cAMP has largely inhibitory effects on components of macrophage activation, yet downstream mechanisms involved in these effects remain incompletely defined. Elevation of cAMP in alveolar macrophages (AMs) suppresses FcγR-mediated phagocytosis. We now report that protein kinase A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA inhibitory peptide 14–22) failed to prevent this suppression in rat AMs. We identified the expression of the alternative cAMP target, exchange protein directly activated by cAMP-1 (Epac-1), in human and rat AMs. Using cAMP analogs that are highly specific for PKA (N6-benzoyladenosine-3′,5′-cAMP) or Epac-1 (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cAMP), we found that activation of Epac-1, but not PKA, dose-dependently suppressed phagocytosis. By contrast, activation of PKA, but not Epac-1, suppressed AM production of leukotriene B4 and TNF-α, whereas stimulation of either PKA or Epac-1 inhibited AM bactericidal activity and H2O2 production. These experiments now identify Epac-1 in primary macrophages, and define differential roles of Epac-1 vs PKA in the inhibitory effects of cAMP.

Leukotrienes (LTs) are lipid mediators that participate in inflammatory diseases and innate immune function. We sought to investigate the importance of LTs in regulating the microbicidal activity of alveolar macrophages (AMs) and the molecular mechanisms by which this occurs. The role of LTs in enhancing AM microbicidal activity was evaluated pharmacologically and genetically using in vitro challenge with Klebsiella pneumoniae. Exogenous LTs increased AM microbicidal activity in a dose-and receptor-dependent manner, and endogenous production of LTs was necessary for optimal killing. Leukotriene B 4 (LTB 4 ) was more potent than cysteinyl LTs. An important role for nicotinamide adenine dinucleotide (NADPH) oxidase in LT-induced microbicidal activity was indicated by the fact that bacterial killing was abrogated by the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10 M) and in AMs derived from gp91phox-deficient mice. By contrast, LT-induced microbicidal activity was independent of the generation of nitric oxide. LTs increased H 2 O 2 production, and LTB 4 was again the more potent agonist. Both classes of LTs elicited translocation of p47phox to the cell membrane, and LTB 4 induced phosphorylation of p47phox in a manner dependent on protein kinase C-␦ (PKC-␦) activity. In addition, the enhancement of microbicidal activity by LTs was also dependent on PKC-␦ activity. Our results demonstrate that LTs, especially LTB 4 , enhance AM microbicidal activity through the PKC-␦-dependent activation of NADPH oxidase. IntroductionPneumonia is the leading cause of death from infection in the United States, 1 and its global mortality is 4.3 million people per year. 2 This problem is compounded by growing numbers of immunosuppressed patients and multidrug-resistant microorganisms. Developing more effective agents for the prevention and treatment of pneumonia requires a better understanding of how innate pulmonary defense mechanisms are regulated.The alveolar macrophage (AM) patrols the epithelial surface of the distal lung and maintains sterility by phagocytosing and killing microorganisms. 3,4 AMs are the first line of defense against invading microorganisms and, as such, are capable of secreting cytokines, lipid mediators, and microbicidal molecules. Among the lipid mediators generated by AMs, the leukotrienes (LTs) play an important role in lung innate immunity, inducing neutrophil recruitment and enhancing macrophage antimicrobial functions. 5 LTs are derived from the metabolism of the cell-membrane fatty acid arachidonic acid (AA) through the enzyme 5-lipoxygenase (5-LO), in concert with its helper protein, 5-LO-activating protein (FLAP). 6 5-LO oxygenates AA to the intermediate 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is either enzymatically reduced by 5-LO to the unstable epoxide leukotriene A 4 (LTA 4 ) or alternatively is reduced to 5-hydroxyeicosatetraenoic acid (5-HETE). LTA 4 can be hydrolyzed to form LTB 4 or can be conjugated with glutathione to form the cysteinyl LTs (cysLTs), LTC 4 , LTD 4 , and ...

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