Immunopharmacology and inflammationInvolvement of P2Y11 receptor in IFN-γ-induced IL-6 production in human keratinocytes
Introduction
The skin is composed of epidermis, dermis, and subcutaneous tissue, and serves to protect internal organs from potentially hazardous external factors, including mechanical and chemical stimuli, UV radiation, etc. In the outer epidermal layer, about 95% of the cells are keratinocytes. The epidermal keratinocytes produce various cytokines (Ansel et al., 1990, Bowman et al., 1997, Elder et al., 1989, Kirnbauer et al., 1991, Kock et al., 1990, Kondo et al., 1993, Neuner et al., 1991, Schwarz and Luger, 1989), in response to environmental stimuli and injury.
IL-6, a well-studied inflammatory cytokine that plays a central role in immune responses, is produced by T cells, macrophages, fibroblasts, keratinocytes, and endothelial cells. In keratinocytes, IL-6 induces proliferation and migration, thereby promoting skin repair (Gallucci et al., 2000, Gallucci et al., 2004). Various stimuli, such as UV radiation and thermal damage, induce IL-6 production in skin (Kawakami et al., 1997, Urbanski et al., 1990). Moreover, it has been suggested that IL-6 is involved in skin diseases, such as psoriasis (Grossmann et al., 1989).
T cell-derived cytokines also have essential roles in immunity. The Th1 cytokine IFN-γ is produced by NK cells and CD4+ T cells, and has pro-inflammatory effects on keratinocytes (Farrar and Schreiber, 1993). IFN-γ production is increased in various skin diseases, and the IFN-γ concentration in serum of patients with psoriasis is higher than that in healthy subjects (el-Barnawi et al., 2001, Gomi et al., 1991, Jacob et al., 2003, Szegedi et al., 2003). The level of IFN-γ is correlated with the clinical severity and activity of psoriasis (Arican et al., 2005). Furthermore, keratinocytes activated by IFN-γ release IL-6 (Gudjonsson et al., 2004), suggesting that IFN-γ and IL-6 may be associated with the onset of psoriasis.
ATP is used as an energy source in cells, but extracellular ATP also acts as an intercellular signaling factor (Burnstock, 1997). Extracellular ATP is degraded to ADP, AMP, and adenosine by ecto-nucleotidases (Burrell et al., 2003; Dubyak and el-Moatassim, 1993; Lazarowski et al., 2000).
Cells, including keratinocytes, release ATP in response to various stimuli, such as mechanical stimuli, hypoosmotic stimuli, and radiation (Azorin et al., 2011, Koizumi et al., 2004, Mizumoto et al., 2003). The mechanisms of this release include a maxi-anion channel, P2X7 receptor/pore, a volume-sensitive outwardly rectifying chloride channel, a member of the ATP-binding cassette protein family, a gap junction hemichannel, and vesicular exocytosis (Fitz, 2007, Hisadome et al., 2002, Ohshima et al., 2010, Pankratov et al., 2006, Pellegatti et al., 2005, Sabirov et al., 2001, Sprague et al., 1998, Stout et al., 2002). ATP released from cells via these pathways activates P2 receptors in an autocrine or a paracrine manner (Corriden and Insel, 2010).
Recently, it has been found that extracellular ATP induces IL-6 production via P2Y receptors (Inoue et al., 2006, Tsukimoto et al., 2010). However, the mechanism of cytokine-induced IL-6 production by keratinocytes is not fully understood. In this study, we investigated the involvement of extracellular ATP and various P2 receptors in IL-6 production induced by the Th1 cytokine IFN-γ in a human keratinocyte cell line, HaCaT.
Section snippets
Reagents
Dulbecco's modified Eagle's medium, penicillin, streptomycin, dithiothreitol (DTT), and polyoxyethylene (Burnstock, 1997) sorbitan monolaurate (equivalent of Tween-20) were purchased from Wako Pure Chemical Industries (Osaka, Japan). ATP, the ecto-nucleotidase apyrase, the maxi-anion channel blockers GdCl3 and arachidonic acid, the anion transporter inhibitors glibenclamide and flufenamic acid (FFA), the gap-junction hemichannel blockers 18-glycyrrhetinic acid (18 GA) and carbenoxolone (CBX),
IFN-γ increased IL-6 production in HaCaT cells
First, we examined the effect of stimulation with IFN-γ on IL-6 production in HaCaT cells. IL-6 production was markedly increased in a time-dependent manner up to 48 h in response to stimulation with IFN-γ (Fig. 1), whereas the cells produced a constant level of IL-6 in the absence of stimulation. These results suggest that IFN-γ induces IL-6 production by HaCaT cells, and in the following experiments, the concentration of IL-6 was assayed at 48 h after stimulation with IFN-γ.
Involvement of P2 receptors in IFN-γ-induced IL-6 production
Before examination
Discussion
Extracellular ATP activates P2 receptors and induces diverse physiological functions in cells (Dixon et al., 1999, Koizumi et al., 2004). ATP is released from cells in response to various stimuli Corriden and Insel, 2010, and extracellular ATP stimulates production and release of IL-6 via P2Y receptors (Inoue et al., 2006, Yoshida et al., 2006). On the other hand, the Th1 cytokine IFN-γ induces IL-6 production in human keratinocytes (Gudjonsson et al., 2004). In this study, we investigated
Conclusion
Involvement of various P2 receptors in IL-6 production induced by IFN-γ was examined in HaCaT cells. It was found that P2Y11 receptor mediates IFN-γ-induced IL-6 production in HaCaT cells, suggesting the importance of purinergic signaling in IFN-γ-induced skin inflammatory conditions.
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