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The early components of human complement (C1, C4, and C2) plus certain serum euglobulins will kill pathogenic strains of Shigella sonnei. Serum from patients with hereditary C3 deficiencies and specific antiserums to C3, C5, and C6 were utilized to demonstrate the absence of requirements for late-acting complement components in this unusual bactericidal system.
Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target cells. It contains three subunits (C8 alpha, C8 beta, C8 gamma) which are encoded in separate genes. In relation to other proteins of the complement system, C8 gamma is unusual in that it is not structurally related to any other component nor does it have an obvious function. Based on weak but significant sequence similarity, it is proposed to be a member of the lipocalin family of widely distributed proteins that bind and transport small hydrophobic ligands. In this study, the human C8 gamma gene has been characterized and found to contain seven exons spanning approximately 1.8 kb. S1 nuclease and anchored PCR were used to identify the transcription initiation site. This site is preceded by putative regulatory elements that include two SP1 binding sites, several glucocorticoid response elements, and two SV40 enhancer core consensus sequences. A comparison to genes of other lipocalins reveals a remarkably close correlation in exon number, lengths, and phases. A close correspondence in exon boundaries is also observed and suggests that C8 gamma contains the same discrete structural elements that define the characteristic beta-barrel shape of the lipocalins. These results establish that C8 gamma is indeed ancestrally related to the lipocalin family and strengthens the likelihood that its role in the complement system is to bind an as yet unidentified ligand.
We evaluated the complement system in 20 patients presenting with a first episode of meningococcal meningitis, meningococcemia, or meningococcal pericarditis. Assays of total serum complement activity were performed prospectively in 12 patients and retrospectively in 8. Six of the twenty patients had a complement deficiency (CH50 greater than 2 S.D. below the normal mean). Three of these six had a deficiency of a terminal-pathway protein (C6 in two and C8 in one), and the other three had deficiencies of multiple complement proteins associated with underlying systemic lupus erythematosus or multiple myeloma. Patients with decreased amounts of complement were similar to normal patients in terms of sex, age, type of infection, and meningococcal serogroup, but 3 of the 6 patients with a complement deficiency were black, as compared with none of the 14 patients with normal function (P = 0.018). Complement deficiency is common in patients with a first episode of meningococcal disease and may be due to either a deficiency in a single terminal protein or a complement-depleting underlying illness.
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.
We have studied synthesis of the complement components and regulatory proteins of the alternative pathway and the membrane attack complex in synovial membrane. RNA was extracted from synovial tissue of patients with rheumatoid arthritis (RA) or osteoarthritis (OA) as well as from normal synovial membrane. Dot blot analysis showed the presence of mRNAs for all the complement components and regulatory proteins (C3, factor B, factor D, C5, C6, C7, C9, factor H, factor I, S-protein, SP-40, 40, DAF, MCP, CR1, CD59), except for properdin, C8 alpha, C8 beta and C8 gamma in all three types of synovial membrane studied. In an attempt to determine which components were synthesised by each cell type, monocytes (mononuclear phagocytes), human umbilical vein endothelial cells (HUVEC), synovial membrane fibroblasts (from normal, OA and RA synovial membrane) and peripheral blood lymphocytes were cultured in vitro and secretion rates of individual components were measured and total cellular RNA analysed by northern blotting. Monocytes secreted properdin, C3, and factor H but not factor B, factor I, C5, C6, C7, C8 or C9. Fibroblasts and endothelial cells secreted factor B, factor H and factor I, but not properdin, C5, C6, C7, C8 or C9. Lymphocytes did not secrete any of these components. mRNAs encoding C3, factor B, factor H, S-protein, SP-40, 40, MCP and DAF were detected in all three other cell types (monocytes, fibroblasts and HU-VEC), but factor I and CD59 mRNAs were not detected in monocytes. C5, C6, C7, C8 alpha, C8 beta, CD8 gamma and C9 mRNAs were not detected in any of the cell types studied.(ABSTRACT TRUNCATED AT 250 WORDS)
We studied the interaction of complement with human platelets. Complement was activated by IgG anti-P1A1 antibody obtained from 3 patients with the post-transfusion purpura syndrome. We used a heparin-plasma buffer system that permits complement activation and also preserves platelet function. With this system complement activation was efficient, and platelet immune alteration was extensive. Anti-P1A1 antibody was effective only in the presence of complement, in which case both platelet lysis and serotonin release (release reaction) in the absence of lysis were observed. Platelet lysis, as assessed by 51Cr loss, required 10-fold more antibody than was necessary to induce platelet aggregation and release of 14C-serotonin. This platelet release reaction required an intact classic complement sequence through C6. The extent of platelet serotonin release parallelled the depletion of C1 and C4 from platelet-rich plasma. Concentrations of antibody insufficient to induce platelet aggregation and serotonin release could still activate C1 and deposit increased C3 on the platelet surface. These studies demonstrated that complement activation by anti-P1A1 antibody can alter human platelets in a nonlytic system. Several phases of complement-mediated human platelet alteration are possible, depending on the concentration of anti-P1A1 antibody.
In chronic membranoproliferative glomerulonephritis (CMPGN) the activation of the complement system through the properdin pathway plays an important role. The clearance of complement components and of the C3-proactivator (C3-PA) have been determined in 18 patients. Hemolytically active C5, C6, C7 and C3-PA were detected in the urine for the first time. The clearances of the complement components did not correlate with the clearances of other serum proteins with similar molecular weights. The specificities of the single complement components in the urine were tested by specific complement inhibitors such as hydrazine, KSCN, and the C4-inactivating factor.
Patients with disseminated Neisseria meningitidis infections (meningococcemia) suffer from a fulminant shock syndrome that is accompanied by extraordinarily high concentrations in serum of tumor necrosis factor (TNF). People with homozygous deficiencies of late complement components (C5, C6, C7, and C8) experience a high incidence of disseminated neisserial infections yet suffer from an attenuated form of the disease. The mechanisms that account for this disparity in host response are unclear, but they may in part be related to differences in the systemic TNF response that are modulated by terminal complement components (C5 to C9). The role of C5 in the modulation of the systemic endotoxin-induced TNF response was studied with matched strains of C5-deficient (B10 D2/Osn) and complement-sufficient (B10 D2/Nsn) mice. Following lipopolysaccharide (LPS) administration, complement-sufficient mice exhibited more rapid increases in pulmonary and hepatic vascular permeabilities than did C5-deficient controls. Complement-sufficient mice developed acute passive hepatic congestion, they appeared more ill than C5-deficient mice, and they exhibited a twofold greater rise in serum TNF activity compared with that by C5-deficient mice. C5-deficient mice reconstituted with normal serum before an LPS injection exhibited pulmonary and hepatic vascular permeability increases and serum TNF levels approaching those observed in complement-sufficient mice. Alveolar and peritoneal macrophages isolated from complement-sufficient and C5-deficient mice and incubated in heat-inactivated serum did not exhibit differences in TNF mRNA expression or secreted TNF activity following stimulation with LPS. However, incubation of macrophages in complement-sufficient mouse serum (before LPS stimulation) resulted in increased TNF mRNA expression and TNF activity compared with those in cells incubated in C5-deficient serum. In vitro studies employing human complement components and peripheral blood monocytes revealed that recombinant C5a, in the presence or absence of LPS, can induce increased concentrations of TNF and that C5b to C9 had no additional modulatory effect on the TNF response. These data suggest that C5 modulates the endotoxin-triggered TNF response. The role of complement components distal to C5 (i.e., C5b to C9) in the endotoxin-triggered TNF response remains unclear.
Endotoxin-stimulated human peritoneal macrophages were cultured in serum-free medium with agarose beads. Monospecific antibodies to human C3c, C3g, C5, C6, C7, C8, C9 and to C9-neoantigen bound to the beads. This shows that activated C3 and the terminal complement complex (TCC), made from complement components C5 to C9, were generated on the beads. De novo synthesis was confirmed by agarose binding of tritium-labelled protein. Moreover, C3-derivatives and C9-neoantigen were detected on normal serum-treated agarose beads but not on beads kept in factor B-depleted or heat-inactivated sera, implying that an intact alternative complement pathway was required for our findings. The macrophages thus synthesize the active complement components of the alternative and terminal pathways in vitro.
Using immunofluorescence (IF) and monoclonal antibodies (MoAbs) to IgG subclasses, terminal complement components, and S-protein/vitronectin, we have extended recent observations concerning reactivity of bullous pemphigoid autoantibodies with intracellular antigens located on the polar tips of basal human keratinocytes (HuK). Using three purified bullous pemphigoid IgG fractions, autoantibody reactivity with these intracellular antigens was present in all four IgG subclasses. When skin sections were used as substrate, an identical IgG subclass distribution of autoantibodies for each bullous pemphigoid IgG fraction was observed, but reactive with the basement membrane zone. All three bullous pemphigoid IgG preparations contained IgG subclass autoantibodies capable of complement fixation. Each IgG fraction resulted in fixation of all of the terminal complement components (C5, C6, C7, C8, and C9) and assembly of the membrane attack complex (MAC) on the polar tips of basal HuK. S-protein/vitronectin was not bound in a similar fashion. Normal IgG fractions yielded consistently negative reactions. Thus, bullous pemphigoid autoantibodies, fixed to polar tips of basal HuK, are found in all four IgG subclasses and will activate complement resulting in generation of MAC.
The transcription regulators, PDR1 and PDR3, have been shown to activate the transcription of numerous genes involved in a wide range of functions, including resistance to physical and chemical stress, membrane transport, and organelle function in Saccharomyces cerevisiae. We report here that PDR1 and PDR3 also regulate the transcription of one or more undetermined genes that translocate endogenous and fluorescent-labeled (M-C6-NBD-PE) phosphatidylethanolamine across the plasma membrane. A combination of fluorescence microscopy, fluorometry, and quantitative analysis demonstrated that M-C6-NBD-PE can be translocated both inward and outward across the plasma membrane of yeast cells. Mutants, defective in the accumulation of M-C6-NBD-PE, were isolated by selectively photokilling normal cells that accumulated the fluorescent phospholipid. This led to the isolation of numerous trafficking in phosphatidylethanolamine (tpe) mutants that were defective in intracellular accumulation of M-C6-NBD-PE. Complementation cloning and linkage analysis led to the identification of the dominant mutation TPE1-1 as a new allele of PDR1 and the semidominant mutation tpe2-1 as a new allele of PDR3. The amount of endogenous phosphatidylethanolamine exposed to the outer leaflet of the plasma membrane was measured by covalent labeling with the impermeant amino reagent, trinitrobenzenesulfonic acid. The amount of outer leaflet phosphatidylethanolamine in both mutant strains increased four- to fivefold relative to the parent Tpe+ strain, indicating that the net inward flux of endogenous phosphatidylethanolamine as well as M-C6-NBD-PE was decreased. Targeted deletions of PDR1 in the new allele, PDR1-11, and PDR3 in the new allele, pdr3-11, resulted in normal M-C6-NBD-PE accumulation, confirming that PDR1-11 and pdr3-11 were gain-of-function mutations in PDR1 and PDR3, respectively. Both mutant alleles resulted in resistance to the drugs cycloheximide, oligomycin, and 4-nitroquinoline N-oxide (4-NQO). However, a previously identified drug-resistant allele, pdr3-2, accumulated normal amounts of M-C6-NBD-PE, indicating allele specificity for the loss of M-C6-NBD-PE accumulation. These data demonstrated that PDR1 and PDR3 regulate the net rate of M-C6-NBD-PE translocation (flip-flop) and the steady-state distribution of endogenous phosphatidylethanolamine across the plasma membrane.
Why LDL entrapped in the subendothelium should trigger events leading to chronic inflammation and to arterial wall injury is a major enigma of modern medicine. Oxidation of LDL in vitro renders the molecule potentially atherogenic, and the concept that oxidation is the major single event underlying the transformation of LDL to a proinflammatory molecule dominates the world literature. Here, an alternative hypothesis on the pathogenesis of atherosclerosis will be presented. We have found that non-oxidative, enzymatic modification of LDL with ubiquitous enzymes (protease + cholesterol esterase + neuraminidase) also transforms the molecule to an atherogenic moiety. Enzymatically altered LDL (E-LDL) shares major properties in common with lipoproteins that have been isolated from atherosclerotic lesions. It activates complement via the alternative pathway and is recognized by a scavenger receptor on human macrophages, thus inducing foam cell formation. Uptake of E-LDL is accompanied by potent induction of MCP-1 synthesis and secretion. In contrast, E-LDL does not stimulate IL-1 or TNF-production and is only a weak inducer of IL-6. Monoclonal antibodies were produced that recognize neoepitopes on E-LDL, but that do not react with native or oxidized LDL. With the use of these antibodies, extensive deposition of E-LDL in very early atherosclerotic lesions was demonstrated. Activated complement components colocalized with E-LDL, corroborating the concept that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development. The pathogenetic relevance of unhalted complement activation in atherogenesis was demonstrated with the use of C6-deficient rabbits. It was found that C6-deficiency markedly protected against development of diet-induced atherosclerosis in the experimental animals. In sum, our hypothesis departs from the mainstream of atherosclerosis research and derives from the recognition that extracellular exposition of free cholesterol in LDL-particles by itself confers pro-inflammatory properties onto the lipoprotein molecule. We believe that the degrading enzymes are ubiquitously present in the extracellular matrix, so the only requirement for atherogenesis to occur is the deposition of large amounts of LDL. Oxidative processes or infections probably play only minor roles, and reduction of LDL plasma levels will predictably represent the single most important prophylactic measure against development and progression of atherosclerosis.
C2, C6, and C7 were measured by hemolytic assay and Factor B and IgG were measured by radial immunodiffusion in samples of normal and inflamed aqueous humor. Normal aqueous humor was found to contain functional C2, C6, and C7, but the small ratios of aqueous humor to serum measurements suggested that there was relatively little of these complement components in normal aqueous humor when compared to serum. The mean values of C2, C6, C7, and Factor B in aqueous humor and the median ratios of aqueous humor to serum measurements for each complement component were higher in patients with inflamed aqueous humor than in patients with normal aqueous humor. A comparison of the ratios of IgG to each complement component in normal and inflamed aqueous humor suggested that levels of IgG and complement increased proportionately in inflamed aqueous humor. Factor B, a component of the alternative pathway, was not detected in normal aqueous humor but measured in five of six samples of aqueous humor from eyes with anterior chamber inflammation.
The membrane attack complex of complement is formed by the molecular fusion of the five terminal complement proteins, C5, C6, C7, C8, and C9. While the assembly process on a target membrane and its modulation by restriction factors present on host cells is now quite well understood the molecular details of the architecture of the complex still need much further clarification. This is especially true for the interaction of the last acting protein C9, which provides the cytotoxic action of the complex, with the precursor C5b-8 complex. Because of this lack of structural details the molecular mechanisms that lead to complement-mediated cell death remain cryptic, however, it is hoped that recent advances in controlling the assembly process and in site-specific modification of the terminal complement proteins by recombinant DNA techniques should change this predicament quickly.
Properdin is the positive regulator of the alternative pathway of complement activation. The 53-kDa protein is essentially composed of six thrombospondin type 1 repeats, all of which contain the WXXW motif, the recognition sequence for C-mannosylation. C-Mannosylation is a post-translational modification of tryptophan residues in which, in contrast to the well known N- and O-glycosylation, the carbohydrate is attached via a C-C bond to C-2 of the indole moiety of tryptophan. C-Mannosylation was first found in human RNase 2 and interleukin-12. The terminal complement proteins C6-C9 also carry this modification as part of their thrombospondin type 1 repeats. We studied the C-mannosylation pattern of human properdin by mass spectrometry and Edman degradation. Properdin contains 20 tryptophans of which 17 are part of a WXXW motif. Fourteen tryptophans were found to be modified 100%. This is the first example of a protein in which the majority of tryptophan residues occurs in the C-mannosylated form. These results show that C-mannosylated proteins occur at several steps along the complement activation cascade. Therefore, this system would be ideal to investigate the function of C-mannosylation.
The seventh component of complement is a single chain plasma glycoprotein that is involved in the cytolytic phase of complement activation. We have determined the structure of the C7 gene, which is encoded by 18 exons whose sizes vary from 56 to 244 bp. For the most part, the exons do not correspond to the protein homology units. However, two intron/exon boundaries occur at junctions between different functional parts of the protein. The first is at a site between the end of the C9 homology unit and the carboxyl-terminal extension which is also a feature of C6. The second of these boundaries occurs between the regions encoding two pairs of cysteine-rich modules (the short consensus repeats and the factor I modules) located in the carboxyl-terminal part of C7. In contrast to the exons, the introns range considerably in size from 0.5 to 8.5 kbp. The complete analysis indicates that the gene encoding C7 is approximately 80 kbp in length. We show here that the C7 gene is highly homologous to that for C6, and also to C8A, C8B, and C9, confirming and extending the published data. With the exception of exon 1, all intron/exon boundaries are preserved with respect to phase when compared with C6.
By using antibodies against C5, C6, C7, C8, and C9, we found that terminal complement components were deposited on IgM-coated sheep erythrocytes (EIgM) kept in serum-free endotoxin-stimulated monocyte cultures for 24 or 48 h. Monoclonal antibodies revealed C9 neoantigens on the EIgM. There was no specific binding of an anti-S protein antibody, which reacts with the SC5b-9 complex, to the EIgM. Controls were native sheep erythrocytes (E) treated similarly which, in contrast to EIgM, do not activate the classical pathway of complement. Cycloheximide (1.0 microgram/ml) in the cell cultures resulted in no specific binding of the anti-C9 antibodies to EIgM. A fraction of the EIgM was lysed during incubation with the monocytes. We conclude that the monocytes secrete C5, C6, C7, C8, and C9, which form the membrane attack complex of complement (C5b-9) on the EIgM.
Treatment of [3H]arachidonic acid ([3H]C20:4)-labeled, antibody-sensitized mouse resident peritoneal macrophages with rabbit serum complement, or C6-deficient rabbit serum + C6, caused hydrolytic release of incorporated [3H]C20:4 from phospholipids, followed by conversion to oxygenated derivatives. The C6 dose-response curve for release of C20:4 plus its metabolites was monotonic, which indicates dependence on channel formation, whereas the dose-response curve for lysis displayed multi-hit behavior. High-performance liquid chromatography demonstrated that the major radiolabeled products in the aqueous phase co-eluted with C20:4, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin E2. Kinetic studies of the release of 6-keto-PGF1 alpha, the major metabolite, displayed biphasic characteristics; a moderate amount of this prostaglandin was released before the onset of cell lysis. Experimental evidence obtained by freeze-thaw or by incubation of these cells with melittin or A23187 indicated that cell lysis does not necessarily result in the production of inflammatory mediators. Furthermore, when macrophages were treated with serum complement, it was apparent that the major part of the release was due to C5b-9 and not to the action of C5a. We conclude that release of C20:4 and its derivatives from complement-treated macrophages does not depend on cytolysis, but is a consequence of insertion and channel formation.