The Investigation And Targeting Immune Intervention Of IL-18/IL-18BP In The Pathogenesis Of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired chronic autoimmune-mediated bleeding disorder in which platelets are opsonized by platelet autoantibodies that target several platelet glycoproteins including GPIIb/IIIa and GPIb/IX and prematurely destroyed in the reticuloendothelial system of the spleen, liver or bone marrow. Apart from phagocytosis, destruction mechanisms include complement activation and T cell abnormalities.It has become evident that T helper 1-(Th1) and Th2 cells might have pathogenetic importance in ITP.T-box expressed in T cells (T-bet) and GATA-binding protein 3 (GATA-3) are two major T transcription factors that regulate the expression of Thl or Th2 cytokine genes respectively and play a crucial role in T-cell differentiation. We, hereby, evaluated the relevance of Th1 type (i.e., IFN-γand T-bet) versus Th2 type (i.e., IL-4 and GATA-3) in the pathogenesis of ITP focusing on the critical role of IL-18.This latter cytokine was originally termed as IFN-y inducing factor that belongs to the IL-1 cytokine superfamily. IL-18 is an inflammatory cytokine, not only predominantly produced by Kupffer cells in the liver, but also expressed in pancreas, kidney, skeletal muscle, lung, osteoblasts, and keratinocytes. Up-regulation of IL-18 has previously been detected in multiple sclerosis (MS), lupus nephritis (LN), atopic dermatitis.Our data strongly support the notion that up-regulation of IL-18 may be an important determinant in the evolution of ITP. The biologic effects of IL-18 by promoting a cytokine imbalance toward a Th1-type immune response may induce ITP. IL-18BP is a constitutively secreted protein able to bind IL-18 with high-affinity, providing a potential mechanism whereby IL-18 activity is regulated. IL-18BP is induced by IFN-y, thus establishing a negative feedback loop that can be observed in vitro and in vivo. Human IL-18BPa and IL-18BPc isoforms are capable of binding to and neutralizing IL-18. The affinity of IL-18BPc isoform is 10 fold less than IL-18BPa isoform. IL-18BPa which was measured in this study is the main IL-18BP in humans. Data showed IL-18BP inhibits IL-18-induced IFN-y and IL-8 production and NF-κB activation in vitro and LPS-induced IFN-y production in vivo. We hypothesized both the onset and aggravation of ITP are promoted by an imbalance of immune response towards a Thl cytokine predominance due to IL-18 and related cytokine up-regulation.Up-to-date, the balance between IL-18 and IL-18BP in patients with ITP is still unknown. In the present study, we have explored the hypothesis that the imbalance of IL-18 and IL-18BP may be of importance in ITP. Plasma levels as well as mRNA expression in peripheral blood mononuclear cells (PBMCs) of IL-18, IL-18BP and other cytokines were measured in patients with active ITP, patients in remission and in healthy volunteers to investigate whether IL-18BP could act as a possible therapeutic approach against ITP.I Effects of IL-18/IL-18BP on the onset of ITP and mechanism of action of dexamethasoneObjective:To evaluate the balance of interleukin (IL)-18 and its endogenous antagonist IL-18 binding protein (IL-18BP) and the effects of high dose dexamethasone (HD-DXM) in patients with immune thrombocytopenia (ITP), plasma IL-18,IL-18BP, IFN-γand IL-4 levels, as well as platelets counts were measured in patients with active ITP (n=37), ITP in remission (n=21),ITP receiving HD-DXM(n=17) and in healthy subjects (n=24) were measured.Methods:(1)37 ITP patients were enrolled in this study. Blood sampling was performed before and after treatment at the end of the second week with high dose dexamethasone (HD-DXM);(2)Plasma IL-18, IL-18BP, IFN-y and IL-4 levels, as well as platelets 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);(3) IL-18, IL-18BP levels as well as IFN-y, IL-4 plasma levels and platelets counts were determined in 17 ITP patients before and after HD-DXM treatment and in 24 healthy subjects;(4) Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IL-18, IL-18BP,IFN-y,IL-4,T-box (T-bet) and GATA-binding protein 3(GATA-3) were studied in all subjects.Results:(1) IL-18 and IFN-γprotein and mRNA levels were significantly increased in patients with active ITP than in control subjects, but the IL-18BP were not significantly elevated in ITP patients, which resulted in an elevated ratio of IL-18/IL-18BP in patients with active disease.(2) HD-DXM administration increased IL-18BP levels significantly (P＜0.01) and reduced IL-18 expression (P＜0.05), which resulting in a downregulation of IL-18/IL-18BP ratio (P＜0.05), IFN-γand T-bet were decreased (P＜0.05) whereas IL-4 and GATA were increased (P＜0.05) after HD-DXM treatment.Conclusions:(1)Persistent overproduction of IL-18 in ITP is associated with disease progression and IL-18BP might inhibit this activity. (2)The HD-DXM-mediated Thl/Th2 cytokine profile alterations observed in this study could be the results of a downregulation of IL-18 and other Thl cytokines by induction of IL-18BP while permitting the production of Th2 cytokines.(3)These findings suggest blockage of IL-18 bioactivities by IL-18BP is likely a promising therapeutic concept.ⅡRecovery of Thl/Th2 balance in immune thrombocytopenia pateints through the actions of the cytokine IL-18BPObjective:To investigate the effects of IL-18BPa/Fc on the production of cytokines in the peripheral blood mononuclear cells (PBMCs) from patients with immune thrombocytopenia (ITP),10 ITP patients and 10 health controls were enrolled in this study.Methods:(1) 10 ITP patients and 10 health controls were enrolled in this study.(2) IFN-γ, IL-2, TNF-α, IL-4, IL-5 and IL-10 levels in the supernatants were determined after 48h of PBMCs culture with or without addition of IL-18BPa/Fc by ELISA.(3) Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IFN-γ, IL-4 and IL-18R were studied in all subjects.(4) Meanwhile, proliferation of PBMCs was examined by CCK-8 assay.(5) Flow cytometry (FCM) was applied to detect the apoptosis of cells by staining with annexinV-FITC/PI.Results:(1) IL-18BPa/Fc (200 ng/mL) had a significant effect on secretion of IL-10 from healthy and ITP patients PBMCs, while diminishing IFN-γrelease from cultures of PBMCs. Notably, IL-18BPa/Fc further reinforced dexamethasone (5 nM)-mediated reduction of PHA-induced IFN-γproduction by an additional 49.9%.(2) Compared with healthy controls, the annexin V% was significantly decreased in ITP patients (ITP:7.1±3.2%, controls:10.9±2.1%, P=0.02). IL-18BPa/Fc significantly increased the annexin V% in ITP patients(p=0.04) but not in controls(p=0.96). The annexin V% in ITP patients in groupⅠ(IL-18BPa/Fc Ong/mL), groupⅡ(IL-18BPa/Fc lOng/mL) were 7.1±3.2% and 9.1±5.1% respectively, The annexin V% in controls in groupⅠand groupⅡwere 10.9±2.1% and 10.8±2.6% respectively.(3) As expected, significant difference was not found between the treated and the untreated group for the growth stimulating activity, indicating the addition of exogenous IL-18BPa/Fc exerted the marginal roles on cell proliferation. The results showed IL-18BPa/Fc indeed had no direct effect of stimulating cells proliferation.(4) After 48h culture with IL-18BPa/Fc, the expression of IL-18R protein and mRNA in PBMCs of ITP patients and controls was unaffected. Responsiveness of human IL-18R to IL-18BPa/Fc was obvious and reproducible. These observations revealed modulation of IL-18R expression was possibly irrelevant to IFN-y downregulation induced by IL-18BPa/Fc.Conclusions:The present data demonstrate that IL-18BPa/Fc may play a therapeutic role in ITP by downregulation of IFN-y and other Thl cytokines while permitting the production of Th2 cytokines (IL-10) via neutralizing the biologic activity of mature IL-18.ⅢAbnormal-expression of IL-18, IL-18BP and Tim-3 in the spleen of ITPObjective:To evaluate the balance of IL-18,IL-18BP and T cell immunoglobulin and mucin domain-3(Tim-3) in spleen of ITP, IL-18, IL-18BP and Tim-3 were measured. Methods:(1) 12 ITP patients spleens and 10 controls spleens were enrolled in this study.(2) IFN-y, IL-4, IL-18 and IL-18BP levels in the supernatants were determined after 48h of PBMCs culture.(3) Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IL-18, IL-18BP, IFN-γ, IL-4, T-box (T-bet) and GATA-binding protein 3(GATA-3) were studied in all subjects.(4) Using immunohistochemistry (IHC) and immunofluorescence, the expression of IL-18, IL-18BP and Tim-3 were studied in all subjects.Results:(1) The expression of IL-18, IL-18BP were much high in ITP patients than in normals using immunohistochemistry (IHC) and immunofluorescence; while the expressions of Tim-3 were declined in ITP than in normals.(2) IL-18 and IFN-y levels were significantly increased in patients with ITP than in control subjects after 48h of spleens lymphocytes culture, but the IL-18BP were not significantly elevated in ITP patients. The level of IL-4 was below the detectable limit of the assay used.(3)Using the REST software, the data are presented as the fold change in gene expression normalized to an endogenous reference gene and relative to healthy controls. The relative amount of mRNA gene expression of IL-18 and IFN-y were increased in active patients spleens compared to healthy controls (P＜0.01). IL-18 was up-regulated in active ITP patients compared to controls (P＜0.01).T-bet mRNA expression was significantly higher in active ITP patients compared to that of the control group.The decrease of IL-4 and GATA-3 was also observed.Conclusions:(1) We observed Thl polarization of the immune response in ITP spleens.(2) The abnormal pathway of Tim-3-galecti-9 may contribute to the ITP pathogenesis.