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Electron microscopic study of the events occurring at the cell membrane during reactive lysis by complement, showed that a foliaceous particle was formed at the C5b-7 stage, that enlarged to a particle with a variable number of arms at the C5b-8 stage. Up to this point, no typical complement lesions were found. At the C5b-9 stages, the particles were completely converted to typical complement lesions, i.e. hollow cylinders projecting from the cell membrane and partly penetrating it. C5b-9 complexes assembled in the fluid phase did not show the typical structure of the lesions, but were amorphous masses of fibres.
We have previously shown that sublytic heterologous complement induces large but transient increases in erythrocyte membrane permeability. We now report that when erythrocytes are bystanders in zymosan-activated autologous serum, they increase their Na+ permeability 10-fold, indicating that autologous complement can also induce transient membrane lesions. When we isolated the effect of the C5b-9 membrane attack complex of complement by using human C5b-9 assembled from purified components, we found there was minimal lysis but efficient Na+ uptake. Suspension of the sublytically damaged erythrocytes in K+ medium caused the cells to lyse, which is consistent with the cells recruiting a compensatory K+ efflux similar to that observed when human erythrocytes were exposed to heterologous complement. Sublytic C5b-9 exposure also became lytic when extracellular Ca2+ was limited and when the cells were exposed to charybdotoxin, an inhibitor of the Ca(2+)-activated K+ channel. This indicates that Ca2+ is required for the functional termination of the C5b-9 lesion. We also show that the membrane hyperpolarization resulting from activation of the Ca(2+)-dependent K+ efflux does not influence the termination of the C5b-9 lesion. Thus, the influx of Ca2+ through the complement lesion initiates at least two apparently independent adaptive responses: (1) a process that terminates the leak; and (2) a K+ efflux that has a volume regulatory function. Our data support the potential of the sublytic C5b-9 lesion to act as a physiologic mediator for autologous erythrocytes.
The extracellular hemoglobins (Hbs) of annelids and tube worms are giant multisubunit proteins of up to approximately 200 polypeptides and molecular masses to at least 3,900 kDa. They differ from all other Hbs in having both O2-binding chains and "linker" chains. The latter are required for assembly and structural integrity of the protein and are deficient in or lack heme. We have determined the nucleotide sequences of the cDNA and gene for linker chain L1 of the hemoglobin of Lumbricus terrestris. The cDNA-derived amino acid sequence has 225 residues and a calculated molecular mass of 25,847 Da. The chain is 21-28% identical to linker chains of the related annelid Tylorrhynchus heterochaetus and the deep-sea tube worm Lamellibrachia sp. A remarkable feature of the linker chains is a conserved 38-39-residue segment that contains a repeating pattern of cysteinyl residues: (Cys-X6)3-Cys-X5-Cys-X10-Cys. This pattern, not present in any globin sequence, corresponds exactly to the cysteine-rich repeats of the ligand binding domains of the low density lipoprotein (LDL) receptors of man and Xenopus laevis. Furthermore, the cysteine-rich segment of linker chain L1 has the sequence Asp-Gly-Ser-Asp-Glu which is characteristic of LDL receptor repeats. Similar cysteine-rich sequences also occur in two other mammalian proteins, complement C9 and renal glycoprotein GP330. The results support the conclusion that the cysteine-rich motif of the LDL receptor and annelid Hbs is a multipurpose protein-binding unit of ancient origin which has been incorporated into diverse unrelated proteins, presumably by the process of exon shuffling.
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Complement activation products, C9 and C3-containing circulating immune complexes (CIC), were evaluated in plasma and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and osteoarthritis. C3 activation products and the fluid phase terminal complement complex were considerably elevated in SF from RA patients reaching levels five- to eighttimes that in plasma, consistant with a local activation of the whole cascade in the joints. The results emphazise the importance of detecting C3 activation by neoepitope expression instead of single fragment determinations. The concentration of native C9 was lower in synovial fluid compared with plasma, consistant with the excessive local complement activation. Increased CIC levels which correlated with the degree of complement activation were also found in the SF from the RA patients.
Antibodies to the ninth component of complement (C9) were found in a patient with hereditary C9 deficiency (C9D) who had received multiple transfusions. The incidence of anti-C9 precipitating antibodies following transfusion was investigated in 63,625 patients treated at Kyushu University Hospital, and 475,886 blood donors at the Fukuoka Red Cross Blood Center were studied to determine the frequency of C9D in Japan. Transfusion reactions in recipients having anti-C9 were also monitored. One hundred and eighty-six subjects (15 patients and 171 donors) with C9D were identified (a prevalence of 0.036%). Four of 15 C9-deficient patients had received transfusions. One of the patients who had received several units of blood within 1 week developed low-titer C9-precipitating antibody. This patient was transfused in the presence of the antibody without evidence of adverse reactions. No precipitating antibody was detected in the remaining three patients who had received several units of blood on a single day. These observations suggest that multiple transfusions may contribute to the development of anti-C9.
Homologous restriction factor (HRF) is a complementary regulatory protein found on the surface of human erythrocytes and other cell types. It has the function of blocking the lytic action of the membrane attack complex (MAC) of complement when incorporated into a membrane. HRF was found to inhibit not only the MAC composed of the proteins C5b-9, but also the C5b-8 complex. When 125I-labelled HRF was allowed to insert into an erythrocyte target, lysis by C5b-8 was reduced by about 80%. When human erythrocytes were treated with antibody to HRF, which bound to and presumably blocked the action of HRF, lysis by C5b-8 increased. This increase was not caused by additional binding of C8 on the cell membrane. When HRF was inserted into a target cell membrane, it had the effect of reducing the amount of C9 that could bind to very low levels, in one experiment from 6000 molecules per cell to about 500 molecules per cell. When this value was compared to the number of C7 molecules that bound, it was found that HRF reduced the amount of C9 bound to the level of C7 that was bound. HRF seemed to reduce the number of molecules of C9 that could bind to the C5b-8 complex so that only one C9 bound per complex.
DNA polymorphisms (RFLPs) of the human complement component C6, C7, and C9 genes were studied in three C7-deficient (C7D) families, one C6-deficient (C6D) family, and one C9-deficient (C9D) family. The 3 loci are closely linked on human chromosome 5. The haplotypes carrying the "silent" allele (C7*Q0, C6*Q0, and C9*Q0) were defined in each family, allowing for the detection of carriers among asymptomatic relatives. This paper describes familial studies on a type of hereditary trait, characterized by recurrent Neisseria infections in individuals homozygous for "silent" alleles at the C6, C7, or C9 loci.
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A 47-year-old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement-sensitivity lysis tests, 80% of her erythrocytes were markedly complement-sensitive (PNH-III). Laser cytofluorimetry with a monoclonal antibody against decay-accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF-negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusion of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patient's erythrocytes. When the patient's serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.
The cytosolic free Ca2+ concentration in rat neutrophils was determined by ratiometric fluorometry and imaging of Fura-2. Transient elevations of cytosolic free Ca2+ concentration were provoked by addition of C9 to neutrophils pre-coated with C5b-8. The rate of rise of the cytosolic free Ca2+ concentration was dependent upon the concentration of C9. These changes in cytosolic free Ca2+ concentration occurred in the absence of cell lysis, since there was no release of Fura-2, and were the result of increased permeability to extracellular Ca2+. More than 96% of the rise in cytosolic free Ca2+ generation by C9 was dependent upon the presence of extracellular Ca2+, but did not occur via channels which were inhibited by the Ca2+ channel blocker SKF96365. The decrease in the permeability of the membrane to Ca2+ after C9 was not triggered by the rise in cytosolic free Ca2+. After attack by C9, individual neutrophils remained responsive to f-met-leu-phe, or further attack by C9, both producing Ca2+ transients. The recovery of the Ca2+ signal was consistent with the complement membrane attack complex generating a series of permeability thresholds in the plasma membrane. These data have implications for the understanding of the mechanisms underlying the inappropriate responsiveness of neutrophils at inflammatory sites.