Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

Abstract

Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We found that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process. These findings provide a better understanding of the role of IL-3 in osteoclastogenesis.

Keywords: APC; BMC; BRC; CMP; Car2; Ctsk; FBS; GAPDH; GM-CSF; GMP; HSC; IL-3; IL-6; Interleukin-3; M-CSF; MMP9; MSC; Osteoclast precursor; Osteoclast progenitor; Osteoclastogenesis; PBS; PE; RANKL; RT-PCR; SCF; SEM; TRAP; allophycocyanin; bone marrow cells; bone remodeling compartment; carbonic anhydrase 2; cathepsin K; common myeloid progenitors; fetal bovine serum; glyceraldehyde 3-phosphate dehydrogenase; granulocyte/macrophage colony stimulating factor; granulocyte/macrophage progenitors; hematopoietic stem cells; interleukin 3; interleukin 6; matrix metalloproteinase 9; mesenchymal stem cells; monocyte/macrophage-colony stimulating factor; phosphate-buffered buffers; phycoerythrin; receptor activator of nuclear factor kappa B ligand; reverse transcription-polymerase chain reaction; scanning electron microscopy; stem cell factor; tartrate resistant acid phosphatase; α-MEM; α-minimal essential medium.

Fig. 1

IL-3 treatment of bone marrow cells generates a population of cells capable of forming osteoclasts. (A) A brief description of the experimental procedure. Bone marrow cells (BMC) were cultured for 24 hours (h) in tissue culture dishes. Nonadherent cells were then moved to new tissue culture dishes and cultured with vehicle (Veh, i.e., PBS) or IL-3 (1ng/ml) for up to 6 days (d). At d 3 and d 6, surviving cells were counted and quantified in panel B. Cells from the 6d cultures were used to perform osteoclast (OC) assays, and the results are shown in panel C. (B) 6×10⁶ cells were added to one 60-mm tissue culture dish and the assay was performed in triplicate. Data are expressed as mean ± S.D. *, p<0.05; **, p<0.01. (C) Different numbers of surviving cells from the 6d culture described in (A) were seeded in 24-well tissue culture plates and cultured with 44ng/ml M-CSF and 100ng/ml RANKL for 5d. The cultures were then stained for TRAP activity. The assay was independently repeated 3 times and a representative area from each condition is shown.

Fig. 2

M-CSF further enhances the osteoclastogenic potential of IL-3-dependent cells and IL-3 inhibits the differentiation of IL-3-dependent progenitors into osteoclasts. (A) A brief description of the experimental procedure. Bone marrow cells (BMC) were cultured for 24 hours (h) in tissue culture dishes. Nonadherent cells were then moved to new tissue culture dishes and cultured with IL-3 for 3 or 6 days (d). Then some of the surviving cells from the 3d and 6d cultures were used to perform osteoclast (OC) assay (OC Assay 1), and remaining cells were continued with 220ng/ml M-CSF for 4 days prior to OC assay (OC Assay 2). (B) OC Assay 1 and OC Assay 2 with cells from 3-day IL-3 treatment. The cells were seeded in 24-well tissue culture plates (2.5×10⁴cells/well) and cultured with 44ng/ml M-CSF (M) and 100ng/ml RANKL (R), or with 44ng/ml M and 100ng/ml R plus 1ng/ml IL-3 for 5d. (C) Quantification of assays in B. Multinucleated TRAP-positive cells (>3nuclei) per representative view area (40 × magnification) was counted. Bars show averages of three replicates ±S.D. **, P<0.01. (D) OC Assay 1 and OC Assay 2 with cells from 6-day IL-3 treatment. The assays were performed as in B. (E) Assays in D were quantified as in C. Bars show averages of three replicates ±S.D. **, P<0.01. The cultures were stained for TRAP activity. The assay was independently repeated 2 times and a representative area from each condition is shown.

Fig. 3

IL-3 promotes the development of osteoclast progenitors. (A) A brief description of the experimental procedure. Bone marrow cells (BMC) were cultured for 24 hours (h) in tissue culture dishes. Nonadherent cells were then moved to new tissue culture dishes and cultured with IL-3 for 0, 3 or 6 days. Then, some of the surviving cells were used to perform flow cytometric assays to determine surface expression of CD11b and CD115 (c-Fms) (Flow a, b and c), and remaining cells were continued with 220ng/ml M-CSF for 4 days prior to flow cytometric assays (Flow d, e and f). (B) Results of flow cytometric assays (a, b, c, d, e and f) as described in (A). The assay was independently repeated 2 times and similar results were obtained. One set of the data is shown.

Fig. 4

IL-3-dependent cells can form functional osteoclasts and a model for the role of IL-3 in osteoclast biology. (A) Bone marrow cells (BMC) were cultured for 24 hours (h) in tissue culture dishes. Nonadherent cells were then moved to new tissue culture dishes and cultured with IL-3 for 5 or 6 days (d). 1×10⁵ of surviving cells were seeded on bone slices in 24-well tissue culture plates and cultured with 44ng/ml M-CSF alone or with 44ng/ml M-CSF and 100ng/ml RANKL for 9d to perform bone resorption assays. (B) BMC were cultured for 24h in tissue culture dishes. Nonadherent cells were then moved to new tissue culture dishes and cultured with IL-3 for 5 or 6d, followed by M-CSF treatment (44ng/ml) for 4d. Then 5×10⁴ of cells were seeded on bone slices in 24-well tissue culture plates and cultured with 44ng/ml M-CSF alone or with 44ng/ml M-CSF and 100ng/ml RANKL for 9d to perform bone resorption assays. These assays were independently repeated 2 times and a representative area is shown. (C) BMC were cultured for 24h in tissue culture dishes. Nonadherent cells were then moved to new tissue culture dishes and cultured with IL-3 for 6d. Then, some of the surviving cells were cultured with 44ng/ml M-CSF alone (control) or with 44ng/ml M-CSF and 100ng/ml RANKL for 5d to form osteoclasts. The remaining cells were continued with M-CSF (44ng/ml) for 5d, followed by treatment with M-CSF alone or M-CSF (44ng/ml) and RANKL (100ng/ml) for 5d. The expression of representative osteoclasts genes (MMP9, Ctsk, TRAP and Car2) was assessed by semi-quantitative RT-PCR. GAPDH was used as control. The assay was independently repeated 2 times. (D) A model for the role of IL-3 in osteoclastogenesis in the context of normal bone remodeling.