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Clin Exp Immunol. May 1999; 116(2): 379–382.
PMCID: PMC1905284

C3 and C4 allotypes in anti-neutrophil cytoplasmic autoantibody (ANCA)-positive vasculitis


In ANCA-associated small vessel vasculitis few genetic factors have proven to be of importance for disease susceptibility, an exception being deficiency of α1-anti-trypsin, the main inhibitor of proteinase 3 (PR3). Alerted by our finding that myeloperoxidase has affinity for C3, and the finding of an increased frequency of the C3F allele in systemic vasculitis in a British cohort, we examined polymorphism of C3 and C4 in patients with ANCA+ small vessel vasculitis. After identification of all patients at our department with a positive ANCA test during the period 1991–95 and a diagnosis of small vessel vasculitis, blood samples were collected after informed consent. The 67 included patients were grouped according to ANCA serology and disease phenotype using the Chapel Hill nomenclature. The gene frequency of C3F was found to be increased (0.32) compared with controls (0.20; P < 0.05) in the PR3-ANCA+ subgroup. The frequency of C4A3 was increased in the group as a whole, but no increase of C4 null alleles was seen. The findings imply a role for the complement system in the pathogenesis of ANCA-associated small vessel vasculitis.

Keywords: ANCA, complement, proteinase 3, vasculitis, Wegener's granulomatosis


Small vessel vasculitis such as Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) are associated with ANCA. Most patients with WG have ANCA with specificity for proteinase 3 (PR3), and most patients with MPA have ANCA with specificity for myeloperoxidase (MPO).

Despite several investigations, few associations have been found between different genetic factors and ANCA+ vasculitis, an exception being the alleles encoding for the main inhibitor of PR3, α1-anti-trypsin (α1-AT). Deficiency states of α1-AT have been found to correlate with PR3-ANCA+ vasculitis, and in one study the PiZ allele was found to correlate with a more severe disease and worse prognosis [1]. Correlation with anti-MPO+ vasculitis is uncertain, and if existing probably weaker [2]. Conflicting data exist concerning the association between HLA antigens and small vessel vasculitis.

C3 has a central position in the complement cascade and is the quantitatively dominating complement factor in serum. The protein is transcribed from a single gene located on chromosome 19. Two common alleles, C3F and C3S, give rise to the three phenotypes C3S, C3FS and C3F which comprise 98% of all C3 phenotypes. The C3F allele is relatively frequent in Caucasoids (gene frequency 0.20) and less common in other races [3]. The phenotypes can be determined by their different electrophoretic mobility [3]. The molecular basis of the difference between C3F and C3S is a single substitution of an arginine residue in C3S for a glycine residue in C3F [4]. No functional differences have been observed between C3S and C3F in ability to interact with control proteins, to solubilize immune complexes, or to bind complement receptors [5,6]. The haemolytic capacity has been shown to be equal in one study [7], and slightly increased for C3S in another investigation [5]. The different C3 phenotypes are not associated with differences in serum level [8].

An increased frequency of the C3F allele has been reported in various conditions, including renal disease. An association has been reported between the C3F allele and the autoantibody nephritic factor (NeF), which in turn is associated with partial lipodystrophy and type II mesangiocapillary glomerulonephritis (MCGN II) [9]. In a report from the UK the gene frequency of C3F among patients with systemic vasculitis was 0.29 compared with 0.19 among control subjects [10].

Some of the complement protein genes, including the genes coding for the highly polymorphic C4, are located in the MHC on chromosome 6. C4 is encoded by two genes yielding two proteins with differences in biologic functions: C4A has been reported to be more efficient in inhibiting immune complex precipitation and C4B being more efficient in inducing haemolysis [11]. Null alleles are not uncommon, but presence of null alleles at both loci on the same chromosome is rare. An increased frequency of C4A null alleles has been found among systemic lupus erythematosus (SLE) patients [12]. One case with WG and partial C4 deficiency has been reported [13].

The aim of the present study was to search for associations between C3 and C4 polymorphism and ANCA+ vasculitis.


All patients with positive ANCA tests at the Department of Nephrology, University Hospital, Lund during the period from March 1991 to March 1995 were identified. During the period between May 1995 and August 1996, blood samples were collected after informed consent from a total of 82 patients. A clinical diagnosis of small vessel vasculitis was made for 67 patients. Included patients were also subgrouped based on PR3-ANCA and MPO-ANCA ELISA serology. Two double-positive patients with equal anti-PR3 and anti-MPO levels were not included in the PR3 and MPO groups, one patient had a clinical diagnosis of WG and one MPA. The clinical diagnosis of small vessel vasculitis was based on the clinical picture at presentation according to the Chapel Hill Consensus Conference as described by Westman et al. [14,15]. The diagnosis of WG was based on demonstration of granulomatous inflammation in five cases, and on non-invasive investigations in 29 cases. In the MPA group, six patients with renal limited vasculitis were included. Of the 67 patients included in the study, 64 were of Scandinavian and two of other Caucasoid origin; 29 were woman and 38 men.

Most of the serum samples were investigated at the Department of Clinical Immunology for the presence of ANCA with indirect immunofluorescence (IIF) and with ELISA for anti-MPO and anti-PR3. The IIF procedure and measurement of anti-MPO with ELISA were performed essentially as described earlier [16]. Measurement of anti-PR3 was performed with commercial ELISA kits (Diastat anti-PR3, c-ANCA, Shield Diagnostics, Dundee, UK; or Immunoscan PR3-ANCA, Euro-Diagnostica AB, Malmö, Sweden). Other samples were investigated for anti-MPO and anti-PR3 at Wieslab (Lund, Sweden) as described earlier [17].

Serum samples for C4 typing were treated with carboxypeptidase B type I (Sigma, St Louis, MO) 130 U/ml [18]. With EDTA added to a final concentration of 20 mm the samples were then treated with neuraminidase from Clostridium perfringens type VI (Sigma) at a concentration of 10 U/ml. The different C4 variants were detected by agarose gel electrophoresis at pH 8.6 and immunofixation was done with rabbit anti-human C4 antibodies (Dakopatts, Glostrup, Denmark) [19].

C3 typing was done on DNA samples extracted from peripheral blood samples. A 286-bp C3 gene fragment containing the single base change between C3S and C3F was amplified by polymerase chain reaction (PCR) using the primers 5′-ATCCCAGCCAACAGGGAG-3′ and 5′-TAGCAGCTTGTGGTTGAC-3′ as described by Botto et al. [4]. The following PCR cycle was run 30 times: (i) denaturation at 94°C for 1 min; (ii) annealing at 56°C for 1 min; and (iii) extension at 72°C for 1 min, with the last extension period prolonged with 15 min. The PCR products were cleaved with Hin61 (MBI Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions. Prior to the enzyme digestion a 504-bp properdin gene fragment with one single Hin61 cleavage site was added as an internal enzyme cleavage control. The cleaved DNA was precipitated and then dissolved before analysis by agarose gel electrophoresis. The DNA fragment derived from the C3S allele is cut by Hin61 into two fragments with 248 and 38 bp sizes, respectively, while the C3F derived fragment remains uncleaved.

Analysis for C3 polymorphism was carried out in a group of 101 blood donors, which served as a control group. As control group for the C4 polymorphism, material from121 persons was used [20].

Fisher's exact test was used to compare frequencies of complement variants in different groups.


Of the 34 patients with WG, two had MPO-ANCA and 31 PR3-ANCA, and of the 33 patients with MPA, 26 had MPO-ANCA and six had PR3-ANCA. Two patients with equal anti-PR3 and anti-MPO levels were not included in the MPO and PR3 groups. The C3 genotype results are presented in Table 1. The C3F gene frequency was 0.28 compared with 0.20 in the control group. When patients were subgrouped according to PR3-ANCA and MPO-ANCA, the gene frequencies in the MPO-ANCA group were almost identical to the control group, but in the PR3-ANCA group the increase of the C3F gene frequency was accentuated (P < 0.05). The total number of patients with the C3FF phenotypes was five (expected 2.0; NS).

Table 1
The gene frequencies for C3S and C3F in the different subgroups and in the whole group of vasculitis patients

Only two C4A phenotypes were represented, C4A3 and C4A6. The frequency of C4A3 was increased (P < 0.05) compared with the control group, but no differences could be seen between the different subgroups (Table 2). There was also an increase of the frequency of C4A6, though not significant. No patient with the C4AQ0 phenotype was found. Concerning C4B, there was an increase in the frequency of C4B3 and a slight increase of C4BQ0 frequency, especially in the WG subgroup, but the differences were not significant (Table 3).

Table 2
The C4A phenotypes in the different subgroups and in the whole group of vasculitis patients
Table 3
The C4B phenotypes in the different subgroups and in the whole group of vasculitis patients


The major finding in the present study is the increased gene frequency of the C3F allele among patients with small vessel vasculitis and PR3-ANCA. When subgrouping based on clinical picture was performed, no statistically significant difference could be seen between patients with WG and those with MPA. No increase of null alleles for C4A or C4B was seen. The method of analysis used can not detect rare C3 allotypes. If existing in the material, they would probably have been registered as C3S and could not explain the C3F excess. Chance and multiple testing is highly unlikely as an explanation for the C3 polymorphism data. At least three other possible explanations have to be considered: aetiological effect of complement factors, linkage disequilibrium or sampling bias in the present study.

A possible and interesting explanation of our finding is that C3 and C4 are important for the development of ANCA and/or small vessel vasculitis. Concerning C3, there is very little direct evidence for a functional difference between different allotypes, but there are some indirect signs and intriguing observations. Presence of the autoantibody NeF, which is associated with partial lipodystrophy and MCGN II, has been reported to be associated with the C3F allele [9]. An increased frequency of the C3F allele has been reported among descendants of Dutch emigrants to Surinam surviving epidemics of typhoid and yellow fever, indicating the C3F allele carries a survival advantage under these circumstances [21]. Increase of the C4A null allele in SLE cohorts indicates a protective role for C4 in SLE pathogenesis. Direct evidence for the involvement of complement in small vessel vasculitis is lacking [22]. However, a role of the complement system as a modulator of immune response might be of importance. Involvement of the complement system does not necessarily mean cytolytic effects by activation of terminal complement components. C3 is important in clearing immune complexes and influencing antibody production [23]. Effects of anaphylatoxins such as C5a could be of importance in amplification of neutrophil-mediated tissue injury. PR3 has been shown to cleave and inactivate C1 inhibitor, which could lead to complement activation via the classical pathway at the inflammatory site, and further neutrophil migration and activation followed by tissue damage [24]. C3dg has recently been shown to interact with MPO, which could be of importance for the clearance and localization of MPO at an inflammatory locus [25]. A dysregulation of inflammation caused by disturbance of normal complement function is in accordance with the hypothesis of small vessel vasculitis as a disease preferentially affecting individuals incapable of normal down-regulation of inflammatory response.

A second possibility is that our results are due to other genes on chromosomes 19 and 6, respectively, that are true carriers of disease susceptibility. At least for C4 and the MHC complex on chromosome 6 there are plenty of candidate genes known to influence immunological processes.

A third possibility is that the skewed gene frequency distribution is a result of biased sampling. In the present study, the identification and inclusion of patients was made retrospectively and the collection of samples prospectively, which introduces a risk of losing patients with severe and lethal disease. Theoretically, a more severe disease among C3S carriers could result in a high frequency of C3F among surviving patients. Complement allele may also influence target organ distribution. If a certain allele would make the kidneys more vulnerable the collection of samples at a renal unit could explain the present findings. The issues concerning sampling all imply a biological effect of complement in small vessel vasculitis and could be settled by prospective multicentre studies.

In conclusion, we have found an increase of the allotype frequency of C4A3 among patients with ANCA+ small vessel vasculitis, and an increase of the C3F allele in patients with PR3-ANCA- but not MPO-ANCA-associated vasculitis. This indicates that genetic factors and the complement system may be of importance in development of the disease, and should increase the interest in studying the complement system in ANCA-associated small vessel vasculitis.


This work was supported by grants from the Swedish Medical Research Council (Project no. 12631), the King Gustaf V 80th Birthday Fund, the Börje Dahlin Trust, the Crafoord Foundation, the Greta and Johan Kock Trusts and the Alfred Österlund Trust. The work was conducted within the framework of the Biomed.2 project no. BMH4-CT96-1005. The authors would like to thank Professor Jörgen Wieslander and Wieslab AB for providing ANCA test results, Mrs Birgitta Gullstrand for excellent laboratory work, and Mrs Eva Gunnefur and Mrs Mona Wendt for help with collection of samples.


1. Segelmark M, Elzouki A-N, Wieslander J, Eriksson S. The PiZ gene of alpha-1-antitrypsin as a determinant of outcome in PR3-ANCA-positive vasculitis. Kidney Int. 1995;48:844–50. [PubMed]
2. Savige JA, Chang L, Cook L, Burdon J, Daskalakis M, Doery J. Alpha-1-antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis. Clin Exp Immunol. 1995;100:194–7. [PMC free article] [PubMed]
3. Alper CA, Propp RP. Genetic polymorphism of the third component of human complement (C′3) J Clin Invest. 1968;47:2181–91. [PMC free article] [PubMed]
4. Botto M, Fong KY, So AK, Koch C, Walport MJ. Molecular basis of polymorphisms of human complement component C3. J Exp Med. 1990;172:1011–7. [PMC free article] [PubMed]
5. Welch TR, Beischel L, Kleesattel A. Functional consequences of the genetic polymorphism of the third component of complement. J Pediatr. 1990;116(Suppl.):S92–S97. [PubMed]
6. Bartók I, Walport MJ. Comparison of the binding of C3S and C3F to complement receptors types 1, 2, and 3. J Immunol. 1995;154:5367–75. [PubMed]
7. Colten HR, Alper CA. Hemolytic efficiencies of genetic variants of human C3′ J Immunol. 1972;108:1184–7. [PubMed]
8. Brönnestam R. Studies of the C3 polymorphism. Relationship between phenotype and quantitative immunochemical measurements. Hum Hered. 1973;23:128–34. [PubMed]
9. Finn JE, Mathieson PW. Molecular analysis of C3 allotypes in patients with nephritic factor. Clin Exp Immunol. 1993;91:410–4. [PMC free article] [PubMed]
10. Finn JE, Zhang L, Agrawal S, Jayne DRW, Oliveira DBG, Mathieson PW. Molecular analysis of C3 allotypes in patients with systemic vasculitis. Nephrol Dial Transplant. 1994;9:1564–7. [PubMed]
11. Schifferli JA, Steiger G, Paccaud J-P, Sjöholm AG, Hauptmann G. Difference in the biological properties of the two forms of the fourth component of human complement (C4) Clin Exp Immunol. 1986;63:473–7. [PMC free article] [PubMed]
12. Fielder AHL, Walport MJ, Batchelor JR, Rynes RI, Black CM, Dodi IA, Hughes GRV. Family study of the major histocompatibility complex in patients with systemic lupus erythematosus: importance of null alleles of C4A and C4B in determining disease susceptibility. Br Med J. 1983;286:425–8. [PMC free article] [PubMed]
13. Lhotta K, Kronenberg F, Joannidis M, Feichtinger H, König P. Wegener's granulomatosis and Henoch–Schönlein purpura in a family with hereditary C4 deficiency. Adv Exp Med Biol. 1993;336:415–8. [PubMed]
14. Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides. Proposal of an International Consensus Conference. Arthritis Rheum. 1994;37:187–92. [PubMed]
15. Westman KWA, Bygren PG, Olsson H, Ranstam H, Wieslander J. Relapse rate, renal survival and cancer morbidity in patients with Wegener's granulomatosis and microscopic polyangiitis with renal involvment. J Am Soc Nephrol. 1998;9:842–52. [PubMed]
16. Nässberger L, Truedsson L, Svantesson H. Occurrence of antibodies against neutrophil granulocyte components in juvenile chronic arthritis. Clin Exp Rheumatol. 1991;9:79–83. [PubMed]
17. Bygren P, Rasmussen N, Isaksson B, Wieslander J. Anti-neutrophil cytoplasm antibodies, anti-GBM antibodies and anti-dsDNA antibodies in glomerulonephritis. Eur J Clin Invest. 1992;22:783–92. [PubMed]
18. Sim E, Cross SJ. Phenotyping of human complement component C4, a class III HLA antigen. Biochem J. 1986;239:763–7. [PMC free article] [PubMed]
19. Awdeh ZH, Alper CA. Inherited structural polymorphism of the fourth component of human complement. Proc Natl Acad Sci USA. 1980;77:3576–80. [PMC free article] [PubMed]
20. Truedsson L, Baskin B, Pan Q, Rabbani H, Vorechovsky I, Smith CIE, Hammarström L. Genetics of IgA deficiency (Review) APMIS. 1995;103:833–42. [PubMed]
21. de Vries RRP, Meera Khan P, Bernini LF, van Loghem E, van Rood JJ. Genetic control of survival in epidemics. J Immunogen. 1979;6:271–87. [PubMed]
22. Mathieson PW, Peters DK. Deficiency and depletion of complement in the pathogenesis of nephritis and vasculitis. Kidney Int. 1993;44(Suppl. 42):S13–S18. [PubMed]
23. Tedder TF, Zhou L-J, Engel P. The CD19/cD21 signal transduction complex of B lymphocytes. Immunol Today. 1994;15:437–42. [PubMed]
24. Leid RW, Ballieux BEPB, van der Heijden I, Kleyburg-van der Keur C, Hagen EC, van Es LA, van der Woude FJ, Daha MR. Cleavage and inactivation of human C1 inhibitor by the human leukocyte proteinase, proteinase 3. Eur J Immunol. 1993;23:2939–44. [PubMed]
25. Segelmark M, Persson B, Hellmark T, Wieslander J. Binding and inhibition of myeloperoxidase (MPO): a major function of ceruloplasmin? Clin Exp Immunol. 1997;108:347–54. [PMC free article] [PubMed]

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