c-ANCA-induced neutrophil-mediated lung injury: a model of acute Wegener's granulomatosis.

Anti-neutrophil cytoplasmic antibodies (c-ANCA) targeting proteinase 3 (PR3) are implicated in the pathogenesis of Wegener's granulomatosis (WG). Fulminant disease can present as acute lung injury (ALI). In this study, a model of ALI in WG was developed using isolated rat lungs. Isolated human polymorphonuclear leukocytes (PMNs) were primed with tumour necrosis factor (TNF) to induce surface expression of PR3. Co-perfusion of TNF-primed neutrophils and monoclonal anti-PR3 antibodies induced a massive weight gain in isolated lungs. This effect was not observed when control immunoglobulin G was co-perfused with TNF-primed PMNs. The c-ANCA-induced oedema formation was paralleled by an increase in the capillary filtration coefficient as a marker of increased pulmonary endothelial permeability. In contrast, pulmonary artery pressure was not affected. In the presence of the oxygen radical scavenger superoxide dismutase and a NADPH oxidase inhibitor, c-ANCA-induced lung oedema could be prevented. Inhibition of neutrophil elastase was equally effective in preventing c-ANCA-induced lung injury. In conclusion, anti-PR3 antibodies induced neutrophil mediated, elastase- and oxygen radical-dependent ALI in the isolated lung. This experimental model supports the hypothesis of a pathogenic role for c-ANCA in WG and offers the possibility of the development of therapeutic strategies for the treatment of lung injury in fulminant WG.

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 alpha-toxin.

OBJECTIVE: Myocardial 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 alpha-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 alpha-toxin-induced vascular and contractile abnormalities. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Isolated hearts from male Wistar rats. INTERVENTIONS: Isolated hearts were perfused with purified staphylococcal alpha-toxin for 60 mins. MEASUREMENTS AND MAIN RESULTS: At a concentration of 0.25 and 0.5 microg/mL, alpha-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 alpha-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 alpha-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. CONCLUSIONS: Staphylococcal alpha-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.

Endotoxin-induced myocardial tumor necrosis factor-alpha synthesis depresses contractility of isolated rat hearts: evidence for a role of sphingosine and cyclooxygenase-2-derived thromboxane production.

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)-alpha into the perfusate and preceded by myocardial TNF-alpha mRNA upregulation as quantified by real-time polymerase chain reaction. The cardiodepression was abrogated when LPS was perfused with a TNF-alpha 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) A(2) 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-alpha 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-alpha 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 TxA(2) appears to facilitate TNF-alpha synthesis in response to LPS.

E. coli hemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid.

Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B(4) and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB(4) and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB(5) and 5-, 8-, 9-, and 12-hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB(5) and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB(4) and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.

Staphylococcal alpha-toxin provokes coronary vasoconstriction and loss in myocardial contractility in perfused rat hearts: role of thromboxane generation.

BACKGROUND: Cardiac performance is severely depressed in septic shock. Endotoxin has been implicated as the causative agent in Gram-negative sepsis, but similar abnormalities are encountered in Gram-positive sepsis. We investigated the influence of the major exotoxin of Staphylococcus aureus, staphylococcal alpha-toxin, in isolated perfused rat hearts. METHODS AND RESULTS: Alpha-toxin 0.25 to 1 microg/mL caused a dose-dependent increase in coronary perfusion pressure that more than doubled. In parallel, we noted a decrease in left ventricular developed pressure and the maximum rate of left ventricular pressure rise (dP/dt(max)), dropping to a minimum of <60% of control. These changes were accompanied by a liberation of thromboxane A(2) and prostacyclin into the coronary effluent. The release of creatine kinase, lactate dehydrogenase, potassium, and lactate did not surpass control heart values, and leukotrienes were also not detected. Indomethacin, acetylsalicylic acid, and the thromboxane receptor antagonist daltroban fully blocked the alpha-toxin-induced coronary vasoconstrictor response and the decrease in left ventricular developed pressure and dP/dt(max), whereas the lipoxygenase inhibitor nordihydroguaiaretic acid, the platelet activating factor antagonist WEB 2086, and the alpha-adrenergic antagonist phentolamine were entirely ineffective. Inhibition of nitric oxide synthase even enhanced the alpha-toxin-induced increase in coronary perfusion pressure and the loss in myocardial performance. CONCLUSIONS: Purified staphylococcal alpha-toxin provokes coronary vasoconstriction and loss in myocardial contractility. The responses appear to be largely attributable to the generation of thromboxane and are even enhanced when the endogenous nitric oxide synthesis is blocked. Bacterial exotoxins, such as staphylococcal alpha-toxin, may thus be implicated in the loss of cardiac performance encountered in Gram-positive septic shock.