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Simvastatin, ein HMG-CoA-Reduktase-Inhibitor, wird im klinischen Alltag zur Senkung des Cholesterin-Spiegels eingesetzt. Darüber hinaus berichteten aktuelle Studien über protektive Eigenschaften von Statinen unter inflammatorischen Bedingungen. In dieser Studie wurden die Effekte von Simvastatin auf die Leukozyten-vermittelte Schädigung nach Stimulation mit Staphylococcus aureus alpha-Toxin untersucht. Aortale Gefäßsegmente von Ratten, vorbehandelt mit NaCl oder Simvastatin (100 μg/kg) intraperitoneal appliziert, wurden isoliert. Die Gefäßsegmente wurden mit Staphylococcus aureus alpha-Toxin (0,1–1 μg/ml) aktiviert und die PMN-Adhäsion quantifiziert. Zusätzlich wurde in der Organbadanlage die PMN-induzierte Vasokontraktion und endotheliale Dysfunktion untersucht. Die Adhäsion von PMN an das vaskuläre Endothel wurde durch die Stimulation mit alpha-Toxin signifikant gesteigert. Die Vorbehandlung mit Simvastatin führte zu einer signifikant geringeren Adhäsion nach alpha-Toxin Stimulation (p<0,05). Im Organbad führte die Inkubation von PMN mit Gefäßringen zu keiner Vasokontraktion bzw. endothelialen Dysfunktion. Nach Stimulation der Gefäßringe im Organbad mit alpha-Toxin 0,5 μg/ml und anschließender Koinkubation mit PMN (106Zellen/ml) zeigte sich eine signifikante Vasokontraktion (119±8 mg, p<0,05). Dies führte gleichzeitig zur deutlichen Einschränkung der endothelialen Relaxation in diesen Gefäßringen nach Acetylcholingabe (21±4% Relaxation, p<0,01). Die endotheliale Dysfunktion konnte in Gefäßringen von mit Simvastatin vorbehandelten Tieren in einem geringeren Ausmaß beobachtet werden (75±5% Relaxation, p<0,05). Auch die PMN-vermittelte Vasokontraktion zeigte sich in den Gefäßringen von Simvastatin vorbehandelten Tieren ebenfalls in einem deutlich geringeren Ausmaß. Die Ergebnisse dieser Untersuchung zeigen, dass Simvastatin die durch alpha-Toxin induzierten PMN-Endothel-Interaktionen an der Gefäßwand signifikant reduziert. Dadurch kommt es zu einer geringeren vaskulären Schädigung welche bei Patienten mit Sepsis oder Endokarditis von Bedeutung sein könnte. Die Zukunft wird zeigen, ob Statine eine therapeutische Anwendung auch zur Reduktion vaskulärer Schädigungen durch bakterielle Toxine finden.

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.

At present, the exact mechanism of the pathogenic effect of anti-PR-3 antibodies remains unknown. Interaction of anti-neutrophil cytoplasmic antibodies (ANCAs) with human umbilical vein endothelial cells (HUVECs) may play a key role. Recently we were able to show that ANCAs recognize their target antigen, PR-3, translocated into the membrane of HUVECs. The objective of this study was to investigate regulation, i.e. signal transduction pathways, of PR-3 expression in endothelial cells. HUVECs were isolated according to the method of Jaffe et al. and cultured under standard conditions. A cyto-enzyme-linked immunosorbent assay (ELISA) with unfixed cells was performed. Membrane-expressed PR-3 was detected by affinity-purified and monoclonal anti-PR-3 Ab. Tumour necrosis factor alpha (TNF-alpha)-induced membrane expression of PR-3 could be blocked with the RNA synthesis inhibitor actinomycin D, the protein kinase C (PKC) and proteinase A (PKA) inhibitor staurosporine, the specific PKC inhibitor calphostin C, the c-AMP-dependent PKA inhibitor KT5720 and the tyrosine kinase inhibitor genistein in a dose-dependent manner. The effect of calphostin C was the most significant. In addition, the effect of phorbol 12-myristate 13-acetate (PMA), a mediator of intracellular second messengers, was investigated. In our study, pretreatment of cells with PMA for 48 h led to a down-regulation of PR-3 expression. This effect, however, could be overridden by TNF-alpha stimulation, i.e. TNF-alpha-induced membrane expression of PR-3 was resistant to down-regulation of PKC. In conclusion, our data suggest that translocation of PR-3 in HUVECs is an active process depending on protein synthesis. PR-3 expression by HUVECs may involve a PKC reactive to cytokines such as TNF-alpha which induces PR-3 expression at a transcriptional level.

Botulinum C2 toxin, a binary toxin that ADP-ribosylates nonmuscle G-actin, was used as a selective tool to evaluate the role of actin-dependent cytoskeletal rearrangement in ligand-evoked lipid mediator generation. Human neutrophils (PMN) were preincubated with varying concentrations of the toxin for 30 min. Lipoxygenase products of arachidonic acid were measured by chromatographic techniques in the presence of exogenous arachidonic acid to probe PMN 5-lipoxygenase activity. Formation of platelet-activating factor (PAF) was assayed by the bioincorporation of [3H]acetate. Stimulation was performed with the soluble chemotactic ligands formyl-methionyl-leucyl-phenylalanine (FMLP) and PAF, as well as opsonized zymosan. PMN pretreatment with C2 toxin in the range between 200/400 and 800/1600 ng/ml C2I/II caused a dose-dependent suppression of the basal F-actin content and of stimulus-induced actin assembly. Phosphoinositide hydrolysis (measured as liberated inositol phosphates) and PAF generation in response to FMLP and exogenous PAF were markedly increased at these toxin doses. Minor C2 toxin concentrations (range, approximately 25/50 to 200/400 ng/ml C2I/II) were sufficient to amplify stimulus-induced formation of leukotriene B4 and its omega-oxidation products, nonenzymatic hydrolysis products of leukotriene A4, and 5-hydroxyeicosatetraenoic acid (5-HETE). With increasing toxin doses, leukotriene generation declined and 5-HETE became the predominant metabolite. In contrast to the soluble ligands, the zymosan-effected generation of PAF and leukotrienes was dose-dependently inhibited by C2 toxin concentrations of greater than 200/400 ng/ml, paralleled by a loss of motile and phagocytotic functions in these cells. We conclude that selective inhibition of actin assembly amplifies PAF and 5-lipoxygenase product formation in response to soluble chemoattractants with distinct dose dependences. The augmentation of PAF generation may be linked to amplified second messenger levels at higher doses of C2 toxin, whereas the sensitivity of the 5-lipoxygenase metabolism to low concentrations may indicate toxin effect on a small, functionally specified, actin pool. The present data support an important role of cytoskeletal rearrangement in temporal and/or spatial limitation of chemoattractant-evoked PMN activation.

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA-positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody-related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA (10 microM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha-priming and AA incubation was ineffective. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-lipoxygenase pathway was demasked, with the appearance of excessive quantities of leukotriene (LT)B4, LTA4, and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-lipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4-related autocrine loop of cell activation. In contrast, the increased synthesis of platelet-activating factor in response to TNF-alpha-priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5-lipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.

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 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.