C1 subcomponent complexes: basic and clinical aspects.

C1 subcomponents form a variety of complexes that can be detected in normal and pathological sera. Since aberrations of C1 subcomponents in disease could reflect in vivo interactions with influence on complement function, studies of C1 subcomponent complexes might provide insight into pathogenetic mechanisms. C1 inhibitor (C1Inh)-dependent dissociation of the C1q(C1r-C1s)2 complex gives rise to C1Inh-C1r-C1s or C1Inh-C1r-C1s-C1Inh complexes. Increased concentrations of C1Inh-C1r-C1s probably signify prevention of C1 activation, while C1Inh-C1r-C1s-C1Inh appears to be a clinically useful marker of efficient classical pathway activation. "Free" C1q as found in some pathological sera, and in joint fluids of patients with rheumatoid arthritis could be a result of C1Inh-dependent dissociation of C1q(C1r-C1s)2. The presence in serum of zymogen (C1r-C1s)2 is an expected finding in various conditions with low C1q concentrations without evidence of C1 activation. It is not excluded that circulating (C1r-C1s)2 might sometimes be acquired due to factors capable of interacting with the collagenous part of the C1q molecule.

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 to 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 Clq are probably of low affinity. By Western blot analysis, IgG reactive with 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 Cl inhibitor, C1r and C1s. Different binding specificities 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 SLE-like disease.

C1 subcomponent complexes and C2 cleavage in active systemic lupus erythematosus.

We studied the activation and C1 inactivator-dependent dissociation of the first component of complement, the C1q(C1r-C1s)2 complex, in relation to recruitment of the classical activation pathway in the circulation of 24 patients with systemic lupus erythematosus (SLE). The patients were divided into three groups on a clinical basis, and were investigated during flares of disease activity. Group I had mild symptoms, group II major extrarenal manifestations, and group III manifest renal disease. High serum concentrations of trimer complexes containing C1 inactivator, activated C1r and zymogen C1s(C1 IA-C1r-C1s) were found in the majority of the patients. Some patients with high C1 IA-C1r-C1s concentrations showed no evidence of classical pathway activation, indicating that C1 activation was controlled by the action of C1 IA at the C1r level. By contrast, formation in serum of tetramer complexes in which C1 IA was firmly bound to both C1r and C1s (C1 IA-C1r-C1s-C1 IA) was associated with C2 and C3 cleavage in EDTA plasma, and with manifest hypocomplementemia. Low C1 IA-C1r-C1s-C1 IA values were observed in conjunction with substantial C2 cleavage in a few patients. Thus, C1 IA-C1r-C1s-C1 IA may not always be a sensitive indicator of classical pathway activation. Efficient recruitment of the classical pathway was related to disease severity, with some overlap between the clinical groups. In conclusion, C1 dissociation with formation of C1 IA-containing complexes was consistently found in patients with active SLE. The results suggested that C1 IA-dependent control of C1 activation was of biological significance in the disease.

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.

Common epitopes in Clq and collagen type II.

An epitope common for collagen type II and Clq was demonstrated by specific binding of a monoclonal anti-collagen type II antibody, MAb B1, to purified Clq. This was further substantiated by the affinity shown between F(ab')2 fragments of anti-Clq antibodies and rat chondrosarcoma collagen type II. The interaction between MAb B1 and Clq was demonstrated in hemolytic assays, in an enzyme-linked biotin-avidin assay and by the binding of Clq to MAb B1 immobilized on Sepharose 4B beads. MAb B1 recognized only purified Clq and not the macromolecular Cl complex, indicating that the epitope for MAb B1 was situated in the collagen-like region in Clq, where Clq and Cls are anchored. The binding of the purified collagen-like fragment of Clq to radiolabelled MAb B1 confirmed these findings. The affinity between MAb B1 and Clq was significantly increased if Clq was first reacted with heat aggregated IgG, indicating a demasking of the reactive epitope on binding to the aggregated IgG. The present findings raise the question of the pathogenetic significance of the presence of anti-collagen type II antibodies and free Clq, both of which are frequently seen in high amounts in rheumatoid arthritis.

C1 dissociation. Spontaneous generation in human serum of a trimer complex containing C1 inactivator, activated C1r, and zymogen C1s.

Activation of the C1 complex in the presence of C1 inactivator (C1 IA) is known to result in the formation of tetramer C1 IA-C1r-C1s-C1 IA complexes that are dissociated from C1q. Both C1r and C1s of the tetramers are present in their activated forms. The present investigation concerned the generation of trimer complexes containing C1 IA, activated C1r, and zymogen C1s (C1 IA-C1r-C1s). C1 IA-C1r-C1s were released from C1q and were formed in high concentration during prolonged incubation (1 to 3 days) of normal serum at 37 degrees C without addition of activators. By contrast, dissociation of C1 with formation of C1 IA-C1r-C1s-C1 IA was complete within 30 min at 37 degrees C, when the serum was treated with heat-aggregated IgG (1 g/liter). On size exclusion chromatography (TSK-4000), C1 IA-C1r-C1s and C1 IA-C1r-C1s-C1 IA emerged with apparent m.w. of 320,000 and 460,000, respectively. The composition of the complexes was examined by absorption of serum with F(ab')2 anti-C1s- or anti-C1r-coated Sepharose beads. Eluates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with immunoblotting. Under nonreducing conditions, heat-aggregated IgG-treated serum showed high concentrations of C1 IA-C1r (m.w. 202,000) and C1 IA-C1s (m.w. 194,000), while serum incubated at 37 degrees C without activators showed high concentrations of C1 IA-C1r but no C1 IA-C1s. Under reducing conditions, heat-aggregated IgG-treated serum showed m.w. 120,000 and 110,000 complexes of C1 IA and the C1r and C1s light chains, respectively. Uncleaved C1s and the m.w. 120,000 complex was found in serum that was incubated at 37 degrees C without activators. Consistent with results obtained by size exclusion chromatography, analysis by crossed immunoelectrophoresis and by electroimmunoassay showed that C1s could be released from C1 IA-C1r-C1s in the presence of EDTA.

C1 activation and dissociation in disease.

The composition of complexes containing C1 inactivator (C1 IA), C1r and C1s was investigated in normal serum after activation of C1 under various conditions. Analyses were performed with PAGE of eluates from Sepharose beads coated with F(ab')2 fragments of anti C1s followed by immunoblotting with anti C1 IA, anti C1s or anti C1r. Eluates obtained from serum treated with aggregated IgG (AGG) contained C1 IA in complex with C1r and C1s with both subcomponents in activated form. Eluates from serum incubated at 37 degrees C for 1, 2 or 3 days without activators showed C1 IA complexed with activated C1r and with C1s in proenzyme state associated to the complex. On analysis of serum, treated as mentioned above, by a variant of the electroimmunoassay using an intermediate gel containing anti-C1 IA and with anti-C1s in the anodal gel the two types of C1r--C1s--C1 IA complexes could be distinguished. Investigation of fresh sera and synovial fluids from patients with rheumatoid arthritis in this assay showed complexes containing C1 IA and C1r-C1s in activated form in the synovial fluids, while C1 IA-activated C1r-proenzyme C1s complexes were found in the corresponding sera.

C1 activation, with C1q in excess of functional C1 in synovial fluid from patients with rheumatoid arthritis.

Free Clq, in functionally active form was present in increased amounts in the synovial fluid of patients with rheumatoid arthritis. The presence of free Clq was associated with low concentrations of hemolytic C1, low C4 and raised amounts of C3dg/d fragments in the synovial fluid. The findings suggested intra-articular C1 activation with dissociation of C1 into free C1q and complexes containing C1r, C1s, and C1 inactivator. However, the immunochemical properties of synovial fluid C1r-C1s-C1 inactivator complexes appeared to differ from those of the complexes formed in serum, which hampered quantification with the assay used. Control patients with osteoarthritis or spondylarthritic syndromes did not show evidence of intra-articular complement activation, even though 1 patient with Reiter's disease had unexplained low concentrations of synovial fluid C4 and C3. The concentrations of circulating complement components were largely normal in the patients. Slightly increased concentrations of free C1q and C1r-C1s-C1 inactivator complexes in serum and C3dg/d fragments in EDTA plasma were observed, particularly in the patients with rheumatoid arthritis.

C1 dissociation in serum: estimation of free C1q by electroimmunoassay.

A two-stage electroimmunoassay was developed for measuring macromolecular C1 (C1qrs) and free C1q. The method was based on Ca2+ dependent fixation of C1qrs to agarose, followed by immune precipitation of dissociated C1s in the presence of EDTA. Free C1q was estimated from the increase in C1qrs resulting from saturation of C1q in the samples with purified C1r-C1s. The assay system was studied under various experimental conditions. Combined analysis by electroimmunoassay and crossed immunoelectrophoresis indicated that part of the free C1q in undiluted normal serum could be attributed to physiological C1 activation. Owing to concentration dependent C1qrs dissociation the proportion of free C1q increased with the dilution of serum. Results obtained with serum and with purified C1qrs were consistent with the formation of an equimolar C1q:C1r-C1s complex. However, the capacity for C1r-C1s binding appeared to be higher in the purified system than in serum. Serum concentrations of free C1q were high in some of the patients with disease conditions characterized by increased C1 activation, such as systemic lupus erythematosus or primary biliary cirrhosis.

Correlation among complement activation, protease inhibitors, and clinical course in acute pancreatitis in man.

Changes in complement levels and protease inhibitors were measured in plasma/serum and peritoneal fluid during 15 attacks of acute pancreatitis. The abnormalities found in the complement system and the protease inhibitors were most pronounced in severe attacks, especially in the peritoneal fluid. Depressed levels of C1q, C3, properdin, and factor I were found in blood on admission in severe attacks. A decrease during the first days of illness was found for C1q, C3, C4, properdin, factor I, and factor H levels in blood. There was a discrepancy between the low C1q and the high C1r and C1s levels in blood. Complexes of C1r-C1s-C1 inactivator and factor B conversion products were found, especially in the peritoneal fluid, denoting an activation of the complement system. High levels of trypsin in complex with alpha 1-protease inhibitor were found, both in blood and in peritoneal fluid, denoting the liberation of active trypsin in acute pancreatitis. The levels of the functional alpha 2-macroglobulin were low, especially in the peritoneal fluid. It is concluded that both classical and alternative complement activation take place in acute pancreatitis, starting in the peritoneal cavity. The magnitude of activation depends on the severity of the disease. Trypsin-induced activation of complement components may explain some of these changes.

Complement components and activation in primary biliary cirrhosis.

Total complement activity was normal in 18 patients with primary biliary cirrhosis using two hemolytic assays capable of distinguishing between defects in classical and alternative pathways. Activation of the classical pathway was demonstrated in all patients by formation of complexes between C1r, C1s, and C1 inactivator. Large amounts of free C1q, not in complex with C1r and C1s, were demonstrated in the majority of patient sera. Furthermore, C4 levels were within the normal range or slightly subnormal. No evidence for alternative pathway activation was found. Increased mean levels of several complement components, in particular C1 inactivator, C2, C3, factor B, factor H, were noted. A significant correlation between these complement factors, derived mainly from the liver, and ceruloplasmin suggests that this elevation might be secondary to cholestasis. In contrast, no significant correlation with levels of early reacting acute phase reactants, immunoglobulins, or circulating immune complex-like material were observed. It is concluded that activation of the complement system by the classical pathway is common in patients with primary biliary cirrhosis.

Frequent occurrence of anti-rabbit IgM in IgA deficiency.

Analysis of IgA deficient sera revealed impaired hemolysis of sensitized sheep erythrocytes when tested by a hemolysis in gel (HIG) assay developed for detection of complement deficiencies. All sera were normal in a test for the alternative pathway. The impaired hemolysis was not related to complement aberrations but was caused by antibodies to rabbit IgM, demonstrated in 14 of 21 IgA deficient sera, by use of HIG technique and by agglutination. The presence of these antibodies was not related to age, sex or disease. One serum was further examined and the antibodies were shown to be of IgG class.

Homozygous deficiency of C4 in a child with a lupus erythematosus syndrome.

A complete, selective lack of C4 was found in a girl who at 2 years of age presented with an atypical rash and low titres of antinuclear antibodies (less than 1/25). Rheumatoid factors were also found. The deficiency has been followed for 5 years. Tests for Chido and Rodgers antigens on the erythrocytes were negative. A possible proneness to bacterial infections has been noted with recurrent otitis media and purulent parotitis. At the age of 5, the patient developed polyarthritis of large joints and signs of glomerulonephritis. These symptoms responded well to high-dose steroid treatment. At present, there are initial signs of sclerodactylia and some persistent exanthema and parotic swelling. IgM levels were remarkably high with 19 S IgM at about 7 g/l and 7 S IgM at about 1.5 g/l. In the large kindred studied, lower immunochemical and functional C4 values were found in carriers of the genetical defect than in the rest of the family members. The C4 deficiency gene(s) segregated with HLA A2, Cw3, B40, BfS on the paternal, and with Aw30,-, B18, BfF1 on the maternal side of the family.

Immunosuppression and the additive effect of plasma exchange in treatment of rapidly progressive glomerulonephritis.

Attempts were made to evaluate the separate effect on kidney function of immunosuppressive treatment (IS) and plasma exchange (PE) in 27 patients with rapidly progressive glomerulonephritis (RPGN). Twenty-four of the patients were treated with PE. Initial IS was supplemented with PE within 6-12 days in 5 patients, and after at least 14 days in 13. Because of suspected septicemia, 2 patients were first treated with PE, and IS was not initiated until the possibility of septicemia had been excluded. In 4 severely ill patients wih rapid clinical deterioration, both treatments were started simultaneously. Twenty patients improved during one or both treatments, 4 with IS alone, 2 with IS and doubtfully with PE, 3 with IS and probably also with PE, 5 both with IS and PE and one with PE alone. In 5 patients the individual effects of IS and PE could not be evaluated. In another 2 patients the combined treatment seemed to influence the course favourably. In the remaining 7 patients the effect of the treatment was doubtful or nil. Two further patients with Goodpasture's syndrome were treated. They were admitted late, and both kinds of treatment were instituted simultaneously. One of them died in respiratory insufficiency, the other remained oliguric while the pulmonary changes faded. Thus, PE added a positive effect to IS in several patients with RPGN. The treatment had few and mostly mild side-effects.

Binding of purified C1 subcomponents, C1 inactivator and their complexes to immobilized heparin.

Under specified conditions purified C1q, activated C1r and C1s and C1r-C1s complexes were bound independently of Ca2+, to heparin-Sepharose, and could be eluted by an increasing salt gradient. Zymogen C1r and C1s, C1r-C1s complexes, C1 inactivator, and C1r-C1s-C1 activator complexes were not bound. However, at lower conductance Ca2+ independent binding of C14 occurred, which was utilized in the purification of C14 and C1s. In the presence of C1t (serum amyloid P component), C1s was firmly retained on heparin-Sepharose, which was probably due to formation of a C1s-C1t complex.

Screening for deficiencies in the classical and alternative pathways of complement by hemolysis in gel.

Two assays based on hemolysis in gel were assessed for screening complement (C) component deficiencies. In one assay sensitized sheep erythrocyte (EA) were incorporated in agarose gel containing Ca2+ and Mg2+, in the other guinea pig erythrocytes (GpE) were used in the presence of Mg2+and EGTA. With few exceptions, fresh samples from healthy individuals produced homogeneous areas of complete hemolysis in both assays. Clearly aberrant patterns were observed in approximately 4% of healthy blood donors. Sera from patients having complete deficiencies of Clq, C2 or C4 produced clear lysis of GpE only, whereas in sera lacking C3 or C8 lysis was grossly impaired in both assays. Properdin deficient serum produced very slight lysis of GpE but normal lysis of EA. Reconstitution of these C-deficient sera gave normal lysis. Together, the two assays supplement immunochemical C3 and C4 determinations for screening out C disorders.