Adenosine A2A receptors promote adenosine-stimulated wound healing in bronchial epithelial cells.

Adenosine produces a wide variety of physiological effects through the activation of specific adenosine receptors (A(1), A(2A), A(2B), A(3)). Adenosine, acting particularly at the A(2A) adenosine receptor (A(2A)AR), is a potent endogenous anti-inflammatory agent and sensor of inflammatory tissue damage. The complete healing of wounds is the final step in a highly regulated response to injury. Recent studies on epidermal wounds have identified the A(2A)AR as the main adenosine receptor responsible for altering the kinetics of wound closure. We hypothesized that A(2A)AR promotes wound healing in bronchial epithelial cells (BECs). To test this hypothesis, the human BEC line BEAS-2B and bovine BECs (BBECs) were used. Real-time RT-PCR of RNA from unstimulated BEAS-2B cells revealed transcriptional expression of A(1), A(2A), A(2B) and A(3) receptors. Western blot analysis of lysates from BEAS-2B cells and BBECs detected a single band at 44.7 kDa in both the BECs, indicating the presence of A(2A)AR. In a wound healing model, we found that adenosine stimulated wound repair in cultured BBECs in a concentration-dependent manner, with an optimal closure rate observed between 4 and 6 h. Similarly, the A(2A)AR agonist 5'-(N-cyclopropyl)carboxamidoadenosine (CPCA) augmented wound closure, with a maximal closure rate occurring between 4 and 6 h. Inhibition of A(2A)AR with ZM-241385, a known A(2A)AR antagonist, impeded wound healing. In addition, ZM-241385 also attenuated adenosine-mediated wound repair. Kinase studies revealed that adenosine-stimulated airway repair activates PKA by ligating A(2A)AR. Collectively, the data suggest that the A(2A)AR is involved in BEC adenosine-stimulated wound healing and may prove useful in understanding purinergic-mediated actions on airway epithelial repair.

IL-8 inhibits isoproterenol-stimulated ciliary beat frequency in bovine bronchial epithelial cells.

Mucociliary clearance is a critical host defense that protects the lung. The mechanisms by which mucociliary function is altered by inflammation are poorly defined. Chronic exposure to cigarette smoke decreases ciliary beating and interferes with proper airway clearance. Bronchoalveolar lavage (BAL) fluid from smokers and ex-smokers has increased amounts of IL-8, which has played a critical role in airway inflammation. We hypothesized that IL-8 might interfere with stimulated ciliary beating in airway epithelium. To test this hypothesis, we stimulated bovine ciliated bronchial epithelial cells (BBECs) with a known activator of ciliary beat frequency (CBF), isoproterenol (ISO; 100 microM), in the presence or absence of IL-8 (100 pg/mL). We measured CBF digitally using the Sisson-Ammons Video Analysis (SAVA) system. CBF increased in untreated cells exposed to ISO (approximately 3 Hz) over baseline. In contrast, cells pre-incubated with IL-8 failed to respond to ISO. Pretreatment with IL-8 also blocked ISO-stimulated cAMP-dependent kinase (PKA) activation, which is known to control ISO-stimulated CBF. In addition, IL-8 pretreated cells revealed a marked decrease in PKA activity when cells were stimulated with forskolin (FSK; 10 microM). Cells were assayed specifically for cAMP-phosphodiesterase (PDE) activity. ISO-stimulated cells demonstrated an increase in PDE activity as compared to control. Pretreatment with IL-8 had no effect on ISO-stimulated PDE activity. Collectively, these data suggest that IL-8 appears to mediate its effect at the level of adenylyl cyclase. It is also possible that IL-8 may not only act as a chemotactic agent, but also as a potential autocrine/paracrine inhibitor of PKA-mediated stimulation of ciliary motility. In conclusion, IL-8 inhibits beta-agonist dependent ciliostimulation and such inhibition of stimulated ciliary activity may contribute to the impaired mucociliary clearance seen in airway diseases. Furthermore, since IL-8 levels are increased in the airway of cigarette smokers, it is likely they may be more resistant to the cilio and muco-ciliostimulating effects of beta-agonists.

Cytokines potentiate human eosinophil superoxide generation in the presence of N(omega)-nitro-L-arginine methyl ester.

The eosinophilic (EOS) leukocyte has been implicated as a primary effector cell in inflammatory and allergic diseases. Cytokines are among the mediators of inflammatory and allergic diseases which modulate the effector functions of EOS. Certain cytokines, elevated in patients with various allergies, are thought to modulate EOS reactive oxygen species superoxide anion and nitric oxide (NO) responses. Though EOS transcribe and translate mRNA for inducible NO synthase, the effects of cytokines on NO generation remain largely unknown. Thus, we have investigated effects of IL-3, IL-5, GM-CSF, IL-8, RANTES and the proinflammatory cytokines TNF-alpha and IFN-gamma, on superoxide anion and NO generation by clone 15 HL-60 human eosinophilic cells. Cytokine treatments (3 and 18 h) resulted in production of small amounts of superoxide anion which were enhanced by the NO inhibitor L-NAME. In the presence of L-NAME, PMA (1 nM) stimulation significantly increased superoxide anion generation following 3 h treatments with IL-3, TNF-alpha or IFN-gamma. Eighteen hour cytokine treatments with GM-CSF, IL-8, RANTES, IFN-gamma or TNF-alpha primed the cells for enhanced reactive oxygen species following exposure to an EOS stimulant. Inhibition of NO synthesis resulted in increased levels of superoxide anion. Collectively, these results suggest that an environment of proinflammatory cytokines may potentiate the generation of reactive oxygen species by EOS. These results further suggest that at an inflammatory site or during an allergic response, EOS may concomitantly synthesize NO and generate superoxide anion, fractions of which may rapidly react to form the potent oxidant peroxynitrite.

Regulation of 10P2 murine mast cell proliferation and secretory function by stem cell factor or IL-9.

Mast cells are effectors of inflammatory responses. When triggered by immunological or nonimmunological mechanisms, mast cells release potent biological mediators from preformed stores and synthesize others de novo. In previous investigations from this laboratory, the signal transduction pathways of cloned 10P2 cytokine-independent mast cells were explored. Results suggested that 10P2 cells undergo activation-secretion coupling assessed as release of stored [14C]serotonin (5-HT) when challenged with IgE-specific antigen, influx of extracellular calcium, release of intracellular calcium stores, or by direct activation of protein kinase C isozymes. In the present investigations, cytokine proliferative effects and modulatory roles on release of stored [14C]5-HT have been explored. Following passive sensitization with anti-dinitrophenol (anti-DNP) IgE and challenge with DNP, mast cells released up to 32% of the stored [14C]5-HT. Pretreatment of cells with 10, 30, or 50 ng/ml stem cell factor (SCF) did not alter the response. SCF did not directly induce [14C]5-HT release. Pretreatment with 25 ng/ml interleukin-9 (IL-9) significantly potentiated the IgE-antigen release by 51.1%, 35.7%, or 31.6% when challenged with 3, 10 or 30 ng/ml DNP-HSA. Treatment of cells with 1-100 ng/ml SCF for 72 hr resulted in significantly enhanced proliferation whereas this did not occur when cells were treated with 1-100 ng/ml IL-9. Collectively, these results suggest that SCF alone has a proliferative effect, does not alter the IgE-specific antigen signal transduction pathway, and does not directly stimulate mast cell degranulation. In contrast, IL-9 potentiates the IgE-antigen signal transduction response but exerts no proliferative response. Reports of effects of orally administered cytokines are now beginning to emerge. This raises the possibility that cytokines may be a future therapeutic approach to treatment of allergic and nonallergic inflammatory diseases. The 10P2 cytokine-independent mast cell line may be a valuable adjunct to existing mast cell models as this avenue of drug discovery is explored.