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Lipoxygenase, cyclo-oxygenase and cytochrome P450 (CYP) products of arachidonic acid (AA) are implicated in pulmonary vasoregulation. The CYP-mediated epoxyeicosatrienoates (EETs) have been described previously as the predominant eicosanoids in human lungs upon stimulation with the Ca(2+) ionophore A23187. In this study, we challenged perfused human lungs with two microbial agents: Escherichia coli haemolysin (ECH) and formyl-methionyl-leucyl-phenylalanine (fMLP). Both stimuli elicited pronounced generation of leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), prostanoids (PTs) and EETs/dihydroxyeicosatrienoic acids (DHETs), as assessed by liquid chromatography-mass spectrometry, paralleled by pulmonary artery pressor response and lung oedema formation. The maximum buffer concentrations of EETs/DHETs surpassed those of LTs plus HETEs and PTs by a factor of four (ECH) or three (AA/fMLP). Dual 5-lipoxygenase/cyclo-oxygenase inhibition caused pronounced reduction of AA/fMLP-induced LT/PT synthesis and oedema formation but only limited attenuation of pulmonary vasoconstriction, while inhibition of CYP epoxygenase clearly attenuated AA/fMLP-induced EET/DHET synthesis and vasoconstriction but not oedema formation, suggesting a major contribution of LTs/PTs to vascular leakage and of EETs/DHETs to pressor response. Consequently, generation of EETs/DHETs is greater than that of LTs plus HETEs and PTs in ex vivo perfused human lungs upon microbial challenge suggesting a substantial contribution of these mediators to inflammatory-infectious pulmonary injury.

Objective: In sepsis, cardiac function is frequently depressed. Microcirculatory disturbances as evidenced in most organs may extend to the coronary circulation and may play a role in the occurrence of cardiac dysfunction. Staphylococcal alpha-toxin and Escherichia coli hemolysin (ECH), pore-forming exotoxins of clinically relevant bacteria, have recently been demonstrated to evoke cardiac dysfunction in isolated rat hearts by activating myocardial eicosanoid metabolism. alpha-Toxin activates synthesis of thromboxane (Tx) A2, ECH of cysteinyl-leukotrienes (Sibelius U, Grandel U, Buerke M, et al: Leukotriene-mediated coronary vasoconstriction and loss in myocardial contractility evoked by low doses of Escherichia coli hemolysin in perfused reat hearts. Crit Care Med 2003; 3:683-688, Sibelius U, Grandel U, Buerke M, et al: Staphylococcal alpha-toxin provokes coronary vasoconstriction and loss in myocardial contractility in perfused rat hearts-Role of Tx formation. Circulation 2000; 101:78-85). We now investigated whether cardiac dysfunction in response to alpha-toxin and ECH is caused by disturbances of regional cardiac perfusion., Design: A prospective, experimental study., Setting: A research laboratory at a university hospital., Subjects: Isolated hearts from male Wistar rats., Interventions: Changes of regional perfusion were investigated by using colored microspheres in isolated rat hearts perfused with alpha-toxin or ECH either at constant coronary perfusion pressure or constant coronary flow rate. Significance of toxin-activated eicosanoid generation was evaluated by pharmacologic interventions., Measurements and Main Results: By eliciting eicosanoid formation, both toxins caused an increase in coronary vascular resistance and a loss in contractile function. In ECH-perfused hearts, reduction of regional perfusion predominantly occurred in subendocardial sections in either perfusion mode (coronary perfusion pressure or coronary flow rate). When synthesis of cysteinyl-leukotrienes was blocked by the 5-lipooxygenase inhibitor MK-886, disturbances of regional perfusion and the associated cardiac dysfunction were largely prevented. Coronary perfusion of alpha-toxin caused a decrease of regional perfusion that was more pronounced in subepicardial layers. Inhibiting the release of TxA2 by blocking the cyclooxygenase with indomethacin attenuated the perfusion abnormalities and the cardiodepression in response to alpha-toxin., Conclusions: Bacterial exotoxins of clinically relevant bacteria may impair cardiac function by eliciting distinct coronary perfusion abnormalities via release of vasoactive eicosanoids.

Objective: hemolysin has been implicated as an important pathogenic factor in extraintestinal infections including sepsis. We investigated the effects of coronary administration of hemolysin on cardiac function in isolated rat hearts perfused at constant flow., 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 hemolysin for 60 min., Measurements and Main Results: Low concentrations of the toxin in the perfusate (0.1-0.2 hemolytic units/mL) caused a dose-dependent coronary vasoconstriction with a marked increase in coronary perfusion pressure, which was paralleled by a decrease in left ventricular developed pressure (and the maximum rate of left ventricular pressure increase). Moreover, 0.2 hemolytic units/mL hemolysin evoked ventricular fibrillation within 10 mins of toxin application. These events were accompanied by the liberation of leukotrienes (LTC4, LTD4, LTE4, and LTB4), thromboxane A2, prostaglandin I2, and the cell necrosis markers lactate dehydrogenase and creatine kinase into the recirculating perfusate. The lipoxygenase inhibitor MK-886 fully blocked the toxin-induced coronary vasoconstrictor response and the loss of myocardial contractility and reduced the release of lactate dehydrogenase and creatine kinase. In contrast to this, the cyclooxygenase inhibitor indomethacin was entirely ineffective. In addition, hemolysin elicited an increase in heart weight and left ventricular end-diastolic pressure, the latter again being suppressed by MK-886., Conclusions: Low doses of hemolysin cause strong coronary vasoconstriction, linked with loss of myocardial performance, release of cell injury enzymes, and electrical instability, with all events being largely attributable to toxin-elicited leukotriene generation in the coronary vasculature. Bacterial exotoxins such as hemolysin thus may be implicated in the cardiac abnormalities encountered in septic shock.

The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.

Staphylococcus aureus alpha-toxin is a pore-forming bacterial exotoxin that has been implicated as a significant virulence factor in human staphylococcal diseases. In primary cultures of rat pneumocyte type II cells and the human A549 alveolar epithelial cell line, purified alpha-toxin provoked rapid-onset phosphatidylinositol (PtdIns) hydrolysis as well as liberation of nitric oxide and the prostanoids PGE(2), PGI(2), and thromboxane A(2). In addition, sustained upregulation of proinflammatory interleukin (IL)-8 mRNA expression and protein secretion occurred. "Priming" with low-dose IL-1beta markedly enhanced the IL-8 response to alpha-toxin, which was then accompanied by IL-6 appearance. The cytokine response was blocked by the intracellular Ca(2+)-chelating reagent 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, the protein kinase C inhibitor bis-indolyl maleimide I, as well as two independent inhibitors of nuclear factor-kappaB activation, pyrrolidine dithiocarbamate and caffeic acid phenethyl ester. We conclude that alveolar epithelial cells are highly reactive target cells of staphylococcal alpha-toxin. alpha-Toxin pore-associated transmembrane Ca(2+) flux and PtdIns hydrolysis-related signaling with downstream activation of protein kinase C and nuclear translocation of nuclear factor-kappaB are suggested to represent important underlying mechanisms. Such reactivity of the alveolar epithelial cells may be relevant for pathogenic sequelae in staphylococcal lung disease.

The effect of Staphylococcus aureus alpha toxin (alpha-toxin) on selectin-mediated neutrophil adhesion was investigated in polymorphonuclear leukocyte- (PMN) induced vasocontraction and endothelial dysfunction. Adherence of human PMNs to rat aortic endothelium increased significantly following stimulation of the endothelium with alpha-toxin (0.1, 0.5, and 1 microg/mL). This effect could be significantly attenuated by monoclonal antibodies directed against P-selectin or fucoidin, a carbohydrate known to block selectins. Unstimulated human PMNs (10(6)cells/mL) were added to organ chambers containing rat aortic rings stimulated with alpha-toxin (0.5 microg/mL). PMNs elicited a significant vasocontraction in alpha-toxin-stimulated, but not in control aortic, rings (142+/-12 mg versus 12+/-4 mg, P < 0.05). This PMN-induced vasocontraction was virtually blunted by pretreatment with MAb directed against P-selectin or fucoidin (P < 0.05). Endothelial function as assessed by endothelium-dependent vasorelaxation to acetylcholine was substantially inhibited after induction of PMN-induced vasocontraction in alpha-toxin-stimulated aortic rings. This endothelial dysfunction was reduced by P-selectin MAb or fucoidin. In contrast, endothelium-independent relaxation to sodium nitrite was not altered by PMN incubation, indicating that vascular smooth muscle function was unaffected. Thus, PMN-endothelial interaction following S. aureus a-toxin activation of the vascular endothelium is at least, in part, mediated by selectins. As a consequence, PMN-induced vasocontraction and endothelial dysfunction occur. Such mechanisms may be involved in microcirculation abnormalities encountered in sepsis or septic shock due to S. aureus infection.

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.

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.

Polymorphonuclear leukocytes (PMN) are essential for resolution of infections with Listeria monocytogenes. The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation. Different Listeria strains, mutated in individual virulence genes, as well as purified LLO were used. Coincubation of human neutrophils with wild-type L. monocytogenes provoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis. Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD- and the avirulent strain L. innocua were used. These effects were fully reproduced by a recombinant L. innocua strain expressing LLO (INN+) and by the purified LLO molecule. LLO secretion was also required for PAF synthesis. However, wild-type L. monocytogenes was more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors. This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD- and by a recombinant L. innocua strain producing listerial PlcA. We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation. Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst. In this way, listerial exotoxins may influence the host defense against infections with L. monocytogenes.

Nitric oxide (NO) is an important vasodilator that is produced by constitutive (cNOS) as well as inducible (iNOS) isoforms of nitric oxide synthase. The pore-forming hemolysin of Escherichia coli (HlyA), an important virulence factor in extraintestinal E. coli infections, was found to be a potent stimulator of NO liberation in isolated endothelial cells, and that it also causes thromboxane generation and related vasoconstriction in rabbit lungs. We investigated the effect of different concentrations of HlyA on pulmonary NO synthesis in buffer-perfused rabbit lungs. NO release into the alveolar as well as the intravascular compartment was monitored on-line by chemiluminescence detection of expired NO and by measurement of (peroxy-)nitrite/nitrate release into the perfusate. HlyA induced a pressor response and an immediate dose-dependent increase of exhalative and intravascular NO liberation, further enhanced by the addition of the NOS substrate L-arginine. The nonspecific NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA), but not the iNOS selective inhibitors aminoguanidine and 2-(2-aminoethyl)-2-thiopseudourea-dihydrobromide, blocked the HlyA-evoked NO liberation into both the alveolar and the intravascular compartments. Enhancement of NO formation (L-arginine) slightly reduced, and inhibition of NO synthesis (L-NMMA) amplified greatly, the HlyA-elicited vasoconstrictor response. Inhibition of the pressor response by a thromboxane receptor antagonist did not interfere with the exotoxin-elicited NO formation. We conclude (1) that marked NO biosynthesis occurs in this model of the septic lung, (2) that the signal transduction in response to HlyA proceeds via activation of cNOS directly related to exotoxin activity and not to secondary changes in shear stress, and (3) that this vasodilator release mitigates the HlyA-induced pulmonary vasoconstriction. These findings may have important implications for therapeutic approaches using NOS inhibitors in sepsis.

Lipopolysaccharides (LPS) of gram-negative bacteria prime rabbit lungs for enhanced thromboxane-mediated vasoconstriction upon subsequent challenge with the exotoxin Escherichia coli hemolysin (HlyA) (Walmrath et al. J. Exp. Med. 1994;180:1437-1443). We investigated the impact of endotoxin priming and subsequent HlyA challenge on lung vascular permeability while maintaining constancy of capillary pressure. Rabbit lungs were perfused in a pressure-controlled mode in the presence of the thromboxane receptor antagonist BM 13.505, with continuous monitoring of flow. Perfusion for 180 min with 10 ng/ml LPS did not provoke vasoconstriction or alteration of capillary filtration coefficient (Kfc) values. HlyA (0.021 hemolytic units/ml) induced thromboxane release and a transient decrease in perfusion flow in the absence of significant changes in Kfc. Similar results were obtained when LPS and HlyA were coapplied simultaneously. However, when the HlyA challenge was undertaken after 180 min of LPS priming, a manifold increase in Kfc values was noted, with concomitant severe lung edema formation, although capillary pressure remained unchanged. Thus, endotoxin primes the lung vasculature to respond with a severe increase in vascular permeability to a subsequent low-dose application of HlyA. Such synergism between endotoxin priming and exotoxin challenge in provoking lung vascular leakage may contribute to the pathogenesis of respiratory failure in sepsis and severe lung infection.

Escherichia coli hemolysin (HlyA) and Staphylococcus aureus alpha-toxin are membrane-perturbating bacterial exotoxins that have been implicated as significant virulence factors in human diseases. We investigated the capacity of these toxins to cause cell activation and mediator release in human endothelial cells, compared with the efficacies of thrombin and the Ca2+ ionophore A23187. Concentration ranges tested were 1 to 1000 ng/ml (HlyA), 0.01 to 10 micro/ml (alpha-toxin), 0.01 to 10 U/ml (thrombin), and 0.01 to 10 microM (A23187). All stimuli caused dose-dependent generation of platelet-activating factor, nitric oxide, and prostaglandin I2. HlyA and thrombin effected time- and dose-dependent accumulation of large quantities of inositol phosphates, with maximum effects at 100 ng/ml and 1 U/ml, respectively. Corresponding time course and dose dependency were noted for HlyA-elicited diacylglycerol formation. In contrast, only the highest concentrations of alpha-toxin (10 microg/ml) and A23187 (10 microM) effected some moderate inositol phosphate accumulation, and this was suppressed in the presence of the platelet-activating factor antagonist WEB 2086. Metabolic and secretory responses elicited by alpha-toxin were dependent on the presence of extracellular Ca2+. We conclude that both HlyA and alpha-toxin are potent inductors of inflammatory and vasodilatory mediators in human endothelial cells. HlyA-elicited effects may proceed predominantly via activation of the phosphatidylinositol hydrolysis-related signal transduction pathway, whereas transmembrane Ca2+ flux appears to be the major event underlying the release of mediators in response to alpha-toxin. These toxin properties may contribute to vasoregulatory and inflammatory disturbances encountered in states of severe infection and sepsis.

Escherichia coli hemolysin (HlyA) has been identified as a potent inductor of phosphoinositide hydrolysis and related metabolic responses in neutrophils (Grimminger and colleagues, 1991, J. Clin. Invest. 88:1531-1539). In isolated perfused rabbit lungs, which harbor a large number of entrapped microvascular leukocytes, we investigated the effect of a low dose of HlyA on lipoxygenase product formation in the presence of exogenous free arachidonic acid (AA), eicosapentaenoic acid (EPA), or both precursor fatty acids. Leukotrienes (LT) and hydroxyeicosatetra(penta)enoic acids (HET[P]E) in the recirculating perfusate were quantified using high-performance liquid chromatography techniques. In the absence of exogenous precursor fatty acid supply, 0.02 hemolytic units/ml HlyA elicited only minor amounts of LTs and 5-HETE. AA, 10 microM, provoked the generation of limited quantities of LTB4, LTE4, and 5-HETE. Combined application of HlyA and AA caused a manifold amplification of 4-series LT and 5-HETE generation, with predominance of cysteinyl-LTs. EPA, 10 microM, elicited the synthesis of 5-series LTs accompanied by marked quantities of 5-HEPE. Dual stimulation with HlyA and EPA provoked exclusive generation of excessive quantities of all 5-series 5-lipoxygenase products. When HlyA was administered in the presence of both AA (10 microM) and EPA (10 microM), the n-3 fatty acid clearly turned out to be the preferred substrate, with ratios of the various 5-series to 4-series products ranging between 1.8 and 14.5. Moreover, the absolute quantities of AA-derived metabolites and the total sum of all 5-lipoxygenase products was markedly reduced under these conditions. We conclude that the HlyA-evoked 5-lipoxygenase product formation in the pulmonary vasculature of the rabbit is critically dependent on the presence of free precursor fatty acids. The profile of LTs suggests neutrophil (PMN)-related transcellular eicosanoid synthesis as a major underlying metabolic pathway. EPA represents the preferred substrate as compared with AA, resulting in a marked suppression of AA metabolite formation. Therapeutic attempts to provide n-3 fatty acids via the intravenous route may have a major impact on lipid mediator profiles in PMN-related inflammatory events.

Escherichia coli hemolysin (HlyA) is a proteinaceous pore-forming exotoxin that is implicated as a significant pathogenicity factor in extraintestinal E. coli infections including sepsis. In perfused rabbit lungs, subcytolytic concentrations of the toxin evoke thromboxane-mediated vasoconstriction and prostanoid-independent protracted vascular permeability increase (11). In the present study, the influence of submicromolar concentrations of free arachidonic acid (AA) and eicosapentaenoic acid (EPA) on the HlyA-induced leakage response was investigated. HlyA at concentration from 0.02 to 0.06 hemolytic units/ml provoked a dose-dependent, severalfold increase in the capillary filtration coefficient (Kfc), accompanied by the release of leukotriene(LT)B4, LTC4, and LTE4 into the recirculating buffer fluid. Simultaneous application of 100 nmol/L AA markedly augmented the HlyA-elicited leakage response, concomitant with an amplification of LTB4 release and a change in the kinetics of cysteinyl-LT generation. In contrast, 50 to 200 nmol/L EPA suppressed in a dose-dependent manner the HlyA-induced increase in Kfc values. This was accompanied by a blockage of 4-series LT generation and a dose-dependent appearance of LTB5, LTC5, and LTE5. In addition, EPA fully antagonized the AA-induced amplification of the HlyA-provoked Kfc increase, again accompanied by a shift from 4-series to 5-series LT generation. We conclude that the vascular leakage provoked by HlyA in rabbit lungs is differentially influenced by free AA versus free EPA, related to the generation of 4- versus 5-series leukotrienes. The composition of lipid emulsions used for parenteral nutrition may thus influence inflammatory capillary leakage.

Both endotoxin (lipopolysaccharides of gram-negative bacteria; LPS) and bacterial exotoxins may induce pulmonary microcirculatory disturbances when infused into the lung vasculature, and synergism between these types of microbial challenge has recently been noted. We now asked whether a bronchoalveolar LPS load in perfused rabbit lungs alters the responsiveness to a subsequent intravascular challenge with Escherichia coli hemolysin (ECH). In control lungs (sham aerosolization) and lungs undergoing LPS nebulization (alveolar deposition of approximately 22 micrograms), normal pulmonary artery pressure (PAP), lung weight, and ventilation/perfusion (V/Q) matching were observed. Intravascular ECH (0.013 hemolytic units/ml buffer fluid) increased PAP by approximately 10 mm Hg and lung weight by approximately 4 g within 10 min, paralleled by V/Q mismatch and a shunt flow of approximately 15%. In lungs "primed" for 3 h by a preceding bronchoalveolar LPS deposition, the same ECH dose provoked a dramatic increase in PAP to 40 to 50 mm Hg, a weight gain of approximately 10 g, and shunt flow of 60%. Both vasoconstrictor response and V/Q mismatch were completely suppressed by preadministration and "rescue" application of the thromboxane receptor antagonist BM13.505. We conclude that a bronchoalveolar endotoxin load, though effecting no changes in pulmonary function by itself and showing no spillover into the vascular compartment, primes the lungs for a manifold increased vascular response to a subsequently infused exotoxin. Enhanced thromboxane-mediated vasoconstriction, largely redistributing perfusate flow from normally ventilated to shunt areas, is suggested as the predominant underlying event.

The impact of Listeria monocytogenes listeriolysin O (LLO) secretion on phosphoinositide metabolism and mediator (platelet-activating factor and prostaglandin I2) generation was investigated in human umbilical vein endothelial cells. Wild-type L. monocytogenes, purified LLO, and an L. innocua strain engineered to secrete LLO all elicited a strong response, whereas mutant strains defective in LLO production were ineffective. Thus, human umbilical vein endothelial cell stimulation by listeriae is linked to production of LLO.

The pore-forming hemolysin of Escherichia coli (HlyA), an important virulence factor in extraintestinal E. coli infections, causes thromboxane generation and related vasoconstriction in perfused rabbit lungs (Seeger, W., H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi. 1989. J. Clin. Invest. 84:220). We investigated the influence of pulmonary vascular "priming" with endotoxin on the responsiveness of the lung to a low-dose HlyA challenge. Rabbit lungs were perfused with Krebs Henseleit buffer containing 0.1-100 ng/ml Salmonella abortus equii lipopolysaccharide (LPS) for 60-180 min. This treatment caused protracted release of tumor necrosis factor into the recirculating medium, but did not induce significant alterations of pulmonary hemodynamics and fluid balance. At a dose of 1 ng/ml, HlyA elicited only moderate thromboxane release (< 200 pg/ml) and pulmonary artery pressure increase (< or = 6 mmHg) in control lungs. Acceleration and potentiation of both the metabolic and vasoconstrictor response occurred in lungs primed with LPS. This priming effect displayed dose (threshold integral of 0.1-1 ng/ml LPS) and time dependencies (threshold integral of 60-90 min LPS incubation). Maximum thromboxane release and pulmonary artery pressure increase surpassed the responses to HlyA in nonprimed lungs by more than 15-fold. Cyclooxygenase inhibition and thromboxane-receptor antagonism blocked these effects. These data demonstrate that LPS priming synergizes with HlyA challenge to provoke vascular abnormalities that are possibly relevant to the pathogenesis of organ failure in severe local and systemic infections.

Escherichia coli hemolysin (ECH), an important pathogenicity factor in extraintestinal E. coli infections, provokes pulmonary hypertension and microvascular leakage in buffer-perfused rabbit lungs. We investigated gas exchange abnormalities in response to low doses of ECH, lipopolysaccharides (LPS), and sequential and combined application of these bacterial agents by using the multiple inert gas elimination technique. In control lungs and after admixture of 100 ng/ml of LPS, unimodal narrow distribution of perfusion and ventilation to midrange ventilation-perfusion (VA/Q) areas was noted. ECH [0.08 hemolytic units (HU)/ml] caused a moderate increase in pulmonary arterial pressure (< 10 mmHg), progressive lung edema formation (approximately 10 g within 20 min), and a broadening of perfusate and gas flow dispersion. Application of 0.08 HU/ml of ECH in lungs "primed" with 100 ng/ml of LPS in a preceding 125-min perfusion period provoked a large increase in pulmonary arterial pressure (> 50 mmHg within 5 min), rapid edema formation (approximately 10 g within 10 min), and severe VA/Q mismatch with predominance of shunt flow. Vasoconstrictor response and VA/Q mismatch, but not edema formation, were largely inhibited by pretreatment of lungs with acetylsalicylic acid or the thromboxane receptor antagonist BM-13.505. In addition, "rescue" application of BM-13.505 rapidly reversed pressure rise and shunt flow due to sequential LPS and/or ECH stimulation, whereas edema formation was not affected. We conclude that the marked pulmonary hypertension in response to low doses of ECH in LPS-primed lungs is paralleled by severe gas exchange abnormalities with predominance of shunt flow. Both the vasoconstrictor response and the development of shunt are closely related to toxin-induced thromboxane generation.

The major pore-forming exotoxin of Staphylococcus aureus, staphylococcal alpha-toxin, causes thromboxane-mediated pulmonary hypertension and prostanoid-independent protracted vascular leakage in perfused rabbit lungs. We asked whether lung responsiveness to the staphylococcal agent would be altered by a preceding period of endotoxin priming. Isolated rabbit lungs were perfused with Krebs-Henseleit buffer in the presence or absence of 100 ng/ml Salmonella abortus equii endotoxin for up to 5 h. The lipopolysaccharide exposure evoked the release of large quantities of tumor necrosis factor into the vascular and alveolar spaces but did not significantly alter pulmonary artery pressure, organ weight, or the repeatedly assessed capillary filtration coefficient (Kfc). Two and 4 h after endotoxin administration, alpha-toxin (10 to 30 ng/ml) was bolus-injected into the pulmonary artery. Toxin-evoked prostanoid generation (TxB2, 6-keto-PGF1 alpha) and pressor responses were markedly accelerated and enhanced in endotoxin-primed lungs, both for the 2 h and the 4 h priming period. No significant influence of endotoxin was noted when applied simultaneously with alpha-toxin. Cyclooxygenase inhibition suppressed the alpha-toxin-evoked pressure rise in both endotoxin-primed and nonprimed lungs. Endotoxin priming did not influence the alpha-toxin-induced protracted increase in Kfc values, assessed in the presence of cyclooxygenase inhibition. We conclude that endotoxin primes rabbit lungs for enhanced prostanoid generation and pulmonary hypertension in response to S. aureus alpha-toxin. Such cooperativity of endotoxin priming and exotoxin triggering may be relevant in critically ill patients suffering from both endotoxemia and gram-positive sepsis.

Nitric oxide (NO) is believed to play an important role in sepsis-related hypotension. We examined the effects of two pore-forming bacterial exotoxins, Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin, on NO formation in cultured porcine pulmonary artery endothelial cells. NO was quantified using a difference-spectrophotometric method based on the rapid and stoichiometric reaction of NO with oxyhemoglobin. Endothelial cyclic guanosine monophosphate levels were also monitored. Both exotoxins increased NO synthesis in endothelial cells in a time- and dose-dependent manner to an extent exceeding that observed with the ionophore A23187 or thrombin. The capacity of exotoxins to induce NO formation may be relevant in patients with severe local or systemic bacterial infections.

Gas exchange conditions in blood-free perfused isolated rabbit lungs were assessed by the use of the multiple inert gas elimination technique. Under baseline conditions, unimodal narrow distribution of perfusion and ventilation to midrange-ventilation-perfusion (VA/Q) areas was noted. Intravascular challenge with staphylococcal alpha-toxin caused a rapid increase in pulmonary arterial pressure (to > 40 mmHg within approximately 15 min) and delayed-onset (> 10-15 min) lung edema formation, with unaltered ventilation pressures. The vasoconstrictor response was paralleled by a progressive, severe leftward shift of perfusion to areas with low-VA/Q ratios, accompanied by a minor fraction of shunt flow. At pulmonary arterial pressures > 40 mmHg, extreme VA/Q mismatch with near absence of perfusate flow to midrange-VA/Q areas was registered. Vasoconstrictor response and VA/Q mismatch, but not the progressive edema formation, were virtually completely suppressed in lungs pretreated with acetylsalicylic acid or the thromboxane receptor antagonist BM 13505. Moreover, "rescue" application of BM 13505 after onset of alpha-toxin-induced pressor response and gas exchange abnormalities completely reversed pressure elevation and loss of VA/Q matching. We conclude that the marked vasoconstrictor response to staphylococcal alpha-toxin is paralleled by severe VA/Q mismatch with predominant perfusion of low-VA/Q areas independent of lung edema formation. Pressor response and VA/Q mismatch, but not vascular leakage, are suppressed by thromboxane inhibition.

S. aureus alpha-toxin and E. coli hemolysin (Hly) represent two prototypes of pore-forming cytolysins. Both are established virulence factors and have been implicated in the development of septic lung failure. Low doses of these agents cause thromboxane-mediated vasoconstriction and edema formation in isolated perfused rabbit lungs. In a preceding investigation, we observed that alpha-toxin causes overt endothelial cell damage in these lungs, as demonstrable by electron microscopy (Seeger W, Birkemeyer RG, Ermert L, Suttorp N, Bhakdi S, Duncker HR: Lab Invest 63:341, 1990). Here, we present results of a parallel study conducted with E. coli hemolysin. Thromboxane-dependent pulmonary hypertension was suppressed by the addition of acetylsalicylic acid to the perfusion fluid in all cases. Administration of 0.2 hemolytic units (HU; i.e., 20 ng/ml protein) resulted in progressive weight gain after a lag period of 10 to 15 minutes, and 30 minutes after toxin application the gravimetrically determined capillary filtration coefficients (Kfc) were increased greater than 10-fold. Perfusion was terminated when the total lung weight gain surpassed 20 gm. 0.12 HU/ml E. coli hemolysin caused 2- to 3-fold increased capillary filtration coefficients values within 110 minutes, concomitant with intermediate quantities of edema formation (9.7 +/- 2.7 gm). Potassium liberation in the absence of lactate dehydrogenase release occurred in all toxin treated lungs. Electron microscopic examination after perfusion fixation revealed interstitial edema formation in areas remote from the blood-gas exchange barrier. Increased numbers of endothelial plasmalemmal vesicles were visualized at the very onset of edema formation in lungs exposed to 0.2 HU/ml, and after a 110-minute exposure to 0.12 HU/ml of the toxin, but not in lungs exhibiting severe edema (greater than 20 gm weight gain). In contrast to our previous results with alpha-toxin, endothelial cells displayed normal electron density here and were not detached from the fused basal lamina. Hence, although both pore formers provoke severe vascular leakage in our experimental model, the underlying pathways probably divert fundamentally from each other.

Escherichia coli hemolysin (Hly) is a proteinaceous pore-forming exotoxin that probably represents a significant virulence factor in E. coli infections. We investigated its influence on human polymorphonuclear neutrophils (PMN), previously identified as highly susceptible targets. Hly provoked rapid secretion of elastase and myeloperoxidase, generation of superoxide, and synthesis of platelet-activating factor (PAF) and lyso-PAF. Concomitantly, marked phosphatidylinositol (PtdIns) hydrolysis with sequential appearance of the inositol-phosphates, inositol-phosphates, inositol triphosphate, diphosphate, and monophosphate, respectively, and formation of diacylglycerol, occurred. The metabolic responses displayed distinct bell-shaped dose dependencies, with maximum events noted at low toxin concentrations of 0.1-0.5 hemolytic units per milliliter. PtdIns hydrolysis and metabolic responses elicited by Hly exceeded those evoked by optimal concentrations of formylmethionyl-leucyl phenylalanine, PAF, leukotriene B4, A23187, or staphylococcal alpha-toxin. The toxin-induced effects were sensitive toward modulators of PMN stimulus transmission pathways (pertussis toxin, the kinase C inhibitor H7, and phorbol myristate acetate "priming"). We conclude that the marked capacity of low doses of Hly to elicit degranulation, respiratory burst, and lipid mediator generation in human PMN probably envolves signal transduction via PtdIns hydrolysis.

Polymorphonuclear leukocytes (PMN) have been identified as preferred target cells for Escherichia coli hemolysin in human blood (Bhakdi, S., Greulich, S., Muhly, M., Eberspächer, B., Becker, H., Thiele, A., and Hugo, F. (1989) J. Exp. Med. 169, 737-754). Leukotriene and 5-hydroxyeicosatetraenoic acid generation was investigated in human PMN challenged with E. coli hemolysin in the absence or presence of free arachidonic acid or eicosapentaenoic acid (EPA). In the absence of exogenous free fatty acids, E. coli hemolysin (0.01-10 hemolytic units/ml) induced moderate generation of leukotriene B4 (LTB4) and its omega-oxidation products. The presence of free arachidonic acid (10 microM) during E. coli hemolysin (0.1 hemolytic unit/ml) challenge evoked the generation of large quantities of these products (greater than 100 pmol/1.5 x 10(7) PMN). In parallel, large amounts of 5-hydroxyeicosatetraenoic acid and nonenzymatic LTA4 hydrolysis products appeared. Product release peaked or plateaued 5-10 min after E. coli hemolysin challenge. The presence of exogenous EPA upon E. coli hemolysin challenge resulted in the exclusive generation of LTB5 and metabolites, LTA5 decay products and 5-hydroxyeicosapentaenoic acid. Dose and time dependences corresponded to those with arachidonic acid provision, and the total of EPA-derived products surpassed that of arachidonic acid metabolites in corresponding experiments approximately 2-fold. Increasing the time between free fatty acid provision and E. coli hemolysin challenge resulted in a rapid decline in the generation of arachidonic acid or EPA metabolites. Thus, subhemolytic doses of E. coli hemolysin evoke marked PMN eicosanoid generation that is dependent on exogenous free fatty acid supply, with total amounts approximating those found in calcium ionophore-stimulated neutrophils.

Escherichia coli hemolysin, a transmembrane pore-forming exotoxin, is considered an important virulence factor. In the present study, the possible significance of hemolysin production was investigated in a model of septic lung failure through infusion of viable bacteria in isolated rabbit lungs; 10(4) to 10(7) E. coli/ml perfusate caused a dose- and time-dependent appearance of hemolysin, accompanied by release of potassium, thromboxane A2, and PGI2 into the perfusate. Concomitantly, marked pulmonary hypertension developed. Inhibitor studies suggested that the pressor response was predominantly mediated by pulmonary thromboxane generation. Administration of hemolysin-forming E. coli additionally caused a protracted, dose-dependent increase in the lung capillary filtration coefficient, followed by severe edema formation. The permeability increase was independent of lung prostanoid generation. An E. coli strain that releases an inactive form of hemolysin completely failed to provoke the described biophysical and biochemical responses. Preapplication of 2 x 10(8) human granulocytes was without effect in the present experimental model. We conclude that the hemolysin produced by low numbers of E. coli organisms can provoke thromboxane-mediated pulmonary hypertension and severe vascular leakage. E. coli hemolysin and, possibly, other related cytolysins may thus contribute directly to the pathogenesis of acute respiratory failure under conditions of sepsis or pneumonia.

Escherichia coli hemolysin, a transmembrane pore-forming exotoxin, is considered an important virulence factor for E. coli-related extraintestinal infections and sepsis. The possible significance of hemolysin liberation for induction of inflammatory lipid mediators was investigated in isolated rabbit lungs infused with viable bacteria (concentration range, 10(4)-10(7)/ml). Hemolysin-secreting E. coli (E. coli-Hly+), but not an E. coli strain that releases an inactive form of the exotoxin, induced marked lung leukotriene (LT) generation with predominance of cysteinyl LTs. Eicosanoid synthesis was not inhibited in the presence of plasma with toxin-neutralizing capacity. Pre-application of 2 x 10(8) human granulocytes, which sequestered in the lung microvasculature, caused a severalfold increase in leukotriene generation in response to E. coli-Hly+ challenge both in the absence and presence of plasma. Data are presented indicating neutrophil-endothelial cell cooperation in arachidonic acid lipoxygenase metabolism as an underlying mechanism. We conclude that liberation of hemolysin from viable E. coli induces marked lipid mediator generation in lung vasculature, which is potentiated in the presence of neutrophil sequestration and may contribute to microcirculatory disturbances during the course of severe infections.

Low doses of Escherichia coli hemolysin cause thromboxane-mediated hypertension and vascular leakage in blood-free perfused rabbit lungs (W. Seeger, H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi, J. Clin. Invest. 84:220-227, 1989). The recirculating buffer medium and bronchoalveolar lavage fluid from lungs exposed to hemolysin (2.5 hemolytic units per ml) in the presence of cyclooxygenase inhibitor were analyzed for leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) by reverse-phase and straight-phase high-pressure liquid chromatographic techniques combined with UV spectrum analysis and post-high-pressure liquid chromatography radioimmunoassay. A rapid release of large amounts of cysteinyl-LTs and leukotriene B4 (LTB4) into the intravascular space was noted (total sum, approximately 4 to 5 micrograms). Similar quantities have hitherto been elicited only by high concentrations of the artificial calcium ionophore A 23187. Moreover, a marked liberation of 5-HETE and 12-hydroxyheptadecatrienoic acid into the buffer medium occurred, whereas LTB4 represented the predominant compound in the lavage fluid. The hemolysin-induced burst of LT and HETE generation preceded the onset of vascular leakage. The outstanding capacity of E. coli hemolysin to produce the liberation of potent lipid mediators is probably relevant to the pathways of vascular injury and amplification of inflammatory events during severe infection with hemolytic E. coli strains.