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PubMed2003

Complement activation following optic nerve crush in the adult rat.

Activation of the complement cascade following peripheral nerve axotomy and following traumatic brain injury has been demonstrated in previous studies. This study investigates the temporal pattern of microglia/macrophages and complement activation following axotomy of sensory CNS neurons, using a standardized experimental crush injury of the optic nerve in adult rats. Numerous ED1-labeled macrophages were found at the lesion site and distal to the injury at 7 days post injury (dpi). Complement C3-mRNA was upregulated 2-28 days post lesion, indicating local synthesis of complement in the optic nerve. Furthermore, increased immunoreactivity (IR) for the end product of the complement cascade, the membrane attack complex (MAC), was detected along disintegrating axons co-labeled with anti-neurofilament distal to the injury. Double-labeling for microglia show MAC-immunoreactivity expressed in their immediate vicinity, indicating a key role of microglia/macrophages in complement activation. The complement regulator Clusterin was upregulated in astrocytes at the lesion site as well as in the distal portion of the injured optic nerve, suggesting activation of a defense response to endogenous complement attack. A crush injury of the optic nerve leads to complement activation at the site of lesion and along the distal portion of the nerve, as well as upregulation of the complement inhibitor Clusterin at least in astrocytes. Reactive microglial cells seem to have a key role in complement activation as a local source of C3. We suggest that the balance between complement activation and their regulators may have impact on axonal degeneration following optic nerve injury.

Animals↗
PubMed1997

Mapping the active site of CD59.

CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. This small (77 amino acid) glycoprotein is a member of the Ly6 superfamily of proteins and is important in protecting host cells from the lytic and proinflammatory activity of the MAC. CD59 functions by binding to C8 and/or C9 in the nascent MAC and interfering with C9 membrane insertion and polymerization. We present data obtained from a combination of molecular modeling and mutagenesis techniques, which together indicate that the active site of CD59 is located in the vicinity of a hydrophobic groove on the face of the molecule opposite to a "hydrophobic strip" suggested earlier. In addition, removal of the single N-linked glycosylation site at Asn18 of CD59 resulted in an enhancement of complement inhibitory activity.

Amino Acid Sequence↗
PubMed2003

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.

Antibodies↗
PubMed2005

Inflammation, the complement system and the diseases of aging.

Inflammation is characteristic of neurodegenerative diseases of aging. Neuropathological evidence of activated microglia and activated astrocytes in lesioned areas, combined with epidemiological evidence of sparing of Alzheimer's disease (AD), Parkinson's disease (PD) and age-related macular degeneration (AMD) in long-term users of anti-inflammatory agents, indicates that inflammation is autodestructive of neurons. Locally produced autodestructive molecules include the membrane attack complex (MAC) of complement and oxygen-free radicals. Stimulation is provided by a variety of inflammatory cytokines. Agents which reduce the intensity of inflammation should have broad spectrum application in degenerative diseases of aging.

Aging↗
PubMed1984

Inherited complement deficiencies.

Isolated genetic deficiencies of individual components of the complementary system have been described in man for all the components of the classical pathway and the membrane attack complex as well as for Factor I, Factor H and properdin. It is only for Factor B and Factor D of the alternative pathway that homozygous deficiency states are not so far known. Complement deficiency states provide the most direct way of looking at the role of the complement system in vivo and emphasize the importance of complement in resistance to bacterial infection and in particular to infection with Neisseria. This association is not unexpected since in vitro studies have shown complement to be an efficient enhancer of phagocytosis and inflammation. The particularly frequent occurrence of neisserial infection may be ascribed to the ability of these organisms to survive in phagocytic cells so that the plasma cytolytic activity provided by complement is needed to kill them. On the other hand the strong association between complement deficiencies and immune-complex diseases--especially systemic lupus erythematosus--was unexpected and seems paradoxical in view of the large part played by complement in the pathogenesis of immune complex mediated tissue damage. The paradox can be explained in part by the necessity for an intact complement system in the solubilization and the proper handling of immune complexes. It is also likely that complement deficiency can allow the persistence of low virulence organisms that produce disease solely by an immune complex mechanism. Recently described deficiencies of complement receptors and their effects in vivo are described.

Blood Donors↗
PubMed2001

BrkA protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition.

Bordetella pertussis produces a 73-kDa protein, BrkA (Bordetella resistance to killing), which inhibits the bactericidal activity of complement. In this study we characterized the step in the complement cascade where BrkA acts, using three strains: a wild-type strain, a strain containing an insertional disruption of brkA, and a strain containing two copies of the brkA locus. Following incubation with 10% human serum, killing was greatest for the BrkA mutant, followed by that for the wild-type strain, while the strain with two copies of brkA was the most resistant. Complement activation was monitored by enzyme-linked immunosorbent assay (ELISA) or Western blotting. ELISAs for SC5b-9, the soluble membrane attack complex, showed that production of SC5b-9 was greatest with the brkA mutant, less with the wild type, and least with the strain containing two copies of brkA. Deposition of complement proteins on the bacteria was monitored by Western blotting. A decrease in deposition on the bacteria of C4, C3, and C9 corresponded with decreased complement sensitivity. Deposition of C1, however, was not affected by the presence of BrkA. These studies show that BrkA inhibits the classical pathway of complement activation and prevents accumulation of deposited C4.

Bacterial Outer Membrane Proteins↗
PubMed1993

Glomerular localization of thrombomodulin in human glomerulonephritis.

BACKGROUND: Thrombomodulin (TM), a glycoprotein expressed on the surface of endothelial cells, transforms protein C into a potent anticoagulant by binding thrombin. TM may be an important regulator of intraglomerular coagulation because functional TM activity was demonstrated in glomeruli isolated from normal human and rat kidneys. The role of TM in glomerulonephritis is unknown. EXPERIMENTAL DESIGN: Sections from 4 normal human kidneys and from kidneys of 100 patients with various forms of glomerulonephritis were studied by light and transmission electron microscopy, and by light and electron immunohistochemical techniques using polyclonal antibodies to recombinant human TM, and monoclonal antibodies to the membrane attack complex of the complement system. The expression of TM was graded from 0 to 4 according to the intensity and extent of the distribution, and the results were compared with the clinicopathologic findings. RESULTS: In normal glomeruli and in glomeruli with minimal abnormalities, a small amount of TM was localized at the vascular pole only (grade 0-1). In membranoproliferative glomerulonephritis and lupus glomerulonephritis, the amount of TM found on the plasma membrane of endothelial cells was significantly increased (grades 2 to 4). The expression of TM was directly correlated with proteinuria (p < 0.001), glomerular hypercellularity (p < 0.01), and number of subendothelial immune deposits (p < 0.01). In contrast, in other forms of glomerular diseases, TM was not increased and no correlation was found with the clinicopathologic parameters. CONCLUSIONS: In membranoproliferative glomerulonephritis and lupus glomerulonephritis, the amount of TM expressed by the plasma membranes of glomerular endothelial cells is increased, and this finding is a marker of disease activity. The significance of an increased expression of an endothelial anticoagulant glycoprotein in diseases characterized by pathologic intraglomerular coagulation is unknown, and requires further studies.

Fluorescent Antibody Technique↗
PubMed1989

In vitro fixation of C3d and C5b-9 on platelets by human platelet reactive antibodies.

Most platelet-reactive autoantibodies and alloantibodies are not able to fix complement in vitro. However, exceptions have been found. These antibodies are usually characterized by the conventional platelet complement fixation test. A recently developed competitive enzyme immunoassay for quantitation of platelet-associated immunoglobulins and a modification thereof allowed the quantitative study of fixation of C3d and the membrane attack complex (C5b-9) on platelets by HLA antibodies, human platelet autoantibodies, and drug-dependent antibodies (ddab). The highest amounts of both complement products were fixed through ddabs, whereas autoantibodies only showed moderate complement fixation. This enzyme immunoassay is a valuable tool for the characterization of the complement-fixing properties of platelet-reactive antibodies.

Antibodies↗
PubMed2004

Complement activation in human prion disease.

The central event in the neuropathological process of prion diseases (PrD) is the accumulation of abnormal prion protein accompanied by severe neuronal loss. Despite the infectious nature of these diseases, no prominent immune response has been detected yet. However, recent studies have shown that complement, a component of the innate immune system, is involved in the early pathogenesis of experimental prion infection. Here we demonstrate, in the diseased human brains, the presence of active compounds of the complement system, like C1q and C3b, in extracellular disease-associated prion protein deposits and the membrane attack complex in neurons. The neuronal localization of the membrane attack complex correlates well with the severity of disease-specific pathology and TUNEL labeling of neurons, irrespective of genotype or molecular phenotype of human prion diseases.

Adolescent↗
PubMed1988

Rapidly progressive glomerulonephritis: classification, pathogenetic mechanisms, and therapy.

Immunopathologic studies over the past two decades have demonstrated that rapidly progressive glomerulonephritis (RPGN) can result from glomerular deposition of anti-GBM antibody, immune complexes, or from some as yet undefined mechanism that does not involve glomerular antibody deposition. The latter process may be cell mediated and resembles a small vessel vasculitis. Most cases of idiopathic RPGN are not accompanied by pathogenic glomerular immunoglobulin deposition. Recent experimental studies of immune mechanisms of glomerular injury have identified several new processes that can induce damage to the capillary wall sufficient to result in crescentic glomerulonephritis (GN). These include direct effects of anti-GBM antibody alone and of the complement C5b-9 (membrane attack) complex, nephritogenic effects of inflammatory effector cells that involve reactive oxygen species and glomerular halogenation, and injury mediated by sensitized lymphocytes independently of antibody deposition. Macrophages have been shown to participate in both intracapillary and extracapillary fibrin deposition and crescent formation as well as to mediate capillary wall damage. The role of resident glomerular cells and cell-cell interactions in glomerulonephritis is still under active investigation. Despite these several advances in understanding immune injury to the glomerulus, therapy for RPGN remains largely empiric. Although the prognosis in RPGN has clearly improved over time, no form of disease-specific therapy has been clearly shown yet to be beneficial in a controlled study. Interpretation of the existing literature on therapy is complicated by the availability of only historical rather than concurrent controls, lack of attention to several variables known to affect disease outcome, and uncertainty regarding bias in favor of reporting positive results. Available data suggests that optimal outcomes may be achieved in anti-GBM nephritis by treatment with steroids, immunosuppression and plasma exchange, particularly when therapy is directed at patients with mild but rapidly progressive disease before oliguria or severe azotemia develop. Pulse steroids are probably the most cost-effective therapy for the idiopathic form of RPGN, but treatment with cytotoxic agents should be considered if clinical or histologic evidence of vasculitis is present.

Glomerulonephritis↗
PubMed1989

Complete deficiency of 20 KDa homologous restriction factor (HRF20) and restoration with purified HRF20.

20 KDa homologous restriction factor (HRF20) is a membrane glycoprotein which inhibits formation of membrane attack complexes of homologous complement. Erythrocytes from a patient who is completely deficient in HRF20 were readily hemolyzed by homologous complement activated by sucrose or by acidification as in paroxysmal nocturnal hemoglobinuria (PNH). After incubating PNH erythrocytes (PNH-E) with purified HRF20, the cells were analyzed by flow cytometry using a monoclonal antibody to HRF20 and shown to have the antigen absorbed. These PNH-E acquired resistance to hemolysis by homologous complement suggesting that HRF20 may be successfully used for treatment of these patients.

Adult↗
PubMed2025

Antibiotic-resistant Acinetobacter baumannii can be killed by a combination of bacteriophages and complement.

Infections caused by multidrug-resistant Acinetobacter baumannii are an emerging global health threat. Although phages have shown promising results in treating bacterial infections, the mechanisms of the combined effect of phages and innate immunity on clearing A. baumannii remain unclear. Here, we report a synergistic effect of the complement system and phages on clearing multidrug-resistant A. baumannii. We show that A. baumannii rapidly adapts and becomes resistant to phage or serum complement by modifying the expression of capsule and lipooligosaccharides, which can be regulated through reversible transposon mutagenesis in the K locus. Compared to the encapsulated phenotype, the non-encapsulated, phage-resistant A. baumannii showed a higher level of membrane attack complex deposition and were susceptible to killing by complement. In contrast, the encapsulated phenotype escaped the complement system by shedding the membrane attack complex to the environment. Thus, while the complement system targets the non-encapsulated phenotype, the phage infects and eliminates the encapsulated subpopulation. These results suggest means of combatting antibiotic-resistant A. baumannii by a simultaneous treatment with phages and complement, a combination which can be supplemented further with antibacterial antibodies.

Acinetobacter baumannii↗
PubMed2003

Chimeric human/murine monoclonal IgM antibodies to HIV-1 Nef antigen expressed on chronically infected cells.

Human IgM antibody (Ab) to gangliosides induced cytolysis of HIV-1-infected cells by homologous human complement. We expected that any human IgM Ab reactive with HIV-1 infected cells could cause complement-mediated cytolysis. The trans-chromosome mouse (TC mouse) contains human chromosomes harboring genes responsible for immunoglobulin production. Spleen cells from TC mice immunized with recombinant Nef were fused with mouse myeloma cells to generate hybridomas, and we selected those that produced human mu-chain-positive Abs reactive with Nef fixed on an ELISA plate. However, the L-chain of the monoclonal Abs (mAbs) were murine lambda in type and were chimeric, and we could not succeed in obtaining mAb with human mu- and human kappa-chains. The chimeric mAbs reacted with the HIV-1 infected cells as seen with flow cytometric analysis, and the surface expression of Nef was also detectable on chronically infected OM10.1 cells which had no detectable gp120. However, although the reaction of the chimeric IgM mAb with HIV-1-infected MOLT4 cells induced C3 deposition on cell surfaces on incubation with fresh human serum, the cells remained unlysed, as determined by 51Cr release assay. The amount of Nef antigen on the cells might not have been high enough to overcome the function of HRF20 (CD59) that restricts formation of membrane attack complexes of homologous complement. However, combination of anti-Nef IgM mAb with other IgM mAbs reactive with the surface of HIV-1-infected cells may induce a synergistic effect in complement mediated cytolysis.

Animals↗
PubMed2005

Complement inhibitors targeted to the proximal tubule prevent injury in experimental nephrotic syndrome and demonstrate a key role for C5b-9.

In glomerular diseases of diverse etiologies, dysfunction of the glomerular barrier to protein passage results in proteinuria, and proteinuria is considered an independent risk factor that plays a direct role in inflammation, interstitial fibrosis, and renal failure. The mechanism by which proteinuria leads to nephrotoxic injury is unclear, but a role for complement in mediating interstitial damage appears likely. We describe a strategy for Ag-specific targeting of complement inhibitors using a single chain Ab fragment and show that complement inhibitors targeted to the tubular epithelium protect against tubulointerstitial injury and renal dysfunction in a rat model of puromycin-induced nephrosis. The targeting of systemically administered complement inhibitors markedly enhanced their efficacy and obviated the need to systemically inhibit complement, thus reducing the risk of compromising host defense and immune homeostasis. Targeted inhibition of complement activation by Crry, and of membrane attack complex (MAC) formation by CD59 was equally therapeutic, demonstrating that the MAC plays a key role in proteinuria-induced tubulointerstitial injury. CD59 activity was dependent on its being targeted to the site of complement activation, and this is the first report of specific inhibition of the MAC in vivo after systemic administration of inhibitor. The data establish the MAC is a valid target for pharmaceutical intervention in proteinuric disorders and provide an approach to investigate the role of the MAC in complement-dependent disease under clinically relevant conditions.

Animals↗
PubMed1992

New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay accelerating factor (DAF)-deficient erythrocytes.

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.

Antibodies, Monoclonal↗
PubMed1995

Immunohistochemical localization of C9 neoantigen and the terminal complement inhibitory protein CD59 in human endometrium.

PROBLEM: Human endometrium expresses complement components, receptors, and regulatory proteins, many of which appear to be expressed in a hormone-dependent manner. Whether terminal complement components are also present in the endometrium is unknown. CD59, a broadly expressed protein that blocks association of C9 with C8 in the membrane attack complex, is localized in reproductive tissue to human spermatozoa, seminal plasma, amniotic fluid, and placenta. The present study examines human endometrium for the presence of CD59 and terminal complement proteins. METHOD: Endometrial biopsies were obtained from six normal women from various phases of the menstrual cycle and analyzed by immunohistochemistry, using MEM-43 anti-human CD59 and anti-human SC5b-9 murine monoclonal antibodies and the immunoperoxidase technique. RESULTS: Both CD59 protein and SC5b-9 (C9 neoantigen) were demonstrated to be present in endometrial glandular epithelium throughout the menstrual cycle. No specific staining was demonstrated in the stromal compartment. CONCLUSION: CD59 protein and terminal complement proteins are expressed in glandular epithelial cells of normal human endometrium, in both proliferative and luteal phases, suggesting that expression is not hormonally dependent. These analyses further support the presence of a functionally active complement system in normal human endometrium.

Antigens↗
PubMed2000

Blockade of complement inhibits obliterative bronchiolitis in rat tracheal allografts.

The role of complement activation in the development of obliterative bronchiolitis, a manifestation of chronic lung allograft rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in intragraft complement components C3 and C5b-9 (membrane attack complex) as well as IgM and IgG deposits were demonstrated during the progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared with syngeneic grafts. A 7-d treatment with recombinant human soluble complement receptor type 1 (sCR1; 20 mg/kg/d, intraperitoneal), an inhibitor of both the classic and alternative complement pathways, significantly decreased epithelial necrosis and intragraft neutrophil infiltration, and reduced obliterative changes by 40%. Immunohistochemical analysis of the grafts showed that sCR1 treatment significantly decreased early C5b-9 and IgG deposits, neutrophil chemoattractant IL-8 immunoreactivity, and ICAM-1 expression. Treatment with sCR1 was associated with increased staining for Th2 cytokines, in particular IL-10, with concomitant downregulation of IL-2 and TNF-alpha immunoreactivity. In contrast, sCR1 treatment did not affect the number of graft-infiltrating CD4(+) and CD8(+) T cells, CD45(+) B cells, ED1(+) and ED3(+) macrophages, or immune activation with expression of IL-2Ralpha or MHC class II. In conclusion, this is the first study to demonstrate that blockade of complement activation attenuates the development of OB and suggests that in addition to T cell-driven responses, humoral and antigen-independent immune responses also operate in the disease process. A blockade of complement activation renders the chemokine milieu unattractive to neutrophils and also modulates the alloimmune response toward Th2 cytokines, which may have an antiproliferative role in fibroproliferative disorders.

Animals↗
PubMed2001

X-linked myopathy with excessive autophagy: a clinicopathological study of five new families.

In 1988, Kalimo et al. (Ann Neurol 23 (1988) 258)described a new type of X-linked myopathy in a Finnish family. The clinical course was characterized by slow progression of muscle weakness without loss of ambulation in childhood and no evidence of cardiac, respiratory, or central nervous system involvement. Muscle fibers were not necrotic and showed excessive autophagic activity and exocytosis of the phagocytosed material. These authors proposed the name X-linked myopathy with excessive autophagy. Subsequently, only one French family has been reported with similar clinical and histopathological data. We report here five new families with a total of eight affected boys with the same clinical and histopathological features as reported in the original families. Histopathological findings of an asymptomatic mother are also reported. Vacuolar changes in muscle fibers result both from invaginations of the sarcolemma along with a variable component of basal lamina and from an autophagic process. The complement C5b-9 membrane attack complex associated with MHC class 1 antigen and calcium deposits is involved in muscle fiber damage. Among the X-linked myopathies, the identification of this new type is of great interest because of its favorable prognosis and unique morphological findings.

Adolescent↗