IFN-γ Pretreatment Research Articles

Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD

Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H2O2), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H2O2 together did not protect them from the H2O2-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H2O2 toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase.

Role of IFN-gamma and IL-6 in a protective immune response to Yersinia enterocoliticain mice

BackgroundYersinia outer protein (Yop) H is a secreted virulence factor of Yersinia enterocolitica (Ye), which inhibits phagocytosis of Ye and contributes to the virulence of Ye in mice. The aim of this study was to address whether and how YopH affects the innate immune response to Ye in mice.ResultsFor this purpose, mice were infected with wild type Ye (pYV+) or a YopH-deficient Ye mutant strain (ΔyopH). CD11b+ cells were isolated from the infected spleen and subjected to gene expression analysis using microarrays. Despite the attenuation of ΔyopH in vivo, by variation of infection doses we were able to achieve conditions that allow comparison of gene expression in pYV+ and ΔyopH infection, using either comparable infection courses or splenic bacterial burden. Gene expression analysis provided evidence that expression levels of several immune response genes, including IFN-γ and IL-6, are high after pYV+ infection but low after sublethal ΔyopH infection. In line with these findings, infection of IFN-γR-/- and IL-6-/- mice with pYV+ or ΔyopH revealed that these cytokines are not necessarily required for control of ΔyopH, but are essential for defense against infection with the more virulent pYV+. Consistently, IFN-γ pretreatment of bone marrow derived macrophages (BMDM) strongly enhanced their ability in killing intracellular Ye bacteria.ConclusionIn conclusion, this data suggests that IFN-γ-mediated effector mechanisms can partially compensate virulence exerted by YopH. These results shed new light on the protective role of IFN-γ in Ye wild type infections.

Soluble bacterial constituents down-regulate secretion of IL-12 in response to intact Gram-positive bacteria

Intact Gram-positive bacteria induce production of large amounts of IL-12 from freshly isolated human monocytes. Here the bacterial structures and signalling pathways involved were studied and compared with those leading to IL-6 production, and to IL-12 production in response to LPS after IFN-γ pre-treatment. Intact bifidobacteria induced massive production of IL-12 (1 ng/ml) and IL-6 (>30 ng/ml) from human PBMC, whereas fragmented bifidobacteria induced IL-6, but no IL-12. IL-12 production induced by intact bifidobacteria was inhibited by pre-treatment with bifidobacterial sonicate, peptidoglycan, muramyl dipeptide, lipoteichoic acid, the soluble TLR2 agonist Pam 3Cys-SK 4, or anti-TLR2 antibodies. Blocking of phagocytosis by cytochalasin, inhibition of the JNK or NF-κB pathways or treatment with Wortmannin also reduced the IL-12 response to intact Gram-positive bacteria. LPS induced moderate levels of IL-12 (0.31 ng/ml), but only from IFN-γ pre-treated PBMC. This IL-12 production was enhanced by Wortmannin and unaffected by blocking the JNK pathway. Thus, intact Gram-positive bacteria trigger monocyte production of large amounts of IL-12 via a distinct pathway that is turned off by fragmented Gram-positive bacteria. This may be a physiological feedback, since such fragments may signal that further activation of the phagocyte via the IL-12/IFN-γ loop is unnecessary.

Sensitivity of tumor cells to proteasome inhibitors is associated with expression levels and composition of proteasome subunits

Sensitivity of tumor cells to induction of apoptosis by proteasome inhibitors varies greatly. This study was undertaken to investigate the sensitivity of neoplastic B cells and solid tumor cells to proteasome inhibition with respect to constitutive expression levels of proteasome subunits. Twelve neoplastic B-cell lines and 12 solid tumor cell lines were assessed for their expression levels of proteasome subunits by using quantitative reverse transcriptase-polymerase chain reaction analysis and were assessed for their sensitivity to the proteasome inhibitors PS-341 and lactacystin by using a flow cytometry assay that detected activated caspases. The neoplastic B-cell lines were categorized into 3 groups representing refractory cell lines, cell lines with moderate sensitivity, and cell lines with high sensitivity. Correlating expression levels of proteasome subunits with sensitivity to proteasome inhibition indicated that refractory B cells exhibited lower expression levels of the standard subunit beta2 and of the immunoproteasome subunit LMP2 compared with sensitive B cell lines. Compared with neoplastic B cells solid tumor cells were less sensitive. They expressed the immunoproteasome subunits LMP2, LMP7 and MECL-1 and the standard subunit beta2 clearly below the median of the expression level of the sensitive B cell lines. IFN-gamma pretreatment enhanced sensitivity to PS-341 in 50% of the tumor cell lines, potentially related to the induction of immunoproteasomes. The results of this study indicated that sensitivity to proteasome inhibition is correlated with expression levels of proteasome subunits, which determine the enzymatic activity of the proteasome. Combining PS-341 with IFN-gamma may enhance its clinical efficacy.

Immunological properties of umbilical cord blood-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are promising candidates for developing cell therapies for intractable diseases. To assess the feasibility of transplantation with human umbilical cord blood (hUCB)-derived MSCs, we analyzed the ability of these cells to function as alloantigen-presenting cells (APC) in vitro. hUCB-MSCs were strongly positive for MSC-related antigens and stained positively for human leukocyte antigen (HLA)-AB and negatively for HLA-DR. When treated with interferon (IFN)-γ, the expression of HLA-AB and HLA-DR, but not the co-stimulatory molecules CD80 and CD86, was increased. hUCB-MSCs did not provoke allogeneic PBMC (peripheral blood mononuclear cell) proliferation, even when their HLA-molecule expression was up-regulated by IFN-γ pretreatment. When added to a mixed lymphocyte reaction (MLR), hUCB-MSCs actively suppressed the allogeneic proliferation of the responder lymphocytes. This suppressive effect was mediated by soluble factors. We conclude that hUCB-MSCs can suppress the allogeneic response of lymphocytes and may thus be useful in allogeneic cell therapies.

Induction of Indoleamine 2,3-Dioxygenase in Vascular Smooth Muscle Cells by Interferon-γ Contributes to Medial Immunoprivilege

Atherosclerosis and graft arteriosclerosis are characterized by leukocytic infiltration of the vessel wall that spares the media. The mechanism(s) for medial immunoprivilege is unknown. In a chimeric humanized mouse model of allograft rejection, medial immunoprivilege was associated with expression of IDO by vascular smooth muscle cells (VSMCs) of rejecting human coronary artery grafts. Inhibition of IDO by 1-methyl-tryptophan (1-MT) increased medial infiltration by allogeneic T cells and increased VSMC loss. IFN-gamma-induced IDO expression and activity in cultured human VSMCs was considerably greater than in endothelial cells (ECs) or T cells. IFN-gamma-treated VSMCs, but not untreated VSMCs nor ECs with or without IFN-gamma pretreatment, inhibited memory Th cell alloresponses across a semipermeable membrane in vitro. This effect was reversed by 1-MT treatment or tryptophan supplementation and replicated by the absence of tryptophan, but not by addition of tryptophan metabolites. However, IFN-gamma-treated VSMCs did not activate allogeneic memory Th cells, even after addition of 1-MT or tryptophan. Our work extends the concept of medial immunoprivilege to include immune regulation, establishes the compartmentalization of immune responses within the vessel wall due to distinct microenvironments, and demonstrates a duality of stimulatory EC signals versus inhibitory VSMC signals to artery-infiltrating T cells that may contribute to the chronicity of arteriosclerotic diseases.

Abrogation of the Antifibrotic Effects of Natural Killer Cells/Interferon-γ Contributes to Alcohol Acceleration of Liver Fibrosis

Chronic alcohol drinking accelerates liver fibrosis in patients with viral hepatitis that cannot be fully explained by ethanol-enhanced liver damage. Here, we identified a novel mechanism by which alcohol accelerates liver fibrosis: inhibition of the antifibrotic effects of natural killer (NK) cells and interferon-gamma (IFN-gamma). Alcohol administration was achieved by feeding mice with a liquid diet containing 5% ethanol for 8 weeks. Liver fibrosis was induced by administration of carbon tetrachloride (CCl(4)) for 2 weeks. Hepatic stellate cells (HSCs) were also isolated and cultured for in vitro studies. CCl(4) treatment induced greater fibrosis and less apoptosis of HSCs in ethanol-fed mice compared with pair-fed mice. Polyinosinic-polycytidylic acid (Poly I:C) or IFN-gamma treatment inhibited liver fibrosis in pair-fed but not in ethanol-fed mice. Poly I:C activation of NK cell cytotoxicity against HSCs was attenuated in ethanol-fed mice compared with pair-fed mice, which was due to reduced natural killer group 2 member D (NKG2D), tumor necrosis factor-related apoptosis-inducing ligand, and IFN-gamma expression on NK cells from ethanol-fed mice. In vitro, HSCs from ethanol-fed mice were resistant to IFN-gamma-induced cell cycle arrest and apoptosis compared with pair-fed mice. Such resistance was due to diminished IFN-gamma activation of signal transducer and activator of transcription 1 (STAT1) in HSCs from ethanol-fed mice caused by the induction of suppressors of cytokine signaling proteins and the production of oxidative stress. Finally, HSCs from ethanol-fed mice were resistant to NK cell killing, which can be reversed by transforming growth factor-beta1 (TGF-beta1) neutralizing antibody. Chronic ethanol consumption attenuates the antifibrotic effects of NK/IFN-gamma/STAT1 in the liver, representing new and different therapeutic targets with which to treat alcoholic liver fibrosis.

Evaluation of IFN-γ effects on apoptosis and gene expression in neuroblastoma—Preclinical studies

Loss of caspase-8 expression and resistance to cytotoxic agents occurs frequently in late stage neuroblastoma (NB). Interferon-γ (IFN-γ) induces caspase-8 in NB cells, sensitizing them to death receptor mediated apoptosis. This study characterizes the kinetics of this phenomenon and examines the effects of IFN-γ on global gene expression to determine whether IFN-γ responses are achievable at physiologically relevant doses and to define the biological effects of this cytokine. Here we examine the IFN-γ responses of 16 NB cell lines. A single < 5-min exposure to IFN-γ (0.5 ng/ml) induced caspase-8 expression in all non-expressing cell lines and in 3/6 cell lines which already expressed high caspase-8. This increase in caspase-8 proteins was observed within 16 h and persisted for up to 9 days. Furthermore, IFN-γ pretreatment of NB cells increased doxorubicin-induced apoptosis nearly 3-fold. Microarray analysis was used to identify additional genes involved in proliferation, signaling and apoptosis whose expression was modulated via IFN-γ. Altered expression of these genes should further enhance the responsiveness of NB cells to chemotherapeutics. Thus, the use of IFN-γ to sensitize NB cells to cytotoxic agents represents an attractive therapeutic strategy and warrants further investigation.

IFNγ pretreatment sensitizes human choriocarcinoma cells to etoposide-induced apoptosis

Choriocarcinoma is a malignant trophoblast-derived tumour, which can arise in any type of gestation. Cell proliferation assays showed that interferon gamma (IFNgamma) alone significantly inhibited proliferation of choriocarcinoma JAR and JEG-3 cells. TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assays and Hoechst staining indicated that IFNgamma alone could not induce apoptosis of JAR and JEG-3 cells, but IFNgamma could enhance the sensitivity of JAR cells to etoposide-induced apoptosis. RT-PCR and western blotting were performed to detect expression of apoptosis-related molecules IFNgammaR, interferon regulatory factor-1 (IRF-1), p53 and pro-caspase 3. In JAR cells, etoposide increased expression of the proteins including IFNgammaR, p53 and pro-caspase 3 as well as IRF-1 mRNA and IFNgamma-pretreatment apparently promoted up-regulation of these molecules expression. In addition, the responses of IRF-1, p53 and pro-caspase 3 expression to IFNgamma pretreatment were dose dependent. IRF-1 knock down assays demonstrated that IRF-1 directly mediated IFNgamma pretreatment enhanced sensitivity of JAR cells to etoposide-induced apoptosis and that pro-caspase 3 was one of the target genes of IRF-1.

Role of strain differences on host resistance and the transcriptional response of macrophages to infection withYersinia enterocolitica

The outcome of a host-pathogen encounter is determined by virulence factors of the pathogen and defense factors of the host. We characterized the impact of host factors [resistant (C57BL/6) or susceptible (BALB/c) genetic background and exposure to interferon (IFN)-gamma] on transcriptional responses of bone marrow-derived macrophages (BMDM) to infection with Yersinia enterocolitica. IFN-gamma treatment more profoundly altered the transcriptome of BMDM than did bacterial infection or genetic background. In BALB/c BMDM, 1,161 genes were differentially expressed in response to Yersinia infection with or without IFN-gamma prestimulation. Fourteen genes (1.2%) could only be induced by BALB/c BMDM in response to Yersinia infection after IFN-gamma pretreatment. These genes inhibit apoptosis, activate NF-kappaB and Erk signaling, are chemotactic to neutrophils, and are involved in cytoskeletal reorganization, hence possibly in phagocytosis. Ten of these genes possess a common module of binding sites for Hox, Pou, and Creb transcription factors in 2 kb of upstream genomic sequence, suggesting a possible novel role of these transcription factors in regulation of immune responses. Fifty-two of one thousand fifty differentially expressed genes (4.9%) were induced more strongly by C57BL/6 BMDM in response to Yersinia infection than BALB/c BMDM. These genes activate NK cells, have antibacterial properties, or are involved in sensing chemokines and lipopolysaccharide (LPS). These data show that host resistance factors modulate a surprisingly small, but identifiable and functionally significant, portion of the macrophage transcriptome in response to Yersinia infection.

Differential Effects of TNF-α and IFN-γ on Gene Transcription Mediated by NF-κB-Stat1 Interactions

Regulation of gene transcription by the cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) involves complex interactions between NF-kappaB and Stat families of transcription factors. The purpose of this study was to identify the spatial promoter requirements that govern cytokine synergy for gene transcription regulated by NF-kappaB and Stat factors. Using a set of transcription reporter-luciferase constructs, we show that the relative orientation of juxtaposed NF-kappaB-Stat (SIE) cis-elements determines the ability of TNF-alpha and IFN- gamma to induce gene transcription. Further, NF-kappaB and Stat1 proteins directly regulate transcription by interacting cooperatively on NF-kappaB-SIE DNA binding in response to TNF-alpha plus IFN-gamma. Coimmunoprecipitation provides evidence for a direct NF-kappaB/Stat1 protein-protein interaction. In contrast, IFN-gamma inhibits TNF-alpha-induced transcription of an NF-kappaB reporter gene in a Stat1-dependent mechanism in 2fTGH fibroblasts. Similarly, Stat1 is inhibitory to NF-kappaB overexpression-induced transcription. IFN-gamma and Stat1-dependent inhibition of NF-kappaB transcription occurs independent of TNF-alpha-induced NF-kappaB DNA binding. Interestingly, IFN-gamma pretreatment of 2fTGH fibroblasts potentiates TNF-alpha induction of Stat1 DNA binding. Further, ChIP analysis was applied to detect cytokine-induced in vivo binding and transcriptional regulation of the human inducible nitric oxide synthase (iNOS) gene by NF-kappaB and Stat1. These data demonstrate complex transcriptional regulatory mechanisms elicited by TNF-alpha and IFN-gamma and have potentially important implications for other genes differentially controlled by cytokines.

Fibrocytes are potent stimulators of anti-virus cytotoxic T cells

Fibrocytes (Fb) are a population of circulating leukocytes reported to be capable of presenting antigen to CD4(+) T lymphocytes. In contrast, no information is available about their capacity to stimulate CD8(+) cytolytic T lymphocyte (CTL) responses. To this end, Fb were isolated from porcine blood to investigate their ability to stimulate CTL responses using a classical swine fever virus model. The isolated Fb (referred to as primary Fb) displayed the phenotype previously reported for mouse and human Fb, particularly in terms of the surface proteins necessary for antigen presentation, major histocompatibility complex (MHC) classes I and II, and CD80/86. These primary Fb endocytosed and degraded antigen efficiently. In absence of exogenous stimuli, endocytosis and MHC II expression were lost when the Fb were passaged and cultured. Treatment of such secondary Fb with interferon-gamma (IFN-gamma) restored the MHC II expression. The primary and secondary Fb were capable of stimulating antigen-specific CD4(+) T lymphocytes relating to previous reports. In addition, an efficient stimulation of virus-specific CD8(+)CTL was measured in terms of CD8(+) T cell proliferation, IFN-gamma production, and cytotoxic activity. This was noted even at low Fb/T lymphocyte ratios, at which dendritic cells were less efficient. Although IFN-gamma pretreatment of Fb was not necessary for this function, it could enhance the Fb activity. These results demonstrate that Fb are efficient, accessory cells for the presentation of viral antigen to specific CD8(+) CTL.

Interferon-γ Down-regulates Adenosine 2b Receptor-mediated Signaling and Short Circuit Current in the Intestinal Epithelia by Inhibiting the Expression of Adenylate Cyclase

Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.

Bcl-xL/Bax ratio is altered by IFNγ in TNFα- but not in TRAIL-induced apoptosis in colon cancer cell line

Apoptosis is a crucial mechanism to eliminate harmful cells in which growth factors and cytokines are key regulators. In HT29-D4 cells, a model of human colon carcinoma, IFNγ presensitization is essential to induce an apoptotic response to TNFα whereas it only slightly enhances TRAIL-induced apoptosis. To compare the transcriptional profiles induced by TNFα and TRAIL and their regulation by IFNγ, we optimized a cDNA array analysis on targeted signaling pathways and confirmed the gene expression modulations by comparative RT-PCR. Although the two TNFSF ligands induced a same strong up-expression of pro-apoptotic Bax gene, the expression of anti-apoptotic Bcl-xL gene was more strongly up-regulated in TNFα- than in TRAIL-stimulated cells. Thus, TRAIL but not TNFα induced apoptotic mitochondrial cascade as highlighted by cytochrome c release into cytosol. IFNγ presensitization of TRAIL-stimulated cells did not induce any change in cytochrome c release, suggesting that the increase of IFNγ/TRAIL-induced apoptosis is independent of this pathway. In contrast, IFNγ pretreatment prevented Bcl-xL gene up-expression in TNFα-stimulated cells and allowed cytochrome c release. Thus, we hypothesize that the Bcl-xL/Bax ratio can block the apoptotic response in TNFα-stimulated cells but allows cell death initiation when it is altered by a crosstalk between IFNγ presensitization and TNFα induced signalings.

IFN-γ primes macrophages for enhanced TNF-α expression in response to stimulatory and non-stimulatory amounts of microparticulate β-glucan

β-(1 → 3)- d-Glucan is an integral cell wall component of a variety of fungi, plants, and bacteria. Like the prototypic inflammatory mediator lipopolysaccharide (LPS), some β-(1 → 3)- d-glucan-containing preparations have been shown to induce the production of proinflammatory cytokines by macrophages. In the present study, we have tested a new microparticulate form of β-(1 → 3)- d-glucan (MG) from Saccharomyces cerevisiae for its ability to induce proinflammatory cytokine secretion in mouse peritoneal macrophages in vitro, and we have examined the effect of IFN-γ. MG was rapidly phagocytized by peritoneal macrophages, and these MG-treated macrophages upregulated TNF-α, IL-6, and IL-1β mRNAs and secreted these proinflammatory cytokines. IFN-γ treatment alone did not induce unstimulated macrophages to produce TNF-α. However, a 4 h IFN-γ pretreatment augmented TNF-α secretion by peritoneal macrophages subsequently treated with an optimally stimulatory dose of MG. IFN-γ pretreatment for 2 h followed by thorough washing and a further 2 h incubation without IFN-γ still resulted in enhanced TNF-α production in response to MG, suggesting that IFN-γ can prime macrophages for a subsequent proinflammatory response. Most interestingly, we found that IFN-γ pretreatment of peritoneal macrophages enhanced the TNF-α response to amounts of MG that were poorly stimulatory or non-stimulatory in the absence of IFN-γ priming. These data suggest that a synergy between IFN-γ and β-glucan may have evolved to lower the threshold of sensitivity of the innate immune response to fungal pathogens.

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFα production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFα production. The pro-inflammatory cytokine, IFNγ, also abolishes suppression of TNFα in LPS tolerance. The effect of LPS tolerance on HKSa and IFNγ-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNγ differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNγ (1 μg/ml). Cells were subsequently stimulated with LPS or HKSa (10 μg/ml) for 24 h. The production of the cytokines TNFα, IL-6, IL-1β, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFα ( 3070±711 pg/ ml and 217±9 pg/ ml , respectively) and IL-6 ( 237±8.9 pg/ ml and 56.2±2.9 pg/ ml , p<0.05, n=3, respectively) in control cells compared to basal levels (<25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% ( p<0.05, n=3) reduction in TNFα. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFα (2.7 fold, from 217 to 580 pg/ml, p<0.05, n=3). In contrast to suppressed TNFα, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p<0.01, n=3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p<0.01, n=3). LPS induced IL-8 production and to a lesser extent IL-1β and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1β, although HKSa stimulation augmented both mediators. In addition, IFNγ pretreatment reversed LPS tolerance as evidenced by increased TNFα levels while IL-6, IL-1β, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNγ. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.

Regulation of STAT6 by interferon- (IFN-) γ in human airway epithelial cells

It is known that the Th1-type cytokine IFN-γ can antagonize respiratory epithelial responses to antigen or IL-4. Therefore, we investigated the effect of IFN-γ on IL-4-induced STAT6 activation and function in cultured primary human airway epithelial cells. Data from Western blot experiments demonstrated that IL-4-induced nuclear phospho-STAT6 was significantly inhibited by 24 h pre-treatment with IFN-γ (64 ± 7.5% inhibition, P<0.0001, n=10). This effect was dependent upon the concentration and duration of the IFN-γ pre-treatment (half maximal effect at ∼40 ng/mL and ∼18 hours). The transcriptional function of STAT6 was also inhibited by IFN-γ, as measured by inhibition of the ability of IL-4 to activate a STAT6-driven luciferase reporter construct and inhibition of eotaxin-3 expression at both the mRNA and protein level. IFN-γ treatment upregulated the mRNA for SOCS-1 and SOCS-3, known inhibitors of STAT6 signaling, and the level of mRNA for IL-13Rα2, a putative decoy receptor, in a dose-dependent fashion (29, 10 and 66-fold respectively, P<0.05, n=3-7). IFN-γ accelerated the decay rate of IL-4-induced eotaxin-3 mRNA by approximately five-fold ( P<0.001, n=4). These studies suggest that the mechanism for IFN-γ inhibition of STAT6 function and eotaxin-3 expression is complex and may involve induction of inhibitory SOCS proteins and IL-13Rα2, as well as the destabilization of eotaxin-3 mRNA. These findings have important implications for the regulation of epithelial cell activation in allergic disease.

Inhibition of interferon- [formula omitted]-activated nuclear factor- [formula omitted]B by cyclosporin A: a possible mechanism for synergistic induction of apoptosis by interferon- [formula omitted] and cyclosporin A in gastric carcinoma cells

We previously reported synergistic induction of apoptosis by IFN-γ plus either cyclosporin A (CsA) or tacrolimus (FK506) in gastric carcinoma cells. In this study, we aimed to elucidate the mechanism for this synergistic induction of apoptosis. IFN-γ plus CsA synergistically induced caspase-3 mediated apoptosis in gastric carcinoma cells. Although IFN-γ induced activation of signal transducer and activator of transcription1 (STAT1) and expression of interferon regulatory factor-1 (IRF-1) mRNA, IFN-γ alone was not able to induce caspase-3 activation and apoptosis. When gastric carcinoma cells were treated with cyclohexamide, a protein synthesis inhibitor, following IFN-γ pretreatment, caspase-3 was activated, and apoptosis was markedly induced. These findings suggest the existence of IFN-γ-induced anti-apoptotic pathway and we evaluated the effect of IFN-γ and CsA on calcium-sensitive nuclear factor-κB (NF-κB) activation. IFN-γ increased intracellular calcium ion concentration ([Ca 2+] i) consisting of a spike and a sustained phase, and the latter was completely abrogated by CsA. Activation of NF-κB occurred in response to IFN-γ, and which was markedly inhibited by either CsA or FK506. NF-κB decoy also enhanced the cytotoxic effect of IFN-γ. These results suggest that IFN-γ may simultaneously induce the STAT1-mediated apoptotic pathway and the anti-apoptotic pathway through calcium-activated NF-κB and that inhibition of the latter by CsA may result in dominance of the apoptosis-inducing pathway.

Nuclear factor kappaB protects pancreatic beta-cells from tumor necrosis factor-alpha-mediated apoptosis.

Recent studies incriminating tumor necrosis factor (TNF)-alpha as the final effector in pancreatic beta-cell death in type 1 diabetes underscore the potential role of TNF-alpha-dependent NF-kappaB activation as an important modulator of pancreatic beta-cell death in autoimmune diabetes. Although nuclear factor (NF)-kappaB activation has been implicated in the protection of target cells against apoptosis by a variety of death effectors, its role in pancreatic islet cell death is not clear. We studied the role of NF-kappaB activation in pancreatic islet cell death by using a gamma-interferon (IFN-gamma)/TNF-alpha synergism model we had previously reported. TNF-alpha induced inhibitor of kappaB (IkappaB) degradation and p65 translocation from cytoplasm to nuclei in MIN6N8 insulinoma cells. The NF-kappaB DNA-binding nuclear complex activated by TNF-alpha contained both the p65 and p50 subunit. IFN-gamma pretreatment did not affect TNF-alpha-induced NF-kappaB activation. Treatment with a proteasome inhibitor blocked p65 translocation and induced susceptibility to TNF-alpha in otherwise resistant insulinoma cells or primary pancreatic islet cells. Specific inhibition of NF-kappaB activation by adenoviral transduction of IkappaB "superrepressor" also sensitized insulinoma cells and primary islet beta-cells to TNF-alpha-induced apoptosis. These results suggest the protective role of NF-kappaB activation against cytokine-mediated pancreatic beta-cell death, contrary to previous reports implicating NF-kappaB as a mediator of pancreatic islet cell death.

The antiviral response to gamma interferon.

A role for alpha/beta interferon (IFN-alpha/beta) in the IFN-gamma antiviral response has long been suggested. Accordingly, possible roles for autocrine or double-stranded-RNA (dsRNA)-induced IFN-alpha/beta in the IFN-gamma response were investigated. Use was made of wild-type and a variety of mutant human fibrosarcoma cell lines, including mutant U5A cells, which lack a functional IFN-alpha/beta receptor and hence an IFN-alpha/beta response. IFN-gamma did not induce detectable levels of IFN-alpha/beta in any of the cell lines, nor was the IFN-gamma response per se dependent on autocrine IFN-alpha/beta. On the other hand, a number of responses to dsRNA [poly(I). poly(C)] and encephalomyocarditis virus were greatly enhanced by IFN-gamma pretreatment (priming) of wild-type cells or of mutant cells lacking an IFN-alpha/beta response; these include the primary induction of dsRNA-inducible mRNAs, including IFN-beta mRNA, and, to a lesser extent, the dsRNA-mediated activation of the p38 mitogen-activated protein (MAP) kinase(s). IFN-gamma priming of mRNA induction by dsRNA is dependent on JAK1 and shows biphasic kinetics, with an initial rapid (<30-min) response being followed by a more substantial effect on overnight incubation. The IFN-gamma-primed dsRNA responses appear to be subject to modulation through the p38, phosphatidylinositol 3-kinase, and ERK1/ERK2 MAP kinase pathways. It can be concluded that despite efficient priming of IFN-beta production, the IFN-alpha/beta pathways play no significant role in the primary IFN-gamma antiviral response in these cell-virus systems. The observed IFN-gamma priming of dsRNA responses, on the other hand, will likely play a significant role in combating virus infection in vivo.