Human Complement System Research Articles

Novel scabies mite serpins inhibit the three pathways of the human complement system

Novel scabies mite serpins inhibit the three pathways of the human complement system

Human complement system activation by a serine protease found in the Premolis semirufa caterpillar venom

Human complement system activation by a serine protease found in the Premolis semirufa caterpillar venom

Crystallographic determination of the disease-associated T1184R variant of complement regulator factor H

The soluble 155 kDa glycoprotein factor H (FH) protects host tissue from damage by the human complement system. It accelerates decay of the alternative-pathway C3 convertase, C3bBb, and is a cofactor for factor I-mediated cleavage of theopsonin C3b. Numerous mutations and single-nucleotide polymorphisms (SNPs) occur in the gene encoding FH and the resulting missense mutations and truncation products result in altered functionality that predisposes to the development of the serious renal condition atypical haemolytic uraemic syndrome (aHUS). Other polymorphisms are linked to membranoproliferative glomerulonephritis and macular degeneration. The two C-terminal modules of FH (FH19-20) harbour numerous aHUS-associated mutations that disrupt the ability of factor H to protect host cells from complement-mediated damage. In this work, the crystal structure of an aHUS-associated T1184R variant of FH19-20 at a resolution of 1.52 Å is described. It is shown that this mutation has negligible structural effects but causes a significant change in the electrostatic surface of these two domains. Mechanisms are discussed by which this mutation may alter FH-ligand interactions, particularly with regard to the extension of a region of this molecule within module 20 that has been associated with the binding of glycosaminoglycans (GAGs) or sialic acid residues.

Activation of the human complement system by pigmented and hypopigmented mycelia of the fungus Fonsecaea pedrosoi

The action of the complement system on pigmented and hypopigmented mycelia of the fungus Fonsecaea pedrosoi, the major aetiological pathogen of the chromoblastomycosis is herein discussed. Fungi were grown in medium Czapeck-Dox at 37°C, for 14 days, without shaking to obtain pigmented mycelium. To obtain hypopigmented mycelium, the fungus was grown at the same conditions, but in the dark and with low oxygenation. Activation was measured by complement consumption and enzyme-linked immunosorbent assay. We also observed by immunofluorescence the deposition of C3, C4 fragments and C9 on the surface of the different forms studied. The results indicate that both forms were able to activate the complement system mainly by the alternative pathway. Pigmented mycelia had the highest consumption results, indicating that the pigment, melanin, may have influence in activation.

Variable Antibody-dependent Activation of Complement by Functionalized Phospholipid Nanoparticle Surfaces

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.

A Complement-Dependent Cytotoxicity-Enhancing Anti-CD20 Antibody Mediating Potent Antitumor Activity In the Humanized NOD/Shi-Scid, IL-2Rγnull Mouse Lymphoma Model

AbstractAbstract 423 Purpose:Engineering the Fc region of monoclonal antibodies (mAb) to enhance effector functions is likely to be a promising approach for next-generation mAb therapy. 113F, a complement-dependent cytotoxicity-(CDC)-enhancing variant of rituximab is such an antibody (Cancer Res 2008;68:3863-72). The first of the three major aims of the present study was to identify tumor-associated factors influencing tumor susceptibility to 113F-induced CDC, especially focusing on complement regulatory proteins (CRPs). The second aim of the present study was to compare 113F-induced CDC against primary lymphoma cells with rituximab in vitro. A current crucial problem in the field of human immunotherapy research, including antibody therapy, is the lack of suitable small animal models for in vivo preclinical testing. With respect to antibody-dependent cellular cytotoxicity (ADCC), we have established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients, in which human immune cells are engrafted and mediate ADCC (Cancer Immunol Immunother 2009;58:1195-206, J Immunol. 2009;183:4782-91). On the other hand, there is no mouse model in which human CDC can be evaluated. Thus, the third and final aim of the present study was to establish a mouse model in which it is human complement that mediates CDC against human tumor cells.Using this model, we assessed the therapeutic potential of 113F in comparison with rituximab. Experimental Design:Rituximab- and 113F-induced human CDC was compared in vitro, and in vivo using NOG mice with human complement. Result:First, we determined that tumor-associated factors influencing tumor susceptibility to 113F-induced CDC included the quantity of CRPs such as CD55 and CD59 on the cell surface, as observed in rituximab-induced CDC. Second, we found that 113F mediated highly enhanced CDC against primary CD20-expressing lymphoma cells from patients, greater than rituximab. Finally, a novel human tumor-bearing mouse model has been developed in which human complement functions in CDC. NOG mouse serum is defective in its capacity to induce CDC against human cells, and endogenous immune cells from NOG mice are unable to mediate ADCC of therapeutic antibodies with an Fc region consisting of human IgG. Thus, we were able to evaluate purely human CDC without interference from endogenous mouse immune cells or complement-mediated mAb induced antitumor effects in this NOG mouse model. The present observation of significant therapeutic efficacy of rituximab together with pooled human serum (PHS) compared to rituximab with inactivated PHS indicated that human complement does function in rituximab-induced CDC in these mice in vivo. The finding of specific localization of human C1q on CD20-expressing tumor cell membranes indicated that human CDC indeed contributed to the antitumor effect in this model. In addition, enhanced therapeutic efficacy of 113F together with PHS compared to rituximab with PHS in this mouse model was observed in vivo. In the cell proliferation assay, viability of the target cell line was not affected by rituximab or 113F alone, and thus no significant difference between these mAbs was observed in vitro. Therefore, the present in vivo observation emphasizes the concept of this type of CDC-enhancing antibody. Furthermore, the more abundant, denser signals of C1q at the tumor cell membrane in these human serum-bearing mice receiving 113F compared with rituximab, are consistent with the Fc region of the former having a much higher C1q binding affinity than the latter. These findings are concordant with the present observation of the greater therapeutic efficacy of 113F compared to rituximab in vivo. Conclusion:This animal model overcomes the limitations of preclinical in vivo investigations of CDC caused by species incompatibilities between humans and mice. This model also makes it possible to reconstitute the human complement system during mAb-based immunotherapy and to perform more appropriate preclinical evaluations of novel therapeutic mAb which mediate CDC. In the present study, highly enhanced human CDC mediated by this type of CDC-enhancing mAb was demonstrated both in vitro and in a humanized mouse model in vivo. In the near future, the efficacy of the type of CDC-enhancing antibody described here will be established in planned clinical trials in humans. Disclosures:No relevant conflicts of interest to declare.

Novel analogues of the therapeutic complement inhibitor compstatin with significantly improved affinity and potency

Compstatin is a 13-residue disulfide-bridged peptide that inhibits a key step in the activation of the human complement system. Compstatin and its derivatives have shown great promise for the treatment of many clinical disorders associated with unbalanced complement activity. To obtain more potent compstatin analogues, we have now performed an N-methylation scan of the peptide backbone and amino acid substitutions at position 13. One analogue (Ac-I[CVW(Me)QDW-Sar-AHRC](NMe)I-NH2) displayed a 1000-fold increase in both potency (IC50=62nM) and binding affinity for C3b (KD=2.3nM) over that of the original compstatin. Biophysical analysis using surface plasmon resonance and isothermal titration calorimetry suggests that the improved binding originates from more favorable free conformation and stronger hydrophobic interactions. This study provides a series of significantly improved drug leads for therapeutic applications in complement-related diseases, and offers new insights into the structure–activity relationships of compstatin analogues.

Bioactive arabinogalactans from the leaves of Opilia celtidifolia Endl. ex Walp. (Opiliaceae)

The leaves of the tree Opilia celtidifolia have a long tradition for being used in Mali and other West African countries against various ailments such as for wound healing. Previous studies on polysaccharides from these leaves showed the presence of pectic-like polymers with an effect on the human complement system as well as the ability to activate macrophages. The present study shows that bioactive arabinogalactans isolated by water of 50°C could be separated into two acidic fractions, Oc50A1 and Oc50A2. The former could, by gel filtration on Sephacryl S-400, be separated into two fractions, which were further purified on a Superdex 200 column to give the fractions Oc50A1.I.pur and Oc50A1.II.pur. These fractions were subjected to chemical and biological studies. The polysaccharides consisted mainly of heavily branched type II arabinogalactans and minor amounts of rhamnogalacturonan I regions. The isolated polymers had a high human complement-fixating ability, as well as the ability to stimulate rat macrophages and dendritic cells (DCs) and to induce B cell proliferation. These effects were especially pronounced for the higher molecular weight fraction of Oc50A1.I.pur. The fractions Oc50A1.I.pur and Oc501.II.pur stimulated secretion of pro-inflammatory cytokines from purified B cells or DCs. Collectively, these results indicate that the arabinogalactan type II polymers present in the leaves of O. celtidifolia may be used to develop medical devices for regulating inflammatory processes.

Complement: a key system for immune surveillance and homeostasis

Nearly a century after the significance of the human complement system was recognized, we have come to realize that its functions extend far beyond the elimination of microbes. Complement acts as a rapid and efficient immune surveillance system that has distinct effects on healthy and altered host cells and foreign intruders. By eliminating cellular debris and infectious microbes, orchestrating immune responses and sending 'danger' signals, complement contributes substantially to homeostasis, but it can also take action against healthy cells if not properly controlled. This review describes our updated view of the function, structure and dynamics of the complement network, highlights its interconnection with immunity at large and with other endogenous pathways, and illustrates its multiple roles in homeostasis and disease.

Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b

The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 Å limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.

Inter alpha inhibitor (IaI) as novel inhibitor of human complement system

Inter alpha inhibitor (IaI) as novel inhibitor of human complement system

Applicability of a kinetic microassay to evaluate modulation of the human complement system activity by snake venoms

Applicability of a kinetic microassay to evaluate modulation of the human complement system activity by snake venoms

NMR characterization and molecular modeling of fucoidan showing the importance of oligosaccharide branching in its anticomplementary activity

Fucoidan is a potent inhibitor of the human complement system whose activity is mediated through interactions with certain proteins belonging to the classical pathway, particularly the protein C4. Branched fucoidan oligosaccharides displayed a higher anticomplementary activity as compared to linear structures. Nuclear magnetic resonance (NMR) characterization of the branched oligosaccharides and saturation transfer difference-NMR experiment of the interaction with the protein C4 allowed the identification of the glycan residues in close contact with the target protein. Transferred nuclear Overhauser effect spectroscopy experiment and molecular modeling of fucoidan oligosaccharides indicated that the presence of side chains reduces the flexibility of the oligosaccharide backbone, which thus adopts a conformation which is very close to the one recognized by the protein C4. Together, these results suggest that branching of fucoidan oligosaccharides, determining their conformational state, has a major impact on their anticomplementary activity.

Clumping Factor A Interaction with Complement Factor I Increases C3b Cleavage on the Bacterial Surface ofStaphylococcus aureusand Decreases Complement-Mediated Phagocytosis

The human complement system is important in the immunological control of Staphylococcus aureus infection. We showed previously that S. aureus surface protein clumping factor A (ClfA), when expressed in recombinant form, bound complement control protein factor I and increased factor I cleavage of C3b to iC3b. In the present study, we show that, compared to the results for the wild type, when isogenic ClfA-deficient S. aureus mutants were incubated in serum, they bound less factor I, generated less iC3b on the bacterial surface, and bound fewer C3 fragments. It has been shown previously that two amino acids in ClfA (P(336) and Y(338)) are essential for fibrinogen binding. However, S. aureus expressing ClfA(P336A Y338S) was less virulent than ClfA-deficient strains in animal models. This suggested that ClfA contributed to S. aureus virulence by a mechanism different than fibrinogen binding. In the present study, we showed that S. aureus expressing ClfA(P336A Y338S) was more susceptible to complement-mediated phagocytosis than a ClfA-null mutant or the wild type. Unlike ClfA, ClfA(P336A Y338S) did not enhance factor I cleavage of C3b to iC3b and inhibited the cofactor function of factor H. Fibrinogen enhanced factor I binding to ClfA and the S. aureus surface. Twenty clinical S. aureus strains all expressed ClfA and bound factor I. High levels of factor I binding by clinical strains correlated with poor phagocytosis. In summary, our results suggest that the interaction of ClfA with factor I contributes to S. aureus virulence by a complement-mediated mechanism.

Renal disease associated with inherited disorders of the complement system

The human complement system is vital for host defense and plays a role in a number of inflammatory disorders. Inherited deficiency or dysfunction of most of the individual complement components occurs uncommonly. The phenotype displayed by such patients varies with the specific component deficiency and ranges from recurrent infections to autoimmune diseases. Most of the latter are associated with glomerulonephritis. The onset of severe lupus erythematosus in a young child, with prominent cutaneous and renal manifestations, especially if a similar disorder is present in another family member, is a clue to the presence of a complement component deficiency. The distinguishing of acquired deficiencies from inherited deficiencies in complement components is sometimes difficult and may require sophisticated laboratory testing.

Scabies Mite Inactivated Serine Protease Paralogs Inhibit the Human Complement System

Infestation of skin by the parasitic itch mite Sarcoptes scabiei afflicts 300 million people worldwide and there is a need for novel and efficient therapies. We have previously identified a multigene family of serine proteases comprising multiple catalytically inactive members (scabies mite-inactivated protease paralogs (SMIPPs)), which are secreted into the gut of S. scabiei. SMIPPs are located in the mite gut and in feces excreted into the upper epidermis. Scabies mites feed on epidermal protein, including host plasma; consequently, they are exposed to host defense mechanisms both internally and externally. We found that two recombinantly expressed SMIPPs inhibited all three pathways of the human complement system. Both SMIPPs exerted their inhibitory action due to binding of three molecules involved in the three different mechanisms which initiate complement: C1q, mannose-binding lectin, and properdin. Both SMIPPs bound to the stalk domains of C1q, possibly displacing or inhibiting C1r/C1s, which are associated with the same domain. Furthermore, we found that binding of both SMIPPs to properdin resulted in prevention of assembly of the alternative pathway convertases. However, the SMIPPs were not able to dissociate already formed convertases. Immunohistochemical staining demonstrated the presence of C1q in the gut of scabies mites in skin burrows. We propose that SMIPPs minimize complement-mediated gut damage and thus create a favorable environment for the scabies mites.

Interpain A, a Cysteine Proteinase from Prevotella intermedia, Inhibits Complement by Degrading Complement Factor C3

Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A) resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the α-chain of C3—the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

Study of the Effect of Anisakis simplex Larval Products on the Early and Late Components in the Classical Complement Pathway

In previous studies, we have reported that the larval products (crude extract [CE] and excretory-secretory [ES]) of Anisakis simplex showed a dose-dependent inhibition of the lysis mediated by classical (CP) and alternative pathways (AP) of the human complement system, with the major inhibition on the CP rather than on AP. This inhibition of hemolysis is due to the consumption of complement factors because the assays performed shortening the preincubation period result in a significant decrease of the inhibitory effect on the lysis of the larval products compared with the standard time. Likewise, we found that the larval products reduce the inhibitory percentages in the CP using C3-deficient sera, but not in the AP, which could indicate that other complement components are implicated in the inhibitory effect in the CP. Hence, we have studied the activity of the larval products of A. simplex on individual components in the CP, using different complement-deficient sera. The investigated complement molecules were C1q, C2, C4, C5, C6, C7, C8, and C9. The larval products showed activity at the C2 level but failed to have a significant effect on the other components. Therefore, CE and ES products from A. simplex interact with C3 and C2 complement proteins, which are early components of the complement system, but not with the late complement components.

Spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides and their ability to activate human complement system in vitro

Spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides and their ability to activate human complement system in vitro

The James Blundell Award Lecture 2007: Do we really understand immune red cell destruction?

We have learned a great deal about immune red blood cell (RBC) destruction since the elaboration of biochemical/immunological interactions of antibodies, complement and macrophages during the past 50 years. We first learned about the direct lysis of RBCs involving complement. We then learned of the role of the macrophage (particularly in the spleen and the liver) in initiating phagocytosis and antibody-dependent cytotoxicity of antibody-coated RBCs. Later, as the complexities of the human complement system were unravelled, we learned that complement-coated RBCs that were not directly haemolysed could interact with macrophages and that specific complement molecules on the RBC membrane could lead to a phagocytic event or the RBC (although heavily coated with complement) could survive normally. The application of isotope-labelling procedures (e.g. (51)Cr) for RBC survival (starting in the 1950s) advanced our knowledge considerably. Advances in knowledge in immunology helped us understand the complexity of the immunoglobulins (e.g. subclasses) and the specific receptors on macrophages and their role in immune haemolysis. Nevertheless, after more than 30 years researching this area, I am sometimes embarrassed to realize how much I cannot explain. Why do some patients have severe haemolytic transfusion reactions because of antibodies that are only detectable by one technique or not detectable by any? How do we explain autoimmune haemolytic anaemia with negative direct antiglobulin tests (DATs)? Why do RBCs strongly coated with immunoglobulin (Ig)G1 or IgG3 sometimes have normal survival? Are cells, other than macrophages, involved in immune RBC destruction? Could the relative amount of cytotoxicity vs. phagocytosis explain different clinical findings and response to treatment? How do we explain 'hyperhaemolysis' in sickle cell disease? Could novel mechanisms involving IgG glycosylation, CD47, 'armed' macrophages, bystander lysis, antibody activated reactive oxygen species, natural killer cells or antibody perturbation of RBC membrane be involved? Why do RBCs die after circulating for 100-120 days in healthy individuals? How should we define a 'clinically significant' antibody; how do we evaluate this? So many questions, so little time!