Background/Purpose:  In rheumatoid arthritis (RA), synovial fibroblasts (SF) are one main contributor of joint destruction since they resist apoptosis and secrete pro-inflammatory cytokines and matrix degrading enzymes. Previous studies already demonstrated the functional expression of several transient receptor potential channels (TRP) such as TRPV1, TRPV2, TRPV4, TRPA1 and TRPM8 in SF. Upon ligation, these receptors increase intracellular calcium but they have also been linked to modulation of inflammation in several cell types. TNF was shown to increase the expression of TRPA1, the receptor for mustard oil (allylisothiocyanate, AITC) and environmental poisons in synovial fibroblasts, but the functional consequences have not been investigated yet.

Methods:  TRPA1 was detected by immunocytochemistry and western blot. Calcium signals were determined fluorometrically (Fura-AM red). Cell viability was assessed by quantification of lactate dehydrogenase (LDH) in culture supernatants. IL-6, IL-8 and MMP-3 were determined by ELISA. Proliferation was determined by cell titer blue incorporation.

Results:  After 72h, TNF up-regulated TRPA1 protein levels in RASF which was accompanied by increased sensitivity to TRPA1 agonists AITC and polygodial. Under unstimulated conditions, polygodial but not AITC elicited modest calcium flux only in the highest concentrations used (50µM and 25µM). TNF pre-incubation substantially lowered the activation threshold for polygodial (from 25µM to1µM) and enabled AITC-mediated calcium flux, which was inhibited by TRPA1 antagonists A967079 (25µM) and HC030031 (50µM). In addition, TNF stimulation strongly enhanced TRPA1 mediated LDH release by AITC and polygodial, a marker for cell death. TNF not only reduced the threshold of AITC and polygodial but also led to a faster onset of LDH release. Furthermore, TRPA1 activation was associated with decreased proliferation of RASFs with TNF pre-incubation enhancing the anti-proliferative effect of AITC and polygodial. Secretion of IL-6, IL-8 and MMP-3 was attenuated by the TRPA1 antagonist A967079 but also polygodial, although the latter mediated this effect by reducing cell viability.

Conclusion:  TNF up-regulates and sensitizes TRPA1 in RASF. Subsequent activation of TRPA1 increased calcium flux and reduced cell viability. Since TRPA1 agonists only showed effects in TNF stimulated RASF, this cation channel might an attractive therapeutic target in chronic inflammation to reduce the activity of pro-inflammatory SF in the joint.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/tumor-necrosis-factor-tnf-sensitizes-rheumatoid-synovial-fibroblasts-to-trpa1-mediated-calcium-flux-anti-proliferation-and-cell-death/