Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp-mediated C3b and C4b degradation

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood-brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+ E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA- E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+ E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum.     

Studies on the interactions between C-reactive protein and complement proteins

Several studies have investigated the interactions between C-reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b-binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme-linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1-INH. Native, ligand-unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea-treatment or by site-directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1-activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.     

Regulation of the alternative pathway of human complement by C1q

The interaction of C1q with C3b and its effect on C3b activities in the alternative pathway of complement (APC) have been studied. Purified C1q markedly inhibited C3b deposition on and lysis of rabbit erythrocytes by the isolated cytolytic APC. It also blocked formation of the C3 convertase, C3b, Bb as well as binding of Factors B and H to sheep erythrocytes (E) bearing C3b. The direct and specific binding of C1q to C3b was clearly demonstrated using the hemagglutination technique at low ionic strength (0.1 M NaCl). C1q concns of 2 micrograms/ml and higher agglutinated, in a dose-dependent fashion, EC3b but not E, EC3bi or EC3d. Addition of C1r and C1s to C1q and formation of C1 did not affect its capacity to agglutinate EC3b. The C1q-mediated agglutination of EC3b was inhibited by EDTA, MgEGTA, C3b and Factor B but not by native C3 or collagen. Heating C1q (56 degrees C) markedly potentiated its agglutinating activity whereas collagenase-treated C1q lost most of its activity. Taken together, these results suggest that C1q binds through its "heads" and in the presence of calcium ions to a site on C3b that is adjacent to the Factor B and Factor H binding sites. This interaction may down-regulate the activity of the alternative pathway of complement on surfaces which activate both the classical and alternative pathways of complement.     

Rat polymeric IgA binds C1q, but does not activate C1

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.     

Interaction of C1q with the receptor calreticulin requires a conformational change in C1q

The interaction between C1q and the chaperone calreticulin was studied under various conditions. When both proteins were present in equal amounts in solution, no interaction could be demonstrated. However, C1q immobilized on a hydrophobic surface, exposed to heat-treatment or bound to immunoglobulins (Igs) showed a strong, rapid and specific binding of calreticulin. The interaction appeared to be a two-step process, and the initial phase of interaction was sensitive to high concentrations of salt but not to a physiological salt concentration. The following strong binding was insensitive to salt and extremes of pH but sensitive to strongly denaturing agents (urea and guanidine). The sensitivity to salt during the initial phase of interaction was practically identical to that observed when calreticulin was bound to type V collagen. Binding between C1q and calreticulin could be inhibited by serum amyloid P component and by proteinase K-digested ovalbumin, and the binding of calreticulin to proteinase K-digested ovalbumin was shown to be inhibited by C1q. The data indicate that C1q binds stably to the peptide-binding site of calreticulin and that the initial binding of calreticulin to C1q involves the collagen-like domain of the C1q molecule. In conclusion, our results suggest calreticulin as a potential receptor for an altered conformation of C1q as occurs during binding to Igs. Thus, the chaperone and protein-scavenging function of calreticulin may extend from the endoplasmic reticulum to the topologically equivalent cell surface, where it may contribute to the elimination of immune complexes and apoptotic cells.     

Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment

We sought to specifically regulate the binding of human C1q, and thus the activation of the first complement component, via the construction of a single chain antibody variable binding region fragment (scFv) targeting the C1q globular heads. Here we describe details of the construction, expression and evaluation of this scFv, which was derived from a high-affinity hybridoma (Qu) specific for the C1q globular heads. The scFv was comprised of the Qu variable heavy chain domain (VH) linked to the Qu variable light chain domain (VL) and was termed scFv-QuVHVL. When mixed with either purified C1q or with human serum as a source of C1, scFv-QuVHVL bound to C1q and competitively restricted the interaction of C1q or C1 with immobilized IgG or with IgG1 antibody-coated cells, and prevented the activation of native C1 in human serum as determined by analyses of C1-mediated C4 deposition and fluid-phase C4 conversion. However scFv-QuVHVL could be manipulated to become a C1 activator when it was irreversibly immobilized onto microtiter ELISA plates, prior to contact with human serum complement. This functional dichotomy can be a useful tool in selectively elucidating, differentiating, inducing or inhibiting specific roles of human C1q and the classical complement pathway in complement-mediated physiological processes. We project that once fully humanized, fluid-phase scFv-QuVHVL could become a useful therapeutic in limiting inadvertent host tissue damage elicited by the classical complement pathway.     

Biochemical and functional characterization of the interaction between pentraxin 3 and C1q

Pentraxin 3 (PTX3) is a recently characterized member of the pentraxin family of acute-phase proteins produced during inflammation. Classical short pentraxins, C-reactive protein, and serum amyloid P component can bind to C1q and thereby activate the classical complement pathway. Since PTX3 can also bind C1q, the present study was designed to define the interaction between PTX3 and C1q and to examine the functional consequences of this interaction. A dose-dependent binding of both C1q and the C1 complex to PTX3 was observed. Experiments with recombinant globular head domains of human C1q A, B, and C chains indicated that C1q interacts with PTX3 via its globular head region. Binding of C1q to immobilized PTX3 induced activation of the classical complement pathway as assessed by C4 deposition. Furthermore, PTX3 enhanced C1q binding and complement activation on apoptotic cells. However, in the fluid-phase, pre-incubation of PTX3 with C1q resulted in inhibition of complement activation by blocking the interaction of C1q with immunoglobulins. These results indicate that PTX3 can both inhibit and activate the classical complement pathway by binding C1q, depending on the way it is presented. PTX3 may therefore be involved in the regulation of the innate immune response.     

Evidence for the binding of human serum amyloid P component to Clq and Fab gamma

In order to clarify the mechanism of interaction of serum amyloid P component (SAP) with complement, the interaction of SAP with C1q and with IgG was studied. It is known that SAP binds Sepharose in the presence of calcium. When purified 125I-C1q was incubated with SAP prior to Sepharose affinity chromatography, 125I-C1q was retained. However, in the absence of SAP, the 125I-C1q was not retained. To further examine the interaction of SAP with C1q, isolated SAP was incubated at varying ratios with C1q in the presence of 1.5 mM Ca2+. These mixtures were subsequently examined via crossed immunoelectrophoresis against goat anti-SAP. A change in the electrophoretic behavior of SAP was observed in the presence of C1q. In other studies, it was observed that SAP might interact with the collagen-like stem of C1q. In these latter studies, 125I-SAP was incubated with pepsin digests of C1q in a microtitre solid-phase binding assay. In addition, a microtitre solid-phase binding assay was utilized in order to investigate the possible binding of isolated 125I-SAP with IgG. Interestingly in the presence of Ca2+, human IgG and Fab gamma, but not Fc gamma, were found to bind 125I-SAP.     

Mannan-binding lectin and C1q bind to distinct structures and exert differential effects on macrophages

While the interaction of complement component C1q with cellular proteins is extensively studied, much less is known about the binding of the structurally related molecule, mannan-binding lectin (MBL) to various cells. Here we show by cytofluorimetry that the interaction of MBL with immunocompetent cells is much more restricted than that of C1q. It is shown that under conditions of physiological ionic strength MBL binds to human monocyte-derived macrophages (Mphi) and monocytoid cell lines, but not to T and B lymphocytes, in contrast to C1q, which interacts with all these cells under the same conditions. As opposed to the binding of C1q, low ionic strength does not improve the interaction of MBL with Mphi. No competition for cellular binding sites was found when MBL and C1q were added simultaneously to the cells. Studying the functional consequences of the interaction, we found that the release of TNF-alpha from Mphi is induced by C1q but not by MBL. Production of complement C3 by Mphi is stimulated by C1q strongly, while the effect of MBL is much weaker. C3 produced upon C1q-mediated triggering is shown to opsonize RBC, resulting in enhanced phagocytosis. These results suggest that cell membrane molecules binding MBL and C1 q are not identical; moreover, biological functions exerted by these proteins are also markedly different.     

C1q-binding proteins and C1q receptors

Aggregated or immobilized complement C1q induces cellular responses in many different cell types. C1q-induced cellular responses may be involved in host defense and in protection against autoimmunity because C1q-deficient humans have infectious complications and a very high incidence of autoimmune disease. The search for the C1q receptor(s), which has been ongoing for 25 years, has led recently to the recognition that proteins identified as binding to C1q may be divided into two groups: C1q-binding molecules that are normally intracellular; and cell surface C1q receptors.     

C1q binding and complement activation by prions and amyloids

C1q binds to many non-self and altered-self-materials. These include microorganisms, immune complexes, apoptotic and necrotic cells and their breakdown products, and amyloids. C1q binding to amyloid fibrils found as extracellular deposits in tissues, and subsequent complement activation are involved in the pathology of several amyloid diseases, such as Alzheimer's disease. Prion diseases, such as scrapie also involve formation of amyloid by polymerization of the host prion protein (PrP). Complement activation is likely to contribute to neuronal damage in the end stages of prion diseases, but is also thought to participate in the initial infection, dissemination and replication stages. Infectious prion particles are likely to bind C1q and activate the complement system. Bound complement proteins may then influence the uptake and transport of prion particles by dendritic cells (DCs) and their subsequent proliferation at sites such as follicular DCs.     

Short leucine-rich glycoproteins of the extracellular matrix display diverse patterns of complement interaction and activation

The extracellular matrix consists of structural macromolecules and other proteins with regulatory functions. An important family of the latter class of molecules found in most tissues is the small leucine-rich repeat proteins (SLRPs). We have previously shown that the SLRP fibromodulin binds directly to C1q and activates the classical pathway of complement. In the present study we further examine the interactions between SLRPs and complement. Osteoadherin, like fibromodulin, binds C1q and activates the classical pathway strongly while moderate activation is seen in the terminal pathway. This can be explained by the interaction of fibromodulin and osteoadherin with factor H, a major soluble inhibitor of complement. Also, chondroadherin was found to bind C1q and activate complement, albeit to a lesser extent. Chondroadherin also binds factor H. We confirm published data showing that biglycan and decorin bind C1q but do not activate complement. In this study a similar pattern is seen for lumican although its affinity for C1q is lower than for biglycan and decorin. Furthermore, using electron microscopy and radiolabeled SLRPs, we demonstrate two different classes of SLRP binding sites on C1q, to head and stalk respectively, where only binding to the head appears to be activating. We propose a role for SLRPs in the regulation of complement activation in diseases involving the extracellular matrix, particularly those characterized by chronic inflammation such as rheumatoid arthritis, atherosclerosis, osteoarthritis and chronic obstructive lung disease.     

The regulatory roles of C1q

C1q binds to immune complexes to elicit complement-dependent microbial killing and enhance phagocytosis. Besides this classical role, C1q also opsonizes apoptotic cells for clearance by phagocytes. C1q deficiency increases susceptibility to microbial infections and is also associated with elevated autoimmunity as characterized by increased apoptotic bodies in tissues. Most complement proteins are of liver origin, but C1q is predominantly synthesized by peripheral tissue macrophages and dendritic cells. Besides being found in the blood, C1q has also been found deposited in extracellular tissues around these cells. In vitro, immobilized C1q inhibits monocyte, macrophage and T-cell production of inflammatory cytokines. It also regulates T-cell activation. Therefore, mounting evidence suggest a major regulatory role for C1q in inflammation and autoimmunity.     

C1q binding by a high affinity anti-fluorescein murine monoclonal IgM antibody and monomeric subunits

Comparative interactions of purified rabbit C1q with 18-2-3, a high affinity (2-3 X 10(10) M-1) anti-fluorescein (anti-F1) murine monoclonal IgM antibody (pentamer) and constitutive monomeric subunits (IgMs) were studied. Using a solid phase radioimmunoassay (SPRIA), based on immobilized polyvalent antigen, it was shown that the mechanism of C1q binding to IgM was characteristically multiphasic while IgMs yielded monophasic binding curves. The latter compared qualitatively and quantitatively with a monoclonal IgG2a anti-fluorescein antibody with the same intrinsic affinity of 2-3 X 10(10) M-1. C1q binding efficiency to antibodies was significantly enhanced when the immunoglobulins interacted with immobilized multivalent antigen. Monoclonal IgM antibody bind identically to six F1-carrier protein conjugates independent of epitope (F1) density. In contrast, the C1q-antibody interaction binding was dependent upon epitope density. An average distance between F1 epitopes of 80 A was optimal for C1q binding by IgM. At low concn of IgM, when fluorescein was bound by antigen-binding sites on adjacent subunits of an intact pentamer, C1q appeared to bind IgM intramolecularly.     

Serum amyloid p component does not circulate in complex with C4-binding protein,fibronectin or any other major protein ligand

Serum amyloid P component (SAP) is a pentameric plasma protein associated with all known kinds of amyloid. The normal physiological function of the protein has not been fully elucidated but it may be involved in clearance of cellular debris and in innate immunity. An important clue to its normal function is the identity of ligands bound to SAP in the circulation. It has been reported that all SAP is complexed with C4-binding protein (C4bp) but other studies have not been able to confirm this. We here study this issue by a combination of crossed immunoelectrophoresis (CIE), size exclusion chromatography, and native polyacrylamide electrophoresis and we show that SAP in serum - analysed under native analysis conditions and free of immobilizing antibodies - does not have any major protein ligand. However, when the protein is aggregated by immobilized antibodies, C4bp and fibronectin clearly bind to SAP. If circulating SAP under normal circumstances bind any protein ligand in vivo, our results strongly suggest that this only occurs to a minor extent.     

Conformationally altered hyaluronan restricts complement classical pathway activation by binding to C1q, C1r, C1s, C2, C5 and C9, and suppresses WOX1 expression in prostate DU145 cells

Linear non-sulfated hyaluronan (HA) does not bind complement proteins yet inhibits their hemolytic function. We have previously induced the complement inhibitory function of HA by heat treatment. However, heated HA readily loses its anti-complementary activity probably due to instantaneous interchain re-association. Here, HA solutions were heated and then freeze-dried. Compared to native HA, heated/freeze-dried HA stably restricted serum complement-mediated hemolysis via the classical pathway, in which serum C1 hemolytic function and C3 activation were blocked. Also, treated HA had a significantly increased binding of component C1q, C1r, C1s, C2, C5, C9, P, D and H. Further, when HA was gel-fractionated by electrophoresis and then freeze-dried, its anti-complementary activity was stably induced. Both native and heated/freeze-dried HA stimulated ERK phosphorylation in prostate DU145 cells. However, treated HA suppressed the expression of tumor suppressors WOX1 and WOX2. Together, HA with an altered conformation stabilizes its inhibition and binding of complement proteins. It may recognize cell surface receptors differently from native HA, thereby differentially regulating the expression of cellular proteins.     

Interactions of histidine-rich glycoprotein with immunoglobulins and proteins of the complement system

This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.     

Immune evasion by acquisition of complement inhibitors: the mould Aspergillus binds both factor H and C4b binding protein

Pathogenic fungi represent a major threat particularly to immunocompromised hosts, leading to severe, and often lethal, systemic opportunistic infections. Although the impaired immune status of the host is clearly the most important factor leading to disease, virulence factors of the fungus also play a role. Factor H (FH) and its splice product FHL-1 represent the major fluid phase inhibitors of the alternative pathway of complement, whereas C4b-binding protein (C4bp) is the main fluid phase inhibitor of the classical and lectin pathways. Both proteins can bind to the surface of various human pathogens conveying resistance to complement destruction and thus contribute to their pathogenic potential. We have recently shown that Candida albicans evades complement by binding both Factor H and C4bp. Here we show that moulds such as Aspergillus spp. bind Factor H, the splicing variant FHL-1 and also C4bp. Immunofluorescence and flow cytometry studies show that the binding of Factor H and C4bp to Aspergillus spp. appears to be even stronger than to Candida spp. and that different, albeit possibly nearby, binding moieties mediate this surface attachment.     

Direct binding of herpes simplex virus type 1 virions to complement C3

Glycoprotein C (gC) of type 1 herpes simplex virus (HSV-1) binds the human complement C3 as purified proteins, or when expressed on the surface of infected cells. However, it is not clear whether the purified HSV virion binds directly to C3. In this study, direct binding of purified virions, HSV-1(KOS) or HSV-1(hrR3), to C3-coated plate was demonstrated by an enzyme-linked immunosorbent assay (ELISA). Captured virions on C3-coated plates were still infectious as determined by adding Vero cells to allow for infection to occur. The binding of virions to C3 was abolished if C3 was heat-inactivated, confirming a requirement for complement. In addition, the interaction was inhibited by preincubation of purified virions with heparin. In conclusion, a direct interaction of C3 with the HSV-1 virions was demonstrated.     

Expression and function of C1q receptors and C1q binding proteins at the cell surface

C1q is the recognition unit of the first component of complement that binds not only IgG and IgM containing immune complexes, but also recognizes foreign structures such as the lipid A of endotoxin, and molecules expressed at the surface of apoptotic cells. In this review, the plasma membrane receptors and binding proteins for C1q are discussed and new data are presented on calreticulin expression on human peripheral blood cells. Although much is known about C1q receptors and binding molecules there are still many questions regarding their role in vivo.