The Changes And Significances Of CD4～+CD25～(high) Treg In The Course Of Rheumatoid Arthritis And RhTNF-α Therapy

【Background】Rheumatoid arthritis (RA) is a chronic, inflammatory polyarthritis and synovitis with frequent progression to joint destruction and disability. The immunol abnormality of RA includes dysfunction of T cell subsets and abnormal activation of T cells. There are numerous evidences showed that T lymphocytes are involved in the pathogenesis of RA, including CD4⁺,CD8⁺ T cells that present in synonial. Although it is still unable to understand the mechanism of autoimmune disease, it has been proved that T cell takes part in the building of peripheral tolerance. Although a number of regulatory T-cells subsets have been described, those with the most potent regulatory function appear to reside within the naturally occurring CD25⁺ subgroup of CD4⁺ T cells, which constitute approximately 10% of CD4⁺ T cells in healthy mice. These cells down-regulate the activation and proliferation of CD25- T-cells, thereby suppressing harmful responses to self-antigens. They are anergic in vitro, and do not proliferate or secret IL-2 in response to stimulation via the T-cell antigen receptor (TCR).There is substantial evidence for the presence of similar CD4⁺CD25⁺ regulatory T-Cells in humans. However, only cells expressing the highest levels of CD25 (termed CD25^high) demonstrate potent regulatory function. There are several reports describing the presence of CD4⁺CD25⁺ regulatory T-cells in RA, with conflicting conclusions, and also one report in juvenile idiopathic arthritis. However , there no data on the size of circulating CD4⁺CD25⁺ T-cell population in individuals with RA on different stages or data on the changes of CD4⁺CD25⁺ T-cell population after using biological agents(rhTNF-α).A number of studies showed that the differentiation and development of CD4⁺CD25⁺ T-cells are regulated by transcriptional factor Foxp3. According to the papers, CD4⁺CD25⁺ Tregs develop into functionally mature subset in thymus, and on the CD4 single positive stage, Foxp3+ cells are the first to be detected. Given that CD4⁺CD25^high Tregs can be expanded in vitro without loss of regulatory function, there would clear therapeutic implications if a deficit in their number and/or function were present. Further study about the role and mechanism of CD4⁺CD25^high Tregs in RA is one thing with vital theoretic and utilized significants.【Objectives】The first purpose of our study is to explore the percentages of CD4⁺CD25^high regulatory T cells in peripheral blood or synovial fluid from patients with rheumatoid arthritis (RA) on different stages, and to study their correlations with the activity of the disease. Second, to explore the expression of Foxp3 on CD4⁺CD25^high Tregs and Foxp3 mRNA in PBMC. Last to examine the changes of CD4⁺CD25^high Tregs of RA patients who received MTX single or MTX + rhTNF-αcombined therapy, and analyze the collations between the changes and disease activity.【Methodes】1. Peripheral blood lymphocytes were obtained from the patients with active RA who had received no previous disease modifying (DMARDs) therapy(n=11),from individuals with stable, well-controlled RA(n=12), from subjects whose disease were poorly improved after DMARDs therapy(n=9), and from healthy controls(n=8). The frequencies of CD4⁺CD25^high Treg were quantified by using flow cytometry(FCM). Meanwhile, the correlations between the percentage of CD4⁺CD25^high Treg cells and the level of Anti-CCP antibody, CRP, ESR and RF were also investigated. Paired blood and synovial fluid was analysed in a small group of RA and other patients.2. Use flow cytometry(FCM)to examine the expression of Foxp3 on CD4⁺CD25^high Tregs and use RT-PCR to examine the level of Foxp3 mRNA in PBMC.3. Use flow cytometry(FCM)to compare the changes of CD4⁺CD25^high Treg between the MTX therapy group and MTX + rhTNF-αcombined therapy group, and analyze the correlation between the changes and activities of the sisease.【Results】1. There was a smaller proportion of CD4⁺CD25^high T cells in the peripheral blood of the active RA patients(mean 5.24%) and poorly-improved RA patients(mean 6.43%)than in patients with stable well-controlled RA or in healthy controls(mean 11.79% and 17.17%,respectively,P<0.01 in each case). The frequency of CD4⁺CD25^high T cells from RA was negatively associated with Anti-CCP antibody(58.0 Ru/mL), ESR(38.8 mm/h) and CRP(2.73μg/L),(P<0.05 for each).On contrast, The frequency of CD4⁺CD25^high T cells from healthy controls was not significantly correlated with the level of Anti-CCP antibody(<5.0 Ru/mL), ESR(4.67 mm/h), CRP(0.15μg/L) and RF(1.37)(all P>0.1). The percentage of CD4⁺CD25^high regulatory T cells from synovial fluid of RA patients(24.32%) was significantly lower than that of AS patients(30.24%)(P<0.05).2. Results by FCM showed that active RA patients'expression of Foxp3 on CD4⁺CD25^high Treg are lower than that of remitted RA patients and healthy controls (97.25% vs 92.88%, P<0.01). On contrast, results by RT-PCR demonstrated that the level of Foxp3 mRNA in PBMC of active RA patients is not significantly lower than that of remitted patients and healthy controls.3. Results by FCM showed that the changes of CD4⁺CD25^high Treg of patients who received MTX therapy is lower than changes of patients of patients received MTX + rhTNF-αcombined therapy, and the changes of the MTX + rhTNF-αgroup is positively correlated with the activities of RA.【Conclusions】1. The results demonstrate a smaller CD4⁺CD25^high regulatory Treg population in peripheral blood of individuals with active RA prior to disease modifying treatment and with poorly-improved RA, and this is negatively associated with the activity of the disease, therefore suggest that CD4⁺CD25^high Treg may be respected for the occur and development of RA.2. The results demonstrate that the expression of Foxp3 may be correlated with the occur and development of RA and their role may relate to CD4⁺CD25^high Treg. But the further mechanism of Foxp3 need deeply studies.3. The results suggest that rhTNF-αmay cure RA by recovering and strengthening peripheral tolerance. On the other hand, rhTNF-αcan also improve activities of RA by increasing CD4⁺CD25^high Treg. Therefore use rhTNF-αto cure autoimmune diseases is of bright promises.4. The study develop a mature site of methods of examining CD4⁺CD25^high Treg and Foxp3, so supply theoretic evidences to further study the pathogenesis of RA and the role of CD4⁺CD25^high Treg in RA.