TNF Produced by Inflammatory Blood Mononuclear Cells Directly Contributes to Cartilage Damage in Arthritis


 Background Anti-TNF therapies are effective at preventing inflammation and structural damage in rheumatoid arthritis (RA). However, the role of TNF in cartilage destruction in RA is not well understood. Therefore, we studied the effects of TNF on cartilage and compared TNF production by different cell types involved in joint pathology.Methods Primary human chondrocytes and cartilage explants were cultured with recombinant TNF. Bovine cartilage was co-cultured with activated human peripheral blood mononuclear cells (PBMCs) or fibroblast-like synoviocytes (FLS). Expression of cytokines and metalloproteinases (MMPs) was assessed by qPCR and MSD, and proteoglycan depletion from cartilage was assessed using histomorphometry and colorimetric detection in tissue culture supernatants. D2E7 was used to block TNF both in vitro and in vivo in a human TNF transgenic (hTNF-Tg) mouse model of arthritis.Results TNF elicited strong pro-inflammatory and catabolic effects on isolated human chondrocytes and cartilage explants leading to upregulation of IL-6 and MMPs, as well as proteoglycan depletion from bovine cartilage explants. In an effort to identify cellular sources of TNF, we challenged chondrocytes, FLS and PBMCs with inflammatory stimuli present in RA joints and found that PBMC that were used to model inflammatory cell infiltration produced significantly higher levels of TNF. Moreover, co-culture with activated PBMCs resulted in proteoglycan depletion from bovine cartilage explants. In sharp contrast with stromal cells, TNF failed to induce high amounts of IL-6 and MMPs in PBMCs, suggesting that different cell populations play distinct roles in the triggering and propagation of joint destruction. TNF blockade protected cartilage from damage both in co-culture systems and in a hTNF-Tg mouse model of arthritis.ConclusionsOur data demonstrate that TNF directly triggers a catabolic program in human chondrocytes leading to cartilage damage and further suggest that neutralization of TNF produced by immune cells infiltrating the inflamed joints, decreases catabolic activity of chondrocytes and fibroblasts, which, in turn, contributes to the cartilage protective effects of anti-TNF biologics in arthritis.

2 Abstract Background Anti-TNF therapies are effective at preventing inflammation and structural damage in rheumatoid arthritis (RA). However, the role of TNF in cartilage destruction in RA is not well understood. Therefore, we studied the effects of TNF on cartilage and compared TNF production by different cell types involved in joint pathology.

Methods
Primary human chondrocytes and cartilage explants were cultured with recombinant TNF. Bovine cartilage was co-cultured with activated human peripheral blood mononuclear cells (PBMCs) or fibroblast-like synoviocytes (FLS). Expression of cytokines and metalloproteinases (MMPs) was assessed by qPCR and MSD, and proteoglycan depletion from cartilage was assessed using histomorphometry and colorimetric detection in tissue culture supernatants. D2E7 was used to block TNF both in vitro and in vivo in a human TNF transgenic (hTNF-Tg) mouse model of arthritis.

Results
TNF elicited strong pro-inflammatory and catabolic effects on isolated human chondrocytes and cartilage explants leading to upregulation of IL-6 and MMPs, as well as proteoglycan depletion from bovine cartilage explants. In an effort to identify cellular sources of TNF, we challenged chondrocytes, FLS and PBMCs with inflammatory stimuli present in RA joints and found that PBMC that were used to model inflammatory cell infiltration produced significantly higher levels of TNF. Moreover, co-culture with activated PBMCs resulted in proteoglycan depletion from bovine cartilage explants. In sharp contrast with stromal cells, TNF failed to induce high amounts of IL-6 and MMPs in PBMCs, suggesting that different cell populations play distinct roles in the triggering and propagation of joint destruction.
TNF blockade protected cartilage from damage both in co-culture systems and in a hTNF-Tg mouse model of arthritis.

Conclusions
Our data demonstrate that TNF directly triggers a catabolic program in human chondrocytes leading to cartilage damage and further suggest that neutralization of TNF produced by immune cells 3 infiltrating the inflamed joints, decreases catabolic activity of chondrocytes and fibroblasts, which, in turn, contributes to the cartilage protective effects of anti-TNF biologics in arthritis.

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However, the manuscript can be downloaded and accessed as a PDF.  FLS/cartilage co-cultures were exposed to 10 ng/ml of TNF or IL-1β for 3 days and thereafter, 5 μg/ml of D2E7 or IgG1 (negative control) were added to some of the wells.  TNF neutralization protects cartilage in vitro and in vivo, in hTNF-Tg mouse model of arthritis (A) Bovine cartilage explants were co-cultured with PBMCs isolated from the whole blood of healthy individuals with or without 10 μg/ml of alarmins: S100A8, S1009 or TNF as a positive control. D2E7 or isotype control (hIgG1) were added at 5 μg/ml to some wells.
After 7 days of culture, supernatants were collected, and levels of PG depleted from the cartilage into the culture media were measured. Results are presented as a fold over PG levels in untreated samples. Data are shown as mean ± SEM of 3 independent experiments.
Dotted line represents untreated control. (B) Human PBMCs alone (left panel, n=4) or in coculture with bovine cartilage (right panel, n=3) were stimulated with 10 μg/ml of alarmins: S100A8 and S1009 or left untreated in the presence of 5 μg/ml of D2E7 or isotype control (hIgG1), supernatants were collected and levels of TNF were measured by MSD assay. Data are shown as mean ± SEM, significant difference between D2E7 and isotype control is shown: * = p<0.05 (two-way ANOVA with Tukey's multiple comparison test). (C) Representative alcian blue stained sections from ankle joints of hTNF-Tg mice treated for 12 weeks with 1 mg/kg once a week of anti-TNFα antibody D2E7 (n=12) or placebo (n=6) are shown on the upper panel (magnification 20x). Staining intensity of the articular cartilage (shown by asterisk) correlates with PG content. The lower panel depicts the results of quantitative image analysis of PG content (stained with alcian blue) in the articular cartilage of talus bones (area of analysis is outlined in red). Data for individual animals are shown as a percent of total cartilage area, significant difference between groups is shown: **= p<0.01 (Mann-Whitney U test).