Ulf Sibelius

ω-3 vs. ω-6 lipid emulsions exert differential influence on neutrophils in septic shock patients: impact on plasma fatty acids and lipid mediator generation

ObjectiveTo compare the effects of a conventional ω-6 lipid infusion and a fish oil based (ω-3) lipid infusion for parenteral nutrition on neutrophil function, lipid mediators, and plasma free fatty acids.Design and settingOpen-label, randomized, pilot study in a university hospital medical intensive care unit and experimental laboratory.Patients and participantsTen patients with septic shock and eight healthy controls.InterventionsPatients (five per group) requiring parenteral nutrition received intravenously either a ω-3 or a ω-6 lipid emulsion for a 10-day period.Measurements and resultsAt baseline levels of plasma free fatty acids were elevated several-fold, including high concentrations of the ω-6 lipid precursor arachidonic acid (AA). Neutrophils isolated from septic patients displayed markedly reduced responsiveness to ex vivo stimulation, including lipid mediator generation [leukotrienes (LT), PAF], respiratory burst, and phosphoinositide hydrolysis signaling. Under the ω-6 lipid infusion regimen abnormalities in plasma free fatty acids and impairment of neutrophil functions persisted or worsened. In contrast, a rapid switch in the plasma free fatty acid fraction to predominance of the ω-3 acids eicosapentaenoic acid and docosahexaenoic acid over AA occurred in response to ω-3 lipid infusion. LTB5, in addition to LTB4, appeared upon neutrophil stimulation originating from these patients, and neutrophil function was significantly improved in the ω-3 lipid group.Conclusionsω-3 vs. ω-6 lipid emulsions differentially influence the plasma free fatty acid profile with impact on neutrophil functions. Lipid-based parenteral nutrition in septic patients may thus exert profound influence on sequelae and status of immunocompetence and inflammation.

Endotoxin-Induced Myocardial Tumor Necrosis Factor-α Synthesis Depresses Contractility of Isolated Rat Hearts Evidence for a Role of Sphingosine and Cyclooxygenase-2–Derived Thromboxane Production

BackgroundAlthough endotoxin (lipopolysaccharides, LPS) is recognized as a mediator of septic cardiodepression, its cardiac effects are still not fully elucidated. Methods and ResultsPerfusion of isolated rat hearts with LPS for 180 minutes resulted in a decline of left ventricular contractility after 90 minutes, whereas coronary perfusion pressure remained unaffected. This cardiodepression was paralleled by a release of tumor necrosis factor (TNF)-&agr; into the perfusate and preceded by myocardial TNF-&agr; mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-&agr; antiserum or the ceramidase inhibitor N-oleoylethanolamine. In contrast, the cardiac release of nitric oxide (NO) was not augmented by LPS. Immunohistochemical studies of LPS-perfused hearts revealed a positive staining for the constitutive (NOSIII) but not for the inducible NO synthase (NOSII). Accordingly, NOSII mRNA levels commenced to increase only at the very end of the LPS perfusion period. Progressive liberation of thromboxane (Tx) A2 and prostacyclin was induced by LPS together with myocardial cyclooxygenase (Cox)-2 mRNA expression. Both nonselective inhibition of Cox by indomethacin and selective inhibition of the inducible Cox-2 by NS-398 abolished prostanoid release. Interestingly, the generation of TNF-&agr; and the associated cardiodepression caused by LPS were reduced by indomethacin, NS-398 and the Tx-receptor antagonist daltroban. ConclusionsLPS depresses contractility of isolated rat hearts by inducing TNF-&agr; synthesis and subsequently activating the sphingomyelinase pathway, whereas no evidence for a role of NOSII- or NOSIII-generated NO was found. Moreover, Cox-2–derived TxA2 appears to facilitate TNF-&agr; synthesis in response to LPS.Background —Although endotoxin (lipopolysaccharides, LPS) is recognized as a mediator of septic cardiodepression, its cardiac effects are still not fully elucidated. Methods and Results —Perfusion of isolated rat hearts with LPS for 180 minutes resulted in a decline of left ventricular contractility after 90 minutes, whereas coronary perfusion pressure remained unaffected. This cardiodepression was paralleled by a release of tumor necrosis factor (TNF)-α into the perfusate and preceded by myocardial TNF-α mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-α antiserum or the ceramidase inhibitor N -oleoylethanolamine. In contrast, the cardiac release of nitric oxide (NO) was not augmented by LPS. Immunohistochemical studies of LPS-perfused hearts revealed a positive staining for the constitutive (NOSIII) but not for the inducible NO synthase (NOSII). Accordingly, NOSII mRNA levels commenced to increase only at the very end of the LPS perfusion period. Progressive liberation of thromboxane (Tx) A2 and prostacyclin was induced by LPS together with myocardial cyclooxygenase (Cox)-2 mRNA expression. Both nonselective inhibition of Cox by indomethacin and selective inhibition of the inducible Cox-2 by NS-398 abolished prostanoid release. Interestingly, the generation of TNF-α and the associated cardiodepression caused by LPS were reduced by indomethacin, NS-398 and the Tx-receptor antagonist daltroban. Conclusions —LPS depresses contractility of isolated rat hearts by inducing TNF-α synthesis and subsequently activating the sphingomyelinase pathway, whereas no evidence for a role of NOSII- or NOSIII-generated NO was found. Moreover, Cox-2–derived TxA2 appears to facilitate TNF-α synthesis in response to LPS.

Human Endothelial Cell Activation and Mediator Release in Response to Listeria monocytogenes Virulence Factors

ABSTRACT The interaction of Listeria monocytogenes with endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-type L. monocytogenes (EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGD hly mutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca2+ suggested LLO-elicited Ca2+ flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGD hly mutant), listerial phosphatidylinositol-specific phospholipase C (EGD plcAmutant), broad-spectrum phospholipase C (EGD plcB mutant) and internalin B (EGD inlB mutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-κB to the stimulation with the LLO-producing strains EGD andListeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-κB activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-κB translocation and the upregulation of cytokine synthesis. We conclude thatL. monocytogenes is a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca2+ flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-κB are suggested as underlying host signaling events. These endothelial responses to L. monocytogenes may well contribute to the pathogenic sequelae in severe listerial infection and sepsis.

Lipoteichoic acid (LTA) from Staphylococcus aureus stimulates human neutrophil cytokine release by a CD14-dependent, Toll-like-receptor-independent mechanism : Autocrine role of tumor necrosis factor-α in mediating LTA-induced interleukin-8 generation

Objective:In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils. Subjects:Isolated human neutrophils from healthy volunteers. Interventions:Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies. Measurements:Measurement of tumor necrosis factor (TNF)-&agr;, IL-1&bgr;, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction. Conclusions:The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-&agr; and IL-1&bgr; and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-&agr;, as it was largely abrogated by neutralizing anti-TNF-&agr; antibodies. In contrast, IL1-&bgr; was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-&agr; generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-&agr; generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Wegener's granulomatosis: antiproteinase 3 antibodies induce monocyte cytokine and prostanoid release—role of autocrine cell activation

Antineutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 [PR3; cytoplasmic ANCA (c‐ANCA)], a leukocyte serine protease, are highly specific for Wegener’s Granulomatosis (WG). A pathogenetic role for c‐ANCA has been proposed as a result of their ability of activating neutrophils, whereas their interaction with monocytes is less well characterized. We investigated the influence of monoclonal anti‐PR3 antibodies (anti‐PR3) and c‐ANCA from WG sera on monocyte cytokine and prostanoid release. We found that PR3 was expressed on the surface of isolated monocytes. Anti‐PR3 challenge provoked a pronounced release of cytokines with early appearance of tumor necrosis factor α (TNF‐α) and interleukin (IL)‐1β and delayed release of IL‐6, IL‐8, and thromboxane A2 (TxA2). The secretory response was reproduced by c‐ANCA but not by human and murine control IgG and anti‐CD14 antibodies. Because F(ab)2 fragments of anti‐PR3 were ineffective, coligation of Fc gamma receptors (FcγR) was apparently mandatory for monocyte activation. Using soluble receptors for TNF‐α and IL‐1β and a Tx receptor antagonist, we noted that the “early” cytokines functioned as inducers of TxA2, which then activated IL‐8 release. In contrast, IL‐6 formation was an independent event. We concluded that anti‐PR3 antibodies are potent inducers of monocyte cytokine and prostanoid release, and TNF‐α, IL‐1β, and TxA2 function as facilitators of the secretory response. These mechanisms may contribute to inflammatory tissue injury in WG.

Increased neutrophil mediator release in patients with pulmonary hypertension - suppression by inhaled iloprost

Polymorphonuclear neutrophils (PMN) have been implicated in various vascular inflammatory processes. We isolated PMN from venous blood samples of 10 patients with severe primary pulmonary arterial hypertension (PPH), 7 patients with pulmonary hypertension secondary to chronic thromboembolism (CTEPH), and 12 healthy controls. When stimulated with the calcium-ionophore A23187, platelet activating factor (PAF) or the microbial agent n-formyl-Methionyl-Leucyl-Phenylalanine (fMLP), significantly increased release of elastase and superoxide anion was noted in both groups with pulmonary hypertension. Moreover, the neutrophils of CTEPH patients responded with an enhanced liberation of leukotriene (LT) B(4) and 5-hydroxyeicosatetraenoic acid (5-HETE). Inhalation of aerosolized iloprost (5 microg) caused a rapid decline in pulmonary vascular resistance, in both PPH and CTEPH. This hemodynamic response was paralleled by a significant suppression of ionophore- and ligand-induced elastase and superoxide release, as well as LTB(4) and 5-HETE formation. The neutrophil inhibitory effect of the inhalation maneuver was fully reproduced by in vitro incubation of neutrophils with 1-10 pg/ml iloprost for 2 hours. This is the first study to demonstrate that circulating neutrophils from patients with PPH and CTEPH possess an enhanced readiness to respond with inflammatory mediator generation to different stimulatory agents ex-vivo, and that PMN respiratory burst, elastase secretion and leukotriene generation are promptly reduced by an iloprost inhalation maneuver. Neutrophils might participate in the inflammatory processes in pulmonary arterial hypertension.

Cell Density Regulates Neutrophil IL-8 Synthesis: Role of IL-1 Receptor Antagonist and Soluble TNF Receptors

Although cytokine synthesis in polymorphonuclear leukocytes (PMN) was shown to be modulated by soluble mediators, the impact of microenvironmental conditions has not been elucidated. In this study, we investigated the effect of cell density on cytokine release from human neutrophils. PMN were cultured at various cell densities (10 × 106 PMN/ml; 60 × 106 PMN/ml), and LPS-induced release of cytokines was quantified by ELISA technique. Upon an increase in PMN density, secretion of the CXC chemokine IL-8 was progressively reduced. This effect was paralleled by a decrease in IL-8 mRNA. In contrast, TNF-α and IL-1β rose proportionally with increasing cell density. The inhibition of IL-8 secretion was reproduced by conditioned media of PMN at high cell density, but was not affected by blocking β2 integrin-dependent adhesion. When analyzing the supernatant of LPS-challenged neutrophils, large amounts of soluble TNFRs p55 and p75 (sTNFRI, sTNFRII), and IL-1R antagonist (IL-1RA), rising constantly with the cell density, were detected. Interestingly, combined blocking of the bioactivities of these mediators completely restored neutrophil IL-8 secretion at high cell densities, with the anti-IL-1RA Ab being the more potent agent. Moreover, combined application of exogenous IL-1RA and sTNFRs to 10 × 106 PMN/ml reproduced the suppression of IL-8 generation. We conclude that neutrophil IL-8 synthesis is autoregulated, being suppressed under conditions of high cell density. IL-1RA and sTNFRs, accumulating under these circumstances, seem to be centrally involved in this regulatory mechanism by interfering with the IL-1β- and TNF-α-dependent IL-8 generation. This feedback mechanism may control further neutrophil recruitment and activation in a neutrophil-rich environment, thereby preventing tissue destruction.

Endotoxin induces proliferation of NSCLC in vitro and in vivo: role of COX-2 and EGFR activation

Lung cancer is frequently complicated by pulmonary infections which may impair prognosis of this disease. Therefore, we investigated the effect of bacterial lipopolysaccharides (LPS) on tumor proliferation in vitro in the non-small cell lung cancer (NSCLC) cell line A549, ex vivo in a tissue culture model using human NSCLC specimens and in vivo in the A549 adenocarcinoma mouse model. LPS induced a time- and dose-dependent increase in proliferation of A549 cells as quantified by MTS activity and cell counting. In parallel, an increased expression of the proliferation marker Ki-67 and cyclooxygenase (COX)-2 was detected both in A549 cells and in ex vivo human NSCLC tissue. Large amounts of COX-2-derived prostaglandin (PG)E2 were secreted from LPS-stimulated A549 cells. Pharmacological interventions revealed that the proliferative effect of LPS was dependent on CD14 and Toll-like receptor (TLR)4. Moreover, blocking of the epidermal growth factor receptor (EGFR) also decreased LPS-induced proliferation of A549 cells. Inhibition of COX-2 activity in A549 cells severely attenuated both PGE2 release and proliferation in response to LPS. Synthesis of PGE2 was also reduced by inhibiting CD14, TLR4 and EGFR in A549 cells. The proliferative effect of LPS on A549 cells could be reproduced in the A549 adenocarcinoma mouse model with enhancement of tumor growth and Ki-67 expression in implanted tumors. In summary, LPS induces proliferation of NSCLC cells in vitro, ex vivo in human NSCLC specimen and in vivo in a mouse model of NSCLC. Pulmonary infection may thus directly induce tumor progression in NSCLC.

Biosynthesis of constitutive nitric oxide synthase-derived nitric oxide attenuates coronary vasoconstriction and myocardial depression in a model of septic heart failure induced by Staphylococcus aureus α-toxin

ObjectiveMyocardial depression, which frequently occurs in the course of septic shock, has been attributed to the cardiodepressant properties of nitric oxide (NO) generated by either the inducible NO synthase (iNOS) or the constitutive isoform (cNOS). We have previously demonstrated that &agr;-toxin from Staphylococcus aureus induces thromboxane-mediated vasoconstriction accompanied by severe cardiodepression in isolated rat hearts. In the present study, we investigated the role of NO in the &agr;-toxin-induced vascular and contractile abnormalities. DesignProspective, experimental study. SettingResearch laboratory at a university hospital. SubjectsIsolated hearts from male Wistar rats. InterventionsIsolated hearts were perfused with purified staphylococcal &agr;-toxin for 60 mins. Measurements and Main Results At a concentration of 0.25 and 0.5 &mgr;g/mL, &agr;-toxin induced a rise in coronary perfusion pressure, depressed myocardial contractility, and caused edema formation. Simultaneously, a time- and dose-dependent rapid release of NO into the perfusate was noted as quantified by a chemiluminescence technique. L-NMMA, a nonselective inhibitor of NOS, but not PBITU, an iNOS-selective inhibitor, blocked NO synthesis, markedly increased the rise in coronary perfusion pressure and the loss in contractility, and enhanced edema formation in response to &agr;-toxin. In contrast, zaprinast, a selective inhibitor of phosphodiesterase type V that is used for stabilization of cyclic guanosine monophosphate, attenuated the toxin-induced coronary vasoconstrictor response and the myocardial depression. l-arginine, the substrate of NOS, had similar, yet less potent, effects as zaprinast and slightly increased the release of NO caused by &agr;-toxin. Immunohistochemical analysis of the myocardium at the end of the perfusion period demonstrated a positive staining for cNOS but not for iNOS. In addition, no up-regulation of iNOS mRNA was detected in the tissue of toxin-exposed hearts. ConclusionsStaphylococcal &agr;-toxin provokes NO biosynthesis via activation of cNOS in rat hearts. NO partly antagonizes the deleterious effects of this pathogenicity factor on coronary vasoregulation and myocardial performance.

Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation

In Wegener’s granulomatosis (WG), a pathogenetic role has been proposed for circulating anti‐neutrophil‐cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti‐PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell‐wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti‐PR3 antibodies induced the release of minor quantities of the chemokine interleukin‐8 (IL‐8), preincubation with anti‐PR3 antibodies, but not with isotype‐matched control immunogloblin G (IgG), resulted in a markedly enhanced IL‐8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti‐PR3 and peaked after 6 h. The anti‐PR3‐related priming was also observed for tumor necrosis factor α (TNF‐α) and IL‐6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA‐IgG derived from WG serum but not with normal IgG. The priming effect of the anti‐PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF‐α. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti‐PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14‐dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.