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Pathogenic role of basic calcium phosphate crystals in destructive arthropathies.

Ea HK, Chobaz V, Nguyen C, Nasi S, van Lent P, Daudon M, Dessombz A, Bazin D, McCarthy G, Jolles-Haeberli B, Ives A, Van Linthoudt D, So A, Lioté F, Busso N - PLoS ONE (2013)

Bottom Line: BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β.Similarly, treatment with anakinra did not prevent BCP crystal effects.The effects are independent of IL-1 and NLRP3 inflammasome.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR-S 606, Hospital Lariboisière, Paris, France.

ABSTRACT

Background: basic calcium phosphate (BCP) crystals are commonly found in osteoarthritis (OA) and are associated with cartilage destruction. BCP crystals induce in vitro catabolic responses with the production of metalloproteases and inflammatory cytokines such as interleukin-1 (IL-1). In vivo, IL-1 production induced by BCP crystals is both dependant and independent of NLRP3 inflammasome. We aimed to clarify 1/ the role of BCP crystals in cartilage destruction and 2/ the role of IL-1 and NLRP3 inflammasome in cartilage degradation related to BCP crystals.

Methodology principal findings: synovial membranes isolated from OA knees were analysed by alizarin Red and FTIR. Pyrogen free BCP crystals were injected into right knees of WT, NLRP3 -/-, ASC -/-, IL-1α -/- and IL-1β-/- mice and PBS was injected into left knees. To assess the role of IL-1, WT mice were treated by intra-peritoneal injections of anakinra, the IL-1Ra recombinant protein, or PBS. Articular destruction was studied at d4, d17 and d30 assessing synovial inflammation, proteoglycan loss and chondrocyte apoptosis. BCP crystals were frequently found in OA synovial membranes including low grade OA. BCP crystals injected into murine knee joints provoked synovial inflammation characterized by synovial macrophage infiltration that persisted at day 30, cartilage degradation as evidenced by loss of proteoglycan staining by Safranin-O and concomitant expression of VDIPEN epitopes, and increased chondrocyte apoptosis. BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β. Similarly, treatment with anakinra did not prevent BCP crystal effects. In vitro, BCP crystals elicited enhanced transcription of matrix degrading and pro-inflammatory genes in macrophages.

Conclusions significance: intra-articular BCP crystals can elicit synovial inflammation and cartilage degradation suggesting that BCP crystals have a direct pathogenic role in OA. The effects are independent of IL-1 and NLRP3 inflammasome.

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Intra-articular BCP crystals induce synovial inflammation and cartilage proteoglycan loss in mice.OCP crystals (200 µg/20 µl) were injected into right knees of C57BL/6 mice whereas 20 µl PBS was injected into the left knees (A–E). Knees were harvested at different times (day 4, 17 and 30 n = 8 mice per group). Sections were stained with fast green/iron hematoxylin (A) and the degree of inflammation was assessed at the different time points (B). Since the inflammation was very low and similar at all time points in the PBS-injected control knees, only data from PBS-injected knees at day 4 was shown in B. OCP crystal deposition in the synovial membrane was evidenced at day 30 after OCP crystals injection by Von Kossa staining (see arrows) (C). Macrophage, endothelial and PMN cells were detected using antibodies for MAC-2, ICAM, and MPO, respectively, at day 4 after OCP injection (D). Isotype controls allowed the identification of giant cells that had engulfed tissue crystal deposits (*) (D). Ratio of Tc uptake between OCP-injected (n = 8) versus PBS controls was calculated (E). Fast green/iron hematoxylin staining of knees injected with 20 µg/20 µl of HA or OCP crystal at day 4 (F). Results are expressed as mean ± S.E.M with significance being at * p<0.05, ** p<0.01, *** p<0.001.
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pone-0057352-g001: Intra-articular BCP crystals induce synovial inflammation and cartilage proteoglycan loss in mice.OCP crystals (200 µg/20 µl) were injected into right knees of C57BL/6 mice whereas 20 µl PBS was injected into the left knees (A–E). Knees were harvested at different times (day 4, 17 and 30 n = 8 mice per group). Sections were stained with fast green/iron hematoxylin (A) and the degree of inflammation was assessed at the different time points (B). Since the inflammation was very low and similar at all time points in the PBS-injected control knees, only data from PBS-injected knees at day 4 was shown in B. OCP crystal deposition in the synovial membrane was evidenced at day 30 after OCP crystals injection by Von Kossa staining (see arrows) (C). Macrophage, endothelial and PMN cells were detected using antibodies for MAC-2, ICAM, and MPO, respectively, at day 4 after OCP injection (D). Isotype controls allowed the identification of giant cells that had engulfed tissue crystal deposits (*) (D). Ratio of Tc uptake between OCP-injected (n = 8) versus PBS controls was calculated (E). Fast green/iron hematoxylin staining of knees injected with 20 µg/20 µl of HA or OCP crystal at day 4 (F). Results are expressed as mean ± S.E.M with significance being at * p<0.05, ** p<0.01, *** p<0.001.

Mentions: To assess the role of BCP crystals in cartilage degradation, we performed intra-articular injections of OCP crystals into mouse right knees and an equivalent volume of PBS into the contralateral knee. Joints were evaluated up to 30 days, using immuno/histology methods. We observed a persistent, significant increase in the degree of inflammation (up to day 30) as compared to PBS controls (which showed almost no inflammation at all time points examined), with peak inflammation observed at day 4 (Figure 1A and 1B). OCP crystals were still present in the joints 30 days post-injection, and were predominantly found within the synovial membrane as evidenced by Von Kossa staining (Figure 1C). At 6h, neutrophils predominated the inflammatory infiltrate (results not shown). At later times (d4 onwards), neutrophils were no longer abundant but the membranes showed prominent macrophage infiltration and the presence of occasional multinucleated giant cells with internalized crystals (Figure 1D). Almost no neovascularisation was found, as evidenced by ICAM staining (Figure 1D). We also assessed inflammation by 99mTechnetium (Tc) scintigraphy. OCP crystal injection induced a small but significant increase in Tc uptake in the knee joints that occurred as an early transient event that peaked at 24 hours, and returned to normal by 72 hours (Figure 1E). Similar histological features of inflammation were also observed with lower doses of OCP and HA crystals (20 µg) into the mouse knee (Figure 1F).


Pathogenic role of basic calcium phosphate crystals in destructive arthropathies.

Ea HK, Chobaz V, Nguyen C, Nasi S, van Lent P, Daudon M, Dessombz A, Bazin D, McCarthy G, Jolles-Haeberli B, Ives A, Van Linthoudt D, So A, Lioté F, Busso N - PLoS ONE (2013)

Intra-articular BCP crystals induce synovial inflammation and cartilage proteoglycan loss in mice.OCP crystals (200 µg/20 µl) were injected into right knees of C57BL/6 mice whereas 20 µl PBS was injected into the left knees (A–E). Knees were harvested at different times (day 4, 17 and 30 n = 8 mice per group). Sections were stained with fast green/iron hematoxylin (A) and the degree of inflammation was assessed at the different time points (B). Since the inflammation was very low and similar at all time points in the PBS-injected control knees, only data from PBS-injected knees at day 4 was shown in B. OCP crystal deposition in the synovial membrane was evidenced at day 30 after OCP crystals injection by Von Kossa staining (see arrows) (C). Macrophage, endothelial and PMN cells were detected using antibodies for MAC-2, ICAM, and MPO, respectively, at day 4 after OCP injection (D). Isotype controls allowed the identification of giant cells that had engulfed tissue crystal deposits (*) (D). Ratio of Tc uptake between OCP-injected (n = 8) versus PBS controls was calculated (E). Fast green/iron hematoxylin staining of knees injected with 20 µg/20 µl of HA or OCP crystal at day 4 (F). Results are expressed as mean ± S.E.M with significance being at * p<0.05, ** p<0.01, *** p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585350&req=5

pone-0057352-g001: Intra-articular BCP crystals induce synovial inflammation and cartilage proteoglycan loss in mice.OCP crystals (200 µg/20 µl) were injected into right knees of C57BL/6 mice whereas 20 µl PBS was injected into the left knees (A–E). Knees were harvested at different times (day 4, 17 and 30 n = 8 mice per group). Sections were stained with fast green/iron hematoxylin (A) and the degree of inflammation was assessed at the different time points (B). Since the inflammation was very low and similar at all time points in the PBS-injected control knees, only data from PBS-injected knees at day 4 was shown in B. OCP crystal deposition in the synovial membrane was evidenced at day 30 after OCP crystals injection by Von Kossa staining (see arrows) (C). Macrophage, endothelial and PMN cells were detected using antibodies for MAC-2, ICAM, and MPO, respectively, at day 4 after OCP injection (D). Isotype controls allowed the identification of giant cells that had engulfed tissue crystal deposits (*) (D). Ratio of Tc uptake between OCP-injected (n = 8) versus PBS controls was calculated (E). Fast green/iron hematoxylin staining of knees injected with 20 µg/20 µl of HA or OCP crystal at day 4 (F). Results are expressed as mean ± S.E.M with significance being at * p<0.05, ** p<0.01, *** p<0.001.
Mentions: To assess the role of BCP crystals in cartilage degradation, we performed intra-articular injections of OCP crystals into mouse right knees and an equivalent volume of PBS into the contralateral knee. Joints were evaluated up to 30 days, using immuno/histology methods. We observed a persistent, significant increase in the degree of inflammation (up to day 30) as compared to PBS controls (which showed almost no inflammation at all time points examined), with peak inflammation observed at day 4 (Figure 1A and 1B). OCP crystals were still present in the joints 30 days post-injection, and were predominantly found within the synovial membrane as evidenced by Von Kossa staining (Figure 1C). At 6h, neutrophils predominated the inflammatory infiltrate (results not shown). At later times (d4 onwards), neutrophils were no longer abundant but the membranes showed prominent macrophage infiltration and the presence of occasional multinucleated giant cells with internalized crystals (Figure 1D). Almost no neovascularisation was found, as evidenced by ICAM staining (Figure 1D). We also assessed inflammation by 99mTechnetium (Tc) scintigraphy. OCP crystal injection induced a small but significant increase in Tc uptake in the knee joints that occurred as an early transient event that peaked at 24 hours, and returned to normal by 72 hours (Figure 1E). Similar histological features of inflammation were also observed with lower doses of OCP and HA crystals (20 µg) into the mouse knee (Figure 1F).

Bottom Line: BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β.Similarly, treatment with anakinra did not prevent BCP crystal effects.The effects are independent of IL-1 and NLRP3 inflammasome.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR-S 606, Hospital Lariboisière, Paris, France.

ABSTRACT

Background: basic calcium phosphate (BCP) crystals are commonly found in osteoarthritis (OA) and are associated with cartilage destruction. BCP crystals induce in vitro catabolic responses with the production of metalloproteases and inflammatory cytokines such as interleukin-1 (IL-1). In vivo, IL-1 production induced by BCP crystals is both dependant and independent of NLRP3 inflammasome. We aimed to clarify 1/ the role of BCP crystals in cartilage destruction and 2/ the role of IL-1 and NLRP3 inflammasome in cartilage degradation related to BCP crystals.

Methodology principal findings: synovial membranes isolated from OA knees were analysed by alizarin Red and FTIR. Pyrogen free BCP crystals were injected into right knees of WT, NLRP3 -/-, ASC -/-, IL-1α -/- and IL-1β-/- mice and PBS was injected into left knees. To assess the role of IL-1, WT mice were treated by intra-peritoneal injections of anakinra, the IL-1Ra recombinant protein, or PBS. Articular destruction was studied at d4, d17 and d30 assessing synovial inflammation, proteoglycan loss and chondrocyte apoptosis. BCP crystals were frequently found in OA synovial membranes including low grade OA. BCP crystals injected into murine knee joints provoked synovial inflammation characterized by synovial macrophage infiltration that persisted at day 30, cartilage degradation as evidenced by loss of proteoglycan staining by Safranin-O and concomitant expression of VDIPEN epitopes, and increased chondrocyte apoptosis. BCP crystal-induced synovitis was totally independent of IL-1α and IL-1β signalling and no alterations of inflammation were observed in mice deficient for components of the NLRP3-inflammasome, IL-1α or IL-1β. Similarly, treatment with anakinra did not prevent BCP crystal effects. In vitro, BCP crystals elicited enhanced transcription of matrix degrading and pro-inflammatory genes in macrophages.

Conclusions significance: intra-articular BCP crystals can elicit synovial inflammation and cartilage degradation suggesting that BCP crystals have a direct pathogenic role in OA. The effects are independent of IL-1 and NLRP3 inflammasome.

Show MeSH
Related in: MedlinePlus