C1q Binding Activity Research Articles

Cardiolipin-protein complexes and initiation of complement activation after coronary artery occlusion.

Specific rabbit anti-cardiolipin (anti-CL) antibodies were used to investigate the hypothesis that cardiolipin, associated with mitochondrial membrane proteins, binds C1 and facilitates activation of the complement cascade following reperfusion of ischemic myocardium. By immunoelectron microscopy, anti-CL localized to subsarcolemmal mitochondria, emerging through breaks in membranes of damaged cardiac myocytes. Anti-CL reacted with > 15 mitochondrial constituents, most of which comigrated with the proteins that bind C1q in transblots of subsarcolemmal mitochondria, fractionated by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate. A subset of the C1q-binding proteins > 24 to 37 kDa served as stable sites for assembly of C3, C5, and C9. Cardiac lymph, collected during the first hour after reperfusion of ischemic myocardium, contained proteins of diverse size that reacted with both anti-CL and C1q. Cardiac lymph, collected before occlusion and 4 to 5 hours after reperfusion, in comparison, had few if any C1q or anti-CL reactive proteins. Treatment with phospholipase suppressed the C1q-binding activity and anti-CL reactivity of the proteins in reperfusion lymph and those with similar properties in mitochondrial extracts. Our data suggest that during ischemia, mitochondria, extruded through breaks in the sarcolemma, unfold and release membrane fragments in which cardiolipin and protein are intimately associated. By binding C1 and supplying sites for the assembly of later-acting complement components, these fragments provide the means to disseminate the complement-mediated inflammatory response to ischemic injury.

Development of a C1q-ABO-ELISA to measure C1q binding by human anti-A alloantibodies

Little is known about the correlation between the amount of anti-A antibodies and the respective complement activating capacity. We have therefore developed a new ELISA procedure for the study of cmplement activation by anti-A antibodies bound to immobilized blood group substance A through the classical pathway. C1q binding capacity of anti-A was then compared to both anti-A immunoglobulin (Ig) isotype serum concentrations and to the hemolysing capacity of O type sera. We have demonstrated that sera with low anti-A IgG and IgM levels produced low C1q binding activity and no hemolysis of human A 1 red blood cells (RBC), whereas higher anti-A Ig levels resulted in either weak or strong C1q binding capacity as well as weak or strong RBC hemolysis. A correlation of r s = 0.755 was observed between C1q-ABO-ELISA and hemolysin assay results, although only 58% of the O type sera containing C1q binding anti-A lysed A 1 type RBC under the used conditions. The C1q-ABO-ELISA is therefore a more sensitive assay for detecting ‘dangerous’ O type donors for blood transfusion to A type recipients than screening tests for hemolytic anti-A or determination of anti-A Ig levels alone.

Residue at position 331 in the IgG1 and IgG4 CH2 domains contributes to their differential ability to bind and activate complement.

A conserved proline residue is found at position 331 in the CH2 domains of human IgG subclasses which fix complement. This residue is replaced by a serine in IgG4 which is inactive. To determine the role of residue 331 in the differential ability of human IgGs to activate the complement cascade, a pair of genetically engineered anti-dinitrophenol IgG1 and IgG4 antibodies with reciprocal mutations at position 331 were tested for their hemolytic activity as well as for their ability to bind C1q, activate C1 and cleave C4. The IgG1 Ser331 mutant was virtually unable to mediate the lysis of trinitrobenzene-sulfonic acid-derivatized sheep red blood cells as a result of a marked defect in C1q binding activity. In contrast, the substitution of Pro for Ser331 in IgG4 bestowed partial hemolytic activity (40%) to the IgG4 Pro331 variant. Under low ionic strength conditions, this mutant was found to be approximately 50 and 75% as active as wild-type IgG1 in the C1q binding and C4b deposition assays, respectively. These results indicate that residue Pro331, which folds into close proximity to a previously identified C1q binding motif (Duncan, A. R., and Winter, G. (1988) Nature 332, 738-740), contributes to the architecture of the IgG1 C1q binding site and that its replacement by a serine residue in IgG4 is largely responsible for the functional inactivity of this isotype.

Single-step purification of F(ab′)2μ fragments of mouse monoclonal antibodies (immunoglobulins M) by hydrophobic interaction high-performance liquid chromatography using TSKgel Ether-5PW

A procedure is described for preparation and single-step purification of F(ab')2 fragments, herein designated as F(ab')2 mu' from mouse monoclonal antibodies of the IgM class. Hydrophobic interaction high-performance liquid chromatography (HPLC) using TSKgel Ether-5PW was well applicable to the purification. The IgM was digested with pepsin at the pepsin-to-IgM ratio of 1:200 (w/w) in 100 mM citrate buffer (pH 4.2) at 37 degrees C for 2 h. The digests were applied to the gel equilibrated with the buffer containing 1 M ammonium sulfate. F(ab')2 mu fragments were adsorbed onto the gel with the same buffer, and eluted by reducing the ammonium sulfate concentration to 0 M. The fraction containing F(ab')2 mu fragments was homogeneous (purity higher than 97%) by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration HPLC. The recovery of the antigen-binding site was 55-72%. The cycle time of the Ether-5PW HPLC was 40 min, and up to 98 mg F(ab')2 mu fragments. The molecular mass of F(ab')2 mu was estimated to be 144-146 kDa. In comparison with IgM, F(ab')2 mu lost entirely the complement C1q binding activity, and the sugar content was greatly reduced. The binding of IgM with non-specific proteins turned to be negligible, when IgM was converted to F(ab')2 mu, suggesting that the fragments are useful for immunological application.

Platelet interactions with C1q in whole blood and in the presence of immune complexes or aggregated IgG

Previous studies identified specific receptors for C1q on human blood platelets in purified systems using monomeric C1q. To assess the physiologic potential of platelet C1q receptors, C1q binding was evaluated in whole blood and in the presence of immune complexes or aggregated IgG. Blood was obtained from healthy volunteers and collected directly into EDTA (1 vol 100m M EDTA:9 vol whole blood) and purified, 125I-labeled C1q or 125I-C1q associated with albumin-anti-albumin immune complexes. Samples were incubated at 22 or 37°C for 60 min, and total cell bound C1q and platelet associated C1q were quantified. Platelet-bound, monomeric C1q or immune complex-associated C1q represented 40–50% of total peripheral blood cell-associated C1q. C1q binding was unaffected by the incubation temperature, but the preincubation of 125I-C1q with immune complexes enhanced binding two- to threefold. This binding was partially inhibited by preincubating platelets with either the collagen-like amino-terminal fragments of C1q (c-C1q) or a monoclonal antibody Fab fragment recognizing platelet Fc receptors. A more complete inhibition was achieved if platelets were preincubated with both agents. Similar observations were made using washed platelets and 125I-C1q associated with aggregated IgG. The role of C1q and platelet C1q receptors in enhancing aggregated-IgG binding to platelets was further supported by experiments demonstrating increased 125I-aggregated IgG binding to platelets not only after preincubation of 125I-aggregated IgG with C1q but also following platelet preincubation with C1q. These data suggest that C1q receptors may participate in the localization and presentation of C1q-associated immune complexes on the platelet surface and demonstrate that platelets contribute significantly to the C1q binding activity of peripheral blood.

Low HIV-1 proviral DNA burden detected by negative polymerase chain reaction in seropositive individuals correlates with slower disease progression

During 1989, 316 members of a cohort of homosexual men were tested for HIV-specific DNA by the polymerase chain reaction (PCR) using a pair of gag-region primers. Of 125 HIV-seronegative subjects, 123 (98.4%) were PCR-negative while 158 (82.7%) of 191 HIV-seropositive subjects were PCR-positive. Fewer of the 33 subjects who were seropositive and PCR-negative were at Centers for Disease Control (CDC) stage IV than the seropositive, PCR-positive subjects (6 versus 25%; P = 0.030). The seropositive, PCR-negative group had higher mean CD4 counts (640 versus 490 x 10(6) cells/l; P = 0.006), higher CD4: CD8 ratios (0.92 versus 0.64; P = 0.004), lower immunoglobulin (Ig) G levels (1290 versus 1645 mg/dl; P = 0.002), lower IgA levels (168 versus 251 mg/dl; P less than 0.001), and lower C1q binding activity (8 versus 14%; P = 0.010) than the seropositive, PCR-positive subjects. The median rate of CD4 cell decline in the 3 years preceding the PCR sample was less marked in the seropositive, PCR-negative group than the seropositive, PCR-positive group (-58 versus -77 x 10(6) cells/l per year; P = 0.028). To control for duration of infection, we restricted the analysis to the subgroups of 11 seropositive, PCR-negative subjects and 34 seropositive, PCR-positive subjects who had seroconverted earlier in the cohort study. Both subgroups had similar durations of infection, yet the same pattern of differences persisted.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2, soluble interleukin-2 receptor and tumor necrosis factor in sera from patients with rheumatoid arthritis

Interleukin-2 (IL-2), soluble interleukin-2 receptor (IL-2R) and tumor necrosis factor (TNF) have been measured in sera from 47 patients affected by classic rheumatoid arthritis (RA) using an enzyme-linked immunosorbent assay. The patients were divided into 4 groups as follows: group A, 18 patients with inactive disease; group B, 19 patients with active disease under treatment with non-steroidal antiinflammatory drugs (NSAID) and second-line drugs; group C, 5 patients with active disease under treatment with NSAID and cyclosporine A (CSA) for at least 4 months; group D, 5 patients in the same condition as patients of group C, but treated with azathioprine (AZA) instead of CSA. IL-2 was undetectable in all patients except two, both characterized by active disease. Soluble IL-2R levels were above the upper limit of the normal range in most of the patients studied, but the mean value ( +/- 1 SD) was significantly higher in patients of group B (1,288 +/- 421 U/ml) than in patients of group A (686 +/- 205 U/ml) and group C (842 +/- 414 U/ml). In two patients affected by active RA treated with pulse methylprednisolone therapy (1 g/day for 3 alternate days) the values of soluble IL-2R dropped from 948 to 662 U/ml and from 660 to 518 U/ml, respectively. No statistically significant correlation was observed between the serum level of IL-2R and the RF titre or percentage of C1q-binding activity, respectively. TNF was found within the normal range in all patients except one, who was characterized by active arthritis, high number of rheumatoid skin nodules and extremely high RF titre.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome

Urticaria, angioedema, and arthritis are cardinal features of hypocomplementemic urticarial vasculitis syndrome (HUVS). Considered to be an immune complex-mediated disorder, HUVS has been differentiated from systemic lupus erythematosus (SLE), based on its clinical manifestations and the C1q precipitin (C1q-p) reaction, which is manifested as gel precipitation of C1q by a small percentage of HUVS IgG molecules. This phenomenon has been attributed to an Fc region abnormality, and the responsible IgG molecules are said to possess C1q-p activity. We purified IgG from 4 HUVS patients and confirmed that HUVS IgG contains C1q binding activity. F(ab')2 fragments from these patients also bound to C1q, as measured by 2 different C1q binding methods at physiologic ionic strength; HUVS IgG Fc fragments did not bind to C1q. Preincubation of HUVS F(ab')2 fragments with antibody to human F(ab')2 prevented subsequent binding to C1q. We conclude that IgG antibodies to C1q are present in HUVS serum, and it is likely that these antibodies are C1q-p. Because the clinical manifestations of HUVS and the presence of anti-C1q antibodies have been described in patients with SLE, our findings support the concept that HUVS is an autoimmune syndrome related to SLE.

C1q binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids.

We have constructed four different deletion mutants of a chimeric mouse-human IgG3 anti-(4-hydroxy-3-nitrophenyl)acetyl/(5-iodo-4 hydroxy-3 nitrophenyl) acetyl (NP/NIP) antibody lacking one or more of the four exons coding for the hinge region. The mutant variants all retained intact hinge region epitopes since they all reacted with IgG3 hinge-specific antibodies. Surprisingly, all the deletion mutants bound C1q equally well or even better than the wild type. Thus the high C1q binding activity of IgG3 compared to IgG1 is apparently not due to the total length of the IgG3 hinge, which is 62 amino acids, nor is it due to the length of the upper hinge which is the stretch from the end of CH1 to the first inter-heavy chain disulfide bond.

Immunologic determinants of head and neck cancer response to induction chemotherapy.

Various measures of immune response were assessed prior to induction chemotherapy (intravenous [IV] cisplatin, fluorouracil [5-FU], and bleomycin) in 43 previously untreated head and neck cancer patients to derive a clinical response prediction model. These were parameters of functional cellular immunity (natural killer [NK] cell activity, lymphocyte blastogenesis response to mitogens), total lymphocyte and lymphocyte subset numbers and percentages, and circulating humoral immunity (total immunoglobulin, immunoglobulin classes, and C1q binding activity [C1q BA]). The C1q BA may reflect levels of circulating immune complexes within peripheral blood. The objective primary tumor response rate was 65% (16 complete responses and 12 partial responses). Univariate logistic regression analysis showed that failure to respond to therapy was significantly related to higher value (vis-à-vis response) of humoral immune parameters total immunoglobulin (Ig), P less than .01; IgG, P less than .01; and C1q BA, P less than .001. No association between cellular immune response measurements and response to chemotherapy was identified. By multivariate logistic regression analysis, only C1q BA levels were predictive of drug therapy responsiveness (P less than .001). Results extend our previous investigations regarding C1q BA measurement in head and neck cancer patients, and show that C1q BA levels add accuracy of prediction of subsequent chemotherapy response to that based solely on standard staging criteria and other parameters of immune status.

Immune complexes and Ross River virus disease (epidemic polyarthritis).

Immune complexes were sought in serum and synovial fluid in Ross River virus disease (epidemic polyarthritis). Multiple samples from 15 patients showing varied degrees of disease activity over a 3 month period were analysed for their content of complement components C3 and C4, and for C1q solid-phase and Raji cell binding activity. Levels of C3 and C1q binding activity were normal. C4 and Raji cell binding activity were normal except for three high levels of Raji cell binding, of which two were accompanied by low levels of C4, with normal C3 and C1q binding. Synovial fluid showed anomalous Raji cell reactivity of uncertain significance. Conglutinin solid-phase binding activity and IgG rheumatoid factor were compared in the serum of 20 patients during active disease and after recovery. The results were identical and within the normal range in both phases. One patient developed IgM rheumatoid factor in a low titre late in his illness. Although these findings do not entirely exclude a role for immune complexes formed at the onset in the circulation or tissues, it is concluded from this and other evidence that circulating complexes are not commonly responsible for the persistence of syndromes in this disease.

Characterization of immune complexes detected by the 125I-C1q binding assay in breast cancer

The aim of the present study was to isolate and characterize immune complexes measured by the 125I-C1q binding assay in breast cancer sera. The C1q binding assay detects immune complexes by their binding to 125I-C1q and subsequent precipitation with PEG. We purified the C1q binding material from these precipitates by superose 6 gel filtration and cation exchange chromatography. The isolation steps were monitored by the C1q binding assay and the purified material was analyzed by SDS-PAGE and immunodiffusion. The C1q binding material isolated from two breast cancer sera contained complexes of IgM, C4b binding protein (C4-bp), and C1q. The C4-bp fraction showed significant C1q binding activity which increased after the addition of IgM. This C4-bp was not different from C4-bp isolated from normal serum. The IgM fraction, however, differed from normal IgM by its binding to C4-bp and C1q and its stronger affinity to the cation exchange column. These unique properties were not due to rheumatoid factor activity. They might be caused by a different glycosylation pattern or by complex formation with an as yet unknown polysaccharide antigen.

The presence of histidine residues at or near the C1q binding site of rabbit immunoglobulin G

Treatment of covalently crosslinked rabbit IgG oligomers with diethylpyrocarbonate resulted in the loss of their C1q binding activity. The inactivation was a first-order process with respect to time in the range 0–8 min, and modifier concentration from 0 to 2.39 mM. Hydroxylamine treatment of diethylpyrocarbonate-treated IgG oligomers led to 80% recovery of their C1q binding activity. Diethylpyrocarbonate treatment of IgG oligomers had little effect on their absorbance at 278 nm, but led to an increase in their absorbance at 242 nm. The apparent p K a of the modified residues was 6.91 ± 0.12. These data are consistent with diethylpyrocarbonate modification of histidine residues leading to loss of C1q binding activity in rabbit IgG oligomers. Modification of four histidine residues per IgG molecule was associated with the loss of C1q binding activity. Thus, there may be two histidine residues at or near the C1q binding sites in the C H2 domains of rabbit IgG.

Isolation and analysis of complement activating aggregates from synovial fluid of patients with rheumatoid arthritis using monoclonal anti-C3d antibodies.

The complement activating aggregates in synovial fluids of patients with rheumatoid arthritis (RA) have been isolated using monoclonal IgM anti-C3d antibodies attached to solid phases, and the content of the material bound has been analysed. High levels of aggregated IgG bearing C3d were found in RA synovial fluids, and IgG was the major immunoglobulin bound from such synovial fluids by anti-C3d Sepharose. A strong correlation was shown between levels of aggregated IgG bearing C3d and complement activation, as judged by C3d levels. Significant (but less strong) relationships were also observed between C3d levels and both complement consuming and C1q binding activity. C3d levels and levels of aggregated IgG bearing C3d were both significantly associated with the numbers of polymorphonuclear leucocytes (PMNs) found in RA synovial fluids. From these results it is concluded that the aggregated immunoglobulins bearing C3d (particularly IgG) isolated from RA synovial fluids are responsible for activating complement and attracting PMNs into the joint space. Radioimmunoassay showed no correlation, however, between levels of aggregated IgG (or IgM) bearing C3d and rheumatoid factor (RF) activity bound by anti-C3d. In addition, the material bound by anti-C3d Sepharose from most synovial fluid polyethylene glycol precipitates did not contain either IgM or IgG RF. Thus both techniques show that the majority of complexes bearing C3d do not contain RF. As the complement fixing aggregates apparently contain only immunoglobulin and complement components the results raise the problem of how the aggregates are formed. It is suggested that RA IgG may remain aggregated after either antigen or antibody (RF) has dissociated from the complex.

Serum immune complexes containing IgA appear to predict erosive arthritis in a longitudinal study in rheumatoid arthritis.

Fifty seven patients with rheumatoid arthritis (RA) were studied longitudinally, and the presence of rheumatoid factor (RF) and various types of immune complexes (IC) was correlated with joint activity and the presence of extra-articular features (EAF). In a cross sectional study it was found that the levels of circulating IC and RF correlated significantly with joint disease activity and the presence of EAF. Longitudinally, levels of IC measured by the C1q binding activity and IC containing IgG and IgM correlated significantly with fluctuations in joint disease activity, whereas IC containing IgG and IgA correlated with the occurrence of EAF. RF and IC levels, however, did not predict the clinical course of the disease. IC containing C3 and C4 were found infrequently and were only present in patients with active rheumatoid vasculitis (RV). The continuous presence of these IC appeared to be linked to the recurrence of vasculitis, irrespective of treatment. Significantly more erosions of hands and feet were found after one year follow up in those RA patients who presented early (disease duration less than one year) who initially had a raised serum IgA IC level (r = 0.72; p less than 0.005).

Binding of complement component C1q by rat adipocyte membranes

Human C1q was found to bind to rat adipocyte membranes with an affinity comparable to that for aggregated immunoglobulin. The binding was ionic strength dependent, and modification of arginyl and histidyl residues in C1q abrogated its binding activity. Treatment of the adipocyte membranes with either high ionic strength buffers, EDTA or trypsin had little effect on their C1q-binding activity.

Early rheumatoid-like synovial lesions in rabbits drinking cow's milk. II. Antibody responses to bovine serum proteins.

Thirty-six percent of Old English rabbits fed pasteurized cow's milk developed early rheumatoid-like synovial lesions. All rabbits drinking milk developed high titres of serum and synovial fluid Clq-binding activity. This has been shown to be due to specific antibody to Clq, produced in response to Clq in cow's milk. In addition, these rabbits responded to other bovine proteins present in cow's milk, including conglutinin, beta-lactoglobulin and IgG. There was no correlation between serum or synovial fluid titres of antibody to bovine milk proteins and synovial lesions.

Significance of circulating C1q-binding activity in chronic liver disease: a study of 133 cases.

Circulating immune complexes (CIC) were measured in 133 biopsy-proven patients with various liver diseases. The correlation between CIC levels and other laboratory findings was investigated in each disease group, in order to assess if the increased C1q-binding activity found in these patients was related to particular features of the disease. CIC levels were not significantly different in HBsAg-positive and HBsAg-negative patients. No correlation was found between CIC levels and serum bilirubin, AST, ALT and C3 levels. A negative correlation with C4 levels and a positive correlation with immunoglobulin levels were found in the majority of the patients, while prothrombin time and albumin levels were negatively correlated to CIC levels only in patients with chronic active hepatitis. Increased CIC levels could represent a response to gut-associated antigens, a passive accumulation due to reduced hepatic function or both.

Influence of ionic strength, EDTA concentration, endogenous C1q and polyanions on the 125I-C1q-binding test

Several parameters of the 125I-C1q-binding test were investigated: ionic strength, pH, concentration of EDTA, influence of serum C1q and the possibility of interference by polyanions. Lowering the ionic strength of the borate buffer resulted in increased precipitation of 125I-C1q in normal human serum. This increase was dependent on the presence of serum proteins, probably immunoglobulins. When the concentration of the EDTA was decreased, increased precipitation of 125I-C1q in normal human serum was also observed. This was prevented by adding NaCl to the EDTA solution. However at very low concentrations of EDTA (too low to chelate all calcium ions in the serum), increased precipitation of 125I-C1q in normal human serum was observed even in the presence of added NaCl. Addition of purified C1q to sera from patients with very low C1q levels had varying effects on the results of the C1q-binding test: (a) it decreased the C1q-binding activity of some sera, probably by competition with 125I-C1q for binding sites on the immune complexes; (b) it increased the C1q-binding activity of other sera, probably by enhancing the precipitation of immune complexes as a consequence of the cross-linking effect of C1q; or (c) it had no influence, possibly due to the opposite effects of (a) and (b). The addition of dextran sulphate resulted in a dose-dependent increase in the 125I-C1q-binding activity of normal human serum. This effect was dependent on the interaction of dextran sulphate with either C1q or low-density lipoproteins and was prevented by addition of polybrene to the assay. However, addition of polybrene to sera with a high C1q-binding activity scarcely influenced binding activity.

Mononuclear phagocytes in rheumatoid arthritis: Fc-receptor expression by peripheral blood monocytes.

Fc receptor expression by enriched monocytes from rheumatoid arthritis (RA) patients and age and sex matched controls (healthy subjects) was compared by measuring the uptake of IgG on monocytes in a competitive radioassay. The association constant (Ka) between IgG and the monocytes and the number of Fc binding sites per cell was calculated from Scatchard plots of 4 degrees C binding data. RA monocytes had increased expression of Fc receptors as compared with those of controls. This increase was particularly pronounced in those RA patients affected by extra-articular disease. There were significant correlations between the numbers of Fc receptors on monocytes and both C1q binding and anticomplementary activity but none between monocyte Fc receptor numbers and serum rheumatoid factors (IgG and IgM). It is considered that monocyte handling of circulating immune complexes is unimpaired in RA and that monocytes make an adaptive response to increased levels of immune complexes.