Selective killing of proinflammatory synovial fibroblasts via activation of transient receptor potential ankyrin (TRPA1)

Background: Studies in rheumatoid arthritis synovial fibroblasts (RASF) demonstrated the expression of several transient receptor potential channels (TRP) such as TRPV1, TRPV2, TRPV4, TRPA1 and TRPM8. 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 and environmental poisons in SF, but the functional consequences have not been investigated yet. Methods: TRPA1 was detected by immunocytochemistry, western blot and cell-based ELISA. Calcium measurements were conducted in a multimode reader. Cell viability was assessed by quantification of lactate dehydrogenase (LDH) in culture supernatants and RealTime-Glo luminescent assays. IL-6 and IL-8 production by SF was quantified by ELISA. Proliferation was determined by cell titer blue incorporation. Results: After 72 h, mimicking proinflammatory conditions by the innate cytokine TNF up-regulated TRPA1 protein levels in RASF which was accompanied by increased sensitivity to TRPA1 agonists AITC and polygodial. Under unstimulated conditions, polygodial elicited calcium flux only in the highest concentrations used (50 mu M and 25 mu M). TNF preincubation substantially lowered the activation threshold for polygodial (from 25 mu M to 1 mu M). In the absence of TNF pre-stimulation, only polygodial in high concentrations was able to reduce viability of synovial fibroblasts as determined by a real-time viability assay. However, following TNF preincubation, stimulation of TRPA1 led to a fast (< 30 min) viability loss by necrosis of synovial fibroblasts. TRPA1 activation was also associated with decreased proliferation of RASFs, an effect that was also substantially enhanced by TNF preincubation. On the functional level, IL-6 and IL-8 production was attenuated by the TRPA1 antagonist A967079 but also polygodial, although the latter mediated this effect by reducing cell viability. Conclusion: Simulating inflamed conditions by preincubation of synovial fibroblasts with TNF up-regulates and sensitizes TRPA1. Subsequent activation of TRPA1 increases calcium flux and substantially reduces cell viability by inducing necrosis. Since TRPA1 agonists in the lower concentration range only show effects in TNF-stimulated RASF, this cation channel might be an attractive therapeutic target in chronic inflammation to selectively reduce the activity of promflammatory SF in the joint.