Receptor activator of nuclear factor κB ligand (RANKL) protein expression by B lymphocytes contributes to ovariectomy-induced bone loss

Abstract

Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen.

FIGURE 1.

B cell RANKL is required for B cell, but not skeletal, development. A, RANKL mRNA expression in CD19⁺ B cells and CD3⁺ T cells in the bone marrow, in intact spleen (spl), and in lumbar vertebrae 5 (L5) (n = 7–17 animals per group). B, serial BMD of RANKL^fl/+ (closed circles), CD19-Cre;RANKL^fl/+ (open circles), RANKL^fl/fl (closed triangles), and RANKL^ΔB (open triangles) mice determined up to 7 months of age (n = 5–15 animals per group). The same cohort of animals was used for each age. C, percentage of B cells (CD19⁺), pre-B cells (CD45R/B220⁺CD43⁻), and T cells (CD3⁺) in the bone marrow of 6-month-old RANKL^fl/fl and RANKL^ΔB mice determined by flow cytometry (n = 5 samples/group). *, p < 0.05 by Student's t test.

FIGURE 2.

Deletion of RANKL from B cells attenuates ovariectomy-induced bone loss. 6-month-old female RANKL^fl/fl and RANKL^ΔB mice were either sham-operated (white bars) or ovariectomized (Ovx) (gray bars) and killed 6 weeks later. A, uterine weight per body weight. B, percentage change of body weight. C and D, changes in BMD in the femur and vertebrae during the 6 weeks after surgery. E and F, bone volume over tissue volume (BV/TV) and trabecular thickness (Tb.Th) of cancellous bone measured in lumbar vertebra 4 by μCT. G and H, BV/TV and Tb.Th of femoral cancellous bone measured by μCT. I, cortical thickness (Ct.Th) of the femoral midshaft measured by μCT. J, μCT images of femoral cancellous bone. All values are the mean of 10–17 animals per group. *, p < 0.05, effect of operation within genotype. #, p < 0.05, effect of genotype within operation.

FIGURE 3.

Deletion of RANKL from B cells blunts ovariectomy-induced osteoclastogenesis. Histological and bone marrow cell counts were performed in the mice described in the legend to Fig. 2. A and B, bone area versus tissue area (BA/TA) and trabecular width (Tb.Wi) measured in L4 vertebra. C and D, osteoclast perimeter per bone perimeter (OcPm/B.Pm) and osteoclast number per bone perimeter (OcN/B.Pm) measured by histomorphometric analysis of lumbar vertebra 1–3 (n = 4–5 animals per group). E, histological sections of lumbar vertebrae stained for TRAP activity (osteoclasts stained red) and toluidine blue (original magnification ×630). F and G, RANKL mRNA levels in sorted CD19⁺ B cells and CD3⁺ T cells measured by RT-PCR (n = 7–8 animals/group). H and I, percentage of CD19⁺ B cells and CD3⁺ T cells in the bone marrow analyzed by flow cytometry (n = 4 animals per group). *, p < 0.05, effect of operation within genotype. #, p < 0.05, effect of genotype within operation.

FIGURE 4.

Ovariectomy increases B cell number but not RANKL gene expression. 7-month-old wild-type C57BL/6 mice were sham-operated or ovariectomized and killed after 2 weeks. A, changes in femoral and spinal BMD (n = 13–14 per group). B, RT-PCR analysis of RANKL mRNA in CD19⁺ B cells isolated from the bone marrow (n = 13–14 mice per group). C and D, percentages of CD19⁺ B cells and CD19⁺ B cells that also express RANKL on their surface in the bone marrow (n = 6 mice per group). E, mean intensity of cell surface RANKL protein detected on CD19⁺ B cells from the bone marrow (n = 6 mice per group). F, amount of sRANKL protein present in bone marrow supernatants normalized to total protein level (n = 9–12 mice per group). *, p < 0.05 using Student's t test.

FIGURE 5.

Deletion of RANKL from T cells does not alter bone mass. A, RANKL mRNA expression in CD19⁺ B cells, CD3⁺ T cells, intact spleen (spl), and L5 vertebra measured by RT-PCR (n = 5–7 mice per group). B, serial BMD of RANKL^fl/+ (closed circles), Lck-Cre;RANKL^fl/+ (open circles), RANKL^fl/fl (closed triangles), and RANKL^ΔT (open triangles) mice measured up to 7 months of age (n = 4–13 animals per group). The same cohort of animals was used for each age. C, percentage of CD19⁺ B cells and CD3⁺ T cells in the bone marrow of 6-month-old RANKL^fl/fl and RANKL^ΔT mice determined by flow cytometry (n = 5–7 mice/group). *, p < 0.05 using Student's t test.

FIGURE 6.

Deletion of RANKL from T cells does not alter ovariectomy-induced bone loss. 6-month-old female RANKL^fl/fl and RANKL^ΔT mice were sham-operated (white bars) or ovariectomized (gray bars) and killed 6 weeks later. A, uterine weight per body weight at time of sacrifice (n = 10–14 animals per group). B and C, percentage of CD19⁺ B cells and CD3⁺ T cells in the bone marrow measured by flow cytometry analysis (n = 5 animals per group). D–F, femoral, vertebral, and total body BMD changes during the 6 weeks after surgery (n = 10–17 animals per group). G and H, bone volume over tissue volume (BV/TV) and trabecular thickness of L4 cancellous bone measured by μCT. I, cortical thickness in femoral midshafts measured by μCT. All μCT values are the mean of 10–13 animals/group. *, p < 0.05, effect of operation within genotype.