Figure 1.
IL-32-treatment induced a dose dependent differentiation of osteoclast precursors.
(A) TRAcP and VNR positive staining of newly-formed multinucleated cells differentiated from human PBMCs in the presence of 25 ng/ml of M-CSF±100 ng/ml of IL-32. Black bar represents 50 µm. (B) The number of newly-generated TRAcP positive multinucleated cells is dependent on the concentration of IL-32. (C) Size of newly-generated multinucleated cells is not increased at doses higher than 50 ng/ml of IL-32. (D) The number of nuclei per newly-generated multinucleated cells is significantly increased in a dose-dependent manner with the optimal response at 100 ng/ml of IL-32. P values represent the statistical significances between each group using Mann-Whitney test.
Figure 2.
Comparison of the effects of IL-32 and soluble RANKL on the differentiation and maturation of osteoclast precursors.
(A) IL-32 was capable of inducing the expression of specific osteoclastic markers as illustrated. PBMCs were exposed to the factors for 72 h prior to Western blot analysis. β-actin was used as an internal control for gel loading. (B) Number of newly formed osteoclasts. Although the number of multinucleated cells formed in response to IL-32 was significantly increased compared to M-CSF-treated cultures, this parameter was significantly decreased when compared with the soluble RANKL-treated cultures. The combined treatment of PBMCs with sRANKL/IL-32 resulted in a highly significant increase in the number of newly formed osteoclasts as compared to either of those factors alone. (C) Size of newly generated osteoclasts. IL-32 increased the size of multinucleated cells compared to sRANKL cultures but was unable to increase the size of the newly formed osteoclasts when combined with sRANKL. (D) Number of nuclei per osteoclast. Compared to sRANKL-treated cultures, multinucleated cells formed in response to IL-32 exhibited more nuclei per cell indicating that the process of cell fusion may have been facilitated. P values represent the statistical significances between each group using Mann-Whitney test.
Figure 3.
Effects of IL-32 on signalling pathways involved during osteoclastogenesis.
Phosphorylation of ERK1/2, JNK, Akt and IkB-α in human PBMCs after 15 minutes exposure to RANKL or IL-32. β-actin was used an internal control of gel loading.
Figure 4.
IL-32 was unable to induce the maturation of osteoclasts in vitro.
(A) F-actin staining of multinucleated cells cultured with M-CSF and sRANKL, M-CSF and IL-32 and M-CSF, sRANKL and IL-32. White bar represents 80 µm. IL-32-treated cultures did not exhibit evidence of F-actin ring formation indicating the lack of anchorage of osteoclasts on dentine surface. (B) Evidence of lacunar resorption on dentine slices cultured with M-CSF and sRANKL, M-CSF and IL-32 and M-CSF, sRANKL and IL-32. Black bar represents 250 µm. Multinucleated cells formed in response to IL-32 were incapable of lacunar resorption. (C) Treatment of PBMCs with M-CSF and IL-32 failed to induce the maturation of newly-formed osteoclasts as evident by the lack of lacunar resorption on dentine slices. However, sRANKL-treatment of PBMCs resulted in a 43.3±9.8% percentage area resorption. Combined treatment of sRANKL and IL-32 resulted in a significant decrease of 2.6 fold in percentage area resorption compared to sRANKL alone. (D) The mean diameter of the lacunar pits formed in response to sRANKL and IL-32 treatment was significantly decreased compared to those generated in response to sRANKL alone. P values represent the statistical significances between each group using Mann-Whitney test.
Figure 5.
Effect of OPG treatment on sRANKL- and IL-32-mediated osteoclastogenesis.
(A) Number of newly-generated osteoclasts in the presence of OPG. OPG significantly decreased the number of osteoclasts generated with sRANKL but OPG inhibition had only a partial effect in PBMC cultures treated with IL-32. (B) Size of the newly-generated osteoclasts. OPG treatment resulted in a decrease in the size of the newly-formed osteoclasts generated with sRANKL and/or IL-32. (C) Number of nuclei per osteoclast. OPG treatment significantly increased the number of nuclei per osteoclast in sRANKL-treated cultures and markedly abrogated this parameter in IL-32-treated cultures. P values represent the statistical significances between each group using Mann-Whitney test.
Figure 6.
Effects of OPG on IL-32/sRANKL-mediated osteoclastogenesis.
(A) The number of multinucleated TRAcP positive cells formed in response to IL-32/sRANKL treatment was significantly reduced in the presence of excess OPG. (B) The size of the newly-formed TRAcP positive multinucleated cells was unaffected by the presence of OPG in IL-32/sRANKL-treated cultures. (C) The number of nuclei noted in TRAcP positive cells formed in response to IL-32/sRANKL treatment was not affected by the OPG treatment. (D) The percentage area lacunar resorption was completely abolished when PBMCs were treated with IL-32/sRANKL and excess OPG. P values represent the statistical significances between each group using Mann-Whitney test.
Figure 7.
Release of soluble mediators in response to treatment of PBMCs with IL-32 after 24 hrs and 48 hrs.
(A) The stimulation of PBMCs with 100 ng/ml of IL-32 induced the release of TNF-α, IL-6, LIGHT, MIP-1α, VEGF. RANKL was undetectable in the supernatant. (B) Levels of IFN-γ and IL-4. The minimum dose detectable was 1.6 pg/ml for TNF-α, 0.70 pg/ml for IL-6, 5.5 pg/ml for LIGHT, 10 pg/ml for MIP-1α, 5 pg/ml for VEGF, 5 pg/ml for RANKL, 8.0 pg/ml for IFN-γ and 10 pg/ml for IL-4. Unstimulated PBMCs were unable to release any of these soluble mediators.
Figure 8.
Schematic representation of downstream pathways activated by RANKL or IL-32 treatment.
The discrepancy observed between IL-32 and RANKL signalling pathways (i.e. increased ERK1/2 and Akt activation by IL-32) may lead to the activation of different downstream targets which in turn could contribute to the inability of cells to express F-actin ring and resorb in response to IL-32.