| Tim-3(T cell immunoglobulin domain and mucin domain-containing molecule-3) was identified to be specifically expressed on polarized Thl cells in 2002. Recent studies have shown that Tim-3 is also expressed on Tc1 cells, dendritic cells and macrophages, and may involve in the pathogenesis of a range of immune related diseases by influencing these cells function.Engagement of Tim-3 with its ligand leads to apoptosis of Thl cells which play role in preventing tissue immuopathology in virus hepatitis and graft rejection. Recent data has bee shown that as a receptor of phosphatidylserine, Tim-3 participates in engulfment of apoptic cells by macrophages. As an important innate immunocytes, macrophages take great roles in clearing apoptosis cells and immune regulation. The abnormal apoptosis of macrophages or macrophages'disability in clearance of apoptosis cells might lead to auotoimmune diseases. In this study, we investigate the role of Tim-3 in regulation for macrophage apoptosis and study the expression of Tim-3 on peripheral blood mononuclear cells(PBMC) in patients with rheumatoid arthristis(RA). PART ONE The role of Tim-3 in activation induced macrophage cell deathActivation induced cell death (AICD) is effective for preventing excess activation of immunocytes, including macrophages. However, little is known about the mechanism of AICD in macrophages. Tim-3 could mediate T cell apoptosis, lead to immune suppression of T cell. But little is known about the role of Tim-3 in AICD of macrophages.AimTo invistigate the role of Tim-3 in activation induced macrophage apoptosis.Methods1 Expression assays of Tim-3 in murine macrophage cell line RAW264.7 and mice peritoneal macrophages1.1 RT-PCR analysis of Tim-3 mRNAPeritoneal macrophages was prepared from 6～8 weeks old BalB/c mice treated with asepsis starch. Total RNA extracted from murine macrophage cell line RAW264.7 cells and mice peritoneal macrophages was used for Reverse Tramscription-Polymerase Chain Reaction (RT-PCR).1.2 Flow cytometry for Tim-3Tim-3 expression on mice peritoneal macrophages and murine macrophage cell line RAW264.7 was detected by flow cytometric analysis.2 Induction of AICD in vitro cultured murine macrophagesMice peritoneal macrophages and RAW264.7 cells were plated in 24 wells-plate at 5×104/well. After treatment of IFN-γ(10ng/ml) for 24 hours, cells were stimulated with LPS(100ng/ml) for another 12 hours.3 Blocking of Tim-3 on AICD of macrophageMice peritoneal macrophages and murine macrophage cell line RAW264.7 cells were cultured in 24 wells-plate at 5×104/well overnight. After pretreatment of Tim-3 blocking antibody (anti-Tim-3:5μg/ml) or control IgG(5μg/ml) forl hour, AICD was induced with IFN-γ(10ng/ml) and LPS(100ng/ml) as previously described.4 Inhibition of STAT1 phosphorylationMurine macrophage cell line RAW264.7 cells were cultured in 24 wells-plate at 5×104/well overnight. After pretreatment of fludarabine(STAT1 phosphorylation inhibitor)(6μM) or PBS for 30 mins, treatment of Tim-3 blocking antibody (anti-Tim-3:5μg/ml) or control IgG(5μg/ml) for1 hour. Then AICD was induced with IFN-γ(10ng/ml) and LPS(100ng/ml) as previously described.5 Detection of apoptosis5.1 Annexin-V/PI stainingMice macrophages (peritoneal macrophages and RAW264.7) were collected and washed with PBS. Cells were re-suspended in ice-cold 1×Binding Buffer at 1×106 cells/ml.5μl of annexin V-FITC solution and 5μl of dissolved PI were added to 500μl of the cell suspensions. After stainning in the dark for 10min at room temperature, the cells were analyzed by flow cytometry.5.2 TUNEL assayRAW264.7 cells were cultivated on asepsis slides for AICD induction using IFN-y and LPS. After washing with PBS, ice-cold 0.1% TritonX100 (containing 1uM sodiocitrate) were added to slides for cell-perforate. Cells were stained with 25μl dihydroxyfluorane coupled terminal-deoxynucleotidyl transferase for 30 minutes in the dark at 37℃.6 Western blot for the expression of p-Ser727-STAT1Total protein of RAW264.7 cells was collected for Western blot. Monoclonal antibody of STAT1 and p-Ser727-STAT1 was prepared for detecting the expression STAT1 and phosphorylated STAT1 on serine 727.Results1 Constructive expression of Tim-3 on murine macrophage cell line RAW264.7 and mice peritoneal macrophagesRT-PCR identified specific Tim-3 mRNA expression in both RAW264.7 and mice peritoneal macrophages. Simmilarly, flow cytometric analysis revealed that the percentage of Tim-3+ cells in mice peritoneal macrophages is 65.9% and percentage of Tim-3+ cells in RAW264.7 is 28.1%.2 AICD induction with RAW264.7 and mice peritoneal macrophagesFlow cytomety analysis showed that both RAW264.7 cells and primary mice peritoneal macrophages could be induced apoptosis with IFN-γ+LPS treatment. The apoptosis of cells treated with IFN-y+LPS(RAW264.7:33.54±0.1747%, peritoneal macrophages:18.41±0.230%)was significantly higher than that of cells treated with IFN-y (RAW264.7:2.443±0.090%, peritoneal macrophage:1.705±0.995%) and LPS (RAW264.7:0.837±0.432%, peritoneal macrophages:1.510±1.210%) (P＜0.05) TUNEL assay displayed same results. These data demonstrated that IFN-y+LPS could induced AICD in cultured mice macrophages.3 Blocking of Tim-3 up-regulated (IFN-y+LPS)-induced AICD of macrophagesFCM showed that apoptosis of RAW264.7 pretreated with anti-Tim-3 antibody (46.29%±1.139%) was significantly higher than that IgG pretreated cells induced by IFN-y+LPS (34.50±1.657%)(P=0.0042). The up-regulation of apoptosis showed anti-Tim-3 antibody dose-depandant. The apoptosis level of cells pretreated with anti-Tim-3:at 5μg/ml,3.5μg/ml,1.5μg/ml,0.5μg/ml were 46.29%±1.139%; 43.07%±0.4096%;41.98%±0.4576% and 36.75%±1.100% respectively. TUNEL assay displayed similar results. Consistently, anti-Tim-3 antibody pretreated mice peritoneal macrophages showed more apoptosis than control(29.45±0.350% vs 19.63±0.730%, P=0.0067).These results indicates that Tim-3 inhibit (IFN-γ+LPS) induced AICD of mice macrophages.4 p-Ser727-STATl involve in Tim-3 mediated regulation on macrophages' AICDResults of Western blot revealed an higher expression of STAT1 and p-Ser727-STAT1 in anti-Tim-3+IFN-γ+LPS treated RAW264.7 cells than IgG+ IFN-γ+LPS group.Flow cytomety analysis showed that after pretreatment with fludarabine(STAT1 phosphorylation inhibitor), the apoptosis level of RAW264.7 in (anti-Tim-3+IFN-γ+LPS) and (IgG+IFN-γ+LPS) group was not significantly differential:the apoptosis ratio between (anti-Tim-3+IFN-γ+LPS) and (IgG+ IFN-γ+LPS group) was decreased after treat with fludarabine (pretreatment with PBS group vs pretreatment of fludarabine:1.258±0.009 vs 0.8438±0.047)(P=0.0130).Conclusion1. Treatment with IFN-γ+LPS could induce apoptosis of RAW264.7 and mice peritoneal macrophages.2. Pretreatment of blocking antibody of Tim-3 could up-regulate AICD of macrophages induced by IFN-γ+LPS.3. Anti-Tim-3 treatment increased the expression of p-Ser727-STATl in macrophages induced by IFN-γ+LPS. Inhibition of STAT1 phosphorylation balanced out increased apoptosis induced by anti-Tim-3 antibody. These results revealed phosphorylation of STAT1 on serine 727 may mediate the increased apoptosis level. Tim-3 may down-regulate apoptosis by inhibition of phosphorylation of STAT1 on serine 727.Innovations and significanceHere, we first show evidence that Tim-3 could involve in macrophage AICD as an anti-apoptosis regulator. Tim-3 might inhibite apoptosis by inhibiting phosphorylation of STAT1 on serine 727.PART TWO Increased Tim-3 expression and its clinical significance on peripheral blood mononuclear cells in patients with RARheumatoid arthritis (RA) is one of the commonest autoimmune diseases, which would leads to mutilation. Excess activation and dis-regulation of immunocytes play pivotal roles in RA. Accumulated dada has been shown that abnormal cell apoptosis involved in RA development. Tim-3 has been shown as a regulator for apoptosis and involved in chronic autoimmune diseases, such as multiple sclerosis and CIA(collogen induced arthritis, a broadly used animal model for RA). It has been shown that -574T＞G and 4259G＞T polymorphism of Tim-3 gene are strongly associated with RA in Korean population, suggesting the involvement of Tim-3 in RA. In this study, we investigated the expression of Tim-3 on peripheral blood mononuclear cells in patients with RA.AimTo study the expression of Tim-3 on the cell subsets of peripheral blood mononuclear cells (PBMC) in RA patients and explore its clinical significance.Methods1 Tim-3 expression on PBMCs from RA patientsFresh heparin-treated blood from RA patients and healthy controls was collected to detect Tim-3 expression on PBMCs with flow cytometric analysis.2 Quantification of plasma TNF-a by ELISAPeripheral blood was immediately centrifuged for 3 mins at 1000rpm, plasma was collected and stored at -80℃. TNF-α(pg/ml) in plasma was measured with an ELISA kit.3 Real-time PCR for quantification of IL-17 mRNATotal RNA was prepared from PBMCs and reversely transcribed into cDNA. Real-time PCR was performed to measure relative expression level of IL-17 mRNA.4 Statistical analysesAll data were analyzed using the GraphPad Prism 5. The Kruskal-Wallis nonparametric H test and Mann-Whitney nonparametric U test were used for comparison between groups. The Pearson correlation analysis was used to calculate the correlation coefficient. Value of P＜0.05 is considered as significant difference.Results1 Augment expression of Tim-3 on PBMCs from RA patients negatively correlates with TNF-a levels and disease activityFlow cytometic results showed that the percentage Tim-3 positive cells in PBMC from RA patients are more than that from healthy individuals. (RA patients vs healthy controls, mean±SEM 29.38±1.321% vs 24.50±1.855%, P=0.0317). Further analysis revealed that the expression of Tim-3 protein on PBMC evaluated by the mean fluorescence intensity (MFI) are identical up-regulated in patients (patients vs controls: 4.926±0.5207 vs 2.907±0.3082, P=0.0043). Further analysis shown the percentage of peripheral Tim-3+ cells negative correlates with the value of Disease Activity Score 28 DAS28(r=-0.4318, P=0.0396) and TNF-αlevel in plasma (r=-0.5459, P=0.0070).2 Tim-3 expression was increased on peripheral T cells and monocytes in patients with RAWe investigated the expression levels of Tim-3 on CD4+T cells, CD8+T cells, CD14+monocytes and CD3+CD16/56+NKT cells from peripheral blood respectively. More large proportion of Tim-3+ cells was detected on CD4+ T cells(RA patients vs healthy controls, mean±SEM 12.92±1.182% vs 9.62±0.864%,P=0.0469), CD8+ T cells(RA patients vs healthy controls, mean±SEM 19.64±1.915% vs 11.70±1.162%, P=0.0029), monocytes(RA patients vs healthy controls, mean±SEM 91.43±3.100% vs 79.25±4.209%,P=0.0209)and NKT cells (RA patients vs healthy controls, mean±SEM49.39±2.574% vs 37.80±2.579%,P=0.0277) in patients with RA than that of healthy controls.3 Tim-3 expression on CD4+ T cells, CD8+ T cells and NKT cells in RA patients negatively correlates with the value of DAS28We correlated Tim-3 expression on each T cell subsets with the value of DAS28. There was an inverse correlation between Tim-3 expression on CD4+ T cells and the value of DAS28 (r=-0.4375, P=0.0368). Similarly, negative correlations were found between DAS28 and Tim-3 expression on CD8+ T cells and NKT cells(r=-0.4234,P=0.0441 and r=-0.5548, P=0.0060,respectively).4 Tim-3 expression on CD4+ T cells positively correlates with IL-17 mRNA expressionWe then evaluated the correlation of IL-17 and Tim-3 expression on CD4+ T cells. As shown in our data, there is a significant correlation with IL-17 expression on PBMC and Tim-3 expression on CD4+ T cells(r=0.5717, P=0.0068), suggesting that Tim-3 might play a role in RA by regulating IL-17 expression.5 Tim-3 expression on CD8+ T cells and NKT negatively correlates with plasma TNF-a in patients with RAThere was a reverse correlation between plasma TNF-a and Tim-3 expression on NKT (r=-0.4874, P=0.0214) and CD8+ T cells (r=-0.4271,P=0.0474).6 Conventional treatment further enhanced Tim-3 expression on CD3+T cells in RA patients with disease remissionWe investigated Tim-3 expression on CD3+T in 6 RA patients which with disease remission on 3-6 month conventional treatment. Flow cytometic analysis shown an increasing tendency of Tim-3 expression on CD3+T cells after treatment. (after treatment vs before treatment:23.01±4.214% vs 14.25±3.137%, P=0.1261).Conclusions1 Compared to controls, Tim-3 expression on PBMCs was significantly increased in RA patients, and Tim-3 expression on CD4+T cells, CD8+T cells, CD14+monocytes and CD3+CD16/56+ NKT cells from peripheral blood were increased in patients respectively.2 Negative correlation between DAS28 and Tim-3 expression was identified in PBMCs and T cell subsets(CD4+T, CD8+T and CD3+CD16/56+NKT cells), indicating the increased Tim-3 expression on various T cells might be vital for RA.3 Positive correlation between Tim-3 expression on CD4+ T cells and systemic IL-17 expression was discovered. It postulated that augmented Tim-3 expression on CD4+ T cells might promote IL-17 secretion by relieving the inhibition of Thl on Thl7 differentiation. Our result revealed Tim-3 expression on CD8+T and NKT cells negatively correlates with plasma TNF-α, indicating Tim-3 may control inflammation by decreasing cytokines production in RA.Innovations and significanceHere, we firstly reported the augment expression of Tim-3 on CD4+ T, CD8+ T cells, NKT cells and PBMC in RA, which inverse correlated with disease activity. These indicated that Tim-3 might be engaged in disease prognosis of rheumatoid arthritis. |
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