Complement activation and C3 binding by serum-sensitive and serum-resistant strains of Pseudomonas aeruginosa

The relationship among complement consumption, C3 deposition, and C3 fragmentation pattern was compared for serum-sensitive (Sers) and serum-resistant (Serr) strains of Pseudomonas aeruginosa. The Sers strains, which were mucoid strains derived from patients with cystic fibrosis, had lipopolysaccharide deficient in O-antigen side chains. These organisms generally activated much less complement per organism than their Serr counterparts, characterized by the presence of lipopolysaccharide with long lipopolysaccharide O side chains. Surprisingly, however, although the Serr strains consumed more total hemolytic complement, less C3 was deposited onto the surface of these strains than onto that of the Sers strains. Maximal C3 binding required the participation of both the classical and alternative complement pathways, although classical complement pathway involvement was more important for Serr strains. Finally, while more than half of the C3 deposited on most Sers strains was in the form of C3b, most of the C3 on the Serr strains was in the form of iC3b, indicating a more rapid and extensive conversion of C3b to iC3b on the surface of these strains. Limited complement activation by Sers mucoid strains of P. aeruginosa may confer a selective survival advantage to these organisms in colonizing the airways of patients with cystic fibrosis.     

Complement activation by immunoglobulin does not depend solely on C1q binding

A matched set of rat chimeric antibodies has been studied for their ability to activate various key stages of the complement cascade. Rat IgM and IgG2b were efficient at all stages from C1q binding to cell lysis. However, for other isotypes, a direct correlation between C1q binding and cell lysis did not apply. IgG2a, which was only modestly efficient at C1q binding, was relatively more so for binding and activation of while C1, and was by far the most effective isotype after IgG2b and IgM for C4 and C3 binding. IgG2c was relatively efficient at binding C1q and C1, but less so for the binding of C4 or for later stages. IgA was efficient at binding C1, but again, this was not reflected in activation of later stages. The results suggest that properties of different isotypes, as well as influencing binding of C1q, may regulate attachment of the C1r2C1s2 tetramer. In addition, distinct features of certain isotypes may favor C4 activation and binding, independent of their ability to activate C1.     

Relationship of mouse erythrocyte binding receptor on peripheral blood mononuclear cells to receptors for IgG Fc and C3

When peripheral blood mononuclear cells separated at 4 degrees C were incubated at 37 degrees C, they shed their surface receptors. Total absorption of the released mouse erythrocyte binding receptors from the supernatant of the cell suspension with mouse erythrocytes did not lead to a change in the IgG Fc and C3 receptor content of the supernatant. On removal of the C3 or IgG Fc receptors, the quantity of mouse erythrocyte binding receptor remained unchanged. These observations indicate that the three receptors are mutually independent structures, or undergo dissociation in the course of the shedding.     

Classical complement pathway activation by antipneumococcal antibodies leads to covalent binding of C3b to antibody molecules

We have examined whether or not a physical relationship exists between antipneumococcal antibodies (Ab) and C3b when Ab activate the classical complement pathway on the surface of pneumococci (Pn). After sensitization with 125I-labeled Ab, Pn were sequentially incubated with purified C1, C4, C2, and biotinylated C3. Ab molecules were then eluted from Pn, and C3b-associated molecules were purified on avidin-Sepharose. Both 125I-labeled immunoglobulin G (IgG) and [125I]IgM bound to C3b; the association was stable to incubation in 1% sodium dodecyl sulfate at 37 degrees C. The association was only partially reversed by incubation in 1 M hydroxylamine-0.5% sodium dodecyl sulfate (pH 10.5), implying that Ab and C3b were linked by amide as well as ester bonds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dithiothreitol and NH2OH eluates from the avidin-Sepharose showed that C3b bound to both heavy and light chains of the Ab. Moreover, the ability to bind to erythrocyte C3b receptors could be transferred to unsensitized Pn by eluates from Pn on which Ab had activated the classical pathway. These covalent complexes of Ab and C3b may be especially important in the opsonization of Pn by Ab and complement.     

Lack of binding of C3 to IgG antibodies during the activation of the classical complement pathway on the red cell

We have studied the interaction between C3 and natural human and rabbit anti-MTX IgM and hyperimmune IgG antibody bound to red cells to which MTX was covalently coupled. IgM Ab molecules bound to the cell surface were measured by their interaction with rabbit anti-human mu-chain IgG Ab: the bound anti-mu-chain or anti-MTX IgG was quantitated with 125I-labelled PA. C3 uptake by EMTX AC142 complexes from purified preparation of C3 was detected by the interaction of bound C3 with rabbit anti-C3 IgG Ab; the bound IgG was then measured with radiolabelled PA. In some experiments the uptake of C3 by the EMTX AC142 complexes was measured by using 125I-labelled C3. To find out if C3 was bound to Ab molecules, anti-MTX Abs were eluted from the cells by excess fluid-phase MTX. After dialysis the eluted anti-MTX IgM or IgG Ab was than reattached to fresh EMTX. All cells were then analyzed for Ab and C3 content by a radioimmunoassay. It was found that when anti-MTX IgM or IgG was eluted from EMTX AC423 complexes no C3 was removed from the cells and that eluted IgM or IgG did not carry with them either C3 antigen or 125I-labelled C3. It was concluded that, during the activation of the classical C-pathway at the red cell surface, no C3 was bound to IgM (rabbit or human) or IgG (rabbit) antibody molecules.     

Complement C3: a molecular mosaic of binding sites.

Many of the biological activities of the complement system are mediated by C3, the third complement component, and its proteolytic fragments. At the same time, several of the molecules which regulate complement activation target their action at the C3 molecule. Accordingly, the C3 molecule is equipped with multiple binding sites for at least 14 other complement or complement-related proteins. As described in this review, major progress has been made recently in the identification of the C3 binding sites and the residues involved. Yet this has exposed only the "tip of the iceberg". A novel technique which may facilitate the elucidation of the active sites in C3 is presented. Finally, based on the current knowledge on the C3 molecule, a hypothetical model of the molecular organization of this molecule and its binding sites is presented.     

EspP,a Serine Protease of Enterohemorrhagic Escherichia coli,Impairs Complement Activation by Cleaving Complement Factors C3/C3b and C5

Hemolytic-uremic syndrome (HUS) is a life-threatening disorder characterized by hemolytic anemia, thrombocytopenia, and renal insufficiency. It is caused mainly by infections with enterohemorrhagic Escherichia coli (EHEC). Recently, Shiga toxin 2, the best-studied virulence factor of EHEC, was reported to interact with complement, implying that complement may be involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate whether or not the serine protease EspP, an important virulence factor of EHEC, interacts with complement proteins. EspP did not have any effect on the integrity of factor H or factor I. However, EspP was shown to cleave purified C3/C3b and C5. Cleavage of the respective complement proteins also occurred in normal human serum (NHS) as a source of C3/C3b or C5 or when purified complement proteins were added to the supernatant of an EspP-producing wild-type strain. Edman degradation allowed unequivocal mapping of all three main C3b fragments but not of the three main C5 fragments. Complement activation was significantly downregulated in all three pathways for C5-depleted serum to which C5, preincubated with EspP, was added (whereas C5 preincubated with an EspP mutant was able to fully reconstitute complement activation). This indicates that EspP markedly destroyed the functional activity, as measured by a commercial total complement enzyme-linked immunosorbent assay (Wieslab). Downregulation of complement by EspP in vivo may influence the colonization of EHEC bacteria in the gut or the disease severity of HUS.Hemolytic-uremic syndrome (HUS) is the most frequent cause of acute renal failure in children. This disorder is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal insufficiency (26). HUS may be caused by different etiological agents; infections with enterohemorrhagic Escherichia coli (EHEC) are, however, the leading cause of HUS in childhood worldwide (13). EHEC produces several virulence factors, among which Shiga toxins (Stxs) 1 and 2 are probably the best characterized.The complement system is an important part of innate immunity and consists of more than 30 proteins in plasma and on host cells (25). Complement is activated by three different pathways—the classical pathway (CP), the mannan-binding lectin (MBL) pathway, and the alternative pathway (AP)—based on different recognition mechanisms. Finally, all three pathways converge in the generation of C3 convertases, which cleave the central component C3, creating C3a and C3b. Nascent C3b has the transient ability to form a covalent ester bond with a variety of target surfaces and finally becomes an essential subunit of both the CP and AP C5-cleaving enzymes.C3b is further cleaved to iC3b by factor I (FI) in the presence of factor H (FH), CR1, or membrane cofactor protein. FI cleavage of C3b to iC3b inactivates and prevents C3b from functioning in the C3 or C5 convertase enzymes. Further cleavage of iC3b by FI in the presence of CR1 results in the formation of C3c. When C3b is not inactivated through cleavage by FI, the C3 convertase develops into a C5 convertase which cleaves C5 into C5a and C5b. Both C3a and C5a have anaphylatoxic and chemotactic activities, recruiting inflammatory cells and triggering degranulation of mast cells. C5b initiates the membrane attack pathway, which results in the terminal complement complex, termed the membrane attack complex when formed on a membrane, consisting of C5b, C6, C7, C8, and polymeric C9 (25).Involvement of complement in diarrhea-negative, atypical HUS has been reported, and mutations in different complement regulator genes, such as those encoding FH, FI, and membrane cofactor protein, as well as gain-of-function mutations in C3 and factor B genes, have been described for affected patients (5, 10, 18). We recently showed that purified Stx2 activates the complement cascade (20) and demonstrated that Stx2 binds FH and that cofactor activity of surface-attached FH is delayed in the presence of Stx2. Thus, our observations indicate that complement may also be involved in the pathogenesis of EHEC-induced HUS (20).Several reports suggest that in addition to Stxs, the serine protease EspP may be an important virulence factor of EHEC bacteria (1, 14). We have shown that EspP is widely distributed among Shiga toxin-producing E. coli strains of human and animal origins and that EspP is associated with EHEC serogroups which cause HUS. This suggests that EspP contributes to the pathogenesis of this disorder (1, 14). However, the exact role of EspP as a virulence factor in the pathogenesis of EHEC-induced HUS is unknown.The aim of this study was to investigate whether or not EspP interacts with complement. In particular, it was envisaged to extend our knowledge on the pathogenesis of EHEC-induced HUS by determining whether or not the serine protease EspP cleaves complement components and thereby modulates complement activity.     

Interference of propamidine with binding of the fifth component of complement to surface-fixed C3b, and with C5 activation.

The effects of propamidine and their dose dependency, on utilization of the third and fifth complement components in immune haemolysis have been compared. While C3 utilization is not disturbed that of C5 is markedly inhibited by propamidine in concentrations as low as 0.5 mM. Both, binding of C5 to surface-fixed C3b and cleavage of C5 by convertases C42 and C3bBb, are also inhibited in the presence of propamidine. Since neither C3 cleavage by these enzymes nor even C5 cleavage by the cobra venom factor-supported convertase CFVBb is significantly reduced a general convertase-inhibiting effect of propamidine is ruled out. Rather the effect on utilization of C5 is the result of interference with binding of C5 is the result of interference with binding of C5 to C3b and hence impairment of its accessibility to the convertases. These findings thus further support the role of surface fixed C3b in C5 activation proposed earlier.     

Control of C3b and C5b deposition by CD46 (membrane cofactor protein) after alternative but not classical complement activation

C3b and C5b deposition following complement activation, and its regulation by CD46 were studied using xenogenic Chinese hamster ovary (CHO) cells as targets and cytofluorometry. Following activation of the alternative pathway, an initial low level of C3b deposition was observed on CHO cell surfaces after a lag time of approximately 4 min. This was followed by a secondary high level of C3b deposition with a slower rate. C3b deposition was maximal within 15 min. When CD46 was expressed (B2 isoform), the kinetics of C3b deposition were essentially unchanged, but the onset of the secondary high C3b deposition was fully prevented. C5b deposition was also observed on CHO but not on CHO.CD46 cells following activation of the alternative pathway. Activation of the classical pathway on CHO and CHO.CD46 cells, using factor B-depleted human serum and anti-CHO antibodies, resulted in almost identical single-peak C3b deposition profiles. Accordingly, no regulation of C5b deposition by CD46 was evident following activation of the classical pathway. These data indicate that CD46 prevents the C3b deposition amplification loop mediated by the alternative C3 convertase and, consequently, inhibits the formation of the alternative C5 convertase. But CD46 prevents neither the spontaneous tick-over C3b deposition leading to the formation of the alternative C3 convertase nor the formation of the functional classical C3 and C5 convertases.     

Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2

Cells infected with herpes simplex virus type 1 (HSV-1), but not HSV-2, express on their surfaces a receptor for the complement component C3b. Receptor activity is markedly enhanced by treatment of the infected cells with neuraminidase. Employing a direct binding assay, consisting of purified HSV glycoproteins immobilized on nitrocellulose and iodinated C3b as a probe, we found that C3b binds directly to gC-1, as well as to gC-2, but not to gB or gD from either serotype. C3b binding was enhanced by treatment of gC-1 or gC-2 with neuraminidase. Endo F or endo H treatment of gC-1 had no effect on C3b binding. However, treatment of gC-2 with these endoglycosidases had a marked negative effect on C3b binding. These results suggest that N-linked oligosaccharides are involved in binding of C3b to gC-2, but not gC-1. Alternatively, removal of N-linked oligosaccharides from gC-2 might adversely affect polypeptide conformation. Glycoprotein C-2 also differs from gC-1 in its effects on the complement cascade. Whereas gC-1 accelerated the decay of the alternative pathway C3 convertase and impaired the efficiency of lysis by the components C5 through C9, gC-2 stabilized the active C3 convertase and had little effect on the late-acting components. The dissimilarity of gC-1 and gC-2 with regard to their effects on the complement cascade may have implications regarding the role of these glycoproteins in confronting the host immune response.     

Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC).

We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.     

Influence of opsonization conditions on C3 deposition and phagocyte binding of large- and small-capsule Cryptococcus neoformans cells.

Previous studies demonstrated that, following opsonization with normal human serum (NHS), phagocytes bind greater numbers of small-capsule Cryptococcus neoformans cells than yeast cells with large capsules. The present study tested the hypothesis that suboptimal deposition of opsonic C3 fragments contributes to this disparity. C neoformans was grown under conditions promoting large or small capsules and was incubated at various concentrations in NHS. At low concentrations of yeast cells (125 cells per microl of NHS), the deposition of C3 fragments per unit of capsule volume and the binding of yeast cells to cultured human monocytes were similar for yeast cells having large and small capsules. However, at higher cell concentrations, large-capsule cells exhibited suboptimal coating with C3 fragments and markedly diminished monocyte binding compared with small-capsule cells. Thus, the inverse correlation between capsule size and phagocyte binding can be overcome by conditions promoting optimal C3 deposition.     

IgG immune complex binding to and activation of liver cells. An in vitro study with IgG immune complexes, Kupffer cells, sinusoidal endothelial cells and hepatocytes

BACKGROUND/AIMS: The aim was to study IgG immune complex (IC) binding to isolated hepatocytes, Kupffer cells (KCs) and sinusoidal endothelial cells (SECs). Further, we wished to analyze the capacity of IgG ICs to induce release of reactive oxygen metabolites by the IC-binding liver cells. METHODS: ICs were formed between (125)I-tyramine-cellobiose-labelled dinitrophenyl-conjugated human serum albumin ((125)I-TC-DNP(10)HSA) and polyclonal rabbit IgG antibodies. Binding of ICs to different rat liver cells in suspension was studied at 4 degrees C. Production of reactive oxygen metabolites was measured by luminol-enhanced chemiluminescence at 37 degrees C. RESULTS: IgG mediated binding of (125)I-TC-DNP(10)HSA to both KCs and SECs, but not to hepatocytes. The binding showed saturation kinetics and was blocked by an excess of unlabelled IgG-ICs. IgG-ICs activated KCs, but not SECs, to a chemiluminescence response. CONCLUSIONS: Both KCs and SECs bind IgG-ICs in vitro, probably via Fc receptor interaction. IgG-ICs activate KCs to produce reactive oxygen metabolites. The binding of IgG-ICs to isolated hepatocytes is small.     

Complement system proteins which interact with C3b or C4b A superfamily of structurally related proteins

Recent cDNA sequencing data has allowed the prediction of the entire amino acid sequences of complement components factor B and C2, the complement control proteins factor H and C4b-binding protein and a partial sequence for the Cab/C4b receptor CR1. These proteins all contain internal repeating units of approximately 60 amino acids, each repeating unit having a characteristic framework of highly conserved residues. The N-terminal Ba and CA portions of factor B and C2 both contain 3 repeating units and the chains of C4b-binding protein and factor H contain 8 and 20 repeating units, respectively, while the precise number of units in CR1 is not known yet. These structurally homologous complement proteins are also functionally related as they all interact with C3b and C4b during activation of the cascade. The repeating units also occur in the functionally unrelated proteins subcomponent C1r, β₂-glycoprotein 1, blood clotting factor XIII and interleukin-2 receptor. In this review Ken Reid and his colleagues propose that this could be a general feature of a superfamily of structurally related proteins.     

PEO enhancement of platelet deposition, fibrinogen deposition, and complement C3 activation.

Whereas it has been commonly thought that adding polyethylene oxide PEO to a surface would diminish the capacity of the surface to cause deposition of platelets and of fibrinogen, and to activate complement C3, we present data showing exactly the opposite. These unexpected results are obtained with low molecular weight (2000) PEO, and are not found with higher molecular weight (20,000) PEO.     

Structural Difference in the Complement Activation Site of Human IgG1 and IgG3

The C1q binding epicentre on IgG molecules involves residues Asp²⁷⁰, Lys³²², Pro³²⁹ and Pro³³¹ in the C_H2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5-iodo-4-hydroxy-3-nitro-phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody-dependent complement-mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10-fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp²⁷⁰, Leu³³⁴, Leu³³⁵. For all these residues, and especially for Asp²⁷⁰, IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.     

Complement component 3 binding to Haemophilus influenzae type b in the presence of anticapsular and anti-outer membrane antibodies.

Antibodies directed against the capsular polysaccharide (polyribosyl ribitol phosphate [PRP]) or the outer membrane proteins (OMP) of Haemophilus influenzae type b (Hib) promote bactericidal activity, complement 3 (C3) binding, and ingestion by phagocytic cells. To assess the relative contribution of anti-OMP to host defense against Hib, we compared the opsonic activities of anti-PRP and anti-OMP as reflected by the amounts of C3 bound to the bacterial surface. Immunoglobulin G (IgG) fractions containing either anti-PRP or anti-OMP were incubated with Hib in the presence of a C5-deficient complement source. C3, total IgG, and IgG subclasses bound to the bacteria were quantified by enzyme-linked immunosorbent assay. The maximum amount of C3 which could be bound to Hib was greater in the presence of anti-PRP than in the presence of anti-OMP. Also, except at low IgG concentrations, the rate of increase in bound C3 as a function of increasing IgG concentration was greater for anti-PRP than for anti-OMP. Hib-bound anti-OMP consisted primarily of IgG1 and IgG3, whereas bound anti-PRP was primarily IgG1 and IgG2. Thus, the potential for C3 binding to Hib is greater in the presence of anti-PRP than in the presence of anti-OMP, probably because of the larger number of binding sites available to the former. Nonetheless, OMP appear to provide important targets for opsonic antibody and would be logical components of a PRP-conjugate vaccine or may be efficacious as vaccines against nontypeable H. influenzae.