Julie Katrine Lindstad

Human genetic deficiencies reveal the roles of complement in the inflammatory network: Lessons from nature

Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to Gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies—natures own knockouts—including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor, cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1β and IL-8 were more dependent on complement than IFN-γ and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-γ inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to Gram-negative bacteria.

CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs

Sepsis is a severe infection‐induced systemic inflammatory syndrome. Inhibition of downstream inflammatory mediators of sepsis, e.g., TNF‐α, has failed in clinical trials. The aim of this study was to investigate the effects of inhibiting CD14, a key upstream innate immunity molecule, on the early inflammatory and hemostatic responses in a pig model of gram‐negative sepsis. The study comprised two arms, whole live Escherichia coli bacteria and E. coli lipopolysaccharide (LPS) (n=25 and n=9 animals, respectively). The animals were allocated into treatment (antiCD14) and control (IgG isotype or saline) groups. Inflammatory, hemostatic, physiological, and microbiological parameters were measured. The proinflammatory cytokines TNF‐α, IL‐1β, IL‐6, and IL‐8, but not the anti‐inflammatory cytokine IL‐10, were efficiently inhibited by anti‐CD14. Furthermore, anti‐CD14 preserved the leukocyte count and significantly reduced granulocyte enzyme matrix metalloproteinase‐9 release and expression of the granulocyte membrane activation molecule wCD11R3 (pig CD11b). The hemostatic markers thrombin‐antithrombin III complexes and plasminogen activator inhibitor‐1 were significantly attenuated. Anti‐CD14 did not affect LPS or E. coli DNA levels. This study documents that CD14 inhibition efficiently attenuates the proinflammatory cytokine response and granulocyte activation and reverses the procoagulant state but does not interfere with LPS levels or bacterial counts in E. coli‐induced sepsis.— Thorgersen, E. B., Hellerud, B. C., Nielsen, E. W., Barratt‐Due, A., Fure, H., Lindstad, J. K., Pharo, A., Fosse, E., Tønnessen, T. I., Johansen, H. T., Castellheim, A., Mollnes, T. E. CD14 inhibition efficiently attenuates early inflammatory and hemostatic responses in Escherichia coli sepsis in pigs. FASEB J. 24, 712–722 (2010). www.fasebj.org

The down-stream effects of mannan-induced lectin complement pathway activation depend quantitatively on alternative pathway amplification

Complement activation plays an important role in human pathophysiology. The effect of classical pathway activation is largely dependent on alternative pathway (AP) amplification, whereas the role of AP for the down-stream effect of mannan-induced lectin pathway (LP) activation is poorly understood. In normal human serum specific activation of LP was obtained after exposure to a wide concentration range of mannan on the solid phase. Reaction mechanisms in this system were delineated in inhibition experiments with monoclonal antibodies. Direct mannose-binding lectin (MBL) independent activation of AP was not observed even at high mannan concentrations since addition of the inhibiting anti-MBL mAb 3F8 completely abolished generation of the terminal C5b-9 complex (TCC). However, selective blockade of AP by anti-factor D inhibited more than 80% of TCC release into the fluid phase after LP activation showing that AP amplification is quantitatively responsible for the final effect of initial specific LP activation. TCC generation on the solid phase was distinctly but less inhibited by anti-fD. C2 bypass of the LP pathway could be demonstrated, and AP amplification was also essential during C2 bypass in LP as shown by complete inhibition of TCC generation in C2-deficient serum by anti-fD and anti-properdin antibodies. In conclusion, the down-stream effect of LP activation depends strongly on AP amplification in normal human serum and in the C2 bypass pathway.

Ornithodoros moubata Complement Inhibitor Is an Equally Effective C5 Inhibitor in Pigs and Humans

Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the inhibitory efficacy of the C5 inhibitor Ornithodoros moubata C inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 μM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 μM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B4 in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate inhibitor for further in vivo studies of sepsis.

Combined Inhibition of Complement (C5) and CD14 Markedly Attenuates Inflammation, Thrombogenicity, and Hemodynamic Changes in Porcine Sepsis

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli–induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin–antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg2+ buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.

A new dynamic porcine model of meningococcal shock.

The objective of this study was to establish a porcine analog of human meningococcal sepsis for pathophysiological investigations and possible future therapy in severe sepsis. Heat-killed Neisseria meningitidis was continuously infused in sublethal concentrations into 10 anesthetized 30-kg pigs (sepsis group). The dose was doubled every 30 min. Six pigs received saline only (control group). The changes described in the succeeding paragraphs were observed in the sepsis group but not in the control group. MAP was aimed to be kept normal by fluid infusion but declined after 3 h in parallel with a decrease in systemic vascular resistance. Pulmonary arterial pressure increased considerably after 30 to 45 min. A massive plasma extravasation was shown by increased hematocrit and a 50% reduction in plasma albumin content. Fluid accumulated in lungs, muscles, and jejunum, as shown by increased wet-dry ratios. Peak inspiratory pressures and fraction of inspired oxygen had to be increased. The cytokines TNF-&agr;, IL-1&bgr;, IL-6, IL-8, IL-10, and IL-12 increased markedly. Neutrophils fell to zero-levels, and platelets were markedly reduced. Thrombin-antithrombin complexes increased notably after 120 min. This is the first large animal model of sepsis using whole Neisseria meningitidis. The model simulates well central aspects of human meningococcal sepsis and could be used for future interventional studies.

The role of bradykinin and the effect of the bradykinin receptor antagonist icatibant in porcine sepsis.

Bradykinin (BK) is regarded as an important mediator of edema, shock, and inflammation during sepsis. In this study, we evaluated the contribution of BK in porcine sepsis by blocking BK and by measuring the stable BK metabolite, BK1-5, using anesthetized pigs. The effect of BK alone, the efficacy of icatibant to block this effect, and the recovery of BK measured as plasma BK1-5 were first investigated. Purified BK injected intravenously induced an abrupt fall in blood pressure, which was completely prevented by pretreatment with icatibant. BK1-5 was detected in plasma corresponding to the doses given. The effect of icatibant was then investigated in an established model of porcine gram-negative sepsis. Neisseria meningitidis was infused intravenously without any pretreatment (n = 8) or pretreated with icatibant (n = 8). Negative controls received saline only. Icatibant-treated pigs developed the same degree of severe sepsis as did the controls. Both groups had massive capillary leakage, leukopenia, and excessive cytokine release. The plasma level of BK1-5 was low or nondetectable in all pigs. The latter observation was confirmed in supplementary studies with pigs undergoing Escherichia coli or polymicrobial sepsis induced by cecal ligation and puncture. In conclusion, icatibant completely blocked the hemodynamic effects of BK but had no beneficial effects on N. meningitidis-induced edema, shock, and inflammation. This and the fact that plasma BK1-5 in all the septic pigs was virtually nondetectable question the role of BK as an important mediator of porcine sepsis. Thus, the data challenge the current view of the role of BK also in human sepsis.

Systemic CD14 Inhibition Attenuates Organ Inflammation in Porcine Escherichia coli Sepsis

ABSTRACT Sepsis is an infection-induced systemic inflammatory response syndrome. Upstream recognition molecules, like CD14, play key roles in the pathogenesis. The aim of the present study was to investigate the effect of systemic CD14 inhibition on local inflammatory responses in organs from septic pigs. Pigs (n = 34) receiving Escherichia coli-bacteria or E. coli-lipopolysaccharide (LPS) were treated with an anti-CD14 monoclonal antibody or an isotype-matched control. Lungs, liver, spleen, and kidneys were examined for bacteria and inflammatory biomarkers. E. coli and LPS were found in large amounts in the lungs compared to the liver, spleen, and kidneys. Notably, the bacterial load did not predict the respective organ inflammatory response. There was a marked variation in biomarker induction in the organs and in the effect of anti-CD14. Generally, the spleen produced the most cytokines per weight unit, whereas the liver contributed the most to the total load. All cytokines were significantly inhibited in the spleen. Interleukin-6 (IL-6) was significantly inhibited in all organs, IL-1β and IP-10 were significantly inhibited in liver, spleen, and kidneys, and tumor necrosis factor, IL-8, and PAI-1 were inhibited only in the spleen. ICAM-1 and VCAM-1 was significantly inhibited in the kidneys. Systemic CD14-inhibition efficiently, though organ dependent, attenuated local inflammatory responses. Detailed knowledge on how the different organs respond to systemic inflammation in vivo, beyond the information gained by blood examination, is important for our understanding of the nature of systemic inflammation and is required for future mediator-directed therapy in sepsis. Inhibition of CD14 seems to be a good candidate for such treatment.

Properdin binding to complement activating surfaces depends on initial C3b deposition

Significance The role of properdin in stabilization of the alternative pathway C3 convertase is indisputable, whereas its role as pattern recognition molecule remains controversial. Properdin lacks the structural homology shared by other pattern recognition molecules of the complement system, and has its major function in stabilizing the C3bBb convertase. We found that properdin binding was completely abolished by C3 inhibition after the exposure of human serum to myeloperoxidase, human umbilical vein endothelial cells, and Neisseria meningitidis, showing that properdin is not a pattern recognition molecule for these targets. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that properdin typically binds a complement-activating surface subsequent to C3b to stabilize the alternative pathway C3 convertase. Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.