Both clinical and experimental evidence suggests that sex hormones play the critical role in the pathogenesis and disease severity in human SLE and murine models of lupus. 34 Oestrogen has been documented to display potent immunoregulatory properties; however, the exact roles of the sex steroids in autoimmune diseases remain unclear. Many studies have demonstrated aggravation of lupus disease in MRL-lpr/lpr and NZB/W F1 mice upon continuous exposure to physiological levels of oestrogen.25,26 However, Steinberg et al. suggested that MRL-lpr/lpr mice have a small response to gonadal hormone, 25 and autoimmunity in these mice is not strongly influenced by this hormone. In our study, we found that TAM treatment of MRL-lpr/lpr mice had a beneficial effect on both female and male (data not shown) mice. While the association between oestrogen metabolites and autoimmunity has been well documented, the mechanisms by which exogenous oestrogen can affect the disease process remain unclear. Oestrogen has been demonstrated to suppress T-cell function and yet exacerbate humoral immunity. 36 Clearly, the effects of oestrogen and metabolites on autoimmunity are complex, involving multiple cell types, signal pathways and various soluble mediators. The data here demonstrated that TAM, a potent oestrogen antagonist, could alleviate disease severity and decrease DN T-cell numbers in MRL-lpr/lpr mice.
The individual role of type 1 and type 2 T helper (Th1 and Th2) cell subsets, exhibiting different capacities of cytokine secretion, in the development and acceleration of SLE have not been well defined. Because several cytokines produced by Th2 cells, such as IL-4, IL-5, IL-6 and IL-10, are known to promote antibody production by B cells, 37 it has been speculated that Th2 cells and related cytokines may play an active role in the development of autoantibody-mediated autoimmune diseases such as SLE. Dayan et al. have shown that the beneficial effects of TAM and anti-oestradiol antibody on experimental murine lupus are associated with cytokine modulation, 28 but the data here did not show any difference in cytokine production from splenocytes stimulated with Con A after TAM treatment in MRL-lpr/lpr mice. In contrast to the non-autoimmune BALB/c mice used in Dayan's study, lupus-prone MRL-lpr/lpr mice were used in this study. Many studies have demonstrated multiple cytokine production defects in MRL-lpr/lpr mice, which may be the reason why the cytokine change was not as obvious as in the previous study. TAM may modulate autoimmune response via several different mechanisms. The pathogenesis of autoantibody production in MRL lupus largely requires CD4+αβ T cells, which provide the help to autoreactive B cells;38,39 however, the individual Th1 and Th2 cell subsets in this process remain unclear. The appearance of autoantibody is the hallmark of both human and murine lupus, suggesting a requirement for cytokines produced by Th2 cells in autoreactive B-cell activation. The most interesting finding of the data is that the number of double negative T cells in TAM-treated MRL-lpr/lpr mice was significantly lower than that of control group. Lymphadenopathy in MRL-lpr/lpr mice consists primarily of T cells with unusual phenotypes (CD4– CD8–, CD2–, IL-2R–, TCR-αβ+, CD3+, and B220+). 40 The lpr DN T-cell population is distinct from other T cells with the characteristic that little IL-2 is produced in response to mitogenic stimulation or T-cell receptor (TCR)/CD3 engagement. 41 This extremely low level of IL-2 production by lpr DN T cells was caused by both the increased instability of mRNA and the reduced activation of IL-2 gene promoter. 42 The IL-2 level secreted by lymph node cells was significantly higher in the TAM-treated group, and lymph node cells contain more than 80% of DN T cells, so in vivo treatment of TAM may affect DN T-cell function, including cell numbers and IL-2 production function. Recently, Radavanyi et al. found that IL-2 signalling may prime activated T cells to TCR-mediated apoptosis and that this pathway may not require fas expression, as they chose MRL-lpr/lpr mice as their animal model. 43 Their investigation also revealed that the defect of activation induced apoptosis in T cells from lpr mice was age-related. In our study, we begin to treat mice with TAM at 8 weeks of age; the age that was the oldest group of Radavanyi's study. We have designed a new animal study, and mice will be treated with TAM as early as 5 weeks of age, so we can determine whether there will be a large reduction in lymphadenopathy. In addition, TAM did not interfere with T-cell activation as determined by CD25 and CD69 expression in the presence of Con A stimulation in vitro, and the data is similar to that in human T cells. 22 The data here also demonstrated that DN T cells did respond to IL-2-activated activation but poorly to cross-linking anti-CD3 activity or phytohaemagglutinin (PHA) (data not shown). To further understand the effect of TAM on IL-2-activated proliferation of DN T cells, the T cells were pretreated with TAM and then stimulated with IL-2 (1·0 ng/ml). Interestingly, pretreatment of TAM on DN T cells can effectively inhibit their proliferative response in a dose-dependent manner. In contrast, the inhibitory effect on CD4+ T cells was not observed until a rather high dose of TAM was given. We therefore speculated that TAM might potentially influence DN T cells and modulate the immune function both in vivo and in vitro. After TAM treatment, the cell cycle of activated T cells may show sustained accumulation in S phase, and the cells are then susceptible to death. Another possibility is that the oestrogen levels in control group mice may be much higher than that of TAM-treated mice. Higher oestrogen can inhibit T-cell function; 36 subsequently the ability of thymidine incorporation was much lower in control-group mice.
In conclusion, the present study further demonstrated the beneficial effects of TAM on an animal model of lupus. The exact immunoregulatory mechanisms of TAM remain to be elucidated. Nevertheless, as TAM is a relatively safe drug without serious adverse reactions, it might be effective in the treatment of human SLE and related disorders.