Factors secreted by monosodium urate crystal-stimulated macrophages promote a proinflammatory state in osteoblasts: a potential indirect mechanism of bone erosion in gout

Abstract

Background: Tophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts.

Methods: Conditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay.

Results: In MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE₂ and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE₂ secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs.

Conclusions: Factors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

Keywords: Bone; Gout; Osteoblast; Tophus; Urate.

Fig. 1

Conditioned medium from MSU crystal-stimulated RAW264.7 macrophages—proinflammatory factors and effect on MC3T3-E1 cell viability. RAW264.7 macrophages were cultured with MSU crystals for 24 h. A The concentrations of proinflammatory mediators secreted from MSU crystal-stimulated RAW264.7 cells, measured by ELISA. B Conditioned media preparations were added to MC3T3-E1 cells at 40% of the final culture volume. MC3T3-E1 cell viability was determined by the alamarBlue® assay. Means (SEM) of data pooled from three biological repeats are presented. Data were analyzed by one-way ANOVA with post hoc Dunnett’s test. **p < 0.01 and ***p < 0.001 versus control conditioned medium. MSU CM, concentrations of MSU crystals used to prepare conditioned medium from RAW264.7 cells

Fig. 2

Effects of conditioned medium from MSU crystal-stimulated RAW264.7 macrophages on osteoblastic differentiation of MC3T3-E1 cells. RAW264.7 macrophages were cultured with the indicated concentrations of MSU crystals for 24 h. Conditioned medium from the RAW264.7 cultures was added to MC3T3-E1 cells at 40% of the final volume. A Expression of osteoblast marker genes determined by real-time PCR. Means (SEM) of data pooled from three or more biological repeats are presented. Data were analyzed by two-way ANOVA with post hoc Dunnett’s test. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control conditioned medium at that time point. B MC3T3-E1 cells were cultured for 3 weeks in an osteogenic medium supplemented with 40% MSU crystal-stimulated conditioned medium from RAW264.7 cells. The representative photomicrographs show mineralized areas (black) and cells (yellow) in cultures stained with von Kossa stain. The experiment was repeated twice with similar results. MSU CM, concentrations of MSU crystals (mg/mL) used to prepare conditioned medium from RAW264.7 cells

Fig. 3

Effects of conditioned medium from MSU crystal-stimulated RAW264.7 macrophages on factor secretion from MC3T3-E1 cells. RAW264.7 macrophages were cultured with the indicated concentrations of MSU crystals for 24 h. Conditioned medium from the RAW264.7 cultures was added to MC3T3-E1 cells at 40% of the final volume. The graphs on the left present gene expression determined by real-time PCR; the graphs on the right present concentrations of secreted factors determined by ELISA. A Gene expression and secretion of OPG. B Gene expression and secretion of IL-6. C Gene expression of Ptgs2, encoding the COX-2 enzyme, and secretion of PGE₂. Means (SEM) of data pooled from three or more biological repeats are presented. Data were analyzed by two-way ANOVA with post hoc Dunnett’s test. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control conditioned medium at that time point; MSU CM, concentrations of MSU crystals used to prepare conditioned medium from RAW264.7 cells

Fig. 4

Secretion of proinflammatory mediators from THP-1 monocytes stimulated with MSU crystals. THP-1 monocytes were cultured with MSU crystals for 20 h for preparation of MSU crystal-stimulated and control conditioned media. Concentrations of proinflammatory mediators were determined by ELISA. Means (SEM) of data pooled from three biological repeats are presented. Data were analyzed by one-way ANOVA with post hoc Dunnett’s test. *p < 0.05 and **p < 0.01, versus control without MSU crystals

Fig. 5

Effects of conditioned medium from MSU crystal-stimulated THP-1 monocytes on gene expression in HOBs. THP-1 monocytes were cultured with MSU crystals for 20 h. Conditioned medium from THP-1 cultures was added to HOB cells at 40% of the final volume. Expression of osteoblast marker genes (A) and genes encoding proinflammatory mediators (B) was determined by real-time PCR. Means (SEM) of data pooled from three or more biological repeats are presented. Data were analyzed by two-way ANOVA with post hoc Dunnett’s test. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control conditioned medium at that time point. MSU CM, concentrations of MSU [mg/mL] used to prepare conditioned medium from THP-1 cells

Fig. 6

Effects of conditioned medium from MSU crystal-stimulated THP-1 monocytes on factor secretion from HOBs. THP-1 monocytes were cultured with MSU crystals for 20 h. Conditioned medium from THP-1 cultures was added to HOB cells at 40% of the final volume. The concentrations of IL-6, PGE₂, and OPG secreted from HOBs were measured by ELISA. Means (SEM) of data pooled from three or more biological repeats are presented. Data were analyzed by two-way ANOVA with post hoc Dunnett’s test. *p < 0.05, **p < 0.01, and ***p < 0.001 versus control conditioned medium at that time point. MSU CM, concentrations of MSU [mg/mL] used to prepare conditioned medium from THP-1 cells

Fig. 7

Effects of COX-2 inhibition on HOB response to conditioned medium from MSU crystal-stimulated THP-1 monocytes. Conditioned medium was collected from THP-1 monocytes cultured with or without 0.5 mg/mL MSU crystals for 20 h. The COX-2-selective inhibitor SC-236 was added to HOB cells for 1 h prior to the addition of conditioned medium at 40% of the final volume. A Gene expression analyzed by real-time PCR. B Secretion of PGE₂ and IL-6 from HOBs. Data shown are pooled from three biological repeats and are presented as mean (SEM). Data were analyzed by two-way ANOVA with post hoc Sidak’s test. **p < 0.01 and ***p < 0.001 versus control conditioned medium at that time point. Cnt CM and MSU CM, conditioned medium from THP-1 cells incubated without MSU and with 0.5 mg/mL MSU, respectively