Lutz Koch

LPS- and LTA-Induced Expression of IL-6 and TNF-α in Neonatal and Adult Blood: Role of MAPKs and NF-κB

As nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) seem to be critical mediators in the inflammatory response, we studied the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on (a) the activation of NF-κB and MAPKs and (b) the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) with or without the specific inhibitors of these intracellular signal transduction pathways in neonatal cord and adult blood. TNF-α and IL-6 concentrations showed a sharp increase in the supernatants of cord and adult whole blood after stimulation. TNF-α concentrations were significantly higher, whereas IL-6 concentrations were tendentially lower in adult blood after stimulation. Stimulation with LPS or LTA resulted in a significantly decreased activation of p38 MAPK in neonatal compared with adult blood. Although LTA failed to induce additional ERK1/2 phosphorylation, LPS stimulation mediated the moderately increased levels of activated ERK1/2 in neonatal monocytes. The addition of the p38 MAPK inhibitor SB202190 significantly decreased IL-6 and TNF-α production upon LPS or LTA stimulation. Furthermore, the inhibition of ERK1/2 was able to reduce LPS-stimulated TNF-α production in neonatal blood. We conclude that p38 MAPK as well as ERK1/2 phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.

Protein C Concentrate Controls Leukocyte Recruitment during Inflammation and Improves Survival during Endotoxemia after Efficient in Vivo Activation

Anti-inflammatory properties of protein C (PC) concentrate are poorly studied compared to activated protein C, although PC is suggested to be safer in clinical use. We investigated how PC interferes with the leukocyte recruitment cascade during acute inflammation and its efficacy during murine endotoxemia. We found that similar to activated protein infusion, intravenous PC application reduced leukocyte recruitment in inflamed tissues in a dose- and time-dependent manner. During both tumor necrosis factor-α induced and trauma-induced inflammation of the cremaster muscle, intravital microscopy revealed that leukocyte adhesion and transmigration, but not rolling, were profoundly inhibited by 100 U/kg PC. Moreover, PC blocked leukocyte emigration into the bronchoalveolar space during lipopolysaccharide (LPS) induced acute lung injury. PC was efficiently activated in a murine endotoxemia model, which reduced leukocyte infiltration of organs and strongly improved survival (75% versus 25% of control mice). Dependent on the inflammatory model, PC provoked a significant inhibition of leukocyte recruitment as early as 1 hour after administration. PC-induced inhibition of leukocyte recruitment during acute inflammation critically involves thrombomodulin-mediated PC activation, subsequent endothelial PC receptor and protease-activated receptor-1-dependent signaling, and down-regulation of intercellular adhesion molecule 1 leading to reduced endothelial inflammatory response. We conclude that during acute inflammation and sepsis, PC is a fast acting and effective therapeutic approach to block leukocyte recruitment and improve survival.

Neutrophil Chemotaxis in Cord Blood of Term and Preterm Neonates Is Reduced in Preterm Neonates and Influenced by the Mode of Delivery and Anaesthesia

Bacterial infections, even without any perinatal risk factors, are common in newborns, especially in preterm neonates. The aim of this study was to evaluate possible impairment of neutrophil chemotaxis in term and preterm neonates compared with adults as well as neonates with different modes of delivery and anaesthesia. We analysed the expression of the adhesion molecule L-Selectin as well as shape change, spontaneous and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced transmigration of neutrophils in a flow cytometric assay of chemotaxis after spontaneous delivery with Cesarian Section (CS) under spinal anaesthesia (mepivacaine, sufentanil), epidural anaesthesia (ropivacaine or bupivacaine, sufentanil) or general anaesthesia (ketamine, thiopental, succinylcholine). Chemokinesis was higher (p=0.008) in cord blood neutrophils than in the adult ones, whereas those could be more stimulated by fMLP (p=0.02). After vaginal delivery neutrophils showed a higher spontaneous and fMLP-stimulated chemotactic response compared to neonates after CS without labor. Comparing different types of anaesthesia for CS, spinal anaesthesia resulted in less impairment on chemotaxis than general anaesthesia or epidural anaesthesia. The new flow cytometric assay of neutrophil chemotaxis is an appropriate and objective method to analyse functional differences even in very small volumes of blood, essential in neonatology. Term neonates do not show reduced chemotaxis compared to adults. Preterm neonates present with reduced chemotaxis and chemokinesis, confirming the well known deficits in their neutrophil function. The side effects of maternal drugs on the neonatal immune system have to be considered especially when the immune response is already impaired, as in preterm infants.

CXCL1-Triggered Interaction of LFA1 and ICAM1 Control Glucose-Induced Leukocyte Recruitment during Inflammation In Vivo

It is well acknowledged that proinflammatory stimulation during acute hyperglycemia is able to aggravate inflammatory diseases. However, the mechanisms of proinflammatory effects of glucose are controversially discussed. We investigated leukocyte recruitment after intravenous injection of glucose in different inflammatory models using intravital microscopy. Flow chamber experiments, expression analysis, functional depletion, and knockout of key adhesion molecules gave mechanistic insight in involved pathways. We demonstrated that a single injection of glucose rapidly increased blood glucose levels in a dose-dependent manner. Notably, during tumor necrosis factor (TNF) α-induced inflammation leukocyte recruitment was not further enhanced by glucose administration, whereas glucose injection profoundly augmented leukocyte adhesion and transmigration into inflamed tissue in the trauma model, indicating that proinflammatory properties of glucose are stimulus dependent. Experiments with functional or genetic inhibition of the chemokine receptor CXCR2, intercellular adhesion molecule 1 (ICAM1), and lymphocyte function antigen 1 (LFA1) suggest that keratino-derived-chemokine CXCL1-triggered interactions of ICAM1 and LFA1 are crucially involved in the trauma model of inflammation. The lacking effect of glucose on β 2 integrin expression and on leukocyte adhesion in dynamic flow chamber experiments argues against leukocyte-driven underlying mechanisms and favours an endothelial pathway since endothelial ICAM1 expression was significantly upregulated in response to glucose.

Lipopolysaccharide-Induced Expression of Th1/Th2 Cytokines in Whole Neonatal Cord and Adult Blood: Role of Nuclear Factor-Kappa B and p38 MAPK

Background: Sepsis continues to be a leading cause of morbidity and mortality in newborns. Objective: As both nuclear factor-kappa B (NF-ĸB) and p38 mitogen-activated protein kinase (MAPK) appear to be critical mediators in inflammatory response, we studied the effects of lipopolysaccharide (LPS) on expression and function of NF-ĸB and p38 MAPK in whole neonatal cord and adult blood. Methods:Th1/Th2 cytokine concentrations and phosphorylation of NF-ĸB and p38 MAPK were determined by flow-cytometric analysis. Results: Tumor necrosis factor-alpha (TNF-α), IL-6, and IL-10 concentrations were significantly elevated in supernatants of neonatal and adult blood after LPS stimulation for 4 h. IFN-γ, IL-4, and IL-2 showed no significant alterations. Furthermore, TNF-α concentrations were significantly higher in adult compared to neonatal blood after LPS stimulation. Stimulation with LPS resulted in significantly decreased activation of p38 MAPK in neonatal blood, whereas NF-ĸB showed no difference. Following inhibition of p38 MAPK with the specific inhibitor SB-202190, levels of TNF-α and IL-6 significantly decreased in neonatal and adult blood, whereas pharmacological inhibition of NF-ĸB with SC-514 showed no significant effect on cytokine expression. Conclusions: We conclude that p38 MAPK phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.

Lipopolysaccharide-induced activation of coagulation in neonatal cord and adult blood monitored by thrombelastography.

INTRODUCTION Although precisely balanced hemostasis in newborns is rapidly changing during early development. During gram-negative sepsis, lipopolysaccharide (LPS) activates toll-like receptor (TLR) 4 and induces complex responses of immune system and hemostasis. In the present study we compared LPS-induced activation of coagulation in cord blood (CB) samples of healthy newborns to whole blood (WB) samples from healthy adult volunteers. MATERIALS AND METHODS Samples were incubated with LPS for various incubation periods (0-2-3-4 hrs), and coagulation was measured by rotation thrombelastography (TEG). RESULTS All parameters (clotting time (CT), clot formation time (CFT), maximal clot formation (MCF), and angle alpha) were affected with increasing incubation period with LPS (100 ng/ml) in adult and cord WB. In the absence of LPS, CTs and CFTs were significantly shorter and MCFs and angels alpha were significantly longer in cord than in adult WB samples. Reduction of CT due to LPS incubation was significantly stronger in CB than in adult WB samples. Pyrrolidine dithiocarbamate, a specific inhibitor of the NFkappaB pathway, inhibited the effects of LPS on CT in adult and cord WB. CONCLUSION In summary, TEG proved to be a sensitive and reliable tool for the determination of LPS-induced tissue factor mediated activation of hemostasis in whole blood samples from adults and neonates.

Gene Expression Profiles of Adult Peripheral and Cord Blood Mononuclear Cells Altered by Lipopolysaccharide

Background: Neonatal Gram-negative sepsis is often characterized by a fulminant clinical course, compared to adults, resulting in higher morbidity and mortality. Genome-wide gene expression analysis can provide insights into the molecular alterations in sepsis. Objectives: To evaluate in vitro activation of the neonatal and adult immune system, gene expression patterns were compared in mononuclear cells from cord (CBMNC) and adult peripheral blood (APBMNC). Methods: To better understand the influence of early molecular signals on the effects of sepsis, Affymetrix gene profiling (8,475 genes) was done on RNA isolated from CBMNC and APBMNC without and after incubation with 100 ng/ml lipopolysaccharide (LPS). Results: We demonstrated significant alterations in the expression of 108 CBMNC and APBMNC genes compared with basal levels, 188 significant changes in CBMNC and 97 in APBMNC, including cytokines, chemokines and immunoregulatory genes. Furthermore, we found 5 genes showing a significant interaction effect between cell type and LPS stimulation, including tumor necrosis factor receptor superfamily, member 6 (FAS), absent in melanoma 2, malic enzyme 1, hemoglobin Ε 1, and trans-prenyltransferase. Conclusions: These results provide further support for a marked difference in the pathogenesis of neonatal and adult sepsis and may stimulate additional studies to investigate some of the altered genes as potential new targets for diagnostic tools and therapeutic strategies.

Stable Precursor Fragments of Vasoactive Peptides in Umbilical Cord Blood of Term and Preterm Infants

Background:Though various neurohormonal systems are concurrently activated during birth, their biological effectors are not always easy to measure due to their short half-life in vivo, instability in biological samples, or very low concentrations. Methods: Using a recently discovered chemiluminescence assay, we measured the stable precursor fragments mid-regional pro-atrial natriuretic peptide (MR-proANP), mid-regional pro-adrenomedullin (MR-proADM), C-terminal pro-endothelin-1 (CT-proET-1) and C-terminal pro-vasopressin (CT-proAVP or copeptin) immediately after birth in umbilical venous cord blood from 119 infants with a gestational age of 23–42 weeks and evaluated their possible functions. Results:Cord blood levels of MR-proANP, MR- proADM, CT-proET-1, and CT-proAVP were considerably higher compared with normal adult levels. The CT-proAVP concentrations were 10-fold higher in term, and 70-fold higher in extremely preterm infants. MR-proANP showed 4-fold higher levels in term infants and 20-fold higher levels in extremely preterm infants. Levels of MR-proADM and CT-proET-1 were 2- to 3-fold higher in preterm and term infants. All four parameters showed significantly decreased values with increasing gestational age and a significant correlation between CT-proET-1 and MR-proADM. Conclusion:Our results indicate that vasoactive and natriuretic mediators play a functionally relevant role in circulatory transition from fetal to neonatal life.

RAGE Controls Activation and Anti-Inflammatory Signalling of Protein C

Aims The receptor for advanced glycation endproducts, RAGE, is a multiligand receptor and NF-κB activator leading to perpetuation of inflammation. We investigated whether and how RAGE is involved in mediation of anti-inflammatory properties of protein C. Methods and Results We analyzed the effect of protein C on leukocyte adhesion and transmigration in WT- and RAGE-deficient mice using intravital microscopy of cremaster muscle venules during trauma- and TNFα-induced inflammation. Both, protein C (PC, Ceprotin, 100 U/kg) and activated protein C (aPC, 24 µg/kg/h) treatment significantly inhibited leukocyte adhesion in WT mice in these inflammation models. The impaired leukocyte adhesion after trauma-induced inflammation in RAGE knockout mice could not be further reduced by PC and aPC. After TNFα-stimulation, however, aPC but not PC treatment effectively blocked leukocyte adhesion in these mice. Consequently, we asked whether RAGE is involved in PC activation. Since RAGE-deficient mice and endothelial cells showed insufficient PC activation, and since thrombomodulin (TM) and endothelial protein C receptor (EPCR) are reduced on the mRNA and protein level in RAGE deficient endothelial cells, an involvement of RAGE in TM-EPCR-dependent PC activation is likely. Moreover, TNFα-induced activation of MAPK and upregulation of ICAM-1 and VCAM-1 are reduced both in response to aPC treatment and in the absence of RAGE. Thus, there seems to be interplay of the RAGE and the PC pathway in inflammation. Conclusion RAGE controls anti-inflammatory properties and activation of PC, which might involve EPCR and TM.

Inhibition of LPS-Induced Activation of Coagulation by p38 MAPK Inhibitor

During Gram-negative sepsis, lipopolysaccharide (LPS) activates toll-like receptor (TLR) 4 and induces complex responses of immune system and coagulation. However, the underlying LPS signalling mechanism on coagulation activation remains complex. To determine the role of the intracellular signalling factors p38 mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and c-Jun N-terminal kinase (JNK) in the procoagulant response to LPS, coagulation process of human whole blood exposed to specific inhibitors was measured by thrombelastography. Samples were stimulated with LPS (100 μg/mL) after preincubation with BAY117082 (specific NF-κB inhibitor), SP600125 (specific JNK inhibitor), SB203580 (specific p38 MAPK inhibitor), or vehicle. SB203580 strongly inhibited LPS-induced coagulation activation, whereas BAY117082 and SP600125 showed no significant effect. Activation of p38 MAPK, NF-κB, and JNK and respective inhibitory effects were confirmed by Multi-Target Sandwich ELISA. In conclusion, activation of p38 MAPK is crucial for early LPS-induced activation of coagulation.