RESUMO
Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis. Strategies to block inflammatory mediators such as PAF have been investigated as adjuvant therapies for sepsis. PAF-AH, the enzyme responsible for PAF degradation, showed positive results in pre-clinical studies and phase II clinical trials, but the results of a phase III study were disappointing. In this study, we investigated the potential protective mechanism of PAF-AH in sepsis using the murine model of cecal ligation and puncture (CLP). Treatment with rPAF-AH increased peritoneal fluid levels of the anti-inflammatory mediators MCP-1/CCL2 after CLP. The numbers of bacteria (CFU) in the peritoneal cavity were decreased in the rPAF-AH-treated group, indicating more efficient bacterial clearance after rPAF-AH treatment. Interestingly, we observed increased levels of nitric oxide (NO) after PAF-AH administration, and rPAF-AH treatment did not decrease CFU numbers either in iNOS-deficient mice or in CCR2-deficient mice. We concluded that administration of exogenous rPAF-AH reduced inflammatory injury, altered cytokine levels and favored bacterial clearance with a clear impact on mortality through modulation of MCP-1/CCL2 and NO levels in a clinically relevant sepsis model.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/administração & dosagem , Sepse/tratamento farmacológico , Sepse/microbiologia , Animais , Quimiocina CCL2/biossíntese , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Cavidade Peritoneal/microbiologia , Proteínas Recombinantes/administração & dosagem , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Salmonella typhimurium , Sepse/metabolismoRESUMO
Platelet-activating factor (PAF) is a proinflammatory mediator that plays a central role in acute lung injury (ALI). PAF- acetylhydrolases (PAF-AHs) terminate PAF's signals and regulate inflammation. In this study, we describe the kinetics of plasma and bronchoalveolar lavage (BAL) PAF-AH in the early phase of ALI. Six pigs with oleic acid induced ALI and two healthy controls were studied. Plasma and BAL samples were collected every 2h and immunohistochemical analysis of PAF-AH was performed in lung tissues. PAF-AH activity in BAL was increased at the end of the experiment (BAL PAF-AH Time 0=0.001+/-0.001 nmol/ml/min/g vs Time 6=0.031+/-0.018 nmol/ml/min/g, p=0.04) while plasma activity was not altered. We observed increased PAF-AH staining of macrophages and epithelial cells in the lungs of animals with ALI but not in healthy controls. Our data suggest that increases in PAF-AH levels are, in part, a result of alveolar production. PAF-AH may represent a modulatory strategy to counteract the excessive pro-inflammatory effects of PAF and PAF-like lipids in lung inflammation.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/biossíntese , Pulmão/enzimologia , Síndrome do Desconforto Respiratório/enzimologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Animais , Líquido da Lavagem Broncoalveolar/química , Feminino , Imuno-Histoquímica , Cinética , Pulmão/metabolismo , Ácido Oleico , Síndrome do Desconforto Respiratório/induzido quimicamente , Suínos , Fatores de TempoRESUMO
Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis and in those with systemic inflammatory response syndrome. Strategies to block inflammatory mediators, often with complicated outcomes, are currently being investigated as new adjuvant therapies for sepsis. Here, we determined if administration of recombinant platelet-activating factor (rPAF)-acetylhydrolase (rPAF-AH), an enzyme that inactivates PAF and PAF-like lipids, protects mice from inflammatory injury and death after administration of lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). Administration of rPAF-AH increased plasma PAF-AH activity and reduced mortality in both models. Treatment with rPAF-AH increased peritoneal fluid levels of monocyte chemoattractant protein 1/CCL-2 and decreased interleukin 6 and migration inhibitory factor levels after LPS administration or CLP. Administration of a broad-spectrum antibiotic together with rPAF-AH was more protective than single treatment with either of these agents. The combined treatment was associated with reduced interleukin 6 levels in mice subjected to CLP. We observed acute decreases in plasma PAF-AH activity in mice subjected to CLP or challenged with LPS and in human patients with sepsis. We conclude that alterations in the endogenous PAF-AH contribute to the pathophysiology of sepsis and that administration of exogenous rPAF-AH reduces inflammatory injury and mortality in models relevant to the clinical syndrome. Variations in endogenous PAF-AH activity may potentially account for variable responses to exogenous rPAF-AH in previous clinical trials. Serial measurements of plasma PAF-AH activity in murine models demonstrate dynamic regulation of the endogenous enzyme, potentially explaining the variations in human subjects.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/administração & dosagem , Fator de Ativação de Plaquetas/antagonistas & inibidores , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Animais , Antibacterianos/administração & dosagem , Citocinas/sangue , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Síndrome de Resposta Inflamatória Sistêmica/sangueRESUMO
Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/fisiologia , Plaquetas/enzimologia , Inflamação/metabolismo , Fator de Ativação de Plaquetas/fisiologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Regulação da Expressão Gênica , Fosfolipases A2 , Fator de Ativação de Plaquetas/química , Polimorfismo GenéticoRESUMO
Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.
Assuntos
Animais , /fisiologia , Plaquetas/enzimologia , Inflamação/metabolismo , Fator de Ativação de Plaquetas/fisiologia , /química , /genética , Regulação da Expressão Gênica , Polimorfismo Genético , Fator de Ativação de Plaquetas/químicaRESUMO
Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.
Assuntos
Corpos de Inclusão/metabolismo , Leucócitos/metabolismo , Lipoproteínas LDL/metabolismo , Peroxissomos/metabolismo , Fator de Ativação de Plaquetas/fisiologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/fisiologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/fisiologia , Ciclo-Oxigenase 2 , Sinergismo Farmacológico , Feminino , Humanos , Isoenzimas/metabolismo , Leucócitos/fisiologia , Lipoproteínas LDL/administração & dosagem , Lipoproteínas LDL/farmacologia , Macrófagos Peritoneais/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Oxirredução , Peroxissomos/enzimologia , Peroxissomos/fisiologia , Fosfolipídeos/metabolismo , Fator de Ativação de Plaquetas/administração & dosagem , Fator de Ativação de Plaquetas/metabolismo , Cavidade Pleural/citologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Cavidade Torácica , Fatores de Transcrição/metabolismoRESUMO
Oxidized low-density lipoprotein (LDL) contains inflammatory agents, including oxidatively fragmented phospholipids that activate the platelet-activating factor (PAF) receptor, but in vivo events caused by these pathologically generated agents are not well defined. Injection of PAF-like lipids derived from oxidized LDL, or C(4)-PAF that is a major PAF-like lipid in these particles, into the pleural cavity of mice resulted in rapid monocyte, neutrophil, and eosinophil accumulation. Increased numbers of intracellular lipid bodies in these cells show they were in an inflammatory environment. Leukocyte recruitment was abolished by a PAF receptor antagonist, as expected. PAF-like lipids induced 5-lipoxygenase expression in leukocytes, mRNA expression for monocyte chemoattractant protein-1 (MCP-1) and other chemokines, synthesis of MCP-1, and leukotriene B(4). The 5-lipoxygenase inhibitor zileuton impaired neutrophil influx, while MCP-1 had a more global role, as determined with MCP-1(-/-) mice. The lack of MCP-1 abrogated leukocyte accumulation and lipid body formation both in vivo and in vitro and chemokine transcription in vivo, and reduced in vivo leukotriene B(4) production. Thus, PAF-like phospholipids in oxidized LDL induce an inflammatory infiltrate through the PAF receptor, chemokine transcription, lipid body formation, and 5-lipoxygenase expression in leukocytes. MCP-1 has a key role in this inflammatory response, and 5-lipoxygenase products are essential for neutrophil recruitment into the inflamed pleural cavity.