The Role Of CD₄⁺CD₂₅⁺ Regulatory T Cells In The Pathogenesis Of Vitiligo

Vitiligo is a common dermatological disorder characterized by milky-whi te depigmented macules devoid of identiiable melanocytes. In the recent year s there is a gradual increase in the incidence of vitiligo. The cause of vitilig o is probably associated with genetic and environmental factors. However, t he exact cause of the illness remains unknown, but several hypotheses about i ts pathogenesis are advanced. The hypothesis of autoimmunity is subjected tomore and more concern recently.An exciting new area of immunologic interest has been the identificationof immune-regulatory cells (Tregs) and their role in maintaining immune tole rance. The CD₄⁺ Tregs come from thymus gland, accounting for 5-10% of t he CD₄⁺ T cells in the peripheral blood. Naturally occurring CD₄⁺ Tregs, themajority of which express CD25, can suppress the activation, proliferation an d differentiation of self-activated T cells. Foxp3, a transcriptional factor repor ted recently, is a member of the forkhead family of transcriptional regulators.Foxp3 constitute expresses in CD₄⁺CD₂₅⁺ Tregs and mainly in thymus gland,spleen and lymph nodes. CD₄⁺CD₂₅⁺ Tregs have two major features, includ ing immunotolerance and immunosuppression. They are tolerant to the stimul ation of the high concentration of IL-2, solid-phase coating or soluble anti-CD 3 antibody, and the joint stateof anti-CD3 and anti-CD28 antibodies with no s ecretion of IL-2. The immunosuppression of Tregs shows that the activation of Tregs requires the stimulation of T-cell receptors and some other signals. Once activated, their suppression is not specific. They can cause local immu nosuppression by direct interaction between cells and the secretion of IL-10 a nd TGF-band this immunosuppression has no restriction to MHC. After the activation of Tregs, the expression of CTLA-4 is increased. The anti-CTLA-4 antibody can be used to block the immunosuppression of Tregs and it is gen erally believed that Tregs play effects by cell interactions. The glucocorticoid -induced tumor necrosis factor receptor(GITR), the 18^th member of TNFRSF, i s expressed on the surface of Tregs, with the ligand GITRL. GITR and CTL A-4 can increase the expression of Foxp3. The discovery of Foxp3 as a spe cific marker of Tregs has led to an explosion of research in the function of Tregs. Although the role of Tregs in the pathogenesis of vitiligo was reporte d overseas, their results are inconsistent. However, there is no report of this f ield in China yet.Objectives: To study the role of CD₄⁺CD₂₅⁺ Regulatory T cells in the pathogenesis of vitiligoMethods:1. The blood was taken from vitiligo patients and normal control and density centrifugation was used to separate the PBMC.2. CD₈⁺T cells were separated by Dynabeads, and Militenyi immunity beads were applied to separate the CD₄⁺CD₂₅⁺T cells and CD₄⁺CD₂₅^- T cells.3. The number of CD₄⁺CD₂₅⁺ Tregs in PBMC of vitiligo patients and the normal control was analyzed by flow cytometry.4. Flow cytometry was applied to analyze the number of CD₄⁺CD₂₅⁺ Tregs in PBMC of vitiligo patients both in active stage and post-treatment by NB-UVB.5. RT-PCR was used to analyze the mRNA expression level of Foxp3, CTLA-4 and GITR,and Western-blot was applied to analyze the protein expression level of Foxp3.6. Statistics analysis: The program SPSS 11.0 was used to analyze all the statistical data. When P<0.05, the difference is significant.Results:1. The number of CD₄⁺CD₂₅⁺ Tregs in peripheral blood mononuclear cells(PBMC) of 39 vitiligo patients and normal control was analyzed by flow cytometry. The results showed that the percentage of CD₄⁺CD₂₅⁺ Tregs in PBMC of vitiligo patients in active stage was lower than that of normal control, and the difference is significant. After treated by NB-UVB, the percentage of CD₄⁺CD₂₅⁺ Tregs of vitiligo patients was higher than that of vitiligo patients in the active stage and the difference is also significant. These results suggested that the CD₄⁺CD₂₅⁺ Regulatory T cells might play an important role in the pathogenesis of vitiligo. After the treatment, the percentage of CD₄⁺CD₂₅⁺ Tregs was lower than the normal control, but the difference is not significant.2. The mRNA expression levels of Foxp3, CTLA-4 and GITR were analyzed by RT-PCR, and the protein expression level of Foxp3 was analyzed by Western blotting. The results showed that the expression levels of Foxp3, CTLA-4 and GITR in the patients of the active stage were lower than those of the normal control, and the difference is significant. After treated by NB-UVB, the expression level of Foxp3 of the vitiligo patients was lower than that of normal control, while difference of the number of CD₄⁺CD₂₅⁺ Regulatory T cells between the two groups is not significant.These results demonstrated that after the treatment, the number of CD₄⁺CD₂₅⁺ Regulatory T cells had been normalized but their function has possibly beenchanged.Conclusions:To investigate the role of CD₄⁺CD₂₅⁺ Tregs in the pathogenesis of vitiligo, this study indicated that after the NB-UVB treatment, the number of CD₄⁺CD₂₅⁺ Tregs of vitiligo patients had been restored but the expression level of Foxp3 was lower than that of normal control. It suggested that the function CD₄⁺CD₂₅⁺ Tregs had possibly been changed after the treatment, and the CD₄⁺CD₂₅⁺ Regulatory T cells might play an important role in the pathogenesis of vitiligo.