P2Y6 Receptor-Mediated Proinflammatory Signaling in Human Bronchial Epithelia

Abstract

P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y6 receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl− secretion via both Ca2+- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca2+ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y6 receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca2+, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y6 receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such “cytokine networks” play an important role in sustaining the airway inflammatory disease.

Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

“…FRET imaging experiments were performed using the MetaFluor Imaging System with FRET module (Molecular Devices, Downingtown, PA, USA) as described previously [24,25]. In brief, 16HBE14o- cells were transfected with the Epac-based cAMP sensor with Lipofectamine 2000 according to the manufacturer's protocol.…”

Carbon Monoxide Inhibits Cytokine and Chloride Secretion in Human Bronchial Epithelia

“…Primary human bronchial epithelial (HBE) cells were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and maintained in Bronchial Epithelial Cell Medium (ScienCell Research Laboratories, USA) as described previously [7,10]. To simultaneously measure intracellular calcium concentration ([Ca…”

“…Previously, we reported that bronchial epithelial damage due to poly-L-arginine (PLA), a surrogate that mimics the effects of eosinophil-derived major basic protein, causes the release of nucleotides and the subsequent activation of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) and NF-κB signalling [7]. The pro-inflammatory cytokines IL-6 and IL-8 are released towards the apical compartment following the exposure of airway epithelial cells to PLA [8].…”

Regulation of Intracellular Calcium by Carbon Monoxide in Human Bronchial Epithelial Cells

“…16HBE14o- cells were maintained in Minimum Essential Medium with Earle’s salt supplemented with 10% (vol/vol) fetal bovine serum, 1% (vol/vol) L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin, as previously described [23]. Cells were cultured in plastic flasks coated with fibronectin and collagen (BD Biosciences, San Jose, CA, USA) and were incubated in humidified 5% CO 2 air at 37°C.…”

“…These cells represent a good model for investigating transepithelial Cl - secretion [18, 19], airway inflammation, and airway remodeling [20-22]. Our previous studies showed that activation of P2Y receptors regulated epithelial Cl - secretion via both Ca 2+ - and cAMP-dependent pathways [23] and activated the PLC- D-myo-inositol 1, 4,5-triphosphate (IP 3 )-Ca 2+ signaling cascade in 16HBE14o- cells, which is also responsible for the secretion of at least two pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, when the cells were challenged by cationic proteins [24]. Therefore, we sought to test whether CO would modulate the P2Y receptor-mediated calcium-signaling pathway.…”