First Component Of Complement Research Articles

A Structural Basis for Inhibition of the Complement Initiator Protease C1r by Lyme Disease Spirochetes.

Complement evasion is a hallmark of extracellular microbial pathogens such as Borrelia burgdorferi, the causative agent of Lyme disease. Lyme disease spirochetes express nearly a dozen outer surface lipoproteins that bind complement components and interfere with their native activities. Among these, BBK32 is unique in its selective inhibition of the classical pathway. BBK32 blocks activation of this pathway by selectively binding and inhibiting the C1r serine protease of the first component of complement, C1. To understand the structural basis for BBK32-mediated C1r inhibition, we performed crystallography and size-exclusion chromatography-coupled small angle X-ray scattering experiments, which revealed a molecular model of BBK32-C in complex with activated human C1r. Structure-guided site-directed mutagenesis was combined with surface plasmon resonance binding experiments and assays of complement function to validate the predicted molecular interface. Analysis of the structures shows that BBK32 inhibits activated forms of C1r by occluding substrate interaction subsites (i.e., S1 and S1') and reveals a surprising role for C1r B loop-interacting residues for full inhibitory activity of BBK32. The studies reported in this article provide for the first time (to our knowledge) a structural basis for classical pathway-specific inhibition by a human pathogen.

Blood chromium exposure, immune inflammation and genetic damage: Exploring associations and mediation effects in chromate exposed population

Both genetic damage and inappropriate immune function are relevant to cancer of hexavalent chromium [Cr(VI)]. However, its associations with immune response and genetic damage development are poorly understood. To explore their associations and mediating effects, 1249 participants were included from the Occupational Chromate Exposure Dynamic Cohort, and their blood Cr concentrations were measured as internal exposure. A set of biomarkers including urinary 8-hydroxy-2’ - deoxyguanosine (8-OHdG), micronucleus frequency (MNF) and mitochondrial DNA copy number (mtCN) was developed to evaluate the landscape of genetic damage of Cr(VI). Serum C-reactive protein (CRP) and first component of complement q (C1q) were measured to reflect immune inflammation. Multivariate linear regression and mediation analyses were applied to assess the potential associations and mediation effects. It was found that blood Cr level showed significant dose-dependent relationships with increasing of MNF and urinary 8-OHdG, while negative association with CRP and C1q. Furthermore, a 1-unit increase in CRP was associated with decreases of − 0.765 to − 0.254 in MNF, − 0.400 to − 0.051 in urinary 8-OHdG. 4.97% of the association between blood Cr level and the increased MNF was mediated by CRP. 11.58% of the relationship between concentration of blood Cr and urinary 8-OHdG was mediated by C1q. These findings suggested that Cr(VI) exposures might prompt genetic damage, possibly partial via worsening immune inflammation.

Analysis of Anti-C1q Autoantibodies by Western Blot.

Anti-C1q autoantibodies may be found in many conditions, most commonly in systemic lupus erythematosus (SLE) and hypocomplementemic urticarial vasculitis syndrome (HUVS), and are diagnostic markers as well as disease activity markers in lupus nephritis. Sera from patients with SLE and HUVS show partly distinct autoantibody reactivities to separated protein chains B and C of the first component of complement, C1q. These different binding specificities can be detected by Western blot analysis of the autoantibodies under reducing conditions. Results may help clinicians to differentiate between SLE and HUVS.

Splenic marginal zone lymphomas in acquired C1-inhibitor deficiency: clinical and molecular characterization.

Angioedema due to acquired deficiency of the inhibitor of the first component of complement (C1-INH) is a rare disease known as acquired angioedema (AAE). About 70% of patients with AEE display autoantibodies to C1-INH, the remaining patients have no antibodies to C1-INH. The clinical features of C1-INH deficiency include recurrent, self-limiting local swellings involving the skin, the gastrointestinal tract, and the upper respiratory tract. Swelling is due to accumulation of bradykinin released from high molecular weight kininogen. Patients with angioedema due to acquired C1 inhibitor deficiency (AEE) often have an associated lymphoproliferative disease including Non-Hodgkin Lymphomas (NHL). Among AAE patients with NHL, splenic marginal zone lymphoma (SMZL) has a higher prevalence (66%) compared to general population (2%) In the present study, we focused on patients with SMZL in AAE. We found 24 AAE patients with NHL and, among them 15 SMZL (62.5% of all NHL). We found NOTCH 2 activation in 4 /15 patients (26.6%) with SMZL, while no patients carried MYD 88 or BIRC3 mutations. Restricted immunoglobulin gene repertoire analysis showed that the IGHV1-2*04 allele was found to be over-represented in the group of patients with or without lymphoproliferative disease presenting with autoantibodies to C1-INH (41 of 55 (75%) of patients; p value 0.011) when compared to the control group of patients with AEE without antibodies to C1-INH, (7 of 27 (26%) of patients). Immunophenotyping failed to demonstrate the presence of autoreactive clones against C1-inhibitor. Taken together, these findings suggest a role for antigenic stimulation in the pathogenesis of lymphomas associated with AEE.

The Editor takes a closer look at some of this month's articles

Clinical & Experimental AllergyVolume 43, Issue 4 p. 379-379 Editor's ChoiceFree Access The Editor takes a closer look at some of this month's articles First published: 21 March 2013 https://doi.org/10.1111/cea.12103AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Angioedema: does progesterone work? Angioedema is a distressing and unpleasant condition. Bradykinin-mediated angioedema, including the common ACE inhibitor induced and the rare hereditary (HAE) forms, is potentially life-threatening and difficult to treat. The spectrum of angioedema, particularly the bradykinin associated forms, is reviewed in this issue by Busse and Buckland (pp. 385–394) and new developments in treatment are reviewed by Xu et al. (pp. 395–405). In addition, we report a study by Saule et al. (pp. 475–482), accompanied by a commentary by Hilary Longhurst (pp. 380–382), which describes the potential benefit of progesterone in women with mainly HAE. This group identified over 50 women who had been given progesterone treatment and found a convincing improvement in the frequency and severity of attacks after being started on this hormonal therapy. Time for a double-blind placebo-controlled trial. Anne Gompel Bradykinin binds to the B2 receptor [see Figure 2 in Y.-Y. Xu et al. (pp.395–405)] Probiotics: immunology backs up the clinical findings Building on a previous clinical trial of probiotics in eczema which had shown a reduced risk of eczema at two years 1, Forsberg et al. (pp. 434–442) have now investigated whether there is an immunological explanation. They measured cytokine responses in mononuclear cells after allergen stimulation up to two years of age. They found a general reduction in the generation of both Th1 and Th2 cytokines in the subjects treated with Lactobacillus reuteri suggesting an overall lowering of allergen responsiveness by the probiotic treatment that could explain the reduction in eczema. Anna Forsberg Cat-allergen-induced IL-5 [see Figure 1 in A. Forsberg et al. (pp. 434–442)] Asthma and the environment: intruder in the dust Bacteria as well as eukaryotic cells generate extracellular vesicles (EV) which are thought to facilitate intercellular communication 2. In a thorough and original study involving both mice and humans, Kim et al. (pp. 443–454) have investigated whether such vesicles in house dust can promote an inflammatory response in the lung. They found that repeated administration of purified EV to the airway of mice resulted in a Th1-Th17-mediated neutrophilic inflammatory response. They went on to demonstrate that IgG antibodies against EV were higher in asthmatics compared with control subjects and speculate that bacteria-derived EV could be responsible in part for neutrophilic airway inflammation in asthma. You-Sun Kim Confocal microscopic images of MH-S cells [see Figure 5 in Y.-S. Kim et al. (pp. 443–454)] References 1Abrahamsson TR, Jakobsson T, Bottcher MF et al. Probiotics in prevention of IgE-associated eczema: a double-blind, randomized, placebo-controlled trial. J Allergy Clin Immunol 2007; 119: 1174– 80. CrossrefCASPubMedWeb of Science®Google Scholar 2van der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 2012; 64: 676– 705. CrossrefCASPubMedWeb of Science®Google Scholar Caption to cover illustration C1-inhibitor (C1-INH) inhibits the activation of Factor 12 (FXII), Pre-Kallikrein to Kallikrein, plasminogen to plasmin, the first component of complement (C) from activating C4 and the production of bradykinin from high-molecular weight kininogen (HMWK). C1-INH also affects factor XI, but to a minimal extent. [see figure 2 in Y.-Y. Xu et al. (pp. 396–406)]. This logo highlights the Editor's Choice articles on the cover and the first page of each of the articles. Volume43, Issue4April 2013Pages 379-379 ReferencesRelatedInformation

A review of therapeutic aptamer conjugates with emphasis on new approaches.

The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins for enhanced stability and greater pharmacokinetic lifetimes in vivo. The proteins can include Fc tails of IgG for opsonization, and the first component of complement (C1q) to trigger complement-mediated lysis of antibiotic-resistant Gram negative bacteria, cancer cells and possibly some parasites during vulnerable stages. In addition, the 3' protein adduct may be a biotoxin, enzyme, or may simply be human serum albumin (HSA) or a drug known to bind HSA, thereby retarding kidney and other organ clearance and inhibiting serum exonucleases. In this review, the author summarizes existing therapeutic aptamer conjugate categories and describes his patented concept for PCR-based amplification of double-stranded aptamers followed by covalent attachment of proteins or other agents to the chemically vulnerable overhanging 3' adenine added by Taq polymerase. PCR amplification of aptamers could dramatically lower the current $2,000/gram cost of parallel chemical oligonucleotide synthesis, thereby enabling mass production of aptamer-3'-protein or drug conjugates to better compete against expensive humanized monoclonal antibodies.

C1q limits dendritic cell differentiation and activation by engaging LAIR-1

C1q, the first component of complement, and leukocyte-associated Ig-like receptor 1 (LAIR-1; CD305), an inhibitory receptor expressed on hematopoietic cells, have both been associated with arrest of monocyte-derived dendritic cell (DC) differentiation and inhibition of Toll-like receptor activity in plasmacytoid DCs. Defects in both molecules have been implicated in susceptibility to, and progression of, systemic lupus erythematosus. Inhibitory signaling partners for C1q on monocytes and DCs remain undefined. Because C1q contains collagen-like motifs and LAIR-1 is a universal collagen receptor, we hypothesized that C1q is a functional ligand for LAIR-1. Binding analyses in cell-free systems and on the cell membrane demonstrate that C1q and its collagen tail associate with LAIR-1 and LAIR-2 (CD306), a soluble inhibitor of LAIR-1. Both C1q and its collagen tail trigger phosphorylation of LAIR-1 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in monocytes. Functional analyses show that C1q-mediated inhibition of monocyte-DC differentiation and C1q-mediated inhibition of IFN-α production by plasmacytoid DCs were both reversed by LAIR-2. Moreover, C1q-mediated inhibition of DC differentiation was reversed by LAIR-1 siRNA. Thus, C1q is a functional ligand for LAIR-1 restricting immune cell differentiation and activation. The discovery of C1q interactions with LAIR-1 and LAIR-2 lends much needed insight into molecular mechanisms operating to prevent the loss of tolerance, particularly in systemic lupus erythematosus.

Two paradoxes and a surprise on the road to an understanding of systemic lupus erythematosus

Whereas systemic lupus erythematosus (SLE) as normally encountered results from the coming together of a complex mix of genetic and environmental factors, SLE also develops in virtually all those rare people who lack a functional gene for the first component of complement (C1q). The pathogenic IgG antibodies against double-stranded DNA characteristic of the disease are made in response to nucleosomes – the package of DNA and histone molecules forming the unit structure of chromatin – which are present in apoptotic cells. Analysis of the C1q phenomenon illuminates the arrangements that are normally in place to ensure tolerance is maintained to nucleosomal antigens. Surprisingly in view of the high level of apoptosis occurring in the thymus, it appears that anti-histone helper T cells, which are likely to be required for IgG anti-DNA production, are not deleted in the thymus. It seems rather that tolerance is maintained by non-availability of antigen brought about by the highly efficient C1q-dependent phagocytosis of apoptotic cells. This ‘immunological ignorance’ may be backed up by mechanisms of peripheral tolerance if antigen does become available. Idiopathic SLE may arise when apoptotic cell clearance is sub-optimal, making clearance a promising target for therapy.

Hereditary Angioedema — Therapies Old and New

Hereditary angioedema is a rare syndrome characterized by episodic, nonpruritic, localized subcutaneous and submucosal swelling. Laryngeal edema can precipitate fatal respiratory obstruction. Nearly 50 years ago, Donaldson and Evans linked hereditary angioedema with a deficiency of the plasma inhibitor of the first component of complement, C1 inhibitor — a serine protease inhibitor that inactivates target proteases by means of irreversible binding.1 Its primary targets are the complement proteases C1r and C1s (for which it is the sole plasma inhibitor), the coagulation-system proteases factors XIa and XIIa, and the contact-system protease kallikrein (Figure 1). C1 inhibitor deficiency causes dysregulation in these . . .

Expression of C1q Complement Component in Barrett’s Esophagus and Esophageal Adenocarcinoma

AimsC1q, an element of the first component of complement, is known to be expressed by interdigitating and follicular dendritic cells in the spleen, where it has been suggested that C1q is involved in capturing immune complexes. The present study investigated whether C1q is expressed in Barrett's esophagus and esophageal adenocarcinoma and, if so, whether its expression is associated with dendritic cells. Material and MethodsEndoscopic biopsy or operative surgical specimens were obtained from 15 patients with Barrett's esophagus, 13 patients with esophageal adenocarcinoma and 12 patients whose biopsy specimens did not show the presence of specialized intestinal metaplasia or adenocarcinoma. Barrett's esophagus was diagnosed by the presence of a macroscopic area of columnar-lined esophagus as well as microscopic intestinal metaplasia with goblet cells. Immunohistochemistry utilizing anti-C1q and markers for dendritic cells and macrophages was performed on sections of tissue samples embedded in paraffin. Double immunostaining with C1q/CD83 and C1q/CD68 was used to analyze the possible co-localization of C1q with dendritic cells and macrophages. The expression of C1q by dendritic cells and macrophages was also examined in in vitro studies using reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. ResultsIn all specimens studied, C1q expression was detected as being distributed irregularly throughout the lamina propria. A computerized quantitative analysis showed that C1q expression was significantly higher in tissue specimens without specialized intestinal-type metaplasia than in Barrett's esophagus specimens and specimens with adenocarcinoma. Double immunostaining revealed that dendritic cells and macrophages expressed C1q in all analyzed esophageal specimens. The expression of C1q by dendritic cells and macrophages was also demonstrated in in vitro studies using RT-PCR and Western blotting. ConclusionThe findings suggest that reduced levels of the expression of C1q by dendritic cells and macrophages in the esophagus may play a role in the formation of immune responses associated with the formation of specialized intestinal metaplasia and the development of adenocarcinoma.

Aptamer–biotin–streptavidin–C1q complexes can trigger the classical complement pathway to kill cancer cells

Nucleic acid aptamers are regarded as rivals for antibodies and as such are being investigated for their therapeutic potential. In the present work, it is shown that two different high-affinity DNA aptamers developed previously by Ferreira et al. against MUC1 antigen (designated MUC1-5TR-1 and MUC1-S1.3/S2.2) on MCF7 breast cancer cells can be linked to the first component of complement (C1q) via a biotin–streptavidin system and induce significant killing of MCF7 cells in vitro. Cell viability was assessed by Trypan blue uptake and absorbance at 590 nm of stained cells following buffer washes and lysis in 1% SDS. While the killing effect is demonstrable versus various controls, dependent on aptamer dose, and reproducible, it appears to kill maximally about half of treated MCF7 cells. Possible reasons for the marginal killing effect include antigenic shedding in vitro and membrane-bound complement regulatory proteins (mCRPs) on the cell surface such as CD46, CD55, and CD59 which act to inhibit complement-mediated lysis of cells. Future in vitro research could benefit from application of mCRP-specific aptamers in combination with anti-MUC1 aptamers to overcome surface protective mechanisms while attacking the plasma membrane of MCF7 cells or other MUC1-expressing cancer cells. However, in vivo such a combination could have deleterious effects on normal MUC1-expressing cells as well.

Preliminary characterization of chicken hemaglutination-enhancing factor (81.20)

Abstract Hemagglutination-enhancing factor (HEF) is a chicken serum macroglobulin first described on the basis of the ability of normal chicken serum to cause pronounced aggregation of allogeneic erythrocytes (RBC) sensitized with non-agglutinating levels of alloantisera. Subsequent work showed that HEF was distinct from the first component of complement, but the identity of HEF and its ligand(s) have not been defined. We found that chicken serum agglutinated homologous RBC and sheep RBC lightly sensitized with specific IgY but not sheep RBC sensitized with rabbit IgG. Thus, the sole ligand of HEF appears to be antigen-bound IgY. HEF activity was found to be heat resistant (56o for 30 min.), unaffected by chelation with 10mM EDTA, and was not inhibited by monosaccharide ligands of C-type lectins. An isolate was obtained from chicken serum following salt precipitation, gel filtration and ion exchange chromatography that contained potent HEF activity, and consisted almost exclusively of IgM. These findings suggest that HEF is a naturally occurring IgM antibody that has the capacity to interact with antigen-bound IgY. Supported in part by the Ralph & Louise Bricker Endowed Fund

Antibodies against C1q in anti-glomerular basement membrane nephritis

The prevalence of antibodies against the collagen-like region of the subcomponent of the first component of complement, C1q, was investigated in 11 patients with anti-glomerular basement membrane (GBM) nephritis. Anti-C1q antibodies (anti-C1qAb) were detected in seven patients. IgG anti-C1qAb were found in four and IgA anti-C1qAb in five patients. During follow up of the patients a relationship was observed between the levels of IgG anti-C1qAb and the levels of anti-GBM antibodies (anti-GBMAb). Gelfiltration experiments indicated that both IgG anti-C1qAb as well as IgG anti-GBMAb were monomeric and that binding also occurred with the F(ab')2 fragments of the antibodies. Although anti-C1qAb and anti-GBMAb are both directed against a collagen-like structure, it was demonstrated by means of inhibition experiments that anti-C1qAb and anti-GBMAb are directed against different antigenic sites. Comparison of patients with anti-GBM nephritis with and without anti-C1qAb revealed that there were no differences in disease activity or disease severity. Therefore, the results of this study suggest that anti-C1qAb do not play a direct pathogenetic role in anti-GBM nephritis.

Tobacco Smoke and Elevated Shear Stress Enhance Classical Pathway Complement Activation on Human Endothelial Cells.

Cigarette smoke and hemodynamic stress contribute to inflammatory processes associated with the development of atherosclerosis. Endothelial cells are intimately involved in vascular pathology. We recently demonstrated classical pathway complement activation on human umbilical vein endothelial cells (HUVEC) and on endothelial cell lines including human bone marrow microvascular endothelial cells (BMEC). Complement components have been identified in atherosclerotic lesions, and complement derived inflammatory mediators likely play a role in vascular injury. Endothelial cells express several complement receptors, including gC1qR/p33. gC1qR/p33 recognizes the globular domain of the first component of complement, C1q, and directly activates the classical pathway. The present study was designed to explore the effects of two known atherosclerotic risk factors, tobacco smoke and shear stress, on classical pathway complement activation on endothelial cells. BMEC were grown to confluence on type I collagen, and treated for 24 hours with tobacco smoke extract (0.015 or 0.03 puffs/ml) from 2R4F research cigarettes. BMEC monolayers, in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% human plasma (anticoagulated with 0.32 % sodium citrate), were exposed to constant shear stress at 18 dynes/cm2 for 1 hour at room temperature using a cone and plate shearing device (courtesy of Dr. David Varon, BDR Technologies Ltd., Israel). BMEC monolayers were subsequently fixed with 0.5% glutaraldehyde. C1q dependent C4 activation (C4d deposition) was quantified on the cell surface using an ELISA approach. BMEC activation was characterized using endothelial cell intracellular adhesion molecule 1 (ICAM-1, CD54). Compared to background (0.26±0.13, O.D. 405 nm, n=5), low baseline levels of C4d deposition were noted on unstimulated BMEC (0.37±0.15, O.D. 405 nm, n=5, P<0.05). However, moderately increased C4d deposition was observed after treatment with tobacco smoke (0.03 puffs/ml) or exposure to shear stress (Table). Moreover, combined effects from tobacco smoke (0.03 puffs/ml) and shear stress resulted in an ~ 2-fold increase in C4d deposition. This was accompanied by a 50% increase in surface gC1qR/p33 and a 33% increase in ICAM-1 expression (Table). Tobacco smoke at 0.015 puffs/ml with or without shear stress had no effect on BMEC activation or C4d deposition. Similar results were obtained with HUVEC. In summary, these data provide further insight into the atherogenic effects of tobacco smoke by suggesting that high dose tobacco smoke in combination with elevated shear stress leads to enhanced endothelial cell surface classical pathway complement activation.TableC4d deposition, gC1qR/p33 and ICAM-1 expression on BMEC treated with tobacco smoke and shear stress.C4d*gC1qR/p33*ICAM-1*Baseline111Tobacco smoke 0.03 puffs/ml1.25±0.2 (n=5, P<0.05)NANAShear 18 dynes/cm21.45±0.4 (n=5, P<0.05)NANATobacco smoke plus shear1.92±0.5 (n=5, P<0.05)1.51±0.4 (n=4, P<0.05)1.33±0.2 (n=4, P<0.05)* Results are expressed relative to baseline. P values were obtained by ANOVA. NA:

The Crystal Structure of the Bacillus anthracis Spore Surface Protein BclA Shows Remarkable Similarity to Mammalian Proteins

The lethal disease anthrax is propagated by spores of Bacillus anthracis, which can penetrate into the mammalian host by inhalation, causing a rapid progression of the disease and a mostly fatal outcome. We have solved the three-dimensional structure of the major surface protein BclA on B. anthracis spores. Surprisingly, the structure resembles C1q, the first component of complement, despite there being no sequence homology. Although most assays for C1q-like activity, including binding to C1q receptors, suggest that BclA does not mimic C1q, we show that BclA, as well as C1q, interacts with components of the lung alveolar surfactant layer. Thus, to better recognize and invade its hosts, this pathogenic soil bacterium may have evolved a surface protein whose structure is strikingly close to a mammalian protein.

Complement-coated antibody-transfer (CCAT); serum IgA1 antibodies intercept and transport C4 and C3 fragments and preserve IgG1 deployment (PGD)

In periodontal disease, IgG1 and IgA1 antibodies produced in situ deposit on antigens in the affected tissues. Thus, there is an interest in the effect of co-deposited IgA1 antibodies on complement activation by IgG1-immune complexes. In the present study, we first analyzed the effect of IgA1-immune complexes on complement using human IgA1 antibodies to dansyl (with dansylated human serum albumin serving as the immobilized antigen). It was observed that these IgA1-immune complexes when incubated for prolonged times with 33% human serum as a source of complement received C4b and C3b deposition. As C4b and C3b deposited on the IgA1 antibodies and on the antigenic surface, the complement-coated IgA1 antibodies departed. These fluid-phase complement-coated IgA1 antibodies were transferred to antigen-coated microtiter-ELISA plates, where they became bound to the antigens. Thus, the complement-coated IgA1 antibodies retained their antigen-binding function, especially as a proportion of their covalently bound C3b progressively degraded to iC3b and C3d. Genetically engineered carbohydrate-deficient mutant human IgA1 antibodies were used to assess the role of carbohydrate in accepting the C4b and C3b depositions, and these studies indicated that the carbohydrate on the Fc-region of IgA1 played a positive role. Another interesting finding generated by this study was that when IgA1 was co-deposited with IgG1 antibodies, and serum complement was added, the IgG1 antibodies tended to remain on the antigenic surface. The co-deposited IgA1 antibodies not only controlled (reduced) the rate of the consumption of the first component of complement (C1) and of classical complement pathway activation by IgG1-immune complexes (and therein reduced the rate of complement-mediated dissolution of the IgG1-immune complexes), but also the co-deposited IgA1 antibodies simultaneously intercepted/accepted C4b and C3b, then departed, as complement began to cover the antigenic surfaces. The process in which complement-coated IgA1 antibodies transferred to non-complement-coated antigens is termed complement-coated antibody-transfer/transport (CCAT). In this way, IgA1 antibodies extended the efficiency of the complement system by insuring the specific IgA1 antibody-mediated transport of the captured biologically active complement fragments to those antigens stimulating the IgA1 antibody response but not yet neutralized (completely coated) with complement. Simultaneously by impeding the rate of C1 consumption and by intercepting C4b and C3b, IgA1 antibodies slowed C4b and C3b deposition on the antigenic surface and on the co-deposited IgG1 antibodies. Thus, in the presence of ongoing complement activation, the deposition of serum IgA1 antibodies enabled the co-deposited IgG1 antibodies to better maintain their ability to interact with antigens. We termed this latter phenomenon, preservation of IgG antibody deployment (PGD). In summary, co-deposited IgA1 antibodies maximized the efficiency of the complement system, transported their covalently bound complement fragments to specific antigens and sustained the effective deployment of IgG1 antibodies directed to those same antigens.

The critical concentration of C1-esterase inhibitor (C1-INH) in human serum preventing auto-activation of the first component of complement (C1)

C1-esterase inhibitor (C1-INH) was depleted from normal human serum (NHS) at 4 °C by affinity chromatography with a monoclonal anti-C1-INH antibody (mAb 13 E1) coupled to CNBr-activated Sepharose 4B. The C1-INH-depleted serum (C1-INH-depl-HS) had normal levels of C1, C4, and CH 50 and C1-INH concentration was less than 10% of normal (15 μg/ml in C1-INH-depl-HS compared to 230 μg/ml in NHS). C1-auto-activation in C1-INH-depl-HS was followed by measuring C4-consumption in a haemolytic assay and by detection of activated C1s in a C1s-ELISA. After a lag phase of 10–20 min, C1-auto-activation in C1-INH depl-HS occurred and reached its maximum after 40 min at 37 °C. In contrast, neutralization of C1-INH activity in NHS by addition of monoclonal antibodies directed against its C1s-binding site, resulted in an immediate, spontaneous C1-activation without a lag-phase and reached its maximum already after 20 min (mAb 140) and 25 min (mAb 88G2). Addition of highly purified C1-INH or NHS as source of C1-INH to C1-INH-depleted serum to a final concentration of 55 μg/ml (22% of normal C1-INH concentration in HS) was sufficient to control spontaneous C1-activation.

CC1q-R (calreticulin) and gC1q-R/p33: ubiquitously expressed multi-ligand binding cellular proteins involved in inflammation and infection

The first component of complement, C1, is a multi-molecular complex comprising of C1q and the Ca 2+-dependent tetramer C1r 2–C1s 2. The traditional role of C1q within the complex is that of recognition signal—a signal, which is instantly converted into a highly specific intramolecular proteolytic activation of the C1r 2–C1s 2 tetramer thereby triggering activation of the classical pathway. Another important function of C1q is its ability to bind to a wide range of cell types resulting in the induction of cell-specific biological responses. These cells include polymorphonuclear leukocytes, monocytes, lymphocytes, dendritic cells, endothelial cells and platelets. Interaction of C1q with endothelial cells and platelets, for example, leads to cellular activation followed by release of biological mediators and/or expression of adhesion molecules, all of which contribute, directly or indirectly to the inflammatory process. These specific responses are mediated by the interaction of C1q with C1q binding proteins or receptors on the cell surface. To date, four types of putative C1q binding cell surface expressed proteins/receptors have been described. These include cC1q-R/CR, or calreticulin (CR), a 60 kDa protein, which is also known as collectin receptor; gC1q-R/p33, a 33 kDa homotrimeric protein; C1q-Rp (CD93), a 120 kDa, O-sialoglycoprotein; and CR1 (CD35), the receptor for C3b. Although the specific role of each of these molecules in a given C1q-mediated cellular response is yet to be worked out, all of them may, in one form or another, participate in the inflammatory processes associated with vascular or atherosclerotic lesions, autoimmune diseases, or infections. The main focus of our laboratory for the past 20 years has been to elucidate the structure and function of cC1q-R/CR and gC1q-R/p33, both of which have been isolated and characterized on the basis of their ability to bind C1q. The purpose of this article is therefore to provide an up to date overview of these two proteins with particular emphasis on their unique structural and functional features, their multi-faceted nature and most importantly their role in infection and inflammation.

C1q, a subunit of the first component of complement, enhances antibody-mediated apoptosis of cultured rat glomerular mesangial cells.

We have shown previously that IgG2a anti-Thy-1 MoAb (ER4G) induces apoptosis of rat mesangial cells (GMC) in vitro. Since the classical complement pathway plays an essential role in Thy-1 nephritis, we analysed whether C1q, a subunit of the first component of complement, enhances the ER4G-mediated apoptosis of rat GMC. Two different subclasses of anti-Thy-1 MoAb, ER4G (IgG2a) and ER14 (IgG1), were used. It was established that ER4G binds C1q efficiently, while ER14 reacts poorly with C1q. For the experiments of apoptosis, quiescent rat GMC were exposed for 1 h at 37 degrees C to a fixed concentration of anti-Thy-1 MoAb and incubated further for 16 h at 37 degrees C in the presence or absence of C1q. GMC exposed to medium (M-GMC) followed by incubation of the cells with medium alone was used as controls. Apoptosis was assessed by morphological studies and quantitative analysis on FACS using FITC-annexin V (the annexin V methods) or bicolour FACS analysis using FITC-annexin V and propidium iodide (the annexin V/PI method). With the annexin V method, M-GMC revealed 9.4 +/- 1.4% apoptosis. C1q had only marginal effects on apoptosis of M-GMC. GMC exposed to ER4G (ER4G-GMC) and further incubated with medium in the absence of C1q resulted in 25.7 +/- 5.7% apoptosis (P < 0.01 relative to control). Incubation of ER4G-GMC together with 100 microg/ml of C1q significantly increased GMC-apoptosis up to 39.4 +/- 4.9% (P < 0.01 relative to ER4G-GMC incubated in the absence of C1q). This enhancing effect of C1q on apoptosis of ER4G-GMC was time- and dose-dependent. In contrast, C1q did not significantly alter the apoptosis of either GMC exposed to ER14 (ER14-GMC) or to F(ab')2-ER4G (F(ab')2-ER4G-GMC), while ER14-GMC or F(ab')2-ER4G-GMC incubated with medium resulted in significant apoptosis compared with control. These results were supported by morphological studies and bicolour FACS analyses in time course experiments using the annexin V/PI method. The effect of C1q is dependent on the presence of intact C1q-containing globular heads and does not occur with collagen-like fragments of C1q. Furthermore, incubation of ER4G-GMC with anti-mouse K-chain antibodies also increased ER4G-mediated GMC-apoptosis. These results indicate for the first time that C1q enhances antibody-mediated apoptosis of rat GMC in vitro, presumably by its binding to ER4G and probably by additional cross-linking of Thy-1 on the surface of GMC.

Dendritic cells in the arterial wall express C1q: potential significance in atherogenesis.

Dendritic cells (DCs) accumulate in atherosclerotic lesions but their characteristics and their role in atherogenesis are poorly understood. C1q, an element of the first component of complement, is expressed by interdigitating dendritic cells and follicular dendritic cells in the spleen. It has been suggested that C1q is involved in capturing immune complexes in the lymphoid tissue. Immune complexes are also detected in atherosclerotic lesions. The present study investigated whether C1q is expressed by DCs in the arterial wall. Because DCs accumulating within atherosclerotic lesions might originate from monocytes that infiltrate the intima from very early stages of atherosclerosis, C1q expression was also examined in monocyte-derived DCs in vitro. Specimens of the aorta, carotid, mammary, popliteal and tibial arteries were obtained during operation. Expression of C1q in the arterial wall was studied by immunohistochemistry. The nature of cells expressing C1q was studied in sections double stained with antibodies to C1q and cell type specific markers including CD1a and S-100 (for identification of DCs), CD68 (macrophages), CD3 (T-cells), von Willebrand factor (endothelial cells), and smooth muscle alpha-actin (smooth muscle cells). In vitro, DCs were differentiated from human peripheral blood monocytes using GM-CSF and IL-4. Peripheral blood monocytes were differentiated to macrophages using M-CSF. The expression of C1q in monocytes and in vitro monocyte-derived DCs and macrophages was determined by RT-PCR, Western blotting, immunofluorescence microscopy and flow cytometry. In all the arterial specimens studied, DCs expressing C1q were detected. C1q was also found in macrophages, macrophage foam cells and in neovascular endothelial cells in atherosclerotic lesions, but no C1q expression was detected in T-cells and smooth muscle cells. In vitro analysis demonstrated that monocyte-derived DCs and macrophages express C1q but no C1q was detected in monocytes. C1q is expressed by DCs residing in the arterial wall as well as by monocyte-derived DCs in vitro. Expression of C1q occurs during differentiation of monocytes to DCs and macrophages and might be important in binding and trapping immune complexes in atherosclerotic lesions.