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PubMed1999

Functional characterization of soluble and membrane-bound forms of vaccinia virus complement control protein (VCP).

Vaccinia virus secretes a 35 kD protein, vaccinia virus complement control protein (VCP), that inhibits the classical and alternative pathways of complement at several points, indicating that it may be a viral analogue of human complement receptor type 1 (CR1; CD35). Structurally, however, CR1 is composed of 30 short consensus repeats (SCRs), whereas VCP consists entirely of four SCRs. We have begun a structure-function analysis of VCP to define the minimum number of SCRs necessary for function, the functional differences between VCP and CR1, and the potential therapeutic roles for VCP. We addressed these questions by creating and characterizing recombinant soluble and membrane-bound forms of VCP. We have determined that (1) VCP requires all four SCRs to bind C3b, (2) whereas CR1 binds C3b and iC3b, VCP binds C3b but not iC3b, and (3) although normally secreted, if expressed on the membrane of mammalian cells, VCP effectively protects the cells from complement-mediated lysis. Thus, VCP appears to be a compact and unique complement regulatory protein with the ability to inhibit both arms of the complement cascade, but lacking affinity for iC3b. By releasing rather than capturing iC3b-bearing complexes following inactivation of C3b, VCP may 'recycle' its active site locally among infected cells, and thereby enable the virus to evade more efficiently host immune and inflammatory responses. The unique function, compact structure, and capacity of VCP to protect mammalian cells from complement-mediated attack, suggests that it could be used both to better understand the structure-function relationship of complement regulatory proteins, in general, and also to rationally design and develop novel therapeutic agents.

Animals↗
PubMed1993

Complement activation in stored platelet concentrates.

Activation of platelets during preparation and/or storage of platelet concentrates in plastic containers at room temperature has recently been recognized. Many different biologic causes of this activation have been postulated. Activated complement, as a multi-enzyme system, is one of the possible sources of molecules leading to platelet activation. To detect complement activation, functional complement activity and the generation of complement-derived ligands were investigated in platelet concentrate supernatant plasma during 5 days of storage at room temperature. Hemolytic tests for functional classical and alternative pathway activity were used, as was the kinetic test for complement-mediated inhibition of immune complex precipitation. The presence of C3 activation products (C3, C3c, C3dg) was investigated in plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting procedures and on platelets by immunofluorescence. Activation of complement was evident during storage, and C3c and C3d fragments were clearly demonstrated in plasma. The amount of C3d fragments on platelets gradually rose during the first 3 days of storage. At the end of 5 days of storage, the platelets became C3d negative. There are two possible mechanisms of C3d disappearance--shedding and/or further degradation of C3d fragments. Those results indicated that complement activation and the generation of complement-dependent ligand-receptor interaction may be mechanisms for platelet activation in concentrates stored at room temperature.

Binding Sites, Antibody↗
PubMed1984

Complement fixation by pemphigus antibody. I. In vitro fixation to organ and tissue culture skin.

Although complement is often detected in the intercellular substance of pemphigus skin lesions, the ability of pemphigus antibodies to fix complement in vitro is controversial. The purpose of this study was to test in vitro complement fixation abilities of pemphigus antibodies further using organ and tissue culture methods. Epidermal cell monolayers from mouse tail were incubated with the purified IgG fraction of pemphigus serum followed by purified Clq. Binding of Clq, as well as IgG was demonstrated by immunofluorescence methods. When purified Clq was replaced with normal human serum as a complement source, positive C3 and C4 staining were also evident. When purified IgG of normal human serum was used in place of pemphigus IgG, similar immunofluorescence staining was not observed. Further evidence for complement fixation in vitro by pemphigus antibodies was obtained using organ cultures. Organ culture of normal human skin and monkey esophageal mucosa cultured in purified pemphigus IgG showed intercellular substance binding of IgG. No binding was observed when normal IgG was substituted for pemphigus IgG. Additional organ culture sections were then treated with complement (fresh normal human serum) and tested by in vitro complement staining. Fixation of Clq, C4, and C3 was noted in intercellular substance areas of organ cultured skin and mucosa incubated with pemphigus IgG but not those incubated with normal IgG. Prior treatment of pemphigus IgG organ cultured skin sections with unlabeled anti-C3, blocked positive C3 staining. These results suggest that some pemphigus antibodies are capable of activating complement in vitro.

Animals↗
PubMed1993

Complement and the recruitment of mononuclear osteoclasts.

AIM: To investigate the hypothesis that complement mediates the recruitment of mononuclear osteoclast precursors to the exposed mineralised bone surface. METHODS: Synthetic hydroxyapatite was incubated in vitro with fresh human serum, with and without complement activation inhibitors. Assays for complement components and the generation of the C3 breakdown product C3d were done. C3 deposition in human fetal tibia primary spongiosa was localised immunohistochemically and complement receptors CR1, CR2, CR3, and CR4 were localised cellularly. Immunohistochemical and enzyme histochemical characterisation of the mononuclear and multinuclear osteoclasts was made with emphasis on their association with complement C3 deposition. RESULTS: Components of complement bind to synthetic hydroxyapatite crystals and, at lower concentrations, C3d was generated in the fluid phase. C3 was deposited in a focal and linear distribution on newly formed bone trabecular surfaces in the primary spongiosa. In a similar distribution CD61, CD68, and tartrate resistant acid phosphatase positive mononuclear osteoclasts were shown in close apposition to the bone trabecular surface. These mononuclear osteoclasts, unlike multinucleate osteoclasts, expressed the complement receptors CR3 and CR4. CR1 and CR2, however, could not be shown on either mononuclear or multinuclear osteoclasts. CONCLUSION: It is suggested that C3 deposition on mineralised bone surfaces mediates the recruitment of mononuclear osteoclasts to this site. As the mononuclear osteoclasts fuse to form the multinucleate osteoclast, complement receptor expression is lost.

Bone Development↗
PubMed1989

Cardiac anaphylaxis. Complement activation as an amplification system.

Complement is activated, and C3a and C5a anaphylatoxins are generated during hypersensitivity reactions clinically associated with cardiopulmonary collapse. The administration of C3a or C5a to nonsensitized isolated guinea pig hearts mimics the events caused by antigen challenge of sensitized hearts (i.e., cardiac anaphylaxis) in the absence of complement. Thus, complement-derived anaphylatoxins may participate in immediate hypersensitivity reactions in which the heart is a target organ. To assess the contribution of complement activation and anaphylatoxin generation to cardiac dysfunction, we have elicited anaphylaxis in isolated guinea pig hearts in the presence of complement and found that the ensuing dysfunction is markedly enhanced. This amplification is most likely attributable to anaphylatoxin formation because 1) inactivation of C3 or selective C3 depletion, i.e., the loss of the component responsible for the formation of the anaphylatoxins C3a and C5a, prevents complement-induced exacerbation of cardiac anaphylaxis, whereas reconstitution with C3 and C5, or even only C3, restores it; in fact, the greater the C3 content at the time of antigen challenge, the more intense the anaphylactic crisis; and 2) the severity of cardiac anaphylaxis is markedly reduced by preexposure to C3a, and this reduction is directly related to the dose of C3a injected and to the amount of endogenous cardiac histamine depleted by C3a before antigen challenge. Complement-derived anaphylatoxins appear to promote the same mediator release that has been initiated by the antigen-antibody reaction; thus, complement activation functions as an amplification system in cardiac anaphylaxis.

Anaphylatoxins↗
PubMed2005

Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.

Previous studies indicate a pivotal role for complement in mediating both local and remote injury following ischemia and reperfusion of the intestine. Here, we report on the use of a mouse model of intestinal ischemia/reperfusion injury to investigate the strategy of targeting complement inhibition to sites of complement activation by linking an iC3b/C3dg-binding fragment of mouse complement receptor 2 (CR2) to a mouse complement-inhibitory protein, Crry. We show that the novel CR2-Crry fusion protein targets sites of local and remote (lung) complement activation following intestinal ischemia and reperfusion injury and that CR2-Crry requires a 10-fold lower dose than its systemic counterpart, Crry-Ig, to provide equivalent protection from both local and remote injury. CR2-Crry has a significantly shorter serum half-life than Crry-Ig and, unlike Crry-Ig, had no significant effect on serum complement activity at minimum effective therapeutic doses. Furthermore, the minimum effective dose of Crry-Ig significantly enhanced susceptibility to infection in a mouse model of acute septic peritonitis, whereas the effect of CR2-Crry on susceptibility to infection was indistinguishable from that of PBS control. Thus, compared with systemic inhibition, CR2-mediated targeting of a complement inhibitor of activation improved bioavailability, significantly enhanced efficacy, and maintained host resistance to infection.

Animals↗
PubMed2001

Regulation of complement classical pathway by association of C4b-binding protein to the surfaces of SK-OV-3 and Caov-3 ovarian adenocarcinoma cells.

The role of fluid-phase regulators of complement is to inhibit excessive complement activation and maintain homeostasis in blood. By binding to and inactivating complement components on cell surfaces, they can also protect autologous cells from complement-mediated cytotoxicity and phagocytosis. In this study, we wanted to find out whether C4b-binding protein (C4bp), a fluid-phase regulator of the classical complement pathway, could directly bind to cell surfaces in a functionally active form. After screening several malignant cell lines, we observed that the ovarian adenocarcinoma cell lines SK-OV-3, Caov-3, and SW626 were capable of binding C4bp. Binding tests with recombinant deletion mutants suggested that the primary binding site on C4bp is located on the alpha-chain complement control protein 4 domain. Functional tests showed that tumor cell-bound C4bp retained its cofactor activity for factor I-mediated inactivation of C4b, thus increasing the control of classical complement pathway activation on the surfaces of these cells. These results demonstrate a novel mechanism of complement regulation on cell surfaces, particularly on those of malignant ovarian tumor cells.

Adenocarcinoma↗
PubMed2003

Complement activation contributes to hypoxic-ischemic brain injury in neonatal rats.

Conflicting data have emerged regarding the role of complement activation in the pathophysiology of cerebral ischemia. On the basis of considerable evidence implicating inflammatory mediators in the progression of neonatal brain injury, we evaluated the contribution of complement activation to cerebral hypoxic-ischemic (HI) injury in the neonatal rat. To elicit unilateral forebrain HI injury, 7-d-old rats underwent right carotid ligation followed by 1.5-2 hr of exposure to 8% oxygen. Using immunoprecipitation and Western blot assays, we determined that HI induces local complement cascade activation as early as 8 hr post-HI; there was an eightfold increase in the activation fragment inactivated C3b at 16 hr. With immunofluorescence assays and confocal microscopy, both C3 and C9 were localized to injured neurons 16 and 24 hr post-HI. To investigate the contribution of systemic complement to brain injury, we administered the complement-depleting agent cobra venom factor (CVF) 24 hr before HI lesioning and evaluated both acute HI-induced complement deposition and the extent of resulting tissue injury 5 d after lesioning. CVF depleted both systemic and brain C3 by the time of surgery and reduced infarct size. Analysis of lesioned, CVF-treated animals demonstrated minimal neuronal C3 deposition but no reduction in C9 deposition. C3-immunoreactive microglia were identified in injured areas. These results indicate that complement activation contributes to HI injury in neonatal rat brain, systemic administration of CVF does not eliminate complement deposition within injured brain, and microglia may represent an important local source of C3 after acute brain injury.

Animals↗
PubMed1988

Accentuated formation of the terminal C5b-9 complement complex in patient plasma precedes development of the adult respiratory distress syndrome.

Extensive studies have been conducted to determine the pathogenesis of the adult respiratory distress syndrome (ARDS) by investigating the role of complement, a mediator of inflammation. Complement activation products have been detected in blood samples from patients during ARDS. However, the individual complement components that have been assessed only indicated generalized inflammation, and none could unequivocally discriminate the onset of this acute inflammatory lung injury. In this two-year prospective study of 87 septic patients, 22 of whom developed ARDS (25%), we determined complement activation by quantifying the terminal complement complex (TCC), C5b-9. The TCC is a stable complement by-product formed following activation of either the classical or alternative pathways. Our results show that plasma TCC concentrations increased an average of 110% (p = 0.002) two days prior to the onset of ARDS and also transiently increased an average of 45% (p = 0.01) immediately preceding its resolution. Furthermore, plasma TCC concentrations were a more sensitive measure of this acute inflammatory lung injury than levels of C3a desarginine, C4a desarginine, C5a desarginine, and total hemolytic complement activity. We conclude that a temporal association exists between accentuated formation of plasma TCC and the development and also resolution of septic ARDS. Therefore, we suggest that researchers include plasma TCC concentrations in clinical studies when they could use a potential early indicator for ARDS.

Complement Activation↗
PubMed2003

Complement activation by direct C4 binding to thyroperoxidase in Hashimoto's thyroiditis.

Biosynthesis of thyroid hormones is an oxidative process that generates reactive oxygen species (ROS) and involves thyroperoxidase (TPO) that is one of the main autoantigens involved in autoimmune thyroid diseases. The ectodomain of TPO consists of a large N-terminal myeloperoxidase-like module followed by a complement control protein (CCP)-like module and an epidermal growth factor-like module. The presence of these two additional gene modules suggests that they may play some crucial, hitherto unsuspected role associated with thyroid function. Because the CCP module is a constituent of the molecules involved in the activation of C4 complement component, we investigated the possibility that C4 may bind to TPO and activate the complement pathway in autoimmune conditions. We showed that TPO via its CCP module directly activated complement without any mediation by Ig. We suggested that this additional complement pathway requires the production of ROS and specially hydroxyl radicals that aggregate TPO and oxidize methionines of C4. Moreover, we found, in patients with Hashimoto's thyroiditis, that thyrocytes overexpress C4 and all the downstream components of the complement pathway. These results indicate that TPO has some as yet unknown function, which may contribute along with other mechanisms to the massive cell destruction observed in Hashimoto's thyroiditis. Investigating this complement pathway, therefore, would provide an excellent means of reaching a better understanding of the etiology of other degenerative diseases.

Acute Disease↗
PubMed1997

Frequent expression of complement resistance factors CD46, CD55, and CD59 on gastrointestinal cancer cells limits the therapeutic potential of monoclonal antibody 17-1A.

BACKGROUND: One reason for the failure of monoclonal antibody (mab) trials in most cancer patients might be the presence of complement resistance factors that inhibit complement dependent cytotoxicity (CDC) and the release of inflammatory mediators (e.g., anaphylatoxins). METHOD: We have determined the expression of CD46, CD55, and CD59 in five gastric, three colon, and seven pancreatic human cancer cell lines by immunostaining. The complement activating properties of mabs and conjugates with cobra venom factor (CVF) were studied in a 51Cr-release toxicity assay and in an ELISA to determine the release of C3a. RESULT: Virtually all tumor cell lines strongly expressed CD46, CD55, and CD59, except KATOIII gastric cancer cells (CD55 and CD59 negative). In accordance with other studies we could confirm that expression of CD55 and CD59 inhibits a complement activation by mabs. Whereas 17-1A was able to induce a cytotoxic complement activation on KATOIII cells, neither a CDC nor an anaphylatoxin release (C3a) was observed on MKN28 cells (strong expression of CD55 and CD59). Conjugation with CVF, a strong activator of the alternative pathway of complement, could partially restore the complement activation by mabs. A 17-1A-CVF conjugate, although still nontoxic, induced the release of the anaphylatoxin C3a on both cell lines. The same observations were made in PancTuI pancreatic cancer cells treated with a conjugate of the mab CA19-9 and CVF. CONCLUSIONS: Our study shows that complement resistance is a frequent event in gastrointestinal cancer, limiting the potential of monoclonal antibodies. Mabs, when conjugated with CVF, partially retain complement activating properties by releasing C3a, which in vivo will support a cellular immune response.

Antibodies, Monoclonal↗
PubMed1982

Human complement activation by self-associated IgG rheumatoid factors.

IgG rheumatoid factors (IgG-RFs) undergo concentration-dependent self-association into dimers and higher polymers, as previously reported. The interactions of purified IgG-RF from the plasma of 3 patients with rheumatoid arthritis, with guinea pig and human complement were studied. Self-associating IgG-RFs were isolated by affinity columns and gel filtration. These preparations contained no detectable IgM and were composed only of IgG subclasses known to fix complement. Complement utilization of IgG-RF was compared with that of monomeric IgG, heat-aggregated IgG, and soluble rabbit IgG immune complexes. Although incubation of IgG-RF or monomeric IgG with 3 units of guinea pig or human complement resulted in decreased hemolysis of sheep erythrocytes sensitized with IgM hemolysin, these substances were less than 100 times as effective as heat-aggregated IgG or soluble immune complexes. The ability of human or guinea pig complement that had been incubated with IgG-RF to restore hemolytic activity to C4-deficient guinea pig serum served to distinguish Clq binding from complement cascade activation. IgG-RFs and monomeric IgG did not activate guinea pig complement cascade in contrast to aggregated IgG. IgG-RFs, however, activated human complement cascade; monomeric IgG only bound human Clq. These results indicate that self-associated IgG-RFs can activate human complement in fluid phase, but less effectively than aggregated IgG or large-latticed immune complexes.

Arthritis, Rheumatoid↗
PubMed2001

Complement activation and inflammatory processes in Drusen formation and age related macular degeneration.

Recent studies implicate inflammation and complement mediated attack as early events in drusen biogenesis. The investigations described here sought to determine whether primary sites of complement activation could be identified within drusen substructure, and whether known inhibitors of the terminal pathway of complement are present in drusen and/or retinal pigmented epithelial (RPE) cells that lie in close proximity to drusen. Immunohistochemical examination shows two fluid phase regulators of the terminal pathway, vitronectin (Vn, S-protein) and clusterin (apolipoprotein J), to be present in drusen; Vn also accumulates in the cytoplasm of RPE cells that are closely associated with drusen. The membrane associated complement inhibitor, complement receptor 1, is also localized in drusen, but it is not detected in RPE cells immunohistochemically. In contrast, a second membrane associated complement inhibitor, membrane cofactor protein, is present in drusen associated RPE cells, as well as in small, spherical substructural elements within drusen. These previously unidentified elements also show strong immunoreactivity for proteolytic fragments of complement component C3 that are characteristically deposited at sites of complement activation. It is proposed that these structures represent residual debris from degenerating RPE cells that are the targets of complement attack. It is likely that RPE cell debris entrapped between the RPE monolayer and Bruch's membrane serves as a chronic inflammatory stimulus and a potential nucleation site for drusen formation. Thus, the process of drusen biogenesis may be envisaged as a secondary manifestation of primary RPE pathology that is exacerbated by consequences of local inflammatory processes.

Adolescent↗
PubMed1986

Enzyme immunoassay of complement-binding rheumatoid factors.

We describe an enzyme immunoassay for the determination of complement-binding rheumatoid factors. Polystyrene tubes are coated with heat aggregated human IgG. The rheumatoid factors (RFs) of patients heat inactivated sera are allowed to bind to aggregated IgG and thereafter saturated with fresh human complement. The amount of C3 complement bound is measured by indirect enzyme immunoassay. The levels of complement binding RFs were measured in 30 patients with seropositive rheumatoid arthritis (RA), in 19 patients with systemic lupus erythematosus (SLE), and in 30 healthy control subjects. Compared to the controls high levels of complement-binding RFs were found both in RA and in SLE (P less than 0.0005). The mean level of the complement binding RFs was higher (P less than 0.05) in active than in inactive SLE. Even though the 19 S IgM RF bound complement, in RA no correlation was found between the level of complement binding RFs and Waaler-Rose titre, but the level of complement binding RF correlated with the levels of nonagglutinating IgM RF (r = 0.56, P less than 0.01) and IgG RF (r = 0.70, P less than 0.001) that were obtained by enzyme immunoassay.

Adult↗
PubMed1978

Complement system in sodium taurocholate pancreatitis in the rat.

The role of the complement system was studied in Na-taurocholate pancreatitis in rats. Complement activity (CH 50) was determined at various times in the course of pancreatitis. Immediately after induction of acute pancreatitis, serum complement activity declined and massive C 3 deposits could be detected in the vicinity of acinar necroses and necrobioses. After a phase of recovery three to six hours postoperatively a second complement consumption occurred. Lethality rate increased as serum complement activity fell below 50% of preoperative values. The degree of C 3 deposition increased up to six hours. Decline of serum complement activity and deposition within the pancreas seemed to be correlated with histologically demonstrable tissue lesion. The first decline of complement activity in serum is thought to be caused by liberation of complement activating substances within the pancreas due to the detergent action of Na-taurocholate itself. The second decline, however, may be due to the liberation of complement activating and/or destroying enzymes into the blood stream.

Animals↗
PubMed1992

Reconstruction of anti-leprosy drug depleted complement haemolytic activity by addition of zymosan-treated sera (a source of C142) and CratEDTA (a source of C3-C9).

This paper describes the mechanism of in vitro interaction of human serum complement system with anti-leprosy drugs (dapsone and clofazimine) and anti-lepra reaction drugs such as chloroquine. These drugs could inhibit the complement-mediated lysis of erythrocytes both via direct and alternative pathways, but only at hypertherapeutic doses. Attempts were made to restore the drug depleted complement-mediated lysis of erythrocytes by adding zymosan-treated guinea-pig sera (a source of C142) and also by adding Crat-EDTA sera (a source of C3-C9). Destroyed complement-mediated haemolytic activity by dapsone could be restored by early complement (C142) components, while complement-mediated haemolytic activity blocked by clofazimine could be regenerated by adding both late (C3-C9) and early (C142) complement component. However, chloroquine-mediated inhibition of the complement-mediated haemolysis activity could not be appreciably restored by adding both early and late complement reagents.

Animals↗
PubMed1995

Complement and clusterin in the spinal cord dorsal horn and gracile nucleus following sciatic nerve injury in the adult rat.

We provide evidence for activation of the complement cascade in the dorsal horn of the spinal cord and in the gracile nucleus in the brainstem following sciatic nerve transection in the adult rat. Immunocytochemical analyses showed immunoreactivity for endogenous immunoglobulin G as shown by immunostaining with F(ab')2 antibodies, as well as complement factors C1, C1q, C3, C3d and C9 in the appropriate central termination areas of the injured sciatic nerve. Results from double labelling immunocytochemistry showed a strong association between immunoglobulin and complement factors on the one hand and reactive microglia on the other. However, some complement immunoreactivity was also found in the neuropil, possibly representing secreted complement. In situ hybridization with an oligonucleotide probe showed a marked increase in C3 messenger RNA, indicating local synthesis of C3 protein. In parallel with activation of complement, there was an increased immunoreactivity for the putative complement inhibitor clusterin, which co-localized with glial fibrillary acidic protein-positive astrocytes. In situ hybridization showed an increased labelling of clusterin messenger RNA. These findings indicate that complement activation and up-regulation of complement inhibitors are prominent central responses to peripheral sensory nerve injury. These responses may therefore be important elements underlying so-called transganglionic degenerative changes in primary sensory axons and terminals.

Animals↗
PubMed2005

Effects of C-reactive protein and the third and fourth components of complement (C3 and C4) on incidence of atrial fibrillation.

Although C-reactive protein (CRP) is a major cardiovascular risk factor, its association with atrial fibrillation (AF) remains controversial. This study explored whether the CRP-related incidence of AF is modified by serum markers of inflammation in a population-based cohort with follow-up data. Serum CRP and 2 components of the complement system (C3 and C4) were determined in 1,011 healthy patients (25 to 64 years old). The incidence of AF was compared between groups defined by levels of CRP, C3, and C4. The follow-up time was 4 years. A high complement level was defined as either C3 or C4 in the top quartile. High CRP was associated with high complement components. An increased CRP level was associated with an increased incidence of AF. The complement components modified these associations. After risk factor adjustment, those with high CRP and high complement components had a significantly higher risk of AF (relative risk 3.0, 95% confidence interval 2.0 to 4.2) than those with normal CRP and low complement levels. In the absence of a high complement level, a high CRP level was not significantly associated with AF (relative risk 1.1, 95% confidence interval 1.0 to 1.4). In conclusion, high CRP levels are associated with high serum levels of complement components. These proteins increase the CRP-related incidence of AF. In the absence of elevated complement components, no statistically confirmed association was found between a high CRP level and AF.

Adult↗