sphingosine-kinase and Peritonitis

The role of sphingosine-1-phosphate (S1P) and its receptors in the pathogenesis of atherosclerosis has not been investigated.. We hypothesized that the S1P receptor 3 (S1P(3)) plays a causal role in the pathogenesis of atherosclerosis.. We examined atherosclerotic lesion development in mice deficient for S1P(3) and apolipoprotein (Apo)E. Although S1P(3) deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S1P(3)(-/-)/ApoE(-/-) double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S1P(3)-deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Bone marrow-derived S1P(3)-deficient macrophages produced less MCP-1 in response to lipopolysaccharide stimulation. In vitro, S1P was chemotactic for wild-type but not S1P(3)-deficient peritoneal macrophages. In vivo, S1P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S1P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S1P(3) in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S1P(3) deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S1P(3) in the arterial injury response.. Our data suggest that S1P(3) mediates the chemotactic effect of S1P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Cell Movement; Cell Proliferation; Disease Models, Animal; Fingolimod Hydrochloride; Inflammation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Peritonitis; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Thioglycolates

During sepsis, activation of phagocytes leads to the overproduction of proinflammatory cytokines, causing systemic inflammation. Despite substantial information regarding the underlying molecular mechanisms that lead to sepsis, several elements in the pathway remain to be elucidated. We found that the enzyme sphingosine kinase 1 (SphK1) is up-regulated in stimulated human phagocytes and in peritoneal phagocytes of patients with severe sepsis. Blockade of SphK1 inhibited phagocyte production of endotoxin-induced proinflammatory cytokines. We observed protection against sepsis in mice treated with a specific SphK1 inhibitor that was enhanced by treatment with a broad-spectrum antibiotic. These results demonstrated a critical role for SphK1 in endotoxin signaling and sepsis-induced inflammatory responses and suggest that inhibition of SphK1 is a potential therapy for septic shock.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Bacterial Proteins; Cytokines; Endotoxins; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Inflammation; Lipopolysaccharides; Lipoproteins; Macrophages; Macrophages, Peritoneal; Male; Mice; Middle Aged; Neutrophils; NF-kappa B; Peritonitis; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C-delta; RNA Interference; Sepsis; Shock, Septic; Signal Transduction; Up-Regulation; Young Adult

The anti-inflammatory activity of the phytoalexin resveratrol (RSV) was evaluated in C5 anaphylatoxin (C5a)-stimulated primary neutrophils and in a mouse model of acute peritonitis. Pretreatment of human and mouse neutrophils with RSV significantly blocked oxidative burst, leukocyte migration, degranulation, and inflammatory cytokine production. The anti-inflammatory activity of RSV was a function of inhibition of sphingosine kinase (SphK) activity (IC(50) approximately 20 microM) within 5 min of exposure, its membrane localization, and SphK1-mediated Ca(2+) release. As an experimental control, the SphK1 pharmacological inhibitor N,N-dimethyl sphingosine (DMS) was used to compare the inhibitory effect of RSV. We also provide evidence that the SphK inhibitory effect of RSV was mediated via its ability to block phospholipase D (PLD) activity and membrane recruitment. Furthermore, RSV blocked ERK1/2 phosphorylation, which functioned independently of SphK1 in this study. To provide in vivo relevance to these data, C5a-induced model of acute peritonitis was established, and the effects of prior injection of RSV were investigated. Indeed, prior injection of RSV virtually completely attenuated the effects of C5a on vascular permeability, neutrophil migration, release of interleukin 1beta, tumor necrosis factor alpha, interleukin 6, and the chemokine MIP-1alpha. Taken together, these data demonstrate strong anti-inflammatory activity of RSV in vitro and in vivo and highlight SphK1 as a potential target of this remarkable phytoalexin. These data could have tremendous implications for the clinical use of RSV in inflammatory pathologies.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biological Transport, Active; Cell Degranulation; Cell Membrane; Chemokines; Chemotaxis, Leukocyte; Complement C5a; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; In Vitro Techniques; Inflammation; Male; Mice; Mice, Inbred BALB C; Neutrophils; NF-kappa B; Peritonitis; Phospholipase D; Phosphotransferases (Alcohol Group Acceptor); Respiratory Burst; Resveratrol; Stilbenes

The transfection of siRNA in vivo is essential for the study of gene functions, target validation, and for gene therapy. However, the successful delivery of siRNA in whole organisms is still very difficult to achieve. A high-pressure delivery technique, called the "hydrodynamics" method, has been used for siRNA transfection in mice. However, it is a method based on a high-speed and high-volume of i.v. injection, which makes it very difficult to implement in vivo, due to vascular breakage. Here, we systematically investigated ways to optimize the siRNA delivery, in order to avoid strong side effects, while achieving a high-efficiency siRNA-gene knockdown. We show here that the amount of siRNA delivered is crucial, as using too little or too much siRNA minimizes the knockdown effect. We demonstrate that by carefully identifying an optimal-minimal volume, and an optimal amount of siRNA, we achieve a high knockdown effect, with a 100% survival rate. We have previously shown that SphK1 plays a key role in anaphylatoxin (C5a) signaling in neutrophils and macrophages. Our approach, optimizing the dosage of siRNA, allowed us to successfully silence our target gene-product (SphK1) in vivo, and enabled us to validate SphK1 as a key player in our in vivo model of C5a-induced acute peritonitis and systemic inflammation including multi-organ damage, demonstrating that this improved siRNA-silencing method not only allowed us to identify SphK1 as a key therapeutic target, but brings us a step closer to the usage of siRNA for therapeutic intervention.

Topics: Anaphylatoxins; Animals; Capillary Permeability; Cattle; Complement C5a; Cytokines; Gene Deletion; Gene Silencing; Humans; Inflammation; Injections; Male; Mice; Monocytes; Neutrophil Infiltration; Peritonitis; Phosphotransferases (Alcohol Group Acceptor); Pressure; RNA, Small Interfering; Sensitivity and Specificity; Time Factors; Transfection

The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-alpha and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-alpha and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.

Topics: Animals; Capillary Permeability; Chemotaxis, Leukocyte; Complement C5a; Cytokines; Humans; Injections, Intraperitoneal; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Monocytes; Neutropenia; Neutrophil Infiltration; Peritoneal Cavity; Peritoneal Lavage; Peritonitis; Phosphotransferases (Alcohol Group Acceptor); Tumor Necrosis Factor-alpha