Inhibitory Effect Of IFN-gamma Research Articles

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4(+) T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN-gamma-deficient-mice had more forkhead box P3 (FOXP3(+)) cells than WT mice in all secondary lymphoid organs except the thymus. However, T-bet- or IL-4Ralpha-deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3(+) cells (neo-generated inducible Treg (iTreg)) by TGF-beta was significantly inhibited by IFN-gamma in a STAT-1-dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3(+) iTreg generation by IFN-gamma was a T-cell autocrine effect. This inhibitory effect of IFN-gamma on iTreg generation was significantly abrogated after N-acetyl-L-cysteine treatment both in vitro and in vivo, indicating that IFN-gamma regulation of iTreg generation is dependent on ROS-mediated apoptosis. Therefore, our results suggest that autocrine IFN-gamma can negatively regulate the neo-generation of FOXP3(+) iTreg through ROS-mediated apoptosis in the periphery.

IL-23 induces human osteoclastogenesis via IL-17 in vitro, and anti-IL-23 antibody attenuates collagen-induced arthritis in rats

This study demonstrates that IL-23 stimulates the differentiation of human osteoclasts from peripheral blood mononuclear cells (PBMC). Furthermore, in vivo blockade of endogenous IL-23 activity by treatment with anti-IL-23 antibody attenuates collagen-induced arthritis in rats by preventing both inflammation and bone destruction. IL-23 induced human osteoclastogenesis in cultures of PBMC in the absence of osteoblasts or exogenous soluble-receptor activator of NF-kappaB ligand (RANKL). This IL-23-induced osteoclastogenesis was inhibited by osteoprotegerin, anti-IL-17 antibody, and etanercept, suggesting that RANKL, IL-17, and TNF-alpha are involved. In addition, we found the ratio of production levels of IL-17 to those of IFN-gamma from activated human T cells was elevated at 1 to 10 ng/ml IL-23. The inductive effect of IL-17 and the inhibitory effect of IFN-gamma on osteoclastogenesis indicate that the balance of these two cytokines is particularly important. We also demonstrated that IL-23 administered at a later stage significantly reduced paw volume in rats with collagen-induced arthritis, in a dose-dependent manner. Furthermore, anti-IL-23 antibody reduced synovial tissue inflammation and bone destruction in these rats. These findings suggest that IL-23 is important in human osteoclastogenesis and that neutralizing IL-23 after onset of collagen-induced arthritis has therapeutic potential. Thus, controlling IL-23 production and function could be a strategy for preventing inflammation and bone destruction in patients with rheumatoid arthritis.

Interferon-γ inhibits collagen phagocytosis in human fibroblasts by inducing subcortical actin assembly and reducing ability of β1 integrin to bind to collagen

We investigated the possible roles of interferon-gamma (IFN-gamma) in modulation of extracellular and intracellular routes of collagen digestion by human fibroblasts. Human gingival fibroblasts were treated with IFN-gamma, after which matrix metalloproteinase-1 (MMP-1) activation was determined. Following the IFN-gamma treatment, cells were further incubated with either activating antibody for beta1 integrin or actin monomer-sequestering agent latrunculin B before incubation with collagen-coated fluorescent beads. Thereafter, the binding and internalization of the beads were assessed. IFN-gamma had no significant effect on MMP-1 activation, however, it reduced the binding of collagen-coated beads in the minimum affinity range and, subsequently, internalization of the beads. The inhibitory effects of IFN-gamma were partially reversed by adding either the beta1 integrin activating antibody or latrunculin B. Although IFN-gamma does not appreciably moderate the extracellular route of collagen digestion by human fibroblasts, the reduced level of collagen phagocytosis by IFN-gamma in the cells may contribute to fibrosis in inflamed connective tissues. Further, IFN-gamma may decrease the binding of collagen and following phagocytosis in cells by inducing a subcortical actin assembly and reducing the ability of beta1 integrin to bind to collagen.

STAT1 contributes to dsRNA inhibition of liver regeneration after partial hepatectomy in mice†

Increasing evidence suggests that liver regeneration is suppressed in patients with chronic HCV infection; however, the underlying mechanisms remain unclear. Previously, we demonstrated that injection of the synthetic double-stranded RNA (dsRNA) poly I:C to mimic viral infection suppresses liver regeneration in the partial hepatectomy (PHx) model, whereby IFN-gamma contributes to the inhibition. In this study, we examined the role of the IFN-gamma-activated downstream signal (STAT1) and genes (IRF-1, p21(cip1), and SOCS1) in liver regeneration and hepatocyte proliferation. Results show that disruption of the STAT1 gene abolished poly I:C suppression of liver regeneration and the inhibitory effect of poly I:C on liver regeneration was diminished in IRF-1(-/-) and p21(cip1-/-)mice. Treatment with IFN-gamma in vitro inhibited cell proliferation of wild-type mouse hepatocytes, but not STAT1(-/-) hepatocytes. The inhibitory effect of IFN-gamma on cell proliferation was also diminished in IRF-1(-/-) and p21(cip1-/-) hepatocytes, but enhanced in SOCS1(-/-) hepatocytes. Hepatocyte proliferation was unaffected by treatment with poly I:C alone, but when hepatocytes were co-cultured with liver lymphocytes, proliferation was inhibited by IFN-gamma/STAT1-dependent mechanisms. Moreover, in HCV-infected livers with cirrhosis, activation of STAT1 was detected and correlated positively with liver injury (elevated serum levels of AST) but negatively with hepatocyte proliferation (hepatocyte PCNA and Ki-67 positive immunostaining). In conclusion, STAT1 is involved in dsRNA suppression of liver regeneration; not only does STAT1 activation contribute to liver injury, it may also block liver repair through inhibition of hepatocyte proliferation in HCV-infected patients, playing an important role in the pathogenesis of disease.

Inhibitory effect of IFN-gamma on cell proliferation is correlated with prolonged induction of SOCS1 and SOCS3 genes in human melanoma cell lines

Inhibitory effect of IFN-gamma on cell proliferation is correlated with prolonged induction of SOCS1 and SOCS3 genes in human melanoma cell lines

Inhibition of collagen gene expression by interferon‐γ: Novel role of the CCAAT/enhancer binding protein β (C/EBPβ)

By inhibiting collagen synthesis, interferon-gamma (IFN-gamma) plays a key role in maintaining connective tissue homeostasis, but the mechanisms are not well-understood. In addition to intracellular signaling through the canonical JAK-STAT transduction pathway, IFN-gamma was recently shown to regulate gene expression via the CCAAT/enhancer-binding protein beta (C/EBPbeta) as well. Because C/EBPbeta is a crucial mediator of immune and inflammatory responses, and has been implicated in regulation of collagen synthesis by tumor necrosis factor-alpha, we examined its role in the inhibitory effects of IFN-gamma. The results demonstrated that IFN-gamma caused increased C/EBPbeta expression in dermal fibroblasts and enhanced its binding to cognate DNA sequences in the alpha2(I) procollagen gene (COL1A2) promoter in vitro and in vivo. Disruption of C/EBP binding by deletion or site-directed mutagenesis abrogated the inhibition of collagen promoter activity in transient transfection assays, as did cotransfection with dominant negative C/EBPbeta, indicating a functional role of cellular C/EBPbeta in mediating the IFN-gamma response. Rapid phosphorylation of the ERK1/2 MAP kinases induced by IFN-gamma was accompanied by phosphorylation and nuclear translocation of cellular C/EBPbeta, and pretreatment of fibroblasts with ERK1/2 kinase inhibitor blocked C/EBPbeta phosphorylation, as well as inhibition of COL1A2 promoter activity, elicited by IFN-gamma. These results provide compelling evidence for a novel C/EBPbeta-dependent IFN-gamma signaling pathway responsible for inhibition of collagen gene transcription. Taken together with recent reports, the findings indicate that intracellular pathways mediating negative regulation of collagen synthesis in response to distinct inflammatory signals that converge on C/EBPbeta.

Inhibitory mechanism of inteferon-gamma on human fibroblasts from Tenon's capsule.

The inhibitory mechanism of interferon-gamma (IFN-gamma) on the fibroblasts from Tenon's capsule was studied. By using immunohistochemical SP method and pathological image system, the inhibitory effects of IFN-gamma on the expression of transforming growth factor beta receptor I in the in vitro cultured fibroblasts from Tenon's capsule were quantitatively analyzed. The results showed that IFN-gamma could reduce the expression of transforming growth factor beta receptor I in the fibroblasts with the following dose-effect relationship: Y = 1937.5-134.2 Igx (r=-0.971, P<0.01). It was concluded that IFN-gamma could inhibit the expression of transforming growth factor beta receptor I in the fibroblasts from Tenon's capsule. The modulation of the transforming growth factor beta receptor I expression by IFN-gamma may be beneficial to the alleviation of the hyperplasia of scar after trabeculectomy.

Decreased availability of intestinal dopamine in transmural colitis may relate to inhibitory effects of interferon-gamma upon L-DOPA uptake.

The present study aimed to evaluate the relationship between intestinal inflammation, interferon-gamma (IFN-gamma) levels and intestinal levels of dopamine, its precursor l-3,4-dihydroxyphenylalanine (L-DOPA), and the activity of aromatic L-amino acid decarboxylase (AADC) activity along the digestive tract in a rat experimental model of colitis. Colitis was induced by rectal administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS). Catechol derivatives were assayed by means of HPLC-EC. It is shown that dopamine and noradrenaline levels in the distal colon (inflamed mucosa), but not in the ileum (non-inflamed mucosa), of TNBS-treated rats were markedly lower than in control animals. A slight decrease in L-DOPA tissue levels, no changes in AADC activity and an increase in plasma IFN-gamma levels accompanied this decrease in dopamine levels. Exposure of Caco-2 cells, a human intestinal epithelial cell line, to human IFN-gamma resulted in a concentration-dependent and long-lasting inhibition of L-DOPA uptake, which most likely explains the decrease in dopamine levels in the inflamed mucosa. Changes in tissue levels of noradrenaline and dopamine in experimental colitis in the rat follow a similar pattern to that observed in patients with Crohn's disease and ulcerative colitis. In this model of experimental colitis, the decrease in dopamine levels is most likely explained by the inhibitory effect of IFN-gamma on L-DOPA uptake by intestinal epithelial cells.

Interferon-gamma inhibits transforming growth factor-beta production in human airway epithelial cells by targeting Smads.

Because interferon (IFN)-gamma may attenuate pulmonary fibrosis, we hypothesized that IFN-gamma may regulate transforming growth factor (TGF)-beta production by airway epithelial cells. Human bronchial epithelial cells (HBECs) were incubated with IFN-gamma +/- TGF-beta1, -beta3, or interleukin (IL)-1beta, platelet-derived growth factor (PDGF), epidermal growth factor, and IL-4. TGF-beta2 protein was measured by enzyme-linked immunosorbent assay and mRNA expression for TGF-beta2, Smad 2, 3, 4, and 7 was evaluated by real-time reverse transcriptase-polymerase chain reaction. Localization of Smads 2, 3, 4, and 7 was evaluated by immunostaining. Exogenous TGF-beta1 and 3, IL-1beta, PDGF, and IL-4 enhanced TGF-beta2 release by HBECs (P < 0.01). IFN-gamma reduced basal and TGF-beta or IL-4-augmented TGF-beta2 release, but had little effect on IL-1beta- or PDGF-augmented TGF-beta2 release. IFN-gamma stimulated Smad 7 protein and mRNA expression. Smad 7-specific siRNA decreased Smad 7 protein expression both in control and IFN-gamma-treated cells. The inhibitory effect of IFN-gamma on TGF-beta2 production was abrogated when the HBECs were treated with Smad 7 siRNA. These results suggest that IFN-gamma down regulates TGF-beta2 production by HBECs by regulating Smad 7. Through this mechanism, IFN-gamma may play an important role in tissue remodeling.

Inhibition of apoptosis by gamma interferon in cells and mice infected with Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis).

The effect of gamma interferon (IFN-gamma) on apoptosis due to infection by Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis) was studied in epithelial cells in culture and in the genital tracts of mice. IFN-gamma concentrations that induce the formation of aberrant, persistent chlamydiae inhibit apoptosis due to C. muridarum infection. In cells treated with an IFN-gamma concentration that leads to the development of a heterogenous population of normal and aberrant Chlamydia vacuoles, apoptosis was inhibited preferentially in cells that contained the aberrant vacuoles. The inhibitory effect of IFN-gamma appears to be due in part to expression of host cell indoleamine 2,3-dioxygenase activity, since inhibition of apoptosis could be partially reversed through coincubation with exogenous tryptophan. Apoptotic cells were observed in the genital tracts of wild-type mice infected with C. muridarum, and a significantly larger number of apoptotic cells was detected in infected IFN-gamma-deficient mice. These results suggest that IFN-gamma may contribute to pathogenesis of persistent Chlamydia infections in vivo by preventing apoptosis of infected cells.

Interleukin-4– and Interleukin-13–Enhanced Transforming Growth Factor- β 2 Production in Cultured Human Bronchial Epithelial Cells Is Attenuated by Interferon- γ

Cytokines derived from lymphocytes are believed to play key roles in a variety of diseases, including airway diseases such as asthma. The current study was designed to evaluate the hypothesis that cytokines derived from Th2 cells, interleukin (IL)-4 and IL-13, might contribute to tissue remodeling by modulating the production of transforming growth factor (TGF)-beta. In addition, the ability of interferon (IFN)-gamma, a cytokine derived from Th1 cells that can antagonize many effects of IL-4 and IL-13, was also assessed for its effects on TGF-beta production. IL-4 and IL-13 both stimulated production of TGF-beta2 release from human bronchial epithelial cells in a time- and concentration-dependent manner. Both with and without acidification, TGF-beta2 were detected. Neither TGF-beta1 nor TGF-beta3 was released. In contrast to the stimulatory effect on human bronchial epithelial cells, neither IL-4 nor IL-13 stimulated release of any TGF-beta isoform from human lung fibroblasts. IFN-gamma reduced both basal, IL-4-, and IL-13-stimulated release of TGF-beta2 in human bronchial epithelial cells. The stimulatory effects of IL-4 and IL-13 and the inhibitory effect of IFN-gamma on TGF-beta2 release were paralleled by mRNA levels, as assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In summary, the Th2-derived cytokines, IL-4 and IL-13, can stimulate production of TGF-beta from airway epithelial cells but not from lung fibroblasts. IFN-gamma, in contrast, can inhibit TGF-beta2 release both under basal conditions and following IL-4 or IL-13 stimulation. The ability of these cytokines to modulate TGF-beta release may contribute to both normal airway repair and to the development of subepithelial fibrosis in asthma.

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.

Interferon-gamma improves muscle flap microcirculation in double-strand RNA-induced inflammation.

Endothelial cell (EC) activation and subsequent expression of leukocyte adhesion molecules are initial events in multiple pathological processes. Viral double-strand ribonucleic acid (dsRNA) induces EC adhesion protein expression and leukocyte adhesion in vitro. Interferon-gamma (IFN-gamma) has been demonstrated to modulate the expression of certain adhesion proteins. The purpose of this study was to measure the inflammatory response to a viral mimetic--a synthetic dsRNA, polyinosinic-polycytidylic acid (poly-I:C)-on the microcirculation of a muscle flap in a rat model and to determine whether IFN-gamma attenuated the response. Two-stage surgery to create a cremaster muscle end-organ tube flap was performed on 18 male Sprague-Dawley rats in three groups. After intra-arterial injection into the abdominal aorta, the reagents (phosphate-buffered saline-bovine serum albumin [PBS-BSA] in groups I and II, and IFN-gamma in group III) were kept for 1 hour in this end-organ system. During the second stage at 16 hours, after injection into the penile vein (PBS-BSA in group I, poly-I:C in groups II and III), the flap was prepared for intravital microscopic measurement. The following parameters were measured: red blood cell velocity; vessel diameter; number of functional capillaries; and number of rolling, sticking, and transmigrating neutrophils and lymphocytes. Wilcoxon's rank sum test was used for statistical comparison. Poly-I:C caused a 70% increase in the main artery diameter and a 7% increase in velocity. But as a consequence of dynamic activation of leukocyte interaction, a 30% drop in functional capillary perfusion was observed. Injury to the entire vascular endothelium was confirmed by a 160% increase in transmigrating leukocytes. Treatment with IFN-gamma inhibited the poly-I:C-induced inflammation, as shown by 88%, 63%, and 85% decreases in rolling, sticking, and transmigrating leukocytes respectively, and by a 28% increase in capillary perfusion. Treating the system with IFN-gamma in advance, inhibited poly-I:C-induced inflammation, shown by marked decreases in rolling, adhering, and transmigrating leukocytes, and a notable increase in perfused capillaries. These observations reflect an inhibitory effect of IFN-gamma on leukocyte adhesion molecule expression in vascular endothelium in response to dsRNA in a muscle flap at the microcirculatory level.

Interferon-gamma regulates the interaction of RBL-2H3 cells with fibronectin through production of nitric oxide.

Interferon-gamma (IFN-gamma) is an important regulatory cytokine in cell proliferation, differentiation, adhesion, mediator release, and gene induction. This diversity of effector roles is achieved by a variety of incompletely understood mechanisms. In the mast cell (MC), IFN-gamma downregulates mediator synthesis and secretion. The present study demonstrates and characterizes for the first time IFN-gamma inhibition of adhesion of the MC analogue RBL-2H3 to the extracellular matrix protein fibronectin (FN). Inhibition requires preincubation of the cells with IFN-gamma for 20 hr, and is statistically significant at 100 U/ml IFN-gamma. Flow cytometry indicates that cell surface expression of very late antigen-4 (VLA-4), VLA-5, and the vitronectin receptor (VNR) remain constant following IFN-gamma treatment, indicating the inhibitory effect of IFN-gamma on adhesion to FN is not achieved through a reduction in integrin receptors for FN. Fluorescent labelling with Texas red phalloidin demonstrated rearrangement of the actin cytoskeleton in response to IFN-gamma was not significant. The tyrosine phosphatase inhibitor vanadate, and the nitric oxide (NO) synthase inhibitor L-NAME, reduced the IFN-gamma effect on adhesion to FN by 62 and 70%, respectively, demonstrating that the IFN-gamma effect is dependent upon the production of NO, potentially though a tyrosine phosphatase dependent mechanism. The NO donors sodium nitroprusside and S-nitrosoglutathione mimicked the effect of IFN-gamma. Thus, following stimulation with IFN-gamma, NO plays an autocrine role in the MC, and is able to modulate integrin function. This adds to the pathways NO is able to inhibit in the mast cell, shows that endogenous NO is able to inhibit these pathways, and suggests NO is impinging upon an element common to many signalling mechanisms in the MC.

Interferon-gamma inhibits the steroidogenic acute regulatory protein messenger ribonucleic acid expression and protein levels in primary cultures of rat Leydig cells.

Interferon-gamma (IFNgamma) is an immunomodulating cytokine that has profound effects on reproductive function. IFNgamma inhibits steroidogenesis both in vivo and in vitro. The mechanism by which IFNgamma inhibits Leydig cell steroidogenesis remains unclear. In the present study, we evaluated the effect of IFNgamma on the expression and regulation of the steroidogenic acute regulatory protein (StAR) gene in primary cultures of rat Leydig cells. StAR facilitates the efficient production of steroid hormone by regulating the translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of the cytochrome P450 side-chain cleavage (P450scc) enzyme system that converts cholesterol to pregnenolone. IFNgamma inhibited hCG-induced StAR messenger RNA (mRNA) levels in a dose-dependent manner. The addition of IFNgamma in a concentration of 500 U/ml decreased hCG-induced 3.8- and 1.7-kilobase StAR mRNA by 78% and 70%, respectively. IFNgamma also reduced hCG-stimulated P450scc mRNA levels by 69%. The inhibitory effects of IFNgamma on StAR mRNA levels were confirmed by ribonuclease protection assay. As early as 12 h after the addition of IFNgamma, hCG-induced StAR mRNA levels decreased by more than 44%. To evaluate the effects of IFNgamma on StAR protein levels, Western blot analyses were performed. hCG in a concentration of 10 ng/ml increased StAR protein by 5.6-fold. Treatment of Leydig cells with IFNgamma (500 U/ml) decreased hCG-induced StAR protein by 44%. In contrast, interleukin-1 and murine tumor necrosis factor-alpha reduced hCG-induced P450scc mRNA expression without inhibiting StAR mRNA or protein levels. In conclusion, IFNgamma inhibits Leydig cell steroidogenesis by down-regulating StAR gene expression and protein production.

A proximal element within the human alpha 2(I) collagen (COL1A2) promoter, distinct from the tumor necrosis factor-alpha response element, mediates transcriptional repression by interferon-gamma.

Previous studies have shown that interferon-gamma (IFN-gamma) inhibits type I collagen gene expression through both transcriptional and post-transcriptional mechanisms (Kahäri et al., 1990). In the present study, using transient cell transfections of human dermal fibroblast cultures with a series of 5' deletion promoter/CAT reporter gene constructs, we have identified the IFN-gamma-response element of the human alpha 2(I) collagen gene (COL1A2) promoter. Specifically, we have identified a segment of the proximal promoter region, located between nucleotides -161 and -125 relative to the transcription start site, as critical for down-regulation of COL1A2 promoter activity by IFN-gamma. This IFN-gamma response element (IgRE) is clearly distinct from the previously described tumor necrosis factor-alpha response element (TaRE) located between nucleotides -265 and -241 of the COL1A2 promoter, a difference which is likely to explain the additive inhibitory effect of these two cytokines. The inhibitory effect of IFN-gamma was dose-dependent and rapidly induced, requiring less than 5 min exposure of fibroblast cultures. Gel mobility shift assays indicated that a highly specific nuclear protein complex bound to this 37-base pair region of promoter. Competition experiments with oligonucleotides spanning discrete segments of this promoter region mapped the binding element within a distinctive pyrimidine-rich sequence. Point mutations within the latter revealed that this element plays a crucial role not only in the IFN-gamma response, but also in the basal activity of the proximal promoter. Substitution mutations within the IgRE of the -161/CAT construct attenuated the promoter response to IFN-gamma, as measured in transient cell transfections, and eliminated specific DNA/protein complex formation, as measured by gel mobility shift assay. UV-crosslinking experiments indicated that two DNA/protein complexes were formed with the IgRE, with molecular weights around 55 kDa and 30 kDa, corresponding to proteins of approximately 30 kDa and approximately 6 kDa, respectively. Our results further clarify the molecular mechanisms involved in the regulation of type I collagen gene expression by IFN-gamma.

Effects of interferon-gamma and tumor necrosis factor alpha on the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes.

To determine the effects of interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), alone or in combination, on the expression of aggrecan, biglycan, and decorin core protein genes in human chondrocytes. Isolated human chondrocytes were cultured on poly(2-hydroxyethyl methacrylate)-coated plastic dishes to prevent the loss of cartilage-specific phenotype, and the effects of IFN gamma and TNF alpha, alone or in combination, on aggrecan, biglycan, and decorin core protein gene transcription and steady-state messenger RNA (mRNA) levels were examined. The addition of IFN gamma (1.5 pM) or TNF alpha (0.3 pM) caused a decrease in the steady-state level of aggrecan mRNA (-25% and -15%, respectively), and the combination of these low-concentration cytokines caused a potent inhibition (-66%). These effects were the result of a decrease (-50%) in the transcription rate of the corresponding gene. At the concentrations used, IFN gamma did not alter the levels of biglycan mRNA or the transcription rates of the biglycan core protein gene. In contrast, TNF alpha decreased biglycan steady-state mRNA levels (-62%) and the biglycan core protein gene transcription rate (-18%). The combination of IFN gamma and TNF alpha resulted in a potentiation of the inhibitory effects of TNF alpha on biglycan mRNA levels (-79%) and transcription rate of the biglycan core protein gene (-46%). IFN gamma produced a modest decrease in decorin mRNA levels (-23%) and decorin core protein gene transcription rate (-17%). In contrast, TNF alpha resulted in a marked increase in decorin mRNA levels (+260%) that was not the result of transcriptional regulation. Notably, the combination of IFN gamma and TNF alpha potentiated the inhibitory effects of IFN gamma on decorin mRNA (-80%) and on the transcription of the corresponding gene (-43%). Similar results were obtained in fetal and adult articular chondrocytes. These data demonstrate that 1) the expression of the core protein genes encoding the cartilage proteoglycans aggrecan, biglycan, and decorin is differentially regulated by IFN gamma and TNF alpha; 2) these effects are mediated by transcriptional and posttranscriptional mechanisms; and 3) the combination of the 2 cytokines causes a potent inhibitory effect on the expression of the genes for the core proteins of these 3 proteoglycans, which occurs largely at the transcriptional level. The inhibition of aggrecan, decorin, and biglycan core protein gene expression by the combination of IFN gamma and TNF alpha may contribute to the cartilage destruction that is characteristic of inflammatory joint diseases.

IFN-gamma inhibits double-stranded RNA-induced E-selectin expression in human endothelial cells.

IFN-gamma plays a role in immune regulatory functions as well as in viral defense. We show in this study that IFN-gamma treatment down-regulates the induction by a viral mimetic, polyinosinic-polycytidylic acid (poly(I:C)), of the endothelial cell-specific leukocyte adhesion protein, E-selectin. The inhibitory effect of IFN-gamma on poly(I:C)-induced E-selectin was concentration and time dependent and was specific for dsRNA, in that the induction of E-selectin by TNF-alpha, IL-1 beta, thrombin, or LPS was not inhibited significantly by this pretreatment. IFN-gamma pretreatment reduced poly(I:C)-induced E-selectin mRNA in a protein synthesis-independent manner. Poly(I:C)-induced E-selectin mRNA t1/2 was reduced slightly by IFN-gamma treatment, while the message for VCAM-1 was stabilized. Transient transfection of endothelial cells with an E-selectin promoter-driven reporter gene construct revealed that poly(I:C) stimulation of E-selectin promoter activity was decreased significantly by IFN-gamma pretreatment. Poly(I:C)-induced nuclear factor-kappa B activation following IFN-gamma pretreatment was unaffected, as shown by electrophoretic mobility shift analysis. These results indicate a novel role for IFN-gamma in the regulation of E-selectin gene expression in response to dsRNA by a transcriptional mechanism independent of nuclear factor-kappa B, as well as by a minor decrease in message stability.

The strain difference in the effect of mercuric chloride on antigen-triggered serotonin release from rat mast cells is not mediated via interferon-gamma.

Previous work has shown that in vitro exposure of Brown-Norway (BN) rat peritoneal mast cells to mercuric chloride (HgCl2) causes enhancement of subsequent mediator release induced by cross-linking of surface immunoglobulin E (IgE). This enhancing effect is seen significantly less often with peritoneal cells from Lewis rats. In addition HgCl2 has been shown to suppress interferon (IFN)-gamma production by BN but not Lewis splenocytes. Given that IFN-gamma is known to inhibit mediator release by mast cells, we hypothesized that the strain difference in the effect of HgCl2 on mediator release was mediated via a differential effect on IFN-gamma release from T cells in the mixed peritoneal cell population: IFN-gamma release would be suppressed in the case of the BN rat, releasing the mast cells from inhibition and resulting in the enhancing effect of HgCl2. The aim of the study was to test two predictions of this hypothesis. Exposure of BN rat mast cells to IFN-gamma inhibited subsequent antigen-induced mediator release but did not significantly reduce HgCl2-mediated enhancement of this release. Exposure of Lewis rat mast cells to blocking concentrations of anti-IFN-gamma did not reveal any HgCl2-mediated enhancement of mediator release. These observations provide strong evidence against the hypothesis that the differential effects of HgCl2 on BN and Lewis rat mast cells are mediated via IFN-gamma. In addition the results revealed that BN rat mast cells are significantly more sensitive than Lewis rat mast cells to the inhibitory effects of IFN-gamma on antigen-induced mediator release.

IFN-gamma has a protective role against thyroid-specific autoantibody production in severe combined immunodeficient (SCID) mice xenografted with Graves' thyroid tissue.

We studied the effects of exogenous human IFN-gamma or neutralizing monoclonal antibody (mAb) to IFN-gamma on xenografted human Graves' thyroid tissue in severe combined immunodeficient (SCID) mice to investigate a possible role of IFN-gamma in the pathogenesis of human Graves' disease. Human thyroid tissues from four patients with Graves' disease were xenografted into SCID mice. Two weeks after xenografting, mice were divided into three groups with human IgG levels similar to each other. Mice in the first group were treated with human IFN-gamma daily for 6 weeks; mice in the second (similar) group were treated with an mAb to IFN-gamma; mice in the third group were given mouse IgG only (control group). Blood samples were taken every 2 weeks for human IgG and thyroid-specific autoantibodies (Tg-Ab, TPO-Ab, and thyroid-stimulating antibody). After 6 weeks' treatment, mice were killed, and the thyroid xenograft was examined for thyrocyte HLA-DR expression. Human IgGs were produced equally in all three groups; mice treated with IFN-gamma showed significantly lower amounts of thyroid autoantibodies than those in the control group. Thyrocyte HLA-DR expression was markedly increased in xenografts from mice with IFN-gamma administration. On the other hand, anti-IFN-gamma mAb injection caused only slight suppression of HLA-DR expression on xenografted thyroid cells. In conclusion, IFN-gamma may down-regulate the production of thyroid-specific autoantibodies but not human IgG, at least under these circumstances; there thus may be specific inhibitory effects of IFN-gamma against thyroid-specific autoantibody production of intrathyroidal plasma cells, and this animal model may help to elucidate the possible role of cytokines in the pathogenesis of Graves' disease.