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PubMed2000

Preconditioning reduces myocardial complement gene expression in vivo.

This investigation examined the effect of preconditioning in an in vivo model of ischemia-reperfusion injury. Anesthetized New Zealand White rabbits underwent 30 min of regional myocardial ischemia followed by 2 h of reperfusion. Hearts preconditioned with two cycles of 5 min ischemia-10 min reperfusion (IPC) or with the ATP-sensitive K (K(ATP)) channel opener, diazoxide (10 mg/kg), exhibited significantly (P < 0.05) smaller infarcts compared with control. These treatments also significantly (P < 0.001 to P < 0.05) reduced C1q, C1r, C3, C8, and C9 mRNA in the areas at risk (AAR). The K(ATP) channel blocker 5-hydroxydecanoate (5-HD; 10 mg/kg) attenuated infarct size reduction elicited by IPC and diazoxide treatment. 5-HD partially reversed the decrease in complement expression caused by IPC but not diazoxide. There were no significant differences in complement gene expression in the nonrisk regions and livers of all groups. Western blot analysis revealed that IPC also reduced membrane attack complex expression in the AAR. The data demonstrate that preconditioning significantly decreases reperfusion-induced myocardial complement expression in vivo.

Animals↗
PubMed1987

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis.

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.

Cell Membrane↗
PubMed1989

Distinct restriction of complement- and cell-mediated lysis.

Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

The complement system and systemic sclerosis.

Serum concentrations of the various complement components including the classical and the alternative pathways were determined in 58 control healthy subjects and 80 patients with systemic sclerosis (SSC). The mean concentrations of C1q, C2, C5, C6, C7, C9, and factor B were significantly increased in the SSC patients in comparison to controls, while the increases were not significant for C3 and C8. C4 was an exception in that the mean levels were found to be decreased, with 18 patients having levels < 65% of the mean normal value. Properdin was also found to be decreased, but not significantly. We found a similarity between the pattern of serum complement component concentrations in SSC patients and patients with primary biliary cirrhosis, two disorders frequently associated in the same patients. The significance of complement component patterns in these diseases is discussed.

Complement Activation↗
PubMed1993

Solid-phase synthesis of oligonucleotides containing site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts using 9-fluorenylmethoxycarbonyl as the base-protecting group.

Eight oligodeoxyribonucleotides containing a site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetyl-amino)fluorene (dG-C8-AAF) adduct were prepared successfully by solid-phase DNA synthesis using the 2-cyanoethyl N,N-diisopropylphosphoramidites of dA, dC, dG, dT, and dG-C8-AAF, with 9-fluorenyl-methoxycarbonyl (Fmoc) as the base-protecting group. The oligonucleotides were deprotected and released from the support by 1:9 piperidine/MeOH at room temperature for 22-36 h or by 1:1 diisopropylamine in MeOH at 55 degrees C for 15 h, purified by HPLC, and fully characterized. About 6 mg of HPLC-purified d[GTGGCG(C8-AAF)CCAAGT] and 7 mg of d[GTGATG(C8-AAF)ATAAGT] were obtained from the 10-mumol-scale synthesis, and their 1D 1H NMR spectra were consistent with the presence of a dG-C8-AAF adduct. The dG-C8-AAF oligonucleotides were also deacetylated to afford the corresponding dG-C8-AF oligonucleotides. d[GTGGCG(C8-AAF)CCAAGT] formed stable 1:1 duplexes with both the fully complementary 12-mer and a GC-deleted (across the adduct) 10-mer complement, and identical melting temperatures were observed for both duplexes. The multidimensional NMR study of these duplexes is presently under investigation.

2-Acetylaminofluorene↗
PubMed1997

Mapping the active site of CD59.

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

Amino Acid Sequence↗
PubMed2000

Cleavage of the fifth component of complement and generation of a functionally active C5b6-like complex by human leukocyte elastase.

Activation by complement C3/C5 convertases of the fifth component of human complement, C5, leads to two active cleavage products: C5a, a chemotactic peptide, and C5b, the activated form of C5. Human leukocyte elastase (HLE) has long been known to also release from C5 a chemotactic, C5a-like fragment. This, however, cannot be identical with C5a, since HLE does not cleave peptide bonds at the carboxyl group of arginine, the cleavage site that separates C5a and C5b after the exposure to the complement convertases. Therefore, the question arose whether HLE is capable of releasing a functionally C5b-like product from C5. The results show that this is, indeed, so. Treatment of human C5 with HLE in the presence of C6 leads to the formation on an active C5b6-like complex that lyses non-sensitized guinea pig red cells upon addition of the terminal components C7, C8, and C9. However, since C6 is highly sensitive to the hydrolytic action of HLE, the yield of the activation complex is rather low. The results offer a third possibility for the activation of C5: 1) classical cleavage at Arg74 by complement convertases, 2) oxidation of methionine residues without cleavage, and, as shown here, 3) cleavage by elastase at (a) site(s) distal from Arg74. The three procedures may modulate the relative yield of the two activities generated from C5, C5a-like and C5b-like effects.

Animals↗
PubMed1992

Complement profile in primary biliary cirrhosis.

PBC is a chronic progressive liver disease of unknown etiology. Several abnormalities found in PBC support the hypothesis that it may be considered an autoimmune disease. Despite the complex and interesting relationship that exists between autoimmune disorders and the complement system, very few reports on the level of the serum complement component in PBC have been published, and most of these comprised only a few patients or analyzed only a scant number of the complement components. In the present study, sera of 73 PBC patients were analyzed for the levels of 10 complement components. It was found that the levels of most of the serum complement components, including C1q, C2, C3, C5, C7, properdin and factor B were significantly elevated in patients with PBC in comparison to healthy controls. The level of C4 was slightly lower than that of the normal controls (p = 0.019), while the levels of C6 and C8 were within the normal range. The number of PBC patients with serum levels of C4 and C6 < 60% of normal pooled serum was higher than in the respective control groups (6/69 compared with 0/26 and 4/71 compared with 0/27, respectively). However, the difference was not statistically significant. Thus, our study shows alterations in the levels of most complement components in PBC, the reasons for which are discussed.

Autoimmune Diseases↗
PubMed1970

Lysis of erythrocytes by complement in the absence of antibody.

A new pathway of complement-mediated hemolysis has been described. It is independent of antibody and does not require binding of the first four complement components to the target-cell surface. The actual attack of the target cell begins with the attachment of C5, C6, and C7. The binding reaction is catalyzed by C4, 2, 3, an enzyme which may be formed in cell-free solution. C4, 2, 3 may effect binding of C5, 6, 7 by acting from the fluid phase or from the surface of another cell to which it is specifically bound (EAC 4, 2, 3). In either case, the resulting product is EC5, 6, 7 which is susceptible to lysis by C8 and C9. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were particularly susceptible to lysis by the above described mechanism. PNH cells, but not normal human erythrocytes, could also be lysed through activation of complement by cobra factor. These observations allow the operational distinction of an activation and an attack mechanism of complement.

Animals↗
PubMedDate not supplied

[The complement system. Normal values and congenital deficiencies in the adult Mexican population].

Five hundred mexican mestizos, healthy blood donors were evaluated regarding the hemolytic activity of their serum, to assess the function of the complement (C) system. One of them was hypocomplementemic and his serum was unable to promote hemolysis by either classical or alternative pathway. It had normal protein concentrations of C3 and C4, as well as immunoreactive C1, C4, C5, C6, C7 and C8, and lytic factor D. Factor B was not recognizable. It is possible that a genetic deficiency linked to genes codifying for class III products of the major histocompatibility complex is responsible for our findings.

Adolescent↗
PubMed1993

Membrane defence against complement lysis: the structure and biological properties of CD59.

The complement system is an important branch of the innate immune response, constituting a first line of defence against invading microorganisms which activate complement via both antibody-dependent and -independent mechanisms. Activation of complement leads to (a) a direct attack upon the activating cell surface by assembly of the pore-forming membrane attack complex (MAC), and (b) the generation of inflammatory mediators which target and recruit other branches of the immune system. However, uncontrolled complement activation can lead to widespread tissue damage in the host, since certain of the activation products, notably the fragment C3b and the C5b-7 complex, can bind nonspecifically to any nearby cell membranes. Therefore it is important that complement activation is tightly regulated. Our own cells express a number of membrane-bound control proteins which limit complement activation at the cell surface and prevent accidental complement-mediated damage. These include decay-accelerating factor, complement receptor 1 and membrane cofactor protein, all of which are active at the level of C3/C5 convertase formation. Until recently, cell surface control of MAC assembly had been attributed to a single 65-kD membrane protein called homologous restriction factor (alternatively named C8-binding protein and MAC-inhibiting protein). However a second MAC-inhibiting protein has since been discovered and it is now clear that this protein plays a major role in the control of membrane attack. This review charts the rapid progress made in elucidating the protein and gene structure, and the mechanism of action of this most recently discovered complement inhibitor, CD59.

Animals↗
PubMed1996

The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes.

Two of the hallmarks of Cockayne's syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells with either UV or NA-AAF, the basal transcription factor TFIIH becomes involved in nucleotide excision repair and that the CS gene products are involved in the conversion of TFIIH back to the transcription function. In this view, the CS proteins act as repair-transcription uncoupling factors. If the uncoupling process is defective, RNA synthesis will stay repressed, causing cellular sensitivity. Since transcription is essential for transcription-coupled repair, the CS defect will affect those lesions whose repair is predominantly transcription coupled, i.e., UV-induced cyclobutane pyrimidine dimers.

2-Acetylaminofluorene↗
PubMed1983

Inherited complement component deficiencies in membranoproliferative glomerulonephritis.

Anecdotal reports of complement component deficiencies in patients with immune complex disease led to a systematic study of the levels of seven complement components in serum specimens from 178 patients with glomerulonephritis and 163 normal subjects. Deficiencies were found with significantly higher frequency (22.7%) among 44 patients with membranoproliferative glomerulonephritis (MPGN) types I and III, than among the normal subjects (6.7%, P less than 0.002) or among 134 patients with other glomerulonephritides (5.2%, P less than 0.001). The component deficiencies in MPGN were partial in nine patients and subtotal in one. They could not be ascribed to acquired hypocomplementemia or to a nephrotic syndrome. They were present over long periods, were found in family members, and involved C2, C3, factor B, C6, C7, and C8. Six were presumably the result of null structural genes, two were associated with a structurally abnormal component, and two were of unknown cause. The results give evidence that partial deficiency of one or more complement components is a factor predisposing to MPGN.

Adolescent↗
PubMed1999

Peritoneal mesothelial cells produce complement factors and express CD59 that inhibits C5b-9-mediated cell lysis.

The CD59 membrane protein confers protection from C5b-9-mediated cell lysis. Because evidence exists for complement (C) activation and generation of C5b-9 in the peritoneal cavity during chronic peritoneal dialysis (CPD), we investigated, on mesothelial cell (MC) lines, the expression of CD59 and the production of C components. Four MC lines were obtained from children on CPD, and two from non uremic children. CD59 expression on MCs was investigated with anti-CD59 monoclonal antibody (mAb) and polyclonal goat immunoglobulin G (IgG). MC lines were positive for staining with anti-CD59 mAb. Western blotting analysis of MC membrane demonstrated a band with the same molecular weight as CD59. Incubation of MC with anti-CD59 mAb abrogated the protective effect of CD59 (100% cytotoxicity). C3, C4, and C6 were detected in the supernatants of MC; in non uremic MC supernatants, C5, C7, C8, and C9 were also detectable, and C4 concentration was tenfold higher. CD59 expression confers to MCs protection from C5b-9-mediated lysis. MCs produce C factors. These findings suggest that production of complement components and expression of CD59 on MCs could play a role both in peritoneal cavity infection (decreased complement production) and in peritoneal membrane damage (decreased CD59 expression and reduced remesothelialization owing to MC lysis).

CD59 Antigens↗
PubMed1999

Complement regulators C1 inhibitor and CD59 do not significantly inhibit complement activation in Alzheimer disease.

Proteins characteristic of activated complement are associated with Alzheimer disease (AD) lesions. The classical complement pathway can be activated only when the influence of such endogenous regulators as C1-inhibitor (C1-inh) and CD59 are overcome. We used the techniques of reverse transcriptase-polymerase chain reaction and Western blotting to assess the mRNA and protein levels of C1-inh and CD59 in AD and control brains in comparison with levels of the complement components with which they interact. The inhibitors were only slightly upregulated and then only in heavily affected areas of AD brain such as the entorhinal cortex, hippocampus, midtemporal gyrus and midfrontal gyrus. The ratio of AD to control mRNAs in these four areas was 1.17 for C1-inh and 1.12 for CD59, compared to 3.06 for C1r, 2.67 for C1s, 2.35 for C5, 2.56 for C6, 2.42 for C7, 5. 08 for C8 and 16.3 for C9. Peripheral organ expression of C1-inh and CD59 mRNAs was no different in AD than controls but was slightly upregulated in infarcted heart tissue. Again, the increase was small compared with that of the competitive complement components. These data indicate that the forces which upregulate and activate complement in AD and myocardial infarction are not effectively suppressed by the endogenous regulators, C1-inh and CD59.

Adult↗
PubMed2000

Identification of mutations in rat CD59 that increase the complement regulatory activity.

Formation of the membrane attack complex (MAC) of complement on host cells is inhibited by the glycosylphosphatidylinositol- (GPI-) anchored glycoprotein CD59. Published data on the active site of human CD59 are confusing. To clarify these data, we set out to elucidate the active site of a nonprimate CD59 molecule by site-directed mutagenesis. We also undertook to investigate a region of potential species selectivity, and to this end rat CD59 was chosen for all mutations. Our investigations confirmed the proposal that the active site of CD59 is the major hydrophobic groove, with mutations Y36A, W40A, and L54A ablating complement inhibitory function of CD59. Other mutations reducing the function of rat CD59 were I56E, D24A, and D24R. Importantly, mutations at one residue increased the function of rat CD59. The K48E mutation significantly increased function against human rat or rabbit serum, whereas the K48A mutation increased function against human serum alone. A similar mutation in human CD59 (N48E) had no effect on activity against human or rat serum but completely abolished all activity against rabbit serum. These findings suggest that the alpha-helix of human CD59, adjacent to the hydrophobic groove, influences the interaction between human CD59 and rabbit C8, C9, or both.

Amino Acid Substitution↗
PubMed1996

Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear leukocytes.

Purified human myeloperoxidase (MPO) converted human C5 to an activated form, i.e. the C5 protein adopted a configuration expressing a binding site for C6; the resulting C56 complex then reacted with C7, C8 and C9 forming a hemolytic C5-9 complex. For the activation by myeloperoxidase chloride and hydrogen peroxide were essential. This indicates that the peroxidase acted through the generation of HOCl which had been shown earlier to oxidize and activate C5. Human polymorphonuclear leukocytes (PMN) were stimulated in vitro by incubation with opsonized zymosan; thereafter the supernatants were tested for C5 activating potency. Stimulated PMN release H2O2 and MPO that produces hypochlorite and secondarily various chloramines. As a trap for the labile hypochlorite generated excess taurine was added to the PMN suspensions during the incubation. Hypochlorite is then stoichiometrically converted to the relatively stable taurine chloramine. In order to rule out interfering activities of proteolytic enzymes released from the PMN and known to attack C5, the supernatants were ultracentrifuged, and the ultrafiltrates, containing only low molecular weight compounds, were used for the further studies. They contained taurine chloramine, estimated photometrically, and they activated C5 upon incubation, assayed functionally by reactive lysis. Azide, an inhibitor of myeloperoxidase, and catalase which destroys H2O2, essential for MPO-catalyzed oxidations, prevented the generation of C5 activating potency and of chloramines. Unstimulated PMN produced neither oxidants nor C5 activating potency. When taurine was omitted from the PMN suspensions during stimulations much less oxidant was found in the supernatants and less C5 activating potency. These findings indicate that the C5 activating agent was produced by stimulated PMN through MPO-generated hypochlorite, trapped as taurine chloramine. In the absence of added taurine the hypochlorite formed by MPO oxidized endogenous amines that also activated C5. Further studies suggested that among these was some monochloramine derived from endogenous ammonia. Activation of the terminal complement reaction sequence by MPO released from stimulated PMN may represent a third pathway to complement activation contributing to and reinforcing complement and PMN functions at the site of inflammation or tissue injury.

Complement Activation↗