvum development and that neither IL-13 nor IL-10 influenced the action of IFN-gamma. In contrast, IL-4 cooperated with low concentrations of IFN-gamma (1 and 10 U/ml) to enhance parasite killing. One mechanism that appeared to be involved in the combined activity of IFN-gamma and IL-4 was intracellular Fe(2+) deprivation, but induction of nitric oxide production was not involved. In one cell line, the extents and durations of phosphorylation of STAT1, a transcription factor involved in IFN-gamma signaling, were similar when cells were stimulated with IFN-gamma alone and with IFN-gamma and IL-4 gamma, suggesting that the cooperative effect of the cytokines was not related to STAT1 activation. The effects of the presence of TGF-beta and IL-4 on IFN-gamma function did not appear to involve any alteration in the level of expression of IFN-gamma receptors.

Lower level of synovial fluid interferon-gamma in HLA-B27-positive than in HLA-B27-negative patients with Chlamydia trachomatis reactive arthritis

To compare the synovial fluid (SF) concentrations of various cytokines in rheumatoid arthritis (RA) and in reactive arthritis, and to look for a correlation between cytokine levels and the presence of HLA-B27 antigen in reactive arthritis patients. Concentrations of interleukin (IL) 10, IL-12, IL-18, interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) were determined by commercially available enzyme-linked immunosorbent assays (ELISA) in the SF from 48 patients with reactive arthritis, 33 with RA and 13 with osteoarthritis (non-inflammatory controls). The SF concentrations of IL-10 were significantly lower in patients with reactive arthritis (median 2.3 pg/ml) than in RA patients (median 14.6 pg/ml). The SF levels of IFN-gamma were not significantly different but the ratios of IFN-gamma to IL-10 were significantly higher in patients with reactive arthritis (median 9.2) than in RA patients (median 0.83). When the subset of patients with Chlamydia trachomatis reactive arthritis was considered, the SF concentration of IFN-gamma was significantly lower in HLA-B27-positive (median 2.9 pg/ml) than in HLA-B27-negative patients (median 42.4 pg/ml). After 2 yr of follow-up, two HLA-B27-positive patients, who had low SF levels of IFN-gamma, had a chronic course of arthritis, whereas after 1 yr all HLA-B27-negative patients had complete resolution of arthritis. The lower IFN-gamma concentrations in HLA-B27-positive patients with C. trachomatis reactive arthritis could be related to the tendency of these patients to have more severe or chronic arthritis.

Sensitization of IFN-gamma Jak-STAT signaling during macrophage activation.

A general paradigm in signal transduction is ligand-induced feedback inhibition and the desensitization of signaling. We found that subthreshold concentrations of interferon-gamma (IFN-gamma), which did not activate macrophages, increased their sensitivity to subsequent IFN-gamma stimulation; this resulted in increased signal transducer and activator of transcription 1 (STAT1) activation and increased IFN-gamma#150;dependent gene activation. Sensitization of IFN-gamma signaling was mediated by the induction of STAT1 expression by low doses of IFN-gamma that did not effectively induce feedback inhibition. IFN-gamma signaling was sensitized in vivo after IFN-gamma injection, and STAT1 expression was increased after injection of lipopolysaccharide and in rheumatoid arthritis synovial cells. These results identify a mechanism that sensitizes macrophages to low concentrations of IFN-gamma and regulates IFN-gamma responses in acute and chronic inflammation.

Inhibitory effects of IFN-gamma and acyclovir on the glioblastoma cell cycle.

Glioblastoma multiforme is one of the most aggressive and frequently occurring forms of brain cancer. It originates from astrocytes and is characterized by a loss of cell cycle control frequently involving mutations in tumor suppressor genes, such as p53 and p16. Nucleoside analogs, such as acyclovir (ACV), are currently being used in the treatment of viral diseases, such as those caused by members of the herpes family. Further, ACV in combination with type I interferons (IFN) has been shown to be more effective at lower doses in treatment of viral diseases. We show here that ACV at high concentrations (up to 500 microg/ml) inhibited growth in tissue culture of the human glioblastoma cell lines T98G, SNB-19, and U-373 by as much as 68.3% while inhibiting normal human astrocytes by only 38.3%. Related to this, the tumor cells were more than sevenfold more efficient in phosphorylation of ACV to the active phosphate form than normal human astrocytes. Analogous to treatment of virus-infected cells, suboptimal concentrations of ACV were as effective as high concentrations when used in conjunction with low concentrations of IFN-gamma in inhibition of tumor cell growth. At the cellular level, ACV and IFN-gamma inhibited the cell cycle in both the G1 and S phases. The cooperative effect of ACV and IFN-gamma against the glioblastomas appears to be due to direct inhibition of DNA synthesis by ACV in the S phase of the cell cycle and induction by IFN-gamma of the tumor suppressor gene p21wAF1/CIP1, which in turn acts at the level of proliferating cell nuclear antigen (PCNA) and cyclin E/cyclin-dependent kinase 2 (Cdk2) binding and inhibition of function. These studies show that the combination of IFN-gamma and ACV at suboptimal concentrations elicits significant antiproliferative effects on the glioblastoma cell lines T98G, SNB-19, and U-373 while having very little effect on normal human astrocyte cell proliferation.

Suppression of rat cytomegalovirus replication by antibodies against gamma interferon.

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma.

Reduction in superoxide anion secretion and bactericidal activity of neutrophils from aged rats: reversal by the combination of gamma interferon and growth hormone.

Polymorphonuclear neutrophils (PMN) from bone marrow of 24-month-old rats kill Escherichia coli less efficiently than PMN from 3-month-old rats. Secretion of O2- and killing of E. coli by PMN from both young and old rats can be significantly augmented by preincubation with either 250 U of gamma interferon (IFN-gamma) or 250 ng of growth hormone (GH) per ml. This priming is specific, because neutralizing monoclonal antibodies against either IFN-gamma or GH completely abrogate the enhanced O2- secretion by PMN from young rats. However, in contrast to PMN from young rats, PMN from aged rats are not primed to kill E. coli by 10-fold-lower concentrations of either IFN-gamma (25 U/ml) or GH (25 ng/ml). To explore the mechanism for the reduction in bacterial killing by PMN from old rats, a syngeneic GH-secreting pituitary cell line (GH3) was implanted in vivo. PMN from GH3-treated aged rats, but not control aged rats, could now be primed in vitro for O2- secretion by IFN-gamma (25 U/ml). Although PMN from aged rats do not respond to the lower doses of either IFN-gamma or GH, the combination of both reagents totally restores the ability of PMN to secrete O2- and to kill E. coli. This synergistic priming is observed with PMN from aged rats, but not with those from young rats, and can be detected when both reagents are added simultaneously or when they are added in either sequence. Furthermore, addition of a monoclonal antibody against either IFN-gamma or GH abrogates the synergism of these two molecules. Collectively, these data identify an important alteration in myeloid cells from aged rodents by showing that their PMN are intrinsically unable to respond to low concentrations of IFN-gamma by secreting O2- and killing bacteria. The results also define a previously unrecognized synergism in PMN from aged animals by showing that GH synergizes with IFN-gamma both in vivo and in vitro to restore these suppressed responses.

Interferon-γ Enhances Growth Factor-Dependent Proliferation of Clonogenic Cells in Acute Myeloblastic Leukemia

Abstract Interferon-gamma (IFN-gamma) has been reported to antagonize the stimulatory effect of various conditioned media on the growth of normal hematopoietic progenitor cells and clonogenic blasts from patients with chronic myelogenous leukemia (CML) and acute myeloblastic leukemia (AML). In the present study, using purified recombinant cytokines and homogenous cell populations, we provide evidence for a synergistic or additive effect of IFN-gamma with recombinant human (rhu) hematopoietic growth factors in the stimulation of clonogenic blasts from most AML patients examined. Under conditions of limiting cell concentration, rhuIFN-gamma alone showed little effect on blast proliferation, whereas in conjunction with recombinant human interleukin-3 (rhuIL-3), IFN- gamma significantly enhanced colony formation in 13 of 15 AML cases. Maximal stimulation was obtained at low concentrations of IFN-gamma (2 to 20 pmol/L) in four cases and at higher concentrations (700 to 7,000 pmol/L) in the remainder. IFN-gamma also synergized with recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) in 9 of 13 cases. Within 1 hour of exposure, IFN-gamma induced a twofold to fourfold accumulation of tumor necrosis factor alpha (TNF alpha)- specific transcripts in AML blasts and several AML cell lines that include HL-60 and OCI-AML 1. Further, the synergy between IFN-gamma and IL-3 on AML blasts was partially or completely abrogated by a TNF alpha neutralizing antibody, suggesting that growth enhancement by IFN-gamma may be mediated through TNF alpha production in AML blast culture. Exposure of normal precursors (burst-forming unit-erythroid [BFU-E] and colony-forming unit granulocyte-macrophage [CFU-GM]) to IFN-gamma also resulted in significant growth enhancement, suggesting that the proliferative response elicited by IFN-gamma was not limited to AML blasts. Finally, in M07-E, an IL-3-dependent human “megakaryoblastic” cell line, IFN-gamma also significantly enhanced IL-3-supported colony formation, much in the same way as in primary AML blasts. In contrast, IFN-gamma inhibited growth of all CSF-independent leukemic cell lines tested. This inhibition was partially alleviated by anti-TNF alpha antibody. In summary, our data indicate that IFN-gamma can enhance or antagonize cell proliferation, depending on the cell type. Further, TNF alpha appears to mediate the biologic effect of IFN-gamma either in growth stimulation or growth inhibition.

Interferon-gamma enhances growth factor-dependent proliferation of clonogenic cells in acute myeloblastic leukemia

Interferon-gamma (IFN-gamma) has been reported to antagonize the stimulatory effect of various conditioned media on the growth of normal hematopoietic progenitor cells and clonogenic blasts from patients with chronic myelogenous leukemia (CML) and acute myeloblastic leukemia (AML). In the present study, using purified recombinant cytokines and homogenous cell populations, we provide evidence for a synergistic or additive effect of IFN-gamma with recombinant human (rhu) hematopoietic growth factors in the stimulation of clonogenic blasts from most AML patients examined. Under conditions of limiting cell concentration, rhuIFN-gamma alone showed little effect on blast proliferation, whereas in conjunction with recombinant human interleukin-3 (rhuIL-3), IFN-gamma significantly enhanced colony formation in 13 of 15 AML cases. Maximal stimulation was obtained at low concentrations of IFN-gamma (2 to 20 pmol/L) in four cases and at higher concentrations (700 to 7,000 pmol/L) in the remainder. IFN-gamma also synergized with recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) in 9 of 13 cases. Within 1 hour of exposure, IFN-gamma induced a twofold to fourfold accumulation of tumor necrosis factor alpha (TNF alpha)-specific transcripts in AML blasts and several AML cell lines that include HL-60 and OCI-AML 1. Further, the synergy between IFN-gamma and IL-3 on AML blasts was partially or completely abrogated by a TNF alpha neutralizing antibody, suggesting that growth enhancement by IFN-gamma may be mediated through TNF alpha production in AML blast culture. Exposure of normal precursors (burst-forming unit-erythroid [BFU-E] and colony-forming unit granulocyte-macrophage [CFU-GM]) to IFN-gamma also resulted in significant growth enhancement, suggesting that the proliferative response elicited by IFN-gamma was not limited to AML blasts. Finally, in M07-E, an IL-3-dependent human "megakaryoblastic" cell line, IFN-gamma also significantly enhanced IL-3-supported colony formation, much in the same way as in primary AML blasts. In contrast, IFN-gamma inhibited growth of all CSF-independent leukemic cell lines tested. This inhibition was partially alleviated by anti-TNF alpha antibody. In summary, our data indicate that IFN-gamma can enhance or antagonize cell proliferation, depending on the cell type. Further, TNF alpha appears to mediate the biologic effect of IFN-gamma either in growth stimulation or growth inhibition.

Cytokine interactions in experimental cutaneous leishmaniasis. Interleukin 4 synergizes with interferon‐γ to activate murine macrophages for killing of Leishmania major amastigotes

We investigated the effect of recombinant murine interleukin 4 (IL 4) in the absence or presence of recombinant murine interferon-gamma (IFN-gamma) on adherent bone-marrow macrophages (M phi), peritoneal exudate and resident peritoneal M phi from susceptible BALB/c M phi, which were pulse-infected with Leishmania major amastigotes (AM), IL 4 (5-100 U/ml) failed to activate any of these M phi populations for killing of intracellular AM. However, in the presence of low concentrations of IFN-gamma (10-20 U/ml), which alone caused only a slight or intermediate reduction of the number of intracellular parasites. IL 4 led to a dramatic increase of the parasite elimination by all M phi populations. In the case of resident peritoneal M phi, the synergism of IFN-gamma and IL 4 required the incubation of the M phi with both cytokines or with IFN-gamma alone for at least 10 h prior to infection; adding both cytokines after infection of the M phi did not cause a significant reduction of the intracellular parasite burden. The synergistic effect of IL 4 and IFN-gamma was completely abrogated in the presence of anti-IL 4 antibodies. Furthermore, there was no significant difference between M phi derived from either susceptible BALB/c or from resistant C57BL/6 mice. Evidence is presented that the synergistic action of IL 4 and IFN-gamma occurs via an L-arginine-dependent killing pathway. From these data we conclude that IL 4 provides a strong stimulus for the killing of intracellular L. major AM provided low concentrations of IFN-gamma are present. Also, IFN-gamma is apparently an important priming signal for the activation of resident M phi to eliminate intracellular AM.

Interferon Increases Susceptibility of Murine Pancreatic Beta Cells to Lysis by Allogeneic: Cytotoxic T Lymphocytes

The selective loss of insulin-producing pancreatic beta cells which occurs in IDDM has been postulated to result from lysis by beta cell-specific cytotoxic T lymphocytes (CTL). CTL typically recognise antigen in the context of MHC class I molecules, which are normally present at low levels on beta cells. However, hyperexpression of class I antigens on islet cells has been observed in the early stages of beta cell destruction in IDDM. Since interferon-gamma (IFN-gamma) is known to increase class I expression on a number of cell types, we have investigated the responses of murine beta cells to this cytokine under various conditions. Two color immunostaining followed by FACS analysis showed that on average, only 14.9 +/- 3.1% of cultured beta cells were class I positive. However, a majority of beta cells could be induced to express class I after 24 hours of IFN-gamma treatment, and maximal induction (80-90% positive) occurred after 48 hours. Importantly, increased class I expression on beta cells could be achieved with very low concentrations of IFN-gamma (1-10 U/ml). Expression of class II MHC was never detected under any of the conditions employed to up-regulate class I. Interestingly, although islet cells were only moderately susceptible to lysis by allospecific CTL, this susceptibility was markedly enhanced by prior exposure of the islets to IFN-gamma. Taken together, these results suggest that beta cells are extremely susceptible to up-regulation of class I MHC molecules by IFN-gamma, and that this property may render these cells particularly susceptible to lysis by autologous class I-restricted CTL. Since enhanced expression of class I frequently accompanies inflammatory responses and viral infections, this property of beta cells may account in part for their selective destruction in IDDM.

Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma.

Abstract IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.

Transforming growth factor-beta 2 down-regulates HLA-DR antigen expression on human malignant glioma cells.

Transforming growth factor-beta (TGF-beta) is known to have a potent inhibitory influence on several immune functions. It has recently been demonstrated that TGF-beta 2 is identical to the glioblastoma-derived T cell suppressor factor (G-TsF). In the present study, human malignant glioma cell lines were incubated with various concentrations of TGF-beta 2. An optimal concentration of 1 ng/ml TGF-beta 2 produced a partial but significant decrease of HLA-DR (class II) surface antigen expression on glioma cells expressing this antigen, as well as decreased levels of HLA-DR-specific mRNA. The surface expression of other HLA-related molecules, such as HLA-ABC (class I) and beta 2-microglobulin, was not influenced by TGF-beta 2. The suppressive effect of TGF-beta 2 on HLA-DR expression, both at the surface antigenic and cytoplasmic mRNA levels, could be completely overcome by adding relatively high concentrations (500 U/ml) of interferon (IFN)-gamma to the culture system. However, TGF-beta 2 inhibited the enhancement of HLA-DR surface expression produced by low concentrations of IFN-gamma on some cells which initially did not express these antigens. These results show that TGF-beta 2 can act as a regulator of HLA-DR antigen expression on human glioma cells.

Effect of serotonin (5-HT) and other monoamines on murine macrophages: modulation of interferon-gamma induced phagocytosis.

We have previously shown that serotonin (5-HT) suppresses interferon-gamma (IFN-gamma)-induced Ia expression. In the present report, we show that 5-HT as well as other monoamines, histamine and dopamine, modulate IFN-gamma-induced phagocytosis in murine bone marrow macrophages. The effect of 5-HT on IFN-gamma-induced phagocytosis varied according to the concentration of IFN-gamma to which the macrophages were exposed. At low concentrations of IFN-gamma, 5-HT augmented phagocytosis, whereas at high concentrations of IFN-gamma, 5-HT suppressed phagocytosis. At both low and high IFN-gamma concentrations the response to 5-HT was dose-related and occurred at physiologic concentrations; the half-maximal effect was 6 X 10(-7) M and 3 X 10(-7) M for low and high IFN-gamma concentrations, respectively. Both histamine and dopamine also augmented IFN-gamma (1 U/ml) induced phagocytosis, at half-maximal augmenting concentrations of 7 X 10(-8) M and 4 X 10(-7) M, respectively. The 5-HT effects were blocked by the 5-HT antagonists spiperone, ketanserin, LY53857, mCPP, and PAPP, but not by the histamine antagonists pyrilamine, chlorpheniramine, or cimetidine. Histamine augmentation of IFN-gamma-induced phagocytosis was blocked by the H1 antagonists pyrilamine and chlorpheniramine, but not by the H2 antagonist cimetidine. The dopamine effect was blocked by spiperone and pyrilamine, both of which have been shown to block dopaminergic effects in other systems. This data provides functional evidence that at least part of the modulation of IFN-gamma-induced phagocytosis by 5-HT occurs through a 5-HT receptor-mediated mechanism, and 5-HT, dopamine, and histamine modulate IFN-gamma-induced phagocytosis independently through their respective receptors.