Isolation and characterization of the eighth component of the bovine complement system.

OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.

Characterization of hemolytic and cytotoxic Gallysins: a relationship with arylphorins.

Gallysin-1, an inducible effector protein in the protective response of Galleria mellonella larvae is a 75 kDa component of hemolytically active material (HAM) isolated from immune cell-free hemolymph. The sequence of the first 20 N-terminal amino acids of the antibacterial protein Gallysin-1 is identical to the predicted sequence of the first 20 amino acids of the Galleria arylphorin Lhp76 (larval hemolymph protein 76). A murine monoclonal antibody to the 20 amino acid N-terminal peptide of Gallysin-1 (GYPQYHYDVETRKLDPSLVN) provides additional evidence for a link between Gallysin-1 and Lhp76, and is used to characterize HAM further. HAM, initially characterized as a mixture of two proteins, Gallysin-1 and a 69 kDa component is now identified as a 450-500 kDa heteromultimer, designated Gallysin. In vivo levels of Gallysin rise during the effector phase of an induced immune response. The monoclonal antibody inhibits the hemolytic activity of Gallysin. In addition to a hemolytic activity for mammalian erythrocytes, Gallysin possesses a cytotoxic activity for the human tumor cell line, K562. Lipopolysaccharides (LPS) and a Pseudomonas aeruginosa vaccine induce a cytotoxic activity which reaches its maximum levels in the hemolymph early (2 hours post-vaccination) in the protective response. The partially purified cytotoxic material (Cyt-M) obtained from cell-free hemolymph collected 2 hours after vaccination has hemolytic activity and shows structural similarities to Gallysin and Lhp76. The previously established role of Gallysin-1 as an effector protein in the protective response of Galleria mellonella indicates that arylphorins may play a role in insect immune responses.

Gallysin-1, an antibacterial protein isolated from hemolymph of Galleria mellonella.

An inducible hemolysin with antibacterial properties was isolated from the hemolymph of immune Galleria mellonella larvae. The Galleria-derived lysin, named Gallysin-1, was shown to have an apparent molecular weight of 75,000 and to be relatively heat stable at 56 degrees C. Although Gallysin alone was not bactericidal it caused sufficient damage of the outer cell membranes of Pseudomonas aeruginosa RP4 and Escherichia coli K176 to release beta-lactamase from the periplasm. In the presence of either purified Galleria lysozyme or egg white lysozyme Gallysin-1 had potent antibacterial activity against gram-negative bacteria. Gallysin-1 killed osmotically shocked P. aeruginosa and E. coli that suggests that it can also attack exposed inner cell membranes of gram-negative bacteria. The identification of Gallysin-1 recognizes another distinct member of the bactericidins involved in insect immunity.

Toxicity of complement for chondrocytes. A possible source of cartilage degradation in inflammatory arthritis.

Cartilage from patients with rheumatoid arthritis and from animals with antigen-induced arthritis is frequently contaminated with complement-containing immune complexes. A possible role for complement activation in cartilage degradation was modeled in vitro by exposing cultured bovine chondrocytes to homologous serum, and determining cytotoxicity by monitoring the release of intracellular 51Cr. Complement activation was found to be cytotoxic, having maximal effect at 20-30% serum by 18 h. Serum toxicity was ablated by heat (50 degrees C, 20 min) or methylamine treatment but not by EGTA, suggesting that in these experiments activation occurred by the alternate route. The implications of the results are discussed in relation to ultrastructural evidence for the involvement of complement in the pathogenesis of cartilage degradation in inflammatory arthritis.

Isolation of the fifth component of the bovine complement system.

Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.

Genetic polymorphism of complement factor H in cattle.

Bovine factor H was found to be polymorphic by the combined techniques of SDS-polyacrylamide electrophoresis of bovine plasma and immunoblotting. Three phenotypes (S, SF, F) were identified in a sample population of 149 cattle. Variant S and F differed by an apparent molecular weight of 5000 daltons. Family studies demonstrated Mendelian segregation of variants S and F. The data indicate that these genetic variants of bovine factor H are encoded by two codominant alleles at a single autosomal locus.

Cane sugar factor as an inducing agent of immunity in Galleria mellonella.

The injection of cane sugar factor (CSF) into Galleria mellonella larvae results in an immune response similar to that produced by a formalized Pseudomonas aeruginosa vaccine. Vaccination with CSF is followed by: an increase in the LD50 of Pseudomonas aeruginosa; an in vivo protective response to P. aeruginosa the development of which can be inhibited by cobra venom factor (CVF); an antibacterial activity in hemolymph 24 h after the injection of CSF; the development of a transferrable immune response in hemolymph of donor larvae capable of protecting recipient larvae against a lethal challenge of Pseudomonas aeruginosa; an increase in extracellular lysozyme equal to that induced by Pseudomonas vaccine; a reduction in total hemocyte count during the period of protective immunity; and the presence in hemolymph of new basic proteins, with electrophoretic mobilities and appearance times after the CSF injection, identical to those induced by the formalized vaccine. CSF was shown to be composed primarily of glucose.

The hemolytic activity of Galleria mellonella hemolymph.

A hemolytic activity was identified in the hemolymph of normal and immune Galleria mellonella larvae. The hemolysin was active against sheep, human, guinea pig, and rabbit erythrocytes. Hemolysis occurred in the presence of 0.04M EDTA. Vaccination of the larvae with formalized Pseudomonas aeruginosa increased the hemolytic activity. The increase, and subsequent decline of this activity paralleled the pattern of induced in vivo antibacterial activity that is characteristic of the insect's immune response. The hemolytic activity was distinct from induced phospholipase A-like and phospholipase C-like activities that were detected in immune hemolymph and which were inhibited by EDTA. The hemolytically active material (HAM) was partially purified (apparent molecular weight range 69,000 to 75,000) and was found not to be antibacterial for P. aeruginosa. The physiological role of the HAM is as yet unknown. It is possible that it may act together with other hemolymph components to produce an immune state.

A simple isolation procedure for functionally pure components of the bovine alternative complement pathway (ACP) C3 convertase and bovine conglutinin (K).

A simple multicomponent isolation procedure for bovine C3, factor B, factor D and conglutinin (K) from a single serum sample is described. The components of the alternative pathway C3 convertase were isolated in milligram quantities from 800 ml bovine serum and were found to be functionally pure with respect to each other and to factors H and I.

A simple hemolytic assay for bovine complement component C3.

A simple, one-step, alternative pathway (AP) hemolytic assay for bovine C3 has been developed. Methylamine was used to prepare a bovine serum reagent, R3, functionally depleted of C3. The addition of purified bovine C3 to the R3 reconstituted, in a dose-dependent manner, the hemolytic activity for unsensitized heterologous erythrocytes. The assay was used to determine relative levels of C3 in different bovine serum samples. Human C3 and bovine C3 were interchangeable in the assay. Reconstitution of bovine and human R3 reagents with homologous or heterologous C3, in the presence of different species of erythrocytes, provided evidence that cell surface regulation of the homologous hemolytic AP may not be limited to the assembly and activity of the C3 convertase. The AP assay was more sensitive and less complex to perform than a standard classical pathway assay for bovine C3.

Isolation of bovine complement factor H.

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.

A cobra venom factor (CVF)-induced C3 convertase activity in the hemolymph of Galleria mellonella.

A cobra venom factor (CVF)-induced C3 convertase has been generated from the hemolymph of Galleria mellonella. CVF was immobilized on Sepharose 4B and treated with cell-free hemolymph obtained from either unvaccinated G. mellonella larvae or larvae immunized with formalized Pseudomonas aeruginosa. The C3-cleaving activity was detected by the ability to cleave the alpha-chain of bovine C3 in a manner analogous to the CVF-induced mammalian C3 convertase, CVF,Bb. The insect-derived C3 convertase formed at 28 degrees C but not at 37 degrees C, then once formed was active at both 28 degrees C and 37 degrees C. EDTA did not inhibit the formation and action of the insect derived C3 cleaving activity.

Isolation and characterization of the third component of bovine complement.

C3 was obtained from bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephadex A-50, CM-Sephadex A-50 and Sephacryl S-200. The protein has a molecular weight of 183,000 (alpha-chain 114,000 and beta-chain 69,000). A CVF-induced bovine C3 convertase (Sepharose-CVF.Bb) cleaved C3 into C3a (11,000) and C3b (172,000) as shown by SDS-polyacrylamide gel electrophoresis. Isoelectricfocusing of C3 demonstrated at least three electrophoretic variants with pI 6.55-6.85. The isolated protein promoted the formation and action of a C3 convertase in the presence of purified bovine factors B and D. A monospecific antiserum prepared in rabbits failed to cross react with human C3 or CVF. C3c was identified as a contaminant during the isolation of C3.

Factor D of the alternative pathway of bovine complement: isolation and characterization.

Factor D of the bovine alternative complement pathway has been purified by chromatography on CM-Sephadex C-50, Sephacryl S-200 and hydroxylapatite. The isolated factor D (0.25 mg from 1 litre of bovine serum) had an apparent molecular weight of 27,500 and a pI of 7.2. In whole bovine serum the pI of factor D was also 7.2. The isolated protein caused the Mg++-dependent cleavage of bovine factor B in the presence of cobra venom factor (CVF) to generate a haemolytically active C3-convertase as shown by SDS-polyacrylamide gel electrophoresis and haemolytic diffusion plate assays. Bovine factor D and human factor D were interchangeable in restoring the alternative pathway haemolytic activities of both bovine RD and human RD (factor D deficient sera). The haemolytic activity of bovine serum factor D was completely inhibited by 20 mM diisopropylfluorophosphate (DFP) but only 25% inhibited by 1 mM DFP. Serum heated at 56 degrees C for 10 min completely lost factor D activity but purified factor D was relatively more heat stable.

Alternative pathway of bovine complement: concentration of factor B, hemolytic activity and heritability.

Concentrations of bovine factor B (Bbov) were determined by radial immunodiffusion in sera of 46 Holstein cows and heifers aged one to nine years. Mean values were 34.2 +/- 5.3 mg/100 ml. A hemolytic diffusion plate assay in agarose gel in presence of 10 mM EGTA and 5 mM Mg accurately measured concentrations of purified Bbov but gave higher mean values, i.e. 47.8 +/- 10.2 mg/100 ml, for concentrations of Bbov in whole sera. Hemolytic values obtained by the hemolytic diffusion plate assay, however, weakly correlated (r = .4539, p less than 0.01) with the serum concentration of Bbov measured by radial immunodiffusion. It was concluded that the hemolytic diffusion plate assay was not an accurate technique for the quantitative measurement of Bbov but a good assay for quantitation of the total hemolytic activity mediated via activation of the alternative complement pathway. It is suggested that the difference between the values obtained by the two tests for one particular serum is, to some degree, an expression of the ratio of amplification and restriction of the alternative pathway activity. No significant heritability (offspring and one parent) was detected for the hemolytic activity of serum. A heritability of 0.93 at a significance level of p less than 0.1 was determined for the serum concentration of Bbov.

The in vitro generation of an antibacterial activity from the fat body and hemolymph of non-immunized larvae of Galleria mellonella.

A state of immunity in Galleria mellonella against the pathogen Pseudomonas aeruginosa is known to be induced by the injection of lipopolysaccharide (LPS), isolated from the homologous organism. An in vitro mixture of the LPS and whole or cell-free hemolymph from non-immunized larvae is not antibacterial. In vitro mixtures of fat body and cell-free hemolymph from non-immunized larvae, incubated at 25 degrees C for 20 hours generated a proteinaceous antibacterial activity. The generation of this activity was enhanced by the presence in the incubation mixture of LPS and/or hemocytes from non-immunized larvae. It is suggested that LPS causes the release of a hemocyte factor(s) which acts in conjunction with or directly on the fat body resulting in an enhanced production of antibacterial factors.

The specific in vitro antibody-mediated retention of bovine serum albumin by porcine hyaline articular cartilage.

Porcine hyaline articular cartilage (HAC) has been used to investigate the interaction of bovine serum albumin (BSA) and anti-BSA with articular collagenous tissues in vitro. There was a marked retention of 125I-labelled BSA by plugs of HAC (a) exposed to rabbit anti-BSA for 1-2 h at 37 degrees C prior to a similar incubation with the antigen, or (b) exposed to the antigen and then to antibody. The specifically retained radiolabelled BSA was localized in or at the articular surface of the plugs. In the absence of specific antibody a relatively small amount of the antigen was retained. Exposure of HAC to multiple cycles of antibody and antigen treatment resulted in an increased retention of the 125I-BSA. There was a concomitant increase in the retention of the anti-BSA and the capacity of the treated plugs to fix complement. The forces that maintained the labelled antigen in the tissue were not readily reversed by excess unlabelled BSA. Pre-formed, soluble BSA/anti-BSA complexes did not appear to penetrate the tissue unless the HAC was first exposed to anti-BSA. The results suggest that the antibody-mediated, surface oriented retention of 125I-BSA results from the formation of immune complexes in the tissue.

Transfer of immunity against Pseudomonas aeruginosa P11-1 in Galleria mellonella larvae.

Larvae of Galleria mellonella may be immunized against Pseudomonas aeruginosa by the transfer of whole hemolymph, cell-free hemolymph or hemocytes from insects previously immunized with the lipopolysaccharide of the homologous organisms. Whole immune hemolymph injected as early as 3 hours after vaccination confers protection which persists as long as 40 hours. Hemocytes alone confer good protection when transferred as early as 30 minutes after, and up to about 4 hours after immunization. Transferred protection does not appear to be due to excess immunogen in the hemolymph.

The effect of decomplementation on the infectious course of Sendai virus in mice.

The course of intranasal infection of Sendai virus in CBA and DBA mice was investigated in animals decomplemented with purified cobra venom factor. The mice were decomplemented either immediately before inoculation or at 4 days postinfection. Depletion of complement after the infection had been established had no apparent effect on the course of the viral infection in the two strains of mice. In contrast, both strains of mice were protected completely from the lethal effects of an infectious dose of 1 LD50 of virus when the serum C3 levels were depressed by more than 80% during the early stages of infection. The symptoms of morbidity were less pronounced in these animals and there was a delay in the production of hemagglutination-inhibiting antibody. There was no apparent effect on the growth of the virus in lung tissue. The results suggest that the complement system plays a significant pathogenic role during the course of Sendai virus infections in CBA and DBA mice.