1.
Histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to Ca2+ mobilization and cell proliferation.
Pinheiro, AR, Paramos-de-Carvalho, D, Certal, M, Costa, MA, Costa, C, Magalhães-Cardoso, MT, Ferreirinha, F, Sévigny, J, Correia-de-Sá, P
The Journal of biological chemistry. 2013;(38):27571-27583
Abstract
Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca(2+) ([Ca(2+)]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca(2+)]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca(2+)]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with (10)Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca(2+)]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca(2+)]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.
2.
Poor relevance of a lymphocyte proliferation assay in lamotrigine-induced Stevens-Johnson syndrome or toxic epidermal necrolysis.
Tang, YH, Mockenhaupt, M, Henry, A, Bounoua, M, Naldi, L, Le Gouvello, S, Bensussan, A, Roujeau, JC
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology. 2012;(2):248-54
Abstract
BACKGROUND Prior use of 'lymphocyte transformation test' (LTT) in Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) provided conflicting results, possibly dependent on sampling dates (acute vs. late). OBJECTIVE Evaluation of LTT in patients with SJS or TEN who reacted to lamotrigine (LTG). In a small subgroup we explored the possible role of regulatory T cells (T-reg). METHODS Acute phase samples (9) and post-recovery samples (14) from cases of SJS or TEN to LTG were provided by the RegiSCAR-study group. Controls were persons never exposed to LTG (12), patients exposed without reaction (6), and patients who developed a mild eruption to LTG (6). LTT was performed by measuring (3) H-thymidine incorporation after 3 days of incubation with phytohemmaglutinin, LTG (10 μg/mL) or medium. Stimulation index ≥ 2 was considered positive. In 16 cases LTT was redone after depletion of T-reg by fluorescence activated cell sorting. RESULTS Positive LTT was observed in 3/6 cases of mild eruptions, 1/9 SJS/TEN-cases tested during the acute phase and 3/14 SJS/TEN-cases tested after recovery. We noted a very mild and nonsignificant trend for an increased response after depletion of T-reg in late samples from SJS or TEN patients. CONCLUSIONS AND CLINICAL RELEVANCE With the largest number of LTT performed in patients with SJS or TEN to a single drug, we confirmed that reactive cells are rarely detected in these reactions. Poor reactivity did not seem related to T-reg. Other in vitro assays than those testing proliferation should be evaluated, before raising the hypothesis that specific cells disappeared by undergoing apoptosis during the reaction.