The aim of the present study was to evidence the genes regulated by a combinaison of proinflammatory cytokines (IL-1α, TNF-α, IFN-γ, IL6) in a human chondrocyte model. The mRNA transcription pattern of genes potentially involved in mechanisms of cartilage protection, repair and degradation (growth factors, cytokines, metalloproteinases, metallothioneins, heat shock proteins...) was examined by cDNAarrays.

Biological model:
Normal human chondrocytes in monolayer culture.

Culture conditions:
Confluent cells in 24 wells micro-plates, cultivated in chondrocyte growth medium with 1% FBS, at 37°C, 5% CO2.

Treatment and stimulation:
The conditions compared in this study were:

• Control medium.
• medium with a mix of pro-inflammatory cytokines (IL-1α, TNF-α, IFN-γ, IL6).

Three wells were carried out per experimental condition. After cytokine stimulation, cultures were incubated for 24 hours.

Differential expression analysis :
The analysis of gene expression was performed using standard minichips hBA18-STRESS+ dedicated to inflammation, apoptosis and cellular stress (130 genes) and specially adapted to screening (produced by BIOalternatives).
The total RNA of each culture was extracted using TriReagent (standard protocol) and its quantity and quality were controlled. The multiple 33P-labelled cDNA were prepared by direct reverse-transcription of mRNA, using 33P-dATP and oligodT.
These labelled cDNA targets were hybridized to the specific cDNA probes covalently fixed to the nylon membrane. After extensive washing, the relative amount of each specific target hybridized to its probe was revealed by spot radioactivity counting using a “Cyclone” PhosphorImager (Packard instruments) and ImageQuant TL software (Amersham Biosciences).
The results were expressed in relative expression units (RE) after randomization to housekeeping genes.

The results are shown in Figures 1 & 2

The stimulation of normal human chondrocytes with the pro-inflammatory cytokines mix leads to an "activated" state of the chondrocytes and induced many modifications in the expression pattern of the selected genes.
Among other:

• increase in the expression of transcription factors such as NF-kappa-B and c-jun
• decrease in the expression of the apoptosis regulator BAX,
• increase in the expression of the inflammatory marker PTGS2 coding for COX2,
• increase in the expression of many cytokines such as IL1beta, IL6, IL8, MCP1, CXCL5 and CXCL6,
• increase in the expression of growth factors such as VEGFalpha,
• increase in the expression of matrix proteins such as tenascin,
• increase in the expression of metalloproteinase, such as MMP1 (collagenase) and especially MMP3 (stomelysin) which is involved in the degradation of the articular cartilage matrix.

Chondrocytes also developed ‘defence mechanisms’ in response to the stress induced by the mix of cytokines, including the modulation of the expression of transcripts for metallothioneins (antioxidant proteins 1E, 1G, 1H, 1X, 2A et 3), the superoxide dismutase (SOD1 and SOD2), heat shock proteins (HSP27,HSP90) and other such as TRAIL, which is thought to have a protective role by inducing the proliferation of synovial cells.

Conclusion

This combinaison of cytokines is able to induce a strong inflammatory phenotype in chondrocyte cultures in vitro. This could mimic the inflammation involved in the degradation of the articular cartilage matrix and serve as a model for the assay for inflammation limiting drugs.
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