Dorte Christiansen

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.

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.

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.

Eculizumab treatment during pregnancy does not affect the complement system activity of the newborn

Eculizumab is a humanized IgG2/4 chimeric anti-complement C5 antibody used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) or atypical hemolytic uremic syndrome. The aim of this study was to evaluate whether or not the complement activity in newborns from pregnant women who receive eculizumab is impaired. A novel eculizumab-C5 complex (E-C5) specific assay was developed and revealed that two newborns carried only 6-7% of the E-C5 detected in their eculizumab-treated PNH mothers. Serum from the pregnant women completely lacked terminal complement pathway activity, whereas the complement activity in the serum of the newborns was completely normal. Data from the pregnant women and their newborns were compared with that of healthy age-matched female controls and healthy newborns, as well as a non-treated pregnant woman with PNH and her newborn. These all showed normal complement activity without detectable E-C5 complexes. Furthermore, absence of eculizumab or E-C5 in the newborn could not be explained by lack of eculizumab binding to the neonatal Fc receptor (FcRn), as eculizumab bound strongly to the receptor in vitro. In conclusion, despite binding to FcRn neither eculizumab nor E-C5 accumulates in fetal plasma, and eculizumab treatment during pregnancy does not impair the complement function in the newborn.

Hypothesis: Combined Inhibition of Complement and CD14 as Treatment Regimen to Attenuate the Inflammatory Response

Pattern recognition is an essential event in innate immunity. Complement and Toll-like receptors (TLR), including the CD14 molecule, are two important upstream components of the innate immune system, recognizing exogenous structures as well as endogenous ligands. They act partly independent in the inflammatory network, but also have several cross-talk mechanisms which are under current investigation. Complement is an essential part of innate immunity protecting the host against infection. However, it is a double-edged sword since inappropriate activation may damage the host. Uncontrolled systemic activation of complement, as seen in severe sepsis, may contribute to the breakdown of homeostatic mechanisms leading to the irreversible state of septic shock. Complement inhibition is promising for protection of lethal experimental sepsis, but clinical studies are missing. Lipopolysaccharide (LPS) has been implicated in the pathogenesis of gram-negative sepsis by inducing synthesis of pro-inflammatory cytokines through binding to CD14 and the TLR4/MD-2 complex. Neutralization of LPS or blocking of CD14 has been effective in preventing LPS-induced lethal shock in animal studies, but results from clinical studies have been disappointing, as for most other therapeutic strategies. Based on some recently published data and further pilot data obtained in our laboratory, we hypothesize that inhibition of complement combined with neutralization of CD14 may attenuate the uncontrolled inflammatory reaction which leads to breakdown of homeostasis during sepsis. We further postulate this regimen as an approach for efficient inhibition of the initial innate recognition, exogenous as well as endogenous, to prevent downstream activation of the inflammatory reaction in general.

Combined Inhibition of Complement and CD14 Efficiently Attenuated the Inflammatory Response Induced by Staphylococcus aureus in a Human Whole Blood Model

The complement and TLR systems are activated in sepsis, contributing to an unfavorable inflammatory “storm.” Combined inhibition of these systems has been documented to efficiently attenuate the inflammatory responses induced by Gram-negative bacteria. In this study, we hypothesized that the combined inhibition would attenuate the inflammatory responses induced by Gram-positive bacteria. Staphylococcus aureus bacteria (strains Cowan and Wood), as well as S. aureus cell wall lipoteichoic acid (LTA), were incubated in thrombin-inhibited human whole blood. Complement was inhibited at the level of C3 and C5, and the TLRs by inhibiting CD14 and TLR2. Thirty-four inflammatory markers were measured by multiplex technology and flow cytometry. Thirteen markers increased significantly in response to Cowan and Wood, and 12 in response to LTA. Combined inhibition with the C3 inhibitor compstatin and the anti-CD14 Ab 18D11 significantly reduced 92 (Cowan, LTA) and 85% (Wood) of these markers. Compstatin alone significantly reduced 54 (Cowan), 38 (Wood), and 83% (LTA), whereas anti-CD14 alone significantly reduced 23, 15, and 67%, respectively. Further experiments showed that the effects of complement inhibition were mainly due to inhibition of C5a interaction with the C5a receptor. The effects on inhibiting CD14 and TLR2 were similar. The combined regimen was more efficient toward the bacterial effects than either complement or anti-CD14 inhibition alone. Complement was responsible for activation of and phagocytosis by both granulocytes and monocytes. Disrupting upstream recognition by inhibiting complement and CD14 efficiently attenuated S. aureus–induced inflammation and might be a promising treatment in both Gram-negative and Gram-positive sepsis.

Differential Effect of Inhibiting MD-2 and CD14 on LPS- Versus Whole E. coli Bacteria-Induced Cytokine Responses in Human Blood

BACKGROUND Sepsis is a major world-wide medical problem with high morbidity and mortality. Gram-negative bacteria are among the most important pathogens of sepsis and their LPS content is regarded to be important for the systemic inflammatory reaction. The CD14/myeloid differentiation factor 2 (MD-2)/TLR4 complex plays a major role in the immune response to LPS . The aim of this study was to compare the effects of inhibiting MD-2 and CD14 on ultra-pure LPS - versus whole E. coli bacteria-induced responses. METHODS Fresh human whole blood was incubated with upLPS or whole E. coli bacteria in the presence of MD-2 or CD14 neutralizing monoclonal antibodies, or their respective controls, and/or the specific complement-inhibitor compstatin. Cytokines were measured by a multiplex (n = 27) assay. NFκB activity was examined in cells transfected with CD14, MD-2 and/or Toll-like receptors. RESULTS LPS-induced cytokine response was efficiently and equally abolished by MD-2 and CD14 neutralization. In contrast, the response induced by whole E. coli bacteria was only modestly reduced by MD-2 neutralization, whereas CD14 neutralization was more efficient. Combination with compstatin enhanced the effect of MD-2 neutralization slightly. When compstatin was combined with CD14 neutralization, however, the response was virtually abolished for all cytokines, including IL-17, which was only inhibited by this combination. The MD-2-independent effect observed for CD14 could not be explained by TLR2 signaling. CONCLUSION Inhibition of CD14 is more efficient than inhibition of MD-2 on whole E. coli-induced cytokine response, suggesting CD14 to be a better target for intervention in Gram-negative sepsis, in particular when combined with complement inhibition.

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.

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.

Selective inhibition of TNF-α or IL-1β does not affect E. coli-induced inflammation in human whole blood

Inhibition of the inappropriate and excessive inflammatory response has been a main issue in sepsis-related research. Historically, TNF-alpha and IL-1 beta have been postulated as key mediators in sepsis, but selective inhibition of these cytokines has failed in clinical trials. Recently it was found that inhibition of upstream recognition by complement and CD14 could efficiently reduce Escherichia coli (E. coli)-induced inflammation. An ex vivo model with lepirudin-anticoagulated human whole blood was used to explore the significance of selective inhibition of TNF-alpha and IL-1 beta in E. coli-induced inflammation. The effect of TNF-alpha, IL-1 beta, complement and CD14 on the inflammatory response was assessed by adding highly specific neutralizing agents to these mediators. Proinflammatory cytokines, expression of CD11b and oxidative burst were measured. The controls included relevant isotype-matched immunoglobulins and peptides. Selective inhibition of TNF-alpha or IL-1 beta had no impact on E. coli-induced release of proinflammatory cytokines, CD11b-upregulation or oxidative burst. In contrast, the combined inhibition of complement and CD14 virtually abolished these responses. These data suggest that both TNF-alpha and IL-1 beta are downstream mediators and as single mediators play a limited role within the complex inflammatory reactions induced by E. coli.