C3b Inactivator Research Articles

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

Rare but Relevant Kidney Disorders

The spectrum of predisposing factors to atypical hemolytic uremic syndrome (aHUS) is widening. This syndrome includes cases of HUS that are not linked to Shiga toxin–producing bacteria. Approximately half of the patients with aHUS have mutations in genes that regulate complement system: Either loss-of-function mutations in genes that encode inhibitors of the alternative pathway of the complement system (e.g., factor H or I, CFH or CFI, membrane co-factor protein, factor H–related proteins, C4b-binding protein) or gain-of-function mutations of genes that encode factor B and C3. Antibodies against CFH have been observed in 2 to 10% of patients. Thrombomodulin is a ubiquitous transmembrane endothelial cell glycoprotein that suppresses clot formation, inhibits fibrinolysis, interferes with inflammation, and inactivates some complement-derived anaphylatoxins. Delvaeye et al. studied 152 patients who had aHUS and were recruited from the International Registry of Recurrent and Familial HUS/Thrombotic Thrombocytopenic Purpura and 380 control subjects. They sequenced the entire coding region of the thrombomodulin gene (THBD), which lacks introns. Six heterozygous mutations of THBD were identified in seven (4.6%) unrelated patients. Two of them belonged to families with aHUS. In one of these families, four heterozygous carriers (including adults) were asymptomatic. As in complement defects, the THBD mutation is not sufficient by itself to cause HUS. Low serum C3 levels were measured in four patients. The disease was evident during childhood, from 6 mo to 15 yr of age. Three of seven patients progressed to ESRD. Four had recurrent aHUS. One patient developed a first episode of aHUS after kidney transplantation at 15 yr of age. Further studies will indicate whether there is a risk for recurrence of aHUS after kidney transplantation. In vitro studies on cultured cells showed that thrombomodulin provides protection against complement activation. In addition, thrombomodulin binds to C3 and CFH and negatively regulates complement by accelerating CFI-mediated inactivation of C3b in the presence of co-factors CFH and C4b. Cultured cells that express THBD variants that are associated with aHUS had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and thus were less protected from activated complement. In patients with aHUS, genetic testing should include several genes that encode proteins that regulate complement system, including thrombomodulin.

Functional Characterization ofBorrelia spielmaniiOuter Surface Proteins That Interact with Distinct Members of the Human Factor H Protein Family and with Plasminogen

Acquisition of complement regulator factor H (CFH) and factor H-like protein 1 (CFHL1) from human serum enables Borrelia spielmanii, one of the etiological agents of Lyme disease, to evade complement-mediated killing by the human host. Up to three distinct complement regulator-acquiring surface proteins (CRASPs) may be expressed by serum-resistant B. spielmanii, each exhibiting an affinity for CFH and/or CFHL1. Here, we describe the functional characterization of the 15-kDa CRASPs of B. spielmanii, members of the polymorphic Erp (OspE/F-related) protein family, that bind two distinct host complement regulators, CFH and factor H-related protein 1 (CFHR1), but not CFHL1. CFH bound to the B. spielmanii CRASPs maintained cofactor activity for factor I-mediated C3b inactivation. Three naturally occurring alleles of this protein bound CFH and CFHR1 while a fourth natural allele could not. Comparative sequence analysis of these protein alleles identified a single amino acid, histidine-79, as playing a significant role in CFH/CFHR1 binding, with substitution by an arginine completely abrogating ligand binding. The mutation of His-79 to Arg did not inhibit binding of plasminogen, another known ligand of this group of borrelial outer-surface proteins.

Monomeric CRP contributes to complement control in fluid phase and on cellular surfaces and increases phagocytosis by recruiting factor H

Complement forms the first defense line of innate immunity and has an important role in the non-inflammatory clearance of apoptotic and necrotic cells. Factor H is one essential complement inhibitor that binds to the acute phase reactant C-reactive protein (CRP). By using recombinant proteins, calcium-independent binding of Factor H to monomeric CRP (mCRP), but not to pentameric CRP (pCRP), was shown. In addition to the two known CRP-binding sites, a novel third site was localized within the C-terminus. This region is frequently mutated in the hemolytic uremic syndrome and the mutant proteins show reduced mCRP binding. In this study, we show that mCRP directs Factor H to the surface of apoptotic and necrotic endothelial cells and identify phosphocholine as one binding moiety for this complex. Factor H-mCRP complexes enhance C3b inactivation both in the fluid phase and on the surface of damaged cells and inhibit the production of pro-inflammatory cytokines. By recruiting the soluble complement inhibitor Factor H to the surface of damaged cells, mCRP blocks the progression of the complement cascade beyond the step of the C3 convertase, prevents the formation of inflammatory activation products, and thus contributes to the safe removal of opsonized damaged cells and particles.

Thrombomodulin Mutations in Atypical Hemolytic–Uremic Syndrome

The hemolytic-uremic syndrome consists of the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. The common form of the syndrome is triggered by infection with Shiga toxin-producing bacteria and has a favorable outcome. The less common form of the syndrome, called atypical hemolytic-uremic syndrome, accounts for about 10% of cases, and patients with this form of the syndrome have a poor prognosis. Approximately half of the patients with atypical hemolytic-uremic syndrome have mutations in genes that regulate the complement system. Genetic factors in the remaining cases are unknown. We studied the role of thrombomodulin, an endothelial glycoprotein with anticoagulant, antiinflammatory, and cytoprotective properties, in atypical hemolytic-uremic syndrome. We sequenced the entire thrombomodulin gene (THBD) in 152 patients with atypical hemolytic-uremic syndrome and in 380 controls. Using purified proteins and cell-expression systems, we investigated whether thrombomodulin regulates the complement system, and we characterized the mechanisms. We evaluated the effects of thrombomodulin missense mutations associated with atypical hemolytic-uremic syndrome on complement activation by expressing thrombomodulin variants in cultured cells. Of 152 patients with atypical hemolytic-uremic syndrome, 7 unrelated patients had six different heterozygous missense THBD mutations. In vitro, thrombomodulin binds to C3b and factor H (CFH) and negatively regulates complement by accelerating factor I-mediated inactivation of C3b in the presence of cofactors, CFH or C4b binding protein. By promoting activation of the plasma procarboxypeptidase B, thrombomodulin also accelerates the inactivation of anaphylatoxins C3a and C5a. Cultured cells expressing thrombomodulin variants associated with atypical hemolytic-uremic syndrome had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and were thus less protected from activated complement. Mutations that impair the function of thrombomodulin occur in about 5% of patients with atypical hemolytic-uremic syndrome.

Insights into complement convertase formation based on the structure of the factor B-cobra venom factor complex

Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short-lived protease complexes are formed through pro-convertases, which for the alternative pathway consist of the complement component C3b and the pro-enzyme factor B (FB). Here, we present the crystal structure at 2.2-A resolution, small-angle X-ray scattering and electron microscopy (EM) data of the pro-convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro-peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal-ion dependent adhesion site of the Von Willebrand factor A-type domain. A possible dynamic equilibrium between a 'loading' and 'activation' state of the pro-convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.

The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity

Mutations and polymorphisms in the gene encoding factor H (CFH) have been associated with atypical haemolytic uraemic syndrome, dense deposit disease and age-related macular degeneration. The disease-predisposing CFH variants show a differential association with pathology that has been very useful to unravel critical events in the pathogenesis of one or other disease. In contrast, the factor H (fH)-Ile(62) polymorphism confers strong protection to all three diseases. Using ELISA-based methods and surface plasmon resonance analyses, we show here that the protective fH-Ile(62) variant binds more efficiently to C3b than fH-Val(62) and competes better with factor B in proconvertase formation. Functional analyses demonstrate an increased cofactor activity for fH-Ile(62) in the factor I-mediated cleavage of fluid phase and surface-bound C3b; however, the two fH variants show no differences in decay accelerating activity. From these data, we conclude that the protective effect of the fH-Ile(62) variant is due to its better capacity to bind C3b, inhibit proconvertase formation and catalyze inactivation of fluid-phase and surface-bound C3b. This demonstration of the functional consequences of the fH-Ile(62) polymorphism provides relevant insights into the complement regulatory activities of fH that will be useful in disease prediction and future development of effective therapeutics for disorders caused by complement dysregulation.

The Paramyxoviruses Simian Virus 5 and Mumps Virus Recruit Host Cell CD46 To Evade Complement-Mediated Neutralization

The complement system is a critical component of the innate immune response that all animal viruses must face during natural infections. Our previous results have shown that treatment of the paramyxovirus simian virus 5 (SV5) with human serum results in deposition of complement C3-derived polypeptides on virion particles. Here, we show that the virion-associated C3 component includes the inactive form iC3b, suggesting that SV5 may have mechanisms to evade the host complement system. Electron microscopy, gradient centrifugation, and Western blot analysis indicated that purified SV5 virions derived from human A549 cells contained CD46, a plasma membrane-expressed regulator of complement that acts as a cofactor for cleavage and inactivation of C3b into iC3b. In vitro cleavage assays with purified complement components showed that SV5 virions had C3b cofactor activity, resulting in specific factor I-mediated cleavage of C3b into inactive iC3b. SV5 particles generated in CHO cells, which do not express CD46, did not have cofactor activity. Conversely, virions derived from a CHO cell line that was engineered to overexpress human CD46 contained elevated levels of virion-associated CD46 and displayed enhanced C3b cofactor activity. In comparison with C3b, purified SV5 virions had very low cofactor activity against C4b, consistent with the known preference of CD46 for C3b versus C4b. Similar results were obtained for the closely related mumps virus (MuV), except that MuV particles derived from CHO-CD46 cells had higher C4b cofactor activity than SV5 virions. In neutralization assays with human serum, SV5 and MuV containing CD46 showed slower kinetics and more resistance to neutralization than SV5 and MuV that lacked CD46. Our results support a model in which the rubulaviruses SV5 and MuV incorporate cell surface complement inhibitors into progeny virions as a mechanism to limit complement-mediated neutralization.

Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag.

Identification and characterization of the factor H and FHL-1 binding complement regulator-acquiring surface protein 1 of the Lyme disease spirochete Borrelia spielmanii sp. nov.

Borrelia spielmanii, one of the etiological agents of Lyme disease found in Europe, evades host complement-mediated killing by recruitment of the immune regulators factor H and FHL-1 from human serum. Serum-resistant and intermediate serum-resistant isolates express up to 3 distinct complement regulator-acquiring surface proteins (CRASPs) that bind factor H and/or FHL-1. The present study describes identification and functional characterization of BsCRASP-1 as the dominant factor H and FHL-1 binding protein of B. spielmanii. BsCRASP-1 is a 27.7 kDa outer surface lipoprotein, which after processing has a predicted mass of 24.9 kDa. BsCRASP-1 is encoded by a single copy gene, cspA, that maps to a linear plasmid of approximately 55 kb. Ligand affinity blot techniques revealed that both native and recombinant BsCRASP-1 from different isolates can strongly bind FHL-1, but only weakly factor H. Deletion mutants of recombinant BsCRASP-1 were generated and a high-affinity binding site for factor H and FHL-1 was mapped to its carboxy-terminal 10-amino-acid residue domain. Similarly, the dominant binding site of factor H and FHL-1 was localized to short consensus repeats (SCRs) 5–7. Factor H and FHL-1 maintained cofactor activity for factor I-mediated C3b inactivation when bound to full-length BsCRASP-1 but not to a deletion mutant lacking the carboxy-terminal 10-amino-acid residue domain. In conclusion, BsCRASP-1 binds the host immune regulators factor H and FHL-1, and is suggested to represent a key molecule of B. spielmanii for complement resistance. Thus, BsCRASP-1 most likely contributes to persistence of B. spielmanii and to pathogenesis of Lyme disease.

BhCRASP-1 of the relapsing fever spirochete Borrelia hermsii is a factor H- and plasminogen-binding protein

The spirochete Borrelia ( B.) hermsii is the most frequent tick-borne relapsing fever agent in North America. B. hermsii organisms employ multiple strategies, including acquiring complement regulators and antigenic variation to escape innate and humoral immunity, respectively. Here, we identified a novel member of the complement regulator-acquiring surface protein (CRASP) family, designated BhCRASP-1, that binds the complement regulators, factor H (FH) and FH-related protein 1 (FHR-1) but not FH-like protein 1 (FHL-1). We show that FH when bound to BhCRASP-1 maintains its regulatory capacity to control C3b deposition and C3 convertase activity. BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH, thereby maintaining FH-associated cofactor activity for factor I-mediated C3b inactivation. Heterologous expression of BhCRASP-1 in the serum-sensitive B. burgdorferi B313 strain protects transformed cells from complement-mediated killing, at least partially. Furthermore, we show that BhCRASP-1 concurrently binds plasminogen in addition to FH, however, via distinct, non-overlapping domains. Our findings will be helpful to further elucidate the molecular basis of B. hermsii interactions with host factors in the pathogenesis of relapsing fever.

Subversion of complement activation at the bacterial surface promotes serum resistance and opsonophagocytosis ofFrancisella tularensis

Francisella tularensis (Ft) is resistant to serum but requires complement factor C3-derived opsonins for uptake by phagocytic cells and subsequent intracellular growth. In this study, we show that C3 fragments, deposited on Ft, are detected by anti-C3d and -iC3b mAb and that the classical and the alternative pathways are involved in this event. This was demonstrated using C2-depleted sera and specific inhibitors of the classical-versus-alternative pathways of complement activation. Further, we demonstrate that factor C4b, which is crucial for the classical pathway, is deposited on the surface of Ft. In contrast, the C5b-C9 membrane attack complex (MAC) is not assembled on the surface of Ft, which may explain its resistance to complement killing. Deposition of C3 opsonins leads to enhanced phagocytosis by human immature dendritic cells (DC), which leads to intracellular survival, growth, and DC death. Finally, we show that factor H (fH) can bind to the surface of Ft. We believe our data suggest that important virulence factors for Ft are its ability to bind fH and inactivate C3b to iC3b, which culminates in opsonin-induced uptake for subsequent intracellular growth. C3b inactivation also leads to inefficient MAC assembly, which contributes to the ability of this bacterium to resist complement lysis.

Resistance of the Echinococcus granulosus cyst wall to complement activation: analysis of the role of InsP6 deposits

The larva of the cestode Echinococcus granulosus (hydatid cyst) is protected by the acellular laminated layer (LL). The mechanisms that make this thick coat a poor activator of host complement are incompletely understood. The structure binds, through unknown motifs, the host regulator of the alternative complement pathway (ACP), factor H. A second potential mechanism of ACP regulation, the inhibition of factor B activation, was detected in assays employing purified components (Immunopharmacology 42 : 91). The inhibitor was subsequently identified as myo-inositol hexakisphosphate (InsP(6)), which in the form of nano-deposits is a major component of the LL (Biochem J 362 : 297; J Cell Biochem 93 : 1272; FEBS J 273 : 3192). In this report we show that colloidal InsP(6 )solids inhibit factor B activation, through adsorption and associated impairment of C3b binding. However, this interaction is not relevant in the presence of serum proteins. In serum, InsP(6) deposits instead bind C1q, and initiate complement activation. This activation is curtailed through efficient C3b inactivation, previously shown to be entirely factor H-dependent, and now observed to be independent of the InsP(6) deposits. Therefore the complement resistance of the LL must be based on functional factor H binding sites present on the mucin-based meshwork that is its other major constituent.

Subversion of complement activation at the bacterial surface promotes serum resistance and opsonophagocytosis of Francisella Tularensis

Francisella tularensis (Ft) is resistant to serum but requires complement factor C3-derived opsonins for uptake by phagocytic cells and subsequent intracellular growth. In this study, we show that C3 fragments, deposited on Ft, are detected by anti-C3d and -iC3b mAb and that the classical and the alternative pathways are involved in this event. This was demonstrated using C2-depleted sera and specific inhibitors of the classical-versus-alternative pathways of complement activation. Further, we demonstrate that factor C4b, which is crucial for the classical pathway, is deposited on the surface of Ft. In contrast, the C5b-C9 membrane attack complex (MAC) is not assembled on the surface of Ft, which may explain its resistance to complement killing. Deposition of C3 opsonins leads to enhanced phagocytosis by human immature dendritic cells (DC), which leads to intracellular survival, growth, and DC death. Finally, we show that factor H (fH) can bind to the surface of Ft. We believe our data suggest that important virulence factors for Ft are its ability to bind fH and inactivate C3b to iC3b, which culminates in opsonin-induced uptake for subsequent intracellular growth. C3b inactivation also leads to inefficient MAC assembly, which contributes to the ability of this bacterium to resist complement lysis.

Loss of Self‐Control in the Complement System and Innate Autoreactivity

The complement system performs effective antimicrobial and clean-up functions to keep the body clear from invading microbes and accumulating debris. From its about 35 components many regulate complement activity to prevent self-attack. Our work has focused on regulator defects and dysfunctions that cause autoreactivity, that is, inflammation and damage against self-tissues ("innate autoreactivity"). The major complement regulator, factor H (FH), protects host cells and keeps excessive amplification under control. Mutations and polymorphisms in FH predispose to four different diseases: membranoproliferative glomerulonephritis type II (MPGN II), partial lipodystrophy (PLD), recurrent atypical hemolytic uremic syndrome (aHUS), and age-related macular degeneration (AMD). Loss of the complement regulatory activity (cofactor activity for C3b inactivation and decay accelerating activity) in the N terminus of FH leads to PLD and MPGN II, the latter of which is characterized by C3b and MAC deposition on the glomerular basement membranes of kidneys. Polymorphism in the SCR7-domain in the middle part of FH predisposes to AMD, which is the most common cause of vision loss in elderly people. This polymorphism influences the ability of FH to bind to C-reactive protein (CRP) and to target phagocytic clearance of debris (e.g., eye pigment) and control local inflammation. Finally, the loss of the ability of the FH C terminus to recognize C3b molecules deposited on self-structures predisposes to aHUS, where blood cells, platelets, and endothelial cells, particularly in kidneys, become targets for repeated complement attacks and increased procoagulant activity. The pathogenetic mechanisms of these diseases are being increasingly understood, which in the future will help in designing new therapies.

Human PathogenicBorrelia spielmaniisp. nov. Resists Complement-Mediated Killing by Direct Binding of Immune Regulators Factor H and Factor H-Like Protein 1

Borrelia spielmanii sp. nov. has recently been shown to be a novel human pathogenic genospecies that causes Lyme disease in Europe. In order to elucidate the immune evasion mechanisms of B. spielmanii, we compared the abilities of isolates obtained from Lyme disease patients and tick isolate PC-Eq17 to escape from complement-mediated bacteriolysis. Using a growth inhibition assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum resistant, whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All isolates activated complement in vitro, as demonstrated by covalent attachment of C3 fragments; however, deposition of the later activation products C6 and C5b-9 was restricted to the moderately serum-resistant isolate PMew and the serum-sensitive B. garinii isolate G1. Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates acquired the host alternative pathway regulators factor H and factor H-like protein (FHL-1) from human serum. Both complement regulators retained their factor I-mediated C3b inactivation activities when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding proteins, BsCRASP-1 and BsCRASP-2, were identified, which we estimated to be approximately 23 to 25 kDa in mass. A further factor H binding protein, BsCRASP-3, was found exclusively in the tick isolate, PC-Eq17. This is the first report describing an immune evasion mechanism utilized by B. spielmanii sp. nov., and it demonstrates the capture of human immune regulators to resist complement-mediated killing.

Dual Binding Specificity of a Borrelia hermsii-Associated Complement Regulator-Acquiring Surface Protein for Factor H and Plasminogen Discloses a Putative Virulence Factor of Relapsing Fever Spirochetes

Tick-borne relapsing fever in North America is primarily caused by the spirochete Borrelia hermsii. The pathogen employs multiple strategies, including the acquisition of complement regulators and antigenic variation, to escape innate and humoral immunity. In this study we identified in B. hermsii a novel member of the complement regulator-acquiring surface protein (CRASP) family, designated BhCRASP-1, that binds the complement regulators factor H (FH) and FH-related protein 1 (FHR-1) but not FH-like protein 1 (FHL-1). BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH, thereby maintaining FH-associated cofactor activity for factor I-mediated C3b inactivation. Furthermore, ectopic expression of BhCRASP- 1 converted the serum-sensitive Borrelia burgdorferi B313 strain into an intermediate complement-resistant strain. Finally, we report for the first time that BhCRASP-1 binds plasminogen/plasmin in addition to FH via, however, distinct nonoverlapping domains. The fact that surface-bound plasmin retains its proteolytic activity suggest that the dual binding specificity of BhCRASP-1 for FH and plasminogen/plasmin contributes to both the dissemination/invasion of B. hermsii and its resistance to innate immunity.

The C-terminus of complement factor H is essential for host cell protection

Complement is a powerful self-amplifying system of innate immune defense with the capacity to eliminate microbes directly. Factor H is a central regulator in plasma which protects host tissue from complement mediated damage. Here we characterize the relevance of surface attached factor H, and study the regulatory activity of factor H on endothelial cells. Although these cells expressed membrane bound regulators, cell bound factor H contributed substantially to complement regulatory activity at the cell surface. Blockade of the C-terminus of factor H with monoclonal antibodies inhibited cell binding of this soluble regulator and resulted in enhanced complement activation on the cells. In the absence of factor H, increased deposition and slower inactivation of C3b resulted in higher amount of membrane attack complexes on the cell surface. When the membrane regulators CD55 and CD59 were removed by enzymatic treatment, complement mediated cell lysis was enhanced in the absence of factor H. Importantly, inhibition of the C-terminus did not compromise the regulatory function of factor H in fluid phase. Altogether these data point to a highly relevant, yet so far underestimated role of factor H for complement control at cellular surfaces, and reveal a decisive role of the factor H C-terminus in host cell recognition and protection.

Complement supports progression of HIV and turns neutralising antibodies at low concentration into infection enhancing antibodies

HIV is largely resistant to complement-mediated lysis primarily due to binding of factor H from the fluid phase and secondarily to incorporation of additional complement control proteins (DAF, MCP, CD59) from the host cell into the viral envelope. Since HIV by itself activates the complement system – and even more so, if antibodies are bound to its surface, fragments of C3, i.e. C3b – are deposited on the virus. We could document this situation for exvivo HIV, isolated from human plasma. Depending on the quantity of activation this may reach such a degree that gp120 is not accessible any more. It could be clearly shown that HIV carrying C3b binds to complement receptor typ 1 (CR1) on human erytrocytes (E). Thereby, with the help of the C3b inactivator, C3b is converted into iC3b and further to C3d. HIV-iC3b/HIV-C3d bind to B lymphocytes (CR2, CR1), to FDCs (CR2, CR3, CR1) and to DCs (CR3). HIV-C3d/B cell- and HIV-C3d/FDC – complexes very efficiently infect resting (!) CD4+ T lymphocytes. HIV-iC3b, bound to DCs, causes a productive (!) infection of these cells! HIV – IgG or HIV-IgG-iC3b allows only attachment of HIV to DCs. In both cases HIV can be transmitted to T cells and productively infect these cells. These complement-mediated infection enhancement mechanisms can be overcome by sufficient large quantities of broadly neutralising antibodies. But if these drop to low concentrations, then the fact that these antibodies activate complement and cause opsonisation of HIV with C3 fragments becomes the salient feature and the before neutralising antibodies begin to enhance infection. In our judgment, this complement-deposition by antibodies to surface epitopes is one of the aspects, which make development of a vaccine so difficult.

Complement Factor H Gene Abnormalities in Haemolytic Uraemic Syndrome: From Point Mutations to Hybrid Gene

A typical haemolytic uraemic syndrome (aHUS) is a rare disease involving haemolytic anaemia, thrombocytopenia, and renal failure. There is growing evidence that the disease is associated with defective control of the alternative pathway of complement [1]. Genetic abnormalities in complement regulatory proteins, including complement factor H (CFH), membrane cofactor protein, and complement factor I, have been reported in 30%, 10%, and 5% of patients with aHUS, respectively [2–4]. CFH is a plasma protein produced by the liver that acts as a central regulator in the alternative pathway of complement activation. CFH acts as a cofactor for complement factor I in the inactivation of the central complement protein C3b to form iC3b. CFH also accelerates the decay of the C3 convertase C3bBb of the alternative pathway, and competes with factor B for binding to C3b [5].