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PubMed1985

The cobra complement system: II. The membrane attack complex.

Rabbit erythrocytes are lysed by cobra plasma. The membranes of the lysed rabbit erythrocytes exhibited typical ultrastructural complement lesions with an inner diameter of 72 A. Structures similar to human C5b-8 complexes were also visualized. The proteins found in membranes lysed by cobra plasma resembled closely the proteins of the human membrane attack complex in number and molecular weight. Cobra poly C9 that is resistant to reduction and boiling in SDS could also be demonstrated. These results indicate that the membrane attack complex of cobra complement is very similar to its mammalian counterpart.

Animals↗
PubMed1986

Membrane attack complex of complement in rheumatoid synovial tissue demonstrated by immunofluorescent microscopy.

Rheumatoid and osteoarthritic synovial membranes were examined for evidence of terminal complement pathway activation. Using antiserum highly specific for C9 neoantigen, immunofluorescent microscopy was employed to study synovial membrane specimens from 4 patients with rheumatoid arthritis, one patient with psoriatic arthritis, and 5 patients with osteoarthritis. C9 neoantigen was detected in 3 of the 4 rheumatoid membranes, the one psoriatic membrane, and one of the 5 osteoarthritic membranes. Activation of the terminal pathway of complement through C9 should be added to the spectrum of immunologic events known to occur in some rheumatoid synovial membranes.

Adult↗
PubMed1991

Formation of ion-conducting channels by the membrane attack complex proteins of complement.

The effects of sequential additions of purified human complement proteins C5b-6, C7, C8, and C9 to assemble the C5b-9 membrane attack complex (MAC) of complement on electrical properties of planar lipid bilayers have been analyzed. The high resistance state of such membranes was impaired after assembly of large numbers of C5b-8 complexes as indicated by the appearance of rapidly fluctuating membrane currents. The C5b-8 induced conductance was voltage dependent and rectifying at higher voltages. Addition of C9 to membranes with very few C5b-8 complexes caused appearance of few discrete single channels of low conductance (5-25 pS) but after some time very large (greater than 0.5 nS) jumps in conductance could be monitored. This high macroscopic conductance state was dominated by 125-pS channels having a lifetime of approximately 1 s. The high conductance state was not stable and declined again after a period of 1-3 h. Incorporation of MAC extracted from complement-lysed erythrocytes into liposomes and subsequent transformation of such complexes into planar bilayers via an intermediate monolayer state resulted in channels with characteristics similar to the ones produced by sequential assembly of C5b-9. Comparison of the high-conductance C5b-9 channel characteristics (lifetime, ion preference, ionic-strength dependence) with those produced by poly(C9) (the circular or tubular aggregation product of C9) as published by Young, J.D.-E., Z.A. Cohn, and E.R. Podack. (1986. Science [Wash. DC]. 233:184-190.) indicates that the two are significantly different.

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↗
PubMed1978

Neoantigen of the membrane attack complex of human complement: Occurrence on peripheral blood leukocytes from patients with systemic lupus erythematosus.

Since complement activation and hematological abnormalities occur in systemic lupus erythematosus (SLE), the present study is an investigation of whether the membrane attack complex of complement might be bound to peripheral blood leukocytes (PBL) in vivo. Assembly of the membrane attack complex results in the generation of neoantigen (neoAg) which is complex-specific and not expressed by any of the individual complement proteins. FITC antiserum specific to neoAg was employed to detect the membrane attack complex on PBL from 7 normal donors, 12 patients with SLE, and 2 patients with rheumatoid arthritis (RA): 3 +/- 1% of normal, 25 +/- 13% of SLE, and 23 +/- 11% of RA PBL were positive. The majority of the neoAg positive PBL in SLE were polymorphonuclear neutrophils (PMN) as shown by adherence to plastic, phagocytosis of carbonyl iron, and differential cell counts. The PBL were greater than 98% viable as indicated by the trypan blue exclusion technique. These observations strongly suggest that the membrane attack complex may be bound to viable PBL in patients with SLE and RA, and further raise the possibility that the membrane attack complex, may have a function other than lysis.

Antigen-Antibody Complex↗
PubMed1991

Amplified gene expression in CD59-transfected Chinese hamster ovary cells confers protection against the membrane attack complex of human complement.

Protection against the pore-forming activity of the human C5b-9 proteins was conferred on a nonprimate cell by transfection with cDNA encoding the human complement regulatory protein CD59. CD59 was stably expressed in Chinese hamster ovary cells using the pFRSV mammalian expression vector. After cloning and selection, the transfected cells were maintained in media containing various concentrations of methotrexate, which induced surface expression of up to 4.2 x 10(6) molecules of CD59/cell. Phosphatidylinositol-specific phospholipase C removed greater than 95% of surface-expressed CD59 antigen, confirming that recombinant CD59 was tethered to the Chinese hamster ovary plasma membrane by a lipid anchor. The recombinant protein exhibited an apparent molecular mass of 21-24 kDa (versus 18-21 kDa for human erythrocyte CD59). After N-glycanase digestion, recombinant and erythrocyte CD59 comigrated with apparent molecular masses of 12-14 kDa, suggesting altered structure of asparagine-linked carbohydrate in recombinant versus erythrocyte CD59. The function of the recombinant protein was evaluated by changes in the sensitivity of the CD59 transfectants to the pore-forming activity of human C5b-9. Induction of cell-surface expression of CD59 antigen inhibited C5b-9 pore formation in a dose-dependent fashion. CD59 transfectants expressing greater than or equal to 1.2 x 10(6) molecules of CD59/cell were completely resistant to human serum complement. By contrast, CD59 transfectants remained sensitive to the pore-forming activity of guinea pig C8 and C9 (bound to human C5b67). Functionally blocking antibody against erythrocyte CD59 abolished the human complement resistance observed for the CD59-transfected Chinese hamster ovary cells. These results confirm that the C5b-9 inhibitory function of the human erythrocyte membrane is provided by CD59 and suggest that the gene for this protein can be expressed in xenotypic cells to confer protection against human serum complement.

Animals↗
PubMed1990

Microvascular changes in early and advanced dermatomyositis: a quantitative study.

In adult dermatomyositis 10 muscle specimens with no or minimal histological alterations were compared with 7 that showed typical alterations. Five specimens from patients with inclusion body myositis, 5 from patients with polymyositis, and 8 from normal subjects served as controls. Vascular endothelium, visualized with the lectin Ulex europaeus agglutinin I, and complement membrane attack complex were demonstrated in the same cryostat sections by paired immunofluorescence. Large randomly selected fields were analyzed to determine the number of capillaries per square millimeter of fiber area (capillary density), per 1,000-microns 2 area of each muscle fiber (capillary index), and in 100 x 100-microns grid squares. In dermatomyositis specimens with minimal structural alterations there was focal capillary depletion, the capillary density was significantly reduced, and the frequency distributions of the capillary index and grid count were shifted to the left. In advanced dermatomyositis specimens, the findings were similar but more severe. In both kinds of specimens, clusters of capillaries reacted for complement membrane attack complex. The 2 patients with the highest proportion of vessels positive for membrane attack complex had a fulminant and fatal course. In polymyositis and inclusion body myositis specimens, the capillaries had a normal overall density and none reacted for membrane attack complex. The findings imply that the capillaries are an early and specific target of the disease process in dermatomyositis.

Adult↗
PubMed1988

Restriction of cell lysis by homologous complement: I. An analysis of membrane attack complex formation on target membranes.

The hemolytic efficiency and binding of C9 to homologous and heterologous erythrocytes was evaluated by using a standardized passive sensitization procedure to prepare antigen- and antibody-coated erythrocytes (EA) and human serum for lysis. Heterologous bovine EA were readily lysed by human serum, whereas human EA were quite resistant to lysis. Human EA bound as many C8 and C9 molecules per cell as bovine EA when incubated under identical conditions, but four times as much bound C9 was required to lyse an equal number of human EA compared with bovine EA. The susceptibility of human erythrocytes did not increase when increased volumes of undiluted human serum were used although C9 binding increased to as much as 100,000 molecules per cell. Sodium dodecyl sulfate-resistant polymerized C9 (poly(C9)) was detected on both lysed ghosts and unlysed EA bearing complement proteins C1 through C9 (EAC1-9) after incubation with undiluted human serum; however, the ratio of poly(C9) to monomeric C9 was higher on unlysed cells than on ghosts. Although bovine and human EA bound equal amounts of human C9 at the end point, the rate of lysis and C9 uptake was slower on homologous cells. The rate-limiting step occurred before C9 binding and lysis because the rates of lysis and C9 binding were equal on homologous and heterologous EAC1-8 targets, but the extent of lysis of homologous cells was still lower than lysis of heterologous cells. Human erythrocytes lose restriction against homologous hemolysis during storage in autologous plasma or in isotonic buffers.

Animals↗
PubMed1992

[Evaluation of plasma SC5b-9 (SMAC) in patients with ARDS].

The adult respiratory distress syndrome (ARDS) occurs in patients with various underlying illnesses. It has been suggested that complement activation may play a crucial role in the pathophysiology of ARDS by direct actions of complement fragments and/or by release of mediators including endotoxin, superoxide anion, and arachidonate metabolites. Therefore, complement evaluation was performed in patients with ARDS, with emphasis on SC5b-9 (Membrane Attack Complex). Complement split products, C4d, Bb, and SC5b-9, respectively from the classical, alternative, and terminal complement pathway were measured by enzyme immunoassay (EIA) using respective monoclonal antibodies. Plasma S-protein (vitronectin) concentration was also evaluated by EIA. We could not detect SMAC in normal controls or in a C5-deficient patient with abdominal infection. In contrast, most patients with ARDS and patients at risk showed elevated SMAC, and an increase in the values in association with exacerbation of the disease. However, two patients with ARDS exhibited lower values of SMAC in spite of deterioration and subsequent death. It was suggested that in these two ARDS patients, liver dysfunction may have contributed to the reduced SMAC formation. The finding that S-protein concentration (a MAC inhibitor) in the plasma of these two patients was reduced supports this speculation. It is suggested that simultaneous evaluation of SMAC and S-protein in plasma is helpful in determining the prognosis of critically ill patients.

Adolescent↗
PubMed1990

Thyroid follicular cell function after non-lethal complement membrane attack.

Terminal complement complexes have been identified around thyroid follicles in Graves' disease and Hashimoto's thyroiditis, and the concentrations of such complexes are increased in the sera of these patients, suggesting a role for complement activation and membrane attack complexes (MAC) in autoimmune thyroiditis. This has been investigated further using cultured human and rat thyroid cells. Thyrocytes were resistant to lysis by homologous complement, in contrast to the effects of heterologous (rabbit) complement. The formation of non-lethal amounts of MAC, using reactive lysis or classical pathway activation, significantly reduced cAMP production by these cells in response to thyroid-stimulating hormone (TSH) (P less than 0.01); similar effects were seen with thyroid-stimulating antibodies. Thyroid cells were able to recover rapidly from complement attack after washing and incubation for 30 min. Non-lethal MAC formation also resulted in reactive oxygen metabolite production, detected by luminol-dependent chemiluminescence in three out of five thyroid cell preparations tested. Ionomycin, but not TSH, also stimulated reactive oxygen metabolite production. These results suggest that repeated or continuous sub-lethal complement attack on thyroid cells may exacerbate hypothyroidism in Hashimoto's thyroiditis, or partially counter the effects of thyroid-stimulating antibodies in Graves' disease. Furthermore, the production of reactive oxygen metabolites in these circumstances could increase the intra-thyroidal inflammatory response; oxygen radical scavenging by anti-thyroid drugs (which are concentrated by thyrocytes) may account in part for the amelioration of thyroiditis observed with such treatment.

Animals↗
PubMed1985

The recovery of human polymorphonuclear leucocytes from sublytic complement attack is mediated by changes in intracellular free calcium.

Using polymorphonuclear leucocyte-erythrocyte ghost hybrids entrapping the calcium-activated photoprotein obelin, we have demonstrated that sublytic amounts of the complement membrane attack complex induce a rapid but transient increase in intracellular free calcium ion concentration ([Ca2+]i). This increase in [Ca2+]i occurs prior to, and is required for, rapid removal of membrane attack complexes from the cell surface. The increase in [Ca2+]i is not only due to increased influx from outside the cell, but also results from mobilization of intracellular stores. The possible mechanism of mobilization of calcium, and the importance of an increase in [Ca2+]i as a mediator of recovery processes in nucleated cells, are discussed.

Calcium↗
PubMed1985

Reconstitution of the complement channel into lipid vesicles and planar bilayers starting from the fluid phase complex.

Proteolysis of the fluid phase complement complex SC5b-9 transforms it into an amphiphilic molecule which resembles the membrane attack complex of complement and reconstitutes into lipid vesicles. Complement-containing vesicles prepared in this way can be made to fuse with planar lipid bilayers transferring their protein content to the host membrane. Massive conductance increases can thus be observed, which are due to the insertion of a large number of ionic channels into the membrane. Using low concentrations of vesicles, single channels can be studied.

Animals↗
PubMed1988

Pathogenesis of membranous nephropathy.

Membraneous nephropathy is the most common cause of idiopathic nephrotic syndrome in adults. Recent studies of the pathogenesis of the subepithelial glomerular immune deposits that characterize this disease have revealed new mechanisms of glomerular immune deposit formation involving cell surface antigens and have documented the role of the C5b-9 membrane attack complex of complement in mediating renal injury. Understanding these mechanisms may help us understand the pathogenesis of several other immune-mediated diseases and has implications for possible therapeutic interventions in MN.

Animals↗
PubMed1991

Eosinophilia myalgia syndrome: I. Immunocytochemical evidence for a T-cell-mediated immune effector response.

Specimens of muscle and fascia from 13 patients fulfilling the Centers for Disease Control criteria for the eosinophilia myalgia syndrome (EMS) were studied by quantitative immunocytochemical analysis. The immunolocalization of CD3, CD4, CD8, CD22, and CD56 markers, the gamma delta T-cell receptor, major histocompatibility complex (MHC) class I complex and class II antigens, and complement membrane attack complex (MAC) were examined. The distribution and relative proportions of T cells and T-cell subsets, B cells, macrophages, and eosinophils were determined at perivascular, perimysial, endomysial, and fascial sites of accumulation. At all sites, T cells were predominant, CD8+ cells outnumbered CD4+ cells 6- to 20-fold, and between 60 and 80% of T cells were activated. B cells and eosinophils each accounted for less than 3% of inflammatory cells. Very few cells expressed either the gamma delta T-cell receptor or natural killer cell markers. As in dermatomyositis (DM), MHC class I antigen complex expression was increased on many structurally normal muscle fibers, but in contrast to DM, microvascular MAC deposits were not a feature of EMS. The findings implicate a cellular immune response directed against a connective tissue component in EMS.

Adult↗
PubMed1989

20 KDa homologous restriction factor of complement resembles T cell activating protein.

We previously identified a 20KDa membrane glycoprotein 1F5 antigen which inhibits the assembly of homologous complement membrane attack complexes and we designate it as HRF20 standing for 20KDa homologous restriction factor. The amino acid sequence deduced from its coding base sequence resembles that of T cell activating protein, most conspicuously in cysteine residues, 10 out of 11 of which occupy identical positions in an overall sequence homology of 24.8%. Furthermore, proliferation of human T cells was stimulated by monoclonal antibody to HRF20.

Amino Acid Sequence↗
PubMed1987

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells.

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.

Animals↗
PubMed1979

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Cell Membrane↗