Bone loss in surgically ovariectomized premenopausal women is associated with T lymphocyte activation and thymic hypertrophy

Abstract

Postmenopausal osteoporosis is associated with estrogen deficiency and rapid bone loss. The mechanism by which estrogen deficiency results in bone loss has not been fully explained. Studies in mice rendered acutely estrogen deficient by ovariectomy have suggested that estrogen deficiency results in an activated T-lymphocyte phenotype and increased production of pro-osteoclastic cytokines. The aim of this study was to translate these findings from mouse models that suggest that the T lymphocyte plays an important role in the etiology of postmenopausal osteoporosis. We recruited premenopausal women who underwent ovariectomy for benign gynecologic conditions or for prophylaxis against ovarian cancer and a group of matched control women without ovariectomy (OVX). Subjects provided blood samples to characterize T-lymphocyte phenotype by flow cytometry and for T-lymphocyte culture and collection of conditioned media. Bone mineral density at the lumbar spine and left femoral neck was performed annually for 2 years, and volumetric measurements by computed tomography (CT) of the thymus were obtained during the first 6 months. We enrolled 6 patients who underwent OVX and 13 control women. The OVX subjects had a significant loss of bone mineral density at the lumbar spine and left femoral neck. The volumetric thymus measurements suggested an increase in thymus size in the OVX subjects but did not reach statistical significance owing to the small sample size. The T-lymphocyte phenotype in the OVX subjects demonstrated increased T-lymphocyte activation by flow cytometry compared to the control subjects. Our findings support the hypothesis that estrogen deficiency leads to an activated T-lymphocyte phenotype, which may contribute to the bone loss seen in estrogen deficiency. Larger clinical studies are necessary to confirm these findings.

Figure 1. Bone Mineral Density of Lumbar Spine

Compared to the control group, the OVX group had significant declines in lumbar spine BMD at year 1 (−4%) and year 2 (−6%).

Figure 2. Bone Mineral Density of Left Femoral Neck

The OVX group showed a significant decline in left femoral neck BMD at year 1 (−6%) and year 2 (−3%). A significant increase in left femoral neck BMD was seen at year 2 (+4%) in the control group.

Figure 3. Thymus Size

The OVX group had a trend towards increase in volumetric thymus size 3 months after surgery which persisted up to 6 months following ovariectomy. Volumetric thymus size decreased in the control group.

Figure 4. Markers of T-cell activation and differentiation in subjects undergoing ovariectomy compared to control subjects

Peripheral blood mononuclear cells were collected and T-cells were purified from women before and after ovariectomy (squares) and pre menopausal control subjects (diamonds). Flow cytometry was performed using the indicated cell surface and intracellular markers. Frequencies of both CD69 (A) and TNFα (B) positive cells increased significantly in OVX T-cells at 1 month compared to baseline and remained significantly elevated at 3 months (p<0.05).

Figure 5. Naïve and memory T-cell frequencies in subjects undergoing ovariectomy compared to control subjects

Peripheral blood mononuclear cells were collected and T-cells were purified from women before and after ovariectomy (squares) and pre-menopausal control subjects (diamonds). Flow cytometry was performed using the indicated cell surface markers. A. Compared to the control subjects, there were higher frequencies of CD45RO+ memory T-cells (A) in the ovariectomy group at baseline (p=0.05). B. There were no significant changes in the frequencies of CD45RO+ or CD45RA+ T-cells in control and OVX groups over 3 months follow up period.