A group of bactericidal factors conserved by vertebrates for more than 300 million years

A complement-dependent bactericidal factor (RaRF) specific for the Ra chemotype strains of Salmonella and for the Ra-like strains of other enterobacteria has been found in sera of mice. We show here that the anti-Ra bactericidal factors are present in sera of all species, so far tested, of mammals, birds, reptiles, amphibians, and bony and cartilaginous fish. Certain properties, such as binding specificity, requirement of divalent cations for binding, and sensitivities to heat and reducing agents, of the factor in the representative animals were the same as or similar to those of the mouse RaRF. These results indicate that these factors have been conserved by vertebrates for more than 300 million years as a result of the necessity for the resistance to rough mutants of Gram-negative bacteria.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

Alternative complement pathway activation by C4b deposited during classical pathway activation

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.     

Lysis of sensitized sheep erythrocytes in human sera deficient in the second component of complement

Analysis of C-dependent lysis of sensitized SRBC by C2-deficient sera (C2D) led to the characterization of a C2 bypass pathway. Lysis in the total hemolytic C assay by C2D sera was Ca2+-dependent and required a high concentration of hemolysin to sensitize E. Selective component depletion indicated a requirement for C1 and C4 of the classical pathway (CP) and proteins B, P, and probably D of the alternative pathway (AP). Total hemolytic C could be restored to normal in these C2D sera by utilizing heavily sensitized E or by the addition of a supranormal concentration of B. This system most closely resembles a pathway described by J. E. May and M. M. Frank which requires antibody, C1, and the AP but not C4 or C2. It differs in its requirement for C4. We hypothesize that this pathway represents vestiges of a more primitive C pathway. It becomes evident and possibly clinically important in the setting of C2 deficiency, by allowing C activation, other than the AP, and perhaps in normal individuals, by damaging microorganisms that have evolved means to inhibit early components of the CP.     

Targeting of complement to tumor cells by heteroconjugates composed of antibodies and of the complement component C3b

Tumor cells have adapted several strategies which permit them to grow in an immunologically hostile environment. The C system can potentially destroy these cells; however, its action needs to be specifically potentiated on the surface of the tumor cells. To this end, a heteroconjugate composed of a mouse mAb and of the human C3b C component has been generated by using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The two mAb which were used in this study are V1-10 and TIB219 which bind to the human and mouse transferrin receptors, respectively. The mAb-C3b conjugates were purified by gel filtration and were each composed of one mAb and one C3b. They bound to the human K562 and HL60 or mouse ALB1 cell lines and amplified the killing of these cells by C from 10 to 15% to 70 to 100%. Fresh normal human or mouse sera were used as a source of C. The mAb-C3b conjugates activated primarily the alternative pathway of C since only C3 and factor B but not C4 were cleaved in the sera. After disulfide-linking to the mAb, the C3b became highly resistant to inactivation by factors H and I, probably due to its reduced factor H binding capacity. On the other hand, the conjugated C3b bound factor B better than free C3b and produced more C3 convertases which expressed increased stability. These results suggest that mAb-C3b conjugates may serve as an effective tool for the specific activation of the cytolytic C system on selected cells. As such, they may be used in vitro or in vivo to target the autologous C to tumor cells or to lymphocytes and may promote tumor immunotherapy.     

Major component of Ra-reactive factor, a complement-activating bactericidal protein, in mouse serum

A complement-activating bactericidal protein, Ra-reactive factor was isolated from mouse serum by an affinity method. The m.w. of the isolated RaRF estimated by glycerol density gradient sedimentation (around 300,000) was the same as that of the active material in mouse serum. As evidenced by gel filtration, the intact RaRF was decomposed into high (higher than 200,000) and low (50,000 to 200,000) m.w. components by treatment with 10% acetonitrile. SDS- and acid/urea-PAGE demonstrated that the high m.w. component was completely dissociated into equimolar quantities of two kinds of 28 kDa polypeptides, P28a and P28b, under reducing conditions, indicating that the association of these polypeptides was stabilized by disulfide bonds. The ability to bind specifically to the Ra determinant was retained in the high m.w. component, although the complement-activating potency was lost. The amino acid compositions of P28a and P28b polypeptides were compared with those of related serum proteins. The P28a and P28b polypeptides were found to have the highest homology to rat mannan-binding protein and mouse and human C1q subcomponent of complement.     

A covalent dimer of complement C4b serves as a subunit of a novel C5 convertase that involves no C3 derivatives

A C intermediate, LAC14, was prepared from TNP-aminocaproyl liposomes sensitized with anti-TNP antibody (Ab) and purified human C1 and C4. LAC14, containing radiolabeled C4, was analyzed by SDS-PAGE followed by autoradiography, and yielded a 210-kDa band and a predominant 400-kDa band. The 210-kDa band consisted of monomeric C4b bound to low molecular mass acceptors. The 400-kDa band was comprised of a 200-kDa moiety, as well as beta- and gamma-chains of C4. The 200-kDa moiety contained neither C1 nor sensitizing Ab, but it was largely decreased by treatment with NH2OH to the 90-kDa moiety with the mobility corresponding to the alpha'-chain of C4b. A covalent dimer of C4b, therefore, is the predominant form of C4b deposited on liposomes sensitized with antibody. The C4b-C4b dimer formed rapidly (within 5 min) followed by slow dissociation into monomers. The LAC14 bearing the C4b dimer but not the monomer was lysed, although with relatively low efficiency, by the addition of oxyC2 and EDTA-supplemented C3-deficient serum (C3DS), and, furthermore, LAC142 possessed the ability to convert C5 into C5a and C5b. Moreover, lysis was inhibited not by anti-C3 Ab but by anti-C4 Ab. In other experiments, the dimer served as an element of C3 convertase, as well. These findings imply that the C4b dimer, when complexed with C2, expresses C3/C5 convertase activity without participation of C3, and may provide a molecular mechanism whereby sera from patients with complete C3 deficiency retain the ability to induce C-mediated cytolysis.     

Study of the effect of Anisakis simplex larval products on the early and late components in the classical complement pathway

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

Functional and biochemical properties of the early classical complement system of mice

Mouse serum and EDTA plasma were subjected to low ionicity precipitation, gel filtration, and ion exchange chromatography in an attempt to purify C1, C4, and C2 to functional and chemical homogeneity. In marked contrast to human and guinea pig components, those of the mouse could not be separated by these techniques. Except for partial separation of C1 from C4 and C2 on DE-52 cellulose columuns with EDTA in the eluting buffers, there was no separation of those three components on ion exchange chromatographic columns. Sephadex G-200 gel filtration columns, or with precipitation of euglobulins from serum or plasma. Generation of EAC142 by incubation of EA in whole serum followed first order kinetics when mouse serum was used and second (or greater) order kinetics when human or guinea pig sera were used. Generation of EAC142 by incubation of EA in whole mouse serum followed by incubation in EDTA containing buffers resulted in rapid loss of all three activities from the cell. These experiments indicated that there were significant differences between the early classical C system of mice and those of human and guinea pig. In addition, they indicated that under a variety of in vitro conditions, murine C1, C4, and C2 behaved biochemically and functionally as a unit. The reasons for the major differences in behavior of the murine C components with not become clear until methods to stabilize their function are found so that they can survive multiple purification steps.     

Formation of classical C3 convertase during the alternative pathway of human complement activation

The fluid phase C3 convertase of the alternative pathway of human complement activation has been constructed from the isolated C3 component and from purified factors B and D. The enzyme was able to activate the isolated components C4 and C2 in the presence of C4 but had no effect on C2 in the absence of C4. The C4 and C2 activation was monitored by the loss of their hemolytic activity during the incubation with the alternative fluid phase C3 convertase. The activation of C4 and C2 components by the membrane-bound alternative C3 convertase formed on red cells (EC3bBb) was followed by the formation of C3 convertase of the classic pathway--EC4b2a. This resulted in the enhancement of hemolysis.     

Genetic polymorphism of the second component of human complement (C2)

Summary Proteins were separated by prolonged isoelectric focusing in polyacrylamide gels, whereupon C2 bands were detected by a specific hemolytic assay. This was performed by treating the gel with iodine to increase C2 activity, and then developing C2 bands with an agarose gel overlay containing sensitized sheep cells and diluted human serum as a complement source deficient in functional C2. The gene frequencies observed in a material of 122 unrelated adults were: C2¹:0.97 and C2²:0.03.C2 linkage relations and C2 haplotype associations have been examined a family material. It is concluded that C2 is very closely linked to HLA loci.     

Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

Lysis of tumor cells by antibody and complement. II. Lack of correlation between amount of C4 and C3 fixed and cell lysis.

Two antigenically distinct diethylnitrosamine-induced guinea pig hepatoma cell lines, line-1 and line-10, sensitized with rabbit anti-Forssman or with tumor-specific antibody, were more susceptible to killing by human complement (HuC) than by guinea pig complement (GPC). This difference could not be ascribed to differences in the amount of C1, C4, and C3 fixed: millions of C4 and hundreds of thousands of C3 were detected on cells whether they were killed or not killed by the C sources. Tumor cells sensitized with anti-Forssman IgM antibody generally had more GP C4 and C3 than Hu C4 and C3 bound to their surfaces. Cells sensitized with anti-tumor antibody generally had more Hu C4 and C3 than GP C4 and C3 bound to their surfaces. The resistance to killing of nucleated cells by antibody and C may be due in part to intrinisic properties of the cell.     

Complement components and terminal complement complex in oesophageal smooth muscle of patients with achalasia.

This study investigates whether patients with achalasia exhibit autoimmune reactions with subsequent complement activation within oesophageal smooth muscle, vessels and neurones. Oesophageal muscular biopsies from 8 patients undergoing surgery for achalasia and from 6 patients operated for oesophageal cancer were investigated by immunofluorescence for the presence of the complement components C1q, C4, C3c, C3d, C9 and the C9 neoantigen of the terminal C5b-C9 complement complex. Tissues were also investigated for the expression of immunoglobulins (G,A,M) and of the antigens of rubella and varicella zoster viruses. In addition, sera of both patient groups were tested for the presence of autoantibodies against Auerbach's plexus. The terminal complement complex C5b-C9 was found within muscle cells from all patients with achalasia but in only one specimen from a patient with cancer. Two patients with achalasia also exhibited the terminal complement complex as well as IgM within ganglion cells. Muscle cells stained positive for the complement component C9 in all five patients with achalasia in whom this test was performed but in none of the control tissues. In addition, sera from four patients with achalasia contained antibodies against Auerbach's plexus. Studies for the complement components C1q, C4, C3c and for antigens of rubella and varicella zoster viruses revealed negative results in all patients and controls. The results of this study suggest that a complement activation is involved in the autoimmune pathogenesis of achalasia. However, the triggering mechanism of this phenomenon remains to be determined.     

Influence of serum IgG antibodies to Chlamydia trachomatis on the functional activity of complement components

The immunoenzyme analysis and the method for the determination of IgG-containing immune complexes, carrying C1q component of the complement, were developed. In human blood sera the functional activity of components C3, complex C1r2s2, the content of C1 inhibitor and complement-activating immune complexes were determined. The comparative analysis of the activity of components C3 and C1r2s2, as well as between the content of C1 inhibitor and the activity of complex C1r2s2 for seropositive and seronegative sera, was made. Pronounced correlation for seropositive sera was observed. In addition, for seropositive sera correlation between an increase in IgG immune complexes and a drop in the functional activity of complex C1r2s2, as well as a drop in the functional activity of complex C1r2s2 and a growth in the titers of IgG antibodies to Chlamydia trachomatis, were established. The decreased functional activity of key complement components, simultaneously with the presence of complement-activating immune complexes and high titers of specific antibodies could be the diagnostic criteria of carrier state.     

Role of platelet factors and serum complement in growth of fibroblasts with high-affinity C1q complement receptors

Summary Cultures of human diploid fibroblasts are heterogeneous in that a subpopulation interacts via high-affinity receptors with the globular head regions of the C1q complement protein. Growth and synthetic properties of these cells are characteristic of cells residing in healing wounds and inflammatory lesions. At these sites, fibroblasts are exposed to regulatory molecules such as complement components and factors released from blood platelets. We assessed the effects of native complement proteins and platelet-derived factors on proportions and phenotypic stability of high-affinity and low-affinity receptor cells generated from explants of adult and embryonic connective tissue, using radioligand binding assays and immunofluorescence analysis by flow cytometry. Fibroblasts expressing high-affinity C1q receptors could be generated from explants only when factors from platelets were present in the medium; native complement proteins were not essential. High-affinity receptor cells could be generated only from tissue; they could not be generated by incubating cultures of the low-affinity receptor phenotype in medium containing platelet-derived factors. High-affinity receptor cells, once established from explants in the presence of platelet-derived factors, persisted through many replications in the absence of platelets. We obtained the same fibroblast phenotypes from embryonic skin as from adult gingiva, but the proportion of high-affinity receptor cells from skin was much greater. We conclude that factors derived from platelets are essential for generating cultures containing fibroblasts expressing high-affinity C1q receptors, but not for their maintenance. High-affinity receptor cells may comprise a rapidly dividing subpopulation giving rise only to like progeny or to other, more differentiated cells. Supported by grants DE03301 and DE02600 from the National Institutes of Health, Bethesda, MD.     

Cytotoxic effects of normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation.

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca²⁺, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca²⁺ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg²⁺ These observations also highlight the Mg²⁺-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.     

Functional analysis of Ficolin-3 mediated complement activation

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.     

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.