| Objective:The present study examined the expression of Tim-3 on subsets of peripheral blood mononuclear cells from healthy controls and the percentage of Tim-3 expressing cells to CD4~+T cells and to its subsets in patients with rheumatoid arthritis with high disease activity.Materials and MethodsSubjects:Rheumatoid arthritis (RA) patients with high disease activity were randomly recruited from the Department of Rheumatology of Tongji Hospital, Wuhan, China from 2008 to 2010. The diagnosis of RA patients was established in accordance with the American College of Rheumatology (ACR) criteria of 1987 for RA. Disease activity of patients with RA was evaluated on the basis of DAS28. In all the patients with high disease activity, none of them had received any medication before the study. Healthy persons excluded inflammatory diseases and other conditions served as controls.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from freshly extracted heparinized blood by density gradient centrifugation on Ficoll-Hypaque. Flow cytometric analysis was implemented for the detection of Tim-3 on the subsets of PBMCs. Peripheral blood mononuclear cells were isolated from freshly extracted heparinized blood by density gradient centrifugation on Ficoll-Hypaque; Afterwards, CD4~+T cells were enriched by magnetic selection using a human CD4~+T cell enrichment kit by following the manufacturer's instructions; Purified CD4~+T cells (2×106 cells/ml ) were incubated in AIM-V serum-free medium (Gibco, USA) in 24-well plates in the presence of Phorbol 12-myristate 13-acetate (PMA 20 ng/ml eBioscience, San Diego, CA) and ionomycin (1μg/ml eBioscience, San Diego, CA) for 48 h. Before the termination of the stimulation, monensin, an inhibitor of protein secretion (1μg/ml eBioscience, San Diego, CA) was added into the culture at the final 6 hours; CD4~+T cells cultured for 48 h were harvested from the 24-well plates. Cells were stained with anti-human Tim-3 monoclonal antibody, anti-human interferon-γantibody, FITC-conjugated anti-human IL-4 or FITC-conjugated anti-human IL-17 antibody, respectively. Final flow cytometric analysis was implemented for the detection of Tim-3, interferon-γ, IL-4 and IL-17.Results:1. The expression of Tim-3 on subsets of PBMCs in healthy controls All the subsets of PBMCs, for example, CD4~+T cells (1.18±0.57), CD8~+T cells (3.15±2.83), B cells (8.28±3.94), had an expression of Tim-3. The percentages of Tim-3 expressing cells to NK cells (46.63±30.51) and to monocytes (67.84±24.40) were higher.2. The expression of Tim-3 on the subsets of CD4~+T cells in healthy controls Our study demonstrated Tim-3 is not just expressed on Th1 cells, but also on Th17 cells. The expression of Tim-3 on Th2 cells is can not be determined.3. Compared with healthy controls, the ratio of Tim-3 expressing cells to activated CD4~+T cells from RA patients with high disease activity was lower (2.76±1.79 VS 4.98±3.16, p<0.05), which was also negatively correlated to DAS28 (p<0.05).4. The ratio of Tim-3-expressing cells to Th1 cells or Th17 cells in RA patients with high disease activity and the correlation between the ratio and DAS284.1. The percentage of Th1 or Th17 cells in RA patients with high disease activity 4.1.1. In RA patients with high disease activity, the frequency of Th1 cells was slightly increased as compared to healthy controls, though the difference was not significant ( 14.93±12.83 VS 6.23±1.67, p=0.11).4.1.2. The frequency of Th17 cells was elevated in RA patients with high disease activity (1.37±0.56 VS 0.83±0.88, p<0.05).4.2. Ratio of Tim-3-expressing cells to Th1 cells or Th17 cells in RA patients with high disease activity4.2.1. The percentage of Tim-3-expressing cells to Th1 cells in RA patients with high disease activity was lower (8.29±4.31 VS 16.32±8.11, p<0.05).4.2.2. Compared with RA patient with high disease activity and short disease activity, the alteration was more conspicuous in RA patients with high disease activity and long disease course (6.05±4.78 VS 10.81±1.63, p<0.05).4.2.3. The RA patients with high activity had a significantly lower percentage of Tim-3-expressing cells to Th17 cells than healthy controls (9.67±4.03 VS 23.47±7.03, p<0.05).4.3. Relations between the ratio of Tim-3-expressing cells to Th1 or Th17 cells and DAS28 in RA patients with high disease activity4.3.1. The ratio of Tim-3+Th1 cells is negatively correlated with DAS28 in RA patients with high disease activity (p<0.05).4.3.2. A negative correlation was also found between the percentage of Tim-3+Th17 cells and DAS28 in RA patients with high disease activity (p<0.05).5. After blocking the interaction of Tim-3 and its ligand with anti-Tim-3 antibody, there is no increase of the number of Th1 or Th17 cells.Conclusion:In healthy controls, we further confirmed that the subsets of PBMCs, for example, CD4~+T cells, CD8~+T cells, monocytes, NK cells, B cells, expressed Tim-3. and the expression was more conspicuous on NK cells and monocytes. In subsets of CD4~+T cells, the expression of Tim-3 on Th1 cells is further determined. There are not all the Th1 cells expressing Tim-3. We can also detect the expression on Th17 cells.Compared with healthy controls, the ratio of Tim-3 expressing cells to activated CD4~+T cells from RA patients with high disease activity was lower, which was also negatively correlated to DAS28.Compared with healthy controls, ratio of Tim-3 expressing to Th1 cells or to Th17 cells is lower in RA patients with high disease activity, and this alteration negatively correlated with DAS28 of RA.To sum up, our study showed that both Th1 and Th17 cells had a reduced percentage Tim-3+ cells in RA patients with high disease activity and the alteration were negatively correlated with DAS28 of RA patients, demonstrating that the regulation of Tim-3 on the subsets of Th cells is abnormal in RA patients with high disease activity, which may contribute to the pathogenesis of RA. |
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