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PubMed1991

The relationship of complement-mediated microvasculopathy to the histologic features and clinical duration of disease in dermatomyositis.

Accumulating evidence indicates that a complement-mediated microvasculopathy may play a pathogenic role in dermatomyositis. In a previous study, we demonstrated neoantigens of the C5b-9 complement membrane attack complex in the muscle microvasculature of childhood and adult cases of dermatomyositis. To further characterize the relationship between the vascular complement deposits and histologic changes, quantitative histopathologic analyses were performed on 39 dermatomyositis biopsy specimens (26 adult, 13 children). There was a significant correlation between the percentage of fascicles with fibers having focal myofibrillar loss, a change seen early in the evolution of ischemic muscle fiber damage, and the percentage of fascicles having capillary deposits of membrane attack complex. Conversely, in biopsy specimens with a higher percentage of fascicles with perifascicular atrophy, membrane attack complex deposits were significantly less common. A fascicle-by-fascicle analysis supported these observations. Patients whose biopsy specimens were negative for microvascular membrane attack complex had clinical weakness for a significantly longer time than those patients with vascular complement deposits. These data support the hypothesis that the complement-mediated vasculopathy is a primary immunopathogenic event in the evolution of muscle lesions in dermatomyositis.

Adult↗
PubMed1977

Cell membranes in cytotoxicity.

Silica particles are cytotoxic for macrophages because they damage the membranes around secondary lysosomes in which the particles are engulfed. Hydroxyl groups of silicic acid on the surface of the particles form hydrogen bonds with phosphate ester groups of phospholipids and disrupt a variety of natural and artificial membranes. Asbestos fibers induce secretion of hydrolytic enzymes from cultured macrophages. Magnesium hydroxide groups of chrysotile asbestos interact ionically with ionized sialic acid residues of membrane glycoproteins, increase passive cation flux and produce osmotic lysis. The terminal components of complement (C5b-C9) when inserted into the bilayer structure also increase passive cation flux and produce osmotic lysis. The small complement cleavage product C3a is lytic for several cell types, especially malignant cells. The mechanism by which specifically sensitized thymus-derived (T)-lymphocytes kill tumour cells is discussed. Plasma membranes from effector lymphocytes possess considerable cytolytic potential, which is dependent on the activity of a membrane-associated proteinase.

Animals↗
PubMed1996

Increased release of tumor necrosis factor-alpha and interleukin-6 in women with the syndrome of hemolysis, elevated liver enzymes, and low platelet count.

BACKGROUND: Complement is activated in preeclampsia and complement products are known to activate macrophages. The aim of this study was to determine whether the macrophage derived cytokines, interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha, are released in patients with a form of severe preeclampsia characterized by the syndrome of hemolysis, elevated liver enzymes, and low platelet count (HELLP syndrome). METHODS: Complement activation and plasma levels of cytokines were studied in 11 women with HELLP syndrome and in 11 controls with uncomplicated pregnancies. To further evaluate the connection between complement activation and cytokine release an in vitro study on heparinized whole blood incubated with recombinant C5a was performed. RESULTS: In the HELLP group, complement anaphylatoxin C5a was increased in plasma at delivery (p < 0.01) and one day after delivery (p < 0.05), terminal C5b-9 complement complex was elevated in plasma at delivery (p < 0.001) and one day after (p < 0.01), plasma levels of interleukin-6 were increased one day after delivery (p < 0.01), and plasma concentrations of tumor necrosis factor-alpha were elevated at delivery (p < 0.01), compared with corresponding levels in controls. All parameters normalized within one week. Interleukin-I beta did not differ between the groups. In vitro, recombinant C5a incubated in whole blood gave a dose-dependent release of interleukin-6. No increased release of interleukin-1 or tumor necrosis factor-alpha was seen after incubation. CONCLUSIONS: Since cytokine release occurs in severe preeclampsia, inflammatory mechanisms may participate in the pathophysiology of severe preeclampsia.

Adult↗
PubMed1979

Proteolytic transformation of SC5b-9 into an amphiphilic macromolecule resembling the C5b-9 membrane attack complex of complement.

Proteolysis of fluid-phase SC5b-9 left a major part of the macromolecule intact and caused transition of the molecule from a hydrophilic to an amphiphilic state. The transformed complex exhibited neoantigens characteristic of the C5b-9 membrane attack complex of the complement. It yielded an SDS gel electrophoresis pattern that was similar, but not identical to that of the proteolysed, membrane attack complex. The proteolytically altered SC5b-9 complex bound lipid and incorporated into artificial lipid vesicles to yield a membrane-bound structure resembling the C5b-9 complement lesion.

Antigens↗
PubMed1990

C3, C4, and the terminal complement complex differ from C1q by binding predominantly to the antigenic part of solid phase immune complexes.

The binding of the C components C1q, C4, C3, the terminal C5b-9 complement complex (TCC) and S protein to immune complexes was studied. The hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) conjugated to BSA was adsorbed to polystyrene plates and reacted with a human IgG3-mouse chimeric anti-NIP antibody. After addition of serum a dose-dependent binding of C1q, C4, C3, and TCC to the immune complexes was found. An increase in the amount of NIP-BSA was associated with an increase in the binding of TCC and a decrease in the binding of S-protein. After addition of soluble NIP only 4 to 6% of the anti-NIP antibody remained bound to the Ag. C1q showed diminished binding after addition of NIP, whereas C4, C3, and TCC quantitatively remained bound to the Ag. Binding of TCC to the immune complexes was also found in an alternative assay, in which the anti-NIP antibody was adsorbed to the solid phase before NIP-BSA and an additional layer of anti-NIP antibody were added. The supernatants from the solid phase assay were tested for C3 activation and formation of the fluid phase TCC (SC5b-9). Activation of the C3 was reflected in the fluid phase by a dose-dependent increase in C3 activation products. This was not seen for TCC despite increased binding to the solid phase.

Animals↗
PubMed1993

A biologic basis for altered host defenses in surgically infected abscesses.

OBJECTIVE: This study determines whether there are any differences in several immunologic parameters in circulating peripheral blood leukocytes, serum, and plasma compared with pus leukocytes, and supernatant of various types of abscess. SUMMARY BACKGROUND DATA: Although there have been reports of high lysozyme levels and low complement levels within pus, there has been no systemic comparison of concentrations of these substances and others within pus compared with those within peripheral blood. METHODS: Peripheral blood and abscess pus were collected from 31 patients with abscesses and percent and mean channel of monocytes expressing complement receptor and major histocompatibility antigen HLA-DR, the percent and mean channel of polymorphonuclear leukocytes (PMN) expressing complement receptor, lysozyme level, and levels of total hemolytic complement, iC3b, C5b-9, and immunoglobulins were measured within both pus and peripheral blood. RESULTS: Percent of monocytes expressing HLA-DR and percent of monocytes and PMN expressing complement receptor, total hemolytic complement, and IgM were reduced within pus compared with peripheral blood, whereas the mean channel of monocytes expressing HLA-DR and the mean channel of PMNs expressing CR3, C5b-9, and lysozyme were increased in pus. CONCLUSIONS: There are marked differences in immunologic parameters measured within pus of abscesses versus that seen in peripheral blood.

Abdomen↗
PubMed1994

Complement dependent cytotoxicity of sensory ganglion neurons mediated by the gp120 glycoprotein of HIV-1.

Patients infected with HIV-1 frequently have a sensory neuropathy, but the cause is unknown. We found that the gp120 glycoprotein of HIV-1 bound to the surface of cultured rat dorsal root ganglia (DRG) neurons, and activated the complement cascade to lyse the neuronal cells. Cytotoxicity was measured by a 51Cr-release assay, and deposits of the C5b-9 complement complex were detected on the affected cells. As controls, gp120 or complement alone, or rgp120 plus deactivated complement, did not damage the neuronal cells, and fibroblasts were unaffected. The gp120 glycoprotein can thereby damage DRG neurons by complement dependent mechanisms. This interaction may contribute to the development of the sensory neuropathy in AIDS.

Animals↗
PubMed1990

Assembly of terminal SC5b-9 complement complexes: a new index of blood-membrane interaction.

Activation of the complement system during the course of hemodialysis was recognized more than 20 years ago and since then the generation of C3a and C5a desarg has been used as parameters of blood-membrane interaction. More recently, determination of terminal C5b-9 complement complexes has become feasible. In the present study we determined plasma concentrations of C5b-9 complexes during hemodialysis using Cuprophan or Hemophan membranes. As early as 10 min into dialysis, Cuprophan membranes led to higher arterial plasma concentrations of C5b-9 complexes in comparison to Hemophan-containing devices. With Cuprophan, systemic arterial peak values of 237 +/- 27 U/ml were reached 45 min after the onset of dialysis, while corresponding peak values using Hemophan were only 58 +/- 16 U/ml. Venous concentrations of C5b-9 complexes, measured at the outlet of the dialyzer, were 489 +/- 102 U/ml with Cuprophan and 77 +/- 19 U/ml with Hemophan dialyzers. As an index of red cell lysis, plasma levels of free hemoglobin were evaluated. There was hemolysis with both membranes. Free hemoglobin levels increased threefold with Cuprophan and only twofold with Hemophan membranes. Taken together, plasma concentrations of C5b-9 complexes clearly discern between dialysis membranes of high or low compatibility. The fact that there is simultaneous lysis of red cells might indicate that deposition of C5b-9 complexes on innocent cells occurs which would lead subsequently to an array of diverse pathophysiological reactions.

Adult↗
PubMed1991

Urinary excretion of the C5b-9 membrane attack complex of complement is a marker of immune disease activity in autologous immune complex nephritis.

The urinary excretion of the C5b-9 membrane attack complex of complement correlates with glomerular deposition of antibody in the passive Heymann nephritis (PHN) model of membranous nephropathy (MN). To determine if this parameter can be correlated with antibody deposition in a model of MN induced by an autologous mechanism and thus more analogous to human MN, the relationship of urinary C5b-9 to ongoing glomerular immune complex formation late in autologous immune complex nephritis (AICN) was studied. Based on urinary C5b-9, the animals were divided into two groups at 12 weeks after induction of AICN, those with persistently high urinary C5b-9 excretion and those in whom urinary excretion of C5b-9 returned to undetectable levels. While all rats developed glomerular deposition of rat IgG and significant proteinuria, high C5b-9 excretors had greater proteinuria and prolonged positive staining for glomerular C3. When normal syngeneic kidneys were transplanted into rats (n = 3) from each group, only those with persistent C5b-9 excretion developed subepithelial immune deposits of rat IgG in the transplanted kidney. As in the PHN model of MN, proteinuria was dissociated widely from urinary C5b-9 excretion, glomerular C3 staining, and evidence of circulating antibody. Thus these findings demonstrate that urinary excretion of C5b-9 serves as an index of on-going immunologic disease activity in the AICN model of MN, while proteinuria does not.

Animals↗
PubMed1990

Comparison of channels formed by poly C9, C5b-8 and the membrane attack complex of complement.

The channels formed by poly C9, C5b-8 and C5b-9 were examined using the liposome swelling assay. By plotting the relative rate of swelling of C5b-8-containing liposomes vs the molecular weight of the sugar solute and by applying the Renkin equation, the size of the C5b-8 channel was estimated to be 1.5 mm radius. As increasing amounts of C9 were added during the formation of C5b-9, in C8:C9 ratios of 1:1, 1:2, 1:6 and 1:12, the size of the function channel increased. Poly C9 had a pore that was somewhat larger than C5b-9 at a C8:C9 ratio of 1:12. Using molecular sieving experiments with four different iodinated protein size markers, the channel diameter of poly C9 was estimated at between 90 and 100 A. Monoclonal antibodies to different complement proteins were added to the liposomes to see which might inhibit the channels. C5b-8 containing liposomes could be inhibited by antibodies to C8. Liposomes containing C5b-9 could be inhibited slightly by antibodies to C9 and most strongly by antibodies to the neoantigen of poly C9.

Antibodies, Monoclonal↗
PubMed1986

The pore-forming protein (perforin) of cytolytic T lymphocytes is immunologically related to the components of membrane attack complex of complement through cysteine-rich domains.

Structural, functional and immunological similarities between the ninth component of complement (C9) and the lymphocyte pore-forming protein (PFP, perforin) have recently been described (8-10). PFP is shown here to be immunologically related to all other components of the membrane attack complex (MAC) of human complement, namely, C5b-6, C7, C8, and C9. Polyclonal antibodies raised against purified human C5b-6, C7, C8, or C9 react with other components of the MAC and with mouse lymphocyte PFP. The antigenic epitopes shared by human complement proteins and mouse lymphocyte PFP are limited to cysteine-rich domains. Only complement proteins that have been reduced and alkylated elicit the production of crossreactive antibodies when used as immunogens. The nonreduced forms of complement components or lymphocyte PFP neither react with these antibodies nor give rise to crossreactive antibodies. The homologous domains of complement proteins and lymphocyte PFP may play related functions in their attachment to lipid membranes and assembly of membrane lesions.

Animals↗
PubMed1991

Complement activation induces the expression of decay-accelerating factor on human mesangial cells.

In the present study we evaluated the effect of complement activation by immune complexes (IC) on the expression of decay-accelerating factor (DAF) on human mesangial cells (MC). MC in culture were incubated with an Ag (DNP-Gelatin) that binds to fibronectin present in the MC matrix. Subsequently, MC were incubated with anti-DNP antibodies in the presence of human serum. By immunoperoxidase staining we showed that these incubations resulted in IC formation and deposition of human C3 and terminal complement components (C5b-9) on the mesangial matrix and on the surface of MC. By immunoperoxidase staining and by RIA we showed that IC formation and complement activation significantly increased DAF expression on the MC plasma membrane. The induction of DAF expression was a consequence of deposition of terminal complement components on the MC because, zymosan-activated serum and IC formation in the presence of C5- or C8-deficient serum failed to increase MC DAF expression. Furthermore, the observed increased DAF expression was the consequence of increased DAF synthesis by MC. Thus, both cycloheximide and actinomycin D blocked the increase on MC DAF observed after incubation with IC and serum. MC DAF had biophysical and functional characteristics similar to DAF in other cells. Thus, 1) MC DAF was resistant to trypsin but was removed from the MC membrane by pronase; 2) phosphatidylinositol-specific phospholipase C removed 48 +/- 4% of MC DAF indicating that MC DAF is anchored in the cell membrane by phosphatidylinositol groups; 3) DAF isolated from MC-inhibited complement-mediated hemolysis and demonstrated a molecular mass of 83 kDa. In conclusion, deposition of terminal complement components on human MC trigger new synthesis and membrane expression of DAF. Because DAF protects cells against complement-mediated lysis, we postulate that DAF may protect glomerular cells during IC and complement-mediated glomerulonephritis.

CD55 Antigens↗
PubMed1989

Transcellular transport and membrane insertion of the C5b-9 membrane attack complex of complement by glomerular epithelial cells in experimental membranous nephropathy.

Deposition of the C5b-9 complex of C in glomeruli of rats with experimental membranous nephropathy (MN) is essential for the development of proteinuria. In this investigation C5b-9 was localized in the passive Heymann nephritis (PHN) by immunoelectron microscopy with a mAb specific for C5b-9(m) neoantigen. Its distribution was compared with that in another model of MN induced by successive injections of cationic human IgG and rabbit anti-human IgG into rats. In PHN C5b-9 was found: 1) in the immune deposits (ID), and on the cell membranes of foot processes close to the ID; 2) in clathrin-coated pits of the glomerular epithelial cells (GEC) close to the ID and in membrane vesicles in the cytoplasm, separated from sheep IgG and the gp330 Ag; 3) in high concentration in multivesicular bodies of GEC; and 4) in association with membrane vesicles in the urinary space which presumably are the exocytosed content of membrane vesicular bodies. By contrast, in the cationic IgG-MN model C5b-9 was found mostly in ID, but rarely within the GEC. By freeze-fracture electron microscopy we have further identified 200- to 250-A intramembrane particles in PHN in the cell membranes of the "soles" of the foot processes which resemble membrane inserted human C5b-9(m). Degradation products of C5b-9 were further detected by immunoblotting of a 100,000 x g pellet of PHN rat urine. These results indicate that, in PHN, C5b-9 is inserted into the cell membranes of GEC, and that it is selectively endocytosed and transported across GEC by a cellular mechanism which apparently protects the cell from accumulation of membrane-inserted C5b-9.

Animals↗
PubMed1994

Early accumulation of the terminal complement-complex in the ischaemic myocardium after reperfusion.

The terminal, membrane-damaging complement complex C5b-9 accumulates in the infarcted myocardium. In experimental myocardial infarction, we investigated the time course of C5b-9 deposition and the influence of reperfusion. In a group of 17 rabbits (group 1), the circumflex coronary artery was occluded for different time periods ranging from 0.5 to 29 h without subsequent reperfusion. A second group of 23 rabbits (group 2) underwent coronary artery occlusion for periods ranging from 0.5 to 6 h followed by reperfusion. C5b-9 was determined in transmural myocardial biopsies by immunohistochemistry and by ELISA. In group 1, C5b-9 accumulation in the ischaemic myocardium was found only after 5 to 6 h of coronary artery occlusion. In group 2 (ischaemia and reperfusion), significant C5b-9 deposition was already observed after 30 min of myocardial ischaemia. We conclude that in the absence of reperfusion C5b-9 accumulation occurs as a late event when most of the jeopardized myocardium has probably already become necrotic. In the presence of reperfusion, however, the complement system is activated rapidly and this could play a role in the pathogenesis of reperfusion injury.

Animals↗
PubMed1997

The role of CD11b/CD18 mediated neutrophil adhesion in complement deficient xenograft recipients.

Hyperacute rejection (HAR) of discordant xenografts is dependent on local complement activation. The formation of a functional complex of the complement components C5b-9 (membrane attack complex, MAC) causes endothelial injury and activation leading to coagulation and inflammation. In PVG rats which selectively lack the C6 component of complement, the MAC complex is not formed, whereas early split products of the complement cascade are produced normally. We reported previously that HAR is averted in C6 deficient xenograft recipients, and that subsequent accelerated acute rejection (AAR) can be delayed by inhibition of CD11b/CD18 (Mac-1) dependent neutrophil adhesion using leumedin, a member of a novel class of anti-inflammatory agents. Here we report the in vivo effects of a dose-response study using 2 new members of another class of Mac-1 directed agents designated nactins. Discordant cardiac xenografts from Hartley guinea pigs were heterotopically grafted into PVG(C6-) and PVG(C6+) rats. Experimental animals were divided into 3 groups receiving leumedin (group 1) or nactin (groups 2 and 3). Control animals received intravenous saline solution only. All C6(+) rats rejected their grafts hyperacutely within 10 to 15 min, irrespective of mode or dosage of treatment. C6 deficient controls rejected grafts within 17.7 +/- 3.5 h (n = 10). Treatment with leumedin/nactin prolonged graft survival up to 61.0 +/- 4.7 h (n = 4-6), with dose dependent differences in effectiveness among the 3 compounds tested. Histology showed that treatment was associated with less edema, hemorrhage, and neutrophil infiltrate at 2, 6, and 12 h. The marked decrease in hemorrhage seen in nactin-treated animals may reflect an interaction of Mac-1 with blood coagulation factors. Our data confirm that the neutrophil adhesion pathway is involved in AAR, especially when complement mediated injury due to MAC is restricted.

Acute Disease↗
PubMed1994

Genomic structure of the human complement protein C8 gamma: homology to the lipocalin gene family.

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target cells. It contains three subunits (C8 alpha, C8 beta, C8 gamma) which are encoded in separate genes. In relation to other proteins of the complement system, C8 gamma is unusual in that it is not structurally related to any other component nor does it have an obvious function. Based on weak but significant sequence similarity, it is proposed to be a member of the lipocalin family of widely distributed proteins that bind and transport small hydrophobic ligands. In this study, the human C8 gamma gene has been characterized and found to contain seven exons spanning approximately 1.8 kb. S1 nuclease and anchored PCR were used to identify the transcription initiation site. This site is preceded by putative regulatory elements that include two SP1 binding sites, several glucocorticoid response elements, and two SV40 enhancer core consensus sequences. A comparison to genes of other lipocalins reveals a remarkably close correlation in exon number, lengths, and phases. A close correspondence in exon boundaries is also observed and suggests that C8 gamma contains the same discrete structural elements that define the characteristic beta-barrel shape of the lipocalins. These results establish that C8 gamma is indeed ancestrally related to the lipocalin family and strengthens the likelihood that its role in the complement system is to bind an as yet unidentified ligand.

Amino Acid Sequence↗
PubMed1993

Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane-binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b-9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9-treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

Adult↗
PubMed2000

Molecular basis for a link between complement and the vascular complications of diabetes.

Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycated in vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

Amino Acid Sequence↗