Jutta Schulte

Increased circulating endothelial progenitor cells in septic patients: Correlation with survival

Objective:Endothelial damage and detachment of endothelial cells are known to occur in septic patients. Thus, recruitment of circulating endothelial progenitor cells (cEPCs) to these lesions might have a beneficial effect on the clinical course in septic patients. Therefore, we were interested in whether EPCs, detected by flow cytometry, are increasingly mobilized during sepsis and if this mobilization is associated with clinical outcome. Design:Prospective, nonrandomized study. Setting:Intensive care unit of a university hospital. Patients:Patients with (n = 32) and without (n = 15) sepsis and healthy volunteers (n = 15). Interventions:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and cEPCs were characterized by three-color fluorescence flow cytometry using antibodies against CD133, CD34, and vascular endothelial growth factor receptor-2. Serum concentrations of vascular endothelial growth factor, granulocyte macrophage-colony stimulating factor, and erythropoietin were determined by enzyme-linked immunosorbent assay. Severity of sepsis was assessed according to Acute Physiology and Chronic Health Evaluation II scoring. Measurements and Main Results:In septic patients, the number of cEPCs was significantly higher than in nonseptic intensive care unit patients (p < .05) and healthy controls (p < .02). Nonsurvivors (n = 8), defined as death within 28 days after onset of sepsis, had significantly lower numbers of cEPCs than survivors (n = 24) (p < .0001). The number of cEPCs was correlated with survival in septic patients. Serum vascular endothelial growth factor concentrations were significantly higher in septic patients compared with nonseptic intensive care unit patients and healthy controls (p < .01) and correlated with the cEPC numbers (p < .0001). Similar findings were observed for granulocyte macrophage-colony stimulating factor and erythropoietin. Conclusions:Our data suggest that cEPC enumeration in peripheral blood of septic patients might be a valuable marker to assess the clinical outcome in these patients.

Secreted phospholipases A2 induce the expression of chemokines in microvascular endothelium

Acute respiratory distress syndrome (ARDS) is characterized by alterations in microvascular permeability. In ARDS secreted phospholipase A(2) (sPLA(2)) IB and IIA are found to be highly upregulated. In this study, we therefore investigated the influence of exogenously added sPLA(2)-IB and sPLA(2)-IIA on the production of chemokines and adhesion molecules in lung microvascular endothelial cells (LMVEC). Treatment of LMVEC with sPLA(2)s resulted in a significant increase in the production of chemokines and adhesion molecules due to an increased expression of their mRNA and in an enhanced release of oleic acid. The upregulation of chemokines and adhesion molecules by LPS was stronger in the presence of sPLA(2). Activation of NF-kappaB occurred upon stimulation with sPLA(2). Moreover the MAPkinase pERK seems to be involved since a specific pERK inhibitor, e.g., U0126, but not a p38Kinase inhibitor, e.g., SB203580 prevented sPLA(2)-induced chemokine upregulation. Our data therefore suggest that LMVEC are a highly sensitive target for the direct action of extracellular sPLA(2)s.

Inhibition of LPS-induced chemokine production in human lung endothelial cells by lipid conjugates anchored to the membrane

In acute respiratory distress syndrome (ARDS) induced by endotoxins, a high production of inflammatory mediators by microvascular lung endothelial cells (LMVEC) can be observed. Activation of cells by endotoxins may result in elevated secretion of phospholipase A2 (sPLA2) which is thought to contribute to tissue damage. The present study was undertaken to investigate the role of sPLA2 in chemokine production in human lung microvascular endothelial cells (LMVEC) stimulated with the endotoxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA). In particular, we investigated the effects of sPLA2 inhibitors, specifically, the extracellular PLA2 inhibitors (ExPLIs), composed of N‐derivatized phosphatidyl‐ethanolamine linked to polymeric carriers, and LY311727, a specific inhibitor of non‐pancreatic sPLA2. ExPLIs markedly inhibited LPS and LTA induced production and mRNA expression of the neutrophile attracting chemokines IL‐8, Gro‐α and ENA‐78, as well as of the adhesion molecules ICAM‐1 and E‐selectin. Concomitantly, ExPLIs inhibited the LPS‐induced activation of NF‐κB by LPS but not its activation by TNF‐α or IL‐1. Endotoxin mediated chemokine production in LMVEC seems not to involve PLA2 activity, since LPS stimulation was not associated with activation of intracellular or secreted PLA2. It therefore seems that the inhibitory effect of the ExPLIs was not due to their PLA2 inhibiting capacity. This was supported by the finding that the LPS‐induced chemokine production was not affected by the selective sPLA2 inhibitor LY311727. It is proposed that the ExPLIs may be considered a prototype of potent suppressors of specific endotoxin‐induced inflammatory responses, with potential implications for the therapy of subsequent severe inflammation.

Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factors

Interindividual differences of endothelial cells in response to endotoxins might contribute to the diversity in clinical outcome among septic patients. The present study was conducted to test the hypothesis that endothelial cells (EC) with high and low proinflammatory potential exist and to dissect the molecular basis underlying this phenomenon. Thirty human umbilical vein endothelial cell (HUVEC) lines were stimulated for 24 h with lipopolysaccharide (LPS) and screened for interleukin (IL)‐8 production. Based on IL‐8 production five low and five high producers, tentatively called types I and II responders, respectively, were selected for genome‐wide gene expression profiling. From the 74 genes that were modulated by LPS in all type II responders, 33 genes were not influenced in type I responders. Among the 41 genes that were increased in both responders, 17 were expressed significantly stronger in type II responders. Apart from IL‐8, significant differences in the expression of proinflammatory related genes between types I and II responders were found for adhesion molecules [intercellular adhesion molecule (ICAM‐1), E‐selectin)], chemokines [monocyte chemoattractant protein (MCP‐1), granulocyte chemotactic protein (GCP‐2)], cytokines (IL‐6) and the transcription factor CCAAT/enhancer binding protein‐delta (C/EBP‐δ). Type I responders also displayed a low response towards tumour necrosis factor (TNF)‐α. In general, maximal activation of nuclear factor (NF)‐κB was achieved in type I responders at higher concentrations of LPS compared to type II responders. In the present study we demonstrate that LPS‐mediated gene expression differs quantitatively and qualitatively in types I and II responders. Our results suggest a pivotal role for common transcription factors as a low inflammatory response was also observed after TNF‐α stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients.

Fractalkine is not a major chemoattractant for the migration of neutrophils across microvascular endothelium.

Inflammatory responses during sepsis are determined by leucocyte recruitment into inflamed tissues. Both chemokines and adhesion molecules are believed to be involved in this process. As fractalkine exists as transmembrane protein with cell adhesion properties and as soluble chemotactic factor, the present study was conducted to study the role of fractalkine, produced by microvascular and macrovascular endothelial cells, in neutrophil recruitment. Lung microvascular endothelial cells (LMVECs) stimulated with lipopolysaccharide, tumour necrosis factor‐α or interleukin‐1 (IL‐1) produced much more fractalkine compared with the macrovascular human umbilical vein endothelial cells (HUVECs). No differences were found between microvascular endothelial cells of different organs. Chemotactic activity in supernatants was significantly stronger in stimulated LMVEC when compared with HUVEC. Although recombinant fractalkine induced migration of neutrophils, IL‐8 and monocyte chemoattractant protein‐1 were found to be more strictly required. In vivo fractalkine was strongly upregulated in septic lung and kidney. Our data suggest that fractalkine production per se does not explain the preference for inflammation in the lung of septic patients.

Amelioration of endotoxin-induced sepsis in rats by membrane anchored lipid conjugates.

ObjectiveIn the pathogenesis of septic shock, caused by either bacterial toxins or trauma, increased production of multiple proinflammatory mediators, such as phospholipase A2 (PLA2), cytokines, and chemokines, is known to be of major importance. The present study was undertaken to investigate the influence of a newly designed extracellular PLA2 inhibitor (ExPLI) on synthesis of proinflammatory mediators and mortality rate in a rat sepsis model. DesignProspective, randomized animal study. SettingExperimental laboratory. SubjectsMale Wistar-rats weighing 200–300 g. InterventionsMortality was induced by intraperitoneal bolus administration of lipopolysaccharide 15 mg/kg in 22 rats that were pretreated with NaCl or ExPLI (150 mg/kg). Furthermore, nine rats received a sublethal bolus of lipopolysaccharide (7.5 mg/kg) and nine rats received lipotechoic acid (8 mg/kg) simultaneously with or after ExPLI administration. Blood samples were collected from these rats, and cytokine concentrations were assessed by enzyme-linked immunosorbent assay. Lung and kidney were removed for RNA isolation and immunohistological analysis. Measurements and Main ResultsExPLI treatment significantly reduced lipopolysaccharide-induced mortality of rats (90.9 and 36.4%, p < .05). Up-regulation of tumor necrosis factor-&agr; and interleukin-6 production in serum after endotoxin treatment was significantly inhibited when ExPLIs were administered at the time of or before sepsis induction by using lipopolysaccharide or lipotechoic acid (p < .01). Similarly, messenger RNA expression of secreted PLA2-IIA, interleukin-1, or inducible nitric oxide synthase and the expression of intercellular adhesion molecule-1 were significantly down-regulated in lung and kidney of ExPLI-treated rats, as demonstrated by RNase protection assay, reverse transcription-polymerase chain reaction, or immunohistochemistry. ConclusionsExPLIs may be considered as potentially effective compounds to prevent the production of inflammatory mediators and to improve mortality rate in septic patients.

Effekte von Dopamin auf die zelluläre und humorale Immunantwort von Patienten mit Sepsis

ZusammenfassungZahlreiche In-vitro- und In-vivo-Studien belegen, dass Dopamin neben seinen hämodynamischen Effekten eine Reihe immunmodulatorischer Wirkungen induziert. Dopamin reduziert die Synthese proinflammatorischer und induziert die Synthese antiinflammatorischer Mediatoren. Dopamin hemmt die Synthese neurohypophysärer Hormone und hemmt die Zellproliferation sowie die Thrombozytenaggregation. Es reduziert die Phagozytoseaktivität neutrophiler Granulozyten und induziert Apoptose. Bei hohen Dopaminserumkonzentrationen, wie sie bei einer Sepsis durch vermehrte endogene Synthese, zusätzliche exogene Applikation und verringerte Clearance erreicht werden, könnten diese Effekte zu relevanten Veränderungen pathophysiologischer Abläufe führen. Um die Bedeutung von Dopamin für die zelluläre und humorale Immunantwort von Patienten mit Sepsis hervorzuheben, sind in dieser Übersicht die speziellen Wirkungen von Dopamin zusammengefasst und die zugrunde liegenden Mechanismen dargestellt.AbstractIn vitro and in vivo studies have demonstrated that apart from its hemodynamic action dopamine can modulate immune responses. Dopamine reduces the synthesis of proinflammatory and induces the synthesis of anti-inflammatory mediators. Dopamine inhibits neurohormone synthesis, lymphocyte proliferation and platelet aggregation. It reduces the phagocytic activity of neutrophils and induces apoptosis. Particularly with regard to sepsis, where high serum dopamine levels are reached by enhanced endogeneous production, exogeneous application and impaired clearance, this immunomodulation may have a clinical impact. This review summarizes dopamine-mediated immunomodulating effects to advance the knowledge regarding dopamine as an immune regulator under septic conditions.In vitro and in vivo studies have demonstrated that apart from its hemodynamic action dopamine can modulate immune responses. Dopamine reduces the synthesis of proinflammatory and induces the synthesis of anti-inflammatory mediators. Dopamine inhibits neurohormone synthesis, lymphocyte proliferation and platelet aggregation. It reduces the phagocytic activity of neutrophils and induces apoptosis. Particularly with regard to sepsis, where high serum dopamine levels are reached by enhanced endogenous production, exogenous application and impaired clearance, this immunomodulation may have a clinical impact. This review summarizes dopamine-mediated immunomodulating effects to advance the knowledge regarding dopamine as an immune regulator under septic conditions.

Effekte von Dopamin auf die zelluläre und humorale Immunantwort von Patienten mit Sepsis@@@Effects of dopamine on cellular and humoral immune responses in septic patients

ZusammenfassungZahlreiche In-vitro- und In-vivo-Studien belegen, dass Dopamin neben seinen hämodynamischen Effekten eine Reihe immunmodulatorischer Wirkungen induziert. Dopamin reduziert die Synthese proinflammatorischer und induziert die Synthese antiinflammatorischer Mediatoren. Dopamin hemmt die Synthese neurohypophysärer Hormone und hemmt die Zellproliferation sowie die Thrombozytenaggregation. Es reduziert die Phagozytoseaktivität neutrophiler Granulozyten und induziert Apoptose. Bei hohen Dopaminserumkonzentrationen, wie sie bei einer Sepsis durch vermehrte endogene Synthese, zusätzliche exogene Applikation und verringerte Clearance erreicht werden, könnten diese Effekte zu relevanten Veränderungen pathophysiologischer Abläufe führen. Um die Bedeutung von Dopamin für die zelluläre und humorale Immunantwort von Patienten mit Sepsis hervorzuheben, sind in dieser Übersicht die speziellen Wirkungen von Dopamin zusammengefasst und die zugrunde liegenden Mechanismen dargestellt.AbstractIn vitro and in vivo studies have demonstrated that apart from its hemodynamic action dopamine can modulate immune responses. Dopamine reduces the synthesis of proinflammatory and induces the synthesis of anti-inflammatory mediators. Dopamine inhibits neurohormone synthesis, lymphocyte proliferation and platelet aggregation. It reduces the phagocytic activity of neutrophils and induces apoptosis. Particularly with regard to sepsis, where high serum dopamine levels are reached by enhanced endogeneous production, exogeneous application and impaired clearance, this immunomodulation may have a clinical impact. This review summarizes dopamine-mediated immunomodulating effects to advance the knowledge regarding dopamine as an immune regulator under septic conditions.In vitro and in vivo studies have demonstrated that apart from its hemodynamic action dopamine can modulate immune responses. Dopamine reduces the synthesis of proinflammatory and induces the synthesis of anti-inflammatory mediators. Dopamine inhibits neurohormone synthesis, lymphocyte proliferation and platelet aggregation. It reduces the phagocytic activity of neutrophils and induces apoptosis. Particularly with regard to sepsis, where high serum dopamine levels are reached by enhanced endogenous production, exogenous application and impaired clearance, this immunomodulation may have a clinical impact. This review summarizes dopamine-mediated immunomodulating effects to advance the knowledge regarding dopamine as an immune regulator under septic conditions.