Human primary keratinocytes express functional P2 receptors whose expression is modulated by inflammatory cytokines.

Extracellular nucleotides are agonists at the family of receptors known as P2 receptors. Besides a few reports (Burrell et al., 2003; Greig et al., 2003; 2003a), at present an extensive characterization of P2 receptor expression in human keratinocytes has not yet been carried out. In this study, we have investigated P2 receptor expression and function in human primary keratinocytes. Cells from healthy donors were cultured in vitro and mRNA expression was studied by RT-PCR. We found that human keratinocytes expressed the mRNA for several P2X subtypes (P2X3, P2X4, P2X5, P2X6, P2X7) while P2Y subtypes were less represented (P2Y1, P2Y2). Different cytokines (IL-4, IFN-gamma and TNF-alpha) are thought to play a role in skin diseases, particularly in psoriasis and atopic dermatitis. Human keratinocytes were challenged with cytokines and mRNA expression was analysed at different time-points. IL-4 and IFN-gamma, increased mRNA expression for the P2Y1, P2X3, P2X4 and P2X7 subtypes while, on the contrary, TNF-alpha induced a down-regulation of P2Y2, P2X3, P2X4, P2X5, P2X7. Functional expression and of P2 receptors as been shown by fluorometric analysis. Stimulation of cells with ATP and UTP, in a concentration range from 0.03 to 3000 µM induced a fast and dose-dependent increase in the intracellular calcium concentration ([Ca2+]i). ATP-gammaS, ADP and BzATP also elicited Ca2+ responses. Conversely, UDP-glucose was much less effective. Stimulation of human keratinocytes with the P2X agonist BzATP induced striking morphological changes accompanied by shedding of microvescicles from the cells. Our data show that human keratinocytes express different purinergic receptors whose stimulation is coupled to [Ca2+]i changes, and production of microvesicles. At present, microvesicle content is under investigation.