The relation between the bactericidal activity of complement and the character of the bacterial surfaces

The bactericidal activity of sera not containing antibodies (sera from precolostral piglets and calves) was tested with strains of gram-negative bacteria with different surfaces. The accuracy of the method of bactericidal test was evaluated statistically the bactericidal unit of complement was defined for comparing the activity of sera of different animals and different species. Various methods used for estimating the character of bacterial surface were compared. It was found that the bactericidal activity of piglet sera is directly dependent on the content of complement in the sera tested and the character of the bacterial surface (in the R-form). In selected strains there is a correlation in all criteria characterizing the surfaces of bacteria, and their susceptibility to bactericidal activity of sera; in a group of 37 strains selected at random, correlation with only one of the surface characteristics (stability in solution after heating to 100°C for 1 hour) was found. In calf sera a component was found which increases the effect of complement to some strains (e.g.Shigella shigae). This component may by absorbed from the serum only in the presence of complement. The nature of this factor is discussed.     

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.     

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.     

A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.     

Schistosoma mansoni: complement and cercarial tail loss during in vitro transformation.

When Schistosoma mansoni cercariae are incubated at 37 C in media containing serum, the organisms lose their tails and change into viable, infective schistosomula. Tail loss does not occur in the absence of serum, or when the serum is heat inactivated. In the present studies, tail loss during in vitro conversion was shown to be complement dependent. The capacity of fresh serum to promote tail loss was markedly suppressed or abolished by cobra venom factor, zymosan, Sepharose CL-4B AND anti-C3 antibody. The alternative rather than the classic complement pathway appeared to be responsible since (1) binding of anti-C3 to cercariae required magnesium, but not calcium; (2) both C4-deficient serum and C2-deficient serum supported tail loss; but (3) human serum heated to 50 C for 20 min to inactivate Factor B did not support tail loss. Cercarial tail loss also required the terminal complement components C5 through C8. The extent and rate of tail loss was normal in agammaglobulinemic sera indicating that the complement effect was not antibody dependent.     

Effect of Complement and Viral Filtration on the Neutralization of Respiratory Syncytial Virus

The addition of 10 hemolytic units of guinea pig complement has been shown to enhance the neutralizing capacity of respiratory syncytial (RS) immune sera produced in guinea pigs and ferrets. This same immune sera, when tested without complement, had little or no neutralizing capacity. The addition of complement to RS immune horse serum did not significantly increase its neutralizing capacity. Immune horse serum effectively neutralized RS virus without complement. Other studies indicated that a 50% tissue culture infective dose of between 30 and 100 should be used in RS serum neutralization tests and that incubation should be for 90 to 105 min at room temperature. The neutralizing capacity of guinea pig immune serum was not increased by the use of filtered virus. The rate of virus neutralization, however, was increased with the addition of 10 hemolytic units of complement. The neutralizing capacity of RS immune horse serum was much greater for filtered than for unfiltered RS virus. The addition of complement increased the rate of virus neutralization but did not increase the neutralizing capacity of the horse immune serum.     

Outer surface protein E antibody response and its effect on complement factor H binding to OspE in Lyme borreliosis

Borrelia burgdorferi sensu stricto and B. afzelii, but not B. garinii, are able to escape complement attack by binding factor H via OspE proteins. Recent finding of ospE genes also in B. garinii isolates has raised the question whether, under in vivo-conditions, B. garinii also expresses OspE proteins and consequently induces an antibody response. We set up an IgG ELISA by using recombinant OspE as an antigen. Sixty percent of acute and 64% of convalescent 25 erythema migrans patient samples were positive for anti-OspE antibodies. Anti-OspE antibodies were also found in the sera (83.6%) and cerebrospinal fluids (36%) of patients with neuroborreliosis. Since B. garinii is the major causative agent of neuroborreliosis, the result suggests that OspE is expressed by B. garinii in vivo. Of the 10 acrodermatitis chronica atrophicans patients, 80% had anti-OspE antibodies. Anti-OspE antibody positive sera inhibited factor H binding to Borrelia more efficiently than normal control sera (65% vs. 33.7%). Our results indicate that Borrelia spirochetes, including B. garinii, can induce the production of anti-OspE antibodies. This implies that OspE protein is produced in vivo by B. garinii possibly enabling it to escape complement and cause a CNS infection.     

The generation of chemotactic activity in sera of germ-free newborn piglets by interaction with rough strain ofEscherichia coli

The interaction of sera of newborn precolostral piglets with rough strain ofEscherichia coli or its endotoxin leads to formation of a factor which is chemotactic for rabbit polymorphonuclear neutrophil leucocytesin vitro (as tested using the Boyden's diffusion two-compartment chamber). Smooth strain ofEscherichia coli does not induce chemotaxin formation. The generation of chemotactic factor can be prevented by heating of the serum, addition of EDTA or yeast phosphomannan. The generation of the chemotactic activity is explained by the fixation of piglet complement system which is activated by rough bacteria even in the absence of detectable antibodies in newborn sera.     

Taenia taeniaeformis: evasion of complement-mediated lysis by early larval stages following activation of the alternative pathway

Activation of the alternative pathway of complement by T. taeniaeformis oncospheres and early stage metacestodes, although a factor in host defense against primary infection, does not directly lead to the killing of the parasite larvae observed prior to day 6 post-infection in innately resistant BALB/cByJ inbred mice. Immunogold labelling techniques clearly demonstrated tegument-associated C3 on in vitro-activated oncospheres incubated with non-immune mouse sera. However, C5, a protease necessary for the assembly of the membrane attack complex, was not detected. Early stage larvae cultured from in vitro-activated oncospheres escaped membrane damage and survived incubation in non-immune sera from both BALB/cByJ and taeniid-susceptible C3H/HeDub mice. Comparisons of cobra venom factor-treated and untreated C5-deficient B10.D2osn mice revealed no significant differences in parasite burden and local eosinophil infiltration at 6 days post-infection, suggesting that the terminal arm of the complement system is necessary for the previously reported role of complement in resistance to primary infection in BALB/cByJ and C3H/HeDub mice. An in vivo test of chemotaxis indicated that although both complement-intact mouse strains examined responded to intraperitoneal injections of inulin, there were lower numbers of eosinophils in C3H/HeDub mice than in BALB/cByJ mice, perhaps pointing to possible mouse strain differences in C5a generation/catabolism or eosinophil ability to respond to C5a. Lectin-binding studies showed an affinity of PNA for the exposed surface of taeniid oncospheres and 4-day post-infection metacestodes; however, binding of lectin to the carbohydrate moiety did not inhibit complement activation.     

Complement-dependent cytotoxic factor to megakaryocyte progenitors in sera from patients with idiopathic thrombocytopenic purpura

The influence of sera from patients with idiopathic thrombocytopenic purpura (ITP) was examined on colony formation from megakaryocyte (M) progenitors. Though incubation of marrow cells in Iscove's modified Dulbecco's medium (IMDM) containing 50% sera from several ITP patients stimulated M-colony formation in 8 of 13 cases, incubation in the sera from the patients and in baby rabbit serum as a source of complement significantly suppressed the colony formation. Experiments showed that sera of immunoglobulin G from ITP patients had significant complement-dependent cytotoxicity to M-progenitors in normal marrow cells or in the marrow cells from corresponding patients, but not to CFU-e, BFU-e or CFU-gm. Cytospin preparations of individually collected M-colonies from marrow cells treated with ITP patients' sera and complement revealed a reduction of megakaryocyte colonies containing cells of multilineages. These results indicate that autoantibodies detected in ITP patients can bind not only to platelets and megakaryocytes, but may also bind to M-progenitors.     

Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting.

Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.     

Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement

Abstract Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii . Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.     

Labile Serum Factor and Its Effect on Arbovirus Neutralization

Enhancement of neutralization of Sindbis, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, and St. Louis encephalitis viruses by labile serum factor (LSF) in human serum and plasma was demonstrated. Human serum and plasma could be diluted 1:8 and 1:16 and still retain some LSF activity. Satisfactory storage temperatures for retention of LSF activity were −20 or −56 C. Repeated freeze-thaw cycles of serum did not alter LSF activity, but the activity was completely eliminated by heating at 56 C for 5 min. LSF of human serum equally enhanced neutralization by Sindbis immune mouse and rabbit sera; these results suggest a lack of species specificity. Rehydrated lyophilized gunea pig complement did not restore LSF activity to heated human plasma. Serum components responsible for LSF activity were not dialyzable. Discovery of fresh serum without LSF activity established the need to pretest all sera used as LSF sources.     

Pathogenic complement activation in collagen antibody-induced arthritis in mice requires amplification by the alternative pathway

Immune complex-induced inflammation can be mediated by the classical pathway of complement. However, using mice genetically deficient in factor B or C4, we have shown that the collagen Ab-induced model of arthritis requires the alternative pathway of complement and is not dependent on the classical pathway. We now demonstrate that collagen Ab-induced arthritis is not altered in mice genetically deficient in either C1q or mannose-binding lectins A and C, or in both C1q and mannose-binding lectins. These in vivo results prove the ability of the alternative pathway to carry out pathologic complement activation in the combined absence of intact classical and lectin pathways. C3 activation was also examined in vitro by adherent collagen-anti-collagen immune complexes using sera from normal or complement-deficient mice. These results confirm the ability of the alternative pathway to mediate immune complex-induced C3 activation when C4 or C1q, or both C1q and mannose-binding lectins, are absent. However, when all three activation pathways of complement are intact, initiation by immune complexes occurs primarily by the classical pathway. These results indicate that the alternative pathway amplification loop, with its ability to greatly enhance C3 activation, is necessary to mediate inflammatory arthritis induced by adherent immune complexes.     

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX⁻) or HPX-containing (HPX⁺) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX⁻ sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.     

A comparative study of mammalian and reptilian alternative pathway of complement-mediated killing of the Lyme disease spirochete (Borrelia burgdorferi)

The potential bactericidal activity of the alternative complement pathway of mammalian and reptilian sera to Borrelia burgdorferi sensu stricto (s.s.) was evaluated in vitro. Complement-mediated killing was observed when cultured spirochetes were inoculated into sera from the western fence lizard (Sceloporus occidentalis) and from the southern alligator lizard (Elgaria multicarinata), but not when they were inoculated into serum from either the deer mouse (Peromyscus maniculatus) or from humans. Spirochetes were still alive after 4 hr in lizard serum that had been preheated at 56 C for 30 min to inactivate complement. Furthermore, when lizard serum was chelated with 10 mM ethylenediaminetetraacetic acid to block all complement activation, borreliacidal activity was arrested. When lizard serum was chelated with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 4 mM MgCl2 to block only classical complement pathway activation, >85% of spirochetes were immobilized within 1 hr. Differences in B. burgdorferi s.s. mortality were not observed when chelators with or without MgCl2 were added to serum from either deer mice or humans. Proteins comprising the alternative complement pathway are responsible for the borreliacidal activity observed in the blood of S. occidentalis and E. multicarinata.     

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.