B cells and T cells are critical for the preservation of bone homeostasis and attainment of peak bone mass in vivo

Abstract

Bone homeostasis is regulated by a delicate balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclastogenesis is controlled by the ratio of receptor activator of NF-kappaB ligand (RANKL) relative to its decoy receptor, osteoprotegerin (OPG). The source of OPG has historically been attributed to osteoblasts (OBs). While activated lymphocytes play established roles in pathological bone destruction, no role for lymphocytes in basal bone homeostasis in vivo has been described. Using immunomagnetic isolation of bone marrow (BM) B cells and B-cell precursor populations and quantitation of their OPG production by enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), cells of the B lineage were found to be responsible for 64% of total BM OPG production, with 45% derived from mature B cells. Consistently B-cell knockout (KO) mice were found to be osteoporotic and deficient in BM OPG, phenomena rescued by B-cell reconstitution. Furthermore, T cells, through CD40 ligand (CD40L) to CD40 costimulation, promote OPG production by B cells in vivo. Consequently, T-cell-deficient nude mice, CD40 KO mice, and CD40L KO mice display osteoporosis and diminished BM OPG production. Our data suggest that lymphocytes are essential stabilizers of basal bone turnover and critical regulators of peak bone mass in vivo.

Figure 1

B-cell KO mice display decreased baseline BMD and increase bone resorption, concurrent with decreased BM OPG concentrations. (A) BMD was determined by DXA in μMT/μMT B-cell KO mice (□) and age-matched WT controls (■) at the total body, spine, and femurs. (B) Longitudinal (top) and cross-sectional (bottom) μCT reconstructions of distal femurs from WT and μMT/μMT B-cell KO mice. (C) Elevated biochemical indices of bone resorption (CTx) were observed in B-cell KO mice. (D) Decreased OPG production was detected in μMT/μMT B-cell KO BM compared with equivalent amounts of WT BM. Mean ± SD of 8 mice per group; *P < .05. (E) Real-time RT-PCR quantitation of OPG mRNA expression in 3 independent WT and B-cell KO mice. *P < .01. (F) B-cell reconstitution of μMT/μMT B-cell KO rescues BMD (*P < .01 versus WT) and (G) OPG production (*P < .05 versus WT). Mice were reconstituted at 4 weeks of age and BMD and RT-PCR studies preformed at 12 weeks of age.

Figure 2

B-lineage cells are a major source of OPG production in the BM and spleen. (A) OPG secretion by total BM white cells or the B-cell fraction contained therein was quantitated by ELISA. Mean ± SD of triplicates. Data are representative of more than 6 independent experiments. (B) Relative OPG transcription was quantitated by RT-PCR in total BM white cells or B-cell fraction contained therein. Data are presented as mean ± SD of 3 independent mice per group. (C) OPG production by equivalent numbers (1 × 10⁷ cells) of purified BM B-cell precursors, immature B cells, mature B cells, and plasma cells. (D) B-lineage cell composition as a percentage of total nucleated BM cells. (E) Actual concentration of OPG secreted by specific B-lineage cells per 10⁷ total nucleated BM cells. The total OPG production was 134 ± 26.1 pg/mL/10⁷ total white BM cells. (F) OPG production by equivalent numbers of mature spleen B cells was quantitated relative to plasma cells and non–B-lineage cells (non-B cell). (G) Spleen B-cell composition as a percentage of total nucleated spleen cells. (H) Percentage spleen OPG production for each cell population in the spleen. Mean ± SD of 2 independent experiments measured in triplicate. *P < .05 relative to unstimulated. (I) Comparison of OPG production by equivalent numbers of mature B cells derived from BM, peripheral blood (PB), lymph nodes (LN), or spleen (SP). BM from 5 independent mice was measured in triplicate. *P < .001 relative to BM (ANOVA). (J) OPG production by mature splenic B cells stimulated in vitro with CD40L (500 ng/mL) and/or anti-IgM activating antibody (5 ng/mL).

Figure 3

T-cell–deficient nude mice display decreased baseline BMD and increased bone resorption, concurrent with decreased total BM OPG, and B-cell–specific OPG production. (A) BMD was determined in WT mice at the total body, spine, and femurs, 12 weeks of age (black bars) and 16 weeks of age (white bars), and in nude mice at 12 weeks of age (vertical lines) and 16 weeks (horizontal lines) of age. Data are presented as mean ± SD of 29 nude mice and 18 WT mice. *P ≤ .001 relative to age-matched WT. Percentage changes (Δ) between nude and WT mice at 12 and 16 weeks are indicated above the appropriate bars. (B) μCT reconstructions showing reduced trabecular and cortical structure of representative tibias in nude mice relative to WT. (C) Increased biochemical indices of bone resorption (CTx) were observed in the serum of nude mice compared with WT. *P < .05; n = 9 mice per group. (D) Decreased OPG production by BM derived from nude mice compared with WT (ELISA). (E) Decreased OPG production by purified IgM⁺ B cells from nude mice relative to WT, quantitated in 48-hour–conditioned medium by ELISA; n = 6 mice per group. *P ≤ .01. (F) Real-time RT-PCR quantitation of OPG and RANKL mRNA expression in WT and nude mice. Mean ± SD of 3 independent WT and nude mice. *P < .01.

Figure 4

CD40 KO mice and CD40L KO mice display decreased baseline BMD and increased bone resorption, concurrent with decreased B-cell OPG production. Decreased BMD was observed by DXA in (A) CD40 KO mice and (B) CD40L KO mice relative to age-matched (24 weeks) WT controls of the same genetic background at multiple anatomic positions including total body, spine, and femurs. P < .01, n = 16 (WT), n = 14 (CD40 KO), n = 12 (WT and CD40 KO). μCT reconstructions showing reduced trabecular and cortical structure of representative femurs in (C) CD40 KO mice and in (D) CD40L KO mice relative to their respective WT controls. Increased biochemical indices of bone resorption (CTx) were observed in the serum of (E) CD40 KO mice and (F) CD40L KO mice. Decreased OPG production by IgM⁺ BM B cells derived from (G) CD40 KO mice and (H) CD40L KO mice compared with WT (ELISA). *P ≤ .001. Real-time RT-PCR quantitation of OPG and RANKL mRNA expression in (I) CD40 KO and (J) CD40L KO mice. Mean ± SD of 3 independent WT and CD40 KO mice. *P ≤ .01.