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.     

Membrane receptors of mouse leukocytes. I. Two types of complement receptors for different regions of C3

Mouse leukocytes were studied for membrane receptors for the third C component by rosette formation with C coated erythrocytes (EAC). Methods were devised for the preparation of EAC complexes containing either mouse C3b or mouse C3d. EAC 1-3dmo were prepared from EA treated with whole mouse serum while EAC 1-3bmo were produced from EAC 142hu treated with whole mouse serum containing sodium suramin. The specificity of the EAC complexes for mouse leukocytes was confirmed by inhibition experiments using fluid phase human C3d. Low concentrations of fluid phase human C3d inhibited EAC1-3dmo rosettes but failed to inhibit EAC 1-3bmo rosettes. Eight-fold higher concentrations of fluid phase C3d caused partial inhibition of EAC1-3bmo rosette formation with lymphocytes, but not with other types of murine leukocytes. Thus mouse leukocytes apparently contain the same two types of C receptors as do human and guinea pig leukocytes. Mouse CR1 is specific for a non-C3d region of C3b, (possibly analogous to human C3c) whereas mouse CR2 is specific for both C3d and the C3d region of C3b.     

traT gene sequences, serum resistance and pathogenicity-related factors in clinical isolates of Escherichia coli and other gram-negative bacteria

The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50-70%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.     

The computer analysis of volume distribution curves. Demonstration of two erythrocyte populations of different size in the young guinea pig and analysis of the mechanism of immune lysis of cells by antibody and complement.

Guinea pig, rat and sheep erythrocytes were sized electrically using the hydrodynamic focusing technique. The experimental curves were approximated with a computer by linear and logarithmic normal distributions. Rat and guinea pig erythrocytes from adult animals were best approximated by one linear normal distribution. Two populations (I, II) of erythrocytes with different mean volume could be demonstrated in young guinea pigs by this analysis. Population I erythrocytes are small, have a lower electrophoretic mobility and are mainly present at birth. They are gradually replaced by the larger population II erythrocytes. Both types of erythrocytes are probably the result of separate differentiation pathways. The analysis of erythrocyte volume distribution curves during immune lysis by antibody and complement shows that intact and ghost erythrocytes are measured by electrical sizing. No volume changes were observed up to the EAC1-8 intermediate. After the addition of C9, a C9 dose-dependent part of the erythrocytes swell permanently to spheroids. The spheroid transformation is a temperature-dependent, all or nothing reaction which is independent of protein osmotic forces from the interior of the cell.     

The outer membrane permeability mutation of the virulence-associated plasmid of Salmonella typhimurium is located in a traT-like gene

Abstract The SS-A mutation carried by the virulence-as-associated plasmid of Salmonella typhimurium results in increased outer membrane permeability to hydrophobic compounds. A 7.8-kilobase pair Bam HI- Sal I fragment containing the SS-A mutation was cloned from the virulence-associated plasmid into the cloning vector pACYC184. The cloned DNA segment hybridized with a radioactive probed prepared from the traT gene of R6-5. A similar DNA fragment, cloned from the wild-type virulence-associated plasmid, complemented the SS-A mutant phenotype. Both clones produced a protein that immunologically resembled the R6-5 TraT protein; however, the protein produced by the SS-A containing clone appeared truncated by approximately M _r 1000 indicating an alteration in the primary structure or processing of the protein. We conclude that the mutation producing the SS-A phenotype has occured in a traT -like gene of the Salmonella plasmid.     

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid

The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.     

Activation of therminal components of human complement by a trypsin-activated complex of human factor B and cobra venom factor.

Cleavage of C3 by CVF-B was demonstrated by hemolytic, immunoelectrophoretic and immune adherence reactions. No cleavage of C5 was detected by immunoelectrophoresis, but C5 hemolytic activity, assayed with EAC1423, decreased although less than C3 hemolytic activity. The co-existence of C3 with limiting amounts of C5 did not reduce the final degree of hemolysis of guinea pig erythrocytes (GPE) induced by late-acting components C6 through C9 and CVF-B. Thus, a CVF-B hemolytic system composed of GPE, C5 through C9 and CVF-B provided a method for titration of terminal components of human complement. CVF-B was able to generate hemolytically active sites of C567 on GPE by activation of C5, C6 and C7. The complex C567 in the fluid-phase decayed within 1 min but C567 on GPE was quite stable. Originally insensitive sheep erythrocytes became sensitive to the CVF-B hemolytic system if C3b sites were present, suggesting that cell-bound C3b played a role in orienting the positions of C567 to be fixed. CVF-B could be recovered quantitatively from the supernatant of the reaction mixture in which the hemolytically active intermediate GPEC-5678 had been formed through the interaction between C5 to C8 and CVF-B.     

Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

Ixodes dammini: salivary anti-complement activity

Saliva of the tick Ixodes dammini prevents hemolysis of rabbit erythrocytes by the human alternative pathway of complement. Deposition of C3b to activating surfaces and concomitant C3a release are inhibited. C3b deposition to activating surfaces is inhibited regardless the origin (humans, rat, mouse, guinea pig, and hamster) of the serum. The inhibitor elutes as a single peak upon gel filtration, with an apparent molecular weight of 49,000. Salivary anti-complement may contribute to successful feeding of I. dammini in their natural hosts.     

TraT lipoprotein, a plasmid-specified mediator of interactions between gram-negative bacteria and their environment.

The TraT protein is a cell-surface-exposed, outer membrane lipoprotein specified by large, usually conjugative, F-like plasmids. Two biological activities have been associated with the protein: (i) prevention of self-mating of cells carrying identical or closely related conjugative plasmids, by blocking the formation of stable mating aggregates; and (ii) resistance to the bactericidal activities of serum, possibly by inhibiting the correct assembly or efficient functioning of the terminal membrane attack complex of complement. The protein therefore interacts not only with components of the outer membrane but also with specific external agents. In conjugative plasmids the traT gene lies within the region necessary for the conjugal transfer of DNA (tra), although its expression is not necessarily dependent on the expression of other tra genes. Recently, however, the gene has been discovered in isolation from other tra genes in nonconjugative virulence-associated plasmids, providing further evidence that the TraT protein may have a role in pathogenesis. The nucleotide sequences of several traT genes have been determined, and comparison of the corresponding amino acid sequences suggests that a central region of five amino acid residues flanked by hydrophobic domains determines the specificity of the protein in surface exclusion. Additionally, studies of mutants with different amino acid alterations within the hydrophobic domains have shown that insertion of charged residues disrupts normal outer membrane integrity. This review considers our current knowledge of the distribution, structure, and biological role(s) of the protein. Recent applications of the protein in studies of the unusual permeability properties of the outer membrane and for the transport of foreign antigenic determinants to the bacterial cell surface are also discussed.     

Evidence for a C4b binding site on the C2b domain of C2

We raised murine mAb against human C protein C2. The representative mAb 3A3.3 (IgG1 kappa) recognized an epitope on the C2b domain of C2, as determined by binding and inhibition of binding radioassays. The hemolytic activity of purified human C2 and of C2 in normal human serum was inhibited by the mAb. The rate of decay of the C3-convertase at 30 degrees C was not affected by the mAb. C2 binding to EAC4b was inhibited by intact IgG and the Fab fragment of the mAb; 50% inhibition required 1 microgram/ml of either. The data suggest the presence of a C4b-binding site on the C2b domain of C2 and that the mAb recognizes an epitope at, or adjacent to, this site. The C2b portion of the C2 molecule may be important in assembly of the classical pathway C3-convertase.     

Activation of the fifth and sixth component of the complement system: similarities between C5b6 and C(56)a with respect to lytic enhancement by cell-bound C3b or A2C, and species preferences of target cell

Brief shift of purified C5 and C6 at 0 degrees C to pH 6.4, followed by immediate neutralization, results in the generation of a factor, designated C(56)a, that lyses erythrocytes together with C7, C8, and C9. We compared C(56)a and C5b6 generated by an alternative-pathway convertase, with regard to their action on different target cells. We found tht C(56)a is similar to C5b6 in the following properties: 1) Together with C7, C(56)a forms a stable intermediate on either sheep or guinea pig erythrocytes. 2) Membrane-bound C3b, or A2C incorporated in the membrane, enhances lysis by C(56)a-9, as well as lysis by C5b6-9. We also found that the lysis of EC(56)a7 or EC5b67 intermediates by C8 and C9 depends on the species of the erythrocytes and the species of C8 and C9. Thus, lysis of sheep erythrocytes is more efficient with guinea pig C8 and C9 than with human C8 and C9. In the case of guinea pig erythrocytes, this relationship is reversed, i.e., these cells lyse more efficiently when human C8 and C9 are used. Enhancement of lysis by membrane-bound C3b or A2C does not abrogate this species incompatibility pattern.     

Lipopolysaccharide alteration is associated with induced resistance of Neisseria gonorrhoeae to killing by human serum

On SDS-PAGE, solubilized and proteinase K treated preparations of Neisseria gonorrhoeae strain BS4 (agar) showed differences in silver stained lipopolysaccharide (LPS) patterns, before and after induction to resistance to serum killing by incubation for 3 h at 37 degrees C with low Mr fractions from lysates of guinea pig red blood cells. Preparations from the original serum susceptible gonococci and LPS purified from such bacteria showed two components, but the preparations from the serum resistant gonococci were deficient in the higher Mr component. Furthermore, on immunoblotting with fresh human serum (FHS), the two LPS components of the susceptible gonococci reacted strongly with IgM. With preparations from the serum resistant gonococci there was no reaction in the area corresponding to the higher Mr component and a weaker reaction with the component of low Mr. Purified LPS from the susceptible gonococci neutralized the bactericidal activity of FHS against N. gonorrhoeae strain BS4 (agar) probably by reacting with the relevant antibody, since heated FHS was no longer bactericidal when mixed with a source of complement (human placental serum) after prior reaction with the LPS. These neutralization tests coupled with the results of immunoblotting strongly suggest that increased serum resistance is due to the lack of the high Mr LPS moiety.     

5''-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5''-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups.     

Human erythrocytes inhibit complement-mediated solubilization of immune complexes

Incubation of precipitable immune complexes (IC) with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed human E were added to human serum or guinea pig serum, binding of IC to the E occurred and IC solubilization was significantly inhibited. By contrast, SRBC did not bind IC nor inhibit IC solubilization. Because IC binding to human E is mediated by CR type 1 (CR1) we evaluated whether CR1 was responsible for the inhibition of IC solubilization. Human E were treated with trypsin or anti-CR1 mAb. Both treatments abrogated IC binding to human E but did not affect the ability of the human E to inhibit IC solubilization. Human E inhibited C activation by IC. Thus, incubation of IC in human serum caused significant activation of C3 and C5, but not C4. However, when IC were incubated in whole blood or with isolated human E and serum, C3 activation by IC was inhibited significantly. In addition, we demonstrated that the C3b generated during C activation by IC deposited on both IC and human E. Thus, human E may compete for nascent C3 generated during C activation by IC. In conclusion, human E inhibit both complement-mediated solubilization of IC and C activation by IC.     

CRRP: a guinea pig protein, identified by sequence homology to human CR1, which contains two short consensus repeat motifs and appears not to be transmembrane or secreted.

cDNA from the C4b-binding site of the human C3b/C4b receptor (CR1) was used to find homologous sequences in the guinea pig. This cDNA identified an 18S mRNA species in guinea pig spleen, but not liver. Probing of a guinea pig spleen cDNA library identified clones with identical 1.5-kb inserts, which also hybridized to mRNA in spleen, but not liver. Sequence analysis of the insert revealed a single long open-reading frame coding for a 20,000 Mr protein consisting of two short consensus repeat motifs homologous to human CR1, and unique sequence at the amino- and carboxy-terminals of the short consensus repeats. This sequence did not encode peptides with features of transmembrane domains or signal peptides. Antibody to this complement receptor-related protein-beta galactosidase fusion protein recognized a 20,000 Mr protein in SDS lysates of guinea pig spleen, lymph node, lymphocytes, neutrophils, and peritoneal macrophages. Immunoprecipitation of human serum by this antibody revealed an 180,000 Mr protein reacting both with the anti-guinea pig protein antibody and with anti-human CR1 antibody. Immunoprecipitation of guinea pig serum revealed no protein reacting with the anti-guinea pig protein antibody. Tissue staining of cultured peritoneal macrophages with this antibody showed intracellular staining, as opposed to membrane staining obtained with anti-guinea pig Ig antibody. The lack of membrane expression was confirmed by surface protein radiolabeling experiments and by fluorescent staining of surface proteins. Thus, we have identified a guinea pig protein with homology to human CR1, which may have an unusual property for this class of proteins in that it appears to be intracellular.     

Detection of human B cells and chronic myelocytic leukemic cells by rosette formation with sheep erythrocytes and fresh human sera.

A new method of detecting the C3b receptor is reported. A particular merit of this method is that anti-RBC rabbit antiserum is not required. Rosettes were formed with human B lymphocytes, B lymphoblasts and granulocytes, using sheep erythrocytes (SRBC) sensitized with fresh human serum (FHS). T lymphocytes and T lymphoblasts did not form rosettes. The percentage of cells forming rosettes with this method approximated the percentage of rosettes formed with EACm. However, FHS coated SRBC did not react with most cells of B cell type chronic lymphocytic leukemia (CLL), whereas EACm rosette formations showed a definite reaction. On the other hand, 34--58% of cells of chronic myelocytic leukemia (CML) bound with the indicator red cells. SRBC sensitized with fresh rabbit or guinea pig serum formed rosettes with PBL, tonsil cells, B lymphoblasts and granulocytes. Complement and IgM antibody were required for this reaction, as in EAC rosette formation.     

Complement inhibitor(s) released by leukocytes. III. Evidence for a "new" C1 inhibitor in the supernatants of short-term cultures of mouse spleen and thymus cells.

Mouse spleen or thymus cells in short-term culture release a factor, designated S, that binds to sheep erythrocytes (E). Supernatant-treated sheep erythrocytes (SE) are capable of fixing and transferring the activated first component of guinea pig complement. SEC1, however is not capable of initiating hemolysis by the rest of the complement components. SE is capable of binding but not activating native C1; native C1 bound to SE seems irreversibly inhibited. Evidence is presented that S may be the same factor as the previously described inhibitor released by mouse spleen or thymus cells that inhibits the utilization of C2 by EAC14.     

An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade.

A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.     

Studies on the interaction between protein A and immunoglobulin G. I. Effect of protein A on the functional activity of IgG.

Staphylococcal protein (A (PA) and IgG anti-Forssman immunoglobulin formed complexes that behaved functionally like IgM in their ability to lyse sheep erythrocytes (E) in the presence of whole guinea pig complement (GPC) and to fix purified guinea pig C1. Concanavalin A, a plant lectin that inhibited IgM but not IgG hemolytic activity, inhibited the hemolytic activity of IgG-protein A complexes that behaved like IgM but had no effect on complexes that behaved functionally like IgG. Since Con A is known to bind specifically to glucose and mannose residues, our results suggested that the interaction of protein A with the Fc region of IgG led to exposure of sugar moieties that may participate in complement (C) binding. The production of IgM-like complexes depended on the ratio of protein A to IgG and the empirical formula of these IgM-like complexes was found to be [(IgG)2PA]n. As the ratio of PA to IgG was increased, the resulting complexes tended to behave functionally like IgG but with reduced hemolytic activity and C1 fixing ability. Furthermore, the binding of C1 to EIgG was inhibited by PA and the binding of PA to EIgG was inhibited by C1 indicating that the binding sites for C1 and PA were located near each other or were identical. Our results offer a reasonable explanation for the reported effects of PA or mixtures of PA and IgG in vitro and in vivo.