IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.     

IFN-gamma up-regulates IL-18 gene expression via IFN consensus sequence-binding protein and activator protein-1 elements in macrophages

Constitutive IL-18 expression is detected from many different cells, including macrophages, keratinocytes, and osteoblasts. It has been known that IL-18 gene expression is regulated by two different promoters (p1 promoter and p2 promoter). When RAW 264.7 macrophages were treated with IFN-gamma, IL-18 gene expression was increased in a dose- and time-dependent manner. IFN-gamma activated the inducible promoter 1, but not the constitutive promoter 2. Mutagenesis studies indicated that an IFN consensus sequence-binding protein (ICSBP) binding site between -39 and -22 was critical for the IFN-gamma inducibility. EMSA using an ICSBP oligonucleotide probe showed that IFN-gamma treatment increased the formation of DNA-binding complex, which was supershifted with anti-IFN regulatory factor-1 Ab and anti-ICSBP Ab. Another element, an AP-1 site between -1120 and -1083, was important. EMSA using an AP-1-specific oligonucleotide demonstrated that IFN-gamma or LPS treatment increased the AP-1-binding activity. The addition of anti-c-Jun Ab or anti-c-Fos Ab to IFN-gamma- or LPS-treated nuclear extracts resulted in the reduction of AP-1 complex or the formation of a supershifted complex. Taken together, these results indicate that IFN-gamma increased IL-18 gene expression via ICSBP and AP-1 elements.     

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4

Macrophages release IFN-gamma on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-gamma by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-gamma production in NK cells, was not required for IFN-gamma production by these macrophages. IL-12 alone already induced the expression of IFN-gamma mRNA, but nuclear translocation of STAT4, the release of IFN-gamma protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-kappa B, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-beta) also suppressed macrophage IFN-gamma production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-gamma by macrophages and point to a diversity in the signals required for IFN-gamma production by various cell types.     

IL-18-induced expression of intercellular adhesion molecule-1 in human monocytes: involvement in IL-12 and IFN-gamma production in PBMC

IL-18 time- and concentration-dependently upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in a monocyte population in human PBMC as determined by FACS analysis while the expression of CD11a, CD18, CD29, CD44, and CD62L in monocytes and that of ICAM-1, CD11a, CD18, CD29, CD44, and CD62L in T cells was not influenced by IL-18. IL-18 in the same concentration range stimulated the production of IL-12, TNF-alpha, and IFN-gamma in culture of PBMC; however, IL-18-induced expression of ICAM-1 in monocytes was not inhibited by anti-IL-12, anti-TNF-alpha, or anti-IFN-gamma Ab, suggesting the independence of the upregulating effect of IL-18 on endogenous IL-12, TNF-alpha, and IFN-gamma production. IL-18 also induced the aggregation of PBMC, which was prevented by anti-ICAM-1 and anti-LFA-1 Abs. On the other hand, anti-ICAM-1 and anti-LFA-1 Abs inhibited IL-18-induced production of three cytokines, IL-12, IFN-gamma, and TNF-alpha, by 60 and 40%, respectively. These results strongly suggested that the IL-18-induced upregulation of ICAM-1 and the subsequent adhesive interaction through ICAM-1 on monocytes and LFA-1 on T/NK cells generate an additional stimulatory signaling as well as an efficient paracrine environment for the IL-18-initiated cytokine cascade.     

Expression and release of IL-18 binding protein in response to IFN-gamma.

IL-18 and IL-18 binding protein (IL-18BP) are two newly described opponents in the cytokine network. Local concentrations of these two players may determine biological functions of IL-18 in the context of inflammation, infection, and cancer. As IL-18 appears to be involved in the pathogenesis of Crohn's disease and may modulate tumor growth, we investigated the IL-18/IL-18BPa system in the human colon carcinoma/epithelial cell line DLD-1. In this study, we report that IFN-gamma induces expression and release of IL-18BPa from DLD-1 cells. mRNA induction and secretion of IL-18BPa immunoreactivity were associated with an activity that significantly impaired release of IFN-gamma by IL-12/IL-18-stimulated PBMC. Inducibility of IL-18BPa by IFN-gamma was also observed in LoVo, Caco-2, and HCT116 human colon carcinoma cell lines and in the human keratinocyte cell line HaCaT. Induction of IL-18BPa in colon carcinoma/epithelial cell lines was suppressed by coincubation with sodium butyrate. IFN-gamma-mediated IL-18BPa and its suppression by sodium butyrate were confirmed in organ cultures of intestinal colonic biopsy specimens. In contrast, sodium butyrate did not modulate expression of IL-18. The present data suggest that IFN-gamma may limit biological functions of IL-18 at sites of colonic immune activation by inducing IL-18BPa production. Down-regulation of IL-18BPa by sodium butyrate suggests that reinforcement of local IL-18 activity may contribute to actions of this short-chain fatty acid in the colonic microenvironment.     

Cutting edge: a murine,IL-12-independent pathway of IFN-gamma induction by gram-negative bacteria based on STAT4 activation by Type I IFN and IL-18 signaling

IFN-alphabeta is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections. In this study, we describe an as yet undefined IFN-alphabeta-dependent pathway of IFN-gamma induction in mice. This pathway is based on a synergism of IFN-alphabeta and IL-18, and is independent of IL-12 signaling yet dependent on STAT4. In contradiction to current dogma, we show further that IFN-alphabeta alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains. This pathway participates in the induction of IFN-gamma by Gram-negative bacteria and is therefore expected to play a role whenever IFN-alpha or IFN-beta and IL-18 are produced concomitantly during bacterial, viral, or other infections.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

Induction by IL-1 beta of tissue inhibitor of metalloproteinase-1 in human orbital fibroblasts: modulation of gene promoter activity by IL-4 and IFN-gamma

Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves' disease, is associated with profound connective tissue remodeling and fibrosis that appear to involve the selective activation of orbital fibroblasts. Accumulation of extracellular matrix molecules is a hallmark of this process. Here we report that orbital fibroblasts treated with IL-1beta express high levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important modulator of matrix metalloproteinase activity. These high levels are associated with increased TIMP-1 activity. The induction is mediated at the pretranslational level and involves activating the TIMP-1 gene promoter. IL-1beta activates the ERK 1/2 pathway in these fibroblasts and interrupting this signaling either with PD98059, a chemical inhibitor of MEK, or by transfecting cells with a dominant negative ERK 1 plasmid results in the attenuation of TIMP-1 induction. Surprisingly, treatment with IL-4 or IFN-gamma could also block the IL-1beta induction by attenuating TIMP-1 gene promoter activity. These findings suggest that TIMP-1 expression in orbital fibroblasts following activation with IL-1beta could represent an important therapeutic target for modifying the proteolytic environment. This might alter the natural course of tissue remodeling in TAO.     

IL-1beta down-regulates tissue-type plasminogen activator by up-regulating low-density lipoprotein receptor-related protein in AML 12 cells

Interleukin-1 (IL-1) regulation of tPA in hepatocytes was studied in mouse hepatocyte line AML12. IL-1 induced transient accumulation of tPA mRNA as high as threefold by 2 h after the start of treatment. The cytokine also induced the mRNA for serum amyloid A, a typical acute-phase protein in mice, with more sustained kinetics in a time-dependent manner. In contrast to the induction of mRNA, tPA activity and protein levels in the harvested medium were dramatically diminished by IL-1. IL-1 stimulated the uptake of (125)I-tPA by AML 12. This uptake was inhibited by 39-kDa receptor-associated protein (RAP), but not by the sugar mannan. These results revealed that low-density lipoprotein receptor-related protein (LRP), which is known to be a receptor for tPA and to be blocked by RAP, was up-regulated by IL-1. We also demonstrated, for the first time, that IL-1 transiently increased the mRNA level of LRP threefold by 30 min after the start of IL-1 treatment of AML 12. The receptor-mediated endocytosis of tPA by hepatocytes may thus play a crucial role in the down-regulation of fibrinolysis during the acute-phase response.     

Inhibition of IFN-gamma -induced STAT1 activation by 15- deoxy-Delta 12,14-prostaglandin J2

The inhibitory actions of 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)) on inflammatory gene expression have been attributed to the ability of this prostaglandin to inhibit the activation of NF-kappaB. In this study, we have identified an additional signaling pathway sensitive to inhibition by PGJ(2). We show that PGJ(2) inhibits interferon (IFN)-gamma-stimulated phosphorylation and DNA-binding activity of STAT1. The inhibitory actions on STAT1 phosphorylation are first apparent after a 1- to 2-h incubation and are maximal after a 6-h incubation with PGJ(2), and they correlate with the expression of heat shock protein (HSP)70 in islets. In previous studies, we have correlated the inhibitory actions of PGJ(2) on inducible nitric oxide synthase (iNOS) expression and NF-kappaB activation in response to IL-1 with the increased expression of HSP70. Using overexpression and antisense depletion, we provide evidence that HSP70 does not mediate the inhibitory actions of PGJ(2) on IL-1-induced NF-kappaB or IFN-gamma-induced STAT1 activation or cytokine-stimulated iNOS expression by beta-cells. Last, we show that the inhibitory actions of a short 6-h pulse with PGJ(2) on IL-1 plus IFN-gamma-stimulated iNOS expression and NO production by beta-cells are persistent for extended periods (< or =48 h). These findings suggest that PGJ(2) inhibits multiple cytokine-signaling pathways (IL-1 and IFN-gamma), that the inhibitory actions are persistent for extended periods, and that increased HSP70 expression correlates with, but does not appear to mediate, the inhibitory actions of PGJ(2) on IL-1 and IFN-gamma signaling in beta-cells.     

Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-gamma production by dendritic cells

Mouse splenic dendritic cells (DCs) produce IFN-gamma in response to IL-12. In the present study, we analyzed effects of Th1 and Th2 cytokines on IFN-gamma production by DCs. IL-18 produced by DCs and macrophages acts in an autocrine manner and augments IL-12-induced IFN-gamma production by DCs as also observed in T and NK cells. Surprisingly, IL-4, a Th2 cytokine, also acts synergistically with IL-12 on IFN-gamma production by DCs. In addition, IL-4 markedly enhances IFN-gamma production when DCs are stimulated through CD40 or MHC class II. These results indicate that both Th1 and Th2 cytokines act on DCs during T cell-DC interaction upon Ag presentation. p38 mitogen-activated protein kinase is constitutively activated in mature DCs and is required for IFN-gamma production by DCs. IL-18 but not IL-4 or IL-12 further activates the p38 mitogen-activated protein kinase activity, suggesting that IL-4 and IL-18 enhance IFN-gamma production through distinct intracellular signal transduction pathways in DCs.     

Impaired production of IL-12 in systemic lupus erythematosus. III: deficient IL-12 p40 gene expression and cross-regulation of IL-12, IL-10 and IFN-gamma gene expression.

Interleukin 12 (IL-12) is a heterodimer comprising p35 and p40 subunits which are encoded and regulated separately. The authors previously demonstrated deficient IL-12 production in SLE which correlates negatively with disease activity. The present study was designed to determine whether deficiency of IL-12 and excess production of IL-10 and IL-6 in systemic lupus erythematosus (SLE) are due to aberrant regulation at the gene level. Using semiquantitative RT-PCR assay, it was shown that constitutive expression of IL-12 p35 gene is somewhat impaired in SLE compared with controls and that IL-12 p40 mRNA, which was present at low levels in controls, was undetectable in unstimulated SLE peripheral blood mononuclear cells (PBMC). Gene expression of IL-12 p35 and p40 was significantly increased in response to SAC, with significantly lower SAC-induced expression of p40 in SLE patients than controls. SAC-stimulated IL-12 p35 and p40 mRNAs were significantly augmented by interferon gamma (IFN-gamma). Exogenous IL-12 or IFN-gamma significantly inhibited IL-10 gene expression, without affecting IL-6 mRNA or other proinflammatory cytokine mRNA levels. These observations were further confirmed by studies of protein production at the single cell level using ELISPOT assay. Downregulation of IL-12 p40 expression appears to be the cause of IL12 p70 deficiency in SLE. If this defect could be repaired, normalization of IL-12 and IFN-gamma production should reduce excessive IL-10 and prevent pathology.