Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies.     

Ganglioside modulation of lipopolysaccharide-initiated complement activation

Highly purified preparations of mouse gangliosides have been demonstrated to bind to purified preparations of lipopolysaccharide (LPS). In some instances, the binding has been demonstrated to be dependent upon the presence of sialic acid in the ganglioside preparation. The binding of gangliosides to LPS from the deep rough Salmonella minnesota Re mutant has suggested that the interaction involves the lipid A-2-keto-3-deoxyoctulosanate region of the LPS macromolecule. The interaction between gangliosides and LPS has been demonstrated to result in an abrogation of lipid A dependent activation of the classical pathway of serum complement by Re LPS. Surprisingly, however, the presence of sialic acid containing glycolipids has been shown to enhance significantly the capacity of LPS to initiate activation of the alternative pathway of complement. These data suggest that sialic acid can enhance as well as inhibit the formation of a stable alternative-pathway C3 convertase.     

Complementary Recognition of Alternative Pathway Activators by Decay-Accelerating Factor and Factor H

The alternative complement pathway (ACP) functions as a surveillance mechanism by which microorganisms are opsonized with C3b in the absence of specific antibodies. The effectiveness of the ACP relies on its ability to distinguish self from non-self. This recognition function is mediated by C3 regulatory proteins including serum factor H, membrane cofactor protein (MCP), and membrane decay-accelerating factor (DAF). H activity against bound C3b can be increased by host components such as sialic acid and decreased by microbial polysaccharides. DAF and MCP may also recognize cell surface changes such as the presence of viral glycoproteins, since some virus-infected and tumor cells activate the ACP. In the present study, liposomes containing wild-type and mutant Salmonella minnesota lipopolysaccharide (LPS) were tested for ACP activation in serum. LPS-containing liposomes with bound C3b were then tested for their susceptibility to C3 convertase regulation by H and membrane DAF and for the sensitivity of their bound C3b to the cofactor activity of H. The results indicate that while the shortest mutant, Re595 LPS, did not induce ACP activation, R7 LPS containing an additional disaccharide did. This activation was poorly regulated by DAF but was inhibited by H. The regulatory activity of H for liposome-bound C3b, however, decreased when LPS of greater polysaccharide size was present in the membrane. In contrast the ACP activation induced by the phospholipid phosphatidylethanolamine was effectively inhibited by DAF but only poorly inhibited by H.     

Alternate complement pathway activation by group A streptococci: role of M-protein.

Avirulent strains of group A streptococci readily activate the complement system in normal human serum via the alternate complement pathway (ACP). Virulent M-positive group A streptococci are much less potent as activators of the ACP. The ability of M-positive streptococci to activate the ACP is enhanced by trypsinization or mild peptic digestion. The latter treatment removes the serologically active and antiphagocytic type-specific moieties of M protein, but retains the surface fuzzy layer. The phagocytosis of avirulent streptococci is markedly enhanced by preopsonization in serum chelated with Mg-ethylene glycol tetraacetic acid (classic complement pathway blocked) but not in serum devoid of heat-labile factors. These studies suggest that the function of M protein as a virulence factor may be mediated, at least in part, by its ability to retard interaction of ACP components with structures present on the streptococcal cell surface.     

Species-specific inhibition by glycophorins of complement activation via the alternative pathway

Glycophorin, one of the major glycoproteins of erythrocytes (E), was extracted from human E (glycophorin-Hu) and guinea pig E (glycophorin-GP) and adsorbed to rabbit-E. The adsorption of glycophorin-Hu and glycophorin-GP to rabbit-E made the E resistant to hemolysis by human serum and guinea pig serum, respectively, via the alternative complement pathway (ACP). However, it did not make the rabbit-E resistant to hemolysis by serum heterologous to the glycophorin adsorbed. This species-specific inhibition by glycophorin of ACP activation should play a role in restricting ACP activation on self cell membranes. By recognizing the self-cell surface as the place where the complement reaction must be prevented, ACP will be able to accomplish the discrimination of non-self constituents without diversity of recognition sites for a variety of foreign substances.     

Membrane sialoglycolipids regulate the activation of alternative complement pathway by liposomes containing trinitrophenylaminocaproyldipalmitoylphosphatidylethaolamine.

We found that liposomes associated with trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-Cap-DDPE) activate the alternative complement pathway (ACP) of guinea-pig. The complement-activating capacity (CAC) of liposomes with TNP-Cap-DPPE (TNP-Cap-liposomes) was found to be inhibited by the insertion of sialoglycolipids such as GM3 onto the membrane. However, neutral glycolipids tested had no inhibitory effect on the CAC of the TNP-Cap-liposomes. The minimum amount of sialoglycolipids required for the inhibition of the ACP-activating capacity of TNP-Cap-liposomes was 0.01 or less in molar ratio to dimyristoylphosphatidylcholine in the liposomes. Since the insertion of charged amphiphiles did not affect the status of TNP-Cap-liposomes containing glycolipids with respect to their ACP-activating capacity, the surface potential caused by sialoglycolipids was disregarded as being the factor responsible for restriction of the complement-activating effect. For the inhibitory effect to be manifested, it was demonstrated that the presence of GM3 was required on the same liposome membrane as where the TNP-Cap-triggered ACP activation is taking place. Therefore, sialoglycolipids may inhibit ACP activation by reacting directly on certain nascently activated complement proteins. However, insertion of GM3 could not inhibit liposome lysis via the classical complement pathway. Furthermore, the presence of antibody reaction significantly reduced the inhibitory capacity of GM3 indicating that natural antibody may be responsible for discriminating between self and heterologous surfaces, thus cancelling the glycolipid-mediated restriction of ACP activation in the case of heterologous cell surface, bringing about lysis by the cancellation of the glycolipid-mediated restriction of ACP activation on heterologous cell surfaces.     

Prevention of complement activation on the homologous cell membrane of nucleated cells as well as erythrocytes

A C3b receptor, a glycoprotein with a molecular weight of 205 kDa (gp205) on human erythrocytes (Ehu), has been claimed to restrict the activation of human complement via the alternative complement pathway (ACP), thereby inhibiting the activation of the ACP even on neuraminidase (Nase)-treated Ehu. However, the Nase-treated Ehu were sensitive to hemolysis by guinea pig complement via ACP activation, although the C3b receptors on Ehu can react with guinea pig C3b. Furthermore, HeLa cells which had no detectable C3b receptor did not became reactive with guinea pig ACP. On the contrary, Nase treatment of guinea pig erythrocytes (Egp) as well as guinea pig line 10 tumor cells, which have no detectable C3b receptor, could not make those cells reactive with the homologous guinea pig ACP although they became reactive with human and rabbit ACP. Similarly, rabbit E did not become reactive with the homologous rabbit ACP following Nase treatment. Nase-treated Egp preincubated with human serum in the presence of EDTA were also not lysed by guinea pig serum. Therefore the unreactiveness of Nase-treated cell membrane with homologous ACP was not merely due to the absence of antibody to cell membrane in the homologous serum. There may be a membrane inhibitor which preferentially interacts with homologous complement to circumvent undesirable complement activation on the homologous cell membrane.     

Cytolysis of Sendai virus-infected guinea-pig cells by homologous complement

Guinea-pig line 10 tumor (GPL10) cells, which were transplantable in strain 2 guinea-pigs, gained the capacity to activate the homologous alternative complement pathway (ACP) following infection with ultraviolet-irradiated Sendai virus (UV-SV). The ACP activation of homologous serum on the UV-SV-infected GPL10 cells (UV-SV-GPL10 cells) resulted in cytolysis of the tumor cells. This indicates that UV-SV infection induced not only the capacity to activate complement on the GPL10 cells, but also made them sensitive to complement attack, because GPL10 cells have been demonstrated to be resistant to the reaction of homologous complement via the classical pathway in the presence of rabbit antibody. Furthermore, UV-SV-GPL10 cells prepared with as little as 0.1 plaque-forming unit equivalent of UV-SV per cell showed suppressed growth in syngeneic strain 2 guinea-pigs. The essential role of complement in the elimination of UV-SV-GPL10 cells in vivo was confirmed by demonstrating that UV-SV-GPL10 could grow in guinea-pigs whose complement had been depleted by administration of cobra venom factor.     

Interleukin 6 stimulates synthesis of complement proteins factor B and C3 in human skin fibroblasts

Human interleukin (IL) 6 is a multifunctional cytokine which is synthesized by fibroblasts in response to many stimuli, including bacterial lipopolysaccharide (LPS). During acute-phase response, liver cells secrete a specific group of proteins among which components of the complement system and IL 6 appear to be an important mediator of this response. Human skin fibroblasts also synthesize at least seven proteins of the complement system. Each of these seems to be characteristically regulated by soluble mediators of the inflammatory process. Here we report that in fibroblasts, IL 6 induces increases in the rate of synthesis of factor B and C3, activator proteins of the alternative pathway of complement activation. The increases in factor B and C3 were concentration dependent reaching about 40- and 15-fold, respectively. The protein increases were observed within 4 h after IL 6 addition to the cells and were accompanied by increase in factor B and C3 mRNA. The data suggest that the induction of factor B and C3 by LPS may be mediated, at least in part, by IL 6 induced by LPS. This new function of IL 6 could provide a local protection against invading agents through activation of the antibody-independent alternative pathway of complement activation.     

Activation of the alternative complement pathway of guinea-gip by liposomes incorporated with trinitrophenylated phosphatidylethanolamine

By incorporation of trinitrophenylamino-caproyldipalmitoylphosphatidylethanolamine (TNP-Cap-DPPE) into liposomes composed of an equimolecular mixture of dimyristoylphosphatidylcholine (DMPC) and cholesterol (TNP-Cap-liposomes), liposomes became readily lysed by guinea-pig serum (GPS) in Mg++-EGTA-GVB (gelatin veronal buffered saline containing 2 mM MgCl2 and 10 mM ethyleneglycol-bis (beta-amino-ethyl ether)N-N'-tetraacetate) as well as in GVB++ (gelatin veronal buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2). Since the classical complement pathway (CCP) does not work in Mg++-EGTA-GVB, TNP-Cap-liposome lysis by GPS in Mg++-EGTA-GVB was thought to be mediated by the activation of the alternative complement pathway (ACP). This conclusion was supported by observations that heating of GPS at 50 degrees impaired its lytic activity while C4-deficient GPS was capable of lytic activity, no lysis occurred in EDTA, and there was noted consumption of complement in GPS treated with TNP-Cap-liposomes at 30 degrees. For TNP-Cap-liposome lysis by GPS in Mg++-EGTA-GVB, the epitope density of the TNP hapten was required to be 5% or more of the DMPC. Changing the acyl group of the phosphatidylcholine (PC) significantly influenced the ACP activating capacity of TNP-Cap-liposome. Dipalmitoyl-PC, DMPC and distearoyl-PC facilitated the ACP activating capacity of the TNP-Cap-liposome, while dilauroyl-PC, egg-PC and dioleoyl-PC did not. Furthermore, the length of spacer between TNP and dipalmitoylphosphatidylethanolamine (DPPE) also influenced the ACP activating capacity and maximum activation was noted when the spacer was aminocaproyl. These physicochemical characteristics which increase the ACP activating capacity coincided with those reported to increase the immunogenicity of hapten-sensitized liposomes.     

Opsonization of various capsular (K) E. coli by the alternative complement pathway

A virulence factor of E. coli K-1 is its capacity to avoid opsonization via the alternative complement pathway (ACP). Since it is not known if E. coli with other capsular (K) antigens have similar properties we examined various capsular E. coli for opsonization by the ACP. To assess opsonization we used whole blood luminol-dependent chemiluminescence (CL) and the magnesium salt of ethyleneglycol tetraacetic acid to block the classical pathway. E. coli K-types 6, 7, 27, 30, 42, 53, 57 and 75 were effectively opsonized via the ACP (greater than 65% of CL obtained with unchelated normal serum). K types 2 and 13 were opsonized by the ACP in both the high range greater than 65% and intermediate range 36-65%. Only K-types 1, 3, 5, 12 and 92 were poorly opsonized (less than 35%) by the ACP. The data demonstrate that most E. coli K-types were opsonized via the ACP. The poor opsonization of E. coli K3, 5, 12 and 92 by the ACP may be virulence factor for these bacteria.     

Ontogeny of the third component of complement of Japanese quails

Ontogeny of quail complement was examined in terms of serum C3 level, cytolytic activity of total complement, functional activity of C3 via the classical pathway (CCP) and an alternative route (ACP), and the distribution of C3-bearing cells. In serum, C3 was detected from 7-day-old embryos by rocket immunoelectrophoresis. Thereafter, its concentration increased rapidly and reached the adult level at 4-5 weeks of age. Total activities of serum complement via CCP and ACP, both of which were assayed by cell lysis, were detected first in 10-day-old embryos, and increased sharply after hatching, reaching the maximum level at 5 weeks of age. Deposition of C3 on the cell surface via CCP and ACP was also clearly demonstrated from 10-day-old embryos by haemagglutination and immunofluorescence techniques, respectively. By an immunofluorescence technique using anti-quail C3 monospecific serum, cells belonging to the reticuloendothelial system were indicated to serve major sites for the synthesis of C3 during ontogenesis.     

Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 micrograms/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon gamma (IFN-gamma)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-gamma-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-gamma appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.     

Antibody-independent activation of the classical pathway of human serum complement by lipid A is restricted to re-chemotype lipopolysaccharide and purified lipid A

Incubation of most bacterial lipopolysaccharides (LPS) with normal human sera at 37 degrees C activates the serum complement system, resulting in decreased levels of hemolytic complement. A panel of R-chemotype LPS preparations isolated from Salmonella minnesota rough mutant strains, as well as smooth wild-type LPS from S. minnesota, Escherichia coli O55-B5, Serratia marcescens, and Yersinia enterolitica, were used to examine the effect of LPS polysaccharide chain length on LPS lipid (lipid A)-dependent activation of the classical pathway of complement (CPC). To examine specific lipid A-dependent activation of the CPC, sera deficient in alternative pathway of complement activity were prepared by the removal of factor D. Absorption of normal human sera with formalinized rabbit erythrocytes was found to remove natural antibodies, factors capable of forming LPS complexes which activate the CPC, or both. By using such factor D-depleted formalinized rabbit erythrocyte-absorbed normal human sera, only isolated lipid A and Re-chemotype LPS (R595 LPS) were found to activate the CPC. Thus, the presence of the additional monosaccharide L-glycero-D-mannoheptose in the Rd2 LPS oligosaccharide chain compared with the L-glycero-D-mannoheptose-deficient Re-chemotype LPS structure is sufficient to block lipid A-dependent activation of the CPC by LPS.     

Activation of complement by opportunist pathogens and chemotypes of Salmonella minnesota.

Washed cells of Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella minnesota chemotypes (S, Rb, and Re) were tested for their ability to activate the alternative complement pathway (ACP). Parameters of ACP activation were (i) conversion of C3 in 10 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N1-tetraacetic acid-treated human serum supplemented with 2.5 mM MgCl2, (ii) lysis of glutathione-treated human erythrocytes in the presence of human serum, and (iii) C3 to C9 consumption in C4-deficient guinea pig serum. With the exception of S. minnesota Re and S. aureus, all of the strains were highly active in the test systems when compared with inulin. S. minnesota Re and S. aureus initiated C3 conversion in untreated human serum, suggesting that these microorganisms were capable of activating complement by a mechanism other than the ACP. These results provide direct evidence for ACP activation by opportunist gram-negative bacilli and refute the hypothesis that the lipid A moiety of the lipopolysaccharide cell wall is responsible for ACP activation.     

Bacterial sialic acid modulates activation of the alternative complement pathway of channel catfish (Ictalurus punctatus)

The alternative complement pathway (ACP) provides the non-immune channel catfish (Ictalurus punctatus) with protection against many Gram-negative bacteria. Very little serum bactericidal activity (0-13%) was found against 8 fish pathogens, but a strong bactericidal response (100%) was found against 7 non-pathogens. MgEGTA chelation of catfish serum did not essentially change the bactericidal results. Catfish serum heated at 56 degrees C and serum adsorbed with zymosan had no bactericidal activity. This demonstrated that the ACP was responsible for the bactericidal response. The molecular nature of the microbial surface determines whether or not the ACP will be activated. A relative lack of surface sialic acid has been found to be important for binding complement Factor B of the ACP by susceptible microbial surfaces. This study therefore examined the 15 Gram-negative bacterial fish pathogens and non-pathogens by determining their sialic acid content and their ability to elicit a bactericidal response by the catfish ACP. It was found that there was very little bactericidal activity against the fish pathogens that contained sialic acid but a very strong bactericidal response (100%) against the non-pathogens that lacked sialic acid (p = .0043). A relative lack of sialic acid or no sialic acid therefore correlated with a strong bactericidal response by the catfish ACP. Neuraminidase treatment of the bacterial fish pathogens to remove sialic acid greatly increased the bactericidal response against them by the catfish ACP when compared with untreated bacteria (p = .0431).     

Lipopolysaccharide phase variation determines the complement-mediated serum susceptibility of Coxiella burnetii

Phase variation of Coxiella burnetii is due to variation of the lipopolysaccharide (LPS), a phenomenon analogous to smooth-to-rough LPS variation of gram-negative enteric bacteria. Virulent enterobacteria usually have a smooth LPS and resist serum killing, whereas avirulent rough LPS mutants are sensitive to complement-mediated serum killing. Like gram-negative enterobacteria, smooth LPS phase variants of C. burnetii are virulent, whereas the rough LPS variants are avirulent. We therefore studied the effects of human serum on the LPS variants of the Nine Mile strain of C. burnetii. Analogous to gram-negative enterobacteria, the smooth and intermediate LPS C. burnetii phase variants were resistant to complement-mediated serum killing, whereas the rough LPS variants were killed by serum complement. Although the smooth and intermediate LPS variants were serum resistant, they differed in their interactions with the complement system. The smooth LPS variant activated complement poorly and did not bind C3b; however, the intermediate LPS variant activated complement and bound C3b. The rough LPS variant activated complement via the alternative pathway, whereas the intermediate LPS variant activated the classical pathway. These results provide an explanation for the avirulent nature of the rough LPS variant of C. burnetii and suggest that differences in C. burnetii LPS structure influence the interactions of the LPS phase variants with the complement system.     

Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes.     

Characterization of the susceptibility of Pseudomonas aeruginosa to complement-mediated killing: role of antibodies to the rough lipopolysaccharide on serum-sensitive strains

The mechanism of complement-mediated killing of seven serum-sensitive Pseudomonas aeruginosa strains was examined. All seven strains were sensitive to the bactericidal activity of 20% pooled normal human serum (PNHS) containing magnesium EGTA, which blocks the classical complement pathway (CCP), or 20% PNHS preheated to 50 degrees C for 20 min, which inactivates the alternative complement pathway, suggesting that either pathway was effective against these strains. However, for four of these strains, optimal killing required the function of both pathways. Preabsorption of PNHS with serum-sensitive strains dramatically reduced the killing activity of serum for the homologous strains when a concentration of 10% serum was used, implying a role for antibody in the activation of complement via the CCP. Affinity purification of antibodies to the rough lipopolysaccharide (LPS) on strain 144M resulted in a pool of antibodies which could restore all of the bactericidal activity and most of the C3 activation-deposition activity of serum which had been lost by preabsorption with 144M. Confirmation that the LPS was the target for these bactericidal antibodies was provided by demonstrating that exogenously added 144M LPS inhibited the killing activity of PNHS. These anti-144M LPS-specific antibodies were also bactericidal for the six other serum-sensitive strains examined, suggesting that all seven strains shared an antigenic determinant recognized by these anti-144M LPS-specific antibodies. Results from cross-absorption studies imply that there are bactericidal antibodies in PNHS directed to additional bacterial targets. These studies suggest that part of the bactericidal activity of PNHS is due to binding of antibodies to the rough LPS on serum-sensitive strains, initiating activation of the CCP, and that all seven strains examined shared this bactericidal antibody-binding site.     

A novel membrane glycoprotein capable of inhibiting membrane attack by homologous complement

Neuraminidase-treated human erythrocytes become sensitive to haemolysis by heterologous serum via activation of the alternative complement pathway (ACP), while remaining insensitive to homologous serum because of the presence of inhibitors on the cell membrane. We obtained a monoclonal antibody which renders the neuraminidase-treated erythrocytes sensitive to haemolysis by homologous human serum via the ACP. This antibody reacts with a 20 KDa membrane glycoprotein which interferes with the terminal stage of complement action on cell membranes. The 20 KDa protein is anchored to the membrane via phosphatidylinositol.