In LPS-stimulated human neutrophils, engagement of the adenosine A 2A receptor selectively prevented the expression and release of TNF-α, MIP-1α/CCL3, MIP-1β/CCL4, MIP-2α/CXCL2, and MIP-3α/CCL20. In mice lacking the A 2A receptor, granulocytes that migrated into the air pouch 4 h after LPS injection expressed higher mRNA levels of TNF-α, MIP-1α, and MIP-1β than PMNs from wild-type mice. In mononuclear cells present in the air pouch 72 h after LPS injection, expression of IL-1β, TNF-α, IL-6, and MCP-2/CCL6 was higher in A 2A R knockout mice. In addition to highlighting neutrophils as an early and pivotal target for mediating adenosine anti-inflammatory activities, these results identify TNF-α and the MIP chemokine family as gene products whose expression is pivotally affected by activation of A 2A R in LPS-activated PMNs. Modulation by A 2A R in the production of inflammatory signals by PMNs may thus influence the evolution of an inflammatory response by reducing the activation status of inflammatory cells. Keywords polymorphonuclear leukocytes; experimental animal models; resolution of inflammation 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 (1). Because of their early influence the progression of inflammatory and immune reactions. In response to specific stimuli, PMNs can synthesize and release an array of soluble mediators, including the eicosanoids, leukotriene B 4 , thromboxane A 2 , and prostaglandin E 2 , as well as a number of cytokines/chemokines, such as tumor necrosis factor (TNF)-α, IL-1β, IL-8/CXCL8, and macrophage inflammatory peptides (MIPs) (2-9).Each of these mediators can influence host responses in distinct ways. In particular, chemokines produced by stimulated PMNs, such as IL-8/CXCL8, which specifically attracts and stimulates PMNs, and members of the MIP family, which regulate a wide range of CIHR Author Manuscript CIHR Author ManuscriptCIHR Author Manuscript mononuclear cell functions, including chemotaxis, degranulation, phagocytosis, and inflammatory mediator synthesis (10), contribute to the onset and progression of inflammation, in part through recruitment and/or activation of a range of cell types (11)(12)(13)(14).Although frequent and severe infections seen in patients with defects in PMN function confirm their importance in host defense against infection (15), PMNs also have an enormous destructive capacity and can elicit significant tissue damage; their unchecked activation have been associated with disease states such as ischemia, gram-negative bacterial sepsis and rheumatoid arthritis (16). Elevated numbers of neutrophils in the synovial fluid of patients with arthritis support a role for these cells in joint destruction (17). Unraveling endogenous mechanisms that regulate and specifically limit PMN activation is, in turn, of interest in the context of identifying new and better thera...

Neutrophils, which are often the first to migrate at inflamed sites, can generate leukotriene B4 from the 5-lipoxygenase pathway and prostaglandin E2 through the inducible cyclooxygenase-2 pathway. Adenosine, an endogenous autacoid with several anti-inflammatory properties, blocks the synthesis of leukotriene B4 while it potentiates the cyclooxygenase-2 pathway in fMLP-treated neutrophils, following activation of the A2A receptor. Using the murine air pouch model of inflammation, we observed that inflammatory leukocytes from mice lacking the A2A receptor have less cyclooxygenase-2 induction than wild-type animals. In human leukocytes, A2A receptor activation specifically elicited potentiation of cyclooxygenase-2 in neutrophils, but not in monocytes. Signal transduction studies indicated that the cAMP, ERK1/2, PI-3K and p38K intracellular pathways are implicated both in the direct upregulation of cyclooxygenase-2 and in its potentiation. Together, these results indicate that neutrophils are particularly important mediators of adenosine's effects. Given the uncontrolled inflammatory phenotype observed in knockout mice and in view of the potent inhibitory actions of prostaglandin E2 on inflammatory cells, an increased cyclooxygenase-2 expression resulting from A2A receptor activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine.

In the present study, we characterized the generation of prostaglandin (PG)E 2 in human neutrophils. We found that the Ca 2+ -dependent type IV cytosolic phospholipase A 2 (cPLA 2 ) was pivotally involved in the COX-2-mediated generation of PGE 2 in response to a calcium ionophore, as determined by the use of selected PLA 2 inhibitors. PGE 2 biosynthesis elicited by bacterialderived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA 2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE 2 production becomes favored over that of LTB 4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE 2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA 2 activation. Of the main eicosanoids produced by neutrophils, only LTB 4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE 2 biosynthesis in neutrophils.