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Estrogen deficiency following ovariectomy or menopause results in bone loss. Although evidence strongly suggests that the immune system is involved in the pathogenesis of estrogen-deficient osteoporosis, it is not clear what role, if any, the T-lymphocyte plays in this process. Therefore, we examined the distribution of T-cell subsets in lymphoid organs and tissues, under varying estrogenic states in the rat. Six-month-old female Sprague-Dawley rats, ovariectomized (Ovx) and sham-operated, were randomized 5 d post-surgery into six groups to receive the following treatments: (A) sham/placebo; (B) sham/low-dose E2; (C) sham/high-dose E2; (D) Ovx/placebo; (E) Ovx/low-dose E2; (F) Ovx/high-dose E2. Half of the treated rats (groups A-F) were sacrificed on d 14; the remainder on d 28. Following euthanasia, mononuclear cells were isolated from the thymus, peripheral blood, spleen, lymph node and bone marrow, and were labeled for flow cytometric analysis using mouse anti-rat monoclonal antibodies directed against CD5, CD4, and CD8 antigenic markers. In the thymus, ovariectomy caused a dramatic increase and E2 treatment caused a dose-dependent decrease in weight that was proportional to the number of thymocytes. In the bone marrow, ovariectomy caused a significant reduction in the percentage of all T-cell subsets examined and this effect persisted throughout the duration of the study. Estrogen replacement therapy at the low-dose reversed the effects of ovariectomy and high-dose E2 treatment caused an increase in T-cell subsets in both the sham and Ovx groups, an effect that was more pronounced at d 14 compared with d 28. Although the percentages of some T-cell subsets in the other lymphoid organs/tissues were altered by ovariectomy or E2 treatment at d 0 and 14, all these changes had normalized by d 28 except for CD5 and CD4 cells in peripheral blood. In summary, with the exception of T-lymphocytes in the bone marrow, the effects of varying estrogenic states on T-cells were variable and transient. The influence of estrogen status on bone marrow T-lymphocytes suggests that these cells may play a role in mediating the effects of estrogen on bone turnover and warrant additional studies focusing on the functional role of T-cells in the bone marrow compartment.

Our laboratory has previously demonstrated that the T-lymphocyte is critical in the development of cyclosporin A-induced osteopenia in the rat model. A similar state of osteopenia is induced by estrogen depletion in the ovariectomized (OVX) rat, which is the animal model of postmenopausal bone loss. However, the role of the immune system, and particularly the T-lymphocyte, in estrogen deplete osteopenia has not been elucidated. We used the Rowett athymic nude rat as our model of T-lymphocyte deficiency. In this study, the experimental rats were divided into four groups as follows: (1) sham-operated Rowett heterozygous (rnu/+) euthymic rats (control group); (2) OVX Rowett heterozygous (rnu/+) euthymic rats; (3) sham-operated Rowett homozygous (rnu/rnu) athymic nude rats, which are T-lymphocyte deficient; and (4) ovariectomized Rowett homozygous (rnu/rnu) rats. Rats were weighed, and venous blood was taken in weeks 2, 4, and 6 for determination of serum osteocalcin. Serum 1,25-dihydroxyvitamin D (1,25(OH)2D) was determined on the day of sacrifice. Following sacrifice, histomorphometry was performed on double-labeled proximal tibial metaphyses. Flow cytometric analysis of splenic mononu-clear cell isolates stained for OX19-positive (CD5) T-lymphocytes was performed. T-lymphocyte analysis revealed significant reductions in both athymic nude groups, while OVX euthymic rats demonstrated a diminished number of T-cells relative to their sham-operated counterparts. Histomorphometric data indicated that both OVX groups exhibited a significant loss of trabecular volume, with associated increases in indices for bone formation and resorption, with resorption likely outstripping formation, resulting in osteopenia. Serum osteocalcin was significantly elevated in the ovariectomized euthymic group throughout the experimental period compared with the control group (p < 0.01); it was elevated in the ovariectomized athymic group on week 4 only (p < 0.01 vs. control). It appears that the T-lymphocyte may not be an essential component in the pathogenesis of estrogen deficiency osteopenia. The contribution of circulating T-lymphocytes as well as other T-lymphocyte-rich organs needs to be explored further.