C1q and immune complexes in liver cirrhosis sera

The highest mean value of the serum C1q concentrations among chronic liver diseases was obtained in patients with liver cirrhosis (LC). C1q serum levels seemed to increase along the progression of liver damage. Serum levels of C1q and CH50 in patients with LC and systemic lupus erythematosus (SLE) were simultaneously estimated. No correlation between C1q and CH50 levels was observed in LC sera, while a significant correlation was demonstrated in SLE sera. It could be suggested that the mechanism for the low CH50 in LC sera seemed different from that in SLE sera, and that the activation of classical complement pathway was not the predominant cause in LC sera. Correlations of serum levels between immune complexes and C1q were examined in patients with LC and SLE. It could be deduced from the results of a positive correlation in LC sera and the reverse tendency in SLE sera that the significances of C1q and immune complexes in LC sera differed from those in SLE sera.     

Circulating immune complex-like materials which bind to heat inactivated C1q interfere with the C1q solid phase assay for immune complexes

C1q solid phase assay (C1q SP) was devised based on the fact that immune complexes (IC) and aggregated human globulin (AHG) bind to C1q. Neither IC nor AHG was found to bind to heat inactivated C1q. On the other hand, circulating immune complex (CIC)-like materials in patients' sera were able to bind to heat inactivated C1q, indicating that these CIC-like materials are not true CIC. Gel filtration analysis showed that molecular size of such CIC-like materials was almost the same as monomeric IgG, while true CIC were in heavy fractions. True CIC did not bind to heat inactivated C1q but bind only to native C1q. The CIC-like activity is not due to rheumatoid factors. About 2/3 of CIC positive sera by C1q-SP are not really CIC positive but are due to interference by the CIC-like materials.     

Binding of complement component C5 to model immune complexes and the use of anti-C5 antibodies for determination of C5-containing circulating immune complexes

When normal human or mouse serum is added to micro ELISA plates coated with monomeric or aggregated IgG, complement component C5 binds to IgG. C5 binding was demonstrated with a specific chicken anti-C5 antibody. Hydrazine treatment of the serum or addition of EDTA to the serum abolished the binding of C5. C5-deficient mouse serum was negative for C5 binding, whereas the same serum supplemented with human C5 restored the binding of C5. Chicken anti-C5-coated plates were used for determination of C5-containing circulating immune complexes (CIC). Increased concentrations of CIC were found in sera from patients with rheumatoid arthritis and Bell's palsy.     

A C1q solid phase microenzymatic assay for the detection of soluble immune complexes

Summary The solid phase C1q-binding assay has been adapted to an enzymatic micromethod in which alkaline phosphatase labeled solubleStaphylococcus aureus protein A is used in place of the second antibody. The assay, which is run in microtiter plates, provides a rapid, sensitive (0.030 mg/ml of human heat-aggregated IgG detected) and reproducible method for the measurement of soluble immune complexes in a large number of samples. Soluble immune complexes preparedin vitro with bovine serum albumin (BSA) and anti-BSA antibodies on a wide range of antigen to antibody ratios were all detected with this method. When applied to the screening of unselected patient sera, soluble immune complexes were frequently found in systemic lupus erythematosus (52%) and chronic active hepatitis (57%) and in lower percentages in patients with malignant melanoma (28%), rheumatoid arthritis (30%) and essential mixed cryoglobulinemia (17%). This study was supported in part byConsiglio Nazionale delle Ricerche (CNR), Roma, Italy, ‘Progetto Finalizzato Controllo della Crescita Neoplastica’, grant no. 80.01599.96.     

C1q抗原/C1q抗体与系统性红斑狼疮疾病研究进展

补体系统是固有免疫系统重要的组分之一,不但是机体防御病原体的第一道防线,而且参与清除自身衰老和凋亡细胞等物质在体内的堆积,在词控炎症反应和免疫应答的过程中发挥极其重要的作用.补体分子C1q具有凋节各种细胞反应的能力.近年,随着C1q功能研究的深入,发现C1q抗原/C1q抗体与系统性红斑狼疮疾病有着一定的关联性.     

Circulating immune complexes in systemic lupus erythematosus: a reappraisal of the solid phase C1q radioimmunoassay

Data from a pool of 150 patients with systemic lupus erythematosus (SLE) were used to evaluate the clinical usefulness and specificity of the solid phase C1q radioimmunoassay (SPC1q RIA) for circulating immune complexes (CIC). We found that the assay results correlated with the severity of lupus nephritis but that they did not adequately reflect or predict disease activity so as to directly influence decisions regarding treatment. In addition, the contribution of non-IC reactants to the assay results was found to be of some concern. Finally, we could not demonstrate the presence of anti-DNA antibodies in CIC from patients with SLE.     

Quantification of circulating immune complexes by chicken anti-C4 micro ELISA

A quantitative assay for C4-containing immune complexes (IC) by a solid phase anti-C4 micro ELISA is described. It is based upon the use of an affinity purified chicken anti-human C4 antibody to capture the immune complex, and protein A-alkaline phosphatase for detection. The chicken antibody was chosen as capture antibody because it does not react with rheumatoid factor, does not activate the human complement system and is not detected by anti-mammalian IgG antibodies or protein A. Increased levels of C4 containing circulating immune complexes (CIC) were detected in sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and lung cancer, when compared with normal sera. Normal levels of C4 containing immune complexes were found in sera from patients with Bell's palsy.     

Evaluation of the C1q solid-phase binding assay for immune complexes. A clinical and laboratory study.

The solid-phase C1q binding assay for circulating immune complexes has been evaluated. The assay provides a rapid, sensitive (detecting as little as 1 microgram of aggregated IgG) and reproducible procedure for the detection of immune complexes in biological fluids. Using artificially prepared immune complexes, the assay detects complexes at four-times antigen-excess. Gel filtration over Sepharose 6B showed that these complexes were distributed over a range of molecular weights from greater than 4 x 10(6) to 300,000 daltons. Using radiolabelled anti-BSA, antigen (BSA) could be detected in these complexes. Screening of gel-filtered SLE showed that the assay detects complexes of both high and low molecular weight, but does not detect all complexes in the SLE sera. Clinical studies showed that immune complexes are frequently found in the sera of patients with SLE and measurement of the concentrations of complexes provides a more sensitive index of disease activity than either serum C3 or C4 concentrations or DNA binding capacity. In patients with RA concentrations of immune complexes were generally higher in synovial fluid than serum, although a patient with systemic rheumatoid disease with hypocomplementaemia had an extremely high level of circulating immune complexes. The assay only infrequently detects circulating immune complexes in glomerulonephritis and in renal transplant recipients. It is concluded that the assay provides a useful clinical tool, but detects only a limited species of immune complexes. It can be used in the detection of antigens in complexes.     

A method of isolating C1q from human serum and its use in the solid-phase immunoenzyme determination of immune complexes

C1q was isolated from human serum by dialysis in 0.24 M EDTA, followed by affinity chromatography on immobilized IgG and removal of IgG traces in a column with anti-IgG antibodies. Microplates were coated with C1q in PBS at 10-20 mg/l, nonspecific binding sites were saturated with human serum albumin. The sera were diluted 16-fold in 0.05 M PBS, 0.01 M EDTA, 0.05% Tween. After incubation with diluted samples the plates were treated with horseradish peroxidase--anti-human IgG conjugates. Enzymic activity was measured by adding p-phenylenediamine (0.2 g/l) in acetate buffer, pH 5.9, containing 0.05% H2O2. The sensitivity of the assay ranged between 2.5 and 300 mg/l.     

C1q governs deposition of circulating immune complexes and leukocyte Fcgamma receptors mediate subsequent neutrophil recruitment

Inflammation induced by circulating immunoglobulin G-immune complexes (ICs) characterizes many immune-mediated diseases. In this work, the molecular requirements for the deposition of circulating ICs and subsequent acute leukocyte recruitment in mice were elucidated. We show that after intravenous injection, preformed soluble ICs are rapidly deposited in the postcapillary venules of the cremaster microcirculation, secondary to increased vascular permeability. This deposition is dependent on complement C1q. IC deposition is associated with leukocyte recruitment. Leukocyte rolling, which is mediated by P-selectin in the exteriorized cremaster muscle, is not further increased in response to ICs. In contrast, leukocyte rolling velocity is significantly decreased and leukocyte adhesion is significantly increased in the presence of ICs. The IC-mediated slow leukocyte rolling velocity and subsequent adhesion and emigration are dependent on Fcgamma receptors (FcgammaRs), particularly FcgammaRIII, with complement C3 and C5 having no detectable role. These studies suggest a regulatory mechanism of IC deposition and leukocyte trafficking in IC-mediated inflammation requiring C1q and FcgammaRs in sequential, noninteracting roles.     

ELISA检测血清C1q抗体水平的研究

目的 研究检测血清C1q抗体方法.方法 建立ELISA检测血清C1q抗体水平的程序,应用棋盘滴定的方法确定ELISA检测人血清C1q抗体系统中包被抗原以及酶标抗体的最适工作浓度.检测92份正常健康体检合格者的血清C1q抗体水平,确定参考值范围.结果 C1q抗原的包被浓度5μg/ml,酶标抗体的工作浓度1：50000.批内和批间重复性试验的变异系数均小于＜15%.血清C1q抗体浓度参考值范围0-30.55 U/ml.结论 小批血清的实验检测表明此方法的特异性较好,操作简单.适于血清C1q抗体水平的检测.     

C1q抗体在狼疮性肾炎诊断中的意义

目的检测系统性红斑狼疮(SLE)患者及非狼疮性肾病综合征患者的C1q抗体(C1qAb)的水平,探讨C1qAb在狼疮性肾炎诊断中的意义,并作C1qAb与抗核抗体(ANA)、抗ds鄄DNA和SLEDAI的相关性分析。方法用ELISA对106例SLE、30例非狼疮性肾病综合征患者和30名健康对照者外周血血清中C1qAb进行检测。同时将SLE患者分为狼疮性肾炎(LN)活动期组(A组,76例)和肾外全身症状组(B组,30例),比较各组C1qAb和其他免疫学指标的阳性率差异。分析C1qAb与ANA、抗ds鄄DNA和SLE病活动指数(SLEDAI)的相关性。结果A组C1qAb滴度为(218.4±179.3)IU/ml,明显高于B组(56±61)IU/ml(P<0.01),与肾病综合征组(33±29)IU/ml及对照组(25±21)IU/ml相比P<0.001。A组的C1qAb阳性率为67.1%,而B组阳性率仅为26.6%;肾病综合征组阳性率为6%。以上结果显示C1qAb与LN关系密切。LN活动期C1qAb水平升高,缓解期可恢复至正常。C1qAb的升高与抗ds鄄DNA的升高可同时出现(r=0.28),C1qAb与SLEDAI呈正相关(r=0.72)。结论C1qAb可作为诊断LN的一个重要实验室指标,与临床活动密切相关。     

Anti-C1q autoantibodies deposit in glomeruli but are only pathogenic in combination with glomerular C1q-containing immune complexes

Anti-C1q autoantibodies are present in sera of patients with several autoimmune diseases, including systemic lupus erythematosus (SLE). Strikingly, in SLE the presence of anti-C1q is associated with the occurrence of nephritis. We have generated mouse anti-mouse C1q mAb's and used murine models to investigate whether anti-C1q autoantibodies actually contribute to renal pathology in glomerular immune complex disease. Administration of anti-C1q mAb JL-1, which recognizes the collagen-like region of C1q, resulted in glomerular deposition of C1q and anti-C1q autoantibodies and mild granulocyte influx, but no overt renal damage. However, combination of JL-1 with a subnephritogenic dose of C1q-fixing anti-glomerular basement membrane (anti-GBM) antibodies enhanced renal damage characterized by persistently increased levels of infiltrating granulocytes, major histological changes, and increased albuminuria. This was not observed when a non-C1q-fixing anti-GBM preparation was used. Experiments with different knockout mice showed that renal damage was dependent not only on glomerular C1q and complement activation but also on Fcgamma receptors. In conclusion, anti-C1q autoantibodies deposit in glomeruli together with C1q but induce overt renal disease only in the context of glomerular immune complex disease. This provides an explanation why anti-C1q antibodies are especially pathogenic in patients with SLE.     

Anti-IgG type test to assay circulating immune complexes from polyethylene glycol precipitates compared with C1q binding and Raji cell tests

Immune complexes (IC) from over 100 normal and patient serum samples were assayed by turbidimetry using anti-IgG as the indicator following a three-step extraction with polyethylene glycol. For 70 patient samples, a high linear correlation was found between the present assay and the C1q-binding test (r = 0.812). Correlation with the Raji cell test was poor (r = 0.176). Both the present test and the Raji cell test showed greater sensitivity for identifying sera with elevated IC levels than the C1q binding test. The present approach may be accomplished using indicators other than turbidimetry, such as light scatter and isotopically-labelled anti-IgG.     

Estimation of immune complexes by a microplate-adapted C1q-Protein A enzyme-linked-immunosorbent-assay (C1q-PA-ELISA)

A microplate-adapted enzyme linked immunosorbent assay (ELISA) for detection of C1q-binding immune complexes (IC) and aggregated IgG (delta IgG) is described. Purified human C1q was adsorbed to the wells of flat-bottomed microtiter plates and EDTA-treated serum samples were subsequently introduced. Bound IC was measured by use of alkaline phosphatase-labelled Protein A followed by the substrate para-nitro-phenyl-phosphate. A dose response was found for both delta IgG and BSA anti-BSA complexes, while variations in the concentration of monomer IgG did not affect the optical density. Elevated levels of IC were found in the majority of sera from patients with rheumatoid arthritis and SLE. The described C1q-PA-ELISA is a simple and inexpensive method for detection of C1q-binding immune complexes. The reproducibility is acceptable and the sensitivity is higher than for most IC-methods based on C1q-binding.     

Role of anti-C1q autoantibodies in the pathogenesis of lupus nephritis

Anti-C1q autoantibodies can be found in the sera of patients with several autoimmune diseases, but also in healthy individuals. Although these anti-C1q autoantibodies were already identified several decades ago, they still puzzle both immunologists and nephrologists. The main reason for this puzzling effect are observations that seemed to indicate quite clearly that anti-C1q should be pathogenic to the kidney and the observation on the other hand that anti-C1q autoantibodies can be found in several disease conditions, as well as in healthy individuals, and are then unrelated to overt renal inflammation. This puzzle is the focus of the current review, which will provide an overview of the historical data, define the clinical interests and, importantly, will try to put several aspects in perspective based on recent observations in patients and in murine models. In addition, the paper will discuss therapeutic intervention possibilities regarding anti-C1q-mediated damage in systemic lupus erythematosus, as well as the therapeutic potential of anti-C1q antibodies in other conditions.     

猪血补体成分在人循环免疫复合物测定中的应用

为更好测定人血清循环免疫复合物（ＣＩＣ），从新鲜猪血中据取高纯度的猪血补体成分Ｃ１ｑ，采用酶联免疫吸附技术建立了猪血Ｃ１ｑ测定ＣＩＣ的方法。人血清ＣＩＣ测定结果用热凝集免疫球蛋白（ＡＨＧ）表示，正常人群血清ＣＩＣ＜３３ｍｇ／ＬＡＨＧ。测定３７例急性肾小球肾炎和１４例川崎病患者，其血清ＣＩＣ浓度在发病急性期普遍升高，经过治疗缓解或病愈后，ＣＩＣ浓度逐步降为正常水平。该法所用的猪血来源丰富，可避免乙型肝炎、艾滋病源的污染，降低了实验背景的测定值，使结果更接近于真实水平，有助于临床对免疫复合物性疾病的诊断及疗效判断。     

C1q deviation test for the detection of immune complexes, aggregates of IgG, and bacterial products in human serum

This report describes a new, rapid, sensitive, and quantitative method for the detection of immune complexes, endotoxins, and other complement activating materials in patients sera utilizing the ability of these substances to react with isolated C1q. The procedure is based on the inhibition of radiolabeled C1q binding to sensitized sheep erythrocytes by C1q-reactive substances in pathological sera. The C1q deviation test may be performed on 50 mu1 of serum, using 1 mug of radiolabeled C1q per sample. The procedure may be completed in 1.5-2 h, it is capable of detecting 5 mug of aggregated human IgG per ml of serum, and its coefficient of variation is 4.2%. Application of the test to the study of 193 sera from 43 patients with Dengue hemorrhagic fever showed a positive correlation between degree of C1q deviation and severity of disease.