Combined genetic deficiency of C6 and C7 in man. (1/127)

By routine screening of sera, a subject was discovered who showed a sub-total deficiency of C6 and C7. No clinical disease was associated with this deficiency which was transmitted through the subject's family as a single genetic characteristic, the C6 deficiency being associated with a silent allele at the structural locus. The propositus was found to have low quantities of an abnormal C6 which was both antigenically deficient and smaller in size than normal C6 (110,000 daltons compared with 140,000 daltons) and small quantities of apparently normal C7. It is concluded that the most likely explanation for this defect is that the subject has a structural mutation in his C6 gene which produces hyopsynthesis not only of C6 but also of the closely linked gene for C7. These findings suggest the possibility that C6 and C7 may function as a single genetic unit and that the primary transcript copied from the genome includes information for both proteins.  (+info)

Reaction of an activated complex of guinea-pig complement components, C56, with unsensitized erythrocytes and with erythrocytes carrying C3b molecule. (2/127)

During the interaction of guinea-pig complement intermediate cells, EAC423, with guinea-pig C5 and C6, an activated complex of C5 and C6, C56, was demonstrated in the fluid phase of the reaction mixture. C56 also was eluted from EAC42356 which had been generated by the interaction of EAC423 with C5 and C6. Both preparations of C56 showed quite similar characteristics and were not distinguished from one another. Both were capable of reacting with unsensitized erythrocytes (E) in the presence of C7 to form EC567. Further, they were able to react with EAC43 in the absence of C7 to form EAC43568 but did react with EAC43 pretreated with C3b inactivator, dithiothreitol or N-bromosuccinimide. These results indicate that guinea-pig C56 generated on EAC423 has a tendency to dissociate into the fluid phase. Nevertheless, the dissociated C56 can bind again to intact C3b molecule on the cells. The ability of cell-bound C3b to combine with C56 may lead to localization of C56 to the cell membrane carrying C3b, resulting in acceleration of attachment of C567 to the membrane. This assumption could be supported by the finding that the replacement of E by EAC43 increased the susceptibility of the cells to lytic action of complement induced by cobra venom factor. Thus, a new function of cell-bound C3b as localizing C56 to the membrane of sensitized cells was indicated.  (+info)

C6 produced by macrophages contributes to cardiac allograft rejection. (3/127)

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.  (+info)

Complement membrane attack complex (C5b-9) mediates interstitial disease in experimental nephrotic syndrome. (4/127)

Accumulating evidence suggests that the generation of complement activation products from filtered complement components in urine with nonselective proteinuria leads to tubulointerstitial disease, resulting in progressive loss of renal function. To elucidate the role of C5b-9 in complement-mediated effects on renal tubular cells exposed to proteinuric urine, equivalent levels of proteinuria were induced (using the aminonucleoside of puromycin) in normocomplementemic and genetically C6-deficient piebald viral glaxo (PVG) rats. Semiquantitative histologic analysis revealed that complement-sufficient animals developed more severe tubulointerstitial disease than did C6-deficient rats. Amelioration of tubulointerstitial damage in C6-deficient animals was confirmed by studies with three independent markers of tubular damage, i.e., vimentin, osteopontin, and proliferating cell nuclear antigen. More tubular epithelial cells expressed osteopontin (an early marker of tubular injury) in normocomplementemic rats, compared with C6-deficient rats, at both days 7 and 12. Staining of vimentin in the tubules, near areas of tubular damage, was increased in normocomplementemic rats at day 12, and more proliferating cell nuclear antigen-positive tubular cells were observed at day 12 in complement-sufficient animals. The tubulointerstitial damage in complement-sufficient rats was also associated with greater accumulation of extracellular matrix (fibronectin) at day 12. These studies document for the first time an important role for C6, and therefore C5b-9, in the pathogenesis of nonimmunologic tubulointerstitial injury induced by proteinuria. These findings suggest that C5b-9 formation resulting from proteinuria contributes to the loss of nephron function by damaging the tubulointerstitium and that prevention of C5b-9 formation in tubules could slow the deterioration of renal function.  (+info)

Function of the factor I modules (FIMS) of human complement component C6. (5/127)

In order to elucidate the function of complement component C6, truncated C6 molecules were expressed recombinantly. These were either deleted of the factor I modules (FIMs) (C6des-748-913) or both complement control protein (CCP) modules and FIMs (C6des-611-913). C6des-748-913 exhibited approximately 60-70% of the hemolytic activity of full-length C6 when assayed for Alternative Pathway activity, but when measured for the Classical Pathway, C6des-748-914 was only 4-6% as effective as C6. The activity difference between C6 and C6des-748-913 for the two complement pathways can be explained by a greater stability of newly formed metastable C5b* when produced by the Alternative Pathway compared with that made by the Classical Pathway. The half-lives of metastable C5b* and the decay of (125)I-C5b measured from cells used to activate the Alternative Pathway were found to be about 5-12-fold longer than those same parameters derived from cells that had activated the Classical Pathway. (125)I-C5 binds reversibly to C6 in an ionic strength-dependent fashion, but (125)I-C5 binds only weakly to C6des-FIMs and not at all to C6des-CCP/FIMs. Therefore, although the FIMs are not required absolutely for C6 activity, these modules promote interaction of C6 with C5 enabling a more efficient bimolecular coupling ultimately leading to the formation of the C5b-6 complex.  (+info)

On the mechanism of cytolysis by complement: evidence on insertion of C5b and C7 subunits of the C5b,6,7 complex into phospholipid bilayers of erythrocyte membranes. (6/127)

The doughnut hypothesis of cytolysis by complement [Mayer, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 2954-2958] describes an annular structure made up of C5b-9 (complement factors C5b, C6, C7, C8, and C9) which becomes inserted in the lipid bilayer of the cell membrane, thus creating a hole. We now present initial explorations of this hypothesis. EAC1-6 and EAC1-7 (sheep erythrocytes carrying rabbit antibody and complement factors C1 through C6 or C1 through C7, respectively), prepared with either 125I-C3 or 125I-C5 were incubated with trypsin and the release of bound 125I was measured. In the case of 125I-C3, all of the radioactivity was released by trypsin from both intermediates. With 125I-C5, trypsin released all of the 125I from EAC1-6, but only 40-55% from EAC1-7. Possible reasons for resistance of the C5b subunit in EAC1-7 to tryptic digestion are discussed; in terms of the doughnut hypothesis it would be due to shielding by lipid molecules as a consequence of insertion into the lipid bilayer. In accord with this interpretation we have also found that C5b in EAC1-7, but not in EAC1-6, resists elution by 0.3 M NaC1. Similarly, we have found that 125I-C7 in EAC1-7 resists stripping by trypsin. Hence, we now propose the hypothesis that hydrophobic polypeptide chains from the C5b and the C7 subunits of C5b,6,7 complex become inserted in the phospholipid bilayer and that subsequent reactions with C8 and C9 open a channel across the membrane.  (+info)

High prevalence of complement component C6 deficiency among African-Americans in the south-eastern USA. (7/127)

Complement component C6 is a part of the membrane attack complex that forms a pore-like structure in cell membranes following complement activation. Deficiency of terminal complement components including C6 predisposes individuals to infection with Neisseriae. Using polymerase chain reaction/single-strand conformation polymorphism analysis followed by DNA sequencing, we screened genomic DNA from 200 randomly chosen blacks and an equal number from whites for three loss-of-function C6 mutations. Ten blacks and two whites were found to be heterozygous for one of the mutations. Two of the mutations, 1195delC and 1936delG, were found exclusively in black individuals. A third previously undescribed mutation, 878delA, was found at equal frequency among the two groups. The difference between the two groups was significant (P = 0.027), indicating that C6 deficiency due to these three mutations is more common among blacks than whites in the local area, principally Jefferson County, Alabama. In addition, three previously undescribed point mutations, two of which result in amino acid substitutions, were identified within exon 6. A review of the county health department records over the past 6 years revealed a higher incidence of meningococcal meningitis in blacks due to serogroups Y and W-135 which paralleled the difference in the estimated prevalence of C6 deficiency. Among black residents of the county (n = 235 598) there were 15 cases of meningitis due to these two serogroups, compared with two cases in the white population (n = 422 604) (P = 0.002). We conclude that C6 deficiency is more common among blacks than whites in the south-eastern United States, with a frequency approaching 1 in 1600 black individuals.  (+info)

Increased ion permeability of planar lipid bilayer membranes after treatment with the C5b-9 cytolytic attack mechanism of complement. (8/127)

The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in whcih components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis.  (+info)