Expression Of IL-18 Binding Protein Research Articles

The role of interleukin-18 and interleukin-18 binding protein in K/BxN serum transfer-induced arthritis.

Interleukin-18 is a proinflammatory cytokine, the activity of which is regulated by its natural inhibitor, IL-18 binding protein (IL-18BP). Elevated circulating levels of IL-18 have been observed in patients with systemic juvenile idiopathic arthritis (sJIA) and adult-onset Still's disease (AOSD), two conditions associated with dysregulated innate immune responses. This study examines the expression and function of IL-18 and IL-18BP in K/BxN serum transfer arthritis (STA), a model that is uniquely dependent on innate immune responses. Naïve and serum transfer-induced arthritis (STA) wild-type (WT) mice were used to examine the articular levels of IL-18 and IL-18BP mRNA by RT-qPCR. The cellular sources of IL-18BP in the joints were determined by using Il18bp-tdTomato reporter knock-in mice. The incidence and severity of arthritis, including mRNA levels of different cytokines, were compared in IL-18BP or IL-18 knock-out (KO) mice and their WT littermates. IL-18 and IL-18BP mRNA levels were significantly increased in arthritic as compared to normal joints. Synovial neutrophils, macrophages, and endothelial cells represented the cellular sources of IL-18BP in arthritic joints, whereas IL-18BP production was limited to endothelial cells in non-inflamed joints. The incidence and severity of arthritis were similar in IL-18BP KO and IL-18 KO compared to their WT littermates. Transcript levels of different inflammatory cytokines were not different in the two KO mouse lines compared to WT mice. Although IL-18 and IL-18BP levels were increased in arthritic joints, our results show that the IL-18/IL-18BP balance is not involved in the regulation of STA.

Abstract 1824: Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity

Abstract Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Although recombinant mIL18 has demonstrated efficacy in mouse tumor models, recombinant human IL18 has not been efficacious in clinical studies, likely due to upregulation of IL18 binding protein (IL18BP), a negative regulator of the IL18 signaling axis. IL18BP is induced 10 - 100 fold after IL18 administration, binds to IL18 with high affinity, and prevents IL18 binding to the IL18 receptor. Analysis of expression data from the TCGA database as well as archived patient samples from select indications revealed that both IL18 and IL18BP are co-expressed in a number of tumor types, suggesting there may be a preexisting pool of IL18/IL18BP complex at the site of the tumor in some patients. Inhibition of IL18/IL18BP binding using a monoclonal antibody may therefore allow release of IL18 in situ, leading to IL18-mediated proinflammatory effects and enhanced anti-tumor immunity. Although the interaction of IL18 with IL18BP is a high affinity interaction, we have been able to identify very high affinity antibodies which can both inhibit complex formation and disrupt existing mouse IL18/IL18BP complexes. These antibodies are active in a number of cell-based functional assays, resulting in potent stimulation of interferon gamma production from cell lines or splenocytes. In vivo, we observed efficacy in a mouse syngeneic tumor model using an anti-mIL18BP antibody. RNA and protein analysis demonstrated expression of both IL18BP and IL18 within the tumors. Combined, these data suggest that inhibition of IL18/IL18BP binding may prove efficacious in the treatment of cancers where existing pools of IL18/IL18BP complexes prevent an IL18-mediated proinflammatory response. Experiments exploring anti-IL18BP therapy in combination with other checkpoint inhibitors such as anti-PD-1 are under current investigation. Citation Format: H. Toni Jun, Deborah A. Witherden, Eric K. Elliot, Christine M. Chidester, Maggie Willen, Bryan R. Peguero, Merrick Chai, Robert A. Horlick, David J. King. Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1824.

Abstract 10491: CSL112 Suppresses Inflammation in Human Monocyte-Derived Macrophages

Introduction: Inflammatory monocytes and tissue-resident macrophages play a key role in the pathogenesis of atherosclerosis. Pro-inflammatory macrophages promote formation of unstable plaques whereas anti-inflammatory macrophages contribute to plaque stabilization. HDL (high-density lipoprotein) stimulates atherosclerosis regression in animal models and alters the inflammatory properties of monocyte-derived cells present in plaques. CSL112, human apoA-I formulated with phosphatidylcholine, is currently in a phase 3 clinical trial (AEGIS-II [ApoA-I Event Reducing in Ischemic Syndromes II]) to prevent major adverse cardiovascular events (MACE) in the 90-day high-risk period following myocardial infarction. Objective: To investigate the effects of CSL112 on human monocyte-macrophage differentiation and pro-inflammatory macrophage activation. Methods: Human blood monocytes from healthy donors were differentiated into macrophages in the absence or presence of CSL112, and then treated with/without the toll-like receptor 2/1 agonist P3CSK4. Gene expression analysis was performed using RNA sequencing technology; secreted cytokines were measured by ELISA. Results: CSL112 did not affect macrophage maturation or cause macrophage polarization towards the M1 or M2 phenotype in the absence of an inflammatory stimulus. After P3CSK4 stimulation, CSL112 exerted broad anti-inflammatory effects by significantly modulating the expression of multiple genes implicated in atherosclerosis. Specifically, CSL112 reduced gene and protein expression of CCL2, CCL4, TNFα and IL-6, decreased the expression of membrane bound receptor expressed on myeloid cells 1 (TREM-1) and inhibited the release of soluble TREM-1. Additionally, CSL112 suppressed P3CSK4-induced IL-18 secretion, while upregulated the expression of IL-18 binding protein, a natural antagonist of IL-18. Conclusion: Our findings demonstrate anti-inflammatory effects of CSL112 that may stabilize vulnerable atherosclerotic plaques and potentially contribute to reduction in MACE in the subacute period following myocardial infarction.

IL-18 binding protein can be a prognostic biomarker for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic, fibrosing interstitial pneumonia that presents with various clinical courses and progression ranging from rapid to slow. To identify novel biomarkers that can support the diagnosis and/or prognostic prediction of idiopathic pulmonary fibrosis, we performed gene expression analysis, and the mRNA of interleukin-18 binding protein was increasingly expressed in patients with idiopathic pulmonary fibrosis compared with healthy controls. Therefore, we hypothesized that the interleukin-18 binding protein can serve as a diagnostic and/or prognostic biomarker for idiopathic pulmonary fibrosis. We investigated the expression of interleukin-18 binding protein in lung tissue, bronchoalveolar lavage fluid, and serum. Additionally, the correlation between interleukin-18 binding protein expression levels and the extent of fibrosis was investigated using mouse models of lung fibrosis induced by subcutaneous bleomycin injections. Serum interleukin-18 binding protein levels were significantly higher in idiopathic pulmonary fibrosis patients (5.06 ng/mL, interquartile range [IQR]: 4.20-6.35) than in healthy volunteers (3.31 ng/mL, IQR: 2.84-3.99) (p < 0.001). Multivariate logistic regression models revealed that the correlation between serum interleukin-18 binding protein levels and idiopathic pulmonary fibrosis was statistically independent after adjustment for age, sex, and smoking status. Multivariate Cox proportional hazard models revealed that serum interleukin-18 binding protein levels were predictive of idiopathic pulmonary fibrosis disease prognosis independent of other covariate factors (hazard ratio: 1.655, 95% confidence interval: 1.224-2.237, p = 0.001). We also demonstrated a significant positive correlation between lung hydroxyproline expression levels and interleukin-18 binding protein levels in bronchoalveolar lavage fluid from bleomycin-treated mice (Spearman r = 0.509, p = 0.004). These results indicate the utility of interleukin-18 binding protein as a novel prognostic biomarker for idiopathic pulmonary fibrosis.

Interleukin-18, Functional IL-18 Receptor and IL-18 Binding Protein Expression in Active and Latent Tuberculosis.

A thorough understanding of the processes modulating the innate and acquired immune response to Mycobacterium tuberculosis (M.tb) infection in the context of gene expression is still a scientific and diagnostic problem. The study was aimed to assess IL-18, IL-18 binding protein (IL-18BP), IL-18R, IFN-γ, and IL-37 mRNA expression in patients with active tuberculosis (ATB) and healthy volunteers with latent M.tb-infection (LTB) or M.tb-uninfected healthy controls (Control). The relative mRNA expression was assessed in the buffy coat blood fraction using the qPCR method. In total, 97 BCG-vaccinated Polish adults were enrolled in the study. The relative expression of IL-18 and IL-18BP mRNA was significantly elevated in the ATB and LTB groups. In ATB, but not LTB individuals, the overexpression of IL-18 and IL-18BP, as well as a significant increase in IFN-γ mRNA expression, might be considered as a manifestation of active tuberculosis disease. No statistically significant differences were observed in the IL-37 mRNA expression among the studied groups. Particularly noteworthy is the outstanding reduction in the relative expression of IL-18R mRNA in the LTB group as compared to the ATB and Control group. Reduced expression of IL-18R in LTB group may, at least partially, prevent the development of a pathological inflammatory reaction and promote the maintenance of homeostatic conditions between host immunity and M.tb.

MiR-92b-5p inhibitor suppresses IL-18 mediated inflammatory amplification after spinal cord injury via IL-18BP up-regulation.

The aim of this study was to investigate the effects of miR-92b-5p inhibitor and interleukin-18-binding protein (IL-18BP) on interleukin-18 (IL-18)-mediated inflammatory response after spinal cord injury (SCI). In this work, microglia was isolated from the newborn C57/B6J mice spinal cord to in vitro culture. The expression of IL-18BP and IL-18 was measured by the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) after transfection of miR-92b-5p into activated microglia. The expression of IL-18BP and IL-18 was determined following miR-92b-5p inhibitor treatment. In addition, the spinal cord injury model was established in mice. The expressions of miR-92b-5p, IL-18BP, and IL-18 were measured by qRT-PCR, and the expressions of inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF-α) and interleukin-1 β (IL-1 β) were determined by Western blot. After intrathecal injection of miR-92b-5p inhibitor, the mRNA expression of miR-92b-5p, IL-18BP, and IL-18 and the expression of iNOS, TNF-α, and IL-1 β in the injured area of the spinal cord of mice were measured. Basso Mouse Scale (BMS) was used to determine the recovery of locomotor function after spinal cord injury and miR-92b-5p inhibition. After miR-92b-5p transfection, the expression of IL-18BP was significantly decreased compared with that of untransfected microglia cells, whereas the level of IL-18 mRNA was significantly increased. However, the level of IL-18BP elevated significantly and the level of IL-18 reduced markedly after treatment with corresponding inhibitors. In addition, compared with the sham operation group (Sham), the RNA level of miR-92b-5p in the SCI group was significantly higher than that in the Sham, but the expression of IL-18BP was evidently declined and the expression of IL-18 was significantly increased in the SCI group. Meanwhile, the expression of miR-92b-5p in miR-92b-5p inhibitor intrathecal injection mice was remarkably lower than that in SCI group, the expression level of IL-18BP was significantly increased, and the RNA expression of IL-18 was weakened accordingly. Moreover, the protein expression of iNOS, TNF-α, and IL-1 β in miR-92b-5p inhibitor-treated mice was significantly lower than that in the SCI group. The locomotor evaluation of miR-92b-5p inhibitor group was dramatically higher than that of the SCI group. Suppressing the expression of miR-92b-5p after SCI can effectively intensify the level of IL-18BP, reduce the expanded inflammatory effect of IL-18, decline the release of iNOS, TNF-α, and IL-1 β, thus alleviate the neuronal injury and improve the locomotor function after SCI.

Remifentanil upregulates hepatic IL-18 binding protein (IL-18BP) expression through transcriptional control

Interleukin (IL)-18 plays an important role in liver ischemia/reperfusion (I/R) injury. We have previously demonstrated that remifentanil protects against liver I/R injury by upregulating the hepatic expression of IL-18-binding protein (IL-18BP), a natural IL-18 inhibitor. The current study was performed to further clarify the effects of remifentanil on IL-18BP expression in the liver as well as investigate the underlying mechanisms. In Sprague-Dawley (SD) rats, we demonstrated that remifentanil significantly increased the expression of IL-18BP in normal rat liver tissue over a 24-h time period with maximal expression at 24 h after treatment. The upregulation of remifentanil on IL-18BP expression displayed similar trends in in vitro cellular studies, including mouse primary hepatocytes, normal human hepatocyte LO2, and mouse hepatoma cells Hep1-6. In LO2 cells, preexposure of the cells to remifentanil significantly inhibited IL-18-activated p65 NF-κB phosphorylation, and the inhibition was absent when the cells were transfected with IL-18BP siRNA, indicating the functional effects of IL-18BP induced by remifentanil. Pretreatment with actinomycin D abolished remifentanil-induced upregulation of IL-18BP mRNA, suggesting that the induction occurred at the transcriptional level. This was further supported by the luciferase reporter assay, which demonstrated that remifentanil treatment significantly increased transcription of the IL-18BP promoter. Both western blot analysis and ChIP assays showed that STAT1 and C/EBP β were activated by remifentanil. Furthermore, remifentanil failed to upregulate IL-18BP expression after silencing STAT1 or C/EBP β gene expression. These findings demonstrate that remifentanil could upregulate hepatic IL-18BP expression through transcriptional activation of the IL-18BP promoter, and STAT1 and C/EBP β are two key transcriptional factors involved in this process.In normal rats and a human hepatic cell line, the authors demonstrate that remifentanil upregulates IL-18BP expression in hepatocytes, and that the underlying mechanism involves activation of the transcription factors STAT1 and C/EBP β. These findings expand our understanding of the pharmacological effects of remifentanil and suggest its potential treatment benefits for IL-18 dysfunction-mediated hepatic diseases.

PKC-β activation inhibits IL-18-binding protein causing endothelial dysfunction and diabetic atherosclerosis.

Clinical observations showed a correlation between accelerated atherosclerosis in diabetes and high plasmatic level of IL-18, a pro-inflammatory cytokine. IL-18 enhances the production of inflammatory cytokines and cellular adhesion molecules contributing to atherosclerotic plaque formation and instability. Previous studies indicated that protein kinase C (PKC)-β inhibition prevented macrophage-induced cytokine expression involved in diabetic (DM) atherosclerotic plaque development. However, the role of PKC-β activation on IL-18/IL-18-binding protein (IL-18BP) pathway causing endothelial dysfunction and monocyte adhesion in diabetes has never been explored. Apoe(-/-) mice were rendered DM and fed with western diet containing ruboxistaurin (RBX), a PKC-β inhibitor. After 20 weeks, atherosclerotic plaque composition was quantified. Compared with non-diabetic, DM mice exhibited elevated atherosclerotic plaque formation, cholestoryl ester content and macrophage infiltration, as well as reduced IL-18BP expression in the aorta which was prevented with RBX treatment. Endothelial cells (ECs) and macrophages were exposed to normal or high glucose (HG) levels with or without palmitate and recombinant IL-18 for 24 h. The combined HG and palmitate condition was required to increase IL-18 expression and secretion in macrophages, while it reduced IL-18BP expression in EC causing up-regulation of the vascular cell adhesion molecule (VCAM)-1 and monocyte adhesion. Elevated VCAM-1 expression and monocyte adherence were prevented by siRNA, RBX, and IL-18 neutralizing antibody. Our study unrevealed a new mechanism by which PKC-β activation promotes EC dysfunction caused by the de-regulation of the IL-18/IL-18BP pathway, leading to increased VCAM-1 expression, monocyte/macrophage adhesion, and accelerated atherosclerotic plaque formation in diabetes.

Pressure overload induces IL-18 and IL-18R expression, but markedly suppresses IL-18BP expression in a rabbit model. IL-18 potentiates TNF-α-induced cardiomyocyte death

Recurrent or sustained inflammation plays a causal role in the development and progression of left ventricular hypertrophy (LVH) and its transition to failure. Interleukin (IL)-18 is a potent pro-hypertrophic inflammatory cytokine. We report that induction of pressure overload in the rabbit, by constriction of the descending thoracic aorta induces compensatory hypertrophy at 4weeks (mass/volume ratio: 1.7±0.11) and ventricular dilatation indicative of heart failure at 6weeks (mass/volume ratio: 0.7±0.04). In concordance with this, fractional shortening was preserved at 4weeks, but markedly attenuated at 6weeks. We cloned rabbit IL-18, IL-18Rα, IL-18Rβ, and IL-18 binding protein (IL-18BP) cDNA, and show that pressure overload, while enhancing IL-18 and IL-18R expression in hypertrophied and failing hearts, markedly attenuated the level of expression of the endogenous IL-18 antagonist IL-18BP. Cyclical mechanical stretch (10% cyclic equibiaxial stretch, 1Hz) induced hypertrophy of primary rabbit cardiomyocytes in vitro and enhanced ANP, IL-18, and IL-18Rα expression. Further, treatment with rhIL-18 induced its own expression and that of IL-18Rα via AP-1 activation, and induced cardiomyocyte hypertrophy in part via PI3K/Akt/GATA4 signaling. In contrast, IL-18 potentiated TNF-α-induced cardiomyocyte death, and by itself induced cardiac endothelial cell death. These results demonstrate that pressure overload is associated with enhanced IL-18 and its receptor expression in hypertrophied and failingrabbit hearts. Since IL-18BP expression is markedly inhibited, our results indicate a positive amplification in IL-18 proinflammatory signaling during pressure overload, and suggest IL-18 as a potential therapeutic target in pathological hypertrophy and cardiac failure.

Imbalance of Interleukin-18 and Interleukin-18 Binding Protein in Children with Henoch-Schönlein Purpura

The balance between interleukin-18 (IL-18) and its endogenous antagonist, IL-18 binding protein (IL-18BP), was evaluated in children with Henoch-Schönlein purpura (HSP). Plasma IL-18 and IL-18BP levels and peripheral blood mononuclear cell IL-18 mRNA expression were significantly higher in patients with active HSP (n = 30) than in healthy controls (n = 20); IL-18BP mRNA expression was similar in active HSP and controls. Plasma levels and mRNA expression of IL-18 and IL-18BP in patients in remission (n = 19) were similar to those in controls. The ratios of IL-18 / IL-18BP plasma levels and IL-18 / IL-18BP mRNA levels in active HSP were significantly higher than in patients in remission and healthy controls. Thus, adequate IL-18BP to block the proinflammatory activity of IL-18 may not be present in active HSP and regulation of the IL-18 / IL-18BP balance might provide a potential therapeutic strategy.

Abstract 8393: IL-18 Binding Protein (IL-18BP) Inhibits β-AR Induced Cardiomyocyte Hypertrophy in vitro and Myocardial Hypertrophy i n vivo

Crosstalk between the sympathetic nervous system and proinflammatory cytokines such as interleukin-18 (IL-18) plays a key role in the pathophysiology of the hypertrophied failing heart. Importantly, IL-18 can directly promote cardiomyocyte hypertrophy. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18, and counters its biological effects. While β-AR stimulation induces IL-18, it is not known whether it also regulates IL-18BP. Here we demonstrate that the β-AR agonist isoproterenol (ISO) upregulates IL-18BP mRNA and protein expression in adult mouse cardiomyocytes in β 2 -AR/Gi-dependent manner. We cloned mouse Il18bp 5’ cis -regulatory region, and identified several putative transcription factor-binding sites, including CREB, C/EBP, NF- k B, and Sp1. While forced expression of dnp65 or kdIKKβ, or pre-treatment with mithramycin (Sp1 inhibitor) had no significant effect, forced expression of dnCREB or C/EBPβ knockdown markedly attenuated ISO-induced Il18bp transcription. ISO induced Il18bp promoter-reporter activity, and the deletion or mutation of CREB and C/EBP motifs reduced the response. ISO induced CREB and C/EBPβ DNA-binding activity, reporter gene activation, and in vivo binding to Il18bp promoter. ISO induced CREB and C/EBPβ activation via PI3K/Akt and ERK1/2. Importantly, ISO-induced hypertrophy (increased cell surface area and protein and not DNA synthesis) was blunted in cardiomyocytes isolated from IL-18BP Tg mice, by the forced expression of IL-18BP, and IL-18 neutralizing Ab. Further, ISO treatment in vivo was associated with Akt and ERK1/2 phosphorylation, CREB and C/EBPβ activation, and IL-18BP expression. Notably, ISO-induced myocardial hypertrophy was markedly attenuated in IL-18BP Tg mice. These data provide the first evidence that ISO induces mouse IL-18BP expression in cardiomyocytes via β 2 -AR/Gi-mediated Akt and ERK1/2-dependent CREB and C/EBPβ activation. Moreover, IL-18BP inhibits ISO-induced cardiomyocyte hypertrophy both in vitro and in vivo , likely via prevention of the biologic effects of IL-18. Thus, catecholamine induced expression of IL-18BP may play a protective role in the hypertrophied failing heart, a disease state characterized by sustained β-AR activation and IL-18 signaling.

β2 adrenergic activation induces the expression of IL-18 binding protein, a potent inhibitor of isoproterenol induced cardiomyocyte hypertrophy in vitro and myocardial hypertrophy in vivo

Both the sympathetic nervous system and the proinflammatory cytokine interleukin-18 (IL-18) play key roles in the pathophysiology of the hypertrophied failing heart. IL-18 binding protein (IL-18BP), a natural inhibitor of IL-18, counters its biological effects. β-AR stimulation induces IL-18 expression, but whether it also regulates IL-18BP is not known. Here we demonstrate that the β-AR agonist isoproterenol (ISO) increases steady state IL-18BP mRNA and protein levels in adult mouse cardiomyocytes in a β2-AR-dependent manner. We cloned mouse Il18bp 5′cis-regulatory region, and identified putative CREB and C/EBPβ transcription factor-binding sites. Forced expression of mutant CREB or C/EBPβ knockdown markedly attenuated ISO-induced Il18bp transcription and deletion or mutation of CREB and C/EBP motifs in the Il18bp promoter reduced ISO-induced promoter-reporter gene activity. ISO induced CREB and C/EBPβ activation in cardiomyocytes via PI3K/Akt and ERK1/2. Importantly, ISO-induced hypertrophy in vitro was dependent on IL-18 induction as it was blunted by IL-18 neutralizing antibodies and forced expression of IL-18BP. Moreover, ISO-induced hypertrophy was markedly attenuated in IL-18 null and IL-18BP transgenic mice. These data support the novel concept that β-AR activation, in addition to inducing cardiomyocyte hypertrophy via IL-18, concomitantly induces a countering effect by stimulating IL-18BP expression, and that ISO-induced cardiomyocyte hypertrophy may result from a net effect of IL-18 and IL-18BP induction.

Administration of IL-18BP by gene therapy reduces inflammation and prevents joint destruction by downregulation of MMP9 in rat AIA: role of MMP9 in bone and joint destruction in arthritis

Background and objectivesInterleukin 18 (IL-18) is a pleiotropic cytokine involved in rheumatoid arthritis (RA) pathogenesis. This study was carried out to evaluate the efficacy of IL-18 binding protein (IL-18BP) gene...

Cloning and characterization of rhesus IL-18 binding protein, a natural antagonist to IL-18

IL-18 is a proinflammatory cytokine that is important for host defense, but is also involved in the pathogenesis of a number of disease processes, ranging from autoimmune disorders to atherosclerosis. IL-18 binding protein (IL-18BP) is a constitutively expressed glycoprotein that specifically neutralizes the effects of IL-18, resulting in decreased production of IFN-γ and reduction in Th1 immune responses. In this study we cloned and sequenced a full-length cDNA of the rhesus IL-18BP (RhIL-18BP) from the spleen of rhesus macaques (Macaca mulatta) and compared its nucleotide and amino acid sequences to the functional murine and human IL-18BP orthologues. In addition, we fused RhIL-18BP to the Fc portion of human IgG1 to make recombinant RhIL-18BP·Fcγ1 in order to facilitate its detection by Western blot analysis and determined the approximate molecular weight of RhIL-18BP·Fcγ1 to be 66 kD. With this fusion protein, we showed that RhIL-18BP was functional and could significantly reduce murine IL-18 and LPS-induced IFN-γ production by murine splenocytes. Furthermore, we demonstrated the expression of IL-18BP in atherosclerotic lesions in a rhesus model of atherosclerosis, underscoring the need to fully understand the role of this protein as a primary negative regulator of IL-18 in multiple disease processes.

Differentiation of Human SH-SY5Y Neuroblastoma Cells by All-TransRetinoic Acid Activates the Interleukin-18 System

The clinical prognosis of children with high-stage neuroblastoma is still poor. Therapeutic approaches include surgery and cellular differentiation by retinoic acid, but also experimental interleukin-based immune modulation. However, the molecular mechanisms of all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells are incompletely understood. Herein, we examined the effect of ATRA on the activity of the interleukin-18 (IL-18) system in human SH-SY5Y neuroblastoma cells. It is shown that SH-SY5Y cells express IL-18 receptor (IL-18R) and the secreted antagonist IL-18-binding protein (IL-18BP), but no IL-18. SH-SY5Y cells are highly sensitive to ATRA treatment and react by cellular differentiation from a neuroblastic toward a more neuronal phenotype. This was associated with induction of IL-18 and reduction of IL-18BP expression, while IL-18R expression remained stable. Thereby, we identified the IL-18 system as a novel target of ATRA in neuroblastoma cells that might contribute to the therapeutic properties of retinoids in treatment of neuroblastoma.

IL-18 binding protein-expressing mesenchymal stem cells improve myocardial protection after ischemia or infarction

IL-18 is a proinflammatory cytokine known to cause tissue injury by inducing inflammation and cell death. Increased levels of IL-18 are associated with myocardial injury after ischemia or infarction. IL-18-binding protein (IL-18BP), the naturally occurring inhibitor of IL-18 activity, decreases the severity of inflammation in response to injury. In the present study, mesenchymal stem cells (MSCs) derived from mice transgenic for over expression of human IL-18BP were tested in rat models of global myocardial ischemia and acute myocardial infarction. Improved myocardial function is associated with production of VEGF, and in vitro, IL-18BP MSCs secreted higher levels of constitutive VEGF compared to wild-type MSCs. Whereas IL-18 increased cell death and reduced VEGF in wild-type MSCs, IL-18BP MSCs were protected. In an isolated heart model, intracoronary infusion of IL-18BP MSCs before ischemia increased postischemic left ventricular (LV) developed pressure to 79.5 + or - 9.47 mmHg compared to 59.3 + or - 7.8 mmHg in wild-type MSCs and 37.8 + or - 5 mmHg in the vehicle group. Similarly, using a coronary artery ligation model, intramyocardial injection of IL-18BP MSCs improved LV ejection fraction to 67.8 + or - 1.76% versus wild-type MSCs (57.4 + or - 1.33%) and vehicle (39.2 + or - 2.07%), increased LV fractional shortening 1.25-fold over wild-type MSCs and 1.95-fold over vehicle, decreased infarct size to 38.8 + or - 2.16% compared to 46.4 + or - 1.92% in wild-type MSCs and 60.7 + or - 2.2% in vehicle, reduced adverse ventricular remodeling, increased myocardial VEGF production, and decreased IL-6 levels. This study provides the concept that IL-18BP genetically modified stem cells improve cardioprotection over that observed with unmodified stem cells.

High-dose dexamethasone regulates interleukin-18 and interleukin-18 binding protein in idiopathic thrombocytopenic purpura

To evaluate the effects of high-dose dexamethasone (HD-DXM) on the balance of interleukin-18 (IL-18) and its endogenous antagonist IL-18 binding protein (IL-18BP) in ITP patients, IL-18, IL-18BP as well as IFN-gamma, IL-4 plasma levels and platelet counts were determined in 17 ITP patients receiving DXM 40 mg/day for four consecutive days and in 24 healthy subjects. Using RT-PCR, the mRNA expression of IL-18, IL-18BP, IFN-gamma, IL-4, T-box (T-bet) and GATA-binding protein 3(GATA-3) were studied in all subjects. The in vitro effects of DXM on IL-18BP and IL-18 of peripheral blood mononuclear cells (PBMCs) were studied by ELISA. HD-DXM administration increased IL-18BP and reduced IL-18 expression significantly (p<0.05), which resulted in a downregulation of IL-18/IL-18BP ratio p<0.05). In vitro, DXM had a significant effect on secretion of IL-18BP while diminishing IL-18 release from cultures of PBMCs. These results suggest that downregulation of IL-18/IL-18BP might account for its clinical efficacy of HD-DXM in active ITP.

Expression of interleukin-18, IL-18BP, and IL-18R in serum, synovial fluid, and synovial tissue in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic immunological disease, the invasive monocytes/macrophages and lymphocytes present in synovial cells and synovial tissue produce many cytokines and inflammatory mediators by paracrine signaling and plays a role in the pathological progress in RA patients. Interleukin-18 (IL-18) is a representative proinflammatory factor and displays multiple biological functions. This study was designed to investigate the expression of IL-18 and its receptor (IL-18R) and IL-18 binding protein (IL-18BP) in serum, synovial fluid, and synovial tissue of patients with RA, and to identify the pathological role of IL-18 in RA. Serum, synovial fluid, and synovial tissue were obtained from RA patients. Samples from patients with osteoarthritis and healthy people were obtained as controls. Levels of IL-18, IL-18BP, and PGE2 in serum and synovial fluid were measured by enzyme-linked immunosorbent assay. The biological activity of IL-18 in serum and synovial fluid was detected on the basis of IFN-gamma secretion from IL-18-responding human myelomonocytic KG-1 cells. NO in serum and synovial fluid was detected by Griess reaction. Expression of IL-18, IL-18BP, IL-18R, iNOS, and COX-2 mRNA and protein in synovial tissues was determined by quantitative reverse transcriptase polymerase chain reaction and Western blot. This study shows the expression levels of IL-18, IL-18R, iNOS, COX-2, and the biological activity of IL-18 in both serum and synovial fluid and tissue of patients with RA were significantly increased compared with the corresponding samples from the two control groups. In addition, expression of IL-18BP in patients with RA was decreased compared with samples from the two control groups. In conclusion, the overexpression of IL-18 and IL-18R may play an important role in the pathogenesis of RA.

Interleukin 18 and interleukin 18 binding protein in patients with idiopathic thrombocytopenic purpura

To evaluate the balance of interleukin IL18 and its endogenous antagonist IL18 binding protein (IL18BP) in patients with idiopathic thrombocytopenic purpura (ITP), plasma IL18, IL18BP, interferon gamma (IFNG) and IL4 levels, as well as platelet counts were measured in patients with active ITP (n = 23), ITP in remission (n = 21) and in healthy subjects (n = 24) by enzyme linked immunosorbent assay (ELISA). Using real-time quantitative polymerase chain reaction, the mRNA expression of IL18, IL18BP, IFNG, IL4, T-box (TBX21) and GATA-binding protein 3(GATA3) were studied in all subjects. The results showed that IL18 and IFNG protein and mRNA levels were significantly increased in patients with active ITP than in control subjects, but that IL18BP were not significantly elevated in ITP patients, which resulted in an elevated ratio of IL18/IL18BP in patients with active disease. During remission stages, the levels of these cytokines were comparable to those of healthy controls. The elevated levels of IL18/IL18BP in plasma during active stages of disease suggest a possible role in the pathogenesis and course of ITP.

Molecular mechanisms of IL‐18BP regulation in DLD‐1 cells: pivotal direct action of the STAT1/GAS axis on the promoter level

Interleukin (IL)-18, formerly known as interferon (IFN)-gamma-inducing factor, is a crucial mediator of host defence and inflammation. Control of IL-18 bioactivity by its endogenous antagonist IL-18 binding protein (IL-18BP) is a major objective of immunoregulation. IL-18BP is strongly up-regulated by IFN-gamma, thereby establishing a negative feedback mechanism detectable in cell culture and in vivo. Here we sought to investigate in D.L. Dexter (DLD) colon carcinoma cells molecular mechanisms of IL-18BP induction under the influence of IFN-gamma. Mutational analysis revealed that a proximal gamma-activated sequence (GAS) at the IL-18BP promoter is of pivotal importance for expression by IFN-gamma-activated cells. Use of siRNA underscored the essential role of the signal transducer and activator of transcription (STAT)-1 in this process. Indeed, electrophoretic mobility shift assay and chromatin immunoprecipitation analysis proved STAT1 binding to this particular GAS site. Maximal expression of IL-18BP was dependent on de novo protein synthesis but unaffected by silencing of interferon regulatory factor-1. Altogether, data presented herein indicate that direct action of STAT1 on the IL-18BP promoter at the proximal GAS element is key to IL-18BP expression by IFN-gamma-stimulated DLD-1 colon carcinoma cells.