Number and function of CD4 + CD25 + FoxP3 + regulatory cells in patients suffering form multiple sclerosis
Duisburg, Essen (2007), III, 93 Bl.
Dissertation / Fach: Biologie; Medizin
Medizinische Fakultät » Universitätsklinikum Essen » Institut für Immunologie
Kreuzfelder, Ernst (Doktorvater, Betreuerin)
Esche, Helmut (GutachterIn)
Multiple sclerosis (MS) is considered to be an autoimmune disorder directed against self antigens of the central nervous system e.g. myelin basic protein (MBP). Natural CD4+CD25+FoxP3+ regulatory T cells (Treg)-mediated active suppression is an essential mechanism in the control of self-antigen reactive cells and in the induction of peripheral tolerance in vivo. In this study, we proved that our isolated Treg has phenotypical and functional characteristics. We found a reduction or loss of suppressive activity of Treg in the patients suffering from multiple sclerosis (MS) compared with healthy individuals. In our study, we set the threshold for CD25 expression of Treg to > 369 channels which was a mixture of CD25high and CD25intemediateexpressing cells, Treg may be defined with the other markers such as FOXP3 and HLA-DR. Analyzing HLA-DR expression on our isolated Treg and CD25high showed that 25 % of our isolated Treg were HLA-DR positive while 37 % of CD4+CD25high cells were HLA-DR positive. This concludes that our CD25intermediate Treg population was also HLA-DR positive. In our study, determination of FOXP3 mRNA message in Treg revealed a 13-fold higher expression compared to the appropriate CD25 negative cell population of MS patients as well as healthy individuals. We studied whether changes in the suppressive function of a mixture of CD25high and CD25intemediate expressing Treg in myelin basic protein (MBP)-and PWMinduced proliferation occurred in untreated MS patients compared with healthy individuals. Suppression of MBP-induced proliferation was observed in 13 out of 29 (45 %) MS patients; this was significantly less compared with 17 out of 19 (89 %) healthy individuals. In pokeweed mitogen-induced co-cultures, Treg from healthy individuals (21 of 23 cases, 91 %) showed a trend to higher suppressive activities (p = 0.086) compared with MS patients (20 of 29 cases, 69 %). We also looked for changes in relative Treg number and found that they were significantly increased in MS patients (mean±SD; 20±8 %) compared with healthy individuals (15±5 %). The exact mechanism of suppression by Treg is not well understood. In order to elucidate the mechanism, we measured tumor necrosis factor (TNF)-α production intracellulary after 5 days in PWM stimulated cultures. We found that in MS patients, TNF-α production had an inverse relation with Treg function. It seems that defect in Treg function may be due to increase in TNF-α production. In contrast to MS patients, we found different results in healthy individuals. We did not find any correlation between percentage of suppression and TNF-α production. This showed that involvement of TNF-α in Treg mediated suppression mechanism might differ in MS patients and healthy individuals. Antibody production by mature B lymphocytes and plasma cells is the final stage of an immune response. Thus, antibody production should also be influenced by Treg function. MBP is one of the target proteins in MS pathogenesis. Therefore, we developed as assay for measurement of anti MBP antibodies. We did not find any significant difference in anti MBP antibodies between healthy individuals and MS patients. In the second part of our study, we investigated the long-term effect of mitoxantrone (MX), a potent disease modifying drug for MS treatment, on the number of Treg and other immune cells in MS patients. We found a persistent and selective reduction of B cell numbers occur during therapy but other lymphocytes subpopulation remains unaffected. We assumed that MX therapy may restore the impaired function of Treg so we measured effect of MX on suppressive activity of Treg. But we did not find a persistent effect of MX on functional activity of Treg up to 9 months compared with baseline. Comparative analysis of Treg mediated suppressive function between healthy individuals and MS patients revealed that MS patients had impaired function, although B cell numbers and anti MBP antibodies between both groups were not different. Thus, it could be that impaired Treg function in MS patients effects interaction between effector cells or interaction between antigen presenting cells but not between B cells. In contrast, we found that MX influences the B cell numbers and not the Treg function. As a summary, it can be demonstrated that MX does not exhibit a persistent effect on the number or suppressive capacity of Treg but its potent effect on MS disease activity is predominantly, although not exclusively, mediated by a suppression of humoral immunity.
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