Prion protein activates and fixes complement directly via the classical pathway: implications for the mechanism of scrapie agent propagation in lymphoid tissue

C1q-deficient and complement depleted mice are highly resistant to intraperitoneal scrapie infection. The molecular mechanisms of complement involvement in scrapie pathogenesis remain unclear. Previous detailed studies have indicated mouse prion protein interactions with human C1q but the question of subsequent complement activation has remained unaddressed. In this investigation, murine prion protein, both recombinant and also from diseased tissue sources, directly activated and fixed complement via the classical but not the alternative pathway. The importance of complexed cupric ions was observed. In addition, evidence of IgG-independent C4 fixation by prion proteins was also shown. Surface plasmon resonance binding studies using variously clustered immobilized recombinant mouse prion protein indicated strong interactions with both purified mouse C1q and also mouse Factor H. Binding, especially by C1q, was dependent upon the volume of immobilized prion protein, suggesting a threshold of clustering density required to support strong interactions. Furthermore, clustered immobilized prion protein appeared capable of promoting polymerization of soluble-phase monomeric prion protein. Direct covalent attachment of complement components to prion proteins via classical pathway activation illustrates a potential mechanism underpinning their trafficking to, and subsequent propagation within, lymphoid tissues.     

Native, amyloid fibrils and beta-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly

Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and beta-oligomers) of recombinant human PrP (90-231 and 121-231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, beta-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that beta-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.     

Complement components, regulators and receptors are produced by human monocyte-derived dendritic cells

Complement and dendritic cells (DCs) are essential components of innate immunity. Both participate in local inflammation and moreover have roles in the initiation of the acquired immunity response and in the maintenance of tolerance. Recent studies have demonstrated the ability of DCs to synthesize C1q, C3, Factor I, Factor B and complement receptors 3 and 4. In this study, we demonstrate that human DCs are a source of other soluble complement proteins including C1q, C4b binding protein (C4BP), C7 and C8. Complement receptors (CR)1 and the CD18 chain (common for CR3 and CR4) were also present on DCs while CR2 was not detected.     

Chemotaxis of human monocyte-derived dendritic cells to complement component C1q is mediated by the receptors gC1qR and cC1qR

Dendritic cells (DCs) are recruited to inflammatory sites where they phagocytose and process antigens for subsequent presentation to the T lymphocytes in the lymphoid tissue. Several leukocyte chemoattractants and their specific receptors have been shown to induce the migration of DC. The complement protein C1q has multiple immune functions including acting as a chemoattractant for neutrophils, eosinophils and mast cells. Therefore, the objective of this study was to determine if soluble C1q can induce chemotaxis of DC. Culturing cells in GM-CSF and IL-4 for 5 to 7 days generated human monocyte-derived DCs. In addition, LPS was added from day 5 to 7 to induce DC maturation. Cells were classified as either immature or mature DC by assessing the cell surface markers by flow cytometry, phagocytosis of dextran-FITC and T cell proliferation in an allogenic MLR. Immature DCs express the C1q receptors (C1qR), gC1qR and cC1qR/CR and, accordingly, display a vigorous migratory response to soluble C1q with maximal cell movement observed at 10-50nM. In contrast, mature DCs neither express C1qR nor do move to a gradient of soluble C1q. Varying the concentration gradient of C1q (checkerboard assay) showed that the protein largely induces a chemotactic response. Finally, blocking gC1qR and cC1qR/CR by using specific antibodies abolished the chemotactic response to C1q but had no effect on a different chemoattractant C5a. These results clearly demonstrate that C1q functions as a chemotactic factor for immature DC, and migration is mediated through ligation of both gC1qR and cC1qR/CR.     

Complement protein C1q induces maturation of human dendritic cells

Maturation of dendritic cells (DCs) is known to be induced by several stimuli, including microbial products, inflammatory cytokines and immobilized IgG, as demonstrated recently. Since immune complexes formed in vivo also contain C1q, moreover apoptotic cells and several pathogens fix C1q in the absence of antibodies, we undertook to investigate whether this complement protein has an impact on various functions of human DCs. Maturation of monocyte-derived immature DCs (imMDCs) cultured on immobilized C1q was followed by monitoring expression of CD80, CD83, CD86, MHCII and CCR7. The functional activity of the cells was assessed by measuring cytokine secretion and their ability to activate allogeneic T lymphocytes. Cytokine production by T cells co-cultured with C1q-matured DCs was also investigated. C1q, but not the structurally related mannose-binding lectin was found to bind to imMDC in a dose-dependent manner and induced NF-kappaB translocation to the nucleus. Immobilized C1q induced maturation of MDCs and enhanced secretion of IL-12 and TNF-alpha, moreover, elevated their T-cell stimulating capacity. As IFN-gamma levels were increased in supernatants of MDC-T cell co-cultures, our data suggest that C1q-induced DC maturation generates a Th1-type response. Interestingly, IL-10 levels were elevated by C1q-treated MDCs but not in the supernatant of their co-cultures with allogeneic T cells. Taken together, these results indicate that C1q-opsonized antigens may play a role in the induction and regulation of immune response. Moreover our data are relevant in view of the role of C1q in removal of apoptotic cells and the association between C1q-deficiency and autoimmunity.     

Chemistry-dependent adsorption of serum proteins onto polyanhydride microparticles differentially influences dendritic cell uptake and activation

The delivery of antigen-loaded microparticles to dendritic cells (DCs) may benefit from surface optimization of the microparticles themselves, thereby exploiting the material properties and introducing signals that mimic pathogens. Following in vivo administration microparticle surface characteristics are likely to be significantly modified as proteins are quickly adsorbed onto their surface. In this work we describe the chemistry-dependent serum protein adsorption patterns on polyanhydride particles and the implications for their molecular interactions with DCs. The enhanced expression of MHC II and CD40 on DCs after incubation with amphiphilic polyanhydride particles, and the increased secretion of IL-6, TNF-α, and IL-12p40 by hydrophobic polyanhydride particles exemplified the chemistry-dependent activation of DCs by sham-coated particles. The presence of proteins such as complement component 3 and IgG further enhanced the adjuvant properties of these vaccine carriers by inducing DC maturation (i.e. increased cell surface molecule expression and cytokine secretion) in a chemistry-dependent manner. Utilizing DCs derived from complement receptor 3-deficient mice (CR3(-/-) mice) identified a requirement for CR3 in the internalization of both sham- and serum-coated particles. These studies provide valuable insights into the rational design of targeted vaccine platforms aimed at inducing robust immune responses and improving vaccine efficacy.     

Pro-inflammatory complement activation by the A beta peptide of Alzheimer's disease is biologically significant and can be blocked by vaccinia virus complement control protein

The amyloid plaque is the hallmark of Alzheimer's disease (AD). The transmembrane domain and a portion of the C-terminus (A beta) of the amyloid precursor protein, are known to form the nucleus of the amyloid plaque. It has been demonstrated recently, using in vitro assays, that the A beta peptide can activate both the classical (antibody-independent) and alternate pathways of complement activation. The proposed complement activation is due to the binding of A beta to the complement components C1q and C3, respectively, which initiate formation of the proinflammatory C5a and C5b-9 membrane attack complex. In this report, we have investigated the in vitro findings for the likely complement-dependent proinflammatory properties of the Alzheimer's disease A beta peptide. We have performed experiments using congenic C5-deficient and C5-sufficient mice injected with synthetic A beta and recombinant polypeptide (C-100) containing A beta. Injection of C-100 into C5-sufficient mice induced a clear increase in the number of polymorphonuclear cells (neutrophils) at the site of injection due to complement activation and the subsequent release of proinflammatory chemtoactic factors. In sharp contrast, the C5-deficient mice did not show any increase in cellular influx. The vaccinia virus complement control protein, an inhibitor of both the classical and alternate pathway can down-regulate the biologically significant activation of complement by A beta, as demonstrated by an in vitro immunassay. The therapeutic down-regulation of A beta-caused complement activation could greatly alleviate the progression of some of the chronic neurodegeneration characteristic of Alzheimer's disease.     

Complement production and regulation by dendritic cells: molecular switches between tolerance and immunity

In recent years it has become clear that the innate and adaptive immune systems are highly integrated and interact at several levels. Dendritic cells (DCs) are on the one hand instrumental for directing and controlling adaptive immunity and on the other hand are specialized in detecting and integrating signals from the microenvironment. In view of the strong link between deficiencies in certain complement components and the development of autoimmunity, interaction between complement and DCs seems to be of fundamental importance. We will discuss the role of C1q, C3, as well as complement regulators in DC biology.     

Complement production and regulation by dendritic cells: Molecular switches between tolerance and immunity

In recent years it has become clear that the innate and adaptive immune systems are highly integrated and interact at several levels. Dendritic cells (DCs) are on the one hand instrumental for directing and controlling adaptive immunity and on the other hand are specialized in detecting and integrating signals from the microenvironment. In view of the strong link between deficiencies in certain complement components and the development of autoimmunity, interaction between complement and DCs seems to be of fundamental importance. We will discuss the role of C1q, C3, as well as complement regulators in DC biology.     

C1q regulation of dendritic cell development from monocytes with distinct cytokine production and T cell stimulation

The causative association of complement C1q deficiency with systemic lupus erythematosus (SLE), which inevitably involves the breakdown of tolerance, remains poorly explained. Its non-hepatic, macrophage and dendritic cell (DC) origin may be highly relevant. In tissues, C1q is produced by DCs and macrophages which deposits around these cells and we ask whether this pericellular form of C1q regulates DC development from monocytes. DCs cultured on immobilized C1q (C1q-DCs) show similar MHC, CD40, CD80, CD86, CD83 and CCR7 expression as normal DCs, but these cells exhibit increased phagocytosis of apoptotic cells and elevated IL-10 but reduced IL-12 and IL-23 production. Intracellularly, C1q-DCs exhibit increased ERK, p38 and p70S6 kinase activity. By mixed leukocyte reaction, C1q-DCs show reduced Th1 and Th17 induction from allogeneic CD4(+) T cells. LPS and IFNγ, which cause normal DCs to induce increased CD25 expression on CD4(+) T cells, attenuate C1q-DC induction of CD25. These imply that the DC pericellular C1q may induce tolerogenic properties in developing DCs.     

C1q as an autocrine and paracrine regulator of cellular functions

Most of the complement proteins in circulation are, by and large, synthesized in the liver. However data accumulated over the past several decades provide incontrovertible evidence that some if not most of the individual complement proteins are also synthesized extrahepatically by activated as well as non-activated cells. The question that is finally being addressed by various investigators is: are the locally synthesized proteins solely responsible for the myriad of biological functions in situ without the contribution of systemic complement? The answer is probably “yes”. Among the proteins that are synthesized locally, C1q takes center stage for several reasons. First, it is synthesized predominantly by potent antigen presenting cells such as monocytes, macrophages and dendritic cells (DCs), which by itself is a clue that it plays an important role in antigen presentation and/or DC maturation. Second, it is transiently anchored on the cell surface via a transmembrane domain located in its A chain before it is cleaved off and released into the pericellular milieu. The membrane-associated C1q in turn, is able to sense danger patterns via its versatile antigen-capturing globular head domains. More importantly, locally synthesized C1q has been shown to induce a plethora of biological functions through the induction of immunomodulatory molecules by an autocrine- or paracrine- mediated signaling in a manner that mimics those of TNFα. These include recognition of pathogen- and danger- associated molecular patterns, phagocytosis, angiogenesis, apoptosis and induction of cytokines or chemokines that are important in modulating the inflammatory response. The functional convergence between C1q and TNFα in turn is attributed to their shared genetic ancestry. In this paper, we will infer to the aforementioned “local-synthesis-for-local function” paradigm using as an example, the role played by locally synthesized C1q in autoimmunity in general and in systemic lupus erythematosus in particular.     

Biochemical and functional characterization of the interaction between pentraxin 3 and C1q

Pentraxin 3 (PTX3) is a recently characterized member of the pentraxin family of acute-phase proteins produced during inflammation. Classical short pentraxins, C-reactive protein, and serum amyloid P component can bind to C1q and thereby activate the classical complement pathway. Since PTX3 can also bind C1q, the present study was designed to define the interaction between PTX3 and C1q and to examine the functional consequences of this interaction. A dose-dependent binding of both C1q and the C1 complex to PTX3 was observed. Experiments with recombinant globular head domains of human C1q A, B, and C chains indicated that C1q interacts with PTX3 via its globular head region. Binding of C1q to immobilized PTX3 induced activation of the classical complement pathway as assessed by C4 deposition. Furthermore, PTX3 enhanced C1q binding and complement activation on apoptotic cells. However, in the fluid-phase, pre-incubation of PTX3 with C1q resulted in inhibition of complement activation by blocking the interaction of C1q with immunoglobulins. These results indicate that PTX3 can both inhibit and activate the classical complement pathway by binding C1q, depending on the way it is presented. PTX3 may therefore be involved in the regulation of the innate immune response.     

Decidual expression and localization of human surfactant protein SP-A and SP-D,and complement protein C1q

Surfactant proteins SP-A and SP-D, and complement protein C1q are soluble innate immune pattern recognizing molecules. SP-A, SP-D and C1q have an overall similar structure composed of an N-terminal triple-helical collagen region that is followed by a trimeric globular domain. While SP-A and SP-D belong to the collectin family (collagen containing lectin), C1q is the first recognition subcomponent of the classical pathway of the complement system. Recently, SP-A, SP-D and C1q have been considered to play important roles in early and late pregnancy. However, their expression in early human decidua has not been examined. Here, we investigated whether SP-A, SP-D and C1q are expressed within first trimester decidua in humans and their expression is associated with trophoblasts and decidual stromal cells. Decidual samples from women undergoing elective vaginal termination of pregnancy during first trimester were obtained from 25 subjects. Immunohistochemical studies using anti-human SP-A, anti-human SP-D and anti-human C1q antibodies were performed on decidual tissue sections along with anti-vimentin and cytokeratin-7 antibodies to identify stromal cells and trophoblasts. The expression was also examined by immunostaining and PCR using decidual and stromal cells. C1q expression was significantly higher when compared to SP-A and SP-D in the first trimester human decidua. Double immunostaining revealed that all stromal cells and trophoblasts expressed SP-A, SP-D and C1q, while only few invasive trophoblasts expressed C1q. Thus, expression of SP-A, SP-D and C1q in human decidua during first trimester suggests potential role of SP-A, SP-D and C1q during the early stages of pregnancy including implantation, trophoblast invasion and placental development.     

Localisation of complement components in association with glomerular extracellular particles in various renal diseases

Summary Intraglomerular extracellular microparticles including so-called virus-like particles and striated membranous structures have been observed in various renal diseases. The presence and localisation of complement components in these extracellular bodies was studied using the protein A-gold electron microscopy method. Ultrastructurally these particles were differentiated into microspherical structures (MSS) and thread-like structures (TS). Both structures showed weak to moderate diffuse labelling with C1s, whilst the intense labelling found with C3d and C9 was confined to individual membrane-like structures of both MSS and TS. Labelling with IgA, IgG, fibrinogen and the complement components C1q, C1r, C3c C4 and C5 showed negative or trace results. There were no differences between the immunolabelling patterns of MSS and TS, nor among different renal diseases in which these structures were found. These findings raise the possibility that formation of so-called virus-like particles such as MSS and TS may be associated with complement activation.     

The tissue pentraxin PTX3 limits C1q-mediated complement activation and phagocytosis of apoptotic cells by dendritic cells

Pentraxins (PTX) and complement belong to the humoral arm of the innate immune system and have essential functions in immune defense to microbes and in scavenging cellular debris. The prototypic long PTX, PTX3, and the first component of the classical complement pathway, C1q, are innate opsonins involved in the disposal of dying cells by phagocytes. Whether the interaction between various innate opsonins impacts on their function is not fully understood. We show here that characterized Toll-like receptor (TLR) ligands elicit the production of C1q and PTX3 by immature dendritic cells (DC). Moreover, these molecules bind to dying cells with similar kinetics, although they recognize different domains on the cell membranes. PTX3 binds in the fluid phase to C1q, decreasing C1q deposition and subsequent complement activation on apoptotic cells. C1q increases the phagocytosis of apoptotic cells by DC and the release of interleukin-12 in the presence of TLR4 ligands and apoptotic cells; PTX3 inhibits both events. Moreover, PTX3 inhibited the cross-presentation of the MELAN-A/melanoma antigen-reactive T cell 1 (MART-1) tumor antigen expressed by dying cells, even in the presence of C1q. These results suggest that interaction of C1q and PTX3 influences the clearance of apoptotic cells by DC. The coordinated induction by primary, proinflammatory signals of C1q and PTX3 and their reciprocal regulation during inflammation influences the clearance of apoptotic cells by antigen-presenting cells and possibly plays a role in immune homeostasis.     

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.     

Cardiac amyloidosis induces up-regulation of Deleted in Malignant Brain Tumors 1 (DMBT1)

BackgroundAmyloidosis is a life-threatening protein misfolding disease and affects cardiac tissue, leading to heart failure, myocardial ischemia and arrhythmia. Amyloid deposits result in oxidative stress, inflammation and apoptosis. The purpose of this study was to examine the role of innate defense components, i.e., Deleted in Malignant Brain Tumors 1 (DMBT1) and the complement system, in different types of cardiac amyloidosis.MethodsExpression of DMBT1 and of the complement proteins C1q, C3d and C4d in cardiac specimens of patients with different types of amyloidosis were determined by immunohistochemistry and correlated with amyloid deposits stained by Congo red dye.ResultsStrong DMBT1 staining adjacent to amyloid deposits was detected in different amyloidosis types, depending on the extent of the deposits. DMBT1 is localized in the endomysium and perimysium, in the endocardium, in the myocytes and in endothelial cells of affected transmural vessels. C1q, C3d and C4d were detected in the amyloid deposits but also in the endomysium and perimysium, in some myocytes, in endothelial cells, in the endocardium, and around the amyloid deposits.ConclusionsUp-regulated DMBT1 and complement activation in cardiac amyloidosis may be part of the activated pathways induced by protein aggregation and the consecutive inflammatory reaction.     

Interactions of histidine-rich glycoprotein with immunoglobulins and proteins of the complement system

This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.     

Labeling of the scrapie-associated prion protein in vitro and in vivo

Prion diseases are a group of infectious neurodegenerative diseases that affect both animals and humans. A characteristic of prion diseases is the aggregation and accumulation of a disease-associated isoform of the prion protein in the brains of infected individuals. The amyloid imaging probe (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) has shown potential in the diagnosis of other amyloid disorders and we hypothesized that this compound would be effective in labeling prion protein plaques in vitro and in vivo. To test this, we compared BSB fluorescence to prion protein immunostaining on infected and uninfected brain tissue sections from scrapie-infected hamsters. We found that both methods labeled the same plaques in infected tissues while not substantially staining uninfected tissues. To test the potential of BSB as an in vivo label for prion aggregates, we perfused scrapie-infected animals with BSB and observed BSB labeled plaques co-stained with an anti-prion protein antibody. These results suggest that BSB may have use as a diagnostic tool for prion diseases. We were unable to detect BSB staining in preclinical scrapie-infected hamsters suggesting that the diagnostic potential of BSB could be limited in cases of prion disease that do not have plaques either due to a preclinical lack of pathology or disease agents like sporadic Creutzfeldt-Jakob disease (CJD), which generally lack prion plaques. However, BSB may be a useful for prion diseases where plaques are present, such as clinical variant CJD.