Sebastiaan Weijer

Anti-Inflammatory Effects of a p38 Mitogen-Activated Protein Kinase Inhibitor During Human Endotoxemia

The p38 mitogen-activated protein kinase (MAPK) participates in intracellular signaling cascades resulting in inflammatory responses. Therefore, inhibition of the p38 MAPK pathway may form the basis of a new strategy for treatment of inflammatory diseases. However, p38 MAPK activation during systemic inflammation in humans has not yet been shown, and its functional significance in vivo remains unclear. Hence, we exposed 24 healthy male subjects to an i.v. dose of LPS (4 ng/kg), preceded 3 h earlier by orally administered 600 or 50 mg BIRB 796 BS (an in vitro p38 MAPK inhibitor) or placebo. Both doses of BIRB 796 BS significantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction of the volunteers. Cytokine production (TNF-α, IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition diminished leukocyte responses, including neutrophilia, release of elastase-α1-antitrypsin complexes, and up-regulation of CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK decreased C-reactive protein release. These data identify p38 MAPK as a principal mediator of the inflammatory response to LPS in humans. Furthermore, the anti-inflammatory potential of an oral p38 MAPK inhibitor in humans in vivo suggests that p38 MAPK inhibitors may provide a new therapeutic option in the treatment of inflammatory diseases.

Role of Toll-Like Receptor 4 in Gram-Positive and Gram-Negative Pneumonia in Mice

ABSTRACT To determine the role of Toll-like receptor 4 (TLR4) in the immune response to pneumonia, C3H/HeJ mice (which display a mutant nonfunctional TLR4) and C3H/HeN wild-type mice were intranasally infected with either Streptococcus pneumoniae (a common gram-positive respiratory pathogen) or Klebsiella pneumoniae (a common gram-negative respiratory pathogen). In cases of pneumococcal pneumonia, TLR4 mutant mice showed a reduced survival only after infection with low-level bacterial doses, which was associated with a higher bacterial burden in their lungs 48 h postinfection. In Klebsiella pneumonia, TLR4 mutant mice demonstrated a shortened survival after infection with either a low- or a high-level bacterial dose together with an enhanced bacterial outgrowth in their lungs. These data suggest that TLR4 contributes to a protective immune response in both pneumococcal and Klebsiella pneumonia and that its role is more important in respiratory tract infection caused by the latter (gram-negative) pathogen.

Improved Host Defense against Pneumococcal Pneumonia in Platelet-Activating Factor Receptor-Deficient Mice

Platelet-activating factor (PAF) is a phospholipid with proinflammatory properties that binds to a specific receptor (PAF receptor [PAFR]) that is expressed on many different cell types. PAFR is able to bind phosphorylcholine, which is present in both PAF and the pneumococcal cell wall. Activation of respiratory epithelial cells in vitro results in up-regulation of PAFR, which, in turn, facilitates invasion of Streptococcus pneumoniae. To determine the role of PAFR in host defense against pneumococcal pneumonia, PAFR-deficient (PAFR(-/-)) and wild-type (wt) mice were inoculated intranasally with S. pneumoniae. PAFR(-/-) mice were relatively resistant to pneumococcal pneumonia, as indicated by delayed and reduced mortality, diminished outgrowth of pneumococci in lungs, and reduced dissemination of the infection (all P<.05, vs. wt mice). PAFR(-/-) mice also had less pulmonary inflammation. These data provide evidence that PAFR is used by S. pneumoniae to induce lethal pneumonia.

Local activation of the tissue factor-factor VIIa pathway in patients with pneumonia and the effect of inhibition of this pathway in murine pneumococcal pneumonia.

Objective:The tissue factor (TF)-factor VIIa (FVIIa) complex not only is essential for activation of blood coagulation but also affect the inflammatory response during sepsis. The objective of this study was to determine the role of TF-FVIIa in pneumonia caused by Streptococcus pneumoniae, the most important causative organism in community-acquired pneumonia and a major cause of sepsis. Design:A controlled, in vivo laboratory study. Setting:Research laboratory of a health sciences university. Patients and Subjects:Patients with unilateral community-acquired pneumonia and female BALB/c mice. Interventions:Bilateral bronchoalveolar lavage was performed in patients with community-acquired pneumonia. In mice, pneumonia was induced by intranasal inoculation with S. pneumoniae with or without concurrent inhibition of TF-FVIIa by subcutaneous injections of recombinant nematode anticoagulant protein (rNAPc2). Measurements and Main Results:Patients with unilateral community-acquired pneumonia demonstrated elevated concentrations of FVIIa, soluble TF, and thrombin-antithrombin complexes in bronchoalveolar lavage fluid obtained from the infected site compared with the uninfected site. Mice with S. pneumoniae pneumonia displayed increased TF expression and fibrin deposits in lungs together with elevated thrombin-antithrombin complex levels in bronchoalveolar lavage fluid; inhibition of TF-FVIIa by rNAPc2 attenuated the procoagulant response in the lung but did not affect host defense, as reflected by an unaltered outgrowth of pneumococci and an unchanged survival. Conclusions:These data suggest that TF-FVIIa activity contributes to activation of coagulation in the lung during pneumococcal pneumonia but does not play an important role in the antibacterial host defense in this murine model.

Macrophages Play a Dual Role during Pulmonary Tuberculosis in Mice

Pulmonary macrophages provide the preferred hiding and replication site of Mycobacterium tuberculosis but display antimicrobial functions. This raises questions regarding the role of macrophages during tuberculosis. We depleted lungs of activated macrophages (activated macrophage(-) mice) and compared this with nonselective macrophage depletion (macrophage(-) mice). Although nonselective depletion of macrophages after infection improved clinical outcome, depletion of activated macrophages led to impaired resistance, reflected by enhanced mycobacterial outgrowth. The production of tumor necrosis factor- alpha and numbers of granuloma decreased after depletion of activated macrophages. Both macrophage(-) and activated macrophage(-) mice showed polarized production of interferon- gamma by splenocytes and lymph-node cells and were able to attract and activate T cells in the lung. These data demonstrate that the dual role of macrophages is associated with the activation state of macrophages and that extensive apoptosis found in patients with tuberculosis could be part of a host defense strategy, as long as these cells are not activated.

Procoagulant protein levels are differentially increased during human endotoxemia.

Summary.  On the basis of plasma interleukin levels it was suggested that there is an inflammatory component to the risk of venous thrombotic disease. Other evidence shows that elevated levels of coagulation factor (F)VIII, FIX, FX and FXI are risk indicators for venous thrombosis, but the reasons for elevation remain unclear. We tested the hypothesis that the elevated levels could reflect an inflammatory reaction by measuring coagulation factor levels during experimental human endotoxemia. Male volunteers received endotoxin (4 ng kg−1), and blood samples were obtained before and at multiple time points after the challenge. Plasma was used for a panel of coagulation tests. Antigen levels of FVIII, von Willebrand factor (VWF), FIX, and FX were increased after endotoxin administration, reaching peak levels between 2 and 5 h. Within 24 h levels normalized, except for FVIII and VWF levels that remained at > 200%. Fibrinogen levels, and to a lesser extent FXI levels, also responded with an increase, but slower. These levels did not return to normal during the observation period. FVII levels were strongly depressed. FVIII, FIX and FX reacted immediately and strongly to endotoxin administration. The time pattern of this response is different from the slower so‐called acute phase response, which appeared to be followed by FXI and fibrinogen. These increased levels of coagulation factors during an inflammatory state provide new ways of explaining why elevated levels of FVIII, FIX and FXI behave as risk indicators disease.

Interleukin-18 Facilitates the Early Antimicrobial Host Response to Escherichia coli Peritonitis

ABSTRACT To determine the role of endogenous interleukin-18 (IL-18) during peritonitis, IL-18 gene-deficient (IL-18 KO) mice and wild-type mice were intraperitoneally (i.p.) infected with Escherichia coli, the most common causative agent found in septic peritonitis. Peritonitis was associated with a bacterial dose-dependent increase in IL-18 concentrations in peritoneal fluid and plasma. After infection, IL-18 KO mice had significantly more bacteria in the peritoneal lavage fluid and were more susceptible for progression to systemic infection at 6 and 20 h postinoculation than wild-type mice. The relative inability of IL-18 KO mice to clear E. coli from the abdominal cavity was not due to an intrinsic defect in the phagocytosing capacity of their peritoneal macrophages or neutrophils. IL-18 KO mice displayed an increased neutrophil influx into the peritoneal cavity, but these migratory neutrophils were less activate, as reflected by a reduced CD11b surface expression. These data suggest that endogenous IL-18 plays an important role in the early antibacterial host response during E. coli-induced peritonitis.

Inhibition of Plasmin Activity by Tranexamic Acid Does Not Influence Inflammatory Pathways During Human Endotoxemia

Objective—Plasmin activates several proinflammatory pathways at the cellular level in vitro. Lipopolysaccharide (LPS) administration to healthy humans results in a rapid generation of plasmin activity, accompanied by activation of a number of inflammatory systems. Methods and Results—To determine the role of early plasmin activity in LPS-induced inflammation in vivo, 16 healthy males received an intravenous bolus injection with LPS (from Escherichia coli, 4 ng/kg) directly preceded by a 30-minute intravenous infusion of tranexamic acid (2 g, n = 8), a plasmin activation inhibitor, or placebo (n=8). LPS injection induced marked increases in the plasma levels of D-dimer and plasmin-&agr;2-antiplasmin complexes, indicative of plasmin activation and generation, respectively, which were strongly attenuated by tranexamic acid (both P <0.01 versus placebo). However, tranexamic acid did not influence LPS-induced coagulation activation, granulocytosis, neutrophil activation (expression of CD11b, CD66b, and L-selectin) or degranulation (plasma concentrations of elastase-&agr;1-antitrypsin and bactericidal permeability-increasing protein), endothelial cell activation (plasma levels of von Willebrand factor and soluble E-selectin), or cytokine release. Conclusion—These data argue against a role of early plasmin generation in the subsequent activation of other inflammatory pathways during human endotoxemia.

Diminished Interferon-γ Production and Responsiveness after Endotoxin Administration to Healthy Humans

To obtain insight in the capacity of the lipopolysaccharide (LPS)-tolerant host to produce interferon (IFN)-gamma and to respond to this cytokine, whole blood was obtained from healthy humans before and 4 h after intravenous injection of LPS (4 ng/kg) and stimulated ex vivo. LPS exposure in vivo resulted in a diminished capacity to produce IFN-gamma after restimulation with LPS, together with a reduced ability to release the IFN-gamma-inducing cytokines interleukin (IL)-12 and IL-18 and with reduced responsiveness toward these cytokines. In addition, IFN-gamma responsiveness was strongly diminished after in vivo LPS exposure, as shown by the fact that blood obtained after LPS injection could not be primed by IFN-gamma for LPS-induced tumor necrosis factor-alpha release and that peripheral blood monocytes could not be stimulated by IFN-gamma to up-regulate major histocompatibility complex type II expression. Experimentally induced immunoparalysis is associated with strongly reduced IFN-gamma production and responsiveness.

P38 mitogen activated protein kinase is involved in the downregulation of granulocyte CXC chemokine receptors 1 and 2 during human endotoxemia.

Chemokine receptors CXC receptor (CXCR) 1 and 2, and their ligands interleukin (IL)-8 and growth-related oncogene alpha (GRO α), are principal regulators of neutrophil activation and migration. To investigate the role of p38 mitogen activated protein kinase (MAPK) in the regulation of CXCR expression during an inflammatory response in vivo, 24 healthy volunteers received an intravenous injection with lipopolysaccharide (LPS) preceded (−3 hr) by a specific p38 MAPK inhibitor (BIRB 796 BS) at a high dose (600 mg) or a low dose (50 mg) or a placebo. The LPS-induced reduction of neutrophil CXCR 1 and 2 expression, as determined by fluorescence-activated cell sorter analysis, was inhibited in volunteers receiving the high dose of the p38 MAPK inhibitor. The kinase inhibitor also dose dependently diminished the LPS-induced rises in plasma IL-8 and GROα levels. These results indicate a principal role for p38 MAPK in regulating factors essential for neutrophil activation and chemotaxis in vivo..