Hilde Fure

The role of complement C3 opsonization, C5a receptor, and CD14 in E. coli-induced up-regulation of granulocyte and monocyte CD11b/CD18 (CR3), phagocytosis, and oxidative burst in human whole blood.

The relative role of complement and CD14 in Escherichia coli‐induced leukocyte CD11b up‐regulation, phagocytosis, and oxidative burst in human whole blood was examined. The highly specific thrombin inhibitor lepirudin was used as anticoagulant, as it does not affect complement activation. Complement inhibition at the level of C3 (anti‐C2 and anti‐factor D) and C5 (C5a receptor antagonist and anti‐C5/C5a) efficiently inhibited CD11b up‐regulation, phagocytosis, and oxidative burst in granulocytes. Monocyte activation was generally less complement‐dependent, but when C3 activation was blocked, a pronounced inhibition of phagocytosis and oxidative burst was obtained. Only the combination of anti‐C2 and antifactor D blocked E. coli C3 opsonization completely. Whole E. coli, disrupted E. coli, and the C3‐convertase activator cobra venom factor up‐regulated CD11b rapidly on both cell types, proportional to their complement activation potential in the fluid phase. In comparison, purified LPS at concentrations comparable with that present in the E. coli preparations did not activate complement. Oxidative burst was induced only by whole bacteria. Finally, the combination of complement inhibition and anti‐CD14 completely blocked E. coli‐induced granulocyte and monocyte CD11b up‐regulation and quantitatively, virtually abolished phagocytosis. The results indicate that complement and CD14, despite differential effects on granulocytes and monocytes, are the two crucial, quantitative factors responsible for E. coli‐induced CD11b, phagocytosis, and oxidative burst in both cell types.

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

Combined inhibition of complement and CD14 abolish E. coli-induced cytokine-, chemokine- and growth factor-synthesis in human whole blood.

The relative role of complement and CD14 in E. coli-induced cytokine synthesis in an in vitro human whole blood model of sepsis was examined. Fresh lepirudin-anticoagulated whole blood was incubated with E. coli for 2h. Monoclonal antibodies or a C5a receptor antagonist were used to block complement. Inflammatory mediators (n=27) were measured by multiplex technology, selected cytokine mRNA by real time PCR, and CD11b, oxidative burst and phagocytosis by flow cytometry. E. coli significantly increased 18 of the 27 inflammatory mediators, including proinflammatory cytokines (TNF-alpha, IL-6, INF-gamma and IL-1beta), chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin and IP-10), growth factors (VEGF, FGF-basic, G-CSF and GM-CSF) and other interleukins (IL-9, IL-15 and IL-17). Notably, the increases in all mediators were abolished by a combined inhibition of CD14 and complement using anti-C2 and anti-factor D in combination, whereas the relative effect of the inhibition of complement and CD14 varied. In comparison, a C5a receptor antagonist and anti-CD14 in combination reduced cytokine synthesis less efficiently. Real time PCR analysis confirmed that the cytokine synthesis was blocked at the mRNA level. Similarly, E. coli-induced CD11b up-regulation, oxidative burst and phagocytosis was totally inhibited by CD14, anti-C2 and anti-factor D in combination after 2h incubation. In conclusion, the combined inhibition of complement using anti-C2, anti-factor D and CD14 almost completely inhibits the E. coli-induced inflammatory response. The combined approach may therefore be a new treatment regimen in Gram-negative sepsis.

Pre-neutralization of C5a-mediated effects by the monoclonal antibody 137-26 reacting with the C5a moiety of native C5 without preventing C5 cleavage

Complement C5a is aetiologically linked to inflammatory tissue damage in conditions like septicaemia, immune complex diseases and ischaemia‐reperfusion injury. We here describe a monoclonal antibody (mAb), 137–26, that binds to the C5a moiety of human C5 and neutralizes the effects of C5a without interfering with C5 cleavage and the subsequent formation of lytic C5b‐9 complex. Mouse anti‐human C5 mAbs were generated and the reactivity with C5 and C5a was detected by ELISA and surface plasmon resonance. The inhibition of C5a binding to C5a receptor was studied using a radioligand binding assay. The effects of the antibody on C5a functions were examined using isolated neutrophils and a novel human whole blood model of inflammation. Haemolytic assays were used to study the effect on complement‐mediated lysis. mAb 137–26 reacted with both solid‐ and solution‐phase C5 and C5a in a dose‐dependent manner with high affinity. The antibody competed C5a binding to C5a receptor and inhibited C5a‐mediated chemotaxis of neutrophils. Furthermore, the antibody effectively abrogated complement‐dependent E. coli‐induced CD11b up‐regulation and oxidative burst in neutrophils of human whole blood. mAb 137–26 was more potent than a C5a receptor antagonist and a previously described anti‐C5a antibody. mAb 137–26 did not inhibit complement‐mediated lysis, nor did it activate complement itself. Together, mAb 137–26 binds both the C5a moiety of native C5 and free C5a, thereby effectively neutralizing the biological effects of C5a. The antibody may have therapeutic potential in inflammatory diseases where C5a inhibition combined with an operative lytic pathway of C5b‐9 is particularly desired.

The key roles of complement and tissue factor in Escherichia coli-induced coagulation in human whole blood

The complement system and the Toll‐like (TLR) co‐receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)‐induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS‐ and Escherichia coli (E. coli)‐induced coagulation in human blood. Fresh whole blood was anti‐coagulated with lepirudin, and incubated with ultra‐purified LPS (100 ng/ml) or with E. coli (1 × 107/ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti‐CD14 F(ab′)2 antibody and a control F(ab′)2. TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme‐linked immunosorbent assay (ELISA). The effect of TF was examined using an anti‐TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84–>99% with compstatin, 55–97% with anti‐CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti‐CD14 alone. The LPS‐ and E. coli–induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS‐ and E. coli–induced PTF1.2 was reduced by 76–81% (P < 0·05) with anti‐TF antibody. LPS and E. coli activated the coagulation system by a complement‐ and CD14‐dependent up‐regulation of TF, leading subsequently to prothrombin activation.

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.

C1-inhibitor efficiently inhibits Escherichia coli-induced tissue factor mRNA up-regulation, monocyte tissue factor expression and coagulation activation in human whole blood

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross‐talk occurs between the complement and coagulation systems. C1‐inhibitor (C1‐INH) can act as a regulator in both systems. Our aim in this study was to examine this cross‐talk by investigating the effects of C1‐INH on Escherichia coli‐induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1‐INH or protease‐inactivated C1‐INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription–quantitative polymerase chain reaction (RT–qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme‐linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1‐INH (1·25–5 mg/ml) reduced both LPS‐ and E. coli‐induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose‐dependently (P < 0·05). Both LPS and E. coli induced marked up‐regulation of TF mRNA levels and surface expression on whole blood monocytes. This up‐regulation was reduced efficiently by treatment with C1‐INH (P < 0·05). C1‐INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1‐INH. C1‐INH inhibited most of the other readouts more efficiently, consistent with additional non‐complement‐dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1‐INH is a broad‐spectrum attenuator of the inflammatory and haemostatic responses.

Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood

The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood.

Lifestyle Changes Followed by Bariatric Surgery Lower Inflammatory Markers and the Cardiovascular Risk Factors C3 and C4

BACKGROUND Morbidly obese patients are at risk of developing insulin resistance and cardiovascular disease. Low-grade systemic inflammation is an important factor for this development. We evaluated the effect of bariatric surgery on markers of inflammation, coagulation and glucose metabolism. METHODS Ninety-seven morbidly obese patients and 17 lean subjects (control group) participated. Anthropometric measurements as well as fasting blood samples were obtained at first admission, prior to surgery, and 1 year after surgery. RESULTS At admission, the morbidly obese group had significantly elevated levels of the complement components C3 and C4 compared to the lean control group (P<0.0001). Levels of C3 and C4 dropped significantly in the morbidly obese group over time (P<0.0001), and, 1 year after the operation, levels were comparable to those of the control group. The same changes were seen for markers of inflammation (high-sensitivity C-reactive protein, tumor necrosis factor-α, interferon-γ, interleukin-1 receptor antagonist, IL-6, and IL-13), coagulation (fibrinogen and plasminogen activator inhibitor-1), and glucose metabolism (leptin and insulin). There was a positive correlation between changes in C3 and body mass index, weight, coagulation parameters, inflammatory parameters, and leptin, respectively. CONCLUSIONS Bariatric surgery in morbidly obese patients reduced weight effectively. Even more importantly, the increased levels of several risk factors associated with diabetes and cardiovascular co-morbidity normalized 1 year after surgery.

CD14 and complement crosstalk and largely mediate the transcriptional response to Escherichia coli in human whole blood as revealed by DNA microarray

Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation.