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PubMed1995

Blockade of C5a and C5b-9 generation inhibits leukocyte and platelet activation during extracorporeal circulation.

Complement activation contributes to the systemic inflammatory response induced by cardiopulmonary bypass. At the cellular level, cardiopulmonary bypass activates leukocytes and platelets; however the contribution of early (3a) versus late (C5a, soluble C5b-9) complement components to this activation is unclear. We used a model of simulated extracorporeal circulation that activates complement (C3a, C5a, and C5b-9 formation), platelets (increased percentages of P-selectin-positive platelets and leukocyte-platelet conjugates), and neutrophils (upregulated CD11b expression). to specifically target complement activation in this model, we added a blocking mAb directed at the human C5 complement component and assessed its effect on complement and cellular activation. Compared with a control mAB, the anti-human C5 mAb profoundly inhibited C5a and soluble C5b-9 generation and serum complement hemolytic activity but had no effect on C3a generation. Additionally, the anti-human C5 mAb significantly inhibited neutrophil CD11b upregulation and abolished the increase in P-selectin-positive platelets and leukocyte-platelet conjugate formation compared to experiments performed with the control mAb. This suggests that the terminal components C5a and C5b-9, but not C3a, directly contribute to platelet and neutrophil activation during extracorporeal circulation. Furthermore, these data identify the C5 component as a site for therapeutic intervention in cardiopulmonary bypass.

Antibodies, Monoclonal↗
PubMed1997

Terminal complement complexes induce cell cycle entry in oligodendrocytes through mitogen activated protein kinase pathway.

Sublytic complement attack through C5b-9 assembly induces oligodendrocytes (OLG) to express proto-oncogenes and to enter the cell cycle from resting G0/G1 phase to S phase. We have investigated whether cell cycle induction by C5b-9 is mediated by mitogen activated protein kinase (MAPK) pathway in OLG. C5b-9 but not C5b6 induced activation of both ERK1 and c-jun NH2 terminal kinases 1 (JNK1) in OLG. The increased ERK1 and JNK1 activities are transient, reaching a maximum around 20 min following exposure to C5b-9. Activation of Raf-1 and MEK1, upstream kinases of ERK1, was shown by increased Raf-1 kinase activity in anti-Raf-1 immunoprecipitates of OLG treated with C5b-9 and ERK1 activity that can be inhibited by PD098,059, a specific MEK1 inhibitor. Requirement for the ERK1 pathway in DNA synthesis was then evaluated using PD098,059. Enhanced DNA synthesis induced by serum complement was completely abolished when OLG were pretreated with PD098,059. On the other hand, c-fos mRNA expression induced by complement was inhibited only 50% by PD098,059, while the c-jun mRNA level was not affected by this MEK1 inhibitor. Interestingly, p70 S6 kinase, an important ribosomal kinase in mitogenesis, was also activated by C5b-9. These findings indicated that the MAPK pathways appears to play a major role in inducing OLG to enter the S phase of the cell cycle from the resting G1/G0 phase.

Animals↗
PubMed1991

Inhibition of homologous complement by CD59 is mediated by a species-selective recognition conferred through binding to C8 within C5b-8 or C9 within C5b-9.

The capacity of the human complement regulatory protein CD59 to interact with terminal complement proteins in a species-selective manner was examined. When incorporated into chicken E, CD59 (purified from human E membranes) inhibited the cytolytic activity of the C5b-9 complex in a manner dependent on the species of origin of C8 and C9. Inhibition of C5b-9-mediated hemolysis was maximal when C8 and C9 were derived from human (hu) or baboon serum. By contrast, CD59 showed reduced activity when C8 and C9 were derived from dog or sheep serum, and no activity when C8 and C9 were derived from either rabbit or guinea pig (gp) serum. Similar specificity on the basis of the species of origin of C8 and C9 was also observed for CD59 endogenous to the human E membrane, using functionally blocking antibody against this cell surface protein to selectively abrogate its C5b-9-inhibitory activity. When E bearing human CD59 were exposed to C5b-8hu, CD59 was found to inhibit C5b-9-mediated lysis, regardless of the species of origin of C9, suggesting that the inhibitory function of CD59 can be mediated through recognition of species-specific domains expressed by human C8. Consistent with this interpretation, CD59 was found to bind to C5b-8hu but not to C5b67hu or C5b67huC8gp. Although CD59 failed to inhibit hemolysis mediated by C5b67huC8gpC9gp, its inhibitory function was observed for C5b67huC8gpC9hu, suggesting that, in addition to its interaction with C5b-8hu, CD59 also interacts in a species-selective manner with C9hu incorporated into C5b-9. Consistent with this interpretation, CD59 was found to bind both C5b67huC8gpC9hu and C5b-8huC9gp, but not C5b67huC8gpC9gp. Taken together, these data suggest that the capacity of CD59 to restrict the hemolytic activity of human serum complement involves a species-selective interaction of CD59, which involves binding to both the C8 and C9 components of the membrane attack complex. Although CD59 expresses selectivity for C8 and C9 of human origin, this "homologous restriction" is not absolute, and this human complement regulatory protein retains functional activity toward C8 and C9 of some nonprimate species.

Antigens, Differentiation↗
PubMed1987

Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing.

We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels.

Blood Bactericidal Activity↗
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↗
PubMed1975

The membrane attack mechanism of complement. Isolation and subunit composition of the C5b-9 complex.

Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.

Animals↗
PubMed1996

Protein SIC, a novel extracellular protein of Streptococcus pyogenes interfering with complement function.

The human pathogen Streptococcus pyogenes possesses a chromosomal region, the mga regulon, that contains co-regulated genes important to the virulence of these bacteria. A novel gene located in the mga regulon of a S. pyogenes strain of serotype M1 was cloned and sequenced. It translates into a protein of 305 amino acid residues, including a signal sequence of 32 amino acids and a central region consisting of three tandem repeats. The sequence represents a novel structure with no significant homology to any previously published sequence. The protein was purified from the streptococcal culture media where it is present in substantial amounts. Affinity chromatography of human plasma on Sepharose coupled with the protein specifically absorbed two plasma proteins which were identified as clusterin and histidine-rich glycoprotein (HRG). The interactions between the streptococcal protein and the plasma proteins were further characterized using purified clusterin and HRG. Inhibition experiments indicated that they have affinity for overlapping or closely located sites in the streptococcal protein. Both clusterin and HRG are regulators of the membrane attack complex (C5b-C9) of complement. When the streptococcal protein was added to serum, complement-mediated lysis of sensitized sheep erythrocytes and guinea pig erythrocytes was inhibited. In addition, the streptococcal protein was incorporated into C5b-C9 in serum, indicating the location of its action. The name, protein SIC, streptococcal inhibitor of complement-mediated lysis, is therefore suggested for this novel protein. The occurrence of protein SIC and its gene was investigated in a collection of S. pyogenes strains comprising 55 different M serotypes. Only M1 and M57 strains were positive in this screening, indicating that protein SIC could be a virulence determinant. Thus, during recent years, the M1 serotype has been connected with a world-wide increase of severe and toxic S. pyogenes infections.

Amino Acid Sequence↗
PubMed1998

Complement activation in patients with acute myocardial infarction treated with streptokinase.

Twenty patients with acute myocardial infarction (AMI) treated with streptokinase (SK) and 11 patients with AMI not treated with SK were clinically and echocardiographically evaluated in the intensive coronary care unit (ICCU). Blood samples, in which complement components C4d, C5b-9, and Bb were measured, were taken immediately after arrival of the patient at the ICCU, 1 hour after SK infusion, and 24 hours later from the SK group, and similar time-matched samples were taken from the control group. The classic, alternative, and common complement pathways were found to be activated in AMI, but this activation was statistically significant only in the SK-treated group. Activation was not related to the site of the AMI nor to the extent of its damage, as assessed by Killip class and ejection fraction. However, in the SK-treated group and even more so in non-SK group, the C5b-9 increment was significantly higher in patients diagnosed with reperfusion, based on ST-segment recovery of > or =50% reduction from the peak ST-segment level. There was little change in complement levels when reperfusion did not occur. Detection of reperfusion without coronary angiography would have important practical clinical implications, and complement activation may predict its occurrence. However, more studies of this phenomenon are needed.

Aged↗
PubMed1991

Human umbilical vein endothelial cells synthesize functional C3, C5, C6, C8 and C9 in vitro.

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.

Antibodies, Monoclonal↗
PubMed1995

Calcium-loaded erythrocytes have a defect in complement regulation distinct from that resulting from exposure to 2-aminoethylisothiouronium bromide.

Calcium-loaded red blood cells (RBCs) previously have been shown to have an increased sensitivity to complement-mediated hemolysis and particularly to lysis mediated by the C5b-9 membrane attack complex (MAC) of complement. Because RBCs exposed to 2-aminoethylisothiouronium bromide (AET) also have been shown to be particularly sensitive to the MAC, a direct comparison of calcium-loaded and AET-treated RBCs was performed. Calcium-loaded and AET-treated RBCs shared a marked increase in sensitivity to lysis by the MAC in two different assays. However, measurements of C5b-7 and C9 binding suggested that different mechanisms were responsible. AET-treated RBCs showed an increase in C9 binding and an increased C9/C7 ratio consistent with functional loss of CD59/membrane inhibitor of reactive lysis (MIRL). In contrast, calcium-loaded RBCs had minimally increased C9 binding that resulted in C9/C7 ratios that were less than those for untreated RBCs, suggesting that CD59/MIRL inactivation had not occurred. When RBCs were incubated in acidified serum, AET-treated cells demonstrated a marked increase in C3b binding and hemolysis that was observed in neither control nor calcium-loaded RBCs. These results suggest that the underlying lesions responsible for an increase in susceptibility to complement-mediated hemolysis are different for calcium-loaded and AET-treated RBCs.

Adult↗
PubMed1996

In vitro expression of the beta subunit of human complement component C8.

Human C8 is one of five components of the cytolytic C5b-9 complex of complement. It is an oligomeric protein composed of three subunits (alpha, beta, gamma) encoded in separate genes. These are arranged as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. Biosynthesis studies and analyses of humans with hereditary C8 deficiencies suggest that C8 alpha-gamma synthesis and secretion can occur independently of C8 beta, but that serum levels of C8 beta are dependent on C8 alpha-gamma. One aim of the present study was to determine if functional human C8 beta could be synthesized in the absence of C8 alpha-gamma. Human C8 beta expression constructs were prepared and used to produce recombinant C8 beta (rC8 beta) in insect and COS-7 cells. Both cell types secreted rC8 beta that was similar in size to human C8 beta and exhibited similar ability to associate with human C8 alpha-gamma and form functional C8. A mutant form of C8 beta in which N-glycosylation sites were eliminated was also expressed and found to be functionally similar to rC8 beta and human C8 beta. These results indicate that C8 alpha-gamma is not required for intracellular processing and secretion of C8 beta. Furthermore, N-linked carbohydrate on C8 beta is not necessary for association with C8 alpha-gamma or for C8 activity.

Animals↗
PubMed1999

mRNA expression of complement components and regulators in rat arterial smooth muscle cells.

The presence of C5b-9 complexes, some complement regulators, and abundant cytokines in atherosclerotic lesions has been reported. However, it is unclear whether these complement-associated proteins are produced by vascular smooth muscle cells (SMCs) and how they are influenced by the cytokines. In the present study, we demonstrated, by the reverse transcription-polymerase chain reaction method, the mRNA expression of complement components (C3, C4, and C5) and membrane regulators (decay-accelerating factor, membrane cofactor protein, Crry, and CD59) in cultured SMCs derived from the rat carotid artery. The expression of C9 mRNA was also induced upon stimulation by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and/or lipopolysaccharide (LPS). Northern blot analysis showed that the mRNA expression of C3, C4, DAF and Crry was up-regulated, but that of CD59 was down-regulated by IFN-gamma, TNF-alpha and/or LPS alone or by synergy. The increase of C3 mRNA by TNF-alpha or LPS and that of C4 mRNA by IFN-gamma was induced in a dose-dependent manner. The results indicate that the arterial SMCs of rat have the ability to produce complement components and regulators, which is affected by cytokines and/or LPS. Since atherosclerosis is characterized by the intimal proliferation of SMCs, the complement system including its regulators may be involved in the pathogenesis of the disease.

Animals↗
PubMed1988

Complement-independent lysis of human red blood cells by cold hemagglutinins.

Hemolysis mediated by human antibodies is generally ascribed to the attack of red blood cells (RBC) by complement. We here extend earlier in vitro observations which indicate that potent cold agglutinins can directly cause lysis of RBC without the participation of complement. We have noted that EDTA plasma taken from patients with cold agglutinin disease is frequently reddish if the plasma is not immediately separated from the cells at 37 degrees C. Moreover, eluates prepared in such cases from plasma or heat-inactivated serum (30 min at 56 degrees C) by absorption (at 4-20 degrees C) and elution (at 37 degrees C) are usually contaminated with hemoglobin, and a large number of RBC used for absorption is lost during the procedure. To characterize this phenomenon further, we examined the effect of different hemagglutinating antibodies in vitro on normal RBC in the absence of complement. Hemolysis (5-17%) of RBC only occurred after treating the cells with potent antibodies at low temperatures (0-20 degrees C). This hemolysis increased 2- to 3-fold when the RBC were treated with an enzyme and decreased with rising temperature. Unlike cells hemolyzed by complement activation, no C5b-9 complexes could be detected on RBC damaged by this mechanism.

Cold Temperature↗
PubMed1984

Function of subunits within the eighth component of human complement: selective removal of the gamma chain reveals it has no direct role in cytolysis.

The eighth component of human complement (C8) consists of a disulfide-linked alpha-gamma dimer that is noncovalently associated with beta. Previous results from this laboratory established that two of these subunits have distinct roles in the cytolytic function of C8. Binding of C8 to the precursive C5b-7 complex is mediated strictly through beta while interaction between C8 and the lipid bilayer of target membranes occurs primarily through alpha. In the present study, we examined the importance of the gamma subunit in cytolysis by characterizing functional properties of C8', a derivative of C8 that lacks gamma. Preparation of this derivative was accomplished by limited cleavage of disulfide bonds in purified alpha-gamma and separation of alpha from gamma. When mixed, purified alpha and beta combined to form C8'. When tested for functional similarity to normal C8, the following results were obtained. (1) Specific and saturable binding of C8' to the C8 binding site on C5b-7 could be achieved. Significantly, the resulting C5b-8' supported subsequent C9 binding and cell lysis, which was equivalent to that observed with C5b-8. (2) Complement activation of (C8 + C9)-depleted serum containing C8' resulted in formation of SC5b-8'. Inclusion of C9 in the serum resulted in further conversion to SC5b-(8')9. These observations indicate that C8' is functionally similar to C8. Furthermore, they indicate that unlike alpha and beta, the gamma subunit has no direct role in facilitating C8 interaction with the nascent cytolytic complex or in mediating C9 binding and membrane lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Immune attack on the Schwann cell surface in acute inflammatory demyelinating polyneuropathy.

The localization, mode of action, and roles of complement in the Guillain-Barre syndrome have been controversial. We used high-resolution immunocytochemistry to localize complement activation products in early stages of the acute inflammatory demyelinating polyneuropathy (AIDP) pattern of Guillain-Barre syndrome. Three AIDP subjects who were autopsied had had symptoms for 3 to 9 days at the time of death. Immunocytochemistry was performed on etched, epoxy resin-embedded sections, and the next thin section was compared by electron microscopy (thick/thin sections). Many fibers had a rim of the complement activation marker C3d and the terminal complement complex neoantigen C5b-9 along the outer surface of the Schwann cells. Ultrastructural analysis of these C3d-positive fibers showed mild vesicular changes of the outermost myelin lamellae. Vesicular degeneration was seen before the invasion of macrophages into the myelin, and was the predominant change in the subject with symptoms for 3 days. C3d staining was not found on myelin membranes. The results suggest that at least some forms of AIDP are complement mediated. We speculate that complement is activated by antibody bound to epitopes on the outer surface of the Schwann cell and that the resulting complement activation initiates the vesiculation of myelin.

Acute Disease↗
PubMed1992

Expression of decay-accelerating factor on synovial lining cells in inflammatory and degenerative arthritides.

The decay-accelerating factor (DAF) is a complement regulatory cell surface protein that protects cells from complement-mediated lysis. We analysed synovial tissue biopsies from patients with chronic arthritides for the presence of DAF using immunohistochemistry. DAF was expressed in the synovial lining cell layer both in rheumatoid arthritis (RA) and in osteoarthritis (OA). DAF was also on vascular endothelial cells of synovial tissue. A significant correlation was found between the expression of DAF and of HLA-DR in the lining layer, suggesting that DAF may be induced during a local inflammatory response. In addition, C5b-9 terminal complement complexes were found in several DAF-positive cases, suggesting that complement activation might, in itself, induce DAF expression. We propose that the occurrence of DAF may represent a physiological mechanism for local complement regulation in synovial tissue.

Adult↗
PubMed2001

Structure and composition of drusen associated with glomerulonephritis: implications for the role of complement activation in drusen biogenesis.

PURPOSE: The ocular fundi of many patients with membranoproliferative glomerulonephritis type II (MPGN-II) are characterised by the presence of deposits within Bruch's membrane that resemble drusen, hallmark lesions associated with age-related macular degeneration (AMD). Glomerulonephritis (GN)-associated drusen appear at a younger age, however, than do drusen in individuals with AMD. In light of recent evidence that immune-mediated events participate in drusen biogenesis and AMD, we examined the structure and composition of drusen in eyes obtained from human donors with two distinct glomerulopathies, both of which involve complement deposition within glomeruli. These features were compared with those of drusen from patients with clinically documented AMD. METHODS: Eyes obtained from two human human donors diagnosed with membranous and post-streptococcal GN, respectively, were analysed histochemically, immunohistochemically and ultrastructurally. RESULTS: Subretinal pigment epithelial (RPE) deposits in both types of GN are numerous and indistinguishable, both structurally and compositionally, from drusen in donors with AMD. GN-associated drusen exhibit sudanophilia, bind filipin, and react with antibodies directed against vitronectin, complement C5 and C5b-9 complexes, TIMP-3 and amyloid P component. Drusen from the membranous GN donor, but not the post-streptococcal GN donor, reacted with peanut agglutinin and antibodies directed against MHC class II antigens and IgG. The ultrastructural characteristics of these deposits were also identical with those of AMD-associated drusen. CONCLUSIONS: The composition and structure of ocular drusen associated with membranous and post-streptococcal/segmental GN are generally similar to those of drusen in individuals with AMD. In view of the recent data supporting the involvement of complement activation in drusen biogenesis and the pathobiology of AMD, further studies of the biological relationships between AMD and diseases associated with complement activation are warranted.

Complement Activation↗