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. 2009 Mar;40(3):251-9.
doi: 10.1165/rcmb.2008-0168OC. Epub 2008 Aug 14.

The adenosine a2a receptor inhibits matrix-induced inflammation in a novel fashion

Kara A Scheibner  1 , Sada Boodoo, Samuel Collins, Katharine E Black, Yee Chan-Li, Paul Zarek, Jonathan D Powell, Maureen R Horton
Affiliations

The adenosine a2a receptor inhibits matrix-induced inflammation in a novel fashion

Kara A Scheibner et al. Am J Respir Cell Mol Biol. 2009 Mar.

Abstract

Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment-induced pro-fibrotic genes TNF-alpha, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1alpha while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis.

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Figures

<b>Figure 1.</b>
Figure 1.
A2 adenosine receptor (A2aR) stimulation modulates low-molecular-weight (LMW) hyaluronan (HA)–induced inflammatory gene expression. (A) MH-S macrophages were stimulated with LMW HA (250μg/ml) + CGS (3μM) simultaneously or pre-stimulated with CGS (3μM) for 10 hours before LMW HA, and enzyme-linked immunosorbent assay (ELISA) for TNF-α on cell cultured media was performed after 18 hours of stimulation. These data represent the average of at least three individual experiments. (B) Northern blot analysis of mRNA harvested from thioglycollate elicited peritoneal macrophages pretreated with CGS for 10 hours before stimulation of LMW HA for 6 hours. (C) Graphic representation of mRNA normalized to actin. These data are representative of at least three individual experiments.
<b>Figure 2.</b>
Figure 2.
Adenosine modulates LMW HA–induced inflammatory gene expression via the A2aR. MH-S macrophages were stimulated with CGS (3μM) for 10 hours before LMW HA (250μg/ml) for 18 hours. Cell cultured supernatants were collected and ELISAs performed for (A) TNF-α, (B) MIP-2, (C) KC, (D) IP-10, (E) MIP-1α, and (F) IL-12. These data represent the average of at least three individual experiments.
<b>Figure 3.</b>
Figure 3.
Adenosine modulates LMW HA–induced gene expression specifically via the A2aR. Thioglycollate elicited peritoneal macrophages were isolated from wild-type (WT) and littermate control A2aR-null mice. (A) Dose response of PEC stimulated with LMW HA fragments for 18 hours, after which cultured supernatants were collected and TNF-α ELISA performed. *P = 0.027; #P = 0.04. (B and C) WT or A2aR-null PEC were pre-stimulated with CGS (A2a-specific agonist), NECA (nonselective adenosine receptor agonist), CCPA (A1-specific agonist), and IB-MECA (A3-specific agonist) for 12 hours before LMW HA (250 μg/ml) for 18 hours. Cultured cell supernatants were collected and ELISA for TNF-α performed. Only statistical difference between samples was for WT PEC CGS and NECA v. CCPA and IB-MECA at 1μm dose agonist, * P < 0.05. (D) WT or A2aR-null PEC were pre-stimulated with CGS (3μM) ± the A2aR-specific antagonist ZM for 12 hours before the addition of LMW HA. TNF-α ELISA was performed on cultured cell supernatants after 18 hours. These data represent the average of at least three individual experiments.
<b>Figure 4.</b>
Figure 4.
Adenosine A2aR engagement inhibits LMW HA–induced NF-κB expression. (A) MH-S macrophages were transfected with an NF-κB–driven luciferase reporter construct overnight, and transfected cells were pre-stimulated with CGS (3μM) for 12 hours before stimulation with LMW HA (250 μg/ml) for 18 hours. Cytoplasmic extracts were isolated and luciferase activity was measured. (B and C) MH-S macrophages were pre-stimulated with CGS (3μM) for 12 hours before stimulation with LMW HA (250 μg/ml) for 2 hours before the addition of actinomycin. RT-PCR for TNF-α or IL-12 p40 was performed on RNA isolated at time 0, 1 hour, 3 hours, and 4 hours after actinomycin. These data are representative of at least three individual experiments.
<b>Figure 5.</b>
Figure 5.
Adenosine A2aR engagement modulates LMW HA–induced gene expression via a PKA-independent EPAC-dependent pathway. (A) TNF-α ELISA of cell cultured supernatants from WT PEC stimulated with LMW HA (250 μg/ml) + CGS (3μM) + the PKA inhibitor H89 (1μM) for 18 hours. EPAC activator (EA) directly inhibits LMW HA–induced protein expression by ELISA of TNF-α (B) and induces IL-12 (C) in MH-S macrophages pre-stimulated with EA for 12 hours before stimulation of LMW HA. There are statistical differences between HA v. HA + EA (all doses) for both TNF-α and IL-12, P < 0.002. (D) MH-S macrophages were transfected with EPAC siRNA for 24 hours and Western analysis was performed for EPAC protein. (E) MH-S macrophages were transfected with the control or EPAC siRNA for 24 hours, stimulated with CGS (μM) for 12 hours, and then stimulated with LMW HA for 18 hours (*P = 0.0007 for HA + CGS control siRNA v. EPAC siRNA). Cell conditioned media was collected and TNF-α protein expression was measured by ELISA. These data represent the average of at least three individual experiments.
<b>Figure 6.</b>
Figure 6.
A2aR-null mice are more susceptible to bleomycin-induced lung injury. A2aR-null or WT mice were given 0.375 U of intratracheal bleomycin and followed for 14 days. (A) Survival curves. (B) Day 7 BAL for total cells count (*P = 0.027 for bleomycin-injured WT v. A2aR-null for total BAL cell count). (C) Day 7 hematoxylin and eosin stain of lung tissue. (D) Day 7 BAL HA concentration (*P = 0.002 for bleomycin-injured WT v. A2aR-null for total BAL HA content).
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