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PubMed1995

Mannose binding protein (MBP) enhances mononuclear phagocyte function via a receptor that contains the 126,000 M(r) component of the C1q receptor.

Mannose-binding protein (MBP), C1q, the recognition component of the classical complement pathway, and pulmonary surfactant protein A (SP-A) are members of a family of molecules containing a collagen-like sequence contiguous with a noncollagen-like sequence, and usually having the properties of a lectin. C1q and SP-A have been shown to enhance monocyte FcR- and CR1-mediated phagocytosis, suggesting that the common structural features of the collagen-like domains may provide a basis for this immunologically important function. Results presented here demonstrate that MBP also enhanced FcR-mediated phagocytosis by both monocytes and macrophages, and stimulated CR1-mediated phagocytosis in human culture-derived macrophages and in phorbol ester-activated monocytes. Furthermore, a monoclonal antibody that recognizes a 126,000 M(r) cell surface protein and inhibits C1q-enhanced phagocytosis, inhibited the MBP-mediated enhancement of phagocytosis. Thus, the receptors that mediate the enhancement of phagocytosis by MBP and C1q share at least one critical functional component, the 126,000 M(r) ClqRP.

Carrier Proteins↗
PubMed2006

Complement, natural antibodies, autoantibodies and tissue injury.

Activation of the classical complement pathway represents an effector mechanism of intestinal ischemia/reperfusion injury. Mice deficient in complement receptors 1 and 2 fail to produce a component of the natural antibody repertoire that binds to ischemia-conditioned tissues and activate complement. In contrast, mice prone to autoimmunity display accelerated and enhanced tissue injury that results from the binding of autoantibodies to injured tissues. Our experiments demonstrate that naturally occurring antibodies and autoantibodies mediate tissue injury only after an organ has been subjected to a stressor such as ischemia.

Animals↗
PubMed2003

Localization of ligand-binding sites on human C1q globular head region using recombinant globular head fragments and single-chain antibodies.

As a charge pattern recognition molecule, human C1q can bind a range of immunoglobulin and non-immunoglobulin ligands via its carboxy-terminal globular domain and activate the classical complement pathway. Each globular domain has a heterotrimeric organization, composed of the carboxy-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Recently, we have found that the recombinant forms of individual ghA, ghB and ghC bind differentially to IgG, IgM, gp41 peptide 601-613 of human immunodeficiency virus-1 (HIV-1), gp21 peptide 400-429 of human T cell lymphotrophic virus-I (HTLV-I), beta-amyloid peptide, and apoptotic cells, suggesting a modular organization of the globular domain. This paper examines the interaction of ghA, ghB and ghC with two known C1q ligands: Klebsiella pneumoniae porin OmpK36 and salivary agglutinin. In addition, we have used a panel of recombinant single-chain antibodies (scFv) specific for ghA, ghB and ghC in order to map sites on the heterotrimeric globular domain which are likely to interact with IgG1, IgG3, IgM, OmpK36, salivary agglutinin and gp41 loop peptide. The combined use of recombinant ghA, ghB, ghC and single-chain antibodies has revealed at least three ligand-binding sites on the globular domain of C1q: one is IgG- and OmpK36-specific, the second (IgM-binding site) is most likely overlapping with IgG/OmpK36 binding site, and the third (the gp41-binding site) seems to be located at the junction between the collagen and globular domains.

Agglutinins↗
PubMed2006

Proteome analysis of myocardial tissue following ischemia and reperfusion--effects of complement inhibition.

Myocardial ischemia-reperfusion injury can be related to complement activation with generation of chemotactic mediators, release of cytokines, leukocyte accumulation, and subsequent severe tissue injury. In this regard, activation of transcription factors (i.e., NFkappaB) and de novo protein synthesis or inflammatory protein degradation seems to play an important role. In the present study, we analyzed the cardiac protein expression following myocardial ischemia (60 min) and reperfusion (180 min) in a rabbit model utilizing two-dimensional electrophoresis and nanoHPLC/ESI-MS/MS for biochemical protein identification. To achieve cardioprotective effects, we used a novel highly selective small molecule C1s inhibitor administered 5 min prior to reperfusion. The reduction of myocardial injury was observed as diminished plasma creatine kinase activity in C1s-INH-248-treated animals (65.2+/-3 vs. 38.5+/-3 U/g protein after 3 h of reperfusion, P<0.05). With proteome analysis we were able to detect 509+/-21 protein spots on the gels of the 3 groups. A pattern of 480 spots with identical positions was found on every gel of myocardial tissue of sham animals, vehicle and C1s-INH-248-treated animals. We analyzed 11 spots, which were identified by mass spectrometry: Superoxide dismutase, alpha-crystallin-chain-B, mitochondrial stress protein, Mn SOD, ATP synthase A chain heart isoform, creatine kinase, and troponin T. All of these proteins were significantly decreased in the vehicle group when we compared to sham-treated animals. Treatment with C1s-INH-248 preserved levels of these proteins. Thus, blocking the classical complement pathway with a highly specific and potent synthetic inhibitor of the activated C1 complex archives cardio-protection by altering and preserving different anti-inflammatory and cytoprotective cascades.

Amino Acid Sequence↗
PubMed2006

Emerging patterns in complement-mediated pathogen recognition.

Protection from acute infection requires an immediate response by the host innate immune system. Two recent Cell papers, including one in this issue, provide new insight into the role of the complement system in response to blood-borne pathogens.

Animals↗
PubMed2004

Complement C1q expression induced by Abeta in rat hippocampal organotypic slice cultures.

Amyloid beta peptide (Abeta) is a major component of senile plaques, one of the principle pathological features in Alzheimer's disease (AD) brains. Fibrillar Abeta has been shown to bind C1 via C1q, the recognition component of the classical complement pathway, resulting in the activation of the complement pathway, thereby initiating an inflammatory cascade in the brain. C1q has also been shown to enhance phagocytic activities of microglia, which could benefit in clearance of apoptotic cells or cellular debris. To begin to define the role of C1q in tissue injury mediated by Abeta, we assessed the appearance of C1q in hippocampal slice cultures treated with freshly solubilized or fibrillar Abeta 1-42. Here we demonstrate a dose- and time-dependent uptake of exogenously applied Abeta by pyramidal neurons in organotypic slice cultures from rat hippocampus. Importantly, when slices were immunostained with antibody against rat C1q, a distinct reactivity for C1q in cells within the neuronal cell layer of cornu ammonis (CA) of hippocampus, primarily the CA1/CA2, was observed in the Abeta-treated slices. No such immunoreactivity was detected in untreated cultures or upon addition of control peptides. ELISA assays also showed an increase in C1q in tissue extracts from slices of the treated group. Similarly, the mRNA level of C1q in slices was increased within 24 h after Abeta treatment. These data demonstrate that upon exposure to Abeta, C1q is expressed in neurons in this organotypic system. The induction of C1q may be an early, perhaps beneficial, tissue or cellular response to injury triggered by particular pathogenic stimuli.

Amyloid beta-Peptides↗
PubMed2006

Mannan binding lectin and its interaction with immunoglobulins in health and in disease.

In humans there are five classes of immunoglobulins (Igs), IgG, IgM, IgA, IgE and IgD, all of which are glycoproteins. The Igs are the major secretory product of the adaptive immune system, and they bind to antigens via variable sequences on their Fab regions. The binding to antigen results in neutralization or agglutination of bound material and also initiates effector functions via the Fc regions, such as opsonisation and activation of the classical complement pathway through binding of C1q. Mannan binding lectin (MBL), the 'recognition' molecule of the lectin pathway of complement activation, is homologous in structure to C1q, and binds in a calcium-dependent manner to terminal mannose and GlcNAc residues which have been identified on the oligosaccharides N-linked to the Igs. MBL binds agalactosylated glycoforms of IgG (IgG-G0), polymeric forms of IgA and certain glycoforms of IgM which have a high incidence of GlcNAc-terminating glycans. This interaction provides a route of clearance of immune complexes from the serum, and a mechanism of complement activation to Ig-coated pathogens. In disease, MBL contributes to inflammation in Rheumatoid Arthritis, a condition in which serum IgG-G0 concentrations can increase significantly compared to healthy individuals. MBL has recently been demonstrated to bind Ig in the B cell receptor complex which expresses abnormal variable region glycosylation, in follicular lymphoma.

Animals↗
PubMed2004

C1q and tumor necrosis factor superfamily: modularity and versatility.

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 ligands via its globular (gC1q) domain and modulate immune cells, probably via its collagen region. The gC1q signature domain, also found in many non-complement proteins, has a compact jelly-roll beta-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family. The members of this newly designated 'C1q and TNF superfamily' are involved in processes as diverse as host defense, inflammation, apoptosis, autoimmunity, cell differentiation, organogenesis, hibernation and insulin-resistant obesity. This review is an attempt to draw structural and functional parallels between the members of the C1q and TNF superfamily.

Amino Acid Sequence↗
PubMed2006

Genetic evidence for the involvement of Fcgamma receptor III in experimental autoimmune myasthenia gravis pathogenesis.

Immune complexes and classical complement pathway play vital roles in experimental autoimmune myasthenia gravis (EAMG). To analyze the role of immune complex receptors in EAMG, FcgammaRIII knockout (KO) mice were immunized with AChR and were found out to be resistant to EAMG induction. This was associated with reduced neuromuscular junction deposits, lymph node cell (LNC) IL-6 production and serum complement levels. EAMG resistance of anti-C1q Ab-administered mice was also associated with reduced LNC IL-6 production and neuromuscular junction deposits, indicating C1q involvement in EAMG resistance. The data provide the first direct genetic evidence for Fcgamma receptor involvement in EAMG pathogenesis.

Animals↗
PubMed2006

Pros and cons of treating murine myasthenia gravis with anti-C1q antibody.

To test the feasibility of classical complement pathway manipulation in experimental autoimmune myasthenia gravis (EAMG) treatment, C57BL/6 (B6) and RIIIS/J mice with EAMG were treated with 10 microg or 100 microg of anti-C1q Ab or isotype Ab. Treatment with 10 microg anti-C1q Ab significantly reduced the clinical severity, decreased lymph node cell IL-6 production and T cell populations. Conversely, administration of 100 microg anti-C1q Ab caused harmful side effects such as increased serum anti-acetylcholine receptor antibody, immune complex, C3 and lymph node B cell levels and kidney C3 and IgG deposits, which reduced the treatment efficacy.

Animals↗
PubMed2006

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.

Animals↗
PubMed2004

Identification of HIV-1 protease cleavage site in human C1-inhibitor.

We have investigated the ability of HIV-1 protease to cleave human complement proteins of the classical complement pathway: C1q, C2 and C4 as well as the regulatory protein, C1-inhibitor. Purified complement proteins were incubated with recombinant HIV-1 protease in vitro and analyzed by SDS-PAGE and immunoblotting assay. The only cleavage site was found in N-terminal region of C1-inhibitor, and it was located between residues Leu-32 and Phe-33 as determined by amino acid sequence analysis of the 85 kDa proteolytic fragment after 12 Edman degradation cycles. The HIV-1 protease cleavage sites were not found in C1q, C2 and C4 protein. HIV-1 protease-susceptible site in N-terminal region of C1-inhibitor is very close to the cleavage sites of some other proteases that are able to induce N-terminal proteolysis of the protein.

Amino Acid Sequence↗
PubMed2001

Generation of C-reactive protein and complement components in atherosclerotic plaques.

C-reactive protein (CRP) and complement are hypothesized to be major mediators of inflammation in atherosclerotic plaques. We used the reverse transcriptase-polymerase chain reaction technique to detect the mRNAs for CRP and the classical complement components C1 to C9 in both normal arterial and plaque tissue, establishing that they can be endogenously generated by arteries. When the CRP mRNA levels of plaque tissue, normal artery, and liver were compared in the same cases, plaque levels were 10.2-fold higher than normal artery and 7.2-fold higher than liver. By Western blotting, we showed that the protein levels of CRP and complement proteins were also up-regulated in plaque tissue and that there was full activation of the classical complement pathway. By in situ hybridization, we detected intense signals for CRP and C4 mRNAs in smooth muscle-like cells and macrophages in the thickened intima of plaques. By immunohistochemistry we showed co-localization of CRP and the membrane attack complex of complement. We also detected up-regulation in plaque tissue of the mRNAs for the macrophage markers CD11b and HLA-DR, as well as their protein products. We showed by immunohistochemistry macrophage infiltration of plaque tissue. Because CRP is a complement activator, and activated complement attacks cells in plaque tissue, these data provide evidence of a self-sustaining autotoxic mechanism operating within the plaques as a precursor to thrombotic events.

Aged↗
PubMed1999

Up-regulated production and activation of the complement system in Alzheimer's disease brain.

We used reverse transcriptase-polymerase chain reaction and Western blotting techniques to measure the levels of complement mRNAs and their protein products in Alzheimer's disease (AD) brain compared with non-AD brain. mRNAs for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 were detected in the 11 regions of brain that were investigated. The mRNA levels were markedly up-regulated in affected areas of AD brain. In the entorhinal cortex, hippocampus, and midtemporal gyrus, which had dense accumulations of plaques and tangles, C1q mRNA was increased 11- to 80-fold over control levels, and C9 mRNA 10- to 27-fold. These levels were substantially higher than in the livers of the same cases. Western blot analysis of AD hippocampus established the presence of all of the native complement proteins as well as their activation products C4d, C3d, and the membrane attack complex. These data indicate that high levels of complement are being produced in affected areas of AD brain, that full activation of the classical complement pathway is continuously taking place, and that this activation may be contributing significantly to AD pathology.

Adult↗
PubMed2002

Anti-inflammatory drugs modulate C1q secretion in human peritoneal macrophages in vitro.

The complement system plays an important role in the humoral immune response. Activation of the classical complement pathway is mediated by its subcomponent, C1q, which is involved in the pathogenesis of several autoimmune disorders. Among the main C1q-synthesising tissues, macrophages have been attributed as the main source. We investigated the effects of anti-inflammatory drugs (methylprednisolone and acetylsalicylic acid (ASA)) on C1q secretion in human peritoneal macrophages in vitro. The macrophages were isolated from peritoneal lavage fluid of patients with end-stage renal disease undergoing continuous ambulatory peritoneal dialysis, and were maintained in culture for up to 6 days. ASA decreased while methylprednisolone increased C1q secretion from human peritoneal macrophages in vitro, which correlated well with the percentage of CD14 positive cells after treatment. We conclude that different response of the macrophages to treatment with methylprednisolone and ASA may point out to the importance of macrophage activation after treatment, as well as an increased abundance of membrane C1q accompanied by increased phagocytosis.

Anti-Inflammatory Agents↗
PubMed1997

Apolipoprotein E4 (ApoE4) but not ApoE3 or ApoE2 potentiates beta-amyloid protein activation of complement in vitro.

Apolipoprotein E4 (ApoE4) increases the risk of late-onset Alzheimer's disease (AD). It binds tightly to beta-amyloid protein (A beta), which is known to activate the classical complement pathway in vitro. Since complement activation is a possible mechanism for promoting inflammation in AD, we tested, utilizing ELISA techniques, whether the various isoforms of ApoE could influence A beta complement activation, or could themselves activate the pathway. A beta applied alone to ELISA plate wells at concentrations of 100-500 ng showed a linear increase in ability to activate serum complement, but all the ApoE isoproteins were inactive. When 200 or 430 ng of A beta were plated and then exposed to solutions of 100-200 ng of ApoE2, ApoE3, ApoE4 or bovine serum albumin (BSA), only ApoE4 significantly enhanced the activation. This ApoE4-specific enhancement of complement activation by A beta may relate to its role in increasing the risk of late-onset AD.

Alzheimer Disease↗
PubMed1999

Complement regulators C1 inhibitor and CD59 do not significantly inhibit complement activation in Alzheimer disease.

Proteins characteristic of activated complement are associated with Alzheimer disease (AD) lesions. The classical complement pathway can be activated only when the influence of such endogenous regulators as C1-inhibitor (C1-inh) and CD59 are overcome. We used the techniques of reverse transcriptase-polymerase chain reaction and Western blotting to assess the mRNA and protein levels of C1-inh and CD59 in AD and control brains in comparison with levels of the complement components with which they interact. The inhibitors were only slightly upregulated and then only in heavily affected areas of AD brain such as the entorhinal cortex, hippocampus, midtemporal gyrus and midfrontal gyrus. The ratio of AD to control mRNAs in these four areas was 1.17 for C1-inh and 1.12 for CD59, compared to 3.06 for C1r, 2.67 for C1s, 2.35 for C5, 2.56 for C6, 2.42 for C7, 5. 08 for C8 and 16.3 for C9. Peripheral organ expression of C1-inh and CD59 mRNAs was no different in AD than controls but was slightly upregulated in infarcted heart tissue. Again, the increase was small compared with that of the competitive complement components. These data indicate that the forces which upregulate and activate complement in AD and myocardial infarction are not effectively suppressed by the endogenous regulators, C1-inh and CD59.

Adult↗