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PubMed1977

Intra-individual variation in the concentrations of IgA, IgG, IgM, and complement component C3 in serum of a normal adult population.

A recent study [Clin. Chem. 22, 1635 (1976)] reported intra-individual variation in 10 serum proteins to be much smaller than interindividual variation. We report results of a similar study involving about 700 apparently healthy adults in whom we estimated the relative magnitude of the intra- and interindividual variation in serum IgA, IgG, IgM and complement component C3. Specimens were collected from each subject weekly for as long as 10 weeks (average, four weeks). The four serum proteins were quantitated by radial immunodiffusion by the maximal-diffusion technique. Traditional 95% reference intervals were computed relative to WHO reference preparations for the immunoglobulins. For C3, the reference interval was computed relative to a commercial reference preparation. We, too, found the ratios of intra-individual to interindividual variation for adults to be so small that the traditional reference intervals do not have the assumed diagnostic sensitivities. Furthermore, these ratios did not change after dividing the study population into subgroups according to sex and age; evidently such subgrouping do not improve the diagnostic sensitivity. The relatively small intra-individual variations were also observed at the extremes of protein concentration ranges.

Adolescent↗

PubMed1984

The specific detection of IgG, IgA and the complement components C3 and C4 in circulating immune complexes.

Four immune complex assays (PEG assays) are described; they are based on the binding of radio-labelled immunospecific antibodies to immune complexes containing IgG, IgA, C3 or C4, and subsequent precipitation with polyethylene glycol. Comparison of the results of the IgA-PEG assay with those of an existing immune complex assay (alpha-IgA-InhBA) showed that the former detects only large-sized (greater than 25 S) material. This was also demonstrated by sucrose density gradient ultracentrifugation of immune complexes in patients' sera as well as in preparations of aggregates containing IgA, C3 or C4. By using aggregates of mixed composition (IgG, IgA, C3 and C4) it was shown that each constituent could be detected by one of the 4 assays. Immune complexes containing the various constituents could also be detected in sera of patients with a variety of disorders. Further studies are needed to establish whether these constituents occur within the same complex or within different complexes.

Animals↗

PubMed1990

Hormonal regulation of complement biosynthesis in human cell lines--II. Upregulation of the biosynthesis of complement components C3, factor B and C1 inhibitor by interleukin-6 and interleukin-1 in human hepatoma cell line.

The effect of interleukin (IL)-6 and IL-1 on the biosynthesis of complement components C3, factor B, C2, C4 and C1 inhibitor (C1 inh), as well as that of albumin, was studied in vitro in human hepatoma-derived cell line, HepG2. Measuring the amounts of secreted complement proteins we detected a significant upregulation of C3 by both hormones. The enhancement of the factor B and especially that of C1 inh production was predominant by IL-6. In our experimental system neither IL-1 nor IL-6 affected the biosynthesis of C2 and C4. Albumin secretion was significantly decreased only in the simultaneous presence of IL-1 and IL-6. Detection of the changes in the amounts of C3- and factor B-specific mRNA of HepG2 cells suggests a pretranslational regulation by these cytokines. The secretion of C3 and factor B was markedly potentiated when IL-1 and IL-6 were added together. However only the gene expression of factor B, but not of C3, was found to reveal synergism. IL-6 enhanced the in vitro production of C3 in mouse hepatocytes as well. This effect was greatly potentiated in the presence of histamine.

Carcinoma, Hepatocellular↗

PubMed1990

Complement component C3 fixes selectively to the major outer membrane protein (MOMP) of Legionella pneumophila and mediates phagocytosis of liposome-MOMP complexes by human monocytes.

Legionella pneumophila is a facultative intracellular bacterial pathogen that parasitizes human monocytes and alveolar macrophages. Previous studies from this laboratory have shown that monocyte complement receptors CR1 and CR3 and complement component C3 in serum mediate L. pneumophila phagocytosis. In this study, we have explored C3 fixation to L. pneumophila. We developed a whole-cell enzyme-linked immunosorbent assay (ELISA) to measure C3 fixation to the bacterial surface. By this assay, C3 fixes to L. pneumophila that are opsonized in fresh nonimmune serum, and C3 fixation takes place via the alternative pathway of complement activation. Immunoblot analysis of opsonized L. pneumophila indicated that C3 fixes selectively to specific acceptor molecules of L. pneumophila. Consistent with this, when nitrocellulose blots of whole L. pneumophila or bacterial components are incubated in fresh nonimmune serum, C3 fixes exclusively to the major outer membrane protein (MOMP) of L. pneumophila, a porin; C3 does not fix to L. pneumophila LPS on these blots. To further explore the role of MOMP in C3 fixation and phagocytosis, we reconstituted purified MOMP into liposomes. By the ELISA, MOMP-liposomes, but not plain liposomes lacking MOMP, avidly fix C3. Consistent with a dominant role for MOMP in C3 fixation, MOMP-liposomes form a C3 complex of the same apparent molecular weight as whole L. pneumophila in nonimmune serum. Opsonized radioiodinated MOMP-liposomes avidly adhere to monocytes, and adherence is dose dependent upon serum. By electron microscopy, opsonized MOMP-liposomes are efficiently phagocytized by human monocytes, and phagocytosis takes place by a conventional appearing form of phagocytosis. This study demonstrates that C3 fixes selectively to the MOMP of L. pneumophila, and that, in the presence of nonimmune serum, MOMP can mediate phagocytosis of liposomes and, potentially, phagocytosis of intact L. pneumophila by human monocytes.

Animals↗

PubMed1992

Structural requirements for thioester bond formation in human complement component C3. Reassessment of the role of thioester bond integrity on the conformation of C3.

A unique thioester bond, formed between the side chains of neighboring C and Q residues, is present in complement components C3 and C4 and the protease inhibitor alpha 2-macroglobulin. This structure is essential for mediating covalent attachment to target acceptors and also for maintaining these proteins in their native conformation. An examination of the residues in the immediate vicinity of the C and Q reveals a very high degree of sequence similarity among the three proteins which crosses species barriers. The following is the sequence flanking the thioester residues in C3, the highly conserved amino acids being underlined and the the thioester-forming residues being indicated by italics: 1005V-T-P-S-G-C-G-E-Q-N-M-I-G-M-T-P-T1021. Through a site-directed mutagenesis and cDNA expression approach, we have examined the importance of the conserved amino acids in the formation, stability, and function of the thioester bond in C3. The behavior of the mutants fell into three categories. The potential loss in peptide backbone flexibility by the replacement of G1009 by A or S was permissive to thioester formation and function as was replacement of M1015 by the still fairly bulky residue F. In contrast, replacement of M1015 by A resulted in an alpha-chain which was highly unstable toward proteolytic degradation. The third category, which included mutant molecules P1007G, P1020G, E1012Q, and Q1013N, displayed an unusual phenotype in which both the autolytic fragmentation and the hemolytic activity characteristics of thioester-intact molecules were absent. However, like their wildtype counterpart, these molecules retained the ability to be cleaved by C3 convertase (C4b2a), a conformation-dependent property that is normally lost in the conversion of native C3 to thioester-hydrolyzed C3(H2O). Since an identical functional profile was obtained when the thioester was deliberately prevented from forming in the mutant C1010A, we conclude that if a stable thioester fails to form during biosynthesis, at least parts of the mature protein can adopt a more native-like conformation than is the case when the thioester is first formed and then hydrolyzed in the mature protein. In view of these new findings, the interpretation of the previously observed correlation between the loss of thioester integrity and the adoption of a C3b-like conformation must be reassessed.

Amino Acid Sequence↗

PubMed1991

Phagocytic function of neutrophils during dialysis in relation to some immunological findings.

Haemodialysis neutropenia and impaired granulocyte function are transitory, but the consequences of altered granulocyte function are observed at the end of haemodialysis. Activity of polymorphonuclear receptor for Fc and C3 complement component, circulating immune complexes and components of complement (C1 inactivator, C4, C3, C3 proactivator) were measured in ten patients before and at the end of haemodialysis. Significant decrease in polymorphonuclear Fc receptor activity (1689 cells/mm3 before and 1277 cells/mm3 after HD) and increase of circulating immune complexes (69.9 micrograms/dl before and 112.7 micrograms/dl after HD) were observed at the end of haemodialysis. A decrease in complement C3 component was observed after haemodialysis (866 mg/l before and 804 mg/l after HD); the other components: C1 inactivator, C4 component, and C3 proactivator, remained unchanged. Increase of circulating immune complexes and decrease of Fc receptor activity correlated with a decrease in phagocytic function of polymorphonuclear leukocytes.

Adult↗

PubMed1980

The nature of secretory agglutinins and aggregating factors. III. Secretory conglutinin-like factor SKF detects a cross-reaction between bacteria and complement component C3.

Secretory conglutinin-like factor (SKF) reacts directly with an unknown surface component of some, but not all, oral gram-positive organisms. The absorption of SKF by bacteria is EDTA-sensitive and cannot be blocked with immunoglobulins. High levels of SKF in EDTA extracts of washed salivary sediment reveal the direct in vivo reaction of SKF with oral bacteria. Mixed aggregation with alexinated erythrocytes showed the SKF corresponds to the secretory bacterial aggregating factor (SBAF) for Streptococcus mutans serotype c and also that for Streptococcus mitis. These reactions represent a cross-reaction between bacteria and complement component C3. SKF/SBAF non-mucin glycoproteins and immunoglobulins possess receptors for bacterial components while mucins are passive carriers of blood group determinants.

Agglutinins↗

PubMed1984

Secretion rates of immunoglobulins, albumin, haptoglobin and complement factors C3 and C4 in the perfused jejunum and ileum of human Salmonella carriers.

Secretion rates of immunoglobulins and other proteins were assessed by luminal perfusion of jejunum and distal ileum, and the jejunal histology was evaluated in eight Danish chronic Salmonella typhi and paratyphi carriers compared to nine healthy controls who previously had suffered from typhoid or paratyphoid fever not followed by a carrier state. The median secretion rates for each protein investigated in the distal ileum as well as in the jejunum revealed no significant differences between the two groups. The secretion rate of secretory IgA was raised in both groups compared to previously investigated normal persons. The histological examination revealed no signs of inflammation or presence of bacteria. It was concluded that no primary humoral immune defect was revealed in the carriers.

Adult↗

PubMed1989

Large intramolecular movement in human complement protein C3 induced by methylamine. A small-angle scattering study using monoclonal antibodies as markers.

The reaction of methylamine with complement protein C3, which involves cleavage of a labile thiol ester bond, yields a large intramolecular rearrangement. This is shown by small-angle neutron and X-ray scattering using a Fab antibody as a marker. For the C3(Fab) 1:1 complex, the methylamine reaction yields an increase in the radius of gyration, R, from 4.6 nm to 6.0 nm. In the absence of Fab the corresponding R values increase from 4.4 nm to 5.1 nm. It is estimated that the methylamine-induced increase in R may correspond to a movement of the epitope to a position 5 nm away from the centre of gravity of the C3 molecule. In agreement with this finding, the maximum distance within the C3(Fab) complex increases from 16 nm to 22 nm as a result of the methylamine reaction. In order to explain this conformational change, it is tentatively suggested that the methylamine-induced cleavage of the C3 thiol ester bond leads to a domain rotation within the C3 molecule. In agreement with this idea, the data is consistent with a model which enables a globular domain within the molecule to rotate without redistributing the molecular mass more than that corresponding to the radii of gyration observed.

Antibodies, Monoclonal↗

PubMed2005

[Complement componenets, C3 and factors B, in the blood of patients with acute ischemic stroke].

Several studies suggest that activation of compliment cascade contributes to the development of the inflammatory immune response in ischemic stroke and results in tissue injury by initiating apoptosis and necrosis. It is proposed that suppression of the compliment activation may have positive effect on stroke severity and outcome. However, the majority of data relevant to involvement of the compliment in the pathogenethic mechanisms of stroke have been obtained in animal models of stroke that does not allow to extrapolate these data to humans. Our previous studies were the first ones demonstrated the involvement of both the classical and the alternative complement activation pathways in the pathogenetic mechanisms of generation and development of stroke in humans. In the present work, using immunoblotting, we determined the levels of key components of the classical and alternative pathways of complement activation, C3 and factor B, in the blood of patients with ischemic stroke. Comparing to healthy controls, patients with ischemic stroke are characterized by the increased level of C3 and decreased level of factor B.

Acute Disease↗

PubMed2002

Immunoglobulin G monoclonal antibodies to Cryptococcus neoformans protect mice deficient in complement component C3.

Passive administration of monoclonal antibodies (MAbs) to the capsular polysaccharide of Cryptococcus neoformans can alter the course of infection in mice. In a murine model of cryptococcal infection, immunoglobulin G1 (IgG1), IgG2a, and IgG2b switch variants of the anti-capsular 3E5 MAb prolong the survival of lethally infected mice, whereas the 3E5 IgG3 MAb does not protect and in some cases enhances infection, shortening the life spans of infected mice. We examined the role of complement component C3 in Ab-mediated protection by determining the efficacy of the four mouse IgG subclasses against C. neoformans in mice genetically deficient in factor C3 as well as mice acutely depleted of C3. Similar to other complement-deficient animal models, C3(-/-) mice and mice depleted of C3 by cobra venom factor were more susceptible to C. neoformans infection than control mice, providing further evidence that complement is important in the host defense against the fungus. In the absence of C3, all IgG isotypes prolonged the lives of mice infected with C. neoformans, indicating that protection by IgG does not require the complement pathways. Furthermore, we observed protection with IgG3 in the complement-deficient mice, suggesting that complement is involved in the lack of protection observed with IgG3 in other mouse models.

Animals↗

PubMed1995

Component C3 of hagfish complement has a unique structure: identification of native C3 and its degradation products.

A protein from hagfish serum that cross-reacted with the third component of hagfish complement (C3) was purified to homogeneity and its structural properties were compared with those of C3 which has a two-subunit chain structure (115 and 72 kDa). This protein (designated C3b), when purified from plasma, consisted of three disulfide-linked polypeptide chains (77, 72 and 30 kDa). On immunoelectrophoresis, purified C3b migrated more rapidly towards the anode than the beta mobility of C3. However, immunochemical analysis revealed that C3b, after the first step in its purification, consisted of two disulfide-linked polypeptide chains (105 and 72 kDa). Treatment of C3b with methylamine, prior to spectrophotometric titration of the free sulfhydryl groups, did not significantly affect the end-point of the titration, suggesting the absence of a thioester bond in this molecule. Analysis of the amino acid sequences of the amino-termini of the subunits of C3b revealed that 77 amino acid residues at the amino-terminus of the native alpha chain were missing from both the 77-kDa and the 105-kDa polypeptides from C3b. These results indicate that the C3b in this study was analogous to mammalian C3b. Furthermore, amino acid sequencing data indicated that most of the native C3 from hagfish serum has an irregular two-subunit (alpha+gamma and beta)-linked structure, as a result of one-sided processing of putative hagfish pro-C3 at the beta-alpha processing site exclusively. Moreover, it appears that only the molecular features of degenerated hagfish C3 (C3b) are altered during its purification to generate a three-chain structure.

Amino Acid Sequence↗

PubMed1984

Accumulation of IgG and complement factor C3 in human arterial endothelium and atherosclerotic lesions.

Endothelial injury is associated with an intracellular accumulation of IgG. Fluorescent antibodies were used to detect IgG and complement factor C3 in biopsies of human arteries. Both proteins were found in endothelial cells with signs of injury, in normal and atherosclerotic arteries. Rhodamine-conjugated anti-IgG and FITC-conjugated anti-C3 were used to count the number of IgG- and C3-containing endothelial cells in non-atherosclerotic cystic arteries. IgG was found in 26% of the cells, with C3 in 38% of the IgG-containing cells. Both IgG and C3 were also observed in the subendothelial intima of cystic arteries and in atherosclerotic lesions of iliac and femoral arteries. The accumulation of IgG and C3 in injured endothelium and in atherosclerotic lesions could be of importance for complement-mediated tissue destruction and for the attraction and activation of white blood cells.

Adult↗

PubMed1982

An equilibrium model of the metastable binding sites of alpha 2-macroglobulin and complement proteins C3 and C4.

Two cyclic structures, the 15-membered thiolactone A and the 5-membered lactam P, have been proposed for the metastable binding sites of the serum proteins C3, C4, and alpha 2-macroglobulin. Neither structure alone adequately explains two unusual reactions of these sites, namely, covalent attachment to nucleophiles with liberation of a thiol group and spontaneous hydrolysis (autolysis) of an internal peptide bond. The metastable binding sites of these proteins were modeled with the 15-membered thiolactone 1 (Khan, S. A., and Erickson, B. W. (1981) J. Am. Chem. Soc. 103, 7374-7376) and the isomeric 5-membered lactam 2, which contains an internal pyroglutamyl (Glp) residue. Under physiologic conditions (phosphate-buffered saline, pH 7.3, 37 degrees C), thiolactone 1 and lactam 2 exist in dynamic equilibrium. Since the molar ratio of 2/1 is 11:1 at equilibrium, lactam 2 is 15 kcal/mol more stable than thiolactone 1. The activation energy for isomerization of 1 into 2 is 18 kcal/mol, which is about 5 kcal/mol lower than that for hydrolysis of the acyclic thiolester N,S-diacetyl-L-cysteine methylamide. Part of the chemistry of the metastable binding sites can be explained by an analogous equilibrium between protein structures A and B. Lactam B may be a key intermediate in the biosynthesis of thiolactone A. Under denaturating conditions, thiolactone A could either bind covalently to a receptive surface or isomerize into lactam B, which could undergo spontaneous hydrolysis of the Glu-Glp peptide bond.

Binding Sites↗

PubMed1995

Tyrosine is a potential site for covalent attachment of activated complement component C3.

Activation of C3 results in the generation of metastable C3b, which has been shown to preferentially react with the hydroxyl groups of carbohydrates and with specific serine and threonine residues in proteins. In this study we have examined the reactivity of metastable C3b with the third type of hydroxyl group present in proteins, tyrosine (Tyr). The results demonstrated that Tyr reacts with the thioester of metastable C3b and that this reactivity was 11-fold better than that of threonine, 47-fold better than serine and 50-fold better than the reactivity of carbohydrates. Model peptides containing Tyr showed even higher reactivity than free Tyr, demonstrating that incorporation into peptide structures does not block C3b attachment. The site of attachment was found to be in the alpha'-chain of C3b and the bond was hydroxylamine sensitive, indicating an ester linkage. The stability of the C3b-Tyr complex was measured under physiological conditions (pH 7.4, 37 degrees C) and compared to the stability of other C3b complexes. C3b-Tyr decayed 50% in 19 hr at 37 degrees C, but C3b bound to a Tyr-containing peptide was more stable, exhibiting a t1/2 of 53 hr. The ester linked complexes C3b-IgG and C3b-glycerol were less stable each exhibiting a t1/2 of approximately 8 hr. As yet, only two specific C3b attachment sites on proteins have been identified, Ser1217 in C4b and Thr144 in IgG1. The present evidence demonstrates that Tyr residues are highly reactive and that the C3b-Tyr linkage is stable. The findings suggest that complexes involving tyrosine residues as the site of attachment will also be found.

Amino Acid Sequence↗

PubMed1995

Phospholipase A2 from plasma of patients with septic shock is associated with high-density lipoproteins and C3 anaphylatoxin: some implications for its functional role.

Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2. The enzyme showed optimal activity on [14C]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [3H]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [3H]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA2.

Amino Acid Sequence↗

PubMed1977

Activation of complement components C3 and factor B in synovial fluids.

Synovial fluids from 106 patients with various types of arthritis were examined for the presence of conversion products of C3 by means of crossed antigen-antibody electrophoresis and for products of factor B by immunoelectrophoresis. C3 conversion was found in all 15 fluids from patients with seropositive rheumatoid arthritis, in 11 of 15 with seronegative rheumatoid arthritis, in the majority with probable and possible rheumatoid arthritis, juvenile rheumatoid arthritis, SLE, pseudogout, gout, Reiter's syndrome, and frequently in other arthritides studied, but in only one of 15 with degenerative arthritis. In 53 synovial fluids a single C3 conversion peak was seen in addition to the native protein and in 18 others two conversion peaks were present. In many synovial fluids showing conversion whole-complement titers and C3 protein concentrations were normal. In both rheumatoid arthritis and crystal synovitis the per cent of C3 conversion, as estimated by planimetry, correlated with synovial fluid leukocyte counts, Factor B conversion was found in 31 fluids and usually occurred in fluids also showing C3 conversion. The findings indicate that in vivo activation of components of the classical and alternative considered mediated by immune complexes. Activation is also commonly present in a wide variety of other inflammatory arthritides and ofter would not be recognized by measuring only concentrations of hemolytic whole complement or C3 by immunodiffusion. The positive association between C3 conversion and synovial fluid polymorphonuclear leukocytes suggests that chemotactic factors generated from complement may be responsible for the attraction of leukocytes into the synovial space in these diseases.

Arthritis↗

PubMed1989

The expression of I-A correlates with the uptake of interferon-gamma by macrophages.

The current studies were designed to examine some of the requirements for I-A expression when macrophages (M phi) were treated with interferon-gamma (IFN-gamma). In order to define the minimum time required for IFN-gamma to induce surface expression of I-A antigen on bone marrow-derived M phi, cells were incubated with IFN-gamma for varying lengths of time, washed and thereafter incubated for 72 h before assaying I-A surface expression. Using saturating amounts of IFN-gamma (300 IRU/ml), we found that between 0 and 30 min of IFN-gamma treatment there is a direct correlation between the length of treatment and the level of I-A surface expression. When the steady state level of RNA for the I-A beta gene was assayed, a low level of I-A beta RNA was seen in cells treated for 10 min with saturating amounts of IFN-gamma (300 IRU/ml) while a 30-min or 60-min exposure of cells to the same concentration of IFN-gamma resulted in a steady increase in the level of I-A beta RNA. Similar results were found when we measured the levels of RNA for the tumor necrosis factor and C3 complement genes, both of which are induced by IFN-gamma in M phi. M phi treated with low amounts of IFN-gamma (3 IRU/ml) for 30 min do not express cell surface I-A. Cells incubated continuously for 72 h with 3 IRU/ml of IFN-gamma expressed a level of I-A on the surface equivalent to the level of I-A expressed on cells treated for only 30 min with 300 IRU/ml of IFN-gamma. Based on the observed correlation between either the IFN-gamma concentration or the length of time the cells were exposed to IFN-gamma, or the level of I-A expression on M phi, we conclude that the expression of I-A correlates with the uptake of IFN-gamma.

Animals↗