The Function And Mechanism Of ICOS-ICOSL Signal In The Immunological Pathogenesis Of Hyperthyroidism

Graves’disease (GD), named as toxic diffuse goiter, is an organ-specific autoimmune disease, caused by the over-stimulation of thyroid cells through the ligation of stimulating anti-thyrotropin receptor (TSHR) and the autoantibody (thyrotrophin receptor antibody, TRAb). The disease is accompanied by thyromegaly, the hyperthyroidism symptoms, infiltrative exophthalmos, pretibial myxedema and the production of TRAb.The abnormal immunologic functions exist in GD patients. Thyroid follicular cells (TFCs) react with auto-reactive T cells via immune molecules abnormal expressed on TFC, then induce the migration, activation, proliferation and differentiation of antigen-specific T cells, and the cytokines released finally mediate the immune injury. On the other hand, B cells differentiation could be promoted, auto-antibody producing specific for thyroid tissue, and lead to the damage the structure and function of thyroid glands.Recently, the discovery of auto-reactive T cells and the role of costimulatory molecules in the autoantibody production were attracted more and more attention. A great deal of research showed that the variance of lymphocyte subsets, and the abnormal expressed costimulatory molecules on its surface, was closely related to the autoantibody production and disease development in GD.Previous studies indicated that: (1) CD4~+CD28~- T cells existed in many autoimmune disease, and the abnormal activation caused disease development. (2) With the coperation of self-T cells and PWM, the IgG secretion by B cells in peripheral blood of GD patients were four fold increased compare to health control. However, in the lack of self-T cells, the IgG secretion was not increased. (3)TRAb could be detected only in the serum of wild-type mouse model of GD, but not in the B cell deficient mouse model. In conclusion, the production of autoantibodies is closely related to the reaction of T cells and the B cells, and the costimulatory molecules abnormal expressed on which might play a very important role in GD, but the exactly immune mechanism is not well known.ICOS was found expressed on activated T cells by Hutloff et al in 1999, and its structure and function were similar with CD28 molecule. It’s the third member of CD28 family, due to its expression was defined on the activated T cells and memory T cells; it was named as ICOS (Inducible Costimular). Different with CD28, ICOS has no MYPPPY motif, so it should have its own ligand. The same year, Yoshinaga and his colleague firstly discover the mouse B7RP-1 molecule and confirmed the B7RP-1 was the specific ligand of ICOS. The ligand of human ICOS, ICOSL (named as hB7RP-1, B7-H2, hLICOS) was mainly found on activated B cells, and can be detected on macrophage and dendritic cells in some extent. Previous study showed that ICOSL expression on mouse fibroblasts and nonlymphoid organ could be induced by TNF-α; IL-1βand the ICOSL expression on endothelial cells could be obviously increased by TNF-α. In all, ICOSL expression on cells and organs could be regulated by various cytokines.ICOS-ICOSL signal plays an important role in the immune regulation. (1)ICOS expression was increased on activated T cells, the reaction of ICOS and ICOSL could promote the T cells’activation and proliferation, then exert an important immunoregulation cooperated with CD28-B7 signal in immune response. By contrast to CD28-B7 signal, ICOS-ICOSL signal inhibit the secretion of cytokine IL-2 but promote the secretion of IL-10 of activated T cells, then promote Th2 polarization. From this, ICOS-ICOSL signal is independent on CD28-B7 signal, both of them probably were independent signals. (2) ICOS-ICOSL signal takes part in humoral immune response. Although ICOS-ICOSL signal didn’t influence development of B cells, it clued to the maturation of B cells. Due to the lack of ICOS-ICOSL signal, T cells dependent B cells response were severely destroyed. In humoral immune response, ICOS-ICOSL signal participate in the cross-talk of T lymphocyte and B lymphocyte, promote the proliferation of activated B cells and the production of antibodies.ICOS-ICOSl signal moderated the effect of humoral immune response. (3) Because of the ICOSL expression on nonlymphoid organ, ICOS-ICOSL signal also plays a key role in local immune and inflammation response.In conclusion, ICOS-ICOSL signal probably takes part in immunological pathogenesis of GD. This study is to explore the functions and mechanisms of ICOS-ICOSL signal in the immunological pathogenesis of hyperthyroidism, based on the detection of abnormal ICOS/ICOSL molecules expression in peripheral blood and thyroid tissue of GD patients.PartⅠ: The abnormal expression of ICOS/ICOSL molecules on lymphocyte subsets in peripheral blood of GD patients and its clinical significanceObjective: To detect the expansion of CD4~+CD28~-T lymphocytes and the abnormal expression of ICOS/ICOSL on T lymphocyte subsets in peripheral blood of GD patients, analysis the correlation between CD4~+CD28~-T lymphocytes expansion or ICOS/ICOSL expression and clinical features of GD patients. To detect the cytokine secretion in the serum of GD patients.Methods: The peripheral blood mononuclear cells (PBMCs) of 105 GD patients were chosen as experimental subject and that of 67 healthy donors as control. Using the method of flow cytometry(FCM), abnormal expansion of lymphocyte subsets in peripheral blood of GD patients was detected. By the methods of FCM and real-time PCR, ICOS/ICOSL expression pattern was detected in peripheral blood of healthy donor and GD patients, as well as of the same patient before and after the treatment. Then statistic analysis the correlation between the CD4~+CD28~- T cells expansion or abnormal expression of ICOS/ICOSL molecules and clinical parameters, therapeutic value and recurrence of disease. Using the skills of real-time PCR and Enzyme-linked immunosorbent assay (ELISA), cytokines such as IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γand IL-17 secretion level were detected in the serum.Results:①The percentage of CD4~+ T cells (P<0.05), the ratio of CD4/CD8 (P<0.05) and the CD28 expression on T cells (P<0.001) in peripheral blood of GD patients were remarkably lower than healthy donor in control group. However, the percentage of CD19~+ B cells was remarkably higher than contro (P<0.001). Further two-color flow cytometry study showed that percentages of both CD4~+CD28~+ (P<0.05) and CD8~+CD28~+ T cells declined markedly (P<0.01).②ICOS expression on CD4~+ T cells (P<0.001) was increased markbly in GD patients than in control, but not on CD8~+ T cells. Meanwhile, ICOSL expression on CD19~+ B cells was also notably increased (P<0.0001).③Further realtime-PCR study showed that expression of both ICOS and ICOSL mRNA of PBMCs in GD patients was remarkably higher than in healthy control.④In GD patient’s serum, the lever of cytokine IL-4, IL-6, IL-10, TNF-α, IFN-γand IL-17 were notably increased than in healthy control, but the IL-2 secretion was declined.⑤In peripheral blood of patients after treatments, the abnormal expansion of autoreactive T cells was detected. Both ICOS expression on CD4~+ T cells and ICOSL expression on CD19~+ B cells were decreased significantly.⑥The percentage of autoreactive T cells or ICOS expression on CD4~+ T cells is positively correlated to free triiodothyronine (FT3) in GD patients’serum.⑦In the patients group with higher TRAb secretion level in serum, the percentage of autoreactive T cells or ICOSL expression on CD19~+ B cells was notably increased than in the patients with lower TRAb.Conclusion: There was abnormal expansion of autoreactive T (CD4~+CD28~- T) cells and abnormal expression of ICOS/ICOSL molecules on lymphocyte subsets in peripheral blood of GD patients. They had correlation with clinical character, implying ICOS-ICOSL signal take part in the immunological pathogenesis of hyperthyroidism.PartⅡ: The expression of ICOS/ICOSL molecules in thyroid tissue of GD patientsObjiective: To detect the ICOS/ICOSL expression on infiltrated lymphocytes and thyroid follicular cells (TFC) in thyroid tissue of GD patients, analysis the effect of cytokines on ICOSL expression of TFC in vitro.Methods: The thyroid tissues of patients were collected from surgical operation, including 10 cases with GD as expression subject, 10 cases with hashimoto disease (HT) and 10 cases with nontoxic goiter (NTG) as control. By the mathods of flow cytometry (FCM), real-time PCR, immunohistochemistry (IHC) and laser scaning confocal, ICOS/ICOSL expression on infliteated T, B cells and TFC was detected. Using primary TFC culture thecnology and FCM, the change of ICOSL expression on TFC was ananlyzed in the absence or presence of cytokines.Results:①Higher expression of ICOS/ICOSL mRNA was found in thyroid tissue of GD patients than of HT and NTG controls.②ICOS/ICOSL expression on infiltrated lymphocytes in thyroid tissue of GD patients were detected by the method of FCM.③Both ICOS expression on infiltrated T cells and ICOSL expression on infiltrated B cells and TFC were detected by the method of IHC in tissue of GD patients. However, ICOSL expression was only found on infiltrated B cells in tissue of HT and NTG controls.④Proinflammatory cytokines IFN-γ, IL-6 and TNF-αlead to significant increasing of ICOSL expression on TFC.Conclusion: There was abnormal expression of ICOS/ICOSL molecules in thyroid tissue of GD patients. ICOS-ICOSL signal promoted Th2 polarization, B cells activation and increased autoantibodies’production. Amount of cytokines caused significant increasing of ICOSL expression on TFC. ICOS-ICOSL signal may exert important role in the initiation, maintainess and exaggeration of autoimmune response in local tissue. PartⅢ: The role of ICOS-ICOSL signal in the biological behavior of primary cultured thyroid follicular cellsObjiective: To investigate the role of ICOS-ICOSL signal in the biological behavior of primary cultured thyroid follicular cells.Methods: By methods of TFC primary culture, MTT assay and radioimmunoassay, ICOS transfected L929 cells(ICOS/L929 cells) and blocking ICOSL antibody named 11C4 were adopted to investigate the role of ICOS-ICOSL signal in primary cultured TFC proliferation, thyroid hormones production and thyroglobulins(Tg) release. Results: Compared with mock transfected L929 (mock-L929) cells (as control), ICOS-L929 cells could induce primary cultured TFC growth markedly, producing more thyroid hormones and more Tg (all P﹤0.01). In the present of ICOSL blocking antibody (11C4), the growth of TFC and the production of thyroid hormones and Tg were inhibited significantly(all P<0.05).Conclusion: According to above results, interaction of ICOS-ICOSL signal can be regarded as direct role in proliferation and differentiation of thyrocytes.In this report, by the method of flow cytometry, PCR et al, the expansion of CD4~+CD28~- T cells(autoreactive T cells), ICOS/ICOSL expression pattern on lymphocyte subsets in peripheral blood and local thyroid organ were incestigated.The correlation between the abnormal augment of CD4~+CD28~- T cells and the abnormal expression of ICOS/ICOSL was analyzed. In vitro study was performed to investigate the influence of ICOS-ICOSL signal on proliferation and function of TFC.In the microenvironment including TFC, infiltrated autoreactive T cells and B cells, ICOS-ICOSL costimulatory signal play an important role in the initiation, maintainess, exaggeration of immune response. It is of important value to study the role of ICOS-ICOSL signal in immune regulation, for the illustration of immunological pathogenesis and exploration of new means to immune intervene.