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We have used a cDNA probe for human complement component 9 (C9), which detects three DNA polymorphisms, to analyse the inheritance of C9 in families informative for C6 protein variants. We found that these genes are closely linked with a lod score of 9.28 at recombination fraction 0.00. There is not indication of allelic association.
Large granular lymphocytes (LGL) from human blood maintained in culture for 2 to 6 weeks with IL-2 were found positive in the K562 cell killing assay. The cytoplasmic granules of the LGL were isolated, lysed and the soluble proteins were passed over a Sepharose-anti-C9 column. The retained protein was eluted with NaCl and found to consist by SDS polyacrylamide gel electrophoresis of essentially one component with a molecular weight of approximately 70 000. The protein did not give a positive precipitation test with anti-human C9 by Ouchterlony analysis, but it reacted reproducibly with anti-human C9 by Western blot analysis. By ELISA the cross reaction with human C9 was less than 1%. The C9 related lymphocyte protein lacked C9 hemolytic activity, but it formed functional pores in liposomes in presence of Ca++. These results suggest that the cytoplasmic granules of human LGL that are capable of killing NK target cells contain C9 related protein which is involved in the cellular cytotoxicity reaction.
Three mAb to human C9, X195, X197, and P40 were used to analyze the roles of the C9a and C9b domains in the reaction of the C9 molecule with sensitized sheep E bearing C1 to C8 (EAC1-8). X195 bound to NH2-terminal (C9a) fragments, and X197 bound to COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin or trypsin. P40 recognized the epitope on the C9b fragment obtained by alpha-thrombin cleavage but did not react with the NH2-terminal or COOH-terminal fragment obtained by trypsin cleavage. In this respect, P40 differed from mAb to C9 reported previously. P40 almost completely inhibited the hemolytic activity of C9. X195 and X197 also inhibited C9 activity, but less effectively than P40. C9 molecules bound to P40 could not bind to EAC1-8 cells. C9 bound to X197 could not bind rapidly to EAC1-8, but prolonged incubation of the C9-X197 complex with EAC1-8 caused considerable lysis of the cells. C9 molecules bound to X195 could bind rapidly to EAC1-8, but their lytic activity was partially inhibited by the bound antibody. From these results, it is concluded that the C9b but not C9a domain contributes to the binding of C9 to EAC1-8 and that the epitope recognized by P40 or a closely adjacent site may be the binding site of C9 molecule to EAC1-8.
Ecto-protein kinases (ecto-PK) are expressed on many cell types, both normal and malignant, yet their functions are largely unknown. An ecto-PK capable of phosphorylating the C9 component of the complement system is described. This C9 ecto-PK could be inhibited by TBB, Emodin and DRB, selective inhibitors of protein kinase CK2. Treatment of Raji human B lymphoma cells with these CK2 inhibitors augmented cell killing by Rituximab (anti-CD20 antibodies) and human complement. Analysis of C5b-7-bearing Raji cells showed that extracellular inhibition of the ecto-CK2 enhanced cell lysis by C8 and C9. Blocking of the membrane complement regulator CD59 with monoclonal antibodies further enhanced the effect of the CK2 inhibitors on Raji cell death by complement. C9 ecto-CK2 activity was increased on cancer cells relative to normal fibroblasts and blood cells. Therefore, ecto-CK2 appears to be an additional factor protecting cells from complement-mediated lysis, probably by phosphorylation/inhibition of complement C9.
As part of a broader analysis of the mechanism(s) by which the most sensitive (type III) paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes are excessively sensitive to reactive lysis by isolated C5b6, C7, C8, and C9, we have compared type III PNH (PNH-III) and normal human E in respect to both total specific binding of 125I-C9 and the proportion of cell-bound C9 appearing in high molecular weight (HMW) complexes. In a previous report, we found that after exposure to purified C5b6 and 125I-C7, specific C7 binding and, by implication, EC5b-7 formation were equal for PNH-III E and normal E. In the present study, C8-dependent binding of 125I-C9 to PNH-III EC5b-7 and normal EC5b-7 was also similar, although lysis of the PNH-III E was up to five times greater; that is, PNH-III E required fewer bound C9 molecules to produce an effective lytic site than did normal E. To quantify radioactivity in monomeric and HMW forms of membrane-bound C9, lysed and unlysed E were subjected to low ionic strength buffers to convert all E to ghosts. These ghosts were solubilized in 0.1 or 2% SDS (without reduction) and electrophoresed on 2.4-11% polyacrylamide gradient gels followed by autoradiography and densitometric scanning. With 0.1% SDS, broad, heterodisperse zones of HMW C9 were recovered from both PNH and normal ghosts; the amounts of C9 incorporated into the HMW complexes were similar for PNH-III E and normal E. In selected experiments, 125I-C7 could be shown in these same HMW bands. When membranes were solubilized in 2% SDS, the overall proportion of HMW C9 complexes compared with dimer and monomer C9 was reduced on both PNH and normal membranes. In many, but not all experiments, more of the highest mol wt C9 complexes were detected from PNH-III E membranes solubilized in 2% SDS than from normal or PNH-II E membranes similarly treated. When antibody-sensitized E were lysed by purified C1-C9, PNH-III EA bound far more C9 than did normal EA, and both lysis and C9 incorporation into HMW complexes were markedly and proportionately increased over normal; however, lytic efficiency of 125I-C9 bound to PNH EA was equal to or less than that bound to normal EA.(ABSTRACT TRUNCATED AT 250 WORDS)
We measured CSF and plasma concentrations of C9, IgG, and albumin in 91 patients with demyelination and 73 controls with other neurologic diseases. The C9 index was reduced and IgG index increased in patients with multiple sclerosis and those with isolated demyelinating lesions, irrespective of disease activity; abnormalities were less marked in patients with isolated lesions than in those with MS. Humoral mechanisms may not be responsible for initiating demyelination, but activation of the complement system could amplify tissue damage and account for some symptomatic recovery.
Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.
We have shown previously that multimeric C9 within C5b-9 (C9:C5b-8 greater than 3:1) is needed for killing of a rough strain of Escherichia coli. We now extend these studies using serum sensitive, rough (R) and serum resistant, wild type (WT) strains of Salmonella typhimurium as well as a mutant S. typhimurium strain (TS) with a temperature sensitive mutation in synthesis of keto-deoxy-octulosonate, a constituent within the deep core structure of Salmonella LPS. Both R and TS required multimeric C9 within C5b-9 to be killed. Addition at 37 degrees C of increasing inputs of C9 to TS or R bearing C5b-9 led to a dose-related increase in C9 binding and killing. In contrast, addition of high inputs of C9 to the same strains at 4 degrees C, a procedure that limits the C9:C5b-8 ratio to 1:1, resulted in low C9 binding and minimal killing. Bactericidal C5b-9 formed at 37 degrees C on R and TS with high inputs of C9 co-sedimented with the bacterial outer membrane on sucrose density gradient analysis. Non-bactericidal C5b-9 on R, WT, and TS co-sedimented near the inner membrane, despite the presumed lack of association between these constituents. Whereas 125I C9 within the non-bactericidal pools immunoprecipitate with anti-C5, 125I C9 within bactericidal pools did not immunoprecipitate with anti-C5, anti-C7, or anti-C9. These findings suggest that bactericidal C5b-9 may be deposited in a unique location or configuration within the bacterial cell wall.
Herpes gestationis is a nosologically undefined uncommon bullous disease in pregnancy. In a patient with herpes gestationis C3, C5 und C9 deposits could be demonstrated by immunohistological methods at the epidermal basement membrane (BM). Deposits of immunoglobulins, properdin and C3PA were absent. Autoantibodies against BM were found in the serum of the mother by using rabbit oesophagus but not with normal human skin as antigen. In the sera of mother and child a factor could be demonstrated which in vitro leads to complement fixation in human epidermal BM. Immunopathological findings may speak in favor of a nosological entity of herpes gestationis with respect to dermatitis herpetiformis and bullous pemphigoid.
The case of a 10-year-old girl with congenital C9 deficiency and poststreptococcal glomerulonephritis (PSGN) is presented. Although her clinical symptoms mimicked those of Henoch-Schönlein purpura, histological examination of a renal biopsy specimen revealed the features commonly described in PSGN.
An intrinsic membrane protein with a m.w. of 65,000 that can bind human C8 has been identified after separation of human erythrocyte membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose sheets. The protein, tentatively designated as the C8-binding protein (C8bp) could be isolated from papain-treated erythrocyte (E) membranes by phenol-water extraction and isoelectric focusing. In a functional assay, with chicken (ch) E as target cells, C8bp inhibited the lysis of ch E C5b67 intermediates by human C8 and C9, whereas the lysis by rabbit C8 and C9 was not affected. Because the decay accelerating factor (DAF) from human erythrocyte membranes also inhibits the activity of C3/C5 convertases in an homologous system, we tested whether or not a DAF activity was present in C8bp. C8bp, however, did not accelerate the decay of the classic C3 convertases. Thus, it appears that C8bp and DAF are two different factors of E membranes with a similar molecular size inhibiting different sites of the activation cascade of complement while they can function synergistically to minimize the self-inflicted damage by complement.
Perforin (P1) is a cytolytic protein with similarity to complement component C9. P1 has been described as a unique component of murine cytolytic T-cell and rat natural killer cell granules Previous studies indicated that human granules and P1 differed from murine granules and P1 in that they appeared to be cytolytically less active and lacked the haemolytic activity characteristic of P1. It has been suggested that P1, like C9, is under the control of the homologous restriction factor. Here we determine the primary structure of human P1, re-examine its functional properties, and address the question of homologous restriction.
Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-alpha plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48-72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.
Ca2+ and Zn2+ prevent antibody-dependent complement-induced permeability changes in tonsil lymphocytes and Lettre cells. Lactate dehydrogenase leaks out from Lettre cells at high complement:cell ratios, under which conditions higher concentrations of Ca2+ and Zn2+ are required for protection. Ca2+ and Zn2+ do not inhibit complement activation or C9 binding to Lettre cells, and prevent leakage through preformed lesions. It is concluded that the extent of complement-induced membrane damage depends on the concentration of extracellular Ca2+, and may be modulated by changes in extracellular Ca2+ or Zn2+.
The molecular mechanism of complement resistance of the human SK-MEL-170 melanoma cell line was investigated. The cells have been shown to express the C3b-cleaving membrane protease p65. To delineate the molecular consequences of the C3b-cleaving activity for the complement cytotoxicity, the molecular events during the initiation (R24 monoclonal antibody, C1), amplification (C4, C3), and membrane attack (C5, C9) phases of complement were studied in comparison to a complement-susceptible human melanoma line (SK-MEL-93-2). No cleavage of C4b and C5b, 2 molecules structurally similar to C3b, was observed on the cells during classical pathway activation indicating the specificity of the p65 protease for the C3b molecule. The rapid degradation of C3b by p65 on the surface of complement-resistant SK-MEL-170 cells generates a M(r) 30,000 C3 alpha'-chain-fragment detectable as early as 1 min after complement activation, whereas no such fragment was present in detectable amounts on complement-susceptible cells. As a result of the rapid C3b proteolysis by p65 on resistant SK-MEL-170 cells, less C5 convertases are formed, which in turn results in the formation of a lower number of terminal complement components and membrane attack complexes. R24 antibody and C1q binding to the resistant cells was slightly lower as to susceptible cells. C4 binding studies, however, revealed that the observed difference in antibody and C1q binding has no influence on the complement resistance of SK-MEL-170 cells: significantly more C4b was bound to complement-resistant (1565 +/- 92 fg/cell) as compared to susceptible cells (715 +/- 31 fg/cell). On extraction of the molecular forms of C4 bound to the cell membranes, an additional high molecular weight C4 species--apparently a C4b-C4b homodimer--appeared only on the resistant SK-MEL-170 cells that may function as a residual back-up C5 convertase. Collectively, these results show that SK-MEL-170 human melanoma cells evade complement-mediated cytolysis despite sufficient activation of early components of the classical complement pathway by p65-mediated rapid degradation of surface-bound C3b, leading to a significant reduction in membrane attack complex formation. Thus, rapid cleavage of surface deposited C3b was established as a powerful mechanism of complement resistance.
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.
Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.
We studied frozen tissue of lesional skin and normal-appearing skin from SLE patients and DLE patients by immunofluorescence. S-protein and late components (C5, C7, C9) were localized at the dermal-epidermal junction of the lesional skin from SLE and DLE patients. All specimens of normal-appearing skin from patients with SLE were free of deposits of S-protein and late components. These results suggest that late components may play a role in the development of skin lesions of SLE and DLE.