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PubMed2001

Systemic complement depletion diminishes perihematomal brain edema in rats.

BACKGROUND AND PURPOSE: The complement cascade is activated after experimental intracerebral hemorrhage (ICH). It remains unclear, however, whether depleting the complement system will improve injury resulting from ICH. This study investigated the effects of systemic complement depletion on brain edema formation after ICH. METHODS: Fifty-six pentobarbital-anesthetized Sprague-Dawley rats were used. Treatment animals were complement-depleted with cobra venom factor (CVF) (intraperitoneally). Control rats received an equal volume of saline injection (intraperitoneally). In both treatment and control rats, autologous blood (100 microL) was infused stereotaxically into the right basal ganglia. Rats were killed 2, 24, or 72 hours later for brain water, ion, and tumor necrosis factor-alpha (TNF-alpha) measurements, for Western blot analysis, and for immunohistochemical studies. Brain edema was quantitated by wet/dry weight. TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Western blot analysis was applied for C9 semiquantification. Immunohistochemistry was used to detect complement C3d, C5a, C9, and myeloperoxidase. RESULTS: Perihematomal brain edema was reduced by systemic complement depletion at 24 hours (78.8+/-0.6% versus 81.5+/-0.8% in control, P:<0.01) and 72 hours (81.5+/-1.5% versus 83.6+/-0.9% in control, P:<0.05), while cerebellar water content was unaffected (78.2+/-0.3% versus 78.0+/-0. 1%). Complement depletion reduced TNF-alpha production 2 hours after ICH. Immunocytochemistry showed that complement depletion significantly reduced perihematomal C9 deposition, C3d production, and the number of C5a- and myeloperoxidase-positive cells. CONCLUSIONS: Complement depletion by CVF attenuates brain edema in ICH, indicating that complement activation plays an important role in ICH-induced brain edema. Preventing complement activation may be effective in the treatment of ICH.

Animals↗

PubMed1995

Evidence for activation of the terminal pathway of complement and upregulation of sulfated glycoprotein (SGP)-2 in the hypoglossal nucleus following peripheral nerve injury.

In a previous study, we found immunoreactivity for complement factors C3, C3d, and C4d, as well as endogenous IgG in the hypoglossal nucleus following hypoglossal nerve transection, suggesting that activation of the complement cascade had taken place in the vicinity of the axotomized motorneurons. In the present study, we found increased immunoreactivity for complement factor C1 and C1q in reactive microglia, indicating an increased potential for initiation of the classical pathway by binding of IgG to C1q. Furthermore, we found immunoreactivity for C9, which contributes to the formation of C5b-9, the final lytic product of the complement cascade close to the axotomized neurons and perineuronal glia. In addition, immunoreactivity and mRNA labeling of sulfated glycoprotein (SGP-2), a putative complement inhibitor, was increased in a subpopulation of the axotomized motorneurons. SGP-2 immunoreactivity was also increased in astroglial cells ipsilateral to the nerve injury. The results lend further support to the hypothesis that the complement cascade is activated in the vicinity of axotomized neurons, which in turn may be protected by complement inhibitors. The balance between activation of complement and complement inhibitors might have an impact on the degenerative components of the axon reaction and, in particular, the events leading to nerve cell death.

Animals↗

PubMed1981

Delayed hypersensitivity reactions to Listeria monocytogenes in rats decomplemented with cobra factor and in C5-deficient mice.

The in vivo effect of cobra factor (CoF), the complement-activating protein of cobra (Naja naja) venom, was investigated, using quantifiable assays for localization of labelled donor lymphoblasts and of host macrophages in intraperitoneal and subcutaneous sites of injection of antigens from Listeria monocytogenes. Both commercially available (Cordis) and highly purified CoF impaired these inflammatory responses, suggesting that the complement-activating protein was itself responsible rather than lymphocytotoxic or other contaminants. CoF had no measurable effect on lymphoblast localization during the first 7 hr, and only a slight effect at 24 hr, whereas macrophage accumulation was reduced by about 50% at 24 hr. This suggests that CoF treatment affected non-specific components of the early inflammatory reaction but had little or no effect on the subsequent immunospecific reaction. The effect of CoF on macrophages may be direct, or via depletion of complement components acting on macrophages, such as factor B and/or C3 or fragments thereof. It does not seem to involve the terminal complement components, C5--C9, since neither delayed-type hypersensitivity (DTH) nor cellular resistance to Listeria was reduced in C5-deficient mice when compared with C5-sufficient congenic controls.

Animals↗

PubMed1985

The killer molecule of complement.

Cell injury by complement occurs as a consequence of activation of either the classical or the alternative pathway on the surface of a cell. It is accomplished by the membrane attack complex (MAC). Its precursor proteins, C5, C6, C7, C8, and C9, are hydrophilic glycoproteins with Mr ranging from 70,000-180,000. When C5 is cleaved by the serine protease C5 convertase which covalently attaches to target cells, nascent C5b is produced and forms together with C6 a soluble and stable bimolecular complex (C5b,6). Upon binding of C5b,6 to C7 a trimolecular complex (C5b-7) is formed which expresses a metastable membrane-binding site. Membrane-bound C5b-7 constitutes the receptor for C8 and the tetramolecular C5b-8 complex binds and polymerizes C9. During the assembly process the proteins undergo hydrophilic-amphiphilic transition and the end product consists of C5b-8 (Mr approximately 550,000) and of tubular poly C9 (Mr approximately 1,100,000). The functional channel size varies but its maximal diameter is approximately 100 A. C9 polymerization appears to involve initial reversible association of several C9 molecules which is followed by temperature-dependent, constrained unfolding. Unfolded C9 monomers then associate laterally with each other and polymerization terminates with closure of the circular structure which consists of 12-18 C9 monomers. Amino acid composition and sequence indicate that the N-terminal half of the single chain C9 molecule is hydrophilic and the C-terminal half rather hydrophobic. Phospholipid-binding and insertion into membranes are functions of the C-terminal portion of the molecule. Control of the MAC is exerted by the S-protein (Mr 80,000) which binds to the forming complex and prevents its attachment to the cell membrane. Control is also exerted by certain species-specific membrane proteins which interfere with C5 convertase and C9 function.

Binding Sites↗

PubMed2002

Complement activated C4d immunoreactive oligodendrocytes delineate small cortical plaques in multiple sclerosis.

C4d-immunoreactive complement-activated oligodendrocytes (C4d-CAOs) have been described in several neurodegenerative diseases but have not been studied in multiple sclerosis (MS). Here we report that such CAOs delineate miniature MS plaques of 300-500 mum diameter. They are devoid of myelin and are surrounded by a rim of activated microglia intermingled with the C4d-CAOs. Although C4d-immunostained periaxonal oligodendroglial processes are often swollen, the axons of passage appear undamaged and extend through the demyelinated plaque area. No immunostaining with other components of the complement cascade (C1q-C9) was observed in association with these miniature plaques. However, in large MS lesions, C1q-C9 immunoreactive fibers were present, indicating complete activation of the complement cascade in these more developed lesions. It is possible that the miniature plaques, bordered by C4d-CAOs, represent the earliest stage of plaque development, preceding even the larger, transient plaques frequently observed in serial MRI studies. The association of CAOs with miniature areas of demyelination suggests a direct attack on oligodendroglial cells by the early complement components as an initiating event in MS. Incomplete complement activation indicates that this step may be reversible, whereas full and persistent activation as observed in large MS lesions may lead to death of oligodendroglia with permanent axonal damage.

Aged↗

PubMed1990

Membrane attack complex of complement in Henoch-Schönlein purpura skin and nephritis.

The present study using direct immunofluorescence with monoclonal antibodies to C5b-9 complex-related antigens was undertaken to determine whether complement activation in Henoch-Schönlein purpura (HSP) causes assembly of the membrane attack complex of complement (MAC) in skin and nephritis lesions. The deposition of C5, C6, C7, C8, C9, and C5b-9 neoantigens was noted in the vascular walls of papillary dermis and/or subpapillary dermal plexus of the vessels in 11 out of 15 patients with HSP. Their presence in vessel walls indicates complement activation which leads to terminal complement activation. There were small deposits of S protein at the same sites in three of the 11 skin specimens. Thus, the majority of C5b-9 demonstrated in HSP skin was the cytolytically active C5b-9 complex, MAC. Granular deposits of C5b-9 related antigens without S protein were also found in the capillary walls and mesangium of the glomeruli of two out of four specimens from patients with HSP nephritis; in the other two S protein was colocalized with the deposition of C5b-9. The results of the present study indicate that complement activation leading to generation of MAC may possibly be involved in the pathogenesis of vascular injury in a significantly large number of skin lesions and of HSP nephritis.

Antibodies, Monoclonal↗

PubMed1977

Role of complement in thrombogenesis. I. Complement-dependent coagulation in a model system.

Chondroitin sulphate was used to isolate from plasma a system that clotted with Russell's viper venom, brain extract and activated contact factors. Clotting appeared to depend on concomitant change in C4, C3 and C1s in the system. Brain extract additionally reacted with C9. Reconstitution of specifically defective plasmas suggested a specific role for each of these complement components in clotting.

Antigen-Antibody Reactions↗

PubMed2002

Membrane attack complex contributes to destruction of vascular integrity in acute lung allograft rejection.

The lung is known to be particularly susceptible to complement-mediated injury. Both C5a and the membrane attack complex (MAC), which is formed by the terminal components of complement (C5b-C9), can cause acute pulmonary distress in nontransplanted lungs. We used C6-deficient rats to investigate whether MAC causes injury to lung allografts. PVG.R8 lungs were transplanted orthotopically to MHC class I-incompatible PVG.1U recipients. Allografts from C6-sufficient (C6(+)) donors to C6(+) recipients were rejected with an intense vascular infiltration and diffuse alveolar hemorrhage 7 days after transplantation (n = 5). Ab and complement (C3d) deposition was accompanied by extensive vascular endothelial injury and intravascular release of von Willebrand factor. In contrast, lung allografts from C6-deficient (C6(-)) donors to C6(-) recipients survived 13-17 days (n = 5). In the absence of C6, perivascular mononuclear infiltrates of ED1(+) macrophages and CD8(+) T lymphocytes were present 7 days after transplantation, but vascular endothelial cells were quiescent, with minimal von Willebrand factor release and no evidence of alveolar hemorrhage or edema. Lung allografts were performed from C6(-) donors to C6(+) recipients (n = 5) and from C6(+) donors to C6(-) recipients (n = 5) to separate the effects of systemic and local C6 production. Lungs transplanted from C6(+) donors to C6(-) recipients had increased alveolar macrophages and capillary injury. C6 production by lung allografts was demonstrated at the mRNA and protein levels. These results demonstrate that MAC causes vascular injury in lung allografts and that the location of injury is dependent on the source of C6.

Acute Disease↗

PubMed1988

Restriction of cell lysis by homologous complement: I. An analysis of membrane attack complex formation on target membranes.

The hemolytic efficiency and binding of C9 to homologous and heterologous erythrocytes was evaluated by using a standardized passive sensitization procedure to prepare antigen- and antibody-coated erythrocytes (EA) and human serum for lysis. Heterologous bovine EA were readily lysed by human serum, whereas human EA were quite resistant to lysis. Human EA bound as many C8 and C9 molecules per cell as bovine EA when incubated under identical conditions, but four times as much bound C9 was required to lyse an equal number of human EA compared with bovine EA. The susceptibility of human erythrocytes did not increase when increased volumes of undiluted human serum were used although C9 binding increased to as much as 100,000 molecules per cell. Sodium dodecyl sulfate-resistant polymerized C9 (poly(C9)) was detected on both lysed ghosts and unlysed EA bearing complement proteins C1 through C9 (EAC1-9) after incubation with undiluted human serum; however, the ratio of poly(C9) to monomeric C9 was higher on unlysed cells than on ghosts. Although bovine and human EA bound equal amounts of human C9 at the end point, the rate of lysis and C9 uptake was slower on homologous cells. The rate-limiting step occurred before C9 binding and lysis because the rates of lysis and C9 binding were equal on homologous and heterologous EAC1-8 targets, but the extent of lysis of homologous cells was still lower than lysis of heterologous cells. Human erythrocytes lose restriction against homologous hemolysis during storage in autologous plasma or in isotonic buffers.

Animals↗

PubMed1986

Membrane attack complex of complement in rheumatoid synovial tissue demonstrated by immunofluorescent microscopy.

Rheumatoid and osteoarthritic synovial membranes were examined for evidence of terminal complement pathway activation. Using antiserum highly specific for C9 neoantigen, immunofluorescent microscopy was employed to study synovial membrane specimens from 4 patients with rheumatoid arthritis, one patient with psoriatic arthritis, and 5 patients with osteoarthritis. C9 neoantigen was detected in 3 of the 4 rheumatoid membranes, the one psoriatic membrane, and one of the 5 osteoarthritic membranes. Activation of the terminal pathway of complement through C9 should be added to the spectrum of immunologic events known to occur in some rheumatoid synovial membranes.

Adult↗

PubMed1999

Complement activation and expression of membrane regulators in the middle ear mucosa in otitis media with effusion.

The aetiopathogenesis of chronic otitis media with effusion (OME) in children is not yet fully understood. OME is characterized by metaplasia of the epithelium and accumulation of sticky, glue-like effusion in the middle ear containing different mediators of inflammation, including activation fragments of the complement system. Here we examined whether the fluid phase complement activation is reflected in the middle ear mucosa and how the mucosa is protected against the cytolytic activity of complement. Mucosal biopsies from 18 middle ears of children with a history of chronic OME were taken. The biopsies were analysed by immunofluorescence microscopy after staining for complement fragments iC3b/C3c, C3d and C9, and regulators membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and protectin (CD59). There was a strong staining for iC3b/C3c, and a weaker one for C3d and C9 on the surface of the middle ear epithelial cells of OME patients but not in controls without OME. MCP was expressed on the hyperplastic three to four outer cell layers of the epithelium, while CD59 was expressed throughout the middle ear mucosa. The results suggest a strong ongoing complement activation and consequent inflammation in the middle ear cavity. Unrestricted complement damage of the epithelial lining is prevented by the strong expression of MCP and CD59.

Antigens, CD↗

PubMed1988

Complement-fixing gastric parietal cell autoantibodies. A good marker for the identification of type A chronic atrophic gastritis.

Using an indirect immunofluorescence (IIF) technique, gastric parietal cell autoantibodies of IgG class (GPCA-IgG) were found in 2% of a normal population, in 5-26% of organ-specific autoimmune subjects and in 100% of patients with pernicious anaemia. With the exception of subjects with alopecia, there was a significantly increased prevalence of GPCA-IgG in autoimmune patients with respect to normal controls. GPCA of IgA class were detected in 22% of GPCA-IgG positive subjects, whereas GPCA of IgM class were uncommon. One-hundred and fifteen subjects underwent gastroscopy and body mucosal biopsy. Histopathological findings of chronic atrophic gastritis (CAG) were present in 71% of GPCA-IgG positive autoimmune patients without pernicious anaemia, in 100% of GPCA-IgG positive patients with pernicious anaemia, and in 20% of GPCA negative autoimmune patients. Complement-fixation test was performed in 46 GPCA-IgG positive subjects without pernicious anaemia using the IIF method. Twenty-nine patients (63%) were found to fix complement fractions till C9 (CF-GPCA) together with properdin factor, and in 25 of them (86%) the histological examination of body gastric mucosa disclosed a CAG (P = 0.0003 versus GPCA-IgG positive/CF negative controls). No significant difference was observed for the prevalence of CAG in GPCA-IgG positive/CF negative subjects with respect to GPCA-IgG negative control group. We conclude that the presence of CF-GPCA represents a useful immunological marker in the identification of CAG, while no predictive value seems to be associated with non-complement fixing GPCA-IgG.

Adolescent↗

PubMed1983

Studies on the mechanism of bacterial resistance to complement-mediated killing. III. C5b-9 deposits stably on rough and type 7 S. pneumoniae without causing bacterial killing.

Gram-positive cocci resist direct killing by serum. The mechanism of resistance was studied by measuring consumption of terminal complement components from serum and uptake of purified, radiolabeled C7 and C9 on rough and encapsulated type 7 Streptococcus pneumoniae. Extensive consumption of C5, C7, and C9 occurred when 5 X 10(8) rough or type 7 pneumococci were incubated for 1 hr in 10% pooled normal human serum (PNHS). Approximately 10,000 molecules of C7 and C9 bound per organism during the same period of incubation. Twenty to 30% of C7 and C9 was released from rough organisms. Release was not due to autolysis since it occurred with glutaraldehyde-fixed organisms as well as in S. pneumoniae that were rendered resistant to autolysis by growth in ethanolamine. Between 10 and 30% of bound 125IC9 counts were eluted from the rough and type 7 organisms by incubation in 1 M NaCl or 0.01 M EDTA, which suggests that bound C5b-9 was not attached by predominantly ionic interactions. Elution of 44 to 74% of 125IC9 from live and glutaraldehyde-fixed organisms by 1% sodium deoxycholate suggests that hydrophobic bonds are involved in C5b-9 attachment. Trypsin cleaved 67 and 55% of 125IC9 counts from live rough and type 7 S. pneumoniae, respectively which indicates that the bound complex is not protected by the cell wall from proteolytic attack. Serum resistance in S. pneumoniae does not represent a failure to form C5b-9 on the bacterial cell wall but apparently reflects a failure of the bound complex to penetrate the thick peptidoglycan layer.

Blood Bactericidal Activity↗

PubMed1993

Expression of the components and regulatory proteins of the alternative complement pathway and the membrane attack complex in normal and diseased synovium.

We have studied synthesis of the complement components and regulatory proteins of the alternative pathway and the membrane attack complex in synovial membrane. RNA was extracted from synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) as well as from normal synovial membrane. Dot blot analysis showed the presence of mRNAs for all the complement components and regulatory proteins (C3, factor B, factor D, C5, C6, C7, C9, factor H, factor I, S-protein, SP-40, 40, DAF, MCP, CR1, CD59), except for properdin, C8 alpha, C8 beta and C8 gamma in all three types of synovial membrane studied. In an attempt to determine which components were synthesised by each cell type, monocytes (mononuclear phagocytes), human umbilical vein endothelial cells (HUVEC), synovial membrane fibroblasts (from normal, OA and RA synovial membrane) and peripheral blood lymphocytes were cultured in vitro and secretion rates of individual components were measured and total cellular RNA analysed by northern blotting. Monocytes secreted properdin, C3, and factor H but not factor B, factor I, C5, C6, C7, C8 or C9. Fibroblasts and endothelial cells secreted factor B, factor H and factor I, but not properdin, C5, C6, C7, C8 or C9. Lymphocytes did not secrete any of these components. mRNAs encoding C3, factor B, factor H, S-protein, SP-40, 40, MCP and DAF were detected in all three other cell types (monocytes, fibroblasts and HU-VEC), but factor I and CD59 mRNAs were not detected in monocytes. C5, C6, C7, C8 alpha, C8 beta, CD8 gamma and C9 mRNAs were not detected in any of the cell types studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis, Rheumatoid↗

PubMed1993

Synthesis and regulation of complement components by human monocytes/macrophages and by acute monocytic leukemia.

Proteins of the complement system (C2, C3) are synthesized by human monocytes and macrophages, thus providing an important local source of these proteins in vivo which serve as a first-line host defense mechanism. In this study, we investigated the production of complement components C2, C4, and C9 by human monocytes/macrophages and by the pathologic cells of acute monocytic leukemia which represent a source of immature monocytic precursors. Human blood monocytes were collected and purified by cytapheresis and elutriation and leukemic cells by Ficoll gradient. Secretion of complement components was measured by a hemolytic assay. The evaluation of the mRNAs of the various complement components in the cells was performed by polymerase chain reaction (PCR) by adding 32P labeled deoxycytidinetriphosphate (dCTP) to the amplification step. Functional C2 was found to increase during in vitro maturation of macrophages up to the fourth week of culture. C2 mRNA was detected after amplification and increased during the maturation. Interferon-gamma (IFN-gamma) mediated a marked increase of the C2 mRNA. We found a decrease in synthesis of C4 mRNA during in vitro differentiation of human monocytes. The effect of IFN-gamma resulted in an increase in C4 mRNA. C9 mRNA was not detected although it was detected in the HepG2 hepatoma-derived cell line. Functional C2 was not detected by leukemic cells after 24 h of culture but little functional C4 was present in the cell supernatants. As they were by human monocytes and macrophages, C2 and C4 mRNAs were detected after amplification but C9 mRNAs were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1981

Homologous species restriction in lysis of erythrocytes by terminal complement proteins.

The cytolytic efficiency of the terminal complement protein complex, C5b-9, varies with the species of origin of C8 and C9. In the present study, we explored the susceptibility of erythrocytes from various species to lysis by C5b6,7 plus C8 and C9 from different species. EC5b6,7 intermediates were prepared on human, guinea pig, rabbit, mouse, and rat erythrocytes with human C5b6 and guinea pig C7. The degree of lysis of these intermediates by C8 and C9 was found to vary widely depending on the species of the proteins and the target cells. In all cases, lysis was least efficient when C8 and C9 were homologous with respect to the target cell species. This effect was mostly attributable to C9. The inefficient lysis in a homologous system is not due to a failure of C9 binding. Rather, the poor lysis in the homologous system may be attributable to inefficient insertion or channel formation.

Animals↗

PubMed1975

Neoantigens of the membrane attack complex of human complement.

The membrane attack complex of complement is a fusion product of five complement proteins: C5b, C6, C7, C8, and C9. The complex causes complement-dependent cell membrane damage. It is assembled following complement activation both on the target cell surface and in the fluid phase. The isolated soluble complex, which has a molecular weight of one million, exhibited reduced expression of the antigenic determinants of the native precursor proteins. Antisera produced to the intact complex contained antibodies to neoantigens which were not detectable on the five precursor proteins. Antisera were rendered neoantigen-specific by adsorption with fresh human serum. Since the adsorbed antisera precipitated the complex, the complex must contain multiple neoantigenic sites. The complex-specific antibodies not only reacted with the soluble complex, but also with the target cell-bound membrane attack complex.

Antigens↗

PubMedDate not supplied

Complement resistance of tumor cells: basal and induced mechanisms.

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement. appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Animals↗