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PubMed1996

Role of complement, chemokines, and regulatory cytokines in acute lung injury.

The roles of complement, proinflammatory cytokines and regulatory cytokines in lung inflammatory injury are becoming defined. Like the proinflammatory cytokines (TNF alpha and IL-1), complement activation products (C5a and/or the membrane attack complex, C5b-9) can directly activate endothelial cells to cause upregulation of adhesion molecules (P-selectin) or can function in a synergistic manner with TNF alpha to cause enhanced upregulation of ICAM-1 and E-selectin. The beta chemokine, MIP-1 alpha, appears to function in vivo as an autocrine activator, enhancing TNF alpha production by pulmonary macrophages, which, in turn, enhances the inflammatory response. Finally, IL-4 and IL-10 have strong regulatory effects by suppressing in vivo production of TNF alpha. There is now compelling evidence to suggest that, in IgG immune-complex-induced lung inflammation in rats, endogenous IL-10 is produced and regulates the intensity of the inflammatory response. Blocking of endogenous IL-10 substantially increases lung TNF alpha production, the recruitment of neutrophils, and the intensity of lung inflammatory injury. Accordingly, the network of cytokines carefully regulates lung inflammatory responses.

Acute Disease↗
PubMed1994

High-dose intravenous immunoglobulin exerts its beneficial effect in patients with dermatomyositis by blocking endomysial deposition of activated complement fragments.

In patients with dermatomyositis (DM) the earliest lesion is microvasculopathy mediated by deposition of C5b-C9 membranolytic attack complex (MAC) on intramuscular capillaries. This leads sequentially to muscle ischemia, necrosis of muscle fibers, and muscle weakness. High-dose intravenous immunoglobulin (IVIG), which can modulate complement-dependent tissue damage in animal models, has been shown to be effective in the treatment of patients with DM. We used an in vitro C3 uptake assay to examine 55 coded sera from 13 patients with DM and 5 patients with other non-complement-mediated neuromuscular diseases, before and after treatment with IVIG or placebo. Patients with active DM had a significantly higher baseline C3 uptake compared with the others (geometric mean 12,190 vs 3,090 cpm). Post-IVIG but not post-placebo sera inhibited the C3 uptake, without depleting the complement components, by 70.6-93.4%. The maximum inhibition of C3 uptake occurred within hours after IVIG infusion, started to rebound 2 d later, and reached pretreatment levels after 30 d. The serum levels of SC5b-9 complex production were high at baseline but normalized after IVIG therapy. Repeat biopsies from muscles of improved patients showed disappearance of C3b NEO and MAC deposits from the endomysial capillaries and restoration of the capillary network. We conclude that IVIG exerts its beneficial clinical effect by intercepting the assembly and deposition of MAC on the endomysial capillaries through the formation of complexes between the infused immunoglobulins and C3b, thereby preventing the incorporation of activated C3 molecules into C5 convertase. These findings provide the first serological and in situ evidence that IVIG modulates complement attack in a human disease.

Adult↗
PubMed2000

Combined glomerular deposition of polymeric rat IgA and IgG aggravates renal inflammation.

BACKGROUND: IgA nephropathy (IgAN) is characterized by deposition in the glomerular mesangium of IgA together with C3, C5b-9, and properdin. IgG deposition as a risk factor in IgAN was recently confirmed by a long-term follow-up of patients with IgAN. We previously reported on an acute model of IgA-mediated glomerular inflammation in Wistar rats. METHODS: To investigate the effect of the combination of IgA and IgG on glomerular injury, Wistar rats were injected with a minimum dose of rat IgG in the presence or absence of a subnephritogenic dose of polymeric rat IgA. Subsequently, glomerular complement activation, influx of inflammatory cells, proteinuria, and hematuria were assessed. RESULTS: Administration of IgG to the rats resulted in maximal proteinuria of 20.3 +/- 12.1 mg/24 h on day 2 and an absence of overt glomerular inflammation. Administration of polymeric rat IgA antibodies to rats resulted in hematuria with a moderate mesangial complement deposition. In the combination group, however, glomerular deposition of C5b-9 was dramatically increased. This was accompanied by increased proteinuria as compared with rats receiving IgA or IgG antibody injections alone on day 7. Microhematuria occurred in rats receiving either polymeric rat IgA or IgG alone or the combination. While both rat IgG and polymeric IgA induced minor mesangial cell (MC) proliferation and MC lysis, the combination resulted in a pronounced, significant increased percentage of aneurysm formation on day 7 after injection. CONCLUSIONS: We conclude that in this model of IgA-induced glomerulopathy, a selective, complement-dependent glomerular inflammation is induced in Wistar rats by glomerular codeposition of rat isotypic monoclonal antibodies.

Animals↗
PubMed1983

Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics.

Lysis of nucleated cells by complement was studied to determine whether the lytic process by C5b-9 conforms to a one-hit mechanism as in the case of erythrocytes. Two nucleated cell lines, Molt 4 and U937, derived from human T lymphocytes and histiocytes, respectively, were employed as targets. The antibody-sensitized cells were used to develop the titration curves, measuring cell death as a function of limiting quantities of human C6 or C5,6 complex in the presence of an excess of other complement components. The cytolysis curves generated in both experiments were sigmoidal, in sharp contrast to the monotonic curves observed in lysis of erythrocytes treated similarly. The sigmoidal curves of cytolysis indicate a cooperative action of several molecules of C6 or acid-activated C5,6 complex, C(56)a. In contrast to the multi-hit characteristics of cytolysis, dose-response measurements of the release of 86Rb indicated that only one effective molecule of C6 per cell is required for assembly of a 86Rb-releasing channel. This divergence indicates that lysis requires formation of several channels or, alternatively, assembly of large channels that are formed by several molecules of C6. Because prior studies with erythrocyte ghosts have shown that only a single effective molecule of C6 is required for assembly of a transmembrane channel, regardless of size, we prefer to interpret the multi-hit characteristics of nucleated cell lysis as an indication of a multi-channel requirement, rather than channel enlargement.

Cell Line↗
PubMed1988

Restriction of cell lysis by homologous complement: II. Protection of erythrocytes against lysis by newly activated complement.

Our previous work revealed that homologous complement (C) was ineffective in lysing antibody-sensitized erythrocytes (EA) even at high concentrations. It was also shown that activation of complement on homologous EA resulted in the binding of C9 and the formation of EA bearing complement proteins C1 through C9 (EAC1-9), yet few hemolytic sites were formed. Instead, as shown here, the formation of homologous EAC1-9 caused the cells to become resistant to lysis even by heterologous complement during a second incubation. In contrast, when homologous EAC1-8 were produced by incubating EA with C9-depleted serum, such intermediates were not protected against lysis by heterologous complement during a second incubation. Furthermore, homologous C9 on EAC1-9 was able to reduce the hemolytic efficiency of heterologous complement without blocking C activation and the formation of new C5b-9 complexes. Protection was not modified when homologous EAC1-9 were produced in one step, by incubation of EA with serum, or sequentially by adding C9 to EAC1-8. The minimum number of 9-sites required to confer a protective effect on EAC1-9 was less than 200 per cell. Thus, in addition to its known effect in heterologous cell killing, homologous C9 is capable of protecting homologous cells against inadvertent complement lysis.

Ammonia↗
PubMed1988

Relative inefficiency of terminal complement activation.

The efficiency of generation of fluid-phase SC5b-9 and membrane C5b-9(m) complexes relative to cleavage of C3 and C5 was studied. Fluid-phase C activation was induced through addition of purified bacterial Ag to human serum. Sephadex beads were used as particulate activators of the alternative pathway. Rabbit or antibody-coated sheep or human E were used to study formation of cytolytic C5b-9(m) complexes. The molar ratios of C3a:C5a generated in the model systems were found to be in the range of 60 to 200:1 in the case of soluble immune complex activators, and 70 to 150:1 with particulate activators and cells. The efficiency of C5 cleavage relative to C3 cleavage increased on surfaces with the density of antibody and/or C3b-binding sites. With soluble immune complexes, the efficiency of subsequent SC5b-9 generation displayed wide variations dependent on Ag and donor with molar ratios of C5a:SC5b-9 ranging from 30:1 for teichoic acid and sometimes approaching 1:1 for streptolysin-O. In contrast, activation on particles or cells always led to C5a:C5b-9 (calculated as the sum of generated moles SC5b-9 and C5b-9(m] ratios approaching 1:1. Hence, there is an overall inefficiency of terminal sequence activation in the C cascade due first to a dissociation at the level of C5 convertase formation/C5-cleavage and second, to a frequent inefficiency of C5b-utilization in the fluid-phase. The results provide an explanation for the very low levels of SC5b-9 found in plasma of healthy individuals and in patients with C-consuming immune complex disease.

Animals↗
PubMedDate not supplied

Change of complement system predicts the outcome of patients with severe thermal injury.

To establish the clinical relevance of the changes in the complement system in patients with thermal injury, we studied 20 patients who had third-degree burns on more than 60% of total body surface area. Their levels of the C3, C4, soluble C5b-9, and functional hemolytic activities of total (CH50) and alternative (AH50) complement pathways were sequentially measured for 2 weeks after thermal injury. All patients showed low C3 levels initially but increased C3 levels in the following days. The increasing trend of C3 levels was prominent in survivors but transient and diminished in nonsurvivors. The change of levels of C3, CH50, and AH50 was closely associated with one another, and their chronological trends related to the survival of patients (P =.0060,.0064 and.0066, respectively). The recovery of C3, AH50, and CH50 to normal or supranormal level during the early treatment period relates to the survival of patients with thermal injury. The failure of recovery of the complement system indicates a poor prognosis for patients and the monitoring of complement system might be beneficial in the care of patients with thermal injury.

Adult↗
PubMed1985

Complement activation and attack on autologous cell membranes induced by streptolysin-O.

Streptolysin-O damages mammalian membranes through generation of large transmembrane channels formed by membrane-inserted polymers of the toxin (S. Bhakdi et al., Infect. Immun. 47:52-60, 1985). We here report that the native toxin binds naturally occurring human serum immunoglobulin G antibodies to form immune complexes with potent complement-activating capacity. Nanomolar concentrations of toxin added to antibody-containing serum cause rapid consumption of C4 and C5 hemolytic activity and 30 to 90% C3 conversion within 10 to 60 min at 37 degrees C. After binding to target membranes, streptolysin-O polymers serve as foci for antibody-dependent complement activation, which proceeds to completion with the formation of terminal C5b-9 complexes on the autologous cells. The binding and insertion of a primarily water-soluble bacterial product into a host cell membrane has thus been shown to generate a stable and hyperactive focus for activation of and self-attack by the complement system. We suggest that this process perpetuates local tissue damage, deviates host complement action away from the invading bacteria, and may possibly play a role in the pathogenesis of poststreptococcal disease.

Antigen-Antibody Complex↗
PubMed2005

cDNA cloning and phylogenetic analysis of the sixth complement component in rainbow trout.

The sixth complement protein (C6) is an essential component of the membrane attack complex (MAC); the end product of the lytic pathway of complement activation. The MAC complex constitutes a supramolecular assembly containing the five precursor proteins C5b, C6, C7, C8, and C9. Once assembled on the target surface it forms transmembrane channels that cause membrane damage and cytolysis of complement-opsonized pathogens. Besides mediating direct pathogen elimination, exposure of cells to sublytic doses of MAC can trigger diverse cellular responses such as, cell activation, induction of apoptosis, cell cycle re-entry and proliferation in various biological settings. The terminal complement components (C6-C9) are structurally related proteins, differing in size and complexity. In order to study their evolution, we report here the cloning and molecular characterization of C6 component in rainbow trout. The deduced amino acid sequence of trout C6 exhibits 55 and 44% identity with zebra fish and human orthologs, respectively. The 'domain' architecture of trout C6 resembles that of mammalian counterparts, and the cysteine backbone is also conserved. Finally, trout C6 gene appears to exist as a single copy in the trout genome, and is expressed in a wide range of trout tissues.

Amino Acid Sequence↗
PubMed1991

[The terminal complement complex (TCC)--a mediator in the development of disease in man?].

Activation of the terminal complement pathway leads to the formation of the terminal complement complexes (TCC). TCC exists in one active membrane form, C5b-9 and in one inactive fluid phase form, SC5b-9. The paper explores the structure and regulatory mechanisms of TCC. In addition, methods of detecting TCC in tissue and in biological fluids, guidelines for sampling and the importance of TCC as an inflammatory mediator in human disease are discussed.

Complement Activation↗
PubMed1990

[Evaluation of the bactericidal effect of the membrane attack complex of serum complement].

The bactericidal activity of serum complement and particularly of the membrane attack complex (MAC-C5b-C9) was studied on E. coli C600 with a simple functional test. The test evaluates the in vitro kinetics of the bactericidal effect and the subsequent counting of surviving germs. Homozygous deficiency of a particular membrane attack complex protein was easily detected from a total loss of bactericidal activity. These results were confirmed on Neisseriae meningitidis A and C, but in this case a more complex protocol was required. Deficits of proteins of the membrane attack complex sequence of complement are often found in sera of patients suffering from recurrent Neisseriae infections. This simple test, adapted to family studies, appears, thus, as a valuable basis for a detection of relatives at risk.

Blood Bactericidal Activity↗
PubMed1997

Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.

The present work was designed to elucidate the in vivo role of complement in the proteinuria-associated tubulointerstitial injury. Rats were intravenously injected with puromycin aminonucleoside, and massive proteinuria was observed within 5 days. Prominent tubulointerstitial injury characterized by proximal tubular degeneration, tubular dilatation, and leukocyte infiltration were observed 7 days after injection. C3 and C5b-9 were observed in the luminal side of proximal tubular cells. Renal function, assessed by inulin and para-aminohippurate clearance, was significantly decreased. To-assess the role of complement in this model, rats were injected with either cobra venom factor or soluble recombinant human complement receptor type 1 starting at day 3. These manipulations significantly improved tubulointerstitial pathology and para-aminohippurate clearance without affecting the degree of proteinuria. Deposition of C3 and C5b-9 was not detected in the kidney of rats depleted of complement by cobra venom factor. In rats treated with soluble complement receptor, C3 was still detected in the tubules, but deposition of C5b-9 was not observed. Soluble complement receptor was detected at the site of C3 deposition and in the urine. These data strongly suggest that complement plays a pivotal role in proteinuria-associated tubulointerstitial injury and that systemic complement depletion or inhibition of complement in the tubular lumen may diminish the tubulointerstitial damage.

Animals↗
PubMed2004

Glomerular deposition and urinary excretion of complement factor H in idiopathic membranous nephropathy.

BACKGROUND/AIMS: The complement system plays an important role in the pathogenesis of membranous nephropathy (MN). In order to elucidate the regulatory mechanism of complement activation, we demonstrated glomerular deposition and urinary excretion of complement factor H, which controls the alternative pathway and the amplification loop at the C3 step, in patients with idiopathic MN. METHODS: Renal biopsy specimens from 20 patients with idiopathic MN were studied immunohistochemically using monoclonal antibodies against complement components including factor H. SDS-PAGE and Western blotting analysis of urine samples were performed, and the urinary excretion of factor H and C5b-9 were measured by quantitative sandwich ELISA. RESULTS: Intense glomerular deposition of factor H was observed with C3b.C3c and C5b-9 at an early stage of the disease. Factor H was detected in Western blots of urine samples, but factor H-like protein 1 (FHL-1) was not. The mean level of urinary factor H was elevated (86.30 +/- 21.93 U/mg urinary creatinine) in comparison to that of normal controls (4.76 +/- 1.03 U/mg urinary creatinine). Urinary factor H level exhibited no correlation with clinical parameters; however, a negative correlation was found between urinary C5b-9/factor H and creatinine clearance (r = 0.662, p < 0.01). CONCLUSION: The source of glomerular and urinary factor H is supposedly a 150-kD protein. There was no evidence to suggest that FHL-1 is synthesized at the site of inflammation. The urinary C5b-9 to urinary factor H ratio is indicative of the degree of ongoing complement activation in the glomeruli and complement-mediated renal injury. These findings suggest that factor H contributes to the control mechanism of in situ complement activation and prevents renal damage in idiopathic MN.

Alternative Splicing↗
PubMed2000

Focal inflammation in the brain: role in Alzheimer's disease.

We hypothesize that amyloid (Abeta) peptide-containing neuritic plaques in the brains of patients with Alzheimer's disease represent chronic inflammatory foci mediated by the actions of the complement system and proinflammatory cytokines. In support of this, in vitro studies show that the (Abeta) peptide is a potent complement activator and that such complement activation leads to the formation of covalent (Abeta)-C3 activation fragment complexes, the generation of the chemokine-like C5a complement activation peptide, and the formation of the proinflammatory C5b-9 complex in functionally active form able to insert into neuronal cell membranes. Other studies show that C5a, together with (Abeta), synergistically augments the release of proinflammatory cytokines from human monocytes. These studies, which provide in vitro support for the hypothesis, are being pursued in an animal model of Alzheimer's disease.

Alzheimer Disease↗
PubMed1993

Alteration of vitronectin. Characterization of changes induced by treatment with urea.

Vitronectin circulates in blood as a 70-kDa monomer that interacts with complexes generated in the terminal steps of the coagulation and complement cascades. Vitronectin complexed to thrombin-antithrombin or C5b-9 is conformationally altered as evidenced by enhanced reactivity with monoclonal antibody 8E6 and binding to heparin; these same alterations also occur when vitronectin is treated with urea (Tomasini, B. R., and Mosher, D.F. (1988) Blood 72, 903-912; Høgåsenk, K., Molnes, T.E., and Harboe, M. (1992) J. Biol. Chem. 267, 23076-23082). We have modified the purifications of native and urea-treated vitronectin to better control conformational state and characterized the alterations induced by urea. After treatment with N-ethylmaleimide to prevent formation of disulfide-linked multimers, purification in 8 M urea, and dialysis against physiological saline, vitronectin was largely oligomeric (approximately 800 kDa) as assessed by gel filtration and polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. Oligomeric urea-treated vitronectin reacted more strongly with the 8E6 antibody, bound biotinylated heparin more strongly, and neutralized the anticoagulant activity of heparin better than monomeric altered vitronectin or native vitronectin. After incubation with urea at 25 degrees C, native vitronectin, treated during purification with dithionitrobenzoic acid to force free sulfhydryls to intramolecular disulfides, exhibited increased reactivity with antibody 8E6, increased binding to heparin, and oligomerization. In addition, incubation in urea caused rearrangement of disulfides as assessed by loss of the light chain of two-chain vitronectin. The transition for these effects occurred between 2 and 4 M urea. Thus, an irreversible conformational alteration occurs upon treatment of vitronectin with urea, resulting in oligomers that bind avidly to heparin.

Antibodies↗
PubMed1990

Regulation of the membrane attack complex of complement. Evidence that C8 gamma is not the target of homologous restriction factors.

Inability of the membrane attack complex of C (C5b-9) to efficiently lyse E from the same species has been attributed to one or more membrane-associated proteins that are collectively called homologous restriction factors. These include a 65,000 Mr protein referred to as the C8 binding protein or homologous restriction factor and a 20,000 Mr protein referred to as P-18, HRF20, CD59 Ag, or MIRL. Both are found on nucleated cells as well as E and both protect against complement-mediated lysis by interfering with C8 and/or C9 function within C5b-9. The exact mechanism by which these factors restrict activity is unknown but studies with purified C8 binding protein suggest they may interact specifically with the gamma subunit of C8. To determine directly if gamma is the target of restriction factors, a derivative of human C8 lacking this subunit was evaluated for its potential to lyse homologous cells. This derivative (C8') was previously shown to be functionally equivalent to normal C8 in a heterologous sheep E system. Here, it is compared to normal C8 by using human E as target cells. Results indicate no difference between the ability of C8 and C8' to incorporate into HuEAC1-7, to mediate subsequent C9 binding and to promote hemolysis. Thus, the presence or absence of gamma has no effect on homologous restriction of C5b-9, therefore gamma cannot be the primary target of homologous restriction factors.

Blood Proteins↗
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↗
PubMed1988

Complement induces a transient increase in membrane permeability in unlysed erythrocytes.

The effects of low concentrations of human serum on antibody-sensitized sheep erythrocytes (EA) were studied. We report that exposure to low concentrations of serum induced a large but transient increase in the membrane permeability of those EA that do not lyse. This change in the permeability of the erythrocyte membrane resulted in net uptake of Na+ and decrease in cell K+, without affecting the total internal cation content. Although exposure to serum also allowed for net uptake of larger molecules like L-glucose, it did not lead to cell swelling. Experiments with sera genetically deficient in one of the terminal complement components showed that C8, but not C9, was required to produce the observed change in membrane permeability. Therefore, we propose that the C5b-8 complex can mediate the transient increase in permeability observed in unlysed erythrocytes during complement activation by whole serum.

Animals↗