C1q binding and C1q deviation assays using enzyme labelled C1q

Methods for the enzyme labelling of C1q are described. The C1q conjugates have been incorporated into suitably modified C1q deviation and C1q binding assays for circulating immune complexes. These assays show the expected high positivity rate in sera from patients with systemic lupus erythematosus. The assays using enzyme labelled C1q confer considerable advantages over their counterparts using 125I.     

The effects of high-intensity exercise on skeletal muscle neutrophil myeloperoxidase in untrained and trained rats

The primary purpose of this study was to examine the effects of high-intensity acute exercise on neutrophil infiltration in different muscle fiber types of untrained rats and to compare postexercise neutrophil accumulation in muscles of untrained and trained animals. The effect of high-intensity acute exercise on blood neutrophil degranulation reaction in trained animals was also elucidated. Neutrophil enzyme myeloperoxidase (MPO) was determined as a measure of neutrophil migration into muscles and blood neutrophil degranulation. Male albino rats were subjected to acute exercise and 5 weeks of training. The used model of intensive acute exercise consisted of 5, 15, and 25 intermittent swimming bouts with the addition of weight (8% of total body mass) for 1-min each, followed by 1.5-min rest intervals. MPO was analyzed in quadriceps muscle (white and red portion) and in soleus muscle 24 h after acute exercise. MPO content in resting blood plasma and neutrophils was determined 48-h following the completion of a training process. In addition, MPO content in the trained rats was measured immediately (in blood plasma and neutrophils) after and 24 h (in muscles) following a single-bout of exercise to exhaustion. The remaining two-third of the trained animals were exposed to a single-bout of nonstop swimming with the addition of 6% body mass until exhaustion. These animals were sacrificed immediately and 24 h after loaded swimming to analyze leukocyte count, MPO content in blood plasma and neutrophils and in muscles, respectively. About 24 h after exercise MPO concentrations in the red portion of quadriceps muscle and in soleus muscle were 4–7-fold higher as compared to the white portion of m. quadriceps. There was an association between the quantity of repetitive bouts of swimming and MPO content in the muscles. The duration of swimming to exhaustion of trained rats was 3.8-fold longer than untrained sedentary control. At rest, plasma MPO concentration was found to be 40% higher in trained rats compared to untrained controls (P < 0.05). Postexercise plasma MPO concentrations were significantly higher both in untrained (+137%; P < 0.05) and trained (+81%; P < 0.05) rats compared to resting values. At rest neutrophil MPO concentration was found to be 33% lower in trained rats compared to untrained controls (P < 0.05). There were no significant differences in muscle MPO concentrations between untrained and trained rats at rest. A single-bout of exercise to exhaustion produced a greater increase in MPO content in untrained compared to trained rats. The data suggest that postexercise neutrophil infiltration is more intensive in red fibers types compared to white fiber types. A smaller neutrophil infiltration in muscles of trained animals after exhaustive exercise suggests a protective effect of previous training to muscle injury.Portions of this paper were presented by V. Morozov in 2003 at the 6th ISEI Symposium on Exercise Muscle Metabolism and Immune Function, Copenhagen.     

Characterization of a non-functional form of C1q found in patients with a genetically linked deficiency of C1q activity

A genetically defective form of C1q was purified from the sera of patients suffering from an immune complex related disease and who were homozygous for the defect. The defective C1q was haemolytically inactive and did not bind to immune aggregates or IgG-Sepharose. It showed the following similarities to the normal C1q molecule: a high glycine content and the presence of hydroxyproline and hydroxylysine; subunits with apparent mol. wts of 70,000 and 56,000, when examined by SDS-polyacrylamide gel electrophoresis under non-reducing conditions; preferential incorporation of 125I-label into only one of the types of chain present in the molecule, in a manner similar to that found for the C-chain of normal C1q. However, the defective molecule had an apparent mol. wt of approximately 155,000 in non-dissociating conditions, which is approximately one-third of the mol. wt of the normal molecule. Also, the material in the defective molecule preparation which corresponded, on the basis of mol. wt, to the disulphide-linked A-chain-B-chain dimer of normal C1q differed from that found in the normal molecule in that it did not appear to be sensitive to reducing agents. Collagenase and pepsin treatment of specific immunoprecipitates containing the radiolabelled defective molecule indicated that it is, like the normal molecule, composed of collagenous and non-collagenous domains.     

C1q-binding proteins and C1q receptors

Aggregated or immobilized complement C1q induces cellular responses in many different cell types. C1q-induced cellular responses may be involved in host defense and in protection against autoimmunity because C1q-deficient humans have infectious complications and a very high incidence of autoimmune disease. The search for the C1q receptor(s), which has been ongoing for 25 years, has led recently to the recognition that proteins identified as binding to C1q may be divided into two groups: C1q-binding molecules that are normally intracellular; and cell surface C1q receptors.     

Glomerular deposition of C1q and anti-C1q antibodies in mice following injection of antimouse C1q antibodies

Anti-C1q autoantibodies are present in the serum of patients with different autoimmune diseases such as systemic lupus erythematosus (SLE). The occurrence of these autoantibodies correlates with renal involvement. In the present study we examined whether injection of rabbit antimouse C1q antibodies in mice leads to deposition in kidneys. Injection of healthy mice with a single dose of rabbit IgG antimouse C1q antibodies resulted in deposition of both C1q and IgG anti-C1q in glomeruli. The pattern of deposition observed in the glomeruli of mice injected with antimouse C1q antibodies both at 24 h and 2 weeks was both glomerular basement membrane (GBM)-associated and mesangial. Injection of control IgG did not have a detectable effect on circulating C1q levels, and no deposition of either C1q or rabbit IgG was seen at 24 h. The deposition of rabbit antimouse C1q and C1q in glomeruli resulted in complement activation, as assessed by C3 deposition, and influx of leucocytes associated with albuminuria in some, but not all mice. In none of the control mice was albuminuria observed. This report is the first to show that anti-C1q antibodies deposit in the healthy glomerulus together with autologous C1q. This deposition is stable for at least 2 weeks, causes complement activation, leucocyte influx and can lead to mild albuminuria.     

Isolation and characterization of macrophage-derived C1q and its similarities to serum C1q

Recently, we have shown that the collagen-like, Fc-recognizing subcomponent C1q of the first complement component is synthesized by human, guinea pig and mouse peritoneal macrophages. To test whether macrophages may contribute to the serum pool of C1q, C1q was purified from guinea pig serum and from guinea pig peritoneal macrophage supernatants and compared for similarities. Both molecules had a similar sedimentation rate (macrophage C1q: 11.3 S, serum C1q: 11.2 S) and showed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions three identical bands with molecular weights of Mr, 29 000, Mr, 27 000 and Mr 23 000 for the A, B and C chains, respectively. Both C1q molecules migrated by immunoelectrophoresis in the gamma region and, in Ouchterlony analysis, showed complete antigenic identity with rabbit anti-serum C1q. These experiments demonstrate the antigenic and protein chemical similarities between serum C1q and C1q secreted by macrophages supporting the idea that macrophages have to be considered as one potential source of serum C1q. Furthermore, macrophage-derived C1q may be of importance in the local microenvironment at an inflammatory site involving macrophages.     

Prevalence and persistence of C1q binding activity in healthy subjects.

Samples of serum from 885 normal healthy blood donors were tested for the presence of soluble immune complex-like material by a solid-phase C1q binding assay. The majority of donors (93%) had low or undetectable levels of C1q binding activity in their sera, but 6% had levels that were clearly outside the normal distribution. When these individuals were retested after several weeks half of them still had elevated levels of C1q binding activity.     

中性粒细胞弹性蛋白酶、髓过氧化物酶在大鼠哮喘中的变化及意义

目的：观察中性粒细胞（PMN）、中性粒细胞弹性蛋白酶（NE）和髓过氧化物酶（MPO）在大鼠哮喘中的表达水平变化及意义。方法:18只大鼠被随机平均分成2组：哮喘组、正常对照组，以卵清白蛋白（OVA）致敏激发法复制大鼠哮喘模型，对血PMN进行分离纯化，免疫组化和比色法检测MPO的表达水平，ELISA法测定NE的蛋白浓度。 结果:（1）免疫组化法显示哮喘组血PMN和支气管壁中MPO的表达水平均显著高于对照组（P<0.01），比色法显示哮喘组支气管肺泡灌洗液（BALF）和肺组织中MPO的活性显著高于对照组（P<0.05，P<0.01）；（2）哮喘组PMN和BALF中NE蛋白浓度显著高于对照组（P<0.01）；（3）哮喘组BALF、支气管壁、肺组织中PMN的计数均显著高于对照组（P<0.01）。 结论:PMN 计数、NE和MPO的表达水平在此实验性哮喘中增加，PMN可能通过分泌NE、MPO参与哮喘炎症过程。     

Redistribution of myeloperoxidase and elastase in human neutrophil granulocytes.

Both myeloperoxidase (MPO) and elastase were found to be redistributed in ethanol-and Bouin-fixed human neutrophil granulocytes. Air-dried and formalin-fixed cells, stained for MPO, appeared with a cytoplasmic fluorescence and unstained nuclei. In elastase air-dried cells a faint perinuclear staining was also detected. Otherwise the redistribution pattern was very similar for MPO and elastase. It was found that both elastase and MPO were most efficiently extracted with Bouin's fixative. A discontinuous fluorescence was seen especially for MPO in ethanol-fixed cells. Nuclear membrane staining was verified with laser scan microscopy. This technique also visualized a scattered fluorescence in the nuclear matrix for MPO.     

Expression and function of C1q receptors and C1q binding proteins at the cell surface

C1q is the recognition unit of the first component of complement that binds not only IgG and IgM containing immune complexes, but also recognizes foreign structures such as the lipid A of endotoxin, and molecules expressed at the surface of apoptotic cells. In this review, the plasma membrane receptors and binding proteins for C1q are discussed and new data are presented on calreticulin expression on human peripheral blood cells. Although much is known about C1q receptors and binding molecules there are still many questions regarding their role in vivo.     

Mannan-binding lectin and C1q bind to distinct structures and exert differential effects on macrophages

While the interaction of complement component C1q with cellular proteins is extensively studied, much less is known about the binding of the structurally related molecule, mannan-binding lectin (MBL) to various cells. Here we show by cytofluorimetry that the interaction of MBL with immunocompetent cells is much more restricted than that of C1q. It is shown that under conditions of physiological ionic strength MBL binds to human monocyte-derived macrophages (Mphi) and monocytoid cell lines, but not to T and B lymphocytes, in contrast to C1q, which interacts with all these cells under the same conditions. As opposed to the binding of C1q, low ionic strength does not improve the interaction of MBL with Mphi. No competition for cellular binding sites was found when MBL and C1q were added simultaneously to the cells. Studying the functional consequences of the interaction, we found that the release of TNF-alpha from Mphi is induced by C1q but not by MBL. Production of complement C3 by Mphi is stimulated by C1q strongly, while the effect of MBL is much weaker. C3 produced upon C1q-mediated triggering is shown to opsonize RBC, resulting in enhanced phagocytosis. These results suggest that cell membrane molecules binding MBL and C1 q are not identical; moreover, biological functions exerted by these proteins are also markedly different.     

C1q,autoimmunity and apoptosis

Deficiency of classical pathway complement components displays a hierarchical association with the development of systemic lupus erythematosus (SLE). Individuals with deficiency of C1q, the first component of the classical pathway of activation, have the highest prevalence of SLE and the most severe manifestations of the disease. However, complement is also implicated in the effector inflammatory phase of the autoimmune response that characterizes SLE. Complement proteins are deposited in inflamed tissues causing consumption of complement. In addition, autoantibodies to C1q develop as part of the autoantibody response. Understanding how C1q deficiency results in the autoimmune phenotype of SLE may provide valuable clues to the role of the complement system in the maintenance of immune tolerance. In this review firstly we discuss the relationship between C1q deficiency and/or consumption and lupus. Secondly, we consider the links between apoptosis and complement. Finally we review the lessons we have learned from a murine model of C1q deficiency discussing the experimental evidence in support of the hypothesis that C1q may critically influence the immune response to self-antigens contained within the surface blebs generated by apoptotic cells.     

C1q solid-phase radioimmunoassay: binding properties of solid-phase C1q and evidence that C1q-binding IgG complexes in systemic lupus erythematosus are not bound to endogenous C1q

The binding properties of C1q solid-phase radioimmunoassay (C1q SPRIA) were examined, using heat-aggregated IgG (HAG) as the model of immune complexes (IC). The free, liquid-phase C1q, which was added to the C1q-coated tubes prior to the addition of HAG, had little inhibitory effect on binding of HAG to the solid-phase C1q, suggesting that the solid-phase C1q has a higher affinity for HAG than the liquid-phase C1q. On the other hand, more than 60% inhibition was seen when HAG was preincubated with the liquid-phase C1q. These binding properties of HAG to the solid-phase C1q in the presence of the liquid-phase C1q were not essentially altered by the heat inactivation or the addition of EDTA, suggesting that these pretreatments are not essential in C1q SPRIA. Next, in similar kinds of experiments, the binding properties of C1q-binding IgG complexes in SLE sera were investigated. In contrast to HAG, the binding capacity of IgG complexes in SLE sera to the solid-phase C1q was not inhibited by the preincubation with excess liquid-phase C1q. These findings suggest that C1q-binding IgG complexes in SLE sera detected by C1q SPRIA may not be bound to endogenous C1q in the circulation.     

Functional affinity constants of the reaction between 125I-labelled C1q and C1q binders and their use in the measurement of plasma C1q concentrations.

Purified 125I-labelled C1q has been found to react with glutaraldehyde-treated red cells (glut-RBC), with a value for the functional affinity constant, K, of 1-3 X 10(8) M-1, based on measurement of concentrations of bound and free reactants at equilibrium. Values of K obtained for other C1q-binders were as follows: diaminopropane, 2 X 10(2) M-1; monomer IgG, 5 X 10(4) M-1; heat-aggregated IgG, 0-5-2-5 X 10(8) M-1; IgG-anti-IgG complexes, 0-31 X 10(8) M-1. The functional rate constant for association (ka) between 125I-labelled C1q and glut-RBC was 5 X 10(5) M-1 S-1 at 37 degrees in 0-17 M NaC1. The rate of dissociation of the C1q-glut-RBC complex was biphasic with rate constants (kd) of 2 X 10(2) S-1 and 2 X 10(5) S-1. Calculated values of K from the ration ka/kd gave values of 2-5 X 10(7) M-1 and 2-5 X 10(10) M-1. It is suggested that the range of values of K reflects the involvement of 1,2 or more binding sites on the C1q molecule. Reduction of the ionic strength of the medium from 0-17 M to 0-14 M increases the rate of association of C1q and glut-RBC eleven-fold, indicating involvement of ionized groups at the binding site. A method is described for measuring plasma C1q concentrations by saturation assay, using 125I-labelled C1q and glut-RBC. Plasma C1q concentrations fell in the range 170-250 microng/ml.     

C1q and its growing family

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and acquired immunity. As a charge pattern recognition molecule of innate immunity, C1q can engage a broad range of self and non-self ligands via its heterotrimeric globular (gC1q) domain and thus trigger the classical pathway. The trimeric gC1q signature domain has been identified in a variety of non-complement proteins that can be grouped together as a C1q family. The X-ray crystal structures of the gC1q domain of a few members of the C1q family reveal a compact jelly-roll beta-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family, hence the C1q and TNF superfamily. This review is an update on the structural and functional aspects of the gC1q domain of human C1q. We also mention the diverse range of proteins that utilize a gC1q domain in order to reflect on its importance as a versatile scaffold to support a variety of functions.     

Effect of EDTA and citrate on the functional activity of the first component of complement, C1, and the C1q subcomponent

The first component of complement, C1, is a calcium-dependent complex of the three distinct subcomponents, C1q, C1r, and C1s. Earlier observations revealed that treatment of C1 with EDTA led to a loss of hemolytic C1 activity even after recalcification. Therefore, it was of interest to study whether EDTA has an additional effect on C1 and its subcomponents, beside its chelating capacity. The chelating effect of EDTA was compared to that of citrate. It was found that treatment of C1 or C1 with EDTA followed by addition of Ca++ led to a loss of hemolytic activity up to 90%, depending on EDTA concentration. Even pretreatment of EDTA with varying amounts of Ca++ did not prevent the inactivation of C1 or C1. In contrast, after dissociation of C1 or C1 by citrate, 100% of the original C1q activity is recoverable on addition of C1q deficient serum as source of C1r and C1s. EDTA-treated serum, however, showed a concentration-dependent loss of hemolytic C1q activity, indicating an inhibitory effect of EDTA on C1q. EDTA-treated C1q, fluid phase or bound to EA, was no longer able to form an hemolytically active C1 complex by interaction with C1r and C1s.