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PubMedDate not supplied

Complement resistance of tumor cells: basal and induced mechanisms.

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement. appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Animals↗
PubMed1989

Vitronectin colocalizes with Ig deposits and C9 neoantigen in discoid lupus erythematosus and dermatitis herpetiformis, but not in bullous pemphigoid.

C9 neoantigen immunoreactivity has been found to colocalize with C3 immunoreactivity at the dermal-epidermal junction zone (DEZ) in skin specimens from patients with bullous pemphigoid, lupus erythematosus and dermatitis herpetiformis. The present study was designed to elucidate whether the C9 neoantigen immunoreactivity represents deposition of membrane attack complexes or non-lytic SC5b-9 complexes. Skin specimens from 11 patients with pemphigoid, five patients with discoid lupus erythematosus and from nine patients with dermatitis herpetiformis were studied with immunofluorescence using both monoclonal and polyclonal antibodies against C9 neoantigen and against vitronectin (S-protein), an inhibitor to the membrane attack complex of complement. Specimens from the pemphigoid patients demonstrated C9 neoantigen reactivity along the DEZ without detectable colocalized vitronectin. This suggests deposition of membrane attack complexes in the pemphigoid lesions. Immunoreactivity of both C9 neoantigen and vitronectin was detected in the DEZ in specimens of discoid lupus erythematosus and in the tips of dermal papillae in specimens of dermatitis herpetiformis. The combined presence of C9 neoantigen- and vitronectin immunoreactivity may indicate deposition of C9 as part of the non-lytic SC5b-9 complex. The finding reported here of differential deposition of vitronectin and C9 in different diseases indicates that the presence of C9 neoantigen immunoreactivity in tissue per se does not represent the deposition of membrane attack complexes, but that it may also be C9 deposited as part of the nonlytic SC5b-9 complex.

Autoantigens↗
PubMed1999

C6 produced by macrophages contributes to cardiac allograft rejection.

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.

Animals↗
PubMed1980

A hypocomplementemic vasculitic urticarial syndrome. Report of four new cases and definition of the disease.

We describe four new patients with a unique syndrome of persistent urticaria, with leukoclastic angiitis, severe angioedema, occasional life-threatening laryngeal edema, arthritis, arthralgia, neurologic abnormalities and pronounced persistent hypocomplementemia. The complement abnormalities involved markedly reduced levels of the Clq subunit of the first component of complement (Cl) in the presence of near normal levels of Clr and Cls subunits of Cl; modest to marked depletion of the fourth component of complement (C4), the second component of complement (C2) and the third component of complement (C3); and normal levels of the fifth through ninth components of complement (C5 through C9) and properdin factors B and D. A striking serologic abnormality found in all patients was the presence of low molecular weight (7S) proteins which precipitated with Clq in agarose gels; these previously were shown to be comprised at least in part of immunoglobulin G. The present experience is offered to help to define the clinical, histopathologic and serologic characteristics of this entity, designated hypocomplementemic vasculitic urticarial syndrome, and to emphasize its distinctiveness and prevalence.

Adult↗
PubMed1992

IgA nephropathy in patients with congenital C9 deficiency.

The clinical, histologic, and immunopathological findings of three young Japanese males with congenital C9 deficiency and primary IgA nephropathy are reported. The C9 deficiency was discovered either through mass complement screening, or when low hemolytic activity for CH50 and normal C3 levels were detected in plasma. Hematuria and proteinuria were detected at the age of 8 or 9 years as a result of annual urinary screening tests for school children. Renal biopsy showed focal and segmental mesangial proliferation with small epithelial crescents in one patient, and mild, diffuse mesangial proliferation in two. IgA and C3 were deposited predominantly in the mesangial area, and staining for C9 was negative in these patients. Electron microscopy revealed electron dense deposits predominantly in mesangial and paramesangial zones. Immunohistochemical staining in renal biopsy tissues from two patients showed mesangial staining for C5, C8, and S-protein, but staining for C5b-9 neoantigen was completely negative. These results show that the formation of C5b-9 complex is not essential for the induction of human IgA nephropathy, and also for the proliferation of mesangial and even parietal epithelial cells.

Child↗
PubMed1988

Selected IgG rapidly induces Lambert-Eaton myasthenic syndrome in mice: complement independence and EMG abnormalities.

Antibodies in individual patients with the Lambert-Eaton myasthenic syndrome (LES) differ in their reactivity with mouse motor nerve terminals. Of 26 LES patients' sera injected a single time into mice, 3 caused a highly significant reduction in stimulus-dependent quantal release (m) of acetylcholine (ACh) (to 6, 33, and 42 quanta per impulse at 1 Hz, respectively; mean for 10 control sera, 100 quanta at 1 Hz). The most potent serum (LES-A) was fully effective in mice deficient in complement component C5 and in mice depleted of complement components C3----C9 by cobra venom factor. A single i.v. injection of serum reduced m in direct proportion to log dose. Responses to K+ depolarization and increasing concentrations of Ca2+ were like those observed in human LES. With LES-A serum, or its IgG, m was reduced near maximally in 1 day and plateaued in 3-4 days. Recovery began after day 8; m was in the normal range by day 20-30. Electromyographic (EMG) abnormalities were not seen until m fell below 40 quanta per impulse at 1 Hz. Below 10 quanta, clinical signs of weakness appeared, and the EMG abnormalities were those classically associated with LES: a marked reduction of compound muscle action potential to a single nerve stimulus in rested muscle, a further decrement during stimulation at slow rates, but marked facilitation during rapid repetitive stimulation.

Acetylcholine↗
PubMed1979

Impairment of acute protein reactivity in chronic renal failure.

The acute phase protein response was studied after elective surgery in 13 normal subjects and 9 patients with severe chronic renal failure. Total haemolytic complement reactivity (CH50) and serum concentrations of C1q, C1s, C4, C3, factor B, properdin, C5, C9, C-reactive protein (CRP), caeruloplasmin, alpha1-acid glycoprotein and haptoglobin were measured preoperatively and on days 2, 4 and 6 after operation. Abnormalities were seen in the group with chronic renal failure. Firstly, there was no significant acute phase response of C1s, C3, C5, C9 and CH50 and a significant reduction in the response of factor B. Secondly, CRP showed prolonged elevation in the post-operative period in contrast to the transient rise seen in the control group. With the possible exception of alpha1-acid glycoprotein, the behaviour of the non-complement proteins (caeruloplasmin and haptoglobin) was comparable for the two groups. These defects could impair the physiological response to infection in patients with severe chronic renal failure.

Adult↗
PubMed1988

Role of immunoglobulin G in killing of Borrelia burgdorferi by the classical complement pathway.

The antibody and complement requirements for killing of Borrelia burgdorferi 297 by normal human serum (NHS) and NHS plus immunoglobulin G (IgG) were examined. B. burgdorferi activated both the alternative and classical complement pathways in NHS. In NHS chelated with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 4 mM MgCl2 (Mg-EGTA) to block classical pathway activation, consumption (activation) of total hemolytic complement, complement component 3 (C3), and C9 by B. burgdorferi was observed. Furthermore, challenge of unchelated NHS with 297 cells resulted in the consumption of C4, in addition to an increase in C3 and C9 consumption over that observed in chelated serum. In spite of complement activation, B. burgdorferi was resistant to the nonspecific bactericidal activity of NHS. The addition of human anti-B. burgdorferi IgG to NHS, however, resulted in the complete killing of 297 cells. Bactericidal activity of this serum was abrogated if NHS was immunochemically depleted of C1, indicating that killing was mediated by the classical pathway. The manifestation of bactericidal activity was accompanied by a large increase in total complement and C3 consumption over that observed in NHS alone. Under similar conditions, only a minimal increase in C9 consumption was observed. No increase in total complement consumption was observed if NHS plus anti-B. burgdorferi IgG was treated with Mg-EGTA prior to challenge. The results of these experiments demonstrate that B. burgdorferi is resistant to the nonspecific bactericidal activity of NHS, in spite of classical and alternative complement pathway activation. B. burgdorferi is sensitive to serum, however, in the presence of IgG, which mediates bacterial killing through the classical complement pathway.

Antibodies, Bacterial↗
PubMed2001

Accelerated graft arteriosclerosis in cardiac transplants: complement activation promotes progression of lesions from medium to large arteries.

BACKGROUND: A critical role for the terminal components of complement (C5b-C9) has been demonstrated previously in acute allograft rejection with the use of C6-deficient PVG congenic rat strains. The C6 deficiency prevents the formation of membrane attack complex (MAC) by C5b-C9. Hearts transplanted from PVG.1A (RT1a) rats are rejected acutely (7-9 days) by fully MHC-incompatible C6-sufficient PVG.1L (RT11) recipients, but they survive significantly longer in untreated C6-deficient PVG.1L recipients (19 to >60 days). METHODS: To investigate the contribution of MAC to chronic rejection and accelerated graft arteriosclerosis (AGA) in long-term cardiac allografts, hearts were transplanted heterotopically from PVG.1A donors to C6-sufficient and C6-deficient PVG.1L hosts that were treated with cyclosporine 15 mg/kg/day for 14 days after cardiac grafting. Alloantibody responses in hosts were measured by flow cytometry at 4, 8, 12, and 16 weeks after transplantation. Vigorously contracting grafts were removed at 60 days (n=5) and at 90-128 days (n=12) after surgery for morphological evaluation. Computerized planimetry measurements were made in complete cross-sections of grafts on all assessable arteries larger than 16 microns in diameter. RESULTS: The survival of most (six of seven) cardiac allografts in C6-deficient recipients was prolonged by cyclosporine treatment to greater than 90 days. In contrast, 14 of 25 hearts that were transplanted to C6-sufficient recipients were rejected between 21 and 84 days with severe vascular injury. AGA, defined as smooth muscle cells forming a neointima inside the internal elastic lamina and luminal compromise, affected a greater percentage of arteries in C6-sufficient than in C6-deficient recipients. AGA developed earlier and more frequently in arteries of medium (<100 micron) diameter than those of large diameter in both C6-sufficient and C6-deficient recipients. Serial sections demonstrated the lesions in medium arteries to be located adjacent to the smooth muscle sphincters at the junction of arteriolar branches. CONCLUSIONS: These results demonstrate that MAC promotes the pathogenesis of AGA in long-term cardiac allografts.

Animals↗
PubMed1984

Membrane attack by complement.

Membrane attack by complement involves the self-assembly on membranes of five hydrophilic proteins (C5b, C6, C7, C8 and C9) to an amphiphilic tubular complex comprising approximately 20 subunits. The hydrophilic-amphiphilic transition of the precursor proteins is achieved by restricted unfolding and exposure of previously hidden hydrophobic domains. Restricted unfolding, in turn, is driven by high-affinity protein-protein interactions resulting in the formation of amphilic complexes. Circular polymerization of C9 to a tubular complex (poly C9) constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions.

Animals↗
PubMed1976

Activation of therminal components of human complement by a trypsin-activated complex of human factor B and cobra venom factor.

Cleavage of C3 by CVF-B was demonstrated by hemolytic, immunoelectrophoretic and immune adherence reactions. No cleavage of C5 was detected by immunoelectrophoresis, but C5 hemolytic activity, assayed with EAC1423, decreased although less than C3 hemolytic activity. The co-existence of C3 with limiting amounts of C5 did not reduce the final degree of hemolysis of guinea pig erythrocytes (GPE) induced by late-acting components C6 through C9 and CVF-B. Thus, a CVF-B hemolytic system composed of GPE, C5 through C9 and CVF-B provided a method for titration of terminal components of human complement. CVF-B was able to generate hemolytically active sites of C567 on GPE by activation of C5, C6 and C7. The complex C567 in the fluid-phase decayed within 1 min but C567 on GPE was quite stable. Originally insensitive sheep erythrocytes became sensitive to the CVF-B hemolytic system if C3b sites were present, suggesting that cell-bound C3b played a role in orienting the positions of C567 to be fixed. CVF-B could be recovered quantitatively from the supernatant of the reaction mixture in which the hemolytically active intermediate GPEC-5678 had been formed through the interaction between C5 to C8 and CVF-B.

Animals↗
PubMed2000

Structure, sequence, and promoter analysis of human disabled-2 gene (DAB2).

Disabled-2 (DAB2 for human and Dab2 for other species) is one of two mammalian orthologues of Drosophila Disabled. DAB2 exhibits properties of a tumor suppressor gene: the expression of DAB2 is eliminated in 85-95% of breast and ovarian tumors; homozygous deletions of the gene have been found in some of these tumors; and reintroduction of DAB2 expression suppresses tumorigenicity of carcinoma cells. To study the mechanisms of loss of expression and to detect possible mutations in tumors, we have investigated the genomic structure of the DAB2 gene. The complete DAB2 gene was identified and sequenced from four overlapping BAC clones found to contain the gene. Complement factor 9 (C9) gene was localized next to the DAB2 gene at the 3'-end of the BAC DNA fragments. The human DAB2 gene is about 35 kb in size and consists of 15 exons and 14 introns, producing an approximately 4-kb message. A spliced variant corresponding to mouse Dab2 p93 and a 3'-end spliced variant were also identified. The translation initiation site resides in the second exon, and the noncoding first exon is separated from the second exon by a 14-kb intron. The 420-bp sequence 5' of exon 1 contains a CpG island (39 CpG sites). This 420-bp putative promoter was found to contain the site for transcription initiation, identified by RNase protection assay, and is sufficient for active transcription in epithelial cells. The information about the gene structure of DAB2 will enable us to analyze possible mutations and the mechanisms of loss of DAB2 expression in tumors.

Adaptor Proteins, Signal Transducing↗
PubMed1988

Induction of expression of cell-surface homologous restriction factor upon anti-CD3 stimulation of human peripheral lymphocytes.

Homologous restriction factor (HRF) is a 65-kDa membrane protein that inhibits transmembrane channel formation by the membrane-attack complex of complement and by the complement component C9-related cytolytic lymphocyte protein. Stimulation of resting peripheral human lymphocytes with the anti-CD3 monoclonal antibody OKT3 has been shown to induce cytotoxicity in the CD8+ subpopulation. As demonstrated here, OKT3 stimulation also induces expression of cell-surface HRF by CD4+ and CD8+ T lymphocytes. The small proportion of Leu 19+ natural killer lymphocytes present in peripheral blood mononuclear cells was found to express HRF prior to stimulation. Whereas unstimulated peripheral blood mononuclear cells were susceptible to lysis by the membrane-attack complex or by the C9-related protein, OKT3-stimulated peripheral blood mononuclear cells were relatively resistant to both the membrane-attack complex and C9-related protein. This acquired resistance was abrogated by blocking surface HRF with F(ab')2 anti-HRF, suggesting that resistance was due to lymphocyte-membrane HRF. By using solid-phase anti-HRF, a 65-kDa protein was isolated from the activated peripheral blood mononuclear cells and shown to be capable of conferring upon sheep erythrocytes the characteristic activity of human HRF.

Antibodies, Monoclonal↗
PubMed1992

Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.

The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function.

Amino Acid Sequence↗
PubMed1995

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Amino Acid Sequence↗
PubMed1993

Hemolysis of normal human erythrocytes by autologous serum complement.

Unsensitized normal human erythrocytes (E) were shown to be lysed when incubated with autologous serum in the presence of zymosan (Zy). The hemolysis proceeded slowly with a relatively constant rate for at least 24 h at 37 degrees C. It was shown that the hemolytic reaction is antibody independent and mediated by complement activation through the alternative pathway and that hemolysis is not due to the decay or inactivation of complement regulators present on the E membrane. The mechanism of the phenomenon was studied by use of several kinds of sera genetically deficient in C3, C5, C7 or C9. The reaction was found to be divided into two stages: in the first step, neither E, C5, C7 nor C9 but Zy, serum factors containing C3 and metal ions are necessary, and in the second step, neither C3 nor metal ions but E, C5, C7 and C9 are necessary. Thus, E seem to be lysed by reactive lysis induced by C5 convertase formed on Zy through alternative complement pathway activation.

Complement Activation↗
PubMed1976

C3 metabolism in a patient with deficiency of the second component of complement (C2) and discoid lupus erythematosus.

A patient with a hereditary deficiency of the second component of complement and discoid lupus erythematosus with features of systemic lupus erythematosus was studied. The propositus had a 9-year history of rash and arthralgia. Transient renal disease had completely resolved; there was a history of seizures. Examination of his serum disclosed antinuclear antibodies but no total haemolytic complement activity. C2 was absent. Serum concentrations of C1s, C3, C5 and C9 were elevated; other complement components were present in normal concentration, including C3 pro-activator. The patient's C3 pro-activator was electrophoretically converted by inulin and four of five lipopolysaccharides, but was poorly converted by aggregated human IgG. Two separate turnover studies with radiolabelled C3 showed fractional catabolic rates of 3-03 and 2-48% of the remaining plasma pool/hr (range of three normals: 1-62-2-18%/hr); and estimated C3 synthetic rates of 2-74 and 2-31 mg/kg/hr (range of three normals: 0-89-1-40 mg/kg/hr). Serum complement profiles of the patient's family demonstrated that the C2 deficiency was inherited as an autosomal codominant. One sibling, homozygous for C2 deficiency, and three other siblings, both parents and one daughter, all heterozygous for C2 deficiency, are in good health. Immunofluorescent studies of the patient's diseased skin exhibited substantial deposits of IgG, IgM, C1q, and C4 but not of later acting complement components, properdin, or C3 proactivator. These studies do not support the notion that inflammation in C3-deficient individuals with lupus erythematosus is mediated by the alternative complement pathway.

Adult↗
PubMed1988

Molecular mechanisms of lymphocyte-mediated killing.

Lymphocytes kill tumor cells and other target cells by a contact-dependent mechanism. Killer lymphocytes acquire cytoplasmic granules upon lymphokine stimulation and these granules in turn contain potent cytotoxic mediators, some of which have been characterized recently. These include (i) a pore-forming protein (perforin) of 70 kDa that polymerizes into transmembrane tubules in the presence of calcium and that is structurally related to the terminal components (C5b-6, C7, C8 and C9) of the complement cascade, (ii) a cytokine related to tumor necrosis factor and lymphotoxin that causes DNA fragmentation in target cells, and (iii) a family of serine esterases presently without any known function. The relevance of these mediators in lymphocyte-mediated killing is currently being investigated. The identification and characterization of additional putative mediators of cytotoxicity in the future will undoubtedly provide us with a better understanding of the molecular basis of cell-mediated killing.

Antibody-Dependent Cell Cytotoxicity↗