Prostaglandin E 2 (PGE 2 ), originally discovered as a pro-inflammatory mediator, also inhibits several chemoattractant-elicited neutrophil functions, including adhesion, secretion of cytotoxic enzymes, production of superoxide anions, and chemotaxis. In this study, we have examined the effects of PGE 2 and prostaglandin E (EP) receptor-selective agonists/antagonists on several steps of the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced phospholipase D (PLD) activation pathway in human neutrophils to elucidate the PGE 2 inhibitory mechanism. PGE 2 and EP 2 receptor agonists inhibited the stimulation of the activity of PLD induced by fMLP in a concentration-dependent manner. The fMLP-stimulated translocation to membranes of protein kinase C ␣, Rho, and Arf GTPases was diminished in the presence of PGE 2 or EP 2 agonists. Moreover, PGE 2 and EP 2 agonists decreased the activation of phosphatidylinositol 3-kinase ␥ (PI3K␥) and Tec kinases as well as the tyrosine phosphorylation of proteins stimulated by fMLP. These data provide strong evidence that 1) the inhibitory effects of PGE 2 on the fMLP-induced PLD activation pathway were mediated via EP 2 receptors and that 2) the suppression of PI3K␥ activity was the crucial step in the EP 2 -mediated inhibition of the fMLP-induced signaling cascade.

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PGE(2) and other cAMP-elevating agents are known to down-regulate most functions stimulated by fMLP in human polymorphonuclear neutrophils. We reported previously that the inhibitory potential of PGE(2) resides in its capacity to suppress fMLP-stimulated PI-3Kgamma activation via the PGE(2) receptor EP(2) and hence, to decrease phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] formation. Akt activity is stimulated by fMLP through phosphorylation on threonine 308 (Thr308) and serine 473 (Ser473) by 3-phosphoinositide-dependent kinase 1 (PDK1) and MAPK-AP kinase (APK)-APK-2 (MAPKAPK-2), respectively, in a PI-3K-dependent manner. Despite the suppression of fMLP-induced PI-3Kgamma activation observed in the presence of PGE(2), we show that Akt is fully phosphorylated on Thr308 and Ser473. However, fMLP-induced Akt translocation is decreased markedly in this context. PGE(2) does not affect the phosphorylation of MAPKAPK-2 but decreases the translocation of PDK1 induced by fMLP. Other cAMP-elevating agents such as adenosine (Ado) similarly block the fMLP-induced PI-3Kgamma activation process but do not inhibit Akt phosphorylation. However, Akt activity stimulated by fMLP is down-regulated slightly by agonists that elevate cAMP levels. Whereas protein kinase A is not involved in the maintenance of Akt phosphorylation, it is required for the inhibition of Akt translocation by PGE(2). Moreover, inhibition of fMLP-stimulated PI-3Kdelta activity by the selective inhibitor IC87114 only partially affects the late phase of Akt phosphorylation in the presence of PGE(2). Taken together, these results suggest that cAMP-elevating agents, such as PGE(2) or Ado, are able to induce an alternative mechanism of Akt activation by fMLP in which the translocation of Akt to PI(3,4,5)P(3)-enriched membranes is not required prior to its phosphorylation.

The aim of this study was to assess in human neutrophils theimplication of an adenosine 3′,5′-cyclic monophosphate (cAMP)-dependentpathway in the inhibitory effects of A2a receptor engagement. We foundthat Ro20-1724, a cAMP phosphodiesterase inhibitor, in the presence ofadenosine deaminase (ADA) or A2a receptor antagonists renderedtransient the fMLP-induced sustained increases in cAMP levels. The roleof A2a receptor stimulation was demonstrated by the ability of the A2areceptor agonist, CGS21680, to prevent ADA-mediated reduction of thepersistent cAMP elevation induced by fMLP. Persistent cAMP elevationcorrelated with inhibition of fMLP-induced PLD activation andrecruitment of Arf, RhoA, and PKC to membranes. The suppressive effectof CGS21680 or isoproterenol, a β-adrenergic receptor agonist, wasincreased by Ro20-1724 or by the adenylyl cyclase activator, forskolin, and reversed, at least in part, by the inhibitor of adenylyl cyclase,2′,5′-dideoxyadenosine. The activator of protein kinase A (PKA),Sp-cAMP inhibited fMLP-induced PLD activation and translocation of Arfand RhoA to membranes. In contrast, the suppression by A2a receptorstimulation of fMLP-induced PLD activation and cofactor recruitment wasantagonized by PKA inhibitors, Rp-cAMP and H89. In conclusion, A2areceptor occupancy by extracellular adenosine inhibits fMLP-inducedneutrophil activation via cAMP and PKA-regulated events.