Increase in the Number of Bone Marrow Osteoclast Precursors at Different Skeletal Sites, Particularly in Long Bone and Jaw Marrow in Mice Lacking IL-1RA

Abstract

Recently, it was shown that interleukin-1β (IL-1β) has diverse stimulatory effects on different murine long bone marrow osteoclast precursors (OCPs) in vitro. In this study, interleukin-1 receptor antagonist deficient (Il1rn^-/-) and wild-type (WT) mice were compared to investigate the effects of enhanced IL-1 signaling on the composition of OCPs in long bone, calvaria, vertebra, and jaw. Bone marrow cells were isolated from these sites and the percentage of early blast (CD31^hi Ly-6C^-), myeloid blast (CD31⁺ Ly-6C⁺), and monocyte (CD31^- Ly-6C^hi) OCPs was assessed by flow cytometry. At the time-point of cell isolation, Il1rn^-/- mice showed no inflammation or bone destruction yet as determined by histology and microcomputed tomography. However, Il1rn^-/- mice had an approximately two-fold higher percentage of OCPs in long bone and jaw marrow compared to WT. Conversely, vertebrae and calvaria marrow contained a similar composition of OCPs in both strains. Bone marrow cells were cultured with macrophage colony stimulating factor (M-CSF) and receptor of NfκB ligand (RANKL) on bone slices to assess osteoclastogenesis and on calcium phosphate-coated plates to analyze mineral dissolution. Deletion of Il1rn increased osteoclastogenesis from long bone, calvaria, and jaw marrows, and all Il1rn^-/- cultures showed increased mineral dissolution compared to WT. However, osteoclast markers increased exclusively in Il1rn^-/- osteoclasts from long bone and jaw. Collectively, these findings indicate that a lack of IL-1RA increases the numbers of OCPs in vivo, particularly in long bone and jaw, where rheumatoid arthritis and periodontitis develop. Thus, increased bone loss at these sites may be triggered by a larger pool of OCPs due to the disruption of IL-1 inhibitors.

Keywords: IL-1 signaling; calvaria; early blast; jaw; long bone; monocyte; myeoloid blast; osteoclast; osteoclast heterogeneity; osteoclast precursor; vertebrae.

Figure 1

MicroCT analysis of WT and Il1rn^−/− mice showed no differences in bone microstructure independently of the skeletal site. To assess possible bone destruction at the different skeletal sites, various 3-D microstructural bone parameters, including bone volume fraction (BV/TV), specific bone surface (BS/BV), trabecular thickness (Tb.Th.), and trabecular separation (Tb.Sp.), were measured in all specimens from Il1rn^−/− mice and wild-type (WT) controls (A–D) (n = 3 mice/group). These parameters were comparable between the two mouse strains irrespective of the bone site, suggesting that these Il1rn^−/− mice did not develop structural pathology yet.

Figure 2

Absence of IL-1RA increases the percentage of myeloid blasts in long bone, and the percentage of monocytes in long bone and jaw. Percentage of early blasts, myeloid blasts, and monocytes from long bone (A), calvaria (B), vertebra (C), and jaw (D) were quantified and compared between wild-type (WT) (black bars) and Il1rn^−/− (white bars) mice (n = 6, *p < 0.05 **p < 0.01, ***p < 0.001). Whereas the calvaria and vertebral bone marrow (BM) of Il1rn^−/− mice showed no significant differences in the percentage of various osteoclast precursor subsets, we found a significant increase of both the myeloid blasts and monocyte subsets in the BM of long bone. Similarly, the monocyte subset of OCPs was also significantly increased in the jaw BM of Il1rn^−/− mice as compared to WT controls.

Figure 3

Increased number of osteoclasts generated from long bone, calvaria, and jaw but not vertebra marrow cells of Il1rn^−/− mice. Bone marrow cells from long bone, calvaria, vertebra, and jaw were cultured with 30 ng/mL M-CSF and 20 ng/mL RANKL on bone slices for 6 days. Osteoclasts were stained for tartrate resistant acid phosphatase (TRAcP), counted, and categorized as 3–5 nuclei, 6–10 nuclei, 11–20 nuclei, and >20 nuclei. The number of osteoclasts (≥3 nuclei) was counted for long bone (A), calvaria (B), vertebra (C), and jaw (D) and compared between wild-type (WT) and Il1rn^−/− mice. The total number of osteoclasts is shown as multinucleated cells (MNCs). Osteoclastogenesis was significantly higher in long bone, calvaria, and jaw of Il1rn^−/− osteoclast precursors (OCPs) compared to WT OCPs. In contrast, WT and Il1rn^−/− OCPs isolated from vertebrae formed a comparable number of osteoclasts (n = 6 mice/group, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4

Absence of IL1-RA induces the expression of osteoclast markers particularly in long bone and jaw-derived osteoclasts. Gene expression was measured in wild-type (WT) and Il1rn^−/− osteoclasts derived from long bone, calvaria, vertebra, and jaw bone marrow (BM) that were cultured on bone slices for 6 days. (A) Cultures of Il1rn^−/− osteoclasts from all skeletal sites show no expression of Il1rn. The absence of Il1rn significantly increased the mRNA expression of Il1b in long bone and vertebra-derived osteoclasts (n = 6 mice/ group). (B) Protein levels of IL1-β were determined in the culture supernatants at day 3 of osteoclastogenesis. Whereas IL1-β production was below the detection limit in WT and Il1rn^−/− osteoclasts from calvaria and jaw, Il1rn^−/− osteoclasts derived from long bones and vertebrae showed increased IL1-β levels (n = 3 mice/group). nd = not detected. (C) Of note, the mRNA expression of various specific osteoclast markers was significantly increased exclusively in Il1rn^−/− osteoclasts derived from long bone and jaw BM (n = 6 mice/group). Acid phosphatase 5 (Acp5); nuclear factor of activated T cells 1 (Nfatc1); dendritic cell-specific transmembrane protein (Dcstamp); cathepsin K (Ctsk). All values were normalized for the expression of Beta 2 microglobulin (B2m) as the reference gene. Relative expression is shown (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to WT controls).

Figure 5

Il1rn^−/− osteoclasts show an enhanced mineral dissolution independently of the skeletal site. Absence of IL-1RA increased the in vitro dissolution of the inorganic matrix of bone from all osteoclast precursors irrespective of the isolation site, suggesting that site-specific differences present in vivo can be outweighed in vitro. (A–D) Microphotographs of hydroxyapatite-coated plates of which part is dissolved by osteoclasts derived from various skeletal sites in both wild-type (WT) and Il1rn^−/− mice. Bone marrow (BM) cells from long bone, calvaria, vertebra, and jaw were cultured with 30 ng/mL M-CSF and 20 ng/mL RANKL on hydroxyapatite-coated plates for 8 days. Osteoclasts were stained by TRAcP (in purple) and nuclei were stained by 4’,6-Diamidino-2-Phenylindole (DAPI) (blue). The dissolved area was labeled by asterisks. Scale bar = 100 µm. The dissolved area was quantified and compared between WT and Il1rn^−/− mice for long bone (A), calvaria (B), vertebra (C) and jaw (D) (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001).