Increased bone mass in mice after single injection of anti-receptor activator of nuclear factor-kappaB ligand-neutralizing antibody: evidence for bone anabolic effect of parathyroid hormone in mice with few osteoclasts

Abstract

Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo.

FIGURE 1.

Effects of mRANKL monoclonal antibody OYC1 on BMD, bone structure, and bone histomorphometry in mice. Female 6-week-old C57BL/6N mice (n = 5–6) were injected intraperitoneally (i.p.) with 15 mg/kg anti-mouse RANKL Mab OYC1 or OYC2 or PBS three times per week for 2 weeks. At 12 h after the last administration, femurs were extirpated and fixed with 70% ethanol. A, right femur was partitioned into 20 areas, and BMD of each part was measured by SXA. B, toluidine blue staining of the distal femoral metaphysis. C, histomorphometry was performed on undecalcified sections. Oc.S/BS, N.Oc/BS, ES/BS, Ob.S/BS, MAR. and BFR/BS were measured. Bar, 1 mm. Data are shown as the mean ± S.D. a, p < 0.05; b, p < 0.01 (ANOVA) versus PBS.

FIGURE 2.

Suboptimal dose of OYC1 required to increase bone mass. Various doses (0.5, 1, 1.5, 5, and 15 mg/kg) of OYC1 or PBS were injected subcutaneously (s.c.) in 6-week-old female mice once (n = 5). After 2 weeks, blood samples and femurs were collected, and the femurs were fixed with 70% ethanol. A, femoral trabecular bone mass was measured by SXA analysis. B, bone structure in the trabecular area was analyzed by micro-CT. C, femoral trabecular bone was measured by histomorphometry. Ob.S/BS, Oc.S/BS, N.Oc/BS, and ES/BS were measured in the PBS and OYC1 (5 and 15 mg/kg) groups. D, serum TRAP-5b, ALP, and OYC1 levels were measured by ELISA or pNPP assay. Data are shown as the mean ± S.D. b, p < 0.01 (ANOVA) versus PBS.

FIGURE 3.

Time course of the effect of OYC1 on bone mass (days 4 to 28). A, OYC1 (5 mg/kg) was administered subcutaneously (s.c.) to 6-week-old female mice (n = 5–6) on day 0. The mice were sacrificed on days 4, 7, 14, and 28. Total BMD in femurs was measured by SXA analysis. B, bone structure in the trabecular area was analyzed by micro-CT. C, serum TRAP-5b, ALP, and OYC1 levels were measured by ELISA or pNPP assay. Data are shown as the mean ± S.D. a, p < 0.05; c, p < 0.001 versus PBS (Student's t test); b, p < 0.01 (ANOVA) versus value on day 0.

FIGURE 4.

Time course of the effect of OYC1 on bone mass (days 1 to 4). A, OYC1 (5 mg/kg) was administered subcutaneously to 6-week-old female mice (n = 5–6) on day 0, and mice were sacrificed on days 1–4. Total BMD was measured by pQCT at 1.0 mm from the distal femoral growth plate. B, bone structure in the trabecular area was analyzed by micro-CT. C, serum TRAP-5b, ALP, and OYC1 levels were measured by ELISA or pNPP assay. D, TRAP staining of undecalcified sections of tibia in OYC1-treated mice are shown on the left (bar, 100 μm). and high power fields are shown on the right (bar, 100 μm). Data are shown as the mean ± S.D. a, p < 0.05; b, p < 0.01 (ANOVA) versus value on day 0.

FIGURE 5.

Evaluation of PTH in osteoclast-depleted mice pretreated with OYC1. OYC1 (5 mg/kg) or PBS was administered subcutaneously to 6-week-old female mice (n = 5) on day 0 and PTH (160 μg/kg) or PBS was injected subcutaneously daily from days 4 to 17. Femurs and sera were obtained on day 18. A, total BMD was measured by pQCT at 0.8 mm from the distal femoral growth plate. B, cortical BMD in the femoral diaphysis was measured by pQCT. C, bone structure in the secondary trabecular area was analyzed by micro-CT. D, histological data were obtained under fluorescent light, natural light, and polarization using undecalcified sections of each group. Each arrowhead indicated labeled thickness (blue); osteoblasts (yellow); and osteoclasts (red). MAR, mineral apposition rate; BMU, basic multicellular unit. Bar, 50 μm. E, serum TRAP-5b, osteocalcin, and intact PTH were measured by ELISA. Serum ALP was measured by pNPP assay. Data are shown as the mean ± S.D. a, p < 0.05; b, p < 0.01 (ANOVA) versus PBS.