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Data are presented demonstrating that high concentrations of complement protein C4 in sheep plasma are associated with a particular class I OLA specificity. By way of contrast, a similar association could not be demonstrated between C3 plasma concentrations and OLA specificities. These data support the hypothesis that gene(s) determining C4 plasma concentrations are linked to the ovine MHC.
The genes for the fourth complement component (C4) and sex-limited protein (Slp) of the mouse exhibit a high degree of sequence homology and have probably been derived by recent gene duplication from a common ancestor gene, but their mode of expression is greatly different in most standard mouse strains. Although the C4 gene is constitutively expressed, the Slp gene is expressed only under the influence of androgens or not expressed at all. To map the transcriptional regulatory domains that are responsible for the difference in the promoter activity between the mouse C4 and Slp genes, we constructed various hybrid promoters from the 5'-flanking DNA of the C4 and Slp genes of the FM strain and tested for their transcriptional activity. The hybrid promoters were inserted into the HindIII site located immediately upstream of the chloramphenicol acetyl transferase (CAT) gene of the plasmid pSVOcat. The expression of CAT was assayed in transiently transfected HepG2 cells and in permanently transfected Ltk- cells. The transcriptional regulatory region responsible for the difference between the C4 and Slp gene expression was mapped within the 5'-flanking region of about 400 nucleotides near the promoters of the C4 and Slp genes. By S1 mapping analysis, it was shown that the CAT activities expressed in transfected cells were due to the transcripts correctly initiated from the promoter of the C4 or Slp genes in the chimeric constructs.
Complement activity in 125 cases of classical dengue fever was examined through the measurement of haemolytic activity. During the first 3 d of fever, the classical complement pathway activity (CCPA) was not altered in 109 cases. After 4 d of fever, 9 of 16 patients in the viraemic period had CCPA decreased by 45% (995 +/- 119 units/ml) and serum complement component C4 decreased by 40% (10.4 +/- 0.9 mg/dl). The alternative complement pathway activity was not affected in any case tested throughout both viraemic and convalescent stages. Both CCPA and C4 persistently decreased in 3 of these 9 patients at the convalescent stage. A decrease in serum C3 was also observed in these 3 patients only, and circulating immune complexes (CIC) levels were particularly high in these 3 patients. These results indicate that there is little evidence of complement activation on days 1-3 of viraemia but that complement activation may occur subsequently. It is concluded that both CIC and other unknown factors not related to CIC may contribute to complement activation in some cases (9/125) of classical dengue fever.
The autolytic cleavage reaction of C3 and C4 and the covalent binding reaction of these proteins, are both aspects of the reactivity of an activated thiolester within these proteins. Autolytic cleavage occurs by internal nucleophilic attack on one face of the planar thiolester, while the covalent binding reaction of the activated proteins follows exposure of the opposite face of the thiolester to attack by external nucleophiles. Although the autolytic cleavage reaction does not occur under physiological conditions, the study of this phenomenon has provided valuable evidence in support of the mechanisms postulated for the physiological covalent binding reactions. The ease with which autolysis can be induced and observed in C3, C4, and alpha 2 M has provided a valuable method for detecting the active forms of these proteins in circumstances where other assays are impracticable, as, for example, in the examination of the uptake of active C3 by lymphocytes. Autolytic cleavage has also been used by Karp and colleagues to produce fragments used in characterizing genetic and biosynthetic variants of mouse C4 and the mouse protein Slp, which is structurally similar to C4. Gross structural comparisons made among C3, C4, and alpha 2 M on the basis of alignment of the autolytic cleavage sites and the protease-activation sites in these proteins were useful in predicting how the alpha-, beta-, and gamma-chains of C4, or the alpha- and beta-chains of C3, were aligned in the single polypeptide chain pro-forms of these proteins. The beta-alpha-gamma alignment deduced for C4 was also found by Goldberger and Colten. Similar alignments of cleavage sites have been used as a basis for evolutionary comparisons of complement proteins and alpha 2 M from species other than man. Although autolytic cleavage has been described only for C3, C4, alpha 2 M, and Slp, it is likely that other proteins will be found that exhibit this phenomenon. A possible candidate is pregnancy-associated plasma protein A (PAPP-A) which resembles alpha 2 M in many respects. The autolytic cleavage reaction will serve as a useful indicator in the detection of other proteins that undergo covalent binding by the mechanism discussed above.
The complement system is one potential cytotoxic effector mechanism that might be effective in immunotherapy of cancer using monoclonal antibodies (mAb) directed against tumor antigens. In order to evaluate the treatment outcome from trials using mAb in cancer patients, assessment of complement-dependent cytotoxicity (CDC) may therefore be of interest. Here we describe the elaboration of a CDC assay in vitro using a rat hepatoma cell line, H4-II-E, as target cells sensitised with mAb F12, directed against the tumor-associated ganglioside antigen fucosyl-GMI. Sensitised cells were incubated with various concentrations of fresh serum as complement source for 48 h and cytotoxicity was then assessed by the tetrazolium bromide (MTT) test. A large variation in CDC efficacy was observed between individual serum donors. No differences in CDC could be seen between healthy donors and cancer patients. The CDC showed a strong correlation to the serum concentrations of complement factor C4, supporting the validity of the assay. Our results suggest that there may be significant variations in complement function within and between individuals that might influence the outcome of clinical mAb therapy. The H4/F12 CDC assay described here, together with measurement of individual complement factors. such as C4, should be further validated in cancer patients at various disease stages and phases of treatment.
OBJECTIVES: To determine the inflammatory responses in endovascular abdominal aortic aneurysm (AAA) repair and their relation to clinical findings. DESIGN: Prospective non-randomised study. SETTING: University Hospital, Department of Surgery. PATIENTS AND METHODS: Seven patients treated with an endoluminal procedure (AAA-E) and seven patients undergoing conventional surgery (AAA-C) were included. Inflammatory parameters were assessed by measurements of the cytokines interleukin (IL)-1 beta, IL-6, IL-8 and Tumour Necrosis Factor-alpha (TNF-alpha); analyses of complement proteins C1q, C4, C3, C5a and Terminal Complement Complexes (TCC); haematologic parameters and determination of C-reactive protein (CRP). RESULTS: In six of seven patients in the AAA-E group blood pressure decreases were recorded during introduction of the device. IL-6 and CRP levels were found to be significantly higher in AAA-C patients compared to the AAA-E group. On the other hand, high TNF-alpha levels were recorded in the AAA-E group. Less consumption of the complement proteins C1q, C4 and C3 was observed in AAA-E compared to AAA-C patients. Increased C5a levels were recorded in the AAA-C group, whereas only slight fluctuations were noticed in the AAA-E group. TCC levels were unchanged in both groups. CONCLUSION: Endovascular aortic aneurysm repair induced a significant inflammatory response, mainly involving TNF-alpha and differing from the findings during open AAA repair. These inflammatory responses were probably related to blood pressure decreases during the procedures. On the other hand, conventional repair induced responses related to the more extensive surgical trauma and reperfusion injury.
The prognostic significance of class III major histocompatibility complex complement components, factor B (Bf), C4A and C4B, were studied in a 3-year prospective study of 73 patients with early RA. Patients with C4B null allele had higher disease activity with more radiological progression than patients with C4A null allele or patients without null allele. C4B null allele also associated with increased susceptibility to side effects from antirheumatic treatment. The Bf phenotypes did not associate with the severity of RA. C4B null allele may have prognostic significance in determining a special subgroup of RA patients with a more complicated course of the disease.
Mouse Slp, a duplicate of the fourth complement component (C4) gene, exhibits EDTA-independent complement activity with a hepatic expression that is male specific. To provide an underlying mechanism for the male-specific expression, we have analyzed the promoter activity of the various 5'-flanking sequences and CpG demethylation of the Slp gene. Transient transfections using HepG2 cells indicate that the element TTCCGGGC (nt -124 to -117) regulates the promoter activity. Moreover, CpG at position -121 of this regulatory element is demethylated to a much higher degree in males than in females. This sexually dimorphic DNA demethylation is consistent with the male-specific expression of the Slp gene in DBA/2 males. The regulatory element binds to the different TTCCGGGC-specific nuclear proteins depending on the methylation of the CpG site. In contrast, the corresponding CpG at position -119 of the C4 gene, which is expressed in both males and females, is demethylated at equal and high levels in both sexes. We therefore propose that the DNA demethylation and methylation-sensitive transcription factors may be a part of the regulatory mechanism for the male-specific expression of the Slp gene.
As a collaborative work of three laboratories the polymorphism of the canine fourth complement component (C4) was studied in a total of 131 unrelated dogs from different breeds and mongrels. Using high voltage electrophoresis followed by an immunoblotting technique, we detected eight distinct variants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of canine C4 showed an additional heterogeneity of the alpha and gamma chains which resulted in a total of 11 variants in the population studied. So that more precise information concerning the respective C4 allotypes will be available, a nomenclature is proposed designating not only the migration pattern of the C4 variants in agarose gels but also the heterogeneity of the C4 chains observed in SDS-PAGE.
The authors describe the results of immunological assay of complement factors C3, C4 (the usual path of activation of complement) and of B factor (the alternate path of activation) in 61 multiple sclerosis patients not receiving corticoids, 52 normal controls and 217 patients with other neurological disorders. Hypocomplementaemia (fall in factor C3 related to a fall in total haemolytic activity) was found in 29.5 p. 100 of the patients not on corticotherapy at the first assay, and in 36 p. 100 of the patients when repeated assays were carried out. Hypocomplementaemia is significantly more frequent in multiple sclerosis than in the normal population (0 p. 100) and in neurological patients (9.6 p. 100). In 13.1 p. 100 of the multiple sclerosis patients there was a decrease in B factor: 50.3 p. 100 of the multiple sclerosis patients exhibited no quantitative abnormality of the main factors of complement (normocomplementary multiple sclerosis). The group of multiple sclerosis patients with hypocomplementaemia was characterized by the incidence of other abnormalities in the complement system: cleavage of the C3 factor and a fall in B factor in 60 p. 100 of the cases. A more frequent increase in IgE and measles antibodies was found also while the normocomplementary multiple sclerosis patients more frequently had higher levels of IgA. Genetically, the group with hypocomplementaemia is related to a significant increase in the incidence of the HL-A W18 group while the normocomplementary multiple sclerosis patients appear closely related to the HL-A7 group. Familial investigations show that hypocomplementaemia is usually present in the ascendents and collaterals and that it seems to be transmitted with the HL-A haplotypes. Four families gave evidence of transmission with the W18 group. This transmission sometimes occurs together with transmission of an increase in IgE and/or of measles antibodies. In two pedigrees, one of the ascendents carried in his serum an activator of the alternate path of complement. There does not appear to be any prognostic difference between the two groups. In multiple sclerosis with hypocomplementaemia, the facts suggest a complex immunological abnormality, transmitted genetically to the subject and existing prior to the illness, comprising both elements of deficient and excessive immune response. The recognized presence of a gene of immunological reactivity and of genes of synthesis of complement on the 6th chromosome, in proximity with genes of histocompatability (HL-A and M.L.C.) provides a theoretical basis for this supposition.
The aim of this study was to investigate the relationship between the presence and titre of antibodies against C1q (anti-C1q Ab) and disease activity and renal involvement in patients with systemic lupus erythematosus (SLE). Anti-C1q Ab were measured in 79 patients with SLE (70 women and 9 men; mean age 41.7 years; mean disease duration 8.4 years): 19 patients had active disease with lupus nephritis, 8 active disease without nephritis, 26 inactive disease with nephritis and 26 inactive disease without nephritis. Anti-dsDNA antibodies (EIA and immunofluorescence), antiendothelial cell antibodies (AECA) and complement levels (C3, C4, total haemolytic complement activity) were determined in parallel. Anti-C1q Ab were positive in 49%, anti-dsDNA Ab in 61% and AECA in 19% of the patients, respectively. Significantly higher titres of anti-C1q Ab were found in patients with active disease compared with those with inactive SLE ( P < 0.01). Serum levels of anti-C1q Ab showed a positive correlation with anti-dsDNA Ab and SLEDAI score ( P < 0.01) and a negative correlation with C3 ( P < 0.05), C4 ( P < 0.01) and CH50U ( P < 0.01). The presence of anti-C1q Ab was not different between patients with or without nephritis. In patients with ( P < 0.05) and without nephritis ( P < 0.01) the frequency of anti-C1q Ab was significantly higher in active patients compared with inactive patients. Both anti-C1q and anti-ds-DNA Ab were detectable in 74% of patients with active nephritis but only in 30% of all other patients ( P=0.001). None of the patients with active nephritis was negative for anti-C1q and anti-dsDNA Ab, whereas 37% of the patients without active nephritis were negative for both antibodies ( P < 0.01). Sensitivity, specificity, positive and negative predictive values for active lupus nephritis among SLE patients were 100%, 50%, 51.9% and 100% for anti-dsDNA Ab (EIA) and 74%, 70%, 57% and 89.4% for positive findings of both anti-dsDNA and anti-C1q Ab. The presence and titre of anti-C1q-Ab in SLE are related to disease activity. Absence of anti-dsDNA Ab excludes active nephritis; positive findings of both anti-dsDNA Ab and anti-C1q Ab are of relatively high specificity for active nephritis.
Fusobacterium polymorphum has been isolated from the gingival crevice in humans and has been implicated in the immunopathology of periodontal diseases. The presence of alternative complement pathway factors in gingival crevice material suggests the contribution of this process in the manifestation of the local host response. A complement consumption assay was used to show that the anticomplementary activity of a cell-wall preparation from F. polymorphum in guinea pig complement progressed optimally at 37 degrees C and suboptimally at 0 degrees C. Similar levels of complement consumption were recorded at both temperatures, but a higher concentration of cell walls was required to demonstrate maximum activity in the cold. Augmentation experiments with additional normal and heat-treated complement showed the presence of heat-labile factors that controlled and limited the consumption of complement to a constant proportion of the total amount present. These same experiments indicated that there were no reactive antibodies in the complement used in the assay. Similar tests with C4-deficient guinea pig sera confirmed that F. polymorphum cell walls were capable of generating alternative complement pathway activity in guinea pig sera.
Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.
A C4 haplotype containing C4*A6 without C4*B1 is described. It occurred in the MHC haplotype HLA-A1, Cw5, Bw44, C2*1, BF*S, C4*A6BQO, DR3, GLO 2. This haplotype was observed in five of eight siblings, including the propositus who was in renal failure. The C4A6 was not haemolytically active and this new haplotype is therefore of particular interest since it is functionally equivalent to the rare C4-deficient haplotype C4*AQOBQO.
The c4 gene of phage P1 has been localized to 335 bp of the P1EcoRI-9 fragment, within 50 bp of the EcoRI-9/14 junction. DNA sequence analysis of this fragment reveals a single open reading frame of 66 amino acids. The location of two c4 mutations, both of which produce changes in the predicted amino acid sequence in this reading frame, suggests that the reading frame codes for the c4 repressor. A region with high homology to the E. coli promoter consensus sequence is located approximately 50 bp upstream from the reading frame. Deletion of this potential promoter region abolishes expression of c4, as indicated by the loss of complementation of c4 mutants for lysogeny. Complementation is restored by the introduction of a heterologous promoter (the T7 phi 10 promoter), indicating that c4 expression is absolutely dependent on transcription of the 66-amino acid reading frame.
Two monoclonal antibodies (mAb M4d2 and M4d3) specific for the alpha 2-fragment (C4d), and one antibody (mAb M4c3) specific for the gamma-chain of human complement protein C4, have been tested for cross-reactivity against mammalian complement. These mAb have also been found to react with C4 from guinea-pig (mAb M4d2 and M4c3) as well as from cattle, baboon and rhesus monkey (mAb M4d3 and M4c3) in an activation ELISA. Since reactivity of mAb M4d2 and M4c3 included guinea-pig complement, the specific recognition of mammalian C4 could be confirmed with sera from C4-deficient (def') guinea-pigs. mAb M4d2 or M4d3, but not mAb M4c3, stained glomerular deposits within renal tissue sections from pig, cattle and guinea-pig. In the case of mAb M4d2, specificity of that staining could also be demonstrated in kidney specimens from C4-def' guinea-pigs. It can be concluded that, as in humans, the C4d fragment is also present in mammalian glomeruli. Compared with normal guinea-pigs, the C4-def' and C2-def' animals showed markedly increased glomerular deposits of IgM. It appears that glomerular deposition of complement C4d in mammals: (1) indicates activation via the classical pathway; (2) represents a general phenomenon of renal homeostasis; and (3) seems to be involved in the physiological clearance of immune complexes.
The amino acid sequence of the NH2-terminal regions of the intracellular precursor of the fourth component of guinea pig complement (pro-C4) and isolated alpha, beta, and gamma chains of native C4, synthesized by peritoneal macrophages in culture, were determined with a microradiosequencing technique. Radiolabeled pro-C4 was immunoprecipitated from cell lysates and native C4 from culture media which contained one of six 3H-amino-acids or [35S]methionine. The purity of these proteins was established and molecular weights were estimated by electrophoresis in sodium dodecyl sulfate. Seventeen of the first 22 residues of pro-C4 were identified. Fifteen of these 17 were nonpolar. The eight amino acids ascertained within the first 9 NH2-terminal residues of guinea pig pro-C4 were identical with those reported for human C4 beta chain, and identity with residues determined for guinea pig C4 beta chain was established. These data indicate that beta chain is the NH2-terminal segment of pro-C4, that no residues are cleaved from the NH2 terminus during the conversion to native C4, and that this segment of the pro-C4 molecule is conserved phylogenetically.
Concentrations of fibronectin, immunoglobulins G, M, and A, and C3 and C4 components of complement, and other plasma proteins were determined in synovial fluids from patients with rheumatoid arthritis (RA) and other diseases (non-RA). Fibronectin concentrations were two to three times greater in all synovial fluids than in plasma, and RA synovial fluids had a significantly higher mean concentration than non-RA fluids (883 microgram per ml vs. 588 microgram per ml, respectively, p less than 0.01). The mean concentrations of other synovial fluid constituents were less than their mean plasma concentrations. These results suggest that unlike other plasma constituents, either plasma fibronectin is concentrated in synovial fluids or that a substantial portion of synovial fluid fibronectin may be derived from synovial tissue cells. Both the C3 and C4 complement components were present in lower concentrations in RA than in non-RA synovial fluids. The C3 contents showed a statistically significant negative correlation with the fibronectin contents. Fibronectin was also found in all synovial fluid cryoprotein fractions tested, although its content varied greatly as a percent of the total cryoprotein protein (0.01 to 43 percent). The data show that fibronectin is a consistent constituent of synovial fluid cryoproteins in agreement with our previously reported finding that fibronectin is found in all serum cryoglobulin fraction tested.