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PubMed2005

IL-4 and IL-13 induce protection of porcine endothelial cells from killing by human complement and from apoptosis through activation of a phosphatidylinositide 3-kinase/Akt pathway.

Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-alpha plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48-72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.

Animals↗
PubMed1993

Immunohistochemical localization of C3d fragment of complement and S-protein (vitronectin) in normal and diseased human kidneys: association with the C5b-9 complex and vitronectin receptor.

The localization of C3d, a fragment produced by C3 activation and S-protein (vitronectin), a regulatory factor of C5b-9, was studied immunohistochemically in normal human kidney and renal biopsies from patients with several types of glomerulonephritis. Immunofluorescent staining of the normal kidneys showed that C3d was present along the glomerular basement membrane (GBM), tubular basement membrane (TBM) and arterioles, and that S-protein was present in the GBM, mesangium, TBM, and arterioles. Immunoelectron microscopy of isolated basement membranes showed that C3d was localized exclusively on the epithelial side of the GBM, and that S-protein was present along both the epithelial and endothelial sides. In nephritic tissues, glomerular staining of C3d, C5b-9, and S-protein was increased when compared with that in normal tissues. S-protein, frequently co-localized with C3d and C5b-9 neoantigen, was intensely positive in the immune deposits of glomerular capillaries and the mesangial area, overlapping the background staining of GBM and mesangial matrix. S-protein and its receptor were occasionally co-localized in the glomeruli. These findings indicate that C3d and S-protein are normally present in the glomeruli. Co-staining of C3d, C5b-9 neoantigen, and S-protein within the immune deposits of nephritic kidneys suggests in situ binding of S-protein to locally-formed C5b-9 complex, or merely co-distribution of S-protein with the complex, rather than trapping of large molecular SC5b-9 complex from the circulation.

Complement C3d↗
PubMed1996

Analysis of the gene that encodes the complement regulatory protein, membrane inhibitor of reactive lysis (CD59). Identification of an alternatively spliced exon and characterization of the transcriptional regulatory regions of the promoter.

Membrane inhibitor of reactive lysis (MIRL, CD59) is an 18-kDa glycosylphosphatidylinositol-anchored protein that regulates formation of the membrane attack complex of complement. The purpose of these studies was to characterize the gene that encodes CD59. Our experiments redefined the structural organization of the gene by identifying a previously unrecognized alternatively spliced exon. Analysis by PCR of cDNA derived from a variety of cultured human cell lines and from PBMC showed that transcripts containing the alternatively spliced exon sequence were expressed concordantly with transcripts lacking that sequence. Primer extension studies demonstrated that the transcriptional start site of alternatively spliced CD59 mRNA is the same as that of transcripts without the alternatively spliced exon sequence, suggesting that expression of both forms of CD59 mRNA is regulated similarly. Analysis of the promoter region showed that the first 70 nucleotides immediately 5' of the transcriptional start site of the CD59 gene are essential for both constitutive and PMA-responsive transcription; however, responsiveness to PMA is cell line specific. Together, these studies have redefined the organization of the CD59 gene and identified regions of the promoter involved in constitutive and PMA-inducible transcription.

Antigens, Neoplasm↗
PubMed1991

In vitro demyelination by serum antibody from patients with Guillain-Barré syndrome requires terminal complement complexes.

Serum from 7 patients who had acute-phase Guillain-Barré syndrome with high anti-peripheral nerve myelin antibody activity (54 to 210 units/ml) was compared with serum from 3 patients in the recovery phase (0 to 17 units/ml) and serum from 7 disease control subjects (0 to 24 units/ml) and 7 normal control subjects (0 to 7 units/ml) for its ability to demyelinate rodent dorsal root ganglion cultures. The demyelinating capacity of each serum was quantitated by counting the percent of damaged internodal segments in each of four cultures. All sera from patients in the acute phase GBS caused 50 to 78% demyelination, in contrast with 6 to 19% by the sera from all 3 patients in the recovery phase and all other control subjects. The degree of demyelination correlated with anti-peripheral nerve myelin antibody activity of the sera and demyelination was complement-dependent. Further, cultures were treated with an immunoglobulin M (IgM) fraction of an acute-phase Guillain-Barré syndrome plasma plus normal human serum depleted of complement component C7. Only those cultures treated with IgM and C7-depleted human serum reconstituted with purified C7 resulted in 50.8% demyelination, which was significantly greater than the 14.2 to 16.2% demyelination observed in the presence of heat-inactivated, C7-depleted human serum plus purified C7 or in the absence of C7 or antibody. In summary, our work suggests that anti-peripheral nerve myelin antibody in Guillain-Barré syndrome mediated complement dependent-demyelination of rodent dorsal root ganglion cultures. Further, this in vitro demyelination required generation of activation complexes of the terminal complement cascade.

Acute Disease↗
PubMed1990

Reversible cell damage by T-cell perforins. Calcium influx and propidium iodide uptake into K562 cells in the absence of lysis.

The non-lethal effects of the lymphocyte-derived pore-forming toxin perforin on the human erythroleukaemia cell line K562 were investigated. By using the fluorescent Ca2+ indicator fura-2, perforin was shown to cause intracellular Ca2+ concentration to rise transiently into the micromolar range in the absence of cell death. By fluorescence-activated cell sorting it was demonstrated that K562 cells took up the membrane-impermeant nuclear stain propidium iodide (PI) when exposed to non-lethal doses of perforin. The permeability to PI was short-lived, confirming the transience of the perforin pore. Analogies with non-lethal effects and recovery processes occurring in nucleated cells exposed to the membrane-attack complex of complement are drawn.

Animals↗
PubMed2003

Sublytic terminal complement attack induces c-fos transcriptional activation in myotubes.

Sublytic C5b-9 alters the molecular phenotype of myotubes by inhibiting muscle-specific gene expression. Here, we showed that C5b-9 induced c-fos mRNA and transcription. Using c-fos promoter-CAT constructs and electrophoretic mobility shift assay (EMSA), the minimal c-fos promoter activity was shown to increase within 30-min exposure to serum C5b-9, which also induced the binding of serum response factor (SRF), along with ternary complex factor (TCF) Elk1 and Sap1a to the serum response element. C5b-9 activated ERK1, which in turn activated Elk1 in myotubes. We propose that c-fos gene transcription associated with myotube dedifferentiation is induced by C5b-9 through ERK1-mediated assembly of serum response factor-ternary complex.

Adult↗
PubMed2005

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts.

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. RESULTS: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Acute Disease↗
PubMed2000

Complement mediated vascular endothelial injury in rheumatoid nodules: a histopathological and immunohistochemical study.

OBJECTIVE: To evaluate morphologically and immunohistochemically the role of IgM rheumatoid factor (RF) immune complexes and complement activation in rheumatoid nodule vascular injury, a typical extraarticular manifestation of rheumatoid arthritis. METHODS: Histological features such as cellular infiltration, endothelial alteration, fibrinoid degeneration, and basement membrane alterations were observed in the small vessels in rheumatoid nodules. An immunohistochemical study was also carried out. RESULTS: Distinct colocalization of IgM RF and terminal complement complexes (TCC: C5b-9) was observed on the luminal surface in some of the damaged endothelial cells. Immuno-electron microscopy revealed endothelial vesiculation, typical of the in vitro protective mechanism against complement attack, with deposition of not only TCC but also IgM RF. Most TCC positive endothelial cells simultaneously expressed the major complement regulatory factor, CD59. CONCLUSION: These data suggest that, in rheumatoid nodules, vascular injury mediated by complement activation involves the assembly of IgM RF on the endothelial cell surface.

Aged↗
PubMed1997

Amino acid differences in the N-terminus of C(H)2 influence the relative abilities of IgG2 and IgG3 to activate complement.

The four human IgG isotypes are highly conserved in amino acid sequence, but show differential ability to activate complement (C'): IgG3 and IgG1 are very active, IgG2 is active under certain conditions, and IgG4 is inactive. Although the second constant domain [C(H)2] is critical for C' activation, the individual amino acids that confer isotype-specific activity have not been identified. We have generated a series of mutants between IgG2 and IgG3, resulting in the exchange of the four N-terminal and six C-terminal polymorphic residues within C(H)2. Mutants containing the N-terminus of the C(H)2 of IgG3 were as effective as wildtype IgG3 in C1q binding, C1 activation and terminal complex (MAC) formation, but had reduced ability to effect C'-mediated lysis. IgG2 and mutants containing the N-terminal portion of the C(H)2 of IgG2 were reduced compared to IgG3 in activating C1, binding C1q and inducing assembly of the MAC, and were inactive in mediating lysis of target cells. Thus, the amino acid sequence differences in the N-terminus of C(H)2 play a critical role in determining the relative abilities of IgG2 and IgG3 to bind C1q and activate the C' cascade although additional residues of C(H)2 must be involved in mediating optimal target cells lysis. The sequence of the N-terminus of C(H)2 was less critical in determining C4 and C3 binding. Characterization of domain exchange mutants suggests that intermediate steps may be partly dependent on domains other than C(H)2. IgGs that do not direct target cell lysis nevertheless activate intermediate steps in the pathway, which may contribute to immune complex-associated disorders.

Amino Acid Sequence↗
PubMed2000

Membrane attack complex formation on yeast as trigger of selective release of terminal complement proteins from human polymorphonuclear leukocytes.

It has recently been shown that measurable amounts of complement proteins, C6 and in particular C7, are released from human polymorphonuclear leukocytes (PMNs). The aim of the present study was to investigate the impact of opsonized Candida albicans on this release. Stimulation with opsonized C. albicans led to a rapid and sustained increase of C6 and C7 in the cell culture supernatant beginning within 5 min of placing in co-culture, whereas co-culture with unopsonized C. albicans or C. albicans mock-opsonized with inactivated human serum did not affect the release. In contrast, even after stimulation employing opsonized C. albicans, no release of the complement component C8 and only trace amounts of C9 were detected. The presence of the membrane attack complex (MAC) on C. albicans after opsonization was demonstrated by indirect immunofluorescence. Opsonization of C. albicans with human serum deficient in or depleted of a terminal complement component resulted in only minor stimulation of C6 and C7 release, although C3 deposition on the surface of C. albicans was not affected as determined by direct immunofluorescence. Detailed analyses with inactivated or deficient sera showed that detection of C6 and C7 was not due to insufficient washing of the opsonized yeast prior to co-culture and suggest that only a small proportion of these proteins was derived from the membrane bound and then cleaved off MAC. Thus, these findings imply that MAC on the fungal surface may represent an additional trigger for the release of C6 and C7 from PMNs, suggesting a new role for the terminal complement complex (TCC) on target membranes as modulator of PMN functions locally at the site of inflammation.

Candida↗
PubMed1996

Beneficial effects of systemic immunoglobulin in experimental membranous nephropathy.

To test the hypothesis that systemic administration of immunoglobulin might reduce glomerular injury in membranous nephropathy through mechanisms involving inhibition of complement activation, we studied the passive Heymann nephritis (PHN) model of membranous nephropathy in rats. The daily administration of immunoglobulin goat IgG (600 mg/kg i.p.) reduced proteinuria by 52%. Quantitative immunohistochemical analysis showed that the glomerular deposition of C3c, an indicator of ongoing complement attack, and of C5b-9 was significantly decreased in the immunoglobulin treated group, while deposition of anti-Fx1A was not affected. Electron microscopic analysis demonstrated that the extent of subepithelial immune complexes did not appreciably differ between treated and control animals. Systemic complement levels were not altered by immunoglobulin treatment. These data suggest that the reduction in proteinuria that resulted from systemic immunoglobulin administration was mediated by modifying the effect of complement induced glomerular injury. This interpretation was further supported by in vitro data that documented a significant reduction in C5b-9 induced glomerular epithelial cell lysis in the presence of both goat and rat IgG. These results indicate that systemic administration of immunoglobulin can substantially reduce ongoing complement activation in the glomerulus in PHN rats and that this effect is associated with a significant reduction in glomerular injury.

Animals↗
PubMed1990

Terminal complement complex in plasma of leprosy patients.

A study was carried out to find out the difference in the levels of circulating terminal complement complex between healthy volunteers and the untreated leprosy patients by using double antibody sandwich ELISA. In addition, the levels of terminal complement complex in lepromatous patients with and without reaction were also compared. None of the group showed any significant difference in the levels of circulating terminal complement complex. These findings suggest that complement may not play a direct role in tissue damage in leprosy.

Complement Activation↗
PubMed2001

Generation of C-reactive protein and complement components in atherosclerotic plaques.

C-reactive protein (CRP) and complement are hypothesized to be major mediators of inflammation in atherosclerotic plaques. We used the reverse transcriptase-polymerase chain reaction technique to detect the mRNAs for CRP and the classical complement components C1 to C9 in both normal arterial and plaque tissue, establishing that they can be endogenously generated by arteries. When the CRP mRNA levels of plaque tissue, normal artery, and liver were compared in the same cases, plaque levels were 10.2-fold higher than normal artery and 7.2-fold higher than liver. By Western blotting, we showed that the protein levels of CRP and complement proteins were also up-regulated in plaque tissue and that there was full activation of the classical complement pathway. By in situ hybridization, we detected intense signals for CRP and C4 mRNAs in smooth muscle-like cells and macrophages in the thickened intima of plaques. By immunohistochemistry we showed co-localization of CRP and the membrane attack complex of complement. We also detected up-regulation in plaque tissue of the mRNAs for the macrophage markers CD11b and HLA-DR, as well as their protein products. We showed by immunohistochemistry macrophage infiltration of plaque tissue. Because CRP is a complement activator, and activated complement attacks cells in plaque tissue, these data provide evidence of a self-sustaining autotoxic mechanism operating within the plaques as a precursor to thrombotic events.

Aged↗
PubMed1989

Localization of terminal complement components S-protein and SP-40,40 in renal biopsies.

The terminal complement complex has been implicated in the development of glomerular injury in both experimental and, indirectly, in human glomerulonephritis. Recent data suggests that the terminal complement complex in human glomerulonephritis may be in the cytolytically inactive SC5b-9 form which also contains S-protein and a recently identified protein, SP-40,40. In this study renal biopsies were examined by immunofluorescence to determine the incidence and inter-relation of deposition of the SC5b-9 components C6, C9, S-protein and SP-40,40. All components of SC5b-9 were found in arteries and arterioles, along the tubular basement membrane and in areas of glomerulosclerosis in all biopsies. This deposition was sometimes associated with C3 but never immunoglobulin deposition and correlated with the degree of renal injury. In addition, in biopsies with glomerular deposition of immunoglobulin and C3, the SC5b-9, components co-localized with the immune deposits. Glomeruli without immune deposits or glomerulosclerosis contained none of the SC5b-9 components. The incidence and pattern of distribution of SP-40,40 was similar to that of S-protein, C6 and C9 in all of cases. These data confirm that the terminal complement complex in the kidney is, at least partly, in the SC5b-9 form both in the specific immune glomerular deposition and in the "non-specific" deposition in areas of renal injury. SP-40,40 is also found in the SC5b-9 complex in all forms of renal disease.

Biopsy↗
PubMed2000

Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparin.

Complement (C')-mediated haemolysis in paroxysmal nocturnal haemoglobinuria (PNH) is mainly due to the deficiency of glycosyl phosphatidylinositol-anchored membrane proteins with C'-regulatory activities CD55 and CD59 in PNH-affected red blood cells (RBCs). Hydrophobic insertion of C5b-7 to RBC membranes, initiating the formation of a membrane attack complex, readily results in lysis of PNH RBCs due to the deficiency of CD59. We studied the significance of the electrostatic interactions between C5b-6 and RBC membranes preceding the insertion of C5b-7. In vitro, C'-mediated lysis of PNH RBCs (assessed by sucrose haemolytic assay) was inhibited by heparin, low-molecular weight heparin (LMWH) or protamine, indicating the significance of the electrostatic interactions between C' components and RBC membranes in the process of C'-mediated haemolysis. Neuraminidase-treated PNH RBCs became resistant to C' activation, suggesting that the sialic acid moieties on RBC membranes are involved in the interactions of RBC with C' components. By using biotin-labelled C7, we demonstrated that LMWH as well as heparin inhibited the insertion of C5b-7 to RBCs, although they did not inhibit the incorporation of C7 into membrane-associated C5b-6. Neither heparin nor LMWH could inhibit the procoagulant alteration of PNH RBC membranes induced by C' activation even at concentrations which inhibited the haemolysis completely. Because LMWH inhibited the C'-mediated lysis of PNH RBCs in vitro at the range which induced a limited prolongation of activated partial thromboplastin time of normal plasma, we consider that LMWH may be useful for both the inhibition of haemolysis and the prevention of thrombosis, which often follow a haemolytic attack in PNH.

Anticoagulants↗
PubMed1993

Structural analysis of the light subunit of the Entamoeba histolytica galactose-specific adherence lectin.

Adherence of Entamoeba histolytica trophozoites to colonic mucins and resistance to lysis by the membrane attack complex of complement are mediated by a galactose- and N-acetyl-D-galactosamine-specific cell-surface lectin. This lectin is a heterodimeric glycoprotein of heavy (170 kDa) and light (35/31 kDa) subunits. In this work, the amino acid sequence and membrane anchor of the light subunit were analyzed. The light subunit cDNA encoded a protein with a calculated molecular mass of 32 kDa containing two potential sites for N-linked glycosylation and putative amino- and carboxyl-terminal signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored proteins. No classical carbohydrate-binding domains common to C- or S-type eukaryotic lectins were detected by sequence analysis of either the heavy or light subunits, leaving the location of the ligand-binding site of the lectin unknown. Analysis of restriction enzyme-digested E. histolytica DNA by Southern blotting was consistent with the presence of more than one light subunit gene. Two light subunit isoforms of 31 and 35 kDa were identified by SDS-polyacrylamide gel electrophoresis analysis of affinity-purified lectin, and the isoforms were shown on two-dimensional gel analysis to form distinct 170/35- and 170/31-kDa heterodimers. The amino acid compositions and cyanogen bromide peptide patterns of the two light subunit isoforms were nearly identical. The 35-kDa isoform labeled more efficiently than the 31-kDa isoform with [3H]glucosamine, while only the 31-kDa isoform labeled with [3H]myristate and [3H]palmitate. Nitrous acid deamination released lipid from the 31-kDa isoform, which co-migrated on thin layer chromatography with acylphosphatidylinositol, a component of some GPI anchors. Gas chromatography and mass spectrometry of the deamination product from the 31-kDa subunit identified both myo- and chiro-inositols, supporting the presence of a GPI membrane anchor. The covalent association of a transmembrane protein with a GPI-anchored protein, as suggested by the cDNA sequences of the lectin heavy and light subunits, is novel and suggests unique roles for the two subunits in the pathogenesis of amebiasis.

Amino Acid Sequence↗
PubMed1976

The C5b-9 complex: subunit composition of the classical and alternative pathway-generated complex.

The membrane attack complex of complement (C5b-9) is identical in composition regardless of which pathway of activation was instrumental in its formation. Band V protein was consistently a subunit of the soluble complex. Since band V protein is not required for complement-dependent cytolysis, it probably represents a membrane site equivalent in serum of the nascent C5b-9 complex.

Centrifugation, Density Gradient↗