Michael Fung

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation.

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b‐9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway‐induced C5 activation than previously recognized and used pathway‐selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that  selective  blockade  of  the  alternative  pathway  by  neutralizing  factor  D  in  human  serum  diluted 1 : 2 with mAb 166–32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid‐ and fluid‐phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

Mechanism of complement activation and its role in the inflammatory response after thoracoabdominal aortic aneurysm repair

Background—Complement activation contributes to ischemia-reperfusion injury. Patients undergoing thoracoabdominal aortic aneurysm (TAAA) repair suffer extensive ischemia-reperfusion and considerable systemic inflammation. Methods and Results—The degree and mechanism of complement activation and its role in inflammation were investigated in 19 patients undergoing TAAA repair. Patients undergoing open infrarenal aortic surgery (n=5) or endovascular descending aortic aneurysm repair (n=6) served as control subjects. Substantial complement activation was seen in TAAA patients but not in controls. C1rs-C1-inhibitor complexes increased moderately, whereas C4bc, C3bBbP, C3bc, and the terminal SC5b-9 complex (TCC) increased markedly after reperfusion, reaching a maximum 8 hours after reperfusion. Interleukin (IL)-1&bgr;, tumor necrosis factor &agr; (TNF-&agr;), and IL-8 increased significantly in TAAA patients but not in controls, peaking at 24 hours postoperatively and correlating closely with the degree of complement activation. IL-6 and IL-10 increased to a maximum 8 hours after reperfusion in the TAAA patients, were not correlated with complement activation, and increased moderately in the control subjects. Myeloperoxidase and lactoferrin increased markedly before reperfusion in all groups, whereas sICAM-1, sP-selectin, and sE-selectin were unchanged. No increase was observed in complement activation products, IL-1&bgr;, TNF-&agr;, or IL-8 in a mannose-binding lectin (MBL)–deficient TAAA patient, whereas IL-6, IL-10, myeloperoxidase, and lactoferrin increased as in the controls. Two other MBL-deficient TAAA patients receiving plasma attained significant MBL levels and showed complement and cytokine patterns identical to the MBL-sufficient TAAA patients. Conclusions—The data suggest that complement activation during TAAA repair is MBL mediated, amplified through the alternative pathway, and responsible in part for the inflammatory response.

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.

Effect of complement inhibition and heparin coating on artificial surface-induced leukocyte and platelet activation.

BACKGROUND Exposure of blood to artificial surfaces, as in cardiopulmonary bypass, induces an inflammatory response involving complement, leukocyte and platelet activation. To elucidate the specific role of complement in this process, studies were performed on blood circulated in polyvinyl chloride tubing in the absence and presence of complement inhibitors. Parallel experiments were performed with heparin-coated polyvinyl chloride tubing, which is known to prevent complement and cell activation. METHODS A novel experimental model was used, based on human whole blood anticoagulated with lepirudin. Complement activation products, myeloperoxidase, lactoferrin, and thrombospondin were quantified in enzyme immunoassays. Leukocyte CD11b expression and leukocyte-platelet conjugates were detected by flow cytometry. RESULTS Increased levels of C3 activation products, alternative pathway convertase, and the terminal SC5b-9 complex, combined with unchanged levels of C1rs-C1-inhibitor complexes and marginal changes in C4 activation demonstrated that complement was activated through the alternative pathway. Granulocyte and monocyte CD11b expression and granulocyte-platelet conjugate formation were efficiently attenuated by blocking either factor D, C3, C5, or C5a receptor. In contrast, monocyte-platelet conjugate formation and release of myeloperoxidase, lactoferrin, and thrombospondin were not reduced by complement inhibition. Heparin-coated polyvinyl chloride tubing efficiently reduced all inflammatory markers studied, except for C1rs-C1-inhibitor complexes, which increased, consistent with the enhancing effect of heparin on C1-inhibitor function. This effect did not, however, reduce fluid-phase classic pathway activation induced by heat-aggregated immunoglobulin G. CONCLUSIONS Leukocyte and platelet activation in response to artificial materials occur by mechanisms that vary in their dependence on complement. Heparin coating precludes both the complement-dependent and complement-independent reactions.

Characterization of Natural Human Anti-non-gal Antibodies and Their Effect on Activation of Porcine Gal-deficient Endothelial Cells

Background. The generation of Gal&agr;1-3Gal (Gal) transferase deficient pigs has increased the interest in non-Gal antigens potentially representing important targets for xenoreactive antibody binding leading to xenograft rejection. The present study addressed the levels and immunoglobulin isotypes of preformed human anti-non-Gal antibodies and their potential to activate porcine endothelial cells. Methods. Porcine endothelial cells lacking the Gal epitope (Gal−/−) were used to measure immunoglobulin (Ig) M and IgG subclass anti-non-Gal antibodies, using sera from 80 blood donors and pooled human AB serum. Antibodies specific for the non-Gal Hanganutziu-Deicher (HD) xenoantigen were measured by enzyme-linked immunosorbent assay. Activation of Gal−/− and Gal+/+ endothelial cells by human serum was measured, in the presence or absence of complement inhibitors, by E-selectin cell-surface expression using flow cytometry. Results. Anti-non-Gal antibody levels varied considerably among individual sera and comprised approximately 10% of total anti-porcine antibodies without sex or age differences. Among the IgG subclasses only IgG1 and IgG2 were detected. Human serum-induced E-selectin expression on Gal−/− cells was less than 20% compared with Gal+/+ cells, correlated with anti-HD IgM and IgG antibody levels (P=0.027 and 0.032, respectively), and was largely complement-independent in accordance with the lack of IgG3 anti-non-Gal antibodies. In contrast, E-selectin upregulation on Gal+/+ cells was reduced in complement blocking experiments. Conclusion. Preformed anti-non-Gal antibodies, in particular anti-HD antibodies, were present in all human sera samples, activated porcine endothelial cells, and may therefore play a role in xenograft rejection using organs from GalT−/− pigs.

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.

Meconium is a potent activator of complement in human serum and in piglets.

Meconium aspiration syndrome (MAS) is a clinical condition in the newborn infant with a significant morbidity and mortality. The complex pathophysiology of MAS, leading to both pulmonary and systemic complications, is characterized by an incompletely understood inflammatory reaction. Treatment is symptomatic, mainly limited to airway cleaning and ventilatory support. In this study, we show for the first time that meconium is a potent activator of complement, a key mediator of inflammation. In vitro, meconium activated the alternative complement pathway in human umbilical cord serum as judged by a substantial increase in the alternative pathway convertase C3bBbP. The activation proceeded through C3 (C3bc) and the terminal C5-9 pathway (terminal SC5b-9 complement complex), whereas the classical and lectin pathways were not activated (C1rs-C1-inhibitor complexes and C4bc). The lipid fraction, containing, e.g. free fatty acids, and the water fraction, containing, e.g. bile acids, contributed equally to the complement activation. A blocking antibody to factor D (alternative pathway) completely inhibited the meconium-induced complement activation, whereas blocking antibodies to mannose-binding lectin (lectin pathway) and C2 (classical and lectin pathway) had no effect. In vivo, meconium induced systemic complement activation in a piglet model of MAS, paralleling the increase in lung dysfunction. In conclusion, meconium is a potent activator of the complement system both in vitro and in vivo. Complement may be important in the pathogenesis of MAS, and specific complement inhibition might be a possible treatment approach in MAS.

Role of Complement and CD14 in Meconium-Induced Cytokine Formation

OBJECTIVE. Meconium aspiration syndrome has a complex, poorly defined pathophysiology. Meconium is a potent activator of complement in vitro and in vivo; the latter is associated with a systemic inflammatory response. The complement system and Toll-like receptors are 2 important upstream components of the innate immune system that act partly independently in the inflammatory network. The aim of this study was to investigate the relative role of complement and CD14 in meconium-induced cytokine production. METHODS. Human adult (n = 6) and cord whole blood (n = 6) anticoagulated with lepirudin was collected and distributed into tubes that contained inhibitory antibodies (anti-CD14, anti-C2, anti–factor D, or combinations thereof). The tubes were preincubated for 5 minutes before addition of meconium or buffer and then incubated for 4 hours at 37°C. Complement activation was measured by quantification of the terminal sC5b-9 complement complex by enzyme-linked immunosorbent assay. A panel of 27 inflammatory mediators (cytokines, chemokines, and growth factors) was measured by using multiplex technology. RESULTS. Fourteen of the 27 mediators measured were induced by meconium both in cord and adult blood. In cord blood, 2 additional chemokines were induced and the inflammatory response was, in general, more potent. Blocking of complement or CD14 differentially reduced the formation of most mediators, anti-CD14 being more effective. Notably, the combined inhibition of complement and CD14 almost completely abolished meconium-induced formation of the cytokines and the chemokines and markedly reduced the formation of growth factors. The endogenous lipopolysaccharide content of meconium could not explain the CD14-mediated response. CONCLUSIONS. Meconium-induced triggering of the cytokine network is differentially mediated by complement and CD14. A combined inhibition of these effector mechanisms may be an alternative approach to reduce the inflammatory reaction in meconium aspiration syndrome.

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

BACKGROUND Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. METHODS An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. RESULTS E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. CONCLUSION Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.