María Ángeles García-López

CONTEXT: T regulatory cells have a key role in the pathogenesis of autoimmune diseases in different animal models. However, less information is available regarding these cells in human autoimmune thyroid diseases (AITD). OBJECTIVE: The objective of the study was to analyze different regulatory T cell subsets in patients with AITD. DESIGN: We studied by flow cytometry and immunohistochemistry different T regulatory cell subsets in peripheral blood mononuclear cells (PBMCs) and thyroid cell infiltrates from 20 patients with AITD. In addition, the function of T(REG) lymphocytes was assessed by cell proliferation assays. Finally, TGF-beta mRNA in thyroid tissue and its in vitro synthesis by thyroid mononuclear cells (TMCs) was determined by RNase protection assay and quantitative PCR. RESULTS: PBMCs from AITD patients showed an increased percent of CD4+ lymphocytes expressing glucocorticoid-induced TNF receptor (GITR), Foxp3, IL-10, TGF-beta, and CD69 as well as CD69+CD25(bright), CD69+TGF-beta, and CD69+IL-10+ cells, compared with controls. TMCs from these patients showed an increased proportion of CD4+GITR+, CD4+CD69+, and CD69+ cells expressing CD25(bright), GITR, and Foxp3, compared with autologous PBMCs. Furthermore, a prominent infiltration of thyroid tissue by CD69+, CD25+, and GITR+ cells, with moderate levels of Foxp3+ lymphocytes, was observed. The suppressive function of peripheral blood T(REG) cells was defective in AITD patients. Finally, increased levels of TGF-beta mRNA were found in thyroid tissue, and thyroid cell infiltrates synthesized in vitro significant levels of TGF-beta upon stimulation through CD69. CONCLUSIONS: Although T regulatory cells are abundant in inflamed thyroid tissue, they are apparently unable, in most cases, to downmodulate the autoimmune response and the tissue damage seen in AITD.

CD69 is induced after activation of leukocytes at inflammatory sites, but its physiological role during inflammation remains unknown. We explored the role of CD69 in autoimmune reactivity by analyzing a model of collagen-induced arthritis (CIA) in WT and CD69-deficient mice. CD69-/- mice showed higher incidence and severity of CIA, with exacerbated T and B cell immune responses to type II collagen. Levels of TGF-beta1 and TGF-beta2, which act as protective agents in CIA, were reduced in CD69-/- mice inflammatory foci, correlating with the increase in the proinflammatory cytokines IL-1beta and RANTES. Local injection of blocking anti-TGF-beta antibodies increased CIA severity and proinflammatory cytokine mRNA levels in CD69+/+ but not in CD69-/- mice. Moreover, in vitro engagement of CD69 induced total and active TGF-beta1 production in Concanavalin A-activated splenocyte subsets, mouse and human synovial leukocytes, and Jurkat stable transfectants of human CD69 but not in the parental CD69 negative cell line. Our results show that CD69 is a negative modulator of autoimmune reactivity and inflammation through the synthesis of TGF-beta, a cytokine that in turn downregulates the production of various proinflammatory mediators.

tions In addition, NK cell-dependent tumor rejection is favored in the absence of CD69 (13). However, the functional role of CD69 expressed by T cells and macrophages at inflammatory sites and in autoimmune processes has not been explored.

To better understand the selective migration of lymphocytes in autoimmune thyroid disorders (AITDs), we analyzed thyroid samples and demonstrated an enhanced expression of the chemokines interferon (IFN)-inducible protein (Ip)-10 and regulated on activation normal T lymphocyte expressed and secreted (RANTES) in thyroids from AITD patients. Ip-10 and monokine induced by IFN-gamma (Mig) were expressed in vivo in thyroid follicular cells (TFCs) from AITD thyroids. Interestingly, Ip-10 mRNA, although not basally detected in cultured TFCs, was strongly induced by IFN-gamma and synergistically increased by TNF-alpha addition. Furthermore, high levels of Ip-10 protein were detected in the supernatants of IFN-gamma-stimulated TFCs. Likewise, Mig protein was strongly induced in TFCs by the same stimuli as Ip-10. Unlike Ip-10 and Mig, the expression of RANTES was induced mainly by TNF-alpha. In addition, intrathyroidal lymphocytes from AITD patients showed higher expression of CXCR3, CCR2, and CCR5 chemokine receptors than autologous peripheral blood lymphocytes. T lymphoblasts expressing CXCR3 showed an increased migration to supernatants from stimulated TFCs, which was abolished by specific antibodies to the chemokines Ip-10 and Mig, as well as to their receptor CXCR3. Taken together, these data suggest a potential role of TFCs, through the production of the chemokines Ip-10, Mig and RANTES, in regulating the recruitment of specific subsets of activated lymphocytes in AITDs.