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Immunomodulatory impact of the A_2A adenosine receptor on the profile of chemokines produced by neutrophils

Centre de Recherche en Rhumatologie et Immunologie du CHUQ (CHUL), and Department of Anatomy-Physiology, Faculty of Medicine, Laval University, Quebec, Canada; and
^* School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia, Australia

¹Correspondence: Centre de recherche en rhumatologie-immunologie, 2705 Laurier Blvd., T1-49, Sainte-Foy, QC, G1V 4G2, Canada. E-mail: marc.pouliot{at}crchul.ulaval.ca

SPECIFIC AIMS

Polymorphonuclear neutrophils (PMNs) are the most abundant circulating leukocytes and are usually the first to migrate toward damaged/infected tissues where they accumulate to participate in initial phases of the inflammatory response. Because of their early involvement in the host's response, PMNs can, in addition to directly fighting infections, influence inflammatory and immune reactions. In particular, cytokines and chemokines produced by stimulated PMN can regulate a wide range of inflammatory cell functions including chemotaxis, degranulation, phagocytosis, and inflammatory mediator synthesis, thus contributing to the onset and progression of inflammation.

The A_2A adenosine receptor (A_2AR) takes part in a nonredundant physiological negative feedback mechanism that limits and terminates both tissue-specific and systemic inflammatory responses. No other mechanism of down-regulation of inflammation in vivo appears able to fully compensate a lack of A_2AR, as was clearly demonstrated in mice deficient in the A_2AR that developed extensive tissue damage in response to subthreshold concentrations of inflammatory stimuli. Accumulating evidence indicates that PMNs constitute an early and important target for the modulatory activities of adenosine through activation of the A_2AR. In PMNs, A_2AR engagement has been reported to inhibit their adhesion to endothelial cells, phagocytosis, and generation of superoxide anions.

In the present study, we investigated the effect of adenosine and of A_2AR activation on the profile of cytokines and chemokines produced by PMNs stimulated through toll-like receptor 4 engagement, both in vitro and in vivo. We report here that engagement of A_2AR prevents the generation of selected chemokines, an event likely to mediate, at least in part, anti-inflammatory activities resulting of A_2AR engagement.

In the present study, the specific aims were to 1) establish the use of real-time PCR as an efficient approach to compare mRNA levels; 2) determine the impact of A_2AR activation on the mRNA levels of inflammatory gene products in LPS-stimulated PMNs; 3) determine the effect of A_2AR activation on the release of cytokines/chemokines by LPS-stimulated PMNs; 4) determine the impact in vivo of the absence of a functional A_2AR on the mRNA levels of inflammatory gene products in inflammatory PMNs; and 5) determine the impact in vivo of the absence of a functional A_2AR on the levels of cytokines/chemokines at the injured site.

PRINCIPAL FINDINGS

1. LPS-stimulation of neutrophils stimulates mRNA expression of chemokines: impact of A_2AR engagement
Real-time PCR was used to evaluate the impact of A_2AR activation on mRNA levels of a comprehensive selection of cytokines and chemokines. The level of mRNA for these inflammatory cytokines/chemokines was increased after stimulation with LPS. The results indicate that A_2AR activation has a potent modulatory effect on the mRNA levels of IL-1ß, TNF-, MIP-1/CCL3, and MIP-1ß/CCL4, MIP-2/CXCL2, and MIP-3/CCL20, while not affecting those of IL-8/CXCL8. These results also confirmed the validity of the real-time PCR approach for the comparison of mRNA levels.

2. A_2AR activation prevents the release of TNF- and MIPs from neutrophils
Cytokine production can be regulated at several levels: transcription, mRNA stabilization, translation, storage, and secretion. At this latter level, stimulation of PMNs with LPS provoked the release of TNF-, MIP-1/CCL3 and MIP-1ß/CCL4, MIP-2/CXCL2, MIP-3/CCL20, and IL-8/CXCL8 (Fig. 1 ). Withdrawal of adenosine during stimulation enhanced the release of TNF-, MIP-1/CCL3, and MIP-1ß/CCL4, MIP-2/CXCL2 and, to a lesser extent, MIP-3/CCL20. On the other hand, activation of the A_2AR with CGS 21680 inhibited TNF- secretion, which was comparable to levels found in the supernatants of saline-treated PMNs. A_2AR activation also had a potent inhibitory effect on the release of MIP-1/CCL3, MIP-1ß/CCL4, MIP-2/CXCL2, and MIP-3/CCL20. Activation of the A_2AR had no significant effect on the secretion of IL-8/CXCL8. Secretion of IL-1ß was barely detectable.

View larger version (30K): [in this window] [in a new window]   Figure 1. A2AR activation prevents the release of TNF- and MIPs from neutrophils. A–G) PMNs were stimulated with LPS for up to 20 h, alone or in the presence of ADA and CGS 21680. Cell-free samples were processed for the determination of indicated cytokines/chemokines by specific ELISAs. In each panel, results represent the mean (±SE) of at least n= 3 experiments performed in identical conditions with different donors. H) The impact of A2AR engagement on the release of the indicated soluble factors at t = 20 h is summarized and expressed as % of inhibition (mean±SE, n=3). *Statistically different from LPS+ADA-treated cells.

3. Absence of a functional A_2AR exacerbates the expression of selected chemokines from neutrophils in vivo
The impact of A_2AR activation on the production of chemokines by PMNs in vivo was assessed with the murine air pouch model of inflammation and leukocyte recruitment, using wild-type (A_2AR^+/+) and knockout (A_2AR^–/–) CD1 mice. Dorsal air pouches were injected with LPS for 4 h, which increased the recruitment of leukocytes, predominantly PMNs. mRNA expression of a selection of key inflammatory genes, cytokines, and chemokines were compared in migrated leukocytes from A_2AR^+/+ and A_2AR^–/– mice by real-time PCR. Absence of a functional A_2AR was accompanied by a significant increase of TNF-, MIP-1/CCL3, and MIP-1ß/CCL4), results that are in line with data obtained in vitro with human PMNs. No significant difference between A_2AR^+/+ and A_2AR^–/– mice was observed in lining tissues of the air cavity for any of the genes tested. These results strongly support a role in vivo for A_2AR in regulating the expression of important inflammatory cytokines and chemokines in inflammatory PMNs, particularly that of TNF-, MIP-1/CCL3, and MIP-1ß/CCL4.

4. Absence of a functional A_2AR exacerbates the expression of proinflammatory cytokines/chemokines from mononuclear leukocytes in vivo
As is the normal course of inflammation and wound healing process, PMNs are the first cell type to migrate into the air pouch model after injection of LPS. Within days, mononuclear cells replace PMN and become the most numerous leukocytes present in exudates. We analyzed cell migration and activation at longer time points, up to 96 h after LPS injection. As early as 48 h after LPS injection, the vast majority of cells harvested from exudates were mononuclear cells, as assessed by Giemsa staining. Real-time PCR comparison of mRNA levels in leukocytes collected from the air pouch from A_2AR^+/+ and A_2AR^–/– mice revealed the increased expression of TNF-, IL-6, MCP-2, IL-1ß, and, to a lesser extent, IL-1RA. As observed at 4 h poststimulation, expression in lining tissues was not different for any of the analyzed genes.

CONCLUSIONS AND SIGNIFICANCE

Among the main cytokines/chemokines produced by inflammatory granulocytes, A_2AR activation was found to profoundly and selectively inhibit the generation of TNF-, MIP-1, and MIP-2. Those findings were corroborated in vivo using an inflammatory model of leukocyte recruitment and activation, the murine air pouch, confirming TNF- and MIPs as pivotal gene products targeted by A_2AR actions. We observed, also in vivo, that the profile of cytokines produced by mononuclear cells, which follow granulocytes at the injured site in the normal course of inflammation, is exacerbated in the absence of a functional A_2AR, indicating a cause-and-effect relationship between A_2AR activation and modulation of an inflammatory cell activation.

Considerable evidence now indicates that A_2AR engagement as profound inhibitory consequences on PMN functions; its impact on cell movement appears marginal, however. Results obtained herein showed that adenosine does not significantly affect IL-8—the main chemokine produced by PMNs—either at the expression or release levels. In the murine air pouch model of inflammation, where resident cells appear to serve as the major mechanism for leukocyte recruitment, none of the genes assessed in these tissues was affected in the present study. Accordingly, the number of cells recruited in the air pouches were comparable in A_2AR-knocked out and wild-type mice. Thus, adenosine appears to exert its anti-inflammatory functions predominantly by modulating the profile of inflammatory mediators generated by leukocytes present at sites of injury rather than by affecting their numbers.

Results presented in this study identify granulocytes as an early and pivotal target that can mediate adenosine's anti-inflammatory actions. Early modulation in the generation of inflammatory mediators produced by granulocytes is likely to affect the activation status of subsequent inflammatory cell types, including macrophages, and, in turn, to influence the overall inflammatory response. This study points to adenosine as a potentially important therapeutical target; within an inflammatory context, early intervention may prove beneficial because of the particular responsiveness of granulocytes to A_2AR activation.

View larger version (21K): [in this window] [in a new window]   Figure 2. Modulation of the profile of cytokines/chemokines generated in inflammatory neutrophils by activation of the adenosine A2A receptor. In LPS-stimulated neutrophils, A2AR activation resulted in the suppression of TNF-, MIP-1, MIP-1ß, MIP-2, and MIP-3. IL-8 was largely unaffected. In vivo experiments using mice knocked out for the A2AR confirmed that TNF-, MIP-1, MIP-1ß, and MIP-2 were pivotally targeted by A2AR. Modulation in the release of these important mediators may have a significant effect on the activation status of other inflammatory cells, including monocytes, and in turn influence the overall progression of an inflammatory response. A2AR, adenosine A2A receptor; LPS/LBP, lipopolysaccharide/lipopolysaccharide binding protein; TLR4, Toll-like receptor-4.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4804fje;

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