Ulla Mårtensson

Complement classical pathway components are all important in clearance of apoptotic and secondary necrotic cells.

Inherited deficiencies in components of the classical complement pathway are strong disease susceptibility factors for the development of systemic lupus erythematosus (SLE) and there is a hierarchy among deficiency states, the strongest association being with C1q deficiency. We investigated the relative importance of the different complement pathways regarding clearance of apoptotic cells. Phagocytosis of labelled apoptotic Jurkat cells by monocyte‐derived macrophages in the presence of sera from individuals with complement deficiencies was studied, as well as C3 deposition on apoptotic cells using flow cytometry. Sera from individuals deficient in C1q, C4, C2 or C3 all showed decreased phagocytosis. Mannose binding lectin (MBL) and the alternative pathway did not influence phagocytosis. Notably, the components of the complement classical pathway, including C1q, were equally important in clearance of apoptotic cells. This indicates that deposition of C3 fragments is of major significance; we therefore studied C3 deposition on apoptotic cells. Experiments with MBL‐deficient serum depleted of C1q or factor D confirmed the predominance of the classical pathway. At low dilution, sera deficient of C1q, C4 or C2 supported C3 fragment deposition demonstrating alternative pathway activation. In conclusion, we have found that complement‐mediated opsonization and phagocytosis of apoptotic cells, particularly those undergoing secondary necrosis, are dependent mainly upon an intact classical pathway. The alternative pathway is less important, but may play a role in some conditions. C1q was not more important than other classical pathway components, suggesting a role in additional pathogenetic processes in SLE other than clearance of apoptotic cells.

Western Blot Analysis of Human IgG Reactive with the Collagenous Portion of C1q: Evidence of Distinct Binding Specificities

An enzyme‐linked immunosorbent assay (ELISA) with purified collagenous C1q fragments in the solid phase was used for detection of C1q‐specific immunoglobulins in the sera of twelve patients with systemic lupus erythematosus (SLE) or the SLE‐like disease hypocomplementemic urticarial vasculitis syndrome (HUVS). By clinical criteria, four patients had SLE, and three HUVS. Five patients had overlap syndromes. All patients demonstrated high concentrations of C1q‐specific IgG and markedly low concentrations of circulating C1q. Detection of C1q‐specific IgG in SLE sera was facilitated by employment of saturating concentrations of collagenous C1q fragments in the solid‐phase ELISA. When added to SLE serum, immune complex‐fixed C1q inhibited binding of IgG to the C1q fragments, whereas addition of C1q alone had limited inhibitory effects. Under similar conditions, using approximately equimolar amounts of C1q relative lo solid‐phase C1q fragments, no ELISA inhibition was obtained after addition of C1q or immune complex‐fixed C1q to a HUVS serum. Even in large excess, purified C1q did not inhibit binding of HUVS‐IgG to solid‐phase C1q fragments. Thus, possible interactions between HUVS‐IgG and native C1q are probably of low affinity. By Western blot analysis. IgG reactive wish the B and C chains of C1q was found in the eight patients with evidence of HUVS, five of whom also showed IgG binding to C′‐C′ and A′‐B′ dimers of collagenous C1q fragments. Sera from SLE patients were negative by Western blot analysis. It seems likely that C1q‐specific IgG in SLE primarily recognizes assembled C1q molecules or collagenous C1q fragments expressing conformational epitopes of bound C1q. Interestingly, patients with evidence of HUVS fairly consistently had zymogen (C1r‐C1s)2 complexes in their serum, while patients with SLE showed high concentrations of complexes containing C1 inhibitor, C1r and C1s. Different binding specificites of C1q‐reactive IgG could be of importance with regard to pathogenetic mechanisms in SLE and HUVS. There was no correlation between findings of C1q‐specific IgG and a variety of autoantibodies associated with SLE and SLF‐like disease.

Studies of C1 subcomponents in chronic urticaria and angioedema.

C1q, C1r, C1s, C3, C4 and C-1 IA were determined by electroimmunoassay in sera from 150 patients with chronic urticaria or angioedema. Abnormal C1q and C1s levels were found in about 30% of the patients. In seven sera C1r was not measurable due to the appearance of diffuse precipitates. The levels of C3 and/or C4 were decreased in five sera with aberrations of C1 subcomponents in the electroimmunoassay. None of the patients showed reduced C-1 IA levels in the electroimmunoassay. The presence in sera of abnormal C1 subcomponent complexes was studied by crossed immunoelectrophoresis. Sera from 11% of the patients contained C1r-C1s complexes. Increased amounts of alpha2 complexes (C-1r-C-1-S-C-1 IA) were found in 33% of the patients. A major part of the C1q in sera yielding abnormal C1r precipitates had the same electrophoretic mobility as isolated C1q and was not associated with the C1qrs complex. C1 activity in hemolytic tests was low in these sera as well as in sera with decreased C1q levels. In the esterolytic assay for C-1 IA low values were found in 14 patients. Repeated sampling and family studies in appropriate cases gave no evidence for genetically determined deficiencies of C1q, C1r or C-1 IA.

Hypocomplementaemia caused by C3 nephritic factors (C3 NeF): clinical findings and the coincidence of C3 NeF type II with anti-C1q autoantibodies

Abstract. Skattum L, MBrtensson U, Sjoholm AG (Lund University, Lund, Sweden). Hypocomple‐mentaemia caused by C3 nephritic factors (C3 NeF): clinical findings and the coincidence of C3 NeF type I1 with anti‐Clq autoantibodies. 1 Intern Med 1997; 242: 455‐64.

Human Autoantibodies Against C1q: Lack of Cross-Reactivity with the Collectins Mannan-Binding Protein, Lung Surfactant Protein A and Bovine Conglutinin

The collectins, a group of humoral C‐type lectins, have globular and collagen‐like regions and share structural features with the complement protein C1q. The question was asked if autoantibodies to the collagen‐like region of C1q (anti‐C1qCLR) might cross‐react with collectins, such as mannan‐binding protein (MBP), lung surfactant protein A (SP‐A) and bovine conglutinin (BK). Anti‐C1qCLR antibodies of the systemic lupus erythematosus (SLE) type and anti‐C1qCLR antibodies of the hypocomplementemic urticarial vasculitis syndrome (HUVS) type were investigated. Cross‐absorption and elution experiments combined with antibody detection by enzyme‐linked immunosorbent assay (ELISA) and immunoblot analysis gave no evidence of cross‐reactive anti‐C1qCLR antibodies. However, one serum with HUVS type anti‐C1qCLR antibodies contained anti‐MBP antibodies that were cross‐reactive with SP‐A. Judging from results of ELISA inhibition experiments and immunoblot analysis, four SLE sera contained antibodies to native BK, while two sera with HUVS type anti‐C1qCLR antibodies contained antibodies to epitopes of denatured BK. This might imply that autoimmunity to collagen‐like structures is not restricted to C1qCLR in HUVS and HUVS/SLE overlap syndromes.

Activation of C1, the first component of complement, the generation of C1r-C1s and C1̄ inactivator complexes in normal serum, by heparin-affinity chromatography☆☆☆

Abstract Investigations facilitating heparin-affinity chromatography and immunochemical procedures were utilized to study the interaction and behaviour of heparin with C1, C1 subcomponents and C1 IA in normal serum. Clq bound to heparin-Sepharose independent of divalent cations at 4 and 37° C at physiological pH and salt concentration requiring a high salt concentration for its complete release. MacromolecularC1 bound to heparin-Sepharose in the presence of Ca 2+ at 37°C, activating Clr and Cls in the process as demonstrated by the appearance of C1r-C1s-C1 IA complexes when purified C1 IA was added to the eluted fractions. At 4° C however, proenzyme C1r and C1s were present in the eluted fractions. Proenzyme C1r-C1s and C1r-C1s-C1 IA complexes did not bind directly to heparin-Sepharose. Binding of C1r-C1s in the presence of Ca 2+ was dependent on Clq bound to heparin. High levels of proenzyme C1r-C1s complexes were produced during the 4°C experiments in the presence of Ca 2+ and were collected in the void volume. C1r-C1s-C1 IA complexes generated during the 37°C experiments had no affinity for heparin-Sepharose. C1 IA did not bind directly to heparin. Recirculation of a serum sample collected after Clq was removed by heparinaffinity at 4°C in the presence of EDTA demonstrated that (1) Clr and Cls in the presence of calcium could be reassembled with heparin-bound Clq to form macromolecular Cl. (2) Clr and Cls in non-activated forms did not bind directly to heparin-Sepharose. (3) Clr and Cls contained in the reassembled Cl/heparin complex at 37°C were in active enzymic forms.

Trimer and tetramer complexes containing C1 esterase inhibitor, C1r and C1s, in serum and synovial fluid of patients with rheumatic disease

During activation, the first component of complement C1q (C1r-C1s)2 is dissociated in conjunction with the formation of complexes containing C1 esterase inhibitor (C1-INH). Trimer complexes, with zymogen C1s associated with a firm C1-INH-C1r complex (C1-INH-C1r-C1s) can be distinguished from tetramer complexes C1-INH-C1r-C1s-C1-INH) in which C1-INH is firmly bound to both proteases. In the present study a two-stage electroimmunoassay was developed for the specific measurement of C1-INH-C1r-C1s. In the first step, C1-INH and its complexes were immunoprecipitated with anti-C1-INH during electrophoresis in the presence of Ca2+. In the second step, C1s contained in C1-INH-C1r-C1s was dissociated in the presence of EDTA and was measured by immunoprecipitation with anti-C1s. C1-INH-C1r-C1s were consistently found in normal sera. Normal sera did not contain C1-INH-C1r-C1s-C1-INH as assessed with a previously described ELISA procedure. Sera and synovial fluids from two groups of patients with inflammatory arthritis were investigated. In rheumatoid arthritis patients (n = 15) C1-INH-C1r-C1s complexes were usually found at high concentration both in serum and synovial fluid. C1-INH-C1r-C1s-C1-INH complexes were also present with values that were higher in synovial fluid than in serum, in accord with previous findings of classical pathway activation in the inflamed joints of the patients. Patients with spondylarthritic syndromes (n = 7) had serum and synovial fluid C1-INH-C1r-C1s concentrations that were comparable to those of the rheumatoid arthritis patients. If at all present, C1-INH-C1r-C1s-C1-INH were detected in trace amounts. Thus, C1 activation in patients with spondylarthritic syndromes appeared to be efficiently controlled at the C1r level. Distinguishing between C1-INH-C1r-C1s and C1-INH-C1r-C1s-C1-INH may prove of value in further studies of the activation and control of C1 in disease.

Electroimmunoassay of C1 inactivator and C4 in hereditary angioneurotic edema (HANE): A simplified diagnostic procedure☆

Abstract The laboratory diagnosis of hereditary angioneurotic edema (HANE), can be confirmed or excluded in patients with symptoms suggestive of the condition by immunochemical determination of C 1 IA and C4. Occasionally, esterolytic assay of C 1 IA, quantitation of C3 and other complement components may be required for the diagnosis. The variants of HANE are described on the basis of the laboratory findings, and a diagnostic scheme is proposed. An application of the electroimmunoassay using a single analytic step for the quantitation of C 1 IA and C4 for screening of HANE is presented.