American Society of Hematology

Figure 1.

$IgG isolated from myeloma patients with BD induce osteoclastogenesis. (A-B) Immunoglobulins isolated from patients with BD (BD) or without BD (no BD) or from healthy control subjects were added to preosteoclasts derived from CD14+ cells isolated from PBMCs of healthy donors. After 2 to 4 days, cells were TRAP stained, and TRAP-positive cells with ≥3 nuclei were counted as osteoclasts. Cells were incubated with RANKL (10 ng/mL) as a positive control and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) only as a negative control In panel A, IgG from IgG-myeloma patients (the M component) (5 μg/mL) with (n = 16) and without (n = 10) BD, were used; in panel B, IgGs (5 μg/mL) isolated from the serum of IgA, IgD or nonsecretory patients (uninvolved immunoglobulins) with (n = 17) and without (n = 10) BD were used. Numbers of osteoclasts are presented relative to the RANKL positive control. **P < .01 using ANOVA followed by the Šidák multiple comparisons test. (C) Relative increase in osteoclasts after immunoglobulins (5 μg/mL, n = 4 patients with BD) were added to preosteoclasts that were previously blocked with neutralizing anti-FcγR antibodies. The number of TRAP-positive cells was counted after 1 to 3 days. **P < .01 using mixed model ANOVA followed by Tukey’s multiple comparisons test. (D) Preosteoclasts were stimulated with serum from myeloma patients (n = 4) with and without FcγRII antibody. The number of osteoclasts after 1 to 2 days was evaluated by using TRAP staining. ***P < .001, ****P < .0001 using ANOVA followed by the Šidák multiple comparisons test. (E-F) Isolated immunoglobulins (n = 3 per group) were separated based on size on a Sepharose 6 size-exclusion column, and the different fractions were added to preosteoclasts. The number of mature osteoclasts after 1 to 3 days was evaluated by using TRAP staining. **P < .01 using repeated measurements one-way ANOVA followed by Dunnett’s multiple comparisons test. (G) Amount of nanoparticles over 100 nm (range, 50-200 nm) in size for healthy control subjects (n = 11), no-BD patients (n = 18), and BD patients (n = 20). Error bars represent the standard error of the mean. *P < .05 using ANOVA followed by the Šidák multiple comparisons test. (H) Mass spectrometric analyses of IgG derived from patients with (n = 11) or without (n = 8) BD. P values corrected for multiple comparisons are shown on the y-axis. ns, not significant.$

IgG isolated from myeloma patients with BD induce osteoclastogenesis. (A-B) Immunoglobulins isolated from patients with BD (BD) or without BD (no BD) or from healthy control subjects were added to preosteoclasts derived from CD14⁺ cells isolated from PBMCs of healthy donors. After 2 to 4 days, cells were TRAP stained, and TRAP-positive cells with ≥3 nuclei were counted as osteoclasts. Cells were incubated with RANKL (10 ng/mL) as a positive control and macrophage colony-stimulating factor (M-CSF; 30 ng/mL) only as a negative control In panel A, IgG from IgG-myeloma patients (the M component) (5 μg/mL) with (n = 16) and without (n = 10) BD, were used; in panel B, IgGs (5 μg/mL) isolated from the serum of IgA, IgD or nonsecretory patients (uninvolved immunoglobulins) with (n = 17) and without (n = 10) BD were used. Numbers of osteoclasts are presented relative to the RANKL positive control. **P < .01 using ANOVA followed by the Šidák multiple comparisons test. (C) Relative increase in osteoclasts after immunoglobulins (5 μg/mL, n = 4 patients with BD) were added to preosteoclasts that were previously blocked with neutralizing anti-FcγR antibodies. The number of TRAP-positive cells was counted after 1 to 3 days. **P < .01 using mixed model ANOVA followed by Tukey’s multiple comparisons test. (D) Preosteoclasts were stimulated with serum from myeloma patients (n = 4) with and without FcγRII antibody. The number of osteoclasts after 1 to 2 days was evaluated by using TRAP staining. ***P < .001, ****P < .0001 using ANOVA followed by the Šidák multiple comparisons test. (E-F) Isolated immunoglobulins (n = 3 per group) were separated based on size on a Sepharose 6 size-exclusion column, and the different fractions were added to preosteoclasts. The number of mature osteoclasts after 1 to 3 days was evaluated by using TRAP staining. **P < .01 using repeated measurements one-way ANOVA followed by Dunnett’s multiple comparisons test. (G) Amount of nanoparticles over 100 nm (range, 50-200 nm) in size for healthy control subjects (n = 11), no-BD patients (n = 18), and BD patients (n = 20). Error bars represent the standard error of the mean. *P < .05 using ANOVA followed by the Šidák multiple comparisons test. (H) Mass spectrometric analyses of IgG derived from patients with (n = 11) or without (n = 8) BD. P values corrected for multiple comparisons are shown on the y-axis. ns, not significant.

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