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Basic and translational research
CONCISE REPORT
Interactions between tenocytes and monosodium urate monohydrate crystals: implications for tendon involvement in gout Ashika Chhana,1 Karen E Callon,1 Michael Dray,2 Bregina Pool,1 Dorit Naot,1 Greg D Gamble,1 Brendan Coleman,3 Geraldine McCarthy,4 Fiona M McQueen,5 Jillian Cornish,1 Nicola Dalbeth1 Handling editor Tore K Kvien ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2013-204657). 1
Bone & Joint Research Group, Department of Medicine, University of Auckland, Auckland, New Zealand 2 Department of Histology, Waikato Hospital, Hamilton, New Zealand 3 Department of Orthopaedic Surgery, Middlemore Hospital, Auckland, New Zealand 4 Department of Rheumatology, Mater Misericordiae University Hospital, Dublin, Ireland 5 Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand Correspondence to Dr Nicola Dalbeth, Bone & Joint Research Group, Department of Medicine, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Rd, Grafton, Auckland 1023, New Zealand; [email protected] Received 24 September 2013 Revised 28 February 2014 Accepted 21 March 2014 Published Online First 7 April 2014
ABSTRACT Objectives Advanced imaging studies have demonstrated that urate deposition in periarticular structures, such as tendons, is common in gout. The aim of this study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on tenocyte viability and function. Methods The histological appearance of tendons in joints affected by advanced gout was examined using light microscopy. In vitro, colorimetric assays and flow cytometry were used to assess cell viability in primary rat and primary human tenocytes cultured with MSU crystals. Real-time PCR was used to determine changes in the relative mRNA expression levels of tendon-related genes, and Sirius red staining was used to measure changes in collagen deposition in primary rat tenocytes. Results In joint samples from patients with gout, MSU crystals were identified within the tendon, adjacent to and invading into tendon, and at the enthesis. MSU crystals reduced tenocyte viability in a dose-dependent manner. MSU crystals decreased the mRNA expression of tendon collagens, matrix proteins and degradative enzymes and reduced collagen protein deposition by tenocytes. Conclusions These data indicate that MSU crystals directly interact with tenocytes to reduce cell viability and function. These interactions may contribute to tendon damage in people with advanced gout.
Histology of joint samples from patients with gout Joint samples were obtained from three patients with gout undergoing orthopaedic surgery, and from two cadaveric donors with microscopically proven gout. Paraffin-embedded demineralised slides were prepared as previously described.7 Sections were stained with H&E and examined using polarising light microscopy. The samples were assessed by a musculoskeletal histopathologist (MD) and immunohistochemistry as previously described8 (see online supplementary text).
MSU crystal synthesis Endotoxin-free MSU crystals were prepared by recrystallisation from uric acid (Sigma–Aldrich, St Louis, Missouri, USA) as previously described.9
Tenocyte cell culture Primary rat tenocytes were isolated from tendon fascicles from Wistar rat tails, and cultures of primary human tenocytes were prepared from biceps tendons, obtained from patients undergoing orthopaedic surgery (see online supplementary text). Tendon was chopped and incubated at 37°C with 0.1% collagenase and dispase in 10% fetal bovine serum (FBS)/ Dulbecco’s modified eagle medium : F-12 (DMEM : F-12) until all tissue had been digested. Cells were seeded in 75 cm2 flasks (Corning, Lowell, Massachusetts, USA) with 10% FBS/DMEM : F-12. Cultures were maintained at 37°C with 5% CO2.
INTRODUCTION
Colorimetric assays for cell viability
Imaging studies have demonstrated monosodium urate monohydrate (MSU) crystals and tophi are located within and around tendons in gout.1–4 Tendon rupture in the presence of tophaceous material has also been reported.5 6 To date, the effects of MSU crystals on tenocytes, the main cell found in tendons, has not been described. The aim of this study was to investigate the effects of MSU crystals on tenocyte viability and function.
Tenocytes were seeded into 24-well plates (Greiner Bio-One, Frickenhausen) at 25 000 cells/well in 5% FBS/DMEM : F-12. The following day, media were changed to 1% FBS/DMEM : F-12 and MSU crystals were added for 24 h. MTT, alamarBlue and lactate dehydrogenase (LDH) assays were used to determine changes in cell viability10 11 (see online supplementary text).
Flow cytometry MATERIALS AND METHODS Ethical approvals To cite: Chhana A, Callon KE, Dray M, et al. Ann Rheum Dis 2014;73:1737–1741.
Human sample collection was approved by the Northern Regional ethics committee, and all patients provided written informed consent. All protocols involving animals were approved by the University of Auckland animal ethics committee.
Chhana A, et al. Ann Rheum Dis 2014;73:1737–1741. doi:10.1136/annrheumdis-2013-204657
Primary rat tenocytes were seeded into 24-well plates at 40 000 cells/well in 5% FBS/DMEM : F-12. The next day, media were changed to 1% FBS/DMEM : F-12, and MSU crystals were added for various times. Cells were stained for fluorescein isothiocyanate (FITC)-labelled Annexin V and propidium iodide and analysed by flow cytometry10 11 (see online supplementary text). 1737
Downloaded from http://ard.bmj.com/ on February 25, 2015 - Published by group.bmj.com
Basic and translational research Quantitative real-time PCR Primary rat tenocytes were cultured as described for the viability assays. Purification of total cellular RNA, synthesis of cDNA and real-time PCR was performed as previously described.7 18S rRNA and GAPDH endogenous controls were used to correct for variations in cell numbers between samples (see online supplementary text).
Collagen deposition assay Primary rat tenocytes were seeded into 24-well plates at 75 000 cells/well in 5% FBS/DMEM : F-12. After 2 days, fresh media and MSU crystals were added for 3 days. Cells were then stained with Sirius red11 (see online supplementary text).
analysis of variance with posthoc Dunnett’s and Bonferroni’s multiple comparison tests.
RESULTS MSU crystals are present in tendons of patients with gout In joint samples from patients with gout, MSU crystals and tophaceous material were observed within tendons, adjacent to and invading into tendon, and at the enthesis (figure 1A–C). The inflammatory infiltrate adjacent to and surrounding MSU crystal deposits was predominately histiocytic, including CD68 multinucleated giant cells and macrophages, with scattered lymphocytes including occasional B-cell aggregates (see online supplementary figure S1). Analysis of tendon structure demonstrated that collagen fibrils were disorganised and disrupted at sites of MSU crystal deposition.
Statistical analysis Data were analysed using SAS Software (SAS Institute, Cary, North Carolina, USA) and GraphPad Prism Software (GraphPad Software, San Diego, California, USA). For all experiments, data were pooled from 3 to 5 biological repeats using different rat or patient samples. Data were analysed using one-way or two-way
MSU crystals reduce tenocyte viability in a dose-dependent manner MSU crystals reduced primary rat and primary human tenocyte viability after 24 h of culture in a dose-dependent manner in the MTT assay (figure 2A,B). This result was confirmed in primary
Figure 1 Tendon involvement in joint samples from patients with advanced gout. Photomicrographs of joint samples from two patients, viewed by light microscopy showing (A) tophi within an index finger extensor tendon (top: H&E; middle: eosin only with a polarising filter; bottom: H&E with a polarising filter and red compensator); (B) monosodium urate monohydrate (MSU) crystals adjacent to and invading the index finger extensor tendon (H&E); and (C) tophaceous material at the enthesis (H&E) in an index finger flexor tendon. Scale bar indicates 150 mm. *Indicates the crystalline core of the tophus.
1738
Chhana A, et al. Ann Rheum Dis 2014;73:1737–1741. doi:10.1136/annrheumdis-2013-204657
Downloaded from http://ard.bmj.com/ on February 25, 2015 - Published by group.bmj.com
Basic and translational research rat tenocytes using the alamarBlue assay and the LDH assay, which showed increased tenocyte lysis following culture with MSU crystals for 24 h; there was also a trend for increased LDH release 10 min after the addition of MSU crystals (see online supplementary figures S2A,B). The effects on tenocyte viability were not altered with different MSU crystal lengths (see online supplementary figure S2C), and were not blocked by the addition of increased serum concentrations to cultures (see online supplementary figure S2D). The inhibitory effect was specific to the crystalline form of urate, as soluble uric acid did not reduce tenocyte viability (see online supplementary figure
S2E). A similar effect on viability was observed with calcium pyrophosphate dihydrate crystals, but no effect was observed with basic calcium phosphate or aluminium crystals (see online supplementary figure S2F). Flow cytometry assays demonstrated no significant change in the percentage of early apoptotic cells following culture with various concentrations of MSU crystals for 24 h, although there was a trend for increased apoptosis with the higher concentrations of MSU crystals (figure 2C). When examined over shorter time periods, cell death occurred rapidly without evidence of apoptosis (figure 2D). Furthermore, the addition of a pan-caspase inhibitor to cultures did not alter
Figure 2 Monosodium urate monohydrate (MSU) crystals reduce tenocyte viability in a dose-dependent manner but do not significantly alter levels of tenocyte apoptosis. (A) Primary rat tenocytes and (B) primary human tenocytes were cultured with MSU crystals, and cell viability was assessed after 24 h using the MTT assay. Data shown are mean (SEM), one-way analysis of variance (ANOVA) ( p
Basic and translational research
CONCISE REPORT
Interactions between tenocytes and monosodium urate monohydrate crystals: implications for tendon involvement in gout Ashika Chhana,1 Karen E Callon,1 Michael Dray,2 Bregina Pool,1 Dorit Naot,1 Greg D Gamble,1 Brendan Coleman,3 Geraldine McCarthy,4 Fiona M McQueen,5 Jillian Cornish,1 Nicola Dalbeth1 Handling editor Tore K Kvien ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2013-204657). 1
Bone & Joint Research Group, Department of Medicine, University of Auckland, Auckland, New Zealand 2 Department of Histology, Waikato Hospital, Hamilton, New Zealand 3 Department of Orthopaedic Surgery, Middlemore Hospital, Auckland, New Zealand 4 Department of Rheumatology, Mater Misericordiae University Hospital, Dublin, Ireland 5 Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand Correspondence to Dr Nicola Dalbeth, Bone & Joint Research Group, Department of Medicine, Faculty of Medical and Health Sciences, University of Auckland, 85 Park Rd, Grafton, Auckland 1023, New Zealand; [email protected] Received 24 September 2013 Revised 28 February 2014 Accepted 21 March 2014 Published Online First 7 April 2014
ABSTRACT Objectives Advanced imaging studies have demonstrated that urate deposition in periarticular structures, such as tendons, is common in gout. The aim of this study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on tenocyte viability and function. Methods The histological appearance of tendons in joints affected by advanced gout was examined using light microscopy. In vitro, colorimetric assays and flow cytometry were used to assess cell viability in primary rat and primary human tenocytes cultured with MSU crystals. Real-time PCR was used to determine changes in the relative mRNA expression levels of tendon-related genes, and Sirius red staining was used to measure changes in collagen deposition in primary rat tenocytes. Results In joint samples from patients with gout, MSU crystals were identified within the tendon, adjacent to and invading into tendon, and at the enthesis. MSU crystals reduced tenocyte viability in a dose-dependent manner. MSU crystals decreased the mRNA expression of tendon collagens, matrix proteins and degradative enzymes and reduced collagen protein deposition by tenocytes. Conclusions These data indicate that MSU crystals directly interact with tenocytes to reduce cell viability and function. These interactions may contribute to tendon damage in people with advanced gout.
Histology of joint samples from patients with gout Joint samples were obtained from three patients with gout undergoing orthopaedic surgery, and from two cadaveric donors with microscopically proven gout. Paraffin-embedded demineralised slides were prepared as previously described.7 Sections were stained with H&E and examined using polarising light microscopy. The samples were assessed by a musculoskeletal histopathologist (MD) and immunohistochemistry as previously described8 (see online supplementary text).
MSU crystal synthesis Endotoxin-free MSU crystals were prepared by recrystallisation from uric acid (Sigma–Aldrich, St Louis, Missouri, USA) as previously described.9
Tenocyte cell culture Primary rat tenocytes were isolated from tendon fascicles from Wistar rat tails, and cultures of primary human tenocytes were prepared from biceps tendons, obtained from patients undergoing orthopaedic surgery (see online supplementary text). Tendon was chopped and incubated at 37°C with 0.1% collagenase and dispase in 10% fetal bovine serum (FBS)/ Dulbecco’s modified eagle medium : F-12 (DMEM : F-12) until all tissue had been digested. Cells were seeded in 75 cm2 flasks (Corning, Lowell, Massachusetts, USA) with 10% FBS/DMEM : F-12. Cultures were maintained at 37°C with 5% CO2.
INTRODUCTION
Colorimetric assays for cell viability
Imaging studies have demonstrated monosodium urate monohydrate (MSU) crystals and tophi are located within and around tendons in gout.1–4 Tendon rupture in the presence of tophaceous material has also been reported.5 6 To date, the effects of MSU crystals on tenocytes, the main cell found in tendons, has not been described. The aim of this study was to investigate the effects of MSU crystals on tenocyte viability and function.
Tenocytes were seeded into 24-well plates (Greiner Bio-One, Frickenhausen) at 25 000 cells/well in 5% FBS/DMEM : F-12. The following day, media were changed to 1% FBS/DMEM : F-12 and MSU crystals were added for 24 h. MTT, alamarBlue and lactate dehydrogenase (LDH) assays were used to determine changes in cell viability10 11 (see online supplementary text).
Flow cytometry MATERIALS AND METHODS Ethical approvals To cite: Chhana A, Callon KE, Dray M, et al. Ann Rheum Dis 2014;73:1737–1741.
Human sample collection was approved by the Northern Regional ethics committee, and all patients provided written informed consent. All protocols involving animals were approved by the University of Auckland animal ethics committee.
Chhana A, et al. Ann Rheum Dis 2014;73:1737–1741. doi:10.1136/annrheumdis-2013-204657
Primary rat tenocytes were seeded into 24-well plates at 40 000 cells/well in 5% FBS/DMEM : F-12. The next day, media were changed to 1% FBS/DMEM : F-12, and MSU crystals were added for various times. Cells were stained for fluorescein isothiocyanate (FITC)-labelled Annexin V and propidium iodide and analysed by flow cytometry10 11 (see online supplementary text). 1737
Downloaded from http://ard.bmj.com/ on February 25, 2015 - Published by group.bmj.com
Basic and translational research Quantitative real-time PCR Primary rat tenocytes were cultured as described for the viability assays. Purification of total cellular RNA, synthesis of cDNA and real-time PCR was performed as previously described.7 18S rRNA and GAPDH endogenous controls were used to correct for variations in cell numbers between samples (see online supplementary text).
Collagen deposition assay Primary rat tenocytes were seeded into 24-well plates at 75 000 cells/well in 5% FBS/DMEM : F-12. After 2 days, fresh media and MSU crystals were added for 3 days. Cells were then stained with Sirius red11 (see online supplementary text).
analysis of variance with posthoc Dunnett’s and Bonferroni’s multiple comparison tests.
RESULTS MSU crystals are present in tendons of patients with gout In joint samples from patients with gout, MSU crystals and tophaceous material were observed within tendons, adjacent to and invading into tendon, and at the enthesis (figure 1A–C). The inflammatory infiltrate adjacent to and surrounding MSU crystal deposits was predominately histiocytic, including CD68 multinucleated giant cells and macrophages, with scattered lymphocytes including occasional B-cell aggregates (see online supplementary figure S1). Analysis of tendon structure demonstrated that collagen fibrils were disorganised and disrupted at sites of MSU crystal deposition.
Statistical analysis Data were analysed using SAS Software (SAS Institute, Cary, North Carolina, USA) and GraphPad Prism Software (GraphPad Software, San Diego, California, USA). For all experiments, data were pooled from 3 to 5 biological repeats using different rat or patient samples. Data were analysed using one-way or two-way
MSU crystals reduce tenocyte viability in a dose-dependent manner MSU crystals reduced primary rat and primary human tenocyte viability after 24 h of culture in a dose-dependent manner in the MTT assay (figure 2A,B). This result was confirmed in primary
Figure 1 Tendon involvement in joint samples from patients with advanced gout. Photomicrographs of joint samples from two patients, viewed by light microscopy showing (A) tophi within an index finger extensor tendon (top: H&E; middle: eosin only with a polarising filter; bottom: H&E with a polarising filter and red compensator); (B) monosodium urate monohydrate (MSU) crystals adjacent to and invading the index finger extensor tendon (H&E); and (C) tophaceous material at the enthesis (H&E) in an index finger flexor tendon. Scale bar indicates 150 mm. *Indicates the crystalline core of the tophus.
1738
Chhana A, et al. Ann Rheum Dis 2014;73:1737–1741. doi:10.1136/annrheumdis-2013-204657
Downloaded from http://ard.bmj.com/ on February 25, 2015 - Published by group.bmj.com
Basic and translational research rat tenocytes using the alamarBlue assay and the LDH assay, which showed increased tenocyte lysis following culture with MSU crystals for 24 h; there was also a trend for increased LDH release 10 min after the addition of MSU crystals (see online supplementary figures S2A,B). The effects on tenocyte viability were not altered with different MSU crystal lengths (see online supplementary figure S2C), and were not blocked by the addition of increased serum concentrations to cultures (see online supplementary figure S2D). The inhibitory effect was specific to the crystalline form of urate, as soluble uric acid did not reduce tenocyte viability (see online supplementary figure
S2E). A similar effect on viability was observed with calcium pyrophosphate dihydrate crystals, but no effect was observed with basic calcium phosphate or aluminium crystals (see online supplementary figure S2F). Flow cytometry assays demonstrated no significant change in the percentage of early apoptotic cells following culture with various concentrations of MSU crystals for 24 h, although there was a trend for increased apoptosis with the higher concentrations of MSU crystals (figure 2C). When examined over shorter time periods, cell death occurred rapidly without evidence of apoptosis (figure 2D). Furthermore, the addition of a pan-caspase inhibitor to cultures did not alter
Figure 2 Monosodium urate monohydrate (MSU) crystals reduce tenocyte viability in a dose-dependent manner but do not significantly alter levels of tenocyte apoptosis. (A) Primary rat tenocytes and (B) primary human tenocytes were cultured with MSU crystals, and cell viability was assessed after 24 h using the MTT assay. Data shown are mean (SEM), one-way analysis of variance (ANOVA) ( p
