Chronic inflammation and adenosine receptor activation modulate inflammatory mediators release by dermal fibroblasts

J Arasa1, MC Terencio1,2, F Valcuende3, P Navalón4, M Payá1,2, MC Montesinos1,2. 1University of Valencia, Centre de Reconeixement Molecular i Desenvolupament Tecnològic, Spain, 2University of Valencia, Pharmacology, Spain, 3Hospital La Plana de Vila-real, Dermatology, Spain, 4Hospital General Universitario, Urology, Spain

Fibroblasts are the most abundant cell type in the dermis and play a key role in maintaining proper immune response and regulation of tissue homeostasis. Furthermore, dermal fibroblasts could contribute to psoriasis pathogenesis by producing soluble mediators involved in T-cell recruitment and activation (1, 2). On the other hand, adenosine, a known regulator of inflammation and immunity via interaction with cell-surface receptors, A1, A2A, A2B and A3 (3), mediates in part the anti-inflammatory effect of methotrexate (4). We sought to further evaluate the role of fibroblasts in the inflammatory process of the skin, and its modulation by adenosine receptor activation. Primary cells were isolated from foreskin of healthy donors or from lesional skin biopsies of psoriatic patients and stimulated with TNF-α (10ng/ml) or the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 µg/ml) for 24h. IL-6 and Il-8 levels in cell supernatants were detected by ELISA and prostaglandin E2 (PGE2) levels were determined by RIA. COX-2 expression was detected by western blot of whole cell lysates or immunocytochemistry, using IL-1β (2.5ng/ml) as positive control. After TNF-α stimulation, fibroblasts from healthy donors produced high levels of IL-6 (67.8± 8.6 ng/ml, n=15) and IL-8 (195.1± 19.9 ng/ml, n=15) that were reduced by 30 min pretreatment with 10-7M or 10-6M of the selective adenosine A2A receptor agonist CGS-21680 (28.77± 2.43 ng/ml of IL-6 and 130.6±23.95 ng/ml of IL-8 for 10-6M CGS-21680, n=2 in duplicates). Interestingly, fibroblasts from psoriatic patients elicited a reduced production of IL-6 and, to a lesser extent, IL-8 in comparison to healthy fibroblasts (16.8± 1.6 pg/ml, p<0.0001 and 110.2± 11.6 ng/ml, p<0.05, respectively n=12). Similar results were observed after stimulation with the PKC activator TPA. We further observed that lesional skin fibroblasts released reduced levels of PGE2 after TNF-α stimulation (0.91± 0.19 ng/ml versus 7.56 ± 1.38 ng/ml in healthy fibroblasts, n=9, p<0.001) and TPA stimulation (0.70 ± 0.25 ng/ml versus 8.13 ± 1.54 ng/ml in healthy fibroblasts, n=9, p<0.001), results that correlated with a diminished induction of the enzyme involved in its metabolism COX-2, after TNF-α, TPA or IL-1β stimulation. Our results suggest that adenosine A2A receptor is implicated in the regulation of the inflammatory response in dermal fibroblasts. Moreover, our findings support the view that chronic inflammation, as occurs in psoriasis, could cause a phenotypic change in dermal fibroblasts.

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(3) Blackburn MR, et al. Handb Exp Pharmacol; 193:215-69 (2009).

(4) Chan ESL and Cronstein BN. Nat Rev Rheumatol. 6:175-8 (2010)