ADENOSINE SIGNALLING PATHWAY AS MODULATOR OF THE INFLAMMATORY RESPONSE

Adenosine is an endogenous nucleoside that has been recognised to be a molecule with autocrine/paracrine functions, acting as a signal molecule to preserve host defence and tissue integrity during inflammation and trauma in addition to its important role as homeostatic regulator. The physiological activities of adenosine involve its interactions with four types of receptors, designed as A1, A2A, A2B and A3. Adenosine mediates its anti-inflammatory activity primarily through the A2A receptor (A2AAR). The ecto-5’nucleotidase/CD73 degrades AMP to adenosine and represents a key enzyme for adenosine accumulation at the site of injury. The aim of this research work was to explore different aspects of adenosine signalling pathway in inflammation. Several findings implicate the adenosine signalling pathway as an innate mechanism to attenuate excessive tissue damage and identify CD73 as critical control points for endogenous adenosine generation. It has been shown that CD73 plays an important role in regulating vascular permeability and leukocyte trafficking in inflammatory disease; and a crucial role in the regulation of immune/inflammatory cell function. A better understanding of the role of CD73 enzyme in the development of inflammatory processes can help to identify new therapeutic strategies aimed at strengthening the endogenous anti-inflammatory mechanisms. For this reason we sought to investigate on the role of CD73, the key enzyme in “switching on” adenosine signalling, in the development of inflammation through its pharmacological blockade by using the selective inhibitor, adenosine 5'-(α,β-methylene) diphosphate (APCP; 400 μg/site), in an in vivo model of acute inflammation represented by carrageenan-induced pleurisy in rats. We found that local inhibition of CD73 significantly increased cell accumulation, exudate formation and pro-inflammatory cytokine content into the pleural cavity in the acute phase of inflammation with no differences in the sub-acute phase. The in vivo treatment with APCP induced cells recruited into the pleural cavity to change in a phenotype with increased ability to migrate in vitro either in the presence or in the absence of a chemotactic stimulus. In parallel, these cells showed a reduced CD73 expression and activity compared to cells collected from control group. In addition, APCP, in vitro, strongly increased the ability of cells from control groups to migrate in the presence of a chemotactic stimulus. Local inhibition of CD73 increased also the infiltration of the lung with polymorphonuclear leukocytes (PMNs) and the degree of lung injury 4 hours following carrageenan injection. The interest to explore the role of adenosine signalling pathway in the control of inflammation has been growing further following evidence that adenosine signalling is also involved in the mechanism of action of some well-known anti-inflammatory drugs. With regard to this, we focused our interest on the possible involvement of adenosine signalling in the anti-inflammatory mechanism of nimesulide, in vivo (rat paw oedema) and in vitro (J774A.1cell line); indeed, there is evidence that nimesulide anti-inflammatory effect is the consequence of regulation of the production and actions of a wide range of inflammatory mediators, independently from the sole cyclooxygenase-2 (COX-2) enzyme inhibition. To date, the molecular mechanisms at the basis of nimesulide peculiar pharmacological effects are still unclear. In vivo, in the model of carrageenan-induced rat paw oedema, we found that the anti-inflammatory effect of nimesulide (5 mg/kg i.p.) was inhibited by pre-treatment with the adenosine A2A receptor antagonist, ZM 241385 (3 mg/kg i.p.), and by local administration of the CD73 inhibitor, APCP (400 μg/paw). Furthermore, we observed increased activity of 5'-nucleotidase/CD73 in plasma and paws of nimesulide-treated rats, 4 h following oedema induction that represented the inflammatory peaking point. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin (PG)E2 (PGE2) production by lipopolysaccharide (LPS)-activated J774 macrophage cell line was again reverted by ZM 241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by LPS-activated small interfering RNA (SiRNA) CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide is, in part, mediated by CD73-derived adenosine acting on A2A receptors. There is evidence that A2AAR activation beside anti-inflammatory effects promotes wound healing and extracellular matrix production; given that extracellular matrix and fibroblasts take an active part in the modulation of inflammation beside wound healing, we investigated whether and how extracellular matrix was involved in the anti-inflammatory effect of A2A receptor. Specifically, we evaluated changes in tissue fibroblast growth factor-2 (FGF-2), an important growth factor for fibroblasts that has been shown to facilitate not only tissue regeneration but also to dampen inflammation, following systemic administration of the A2A agonist, CGS 21680, in a rat model of acute inflammation (paw oedema). We observed that CGS 21680 prevented oedema development and inflammation, confirming an anti-inflammatory effect of A2AR. The effect of CGS 21680 was specific, through A2A adenosine receptor stimulation, as revealed by co-administration with ZM 241385 that reverted CGS 21680 inhibitory effect. On the basis of histological analysis showing an increased matrix deposition following rat treatment with CGS 21680, we evaluated whether the beneficial effect of A2A agonist, CGS 21680, was paralleled by changes in FGF-2 expression. Interestingly, we found that the expression of FGF-2 in rat paws, evaluated at each hour following carrageenan injection, was increased following rat treatment with CGS 21680. Immunofluorescence analysis confirmed data obtained by western blot and also showed spots of co-localization between A2AR and FGF-2. In conclusion, in this research work we demonstrate that CD73 regulates cell migration in the acute phase of inflammation and that the anti-inflammatory effects mediated by A2AR activation are paralleled by changes in extracellular matrix morphology. These findings suggest the important role of CD73/adenosine/A2A signalling in the control of the acute phase of inflammation, characterised by PMNs infiltration, but also in the control of a late phase, characterised by re-arrangement of extracellular matrix. In addition, we also demonstrate that CD73/adenosine/A2A axis is involved in the mode of action of nimesulide. Our study may open a path to re-evaluate the mechanism of action of nimesulide and to identify new therapeutic opportunities in COX-2 inhibitors which display a more potent activity on adenosine signalling. Furthermore, these results may give the cue to project an innovative anti-inflammatory strategy based on the manipulation of endogenous anti-inflammatory pathways