| Systemic lupus erythematosus (SLE) is a specific auto-immune disease associated with various factors, and its pathogenesis is too complex to completely elucidate. T cell abnormalities and abnormal production of cytokines is a key event of B cell hyperactivity and antibody synthesis in SLE. To clarify the importance of the abnormalities of thymus and T-cell differentiation in the immune pathogensis and the immunoregulation of immune cells of systemic lupus erythematosus, we carried out this study. Our study consists three segments: The first part: The abnormalities of thymocytes and thymic epithelial cells in BXSB mice. It is well known that stem cells or precursor T cells migrate into the thymus and traffic through the thymus. Bone marrow-dirived precursor T cells participate in a phenomenon termed negative and positive selection and differentiate into functional T cells through the influence of thymic microenvironment. This process of positive/negative selection involves the interaction of precursor T cells with various cell lineages, including thymic stromal cells. Within the thymus, the epithelium is known to play a major role in T cell development. â… .Culture of BXSB thymic epithelial cells This culture conditions were very complicated and much expensive, which favored thymic epithelial cells outgrowth from thymic explants of lupus-prone mice. So it was confined to study the role of thymic epithelial cells in the pathogenesis of SLE. In this report, the thymic epithelial cells of BXSB mice were cultured with merely shearing , shearing and collagenase digestion, shearing and trypsinization in different nutrient media . The cells were identified with microscopy and immunohistochemical methods. The pure thymic epithelial cells were obtained not only in the medium with serum but also in the medium with growth factor. When the concentration of serum was 10%, the growth of thymic epithelial cells was not changed, and the culture was cheap. The thymic epithelial cells grew very well with sheating and collagenase digestion, and there was seldom contaminated by fibroblast. Passage was operated when the primary culture cells spreaded more than 95% the pavement of culture vessel. The purity of cells dyed with keratin were more than 95% in the medium with serum by cutting and collagenase digestion. Shearing and collagenase digestion process was easy and cheap to obtain thymic epithelial cells of BXSB mice. A stable thymic epithelial cells culture system of BXSB systemic lupus erythematosus mice is established. To the best of our knowledge, this is the first study toestablish the culture system of BXSB TEC. â…¡. The influence of TEC-SN of BXSB mice on the haematopoiesis function of marrow cells To determine the effect of the TEC-SN of BXSB mice on the haematopoiesis function, bone marrow cells were cultured with TEC-SN of BXSB mice or TEC-SN of C57 mice culture from 4-week-old and 12-week-old mice, compared with the medium used in the culture system of TEC. The results indicated that thymic epithelial supernatants induced growth of colony forming units of granulocyte/ macrophage (CFU-GM). The CFU-GM was more in the culture with the TEC-SN of 12-week-old BXSB mice, compared with TEC-SN of 4-week-old BXSB mice and C57 mice. This suggest that the effect of TEC-SN of BXSB mice of invasion on the forming of CFU-SN is more powerful than that of the age matched C57mice. â…¢. The effect of TEC-SN and TEC of BXSB mice on the antigen expressions of the marrow cells To determine whether TEC plays a role in the change of the subpopulations of thymocytes, bone marrow cells were cultured with TEC-SN and TEC of 4-week-old and 12-week-old BXSB mice, and then a phenotypic analysis of the bone marrow cells was achieved by flow cytometry. The results indicated that the TEC-SN of 12-week-old BXSB mice up-regulated the CD4-CD8-proportion compared with the TEC-SN of 4-week-old BXSB mice, while the TEC cultured from 12-week-old BXSB mice down-regulated the CD4+CD8+ subpopulation. This result indicated that TEC contributed to the defect in the transformation from DN to DP.â…£.The study on the antigen expressions of thymocytes in BXSB mice To elucidate whether the antigen expressions of thymocytes in BXSB mice are abnormal, we immunophenotyped thymocytes of BXSB mice before (4-week old) and after (12-week old and 20-week old) the onset of proteinuria and autoimmune disease. Thymocytes can be divided into four major subpopulations: CD4+CD8+ (double-positive), CD4-CD8-(double-negative), CD4+CD8-(CD4+) and CD4-CD8+ (CD8+) cells, using two-colour flow cytometry. The autoimmune strains at four weeks of age and age-matched controls (C57) demonstrated normal proportions of thymocyte subsets with approximately 72% double-positive cells, 5-6% double-negative cells, 12-13% CD4+ cells and 4% CD8+ cells. By 20 weeks of age, BXSB mice demonstrated a moderate increase in double-negative cells (approximately 48.11%) and a decrease in double-positive cells (approximately 8.2%). The proportion of Cd3+ and CD71+ thymocytes decreased in the 20-week old BXSB mice, compared with that in the 4-week old and 12-week old BXSB mice. These data indicate that there is abnormality in the prolixferation, maturation and differentiation in the thymus of BXSB after the onset of autoimmune disease. The second part: The expression of immune cell antigen on peripheral blood lymphocytes of patients with systemic lupus erythematosus To study the expression of immune cell differentiating antigens in active and inactive stages of SLE, the expression of T cell differentiating antigens were examined with 6 kinds of monoclonal antibodies using direct and indirect immunofluorescence technique by cytometry from 30 normal controls and 43 SLE patients. The expression of CD2, CD3, CD4 and CD45RA on immunecells were down regulated in active stage of disease of SLE; while the expression of CD8, CD29 were up-regulated. In inactive stage the expression of CD2 and CD8 reached normal, but CD3, CD4, CD29 and CD45RA did not reach normal levels. Recent animal studies have shown that CD4+CD25+ T cells play a crucial role in the suppression of the immune response and that depletion of this subset of T cells might lead to development of autoimmune diseases. This work demonstrated that CD4+CD25+ Tcells were decreased in patients with clinically active SLE, and did not reach normal in inactive SLE patients. The expressions of T cell differentiation antigens were abnormal in SLE patients, especially in active stage of disease. The third part: The effect of immunoregulation of immune cells in SLE â… . The effect of cyclophosphamide and dexamethasone on the differentiation of thymocyte in BXSB mice To determine the effects of cyclophosphamide and dexamethasone treatment on the cellular composition and the mechanism of the treatment to SLE, we examined the distribution of the lymphocyte subsets in thymus of BXSB mice and the index of thymus. Thymocytes were analyzed for cell surface markers, CD4 and CD8 by 2-color flow cytometry using their respective monoclonal antibodies. The treatment of cyclophosphamide and dexamethasone decreased the proteinuria. The proportion of DP cells in treated group was decreased, and the DN cells were increased, the indexes of thymus were not recovery. The therapy of cyclophosphamide decreased CD4+ cells and dexamethasone treatment increased CD8+ cells, the proportions of CD4+ andCD8+ thymocytes was approximate after the therapy. The immune intervention of the cyclophosphamide and dexamethasone may be through the different way. â…¡. The influence of cyclophosphamide and glucocorticoids on the CD89 expression of peripheral neutronphiles in the active SLE patients. To determine the CD89 levels of peripheral neutronphiles in SLE patients with hematuria before and after immuneoregulatory therapy for evaluating the relationship between CD89 level and the formation of renal hematuria, as well as the therapy mechanism of the cyclophosphamide and glucocorticoids in SLE, we applied direct immunofluorescence method and flow cytometry to determine CD89 levels of peripheral blood cells of 20 healthy volunteers and 20 SLE patients before and after therapy. Compared with health controls, CD89 levels of peripheral neutrophiles in SLE were decreased. CD89 levels were elevated after therapy for 1 month. It was revealed that CD89 levels were negatively correlated with the extent of hematuria of SLE either before or after therapy. CD89 levels of peripheral neutrophiles in SLE were decreased. CD89 levels were elevated and hematuria were decrease after immunoregulatory therapy. The decreased CD89 level may play an important role in the pathogenesis of hematuria in SLE. In short, we established the culture system of the TEC of BXSB mice and studied the effect on the differentiation of bone marrow cells to T cells and the hemapoiesis of granular-macrophagic system of the TEC of BXSB mice, for the first time. The influence of dexamethasone and cyclophosphamide on the differentiation of thymocytes of BXSB mice was investigated, which was not reported by now, as we known. The data indicated that the TEC was correlated with the abnomolities of the differentiation of the proliferation, differentiation... |
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