RESUMO
Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule responsible for directing cellular responses to extracellular signals. Once believed to signal exclusively through its ability to bind protein kinase A (PKA), recent research has revealed alternative cAMP-binding targets involved in PKA-independent processes. In this study we addressed the hypothesis that the guanine nucleotide exchange protein directly activated by cAMP (Epac-1) and PKA differentially regulate inflammatory mediator production in distinct phagocytic cell types. To accomplish this, we compared the release of cAMP-regulated polypeptide inflammatory mediators in both macrophages (obtained from the lung and peritoneum) and bone marrow-derived dendritic cells (DCs) stimulated with bacterial endotoxin. Using the highly selective Epac-1 and PKA activating cAMP analogs 8-pCPT-2 -O-Me-cAMP and 6-Bnz-cAMP, respectively, we found that macrophages differ from DCs in the involvement of these distinct cAMP pathways in modulating inflammatory mediator release in response to endotoxin. Whereas the regulation of cytokine and chemokine production in macrophages by cAMP was solely dependent on PKA, we found that both Epac-1 and PKA activation could regulate mediator production in DCs. This finding may be important in the pharmacologic regulation of immune responses through manipulation of cAMP signaling cascades and contributes to our understanding of the differences between these cell types.
Assuntos
AMP Cíclico/metabolismo , Citocinas/biossíntese , Células Dendríticas/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/imunologia , Macrófagos Peritoneais/imunologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinas/imunologia , Células Dendríticas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Especificidade de Órgãos/imunologia , Transdução de Sinais/imunologiaRESUMO
Recent studies have demonstrated a central role for the exchange protein activated by cAMP (Epac) in the inhibition of Fcgamma-receptor-mediated phagocytosis and bacterial killing by prostaglandin E(2) (PGE(2)) in macrophages. However, the subcellular localization of Epac, and its primary target Rap1, has yet to be determined in primary macrophages. Therefore, we used immunofluorescent techniques and phagosome isolation to localize Epac-1 and Rap1 in alveolar macrophages. Epac-1 was predominantly expressed on punctate and tubular membranes throughout the cell body; on the plasma membrane; and co-localized with microtubule organizing centers (MTOCs). Rap1 was abundant on punctate membranes, less abundant on plasma membrane, and also found on MTOCs. Following PGE(2) treatment, Epac-1, but not Rap1, accumulated on the nuclear envelope and disappeared from MTOCs. By immunofluorescent microscopy, both Epac-1 and Rap1 were seen to associate with phagosomes containing IgG-opsonized beads, but this association appeared weak, as we failed to observe such interactions in phagosomes isolated from cells at various time points after bead ingestion. Strikingly, however, Epac-1, but not Rap1, appeared to accumulate on maturing phagosomes, but only after PGE(2) treatment (or treatment with a selective Epac-1 agonist). This association was confirmed in isolated phagosome preparations. The changes in Epac-1 localization were too slow to account for the inhibitory effects of PGE(2) on phagocytosis. However, the appearance of Epac-1 on late phagosomes following PGE(2) treatment might be important for suppressing H(2)O(2) production and inhibiting the killing of intraphagosomal pathogens. The absence of Rap1 on late phagosomes suggests that the effect of Epac-1 might not require Rap1.
Assuntos
Dinoprostona/farmacologia , Fatores de Troca do Nucleotídeo Guanina/análise , Macrófagos Alveolares/metabolismo , Fagocitose/fisiologia , Receptores de IgG/metabolismo , Proteínas rap1 de Ligação ao GTP/análise , Animais , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peróxido de Hidrogênio/metabolismo , Microscopia de Fluorescência , Fagossomos/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais , Fatores de Tempo , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Alveolar macrophages abundantly express PPAR-gamma, with both natural and synthetic agonists maintaining the cell in a quiescent state hyporesponsive to antigen stimulation. Conversely, agonists upregulate expression and function of the cell-surface receptor CD36, which mediates phagocytosis of lipids, apoptotic neutrophils, and other unopsonized materials. These effects led us to investigate the actions of PPAR-gamma agonists on the Fcgamma receptor, which mediates phagocytosis of particles opsonized by binding of immunoglobulin G antibodies. We found that troglitazone, rosiglitazone, and 15-deoxy-Delta12,14-prostaglandin J2 increase the ability of alveolar, but not peritoneal, macrophages to carry out phagocytosis mediated by the Fcgamma receptor. Receptor expression was not altered but activation of the downstream signaling proteins Syk, ERK-1, and ERK-2 was observed. Although it was previously known that PPAR-gamma ligands stimulate phagocytosis of unopsonized materials, this is the first demonstration that they stimulate phagocytosis of opsonized materials as well.
RESUMO
PGI(2) (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI(2), in the regulation of both innate and acquired immunity. As PGI(2) is unstable, we sought to define the effects of various PGI(2) analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, carbaprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the G(alphas) protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI(2) analogs and more closely resembled the effects of PGE(2). Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies differences in immunoregulatory properties of clinically administered PGI(2) analogs. These studies are the first to explore the capacity of PGI(2) to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents.