C1q binding and complement activation by prions and amyloids

C1q binds to many non-self and altered-self-materials. These include microorganisms, immune complexes, apoptotic and necrotic cells and their breakdown products, and amyloids. C1q binding to amyloid fibrils found as extracellular deposits in tissues, and subsequent complement activation are involved in the pathology of several amyloid diseases, such as Alzheimer's disease. Prion diseases, such as scrapie also involve formation of amyloid by polymerization of the host prion protein (PrP). Complement activation is likely to contribute to neuronal damage in the end stages of prion diseases, but is also thought to participate in the initial infection, dissemination and replication stages. Infectious prion particles are likely to bind C1q and activate the complement system. Bound complement proteins may then influence the uptake and transport of prion particles by dendritic cells (DCs) and their subsequent proliferation at sites such as follicular DCs.     

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.     

Functional analysis of mouse ficolin-B and detection in neutrophils

Ficolins and mannan-binding lectin recognize pathogen-associated molecular patterns and initiate the lectin pathway of complement activation via the associated serine proteases. In contrast to human ficolins and mouse ficolin-A, mouse ficolin-B has been considered incapable of complement activation. Dose-dependent binding of recombinant ficolin-B to immobilized GlcNAc, acetylated BSA, acetylated LDL, and fetuin was detected with ficolin-B-specific monoclonal antibodies. Recombinant ficolin-B bound to immobilized acetylated bovine serum albumin interacted with recombinant human mannan-binding lectin-associated serine protease-2, which led to C4 cleavage, thus demonstrating the capability of ficolin-B to activate the lectin pathway. Ficolin-B-specific monoclonal antibodies identified natural ficolin-B protein in lysates of mouse granulocytes isolated from the bone marrow. These results identify mouse ficolin-B as a functional member of the ficolin family activating complement via the lectin pathway.     

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.     

Studies on the interactions between C-reactive protein and complement proteins

Several studies have investigated the interactions between C-reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b-binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme-linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1-INH. Native, ligand-unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea-treatment or by site-directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1-activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.     

Deposition of complement activation products on plastic-adsorbed immunoglobulins. A simple ELISA technique for the detection of defined complement deficiencies

The activation of complement components in human serum has been studied using immunoglobulins adsorbed to microtiter plates. The sequential deposition of complement fragments was detected by a series of mono- and polyclonal antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Antibodies against C1q, C1s, C4b/d, C3b/d, factor B, C5b-9 membrane attack complex (MAC), the regulatory complement proteins C4 binding protein (C4bp) and properdin were reactive. Several lines of evidence suggest that complement activation was via the classical pathway: (1) complement activation was highly isotype-restricted with regard to the adsorbed Igs (human IgG1 and IgG3 as well as mouse IgM, IgG2a and IgG2b isotypes are strong activators in contrast to human IgG2, IgG4, IgA and mouse IgG1); (2) Ca2+ depletion, heat treatment (56 degrees C for 45 min), incubation with 0.5 M KSCN or heat-aggregated immunoglobulins (aggIgG) abrogated serum activity; (3) complement deficient sera (C1q def', C2 def', C6 def' human sera; C2 def', C4 def' guinea pig sera) showed impaired deposition of the complement components that follow the missing component in the cascade of activation. In a clinical study sera from patients with systemic lupus erythematosus (SLE) were investigated in order to measure the effect of hypocomplementemia due to complement consumption. The results obtained suggest that this new and simple assay is well suited for (1) the detection of various inherited complement deficiencies, (2) the semiquantitative evaluation of sera with decreased complement levels, (3) a more detailed study of complement components bound to a solid phase.     

Staphylococcal superantigen-like protein 10 (SSL10) binds to human immunoglobulin G (IgG) and inhibits complement activation via the classical pathway

Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins that share structural similarity with staphylococcal superantigens but exhibit no superantigenic activity. It was previously reported that two members (SSL5 and SSL7) bound to serum components and cell adhesion molecules involved in host immune response; however, the other family members have not been functionally characterized. In this study, we attempted to isolate SSL10-binding proteins from human serum and found that recombinant His-tagged SSL10 bound two major polypeptides of ～50 and ～25 kDa after affinity purification and SDS-polyacrylamide gel electrophoresis. These polypeptides were identified as heavy and light chains of human IgG by peptide mass fingerprinting analysis. The specific interaction between recombinant SSL10 and human IgG was confirmed by far Western blot analysis using immobilized SSL10 and pull-down analysis using SSL10-conjugated Sepharose. Surface plasmon resonance analysis revealed that the dissociation equilibrium constant for the interaction between human IgG and recombinant SSL10 was estimated to be 220 nM. We also found that recombinant SSL10 inhibited the binding of complement component C1q to IgG-Sepharose and hemolysis of IgG-sensitized sheep erythrocytes via the classical complement activation pathway. These results suggest that SSL10 may play a role in the evasion of Staphylococcus aureus from the host immune system via interfering complement activation.     

The alternative pathway is required, but not alone sufficient, for retinal pathology in mouse laser-induced choroidal neovascularization

Human genetic studies have demonstrated that polymorphisms in different complement proteins can increase the risk for developing AMD. There are three pathways of complement activation, classical (CP), alternative (AP), and lectin (LP), which all activate a final common pathway. Proteins encoded by the AMD risk genes participate in the AP (CFB), CP/LP (C2), or in the AP and final common pathway (C3). Here we tested which pathway is essential in mouse laser-induced CNV. CNV was analyzed using single complement pathway knockouts (i.e., eliminating one complement pathway at a time), followed by a double knockout in which only the AP is present, and the CP and LP are disabled, using molecular, histological and electrophysiological outcomes. First, single-gene knockouts were analyzed and compared to wild type mice; C1q(-/-) (no CP), MBL(-/-) (no LP), and CFB(-/-) (no AP). Six days after the laser-induced lesion, mice without a functional AP had reduced CNV progression (P<0.001) and preserved ERG amplitudes, whereas those without a functional CP or LP were indistinguishable from the wild type controls (P>0.3). Second, AP-only mice (C1q(-/-)MBL(-/-)) were as protected from developing CNV as the CFB(-/-) mice. The degree of pathology in each strain correlated with protein levels of the angiogenic and anti-angiogenic protein VEGF and PEDF, respectively, as well as levels of terminal pathway activation product C5a, and C9. The analysis of complement activation pathways in mouse laser-induced CNV allows for the following conclusions. Comparing the single pathway knockouts with those having only a functional AP showed: (1) that AP activation is necessary, but not alone sufficient for injury; and (2) that initial complement activation proceeds via both the LP and CP. Thus, these data indicate an important role for the AP in the generation of complement-dependent injury in the RPE and choroid via amplification of CP- and LP-initiated complement activation. Improving our understanding of the local regulation of this pathway in the eye is essential for developing improved treatment approaches for AMD.     

Mouse Crry/p65 is a regulator of the alternative pathway of complement activation

Like man, mouse has evolved a unique set of regulatory proteins which provide protection from complement-mediated damage to self membranes. The recently described mouse protein Crry/p65 has been shown to inhibit classical complement pathway C3 deposition on cell membranes in which it is expressed. In two distinct experimental systems, we now further delineate the regulatory activity of Crry/p65 and demonstrate its inhibitory effect on alternative complement pathway C3 activation. First, significant inhibition of mouse alternative pathway C3 deposition was demonstrated on neuraminidase-treated human K562 cells expressing recombinant Crry/p65. Second, using a baculovirus technique, recombinant Crry/p65 was synthesized as a soluble molecule and then purified. This molecule was found to inhibit mouse C3 deposition on the surface of zymosan, a potent alternative complement pathway activator. These studies, combined with our earlier findings, demonstrate that Crry/p65 can regulate both the classical and alternative complement pathways. Crry/p65 must, therefore, exert its effects prior to, or at the level of, the C3 convertases, in a fashion similar to that of human membrane cofactor protein and/or decay-accelerating factor. These studies provide further proof of the hypothesis that Crry/p65 is an evolutionarily unique, complement regulatory protein which has developed in mouse.     

Binding of Complement Proteins C1q and C4bp to Serum Amyloid P Component (SAP) in Solid Contra Liquid Phase

Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy. Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')₂ anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate that firm binding of C1q and C4bp to SAP requires that SAP is presented on a solid phase, that C1q and C4bp react with sites distinct from the heparin binding site, and that C1q and collagen I share binding sites on SAP. Immobilized native SAP, aggregated SAP and SAP-heparan-sulphate complexes induced no detectable complement activation.     

Highly specific inhibition of C1q globular-head binding to human IgG: a novel approach to control and regulate the classical complement pathway using an engineered single chain antibody variable fragment

We sought to specifically regulate the binding of human C1q, and thus the activation of the first complement component, via the construction of a single chain antibody variable binding region fragment (scFv) targeting the C1q globular heads. Here we describe details of the construction, expression and evaluation of this scFv, which was derived from a high-affinity hybridoma (Qu) specific for the C1q globular heads. The scFv was comprised of the Qu variable heavy chain domain (VH) linked to the Qu variable light chain domain (VL) and was termed scFv-QuVHVL. When mixed with either purified C1q or with human serum as a source of C1, scFv-QuVHVL bound to C1q and competitively restricted the interaction of C1q or C1 with immobilized IgG or with IgG1 antibody-coated cells, and prevented the activation of native C1 in human serum as determined by analyses of C1-mediated C4 deposition and fluid-phase C4 conversion. However scFv-QuVHVL could be manipulated to become a C1 activator when it was irreversibly immobilized onto microtiter ELISA plates, prior to contact with human serum complement. This functional dichotomy can be a useful tool in selectively elucidating, differentiating, inducing or inhibiting specific roles of human C1q and the classical complement pathway in complement-mediated physiological processes. We project that once fully humanized, fluid-phase scFv-QuVHVL could become a useful therapeutic in limiting inadvertent host tissue damage elicited by the classical complement pathway.     

Human complement regulatory proteins expressed on mouse A9 cells containing a human chromosome 1.

The structural genes of human complement regulatory proteins are clustered on chromosome 1 at position q3.2. Human chromosome 1 was transferred into a mouse fibroblast cell line, A9 [designated as A9(neo-1)], and the surface expression of its gene products participating in complement regulation, namely C3b/C4b receptor (CR1, CD35), decay-accelerating factor (DAF, CD55), membrane co-factor protein (MCP, CD46) and C3d/EB virus receptor (CR2, CD21), were assessed using respective monoclonal antibodies by flow cytometry. CR1 became positive within 7 days of culture. MCP appeared in a small population of cells by Day 3 and, together with DAF, began to increase on Day 7. CR2 appeared on Day 14. The order of the expression was CR1 greater than DAF = MCP greater than CR2. On Day 42, however, all became negative except for MCP, which was markedly diminished. These human regulatory proteins were specifically associated with the presence of human chromosome 1, since none of them were expressed on human chromosome 12-transferred A9 cells [A9(neo-12)]. Intact A9 and A9(neo-12) cells activated human complement via the alternative pathway. The activation of this pathway was suppressed in the A9(neo-1) cells that expressed CR1, DAF and MCP. Slight protective activity was still observed in the 42-day cultured A9(neo-1) cells expressing only trace MCP. These results suggest that human complement regulators, expressed via the transferred human chromosome 1, can protect heterologous cells from complement, overcoming their ability to activate the human alternative pathway.     

Short leucine-rich glycoproteins of the extracellular matrix display diverse patterns of complement interaction and activation

The extracellular matrix consists of structural macromolecules and other proteins with regulatory functions. An important family of the latter class of molecules found in most tissues is the small leucine-rich repeat proteins (SLRPs). We have previously shown that the SLRP fibromodulin binds directly to C1q and activates the classical pathway of complement. In the present study we further examine the interactions between SLRPs and complement. Osteoadherin, like fibromodulin, binds C1q and activates the classical pathway strongly while moderate activation is seen in the terminal pathway. This can be explained by the interaction of fibromodulin and osteoadherin with factor H, a major soluble inhibitor of complement. Also, chondroadherin was found to bind C1q and activate complement, albeit to a lesser extent. Chondroadherin also binds factor H. We confirm published data showing that biglycan and decorin bind C1q but do not activate complement. In this study a similar pattern is seen for lumican although its affinity for C1q is lower than for biglycan and decorin. Furthermore, using electron microscopy and radiolabeled SLRPs, we demonstrate two different classes of SLRP binding sites on C1q, to head and stalk respectively, where only binding to the head appears to be activating. We propose a role for SLRPs in the regulation of complement activation in diseases involving the extracellular matrix, particularly those characterized by chronic inflammation such as rheumatoid arthritis, atherosclerosis, osteoarthritis and chronic obstructive lung disease.     

Monoclonal antibodies to complement components without the need of their prior purification. II. Antibodies to mouse C3 and C4

Rat monoclonal antibodies (MAbs) to mouse complement components C3 and C4 were produced by immunizing rats with cell-bound C3 and C4. This principle involves: a) using mouse thymocytes coated with syngeneic rat antibody isotypes that show high affinity to C1q, b) the intercalation of C1q from serum and c) the subsequent activation of the classical complement pathway leading to deposition of cell-bound complement components. Screening for anti-complement antibodies was performed on antibody coating microtiter plates with mouse serum as source of complement. The reactivity of the MAbs was determined by variations of the ELISA screening system using EDTA-serum to inhibit complement activation by C1 dissociation, serum rendered deficient of functionally active C3 by treatment with cobra venom factor (CVF) or serum of genetically C5-deficient mice. The specificity of the MAbs was confirmed by affinity chromatography followed by SDS-PAGE and immunoblotting. We were able to establish a panel of anti-C3 and anti-C4 MAbs of various isotypes.     

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q‐mediated complement system in the basal chordate

The origin of the classical complement pathway remains open during chordate evolution. A C1q‐like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan‐binding lectin‐associated serine protease‐2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q‐like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q‐mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway.     

Human neutrophil peptide-1 inhibits both the classical and the lectin pathway of complement activation

Human neutrophil peptide-1 (HNP-1) is a member of the alpha-defensin family. Defensins are cationic antimicrobial peptides, which play an important role in the antimicrobial response to microorganisms. In addition, recent studies have revealed the involvement of defensins in inflammation, immunity and wound repair. Defensins are present in the azurophilic granules of neutrophils and are released upon neutrophil stimulation. Previous studies showed that HNP-1 binds to C1q and inhibits the classical complement pathway. In view of the structural and functional similarity between C1q and MBL, we have now examined the interactions between HNP-1 and MBL. We observed a dose-dependent binding of HNP-1 to MBL in calcium-free buffer, indicating that HNP-1 binds to MBL most likely via the collagenous domains. To identify the binding sites in HNP-1 involved in the binding to C1q and MBL, we used a series of overlapping synthetic linear peptides that spanned the entire HNP-1 sequence. Both MBL and C1q showed a dose-dependent binding to the same set of peptides, suggesting a similar binding site in HNP-1 for both MBL and C1q. Strongest binding was observed to peptides containing the C- or N-terminal part of the HNP-1 molecule. Using an ELISA based system, we demonstrated that HNP-1 inhibits activation of both the classical pathway and lectin pathway of complement. Furthermore, we demonstrated that C1q and MBL can form complexes with HNP-1 in solution. Together, the data indicate that HNP-1 interacts with both C1q and MBL efficiently resulting in inhibition of both the classical and the lectin pathway of complement. We conclude that HNP-1 may play a role in protection against tissue injury during inflammatory conditions by inhibiting the early phase of complement activation.     

Structural and functional anatomy of the globular domain of complement protein C1q

C1q is the first subcomponent of the classical pathway of the complement system and a major connecting link between innate and acquired immunity. As a versatile charge pattern recognition molecule, C1q is capable of engaging a broad range of ligands via its heterotrimeric globular domain (gC1q) which is composed of the C-terminal regions of its A (ghA), B (ghB) and C (ghC) chains. Recent studies using recombinant forms of ghA, ghB and ghC have suggested that the gC1q domain has a modular organization and each chain can have differential ligand specificity. The crystal structure of the gC1q, molecular modeling and protein engineering studies have combined to illustrate how modular organization, charge distribution and the spatial orientation of the heterotrimeric assembly offer versatility of ligand recognition to C1q. Although the biochemical and structural studies have provided novel insights into the structure-function relationships within the gC1q domain, they have also raised many unexpected issues for debate.     

Complementing antibody profiles: assessing antibody function on antigen microarrays

Antibody effector functions other than neutralization depend on interactions with soluble and cellular components of the immune system. Antigen recognition is usually oligoclonal, with the different clones of antibodies belonging to different classes, subclasses, glycoforms and having different affinities and epitope specificities. Thus, composition of immune complexes determines biological effects mainly via interactions with FcR and complement proteins. Antibodies are capable of triggering any of the three pathways of complement activation and antigen recognition of complex antigens often results in the activation of more than one pathway. These events can be tracked in a multiplex format using antigen microarrays, where complement products bind to elements of the microarray. By controlling cation concentrations and detecting various complement components (C1q, C4, C3) contribution of the different pathways can be identified. Parallel measurement of antibodies and complement proteins provides a novel way of looking at interactions between antigen and antibodies. We propose the use of immune complex signatures, composite depictions of antibody and complement content of immune complexes characterizing healthy and diseased populations. Normalized interquartile ranges of antibody binding (IgM, IgG) and complement deposition (C4, C3) are projected onto radar charts to produce patterns that can distinguish normal and altered immune responses. We propose that comprehensive interaction studies of serum antibodies and complement with arrays of antigens can generate functional antibody profiles and help better understand immunological disease mechanism.     

Functional characterization of the lectin pathway of complement in human serum

Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum.Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms.We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.     

DOUBLE IMMUNOFLUORESCENCE STUDIES OF IMMUNOGLOBULINS, COMPLEMENT COMPONENTS AND THEIR CONTROL PROTEINS IN PATIENTS WITH IgA NEPHROPATHY

A study on double immunofluorescent staining of immunoglobulins, complement components, and their control proteins in renal tissues from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These biopsy specimens were stained with anti sera to human C1q, C4-binding protein, and β1H globulin by indirect immunofluorescent staining. These samples were then stained with rhodamine-conjugated anti human IgG, IgM, and IgA. Although C1q and C4-binding protein do not combine with IgA, they ubiquitously combine with IgG and/or IgM. β1H gobulin also combine with IgG, IgM and/or IgA. It was demonstrated that the complement system activated in IgA nephropathy was via both alternative and classical pathways. The presence of C4-binding protein in glomeruli appeared to be a more sensitive indicator of classical pathway activation than the presence of C4.