Nucleotides induce IL-6 release from human airway epithelia via P2Y₂and p38 MAPK-dependent pathways

Abstract

Extracellular nucleotides can mediate a variety of cellular functions via interactions with purinergic receptors. We previously showed that mechanical ventilation (MV) induces airway IL-6 and ATP release, modifies luminal nucleotide composition, and alters lung purinoceptor expression. Here we hypothesize that extracellular nucleotides induce secretion of IL-6 by small airway epithelial cells (SAEC). Human SAEC were stimulated with nucleotides in the presence or absence of inhibitors. Supernatants were analyzed for IL-6 and lysates for p38 MAPK activity by ELISA. RNA was analyzed by real-time RT-PCR. Rats (n ϭ 51) were randomized to groups as follows: control, small-volume MV, largevolume MV, large-volume MV-intratracheal apyrase, or small-volume MV-intratracheal adenosine 5Ј-O-(3-thiotriphosphate) (ATP␥S). After 1 h of MV, bronchoalveolar lavage fluid was analyzed for ATP and IL-6 by luminometry and ELISA. ATP and ATP␥S increased SAEC IL-6 secretion in a time-and dose-dependent manner, an effect inhibited by apyrase. Agonists were ranked in the following order: ATP␥S Ͼ ATP ϭ UTP Ͼ ADP ϭ adenosine Ͼ 2-methylthio-ADP ϭ control. SB-203580, but not U-0126 or JNK1 inhibitor, decreased nucleotide effects. Additionally, nucleotides induced p38 MAPK phosphorylation. Inhibitors of Ca 2ϩ signaling, phospholipase C, transcription, and translation decreased IL-6 release. Furthermore, nucleotides increased IL-6 expression. In vivo, large-volume MV increased airway ATP and IL-6 concentrations. IL-6 release was decreased by apyrase and increased by ATP␥S. Extracellular nucleotides induce P2Y2-mediated secretion of IL-6 by SAEC via Ca 2ϩ , phospholipase C, and p38 MAPK-dependent pathways. This effect is dependent on transcription and translation. Our findings were confirmed in an in vivo model, thus demonstrating a novel mechanism of nucleotideinduced IL-6 secretion by airway epithelia. cytokine; inflammation; purine; purinergic; ventilator-associated lung injury; interleukin-6; mitogen-activated protein kinase

Concomitant activation of P2Y2 and P2Y6 receptors on monocytes is required for TLR1/2‐induced neutrophil migration by regulating IL‐8 secretion

“…We have to note that our previous works did not exclude a function of P2Y 2 in LPS-induced IL-8 secretion [14,15]. Other previous studies showed that the activation of this receptor was coupled to MCP-1 release from rat alveolar and peritoneal macrophages [22] and IL-6 release from human airway epithelia [32].…”

“…We have to note that our previous works did not exclude a function of P2Y 2 in LPS-induced IL-8 secretion [14,15]. Other previous studies showed that the activation of this receptor was coupled to MCP-1 release from rat alveolar and peritoneal macrophages [22] and IL-6 release from human airway epithelia [32].Altogether, the results reported in this paper suggest that the P2Y 2 and P2Y 6 receptors may play an important role in the innate immune response resulting from TLR2 activation. As IL-8 secreted by a variety of inflammatory cells including monocytes has been implicated in a number of inflammatory/infectious diseases, such as rheumatoid arthritis [33], inflammatory bowel disease [34], psoriasis [35,36], palmoplantar pustulosis [37,38], asthma and chronic obstructive pulmonary disease [39], targeting P2Y 2 and/or P2Y 6 receptors may reveal as novel therapeutic approaches to fight these diseases.…”

Protective effects of adenosine A2A receptor agonist in ventilator-induced lung injury in rats

“…However, previous in vitro studies have revealed that supernatants from hypoxia-or stretch induced injury contained adenosine that diminished endothelial leakage (14,43). Additional studies in vivo demonstrated prominent increases in pulmonary adenosine levels with mechanical ventilation (13,14). The increased adenosine level was associated with the enhanced expression of ectoapyrase (CD39) and ecto-5′-nucleotidase (CD73) that hydrolyze ATP to adenosine at the cell surface of neutrophils and endothelial cells (43).…”

“…A couple of recent studies have demonstrated that mechanical ventilation in rats and mice increases the level of pulmonary adenosine (13,14), and that mice deficient for extracellular adenosine generation show increased pulmonary edema and inflammation after VILI (14). Once in the extracellular milieu, adenosine exerts its biological effects through binding to the G-protein coupled adenosine receptors including A 1 R, A 2A R, A 2B R, and A 3 R (8,15,16).…”

UDP-glucose promotes neutrophil recruitment in the lung

“…Activation of the P2Y 2 R promotes inhibition of Na + absorption as well as CFTR-dependent and CFTRindependent Cl − secretion, ciliary beating, and mucin secretion [6,7]. In addition, nucleotides within the airway surface liquid (ASL) are potent pro-inflammatory signaling molecules acting on purinergic receptors expressed on immune/ inflammatory cells or by promoting the release of cytokines and other chemoattractants from lung epithelial cells [8][9][10][11]. Thus, the rates of nucleotide release and metabolism in healthy airways are finely regulated to maintain effective MCC without promoting airway inflammation.…”