Dr Sungsin Jo, Hanyang University Institute for Rheumatology Research, 222-1 Wangsimni, Seongdong, Seoul 04763, Republic of Korea
*Contributed equally
Because damage to hyaline cartilage is irreversible, relieving progressive cartilage destruction is an important therapeutic approach for inflammatory arthritis. In the present study, human hyaline chondrocytes were isolated from total knee replacements of 15 patients with osteoarthritis (OA) and three with rheumatoid arthritis (RA). Synovial fluid of OA (n=25) and RA (n=34) were collected to measure tumor necrosis factor α (TNFα) using ELISA. Consistent with previous studies, the synovial fluid exhibited high TNFα levels and hyaline cartilage was severely destroyed in patients with RA. TNFα-treated chondrocytes were used as model for inflammatory arthritis. TNFα did not influence proliferation or extracellular matrix expression in chondrocytes, but induced matrix metalloproteinase (MMP)1, 3 and 13 expression levels in chondrocytes, which was accompanied by activation of nuclear factor-κB signaling. During chondrogenic differentiation, TNFα attenuated mRNA expression levels of anabolic factors (collagen type 2 and aggrecan) and enhanced mRNA expression of catabolic factors (
Hyaline cartilage can be damaged by trauma and is degraded in different forms of arthritis (
Cartilage is an unusual tissue in that chondrocytes can be anabolic (synthesize matrix) or catabolic (degrade matrix) (
Cartilage destruction is a key characteristic of degenerative joint diseases, particularly osteoarthritis (OA); it also features in chronic inflammatory joint diseases, such as rheumatoid arthritis (RA) (
The present study defined specific surface markers of primary chondrocytes derived from human OA hyaline cartilage and investigated the role of TNFα in the chondrogenesis of hyaline cartilage.
The present study was performed in accordance with Hanyang University Hospital Institutional Review Board guidelines and approved by the Ethics Committee of Hanyang University Hospital (approval no. 2017-05-003) and Hanyang University Guri Hospital (approval no. 2018-07-024). Written informed consent was obtained from all subjects.
Between April 2018 and May 2019, 15 patients with OA (9 females and 6 males; mean age, 70.5±8.7 years) and three with RA (all females; mean age, 57.1±11.3 years) were enrolled and surgical samples were obtained from total knee replacement at Hanyang University Guri Hospital. Both OA and RA surgical knee samples were fixed at room temperature (RT) for two weeks with 10% formalin, decalcified with 10% formic acid and embedded routinely in a paraffin block. The paraffin block was sectioned at a thickness of 3.5 µm and stained by hematoxylin and eosin, Safranin O and Toluidine blue. The section slides were deparaffinized in the tissue in 100% neoclear (cat. no. 109843; Merck) for 10 min and rehydration in serial ethanol dilution (100, 90, 80, 70, and 50% ethanol) for 2 min each step, and washed with tap water for 5 min. For H&E staining, the slides were stained with 100% hematoxylin for 3 min, washed with tap water for 10 min, 100% eosin stained for 3 min, washed with tap water, and mounted with a permanent mounting medium (cat. no. H-5000; Vector Lab). For Safranin O staining, the slides were stained with 0.1% fast green stained for 5 min, washed with 1% acetic acid one tapping, washed with tap water for 1 min, 0.1% Safranin O for 6 min, washed with tap water for 1 min, and mounted with a permanent mounting medium. For toluidine blue staining, the slides were stained with 0.1% toluidine blue stained for 2 min, washed with tap water for 10 min, and mounted with a permanent mounting medium. All staining procedures performed at RT. Stained slides were imaged by a Nikon eclipse Ti-U light microscope (Nikon Corporation).
Between February 2017 and May 2019, synovial fluid samples were collected from 34 patients with OA (eight men and 26 women; mean age, 53.7±16.1 years) and 25 with RA (all women; mean age, 68.6±8.5 years) at Hanyang University Hospital for Rheumatic Disease. For synovial fluid analysis, 10 ml fluid was incubated with 1.5 mg hyaluronidase (cat. no. H3506; Sigma-Aldrich) for 15 min at 37˚C, followed by centrifugation at 1,400 x g for 15 min at 4˚C. After centrifugation, the fluids were immediately divided into aliquots and stored at -80˚C for TNFα levels (cat. no. DTA00D; R&D Systems, Inc.) measurement using ELISA according to manufacturer's protocol.
Hyaline cartilage from OA knee joints was scraped using a rongeur and collected in serum-free DMEM (cat. no. L0103-500; Biowest) buffer containing 1 mg/ml Collagenase type 2 (cat. no. C6885; Sigma-Aldrich). The collected tissue samples were incubated at 37˚C with agitation overnight. The next day, the digested hyaline cartilage was filtered through a 70-µm strainer (cat. no. 93070; SPL Life Sciences) and seeded ~60% cell density in DMEM (cat. no. L0103-500; Biowest) supplemented with 10% FBS (cat. no. 16000-044; Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin and streptomycin (cat. no. 15140122; Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO2. Primary chondrocytes at passage 2-5 were used in subsequent experiments. Until the passage 2-5, chondrocytes were cultured at 60% density before full cell density at 37˚C.
Recombinant human TNFα (cat. no. 300-01A; PeproTech, Inc.), golimumab (Janssen Global Services, LLC; 1 µg/ml, 37˚C, 4 weeks) and BAY 11-7082 (cat. no. B5556; Sigma-Aldrich; 10 or 30 µM, 37˚C, 24 h) were obtained.
The chondrocytes were fixed using a fixation/permeabilization solution kit (cat. no. 554715; BD Biosciences) and stained with CD34 (cat. no. 343607; BioLegend, Inc.; 1:100), CD44 (cat. no. 338807; BioLegend, Inc.; 1:100), CD59 (cat. no. 304711; BioLegend, Inc.; 1:100), CD74 (cat. no. 326811; BioLegend, Inc.; 1:100), CD90 (cat. no. 328109; BioLegend, Inc.; 1:100), CD105 (cat. no. 323205; BioLegend, Inc.; 1:100), CD146 (cat. no. 361015; BioLegend, Inc.; 1:100), CD164 (cat. no. 324805; BioLegend, Inc.; 1:100), SOX9-Alexa-647 (cat. no. 565493; BD Pharmingen; BD Biosciences; 1:200), ACAN-PE (cat. no. sc-33695; Santa Cruz Biotechnology, Inc.; 1:100), IgG1-Alexa-647 (cat. no. 557732; BD Pharmingen; BD Biosciences; 1:100), IgG1-PE (cat. no. 400112; BioLegend, Inc.; 1:100), IgG1-APC (cat. no. 400122; BioLegend, Inc.; 1:100), IgG1-FITC (cat. no. 400109; BioLegend, Inc.; 1:100) or IgG2a-APC (cat. no. 400221; BioLegend, Inc.; 1:100) for 30 min at 4˚C. The dilution of the antibody for FACS was ranged from 1:100-1:200. After staining, cells were washed with Perm/Wash Buffer (cat. no. 554715; BD Biosciences) and analyzed by flow cytometry (FACS Canto II; BD Biosciences). Data were analyzed using FlowJo version 10.7 software (FlowJo LLC).
The water-soluble tetrazolium salt (WST) assay was performed with EZ-CYTOX (cat. no. EZ-1000; Dogen Bio Co., Ltd.) according to the manufacturer's instructions. Primary chondrocytes were plated into 96-well plate (1x103 cells/well) and treated with 10 or 25 ng/ml TNFα in 37˚C CO2 incubator for 1-6 days. WST solution was added to the cells, which were incubated for 1 h. The absorbance at 450 nm was measured with a microplate reader (Thermo Fisher Scientific, Inc.).
Chondrocytes were stimulated with 10 ng/ml human TNFα at RT for 1 day and collected for human MMP analysis. The stimulated cells were lysed in 1X RIPA buffer including phosphatase and proteinase inhibitors and assessed according to the manufacturer's protocol (cat. no. ab134004; Abcam). To analyze the array data, comparison of signaling intensities for individual spots was detected using the UVItech system (Cleaver Scientific Ltd.) and analyzed with ImageJ 1.52a version software (National Institutes of Health).
RT-qPCR was performed as previously described (
Immunoblot analysis was performed as previously described (
TCA precipitation was performed as previously described (
The TNFα-stimulated chondrocytes were washed twice with 1X PBS and fixed with 10% formalin at RT for 15 min, followed by permeabilization with 1X PBS containing 0.1% Triton X-100 and 1% BSA (cat. no. BSA-BSH-1XG; Rocky Mountain Biologicals, Inc.) at RT for 1 h, incubation with a primary antibody at 4˚C overnight, washing with 1X PBS and incubation with Cy3-conjugated anti-rabbit antibody (cat. no. 111-165-144; Jackson Immunoresearch) or Alexa 488-conjugated anti-mouse antibody (cat. no. A-11001; Invitrogen) for 1 h. All primary and secondary antibodies were used at 1:100 dilution. The stained cells were washed with distilled water and mounted with DAPI (cat. no. H1200; Vector Laboratories, Inc.; Maravai Life Sciences). In order to visualize stained cells, immunofluorescence images were collected with a confocal microscope (TCS SP5; Leica Microsystems GmbH). Images were captured using LAS version 4.2.1 software (Leica Microsystems GmbH).
NF-κB p65 wild-type and two p65 S536A (substitution of alanine for serine) or S536E (substitution of glutamate for serine 536) mutant promoters in pGL3-Basic were a gift from Dr Heekyoung Chung (Hanyang University, Seoul, South Korea) (
The ‘pellet culture’ method was performed as previously described (
Data were analyzed with GraphPad Prism 6 software (GraphPad Software, Inc.). A two-tailed Student t-test was used to compare data between two unpaired groups. One-way ANOVA with Tukey's post hoc test was used to compare data between more than two groups. All data are expressed as the mean ± SD (n≥3). P<0.05 was considered to indicate a statistically significant difference.
TNFα levels in synovial fluid were significantly higher in patients with RA than OA (mean, 168.7 vs. 64.28 pg/ml;
The present study aimed to detect known chondrocyte surface markers and investigate the effects of TNFα on them (
TNFα-treated chondrocytes were used as a model for inflammatory arthritis. In order to determine whether TNFα may affect the destructive hyaline cartilage of patients with inflammatory arthritis, chondrocytes were stimulated with TNFα. To confirm dose effects of TNFα in chondrocytes, we treated with 1, 5, 10, 25, 50 ng/ml TNFα dose and analyzed MMP expression levels using RT-qPCR and immunoblotting. We confirmed an increase in MMP1, 3, 13 expression in dose-dependently TNFα treatment (
In order to elucidate the effect of TNFα on regenerative capacity in chondrocytes, chondrogenic differentiation of chondrocytes was induced in the presence or absence of TNFα treatment. Chondrocytes treated with TNFα exhibited lower differentiation capacity; they showed weaker intensity of staining and smaller pellet size compared with controls (
To determine whether the modulation of TNFα affected catabolic and anabolic gene expression levels, human chondrocytes were treated with 1 µg/ml golimumab (a TNFα blocker) in the presence of TNFα. TNFα stimulation upregulated p-NF-κB, MMP1, MMP3 and MMP13 in chondrocytes, whereas treatment with golimumab impeded these changes mediated by TNFα (
The present study revealed that TNFα levels were increased in synovial fluid and the destruction of hyaline cartilage was greater in RA compared with in OA. To demonstrate the association between TNFα and hyaline cartilage destruction in OA, chondrocytes were stimulated with TNFα; the results indicated that TNFα induced a metabolic shift in chondrocytes via NF-κB signaling. Based on these findings, a model for the pathogenesis of hyaline cartilage degradation in inflammatory arthritis was proposed. TNFα, an inflammatory cytokine that is increased by excessive inflammation, may modulate anabolic and catabolic factors, thereby revealing potential therapeutic targets for inflammatory arthritis progression.
The precise mechanism by which TNFα suppresses the expression of matrix proteoglycans, such as ACAN and COL2, requires further study. It was hypothesized that MMP 1, 3 and 13 are indicators of TNFα-driven matrix proteoglycan degradation. Although 24-h TNFα exposure did not affect SOX9 and ACAN expression in chondrocytes, exposure to TNF during chondrogenic differentiation resulted in decreased mRNA expression levels of anabolic mediators, such as
When chondrocytes become hypertrophic, they express unique genes, such as
TNFα plays a critical role in bone destruction in several types of inflammatory joint disease (
Blocking studies with an anti-TNFα agent in an arthritis model have shown that TNFα may drive cartilage and bone destruction (
OA is a degenerative joint disease caused by the degradation of hyaline cartilage. Cartilage loss in OA is associated with aging and mechanical stress, as well as inflammation. The primary cause of OA is aging and mechanical stress, which suppress the regenerative capacity of chondrocytes involved in extracellular matrix components comprising hyaline cartilage. Moreover, total knee replacement for OA is a significant health burden, and there are no therapeutic drugs available yet for the regeneration of hyaline cartilage (
In conclusion, the present study showed that TNFα was associated with progressive destruction of hyaline cartilage in OA. Mechanistically, chondrocyte exposure to TNFα increased
The authors would like to thank Dr Dae Hyun Yoo, Dr Sang-Cheol Bae and Dr Jae-Bum Jun of the Hanyang University Hospital for Rheumatic Disease for helping to collect synovial fluid from patients. Immunofluorescence images were analyzed by confocal microscopy (Leica Microsystems GmbH) at Hanyang LINC Analytical Equipment Center (Seoul, Korea).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JP, HP, YLL and SW performed all experiments. SJ, BN, JHY, and YGK designed the experiments. JP, HP, and SJ analyzed experimental data. JHY provided human knee joint samples. YGK and BN provided synovial fluid of patients with OA and RA. SJ and THK wrote the manuscript. JP, HP, and SJ confirm the authenticity of all the raw data. THK conceptualized and supervised the study. All authors read and approved the final manuscript.
Studies involving human materials were performed in compliance with the Helsinki Declaration and were approved by the Ethics Committee of Hanyang University Hospital (approval no. IRB-2017-05-003) and Hanyang University Guri Hospital (approval no. 2018-07-024). All subjects provided written informed consent.
Not applicable.
The authors declare that they have no competing interests.
High TNFα levels in synovial fluid and destruction of hyaline cartilage are observed in RA. (A) TNFα levels in OA (n=25) and RA (n=34) synovial fluid were assessed by ELISA. **P<0.01, unpaired Student's t-test. (B) Knee tissue samples (including hyaline cartilage) from patients with OA and RA were stained with H&E, Safranin O and Toluidine Blue. Overt fibrillation indicated by black arrows; Chondyocyte clustering indicated by blue arrows; changes in chondrocyte morphology/distribution indicated by yellow arrows; matrix destruction indicated by grey arrows. Scale bar, 200 µm. RA, rheumatoid arthritis; OA, osteoarthritis; H&E, hematoxylin and eosin; TNFα, tumor necrosis factor α.
TNFα does not significantly affect proliferation and extracellular molecule expression in OA chondrocytes. (A) CD34, CD44, CD59, CD74, CD90, CD105, CD146 and CD164 surface markers of chondrocytes were evaluated by flow cytometry. (B) Chondrocytes were exposed to TNFα and assessed by water-soluble tetrazolium salt assay. n=5, one-way ANOVA with Tukey's post hoc test. (C) Chondrocytes were exposed to 25 ng/ml TNFα for 24 h and assessed by flow cytometry. IgG was used as a control. OA, osteoarthritis; TNFα, tumor necrosis factor α; ACAN, aggrecan; SOX9, SRY-box transcription factor 9.
TNFα induces the expression of MMPs in OA chondrocytes via NF-κB activation. (A) Chondrocytes were treated with vehicle or 10 ng/ml TNFα for 24 h. MMP protein expression levels in stimulated cell lysates were semi-quantified via a human MMP antibody array (n=2). (B)
TNFα inhibits chondrogenic differentiation of osteoarthritis chondrocytes. Chondrocytes were induced with chondrogenic differentiation media for 28 days in the presence of vehicle, or 10 or 25 ng/ml TNFα. (A) Images were captured after 28 days differentiation. Differentiating chondrocytes were (B) stained by Safranin O and Toluidine Blue and (C) analyzed by reverse transcription-quantitative PCR. Scale bar, 500 µm. Data are presented as the mean ± SD (n=4). *P<0.05; **P<0.01. COL2, collagen type 2; ACAN, aggrecan; MMP, matrix metalloproteinase; SOX9, SRY-box transcription factor 9; TNFα, tumor necrosis factor α.
Blocking TNFα attenuates TNFα-driven degradation of the extracellular matrix of osteoarthritis chondrocytes. (A) Chondrocytes were treated for 24 h and analyzed by immunoblotting. Golimumab was used as an anti-TNF agent (n=3). P-NF-κB protein was semi-quantified with ImageJ and calculated relative to total NF-κB protein. (B) 239T cells were co-transfected with NF-κB promoters and