Rosemarijn Renckens

Matrix Metalloproteinase-9 Deficiency Impairs Host Defense against Abdominal Sepsis

Matrix metalloproteinase (MMP)-9 is involved in extracellular matrix degradation and leukocyte migration. To determine the role of MMP-9 in the innate immune response to peritonitis, MMP-9 gene-deficient (MMP-9−/−) and normal wild-type mice were i.p. infected with Escherichia coli. MMP-9 mRNA and pro-MMP-9 protein levels increased rapidly upon induction of peritonitis. Although MMP-9−/− neutrophils showed a normal phagocytosis of E. coli in vitro, MMP-9−/− mice displayed a reduced resistance against E. coli peritonitis, as indicated by an enhanced bacterial outgrowth in the peritoneal cavity and increased dissemination of the infection. Furthermore, the cytokine response to LPS was not influenced by MMP-9 deficiency. However, during E. coli peritonitis, MMP-9−/− mice showed much higher peritoneal chemokine and cytokine levels compared with wild-type mice. Despite the increased local chemokine concentrations, MMP-9−/− mice displayed a diminished recruitment of leukocytes to the site of infection, indicating that cellular migration was impaired. Moreover, MMP-9−/− mice developed more severe distant organ damage during infection. These data suggest that MMP-9 is an essential component of an effective host response to E. coli peritonitis.

The Acute-Phase Response and Serum Amyloid A Inhibit the Inflammatory Response to Acinetobacter baumannii Pneumonia

BACKGROUND Acinetobacter baumannii is an emerging pathogen in nosocomial pneumonia. Trauma and postsurgical patients display a profound acute-phase protein response and are susceptible to pneumonia. METHODS To study the way in which the acute-phase response induced by sterile tissue injury influences pulmonary host defense, mice were injected subcutaneously with turpentine or saline in both hind limbs either 2 or 5 days before intranasal inoculation with A. baumannii. RESULTS Turpentine-injected mice demonstrated strong increases in levels of the acute-phase proteins serum amyloid A (SAA) and serum amyloid P. The inflammatory response to A. baumannii was significantly impaired in turpentine-injected mice, as shown by decreased local cytokine and chemokine levels, reduced neutrophil influx and lung myeloperoxidase activity, less pulmonary inflammation on histological examination, and lower total protein levels in their bronchoalveolar lavage fluid, which was associated with reduced bacterial clearance of A. baumannii. The late acute-phase protein response still caused lower pulmonary cytokine levels and neutrophil recruitment. Furthermore, previous injection of SAA, a major acute-phase protein, also reduced inflammatory responses to A. baumannii pneumonia. CONCLUSIONS These data suggest that the acute-phase response and SAA inhibit the local inflammatory response to A. baumannii pneumonia, which may facilitate bacterial outgrowth.

Absence of Thrombin-Activatable Fibrinolysis Inhibitor Protects against Sepsis-Induced Liver Injury in Mice

Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase R, has been implicated as an important negative regulator of the fibrinolytic system. In addition, TAFI is able to inactivate inflammatory peptides such as complement factors C3a and C5a. To determine the role of TAFI in the hemostatic and innate immune response to abdominal sepsis, TAFI gene-deficient (TAFI−/−) and normal wild-type mice received an i.p. injection with Escherichia coli. Liver TAFI mRNA and TAFI protein concentrations increased during sepsis. In contrast to the presumptive role of TAFI as a natural inhibitor of fibrinolysis, TAFI−/− mice did not show any difference in E. coli-induced activation of coagulation or fibrinolysis, as measured by plasma levels of thrombin-anti-thrombin complexes and D-dimer and the extent of fibrin depositions in lung and liver tissues. However, TAFI−/− mice were protected from liver necrosis as indicated by histopathology and clinical chemistry. Furthermore, TAFI−/− mice displayed an altered immune response to sepsis, as indicated by an increased neutrophil recruitment to the peritoneal cavity and a transiently increased bacterial outgrowth together with higher plasma TNF-α and IL-6 levels. These data argue against an important part for TAFI in the regulation of the procoagulant-fibrinolytic balance in sepsis and reveals a thus far unknown role of TAFI in the occurrence of hepatic necrosis.

Endogenous Tissue-Type Plasminogen Activator Is Protective during Escherichia coli-Induced Abdominal Sepsis in Mice

Sepsis is associated with enhanced production of tissue-type plasminogen activator (tPA). We investigated the function of endogenous tPA in the immune responses to Escherichia coli-induced abdominal sepsis using tPA gene-deficient (tPA−/−) and normal wild-type (WT) mice. tPA−/− mice demonstrated an impaired defense against E. coli peritonitis as indicated by higher bacterial loads at the primary site of the infection, enhanced dissemination, and reduced survival. The protective function of tPA was independent of plasmin since plasminogen gene-deficient (Plg−/−) mice were indistinguishable from WT mice. Relative to WT mice, tPA−/− mice demonstrated similar neutrophil counts in the peritoneal cavity despite much higher bacterial loads and higher local concentrations of neutrophil attracting chemokines, suggesting a reduced migratory response. In line, tPA−/− mice demonstrated a reduced thioglycolate-induced neutrophil influx into the peritoneal cavity and i.p. injection of WT mice with a replication-defective adenoviral vector expressing tPA caused an enhanced cell migration to the peritoneal cavity during E. coli peritonitis. These findings identify a novel protective function of tPA in abdominal sepsis caused by E. coli that seems independent of its role in the generation of plasmin.

The role of plasminogen activator inhibitor type 1 in the inflammatory response to local tissue injury.

Summary.  Background: The plasma levels of the plasminogen activator‐inhibitor type 1 (PAI‐1) are consistently elevated in patients with sterile tissue injury, often accompanied by a systemic acute phase protein response. It remains unknown, however, whether and to what extent PAI‐1 affects the host response to trauma. Methods and results: By using the well‐established murine model of turpentine‐induced tissue injury we compared local and systemic inflammatory responses in PAI‐1 gene‐deficient (PAI‐1–/–) and normal wild‐type (Wt) mice. Subcutaneous turpentine injection elicited strong increases in PAI‐1 protein concentration in plasma and at the site of injury, but not in liver. PAI‐1 mRNA was locally increased and expressed mainly by macrophages and endothelial cells. PAI‐1 deficiency greatly enhanced the early influx of neutrophils to the site of inflammation, which was associated with increased edema and necrosis at 8 h after injection. Furthermore, PAI‐1–/– mice showed a reduced early interleukin (IL)‐6 induction with subsequently lower acute phase protein levels and a much slower recovery of body weight loss. Conclusion: These findings suggest that PAI‐1 is not merely a marker of tissue injury but plays a functional role in the local and systemic host response to trauma.

Type 3 Deiodinase Is Highly Expressed in Infiltrating Neutrophilic Granulocytes in Response to Acute Bacterial Infection

BACKGROUND Macrophages and polymorphonuclear cells (PMNs) play an important role in the first line of defense against bacteria by infiltrating the infected organ in order to clear the harmful pathogen. Our earlier studies showed that granulocytes express type 3 deiodinase (D3) when activated during a turpentine-induced abscess. We hypothesized that D3 expression by granulocytes may also occur during bacterial infection. METHODS In order to test this hypothesis, we used the following experimental infection models: peritonitis induced by Escherichia coli and acute pneumonia induced by Streptococcus pneumoniae. RESULTS E. coli-induced peritonitis was characterized by infiltration in the liver by inflammatory cells with abundant immunocytochemical D3 expression while no staining was present in hepatocytes of infected or control mice. Acute pneumonia induced by S. pneumoniae resulted in inflamed lungs characterized by numerous infiltrating granulocytes expressing D3 while no D3 staining was present in lung sections without an infiltrate. Serum thyroid hormones were negatively correlated to bacterial outgrowth in both lung and spleen, and thus to the severity of illness. CONCLUSION Infiltrating granulocytes during acute bacterial infection express D3. Our work supports the hypothesis that D3 plays an important role during chemical and bacterial inflammation. Whether the resulting decreased local bioavailability of thyroid hormones or rather the increased local availability of iodide is an important element of the innate immune response remains to be studied.

Acute phase response impairs host defense against Pseudomonas aeruginosa pneumonia in mice.

Objective: Pseudomonas aeruginosa is a common pathogen in hospital-acquired pneumonia. Especially trauma and postsurgical patients display a profound acute phase protein response and are susceptible to acquiring pneumonia. The objective was to study the influence of the acute phase response induced by sterile tissue injury on pulmonary host defense. Design: Laboratory investigation. Setting: Academic medical center. Subjects: Female C57Bl/6 wild-type mice, 8–10 wks old. Interventions: Mice were injected subcutaneously with either turpentine or sterile saline (control) in both hind limbs 1 day before intranasal infection with P. aeruginosa. Measurements and Main Results: The turpentine-induced acute phase response was associated with 100% lethality after induction of pneumonia, whereas control mice all survived the Pseudomonas infection. In addition, turpentine-injected mice demonstrated much higher bacterial loads in their lungs and an increased dissemination of the infection. The acute phase reaction attenuated lung inflammation during pneumonia, as reflected by histopathology, reduced pulmonary levels of proinflammatory cytokines, and a strongly diminished recruitment of neutrophils to the site of infection. Blood neutrophils harvested from turpentine injected mice displayed a reduced capacity to up-regulate their CD11b/CD18 expression upon stimulation with Pseudomonas ex vivo and during Pseudomonas pneumonia in vivo. Administration of a blocking anti-CD11b antibody to turpentine-injected and control mice almost completely abrogated the difference in bacterial outgrowth, whereas inhibition of the sympathetic nervous system did not affect the impaired pulmonary host defense in mice with an acute phase response. Conclusions: These data suggest that a systemic acute phase response might impair host defense against P. aeruginosa pneumonia, possibly in part by inhibition of CD11b/CD18-dependent neutrophil recruitment.

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.

Urokinase-Type Plasminogen Activator Receptor Plays a Role in Neutrophil Migration during Lipopolysaccharide-Induced Peritoneal Inflammation but Not during Escherichia coli—Induced Peritonitis

BACKGROUND Urokinase-type plasminogen activator receptor (uPAR) is expressed on many different cells, including leukocytes. uPAR has been implicated to play a role in neutrophil migration to sites of inflammation. METHODS To determine the role that uPAR plays in neutrophil recruitment in response to bacterial products or intact bacteria, uPAR gene-deficient (uPAR(-/-)) and wild-type (wt) mice were injected intraperitoneally with either Escherichia coli or lipopolysaccharide (LPS) derived from this bacterium. RESULTS uPAR(-/-) mice demonstrated a decreased LPS-induced neutrophil migration into peritoneal lavage fluid, whereas the chemokine and cytokine response was unaltered. In contrast, during E. coli-induced peritonitis, uPAR(-/-) mice had a normal neutrophil migration into the primary site of infection. The unaltered neutrophil trafficking in uPAR(-/-) mice during bacterial infection was corroborated by histological assessment of liver and lung tissue and myeloperoxidase levels in tissue homogenates. uPAR(-/-) mice displayed slightly but significantly lower bacterial loads in the peritoneal cavity, together with a decreased dissemination to the circulation early during the infection. CONCLUSION These data suggest that uPAR, in part, mediates neutrophil migration into the peritoneal cavity on local instillation of LPS but that this function of uPAR can be compensated for during peritonitis caused by intact E. coli.

Plasminogen activator inhibitor type-1-deficient mice have an enhanced IFN-gamma response to lipopolysaccharide and staphylococcal enterotoxin B.

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1−/−) mice demonstrated strongly elevated plasma IFN-γ concentrations after injection of either LPS or SEB. In addition, PAI-1−/− splenocytes released more IFN-γ after incubation with LPS or SEB than Wt splenocytes. Both PAI-1−/− CD4+ and CD8+ T cells produced more IFN-γ upon stimulation with SEB. LPS-induced IFN-γ release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-γ release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-γ release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.