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PubMed2004

Expression of complement factor H by lung cancer cells: effects on the activation of the alternative pathway of complement.

The complement system is important in immunosurveillance against tumors. However, malignant cells are usually resistant to complement-mediated lysis. In this study, we examine the expression of factor H, an inhibitor of complement activation, and factor H-like protein 1 (FHL-1), its alternatively spliced form, in lung cancer. We also evaluate the potential effect of factor H/FHL-1 in the protection of lung cancer cells against the activation of the complement cascade. By Northern blot analysis we demonstrate a high expression of factor H and FHL-1 in most non-small cell lung cancer cell lines, although neuroendocrine pulmonary tumors (small cell lung carcinoma and carcinoid cell lines) had undetectable levels. Western blot analysis of conditioned medium showed the active secretion of factor H and FHL-1 by cells that were positive by Northern blot. Expression of factor H/FHL-1 mRNA was also shown in a series of non-small cell lung cancer biopsies by in situ hybridization. Interestingly, many cultured lung cancer cells were able to bind fluorescence-labeled factor H to their surfaces. Deposition of C3 fragments from normal human serum on H1264, a lung adenocarcinoma cell line, was more efficient when factor H/FHL-1 activity was blocked by specific antibodies. Blocking factor H/FHL-1 activity also enhanced the release of anaphylatoxin C5a and moderately increased the susceptibility of these cells to complement-mediated cytotoxicity. In summary, we demonstrate the expression of factor H and FHL-1 by some lung cancer cells and analyze the contribution of these proteins to the protection against complement activation.

Blood Proteins↗

PubMed2002

Enhancement of the complement activating capacity of 17-1A mAb to overcome the effect of membrane-bound complement regulatory proteins on colorectal carcinoma.

Adjuvant immunotherapy with 17-1A mAb directed against colorectal carcinoma is found to be effective in patients. However, 52 % of the patients treated with mAb 17-1A showed recurrence within 7 years. This high recurrence rate might be due to inhibition of complement activation by membrane-bound complement regulatory proteins (mCRP). The effect of these complement regulatory proteins might be reduced by blocking mCRP, or be overcome by activating more complement at the tumor cell membrane. In this study the complement-activating capacity of the 17-1A mAb was enlarged by conjugating it to cobra venom factor (CVF) or C3b. The most important C3 regulatory protein, CD55, was blocked using a bispecific mAb directed against the 17-1A / Ep-CAM antigen and CD55. Up to a 13-fold increase in C3 deposition was observed due to 17-1A-CVF and 17-1A-C3b, as compared to 17-1A. CD55 was shown to partly inhibit complement activation by these conjugates. The effect of the bispecific anti-17-1A / Ep-CAM*anti-CD55 mAb was compared with 17-1A conjugates with CVF or C3, and bispecific mAb were shown to be equally or more efficient in complement activation than the 17-1A-CVF or 17-1A-C3b conjugates. Therefore, 17-1A conjugates and anti-17-1A / EpCAM*anti-CD55 bispecific mAb may be promising immunotherapeutic agents for patients with colorectal cancer.

Animals↗

PubMed1982

Complement activation in muscle fiber necrosis: demonstration of the membrane attack complex of complement in necrotic fibers.

The membranolytic C5b-9 complement membrane attach complex (MAC) is assembled after activation of either the classic or the alternative complement pathway. The quaternary configuration of the MAC macromolecule presents neoantigenic determinants not present on precursor molecules. Consequently, antibodies specific for these neoantigen(s) do not detect nonspecifically bound native complement precursors of MAC. By means of antibodies rendered specific for MAC neoantigen(s), MAC was localized by the immunoperoxidase reaction in cryostat sections of human muscle. In 66 biopsy specimens containing necrotic muscle fibers (Duchenne dystrophy, 13; other dystrophies, 15; inflammatory myopathies, 31; miscellaneous myopathies, 7) all of the necrotic fibers reacted for MAC neoantigen(s). C3 and C9 were also consistently localized in necrotic fibers, but localization of C1q, C4, and IgG was variable and often did not exceed background staining. None of the nonnecrotic fibers reacted for immunoglobulin or complement. Detection of MAC neoantigen(s) in necrotic fibers in a wide variety of muscle disease unambiguously shows that (1) the lytic complement pathway is consistently activated and participates in muscles fiber necrosis in vivo, and (2) complement reaction products are generated than can stimulate cellular infiltration and phagocytosis of the necrotic fiber. The findings also suggest that cell necrosis in general may involve participation of complement.

Antigens↗

PubMed2006

Complement and complement regulators in the male reproductive system.

Spermatozoa are almost unique among cells in that they must survive transplantation into a foreign host in order to perform their physiological role. The biggest hurdle to overcome is innate immune defence that will target the invaders in the female genital tract. Complement is a major player in innate immunity and is present in the female genital tract. Spermatozoa must therefore evade complement attack if they are to reach their goal. Complement evasion is achieved by the presence of complement regulators both in seminal plasma and on the spermatozoa. Here we discuss the parts played by complement and complement regulators in permitting spermatozoa to survive long enough to reach the oocyte, in clearance of the excess spermatozoa that have outlived their usefulness and in aiding activation of spermatozoa to engage the oocyte. In particular, we focus on the unique distribution patterns of complement regulators on spermatozoa, patterns that strongly suggest roles in spermatozoal development and oocyte binding. An understanding of these roles will inform studies of their contribution to fertility and infertility in man.

Animals↗

PubMed2005

Complement in BuB/BnJ mice revisited: serum C3 levels and complement opsonic activity are not elevated.

With the exception of a few strains such as BuB/BnJ, complement activity in most inbred strains of mice is remarkably similar. The BuB/BnJ strain reportedly has high levels of complement activity and elevated serum levels of C3 and other complement proteins. However, we observed that BuB/BnJ mice have serum C3 levels comparable to those seen in C57BL/6, Balb/c and several other strains of inbred mice. More importantly, using bacteria as a substrate for activation and deposition of complement as a direct biological assay to assess serum complement opsonic activity, we found that BuB/BnJ mice do not have elevated complement activity compared to other inbred mouse strains. In contrast hemolytic assays indicate BuB/BnJ sera to be most active. These results indicate that accepted views regarding mouse serum complement activity need to be re-examined.

Animals↗

PubMed1995

Complement regulation in the rat glomerulus: Crry and CD59 regulate complement in glomerular mesangial and endothelial cells.

The complement regulators, decay accelerating factor, membrane cofactor protein, and CD59 are present in human glomeruli. Crry is the rodent analogue to the former two proteins. In this study, we examined complement regulation in cultured rat glomerular endothelial cells (GEnC) and mesangial cells (MES). Immunoprecipitation of 125I-labeled membrane proteins and Western blotting studies were performed with anti-Crry and anti-CD59. In both GEnC and MES, Crry was present as 53, 65, and 78 kD proteins. The 20 kD CD59 was apparent in GEnC. CD59 was also present in MES, but in relatively smaller quantities. By Northern analyses, 1.8 kb CD59 mRNA was present in GEnC as well as in RNA from isolated rat glomeruli. mRNA for Crry was present in both GEnC and MES as 2.2 kb species. The functional significance of these proteins was evaluated next. Anti-Thy 1.1 IgG was used to activate the complement classical pathway in MES. To inhibit the function of the complement regulators, anti-CD59 and/or anti-Crry F(ab')2 antibodies were added with anti-Thy 1.1. Inhibition of Crry function led to enhanced cytotoxicity, while there was no effect when CD59 function was inhibited. The complement alternative pathway was studied by adding complement in Mg-EGTA buffer. Inhibition of Crry led to productive alternative pathway activation, which was accentuated by anti-CD59 when Crry was incompletely inhibited. Alternative pathway regulation was also evaluated in GEnC. Inhibition of CD59 function alone had no effect in GEnC, while inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. Therefore, Crry is present in both GEnC and MES and restricts the complement alternative pathway in both cell types. Crry also regulates the classical pathway in MES. CD59 is present and functionally active in GEnC, while it appears to have a minor role in MES.

Animals↗

PubMed1995

Enhanced complement susceptibility of avidin-biotin-treated human erythrocytes is a consequence of neutralization of the complement regulators CD59 and decay accelerating factor.

Biotinylation of erythrocytes (E) followed by avidin cross-linking at specific sites has been suggested as a novel means of drug delivery. Upon avidin cross-linking, biotinylated E become complement-activating and highly susceptible to complement lysis, thus bringing about release of entrapped drug. We set out to examine the mechanisms of this biotin-avidin-induced lytic susceptibility, focusing on the effects of biotinylation and avidin cross-linking on the major E complement regulatory molecules, decay accelerating factor (DAF) and CD59. We demonstrate here that biotinylation of E, which does not render them complement activating, partially inhibits DAF but has little effect on CD59. Subsequent cross-linking with avidin causes complete inhibition of DAF and near complete loss of CD59 activity. Following cross-linking, DAF and CD59 become associated in high molecular mass avidin-containing complexes on the membrane. Incorporation of physiological amounts of CD59 into the membranes of biotinylated and avidin cross-linked E is sufficient to render these cells resistant to complement lysis whereas incorporation of DAF has relatively little effect. An understanding of the molecular mechanisms underlying complement susceptibility of biotin-avidin treated E should allow a rational design of strategies for drug delivery using E or other large, potentially complement-activating carriers.

Antibodies↗

PubMed1986

Immobilized doxorubicin increases the complement susceptibility of human melanoma cells by protecting complement component C3b against inactivation.

Human melanoma cells resistant to killing by monoclonal antibody R24 plus human complement became susceptible after treatment with doxorubicin (adriamycin). Treatment with doxorubicin prevented the rapid degradation of surface-bound complement component C3b that has been identified as a protective mechanism of complement-resistant melanoma cells. Doxorubicin caused the increased complement susceptibility as free drug and after immobilization onto glass beads to prevent cellular uptake. Immobilized doxorubicin was more effective than free drug, causing enhanced complement susceptibility at concentrations where the free drug was no longer active. In contrast to free doxorubicin, which exhibited a direct cytotoxic effect leading to cell death within 4 days, immobilized doxorubicin did not affect cell viability. These findings suggest that combination therapy of the complement-activating monoclonal antibody R24 with the complement-enhancing drug doxorubicin may be a promising approach for the treatment of melanoma.

Antibodies, Monoclonal↗

PubMed2005

Characterization of complement receptor 1 domains for prevention of complement-mediated red cell destruction.

BACKGROUND: Complement activation resulting in intravascular hemolysis can cause transfusion-associated mortality. We recently showed that a recombinant soluble form of complement receptor 1 (CR1) effectively reduces complement-mediated red blood cell (RBC) destruction in vitro and more importantly prolongs the survival of transfused human RBCs in mice. To determine CR1-active sites that prevent RBC destruction, structure-function analysis of its extracellular 1930-amino-acid domain has been performed. STUDY DESIGN AND METHODS: Several CR1-truncated soluble proteins were prepared and tested for their ability to prevent complement-mediated RBC destruction in vitro and in mice. RESULTS: A 250-amino-acid region in CR1 that possesses antihemolytic activity and is effective in prolonging survival of transfused RBCs in vivo was identified. Mutation of two critical residues (D109N and E116K) in this 250-amino-acid domain, previously shown to improve complement-inhibitory functions of CR1 derivatives, resulted in a more potent inhibition of complement activation in vitro. In vivo, however, the activity of mutant proteins was comparable to the wild-type molecules. CONCLUSION: Our structure-function studies have characterized smaller CR1-based complement inhibitors for future development of structure-derived transfusion therapeutics. Our studies underscore the importance of testing CR1 inhibitors in vivo.

Animals↗

PubMed1972

Role of antibody and complement in the immune clearance and destruction of erythrocytes. II. Molecular nature of IgG and IgM complement-fixing sites and effects of their interaction with serum.

A model for the immune clearance and destruction of homologous erythrocytes has been further explored. In this model, every IgM anti-erythrocyte antibody molecule in an antibody preparation was shown to fix Cl. About 2000 IgG antibody molecules were required to form a Cl-fixing site on the guinea pig erythrocyte surface. 60 IgM complement-fixing sites per erythrocyte were required for the immune clearance of IgM-sensitized erythrocytes. This number of sites could be detected by a direct agglutination test. 1.4 complement-fixing sites were required for immune clearance of IgG-sensitized cells, a number of molecules which could not be detected by direct agglutination. This number could, however, be detected with the use of a Coombs antiglobulin reagent. Depletion of the late components of complement (C3-9) with cobra venom was associated with the loss of ability to clear IgM-sensitized cells and a marked deficit in the ability to clear IgG-coated cells. Thus, late (C3-9) components of complement as well as an early component (C4) were required for normal clearance of sensitized erythrocytes. There was no evidence that activation of the alternate pathway of complement action could lead to accelerated erythrocyte clearance. In vitro incubation of IgG and IgM-sensitized erythrocytes in fresh serum led to deposition of C3 and C4 on the erythrocyte surface. IgM-sensitized cells treated in this way had a normal survival. IgM-sensitized cells also were shown to remain Coombs positive after their release from the liver. The evidence suggests that the interaction of an IgM site with fresh serum in vitro and in vivo leads to formation of a site which allows for sequestration of cells in the liver. With continued exposure to serum components, this site is destroyed or inactivated. This serum-dependent inactivation is complement-dependent as shown by the use of EDTA-treated and C4-deficient serum. IgG complement-fixing sites are only partially inactivated by incubation in fresh serum, further emphasizing the differences in the biologic activity of IgM and IgG antibodies.

Animals↗

PubMed1989

Differential susceptibility of haemolysin-sensitized erythrocytes to complement-mediated cytotoxicity: a biological manifestation of the relationship between antibody availability and complement fixation.

The influence of the concentration of membrane-bound antibody on the susceptibility of individual human erythrocyte populations to complement-mediated haemolysis has been examined using cold competition analysis. It was observed that under conditions where complement is limiting, the relative susceptibility of erythrocytes to complement lysis is dependent on the availability of sensitizing antibody, even when all target red cells possess sufficient membrane antibody to permit maximal lysis in the presence of excess complement. Furthermore, preliminary evidence is presented which suggests that, where the availability of complement is limiting, sensitized blood group A erythrocytes are more susceptible to complement lysis than are sensitized group B cells. Taken together the data indicate that the quantity, and possibly the distribution, of the membrane antibody dictates the relative susceptibility of erythrocytes to complement haemolysis.

ABO Blood-Group System↗

PubMed1992

Assessment of disease activity and impending flare in patients with systemic lupus erythematosus. Comparison of the use of complement split products and conventional measurements of complement.

OBJECTIVE: To determine whether increased levels of the complement split products generated in the activation of the alternative or classical pathway accompany more severe disease activity in patients with systemic lupus erythematosus (SLE) and whether these measurements are useful in predicting flares of disease. METHODS: Levels of Ba, Bb, SC5b-9, and C4d were measured in 380 plasma samples obtained from 86 SLE patients who were prospectively followed up for 15 months. RESULTS: In the 20 patients who had inactive disease at the initiation of the study, the mean values of all of the complement split products at entry were within the normal range. In the 47 patients with stable or moderate disease activity, levels of Ba were significantly increased, while the mean values for Bb, SC5b-9, and C4d did not differ significantly from those in patients with inactive disease. The mean entry value of each analyte was highest in the group of 19 patients who had the most severe disease activity at initial evaluation. Traditional measurements of complement, i.e., C3, C4, and CH50, followed similar trends, but did not discriminate between the 3 groups of patients as well as did measurements of the split products. Analysis of the disease course in the patients with inactive or stable/moderate disease revealed that an elevated level of C4d had the most sensitivity with regard to subsequent flare, while an elevated Bb level had the highest specificity and the greatest predictive value. CONCLUSION: These data suggest that elevated levels of complement split products, particularly products of alternative and terminal pathway activation, more accurately reflect disease activity than do conventional measurements of complement in SLE and may be useful in the prediction of impending disease flares.

Complement C3 Convertase, Alternative Pathway↗

PubMed1992

Regulation of complement activity by vaccinia virus complement-control protein.

A major protein secreted by vaccinia virus-infected cells has structural similarity to the super-family of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.

Complement Activation↗

PubMedDate not supplied

In vitro activation of human complement by polyacrilonitrile dialyzer membrane assayed by complement binding to bystander red cells.

Quantitation of complement activation by polyacrilonitrile (PAN) dialyzer membrane is complicated by the high adsorptive capacity of the membrane for fluid phase anaphylotoxins. Assays for these anaphylotoxins, therefore, underestimate the degree of complement activation produced by this membrane. Alternative methods of measuring in vitro complement activation by the PAN and Cuprophan membranes were explored by incubating normal human erythrocytes with the membranes in the presence of serum. This led to deposition of C3d on these "innocent bystander" red cells, and provided an independent parameter for measuring complement activation. The PAN membrane caused significantly more C3d deposition on red cells, and thus more complement activation than Cuprophan. The possible significance of complement activation by PAN membrane, in consideration of its property of binding the resultant anaphylotoxins, is discussed.

Acrylic Resins↗

PubMed1989

Complete primary structure and functional characterization of the sixth component of the human complement system. Identification of the C5b-binding domain in complement C6.

Complement C6 is one of five plasma proteins that are incorporated into the lytic terminal complement complex on lipid membranes (C5b-9m) upon activation of the complement cascade. Oligonucleotide probes derived from partial amino acid sequences of purified C6 were used to isolate cDNA clones from a human liver cDNA library. The complete polypeptide structure of mature C6 deduced from the cDNA sequence consists of 913 amino acid residues preceded by a typical 21-residue signal peptide. C6 is most similar in structure to complement C7, sharing 33.5% identical residues with C7 including all 56 cysteine residues. The low density lipoprotein receptor class A and B modules, the thrombospondin type I module at the carboxyl terminus, and the two short consensus repeat modules are arranged in the same way as in C7. In contrast to C7 and other terminal complement proteins, the thrombospondin type I module at the amino terminus occurs as a tandem repeat in C6. The last tandem repeat at the carboxyl terminus of C6 and C7 has been identified as a new distinct module (factor I module), which is closely related to a segment in the heavy chain of complement control factor I. Binding studies with filter-bound C6 fragments generated by proteolysis showed that the C5b-binding domain of C6 was located in the 34-kDa carboxyl terminal fragment consisting of two short consensus repeats and two factor I modules.

Amino Acid Sequence↗

PubMed1994

Complement system activation during orthotopic liver transplantation in man. Indications of peroperative complement system activation in the gut.

Sixteen patients with acute and chronic liver disease undergoing OLT were studied regarding the role of the liver and the gut in complement activation. Also, the relation between complement activation and clinical manifestations during the liver transplantation reperfusion period was investigated. Blood samples for measurement of complement anaphylatoxin C3a (C3a), complement anaphylatoxin C5a (C5a), and terminal C5b-9 complement complex (TCC) were taken simultaneously from the central venous catheter and the radial arterial line before starting the operative procedure, 1 min before declamping, and 1-2 min, 5 min, 30 min and 6-12 hr after declamping. Simultaneous blood sampling from the radial arterial line, central venous catheter, portal vein, and hepatic vein was performed 1-2 min and 5 min after completed unclamping. Elevated plasma levels of C3a and TCC were found upon reperfusion, while C5a levels remained unchanged throughout the operation compared with the preoperative levels. The levels of C3a in the portal vein were higher compared with the levels in the simultaneously obtained samples from the radial artery. The results indicate complement cascade activation located to the gut during the reperfusion phase of OLT. Seventy-five percent of the patient studied suffered from the postreperfusion syndrome, indicated by profound hypotension upon reperfusion of the transplanted liver. There was a significant correlation between high concentration of C3a anaphylatoxin and development of profound hypotension.

Adult↗

PubMed2006

Kaposi's sarcoma-associated herpes virus complement control protein: KCP--complement inhibition and more.

The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure, as well as priming the adaptive immune response through opsonisation, leukocyte recruitment and enhancing humoral immune responses. Its importance is not only shown through recurring fulminant infections in individuals with complement component deficiencies, but also through the many complement evasion strategies discovered for a wide range of infectious microbes (including acquisition of endogenous host complement inhibitors and expression of own homologues). Knowledge of these mechanisms at a molecular level may aid development of vaccines and novel therapeutic strategies. Here, we review the structure-function studies of the membrane-bound complement inhibitor KCP that is expressed on the surface of Kaposi's sarcoma-associated herpesvirus (KSHV) virions and infected cells. KCP accelerates the decay of classical C3 convertase and induces the degradation of activated complement factors C4b and C3b by a serine proteinase, factor I. Molecular modeling and site-directed mutagenesis have identified sites on the surface of KCP required for complement inhibition and support the hypothesis that KCP has evolved to mimic the structure and function of endogenous human inhibitors. KCP additionally enhances virion binding to permissive cells through a heparin/heparan sulfate-binding site located at the N-terminus of the protein.

Amino Acids↗

PubMed1996

The role of serum complement on the organ distribution of intravenously administered poly (methyl methacrylate) nanoparticles: effects of pre-coating with plasma and with serum complement.

PURPOSE: The organ distribution of radiolabeled poly (methyl methacrylate) (PMMA) nanoparticles coated with plasma proteins and serum complement in rats was studied in order to determine the effect of serum complement on the particle phagocytosis by the organs of the reticulo-endothelial system (RES). METHODS: PMMA-nanoparticles were coated overnight with plasma proteins or serum complement, and injected into Wistar rats. The body distribution of nanoparticles was measured by means of scintillation counting of organ samples. In addition, proteins adsorbed to the particle surface were inactivated by heat treatment prior to injection, and the particle's distribution was measured as described above. RESULTS: Whereas uncoated nanoparticles (control group) were mainly taken up by the Kupffer cells in the liver, incubation of the particles in plasma for 12 h followed by heat inactivation reduced the particle concentrations in the liver to merely 22% after 30 min. After 120 min, liver concentrations were still lower than the control group, and almost 30% of the administered dose of the heat-inactivated particle group was present in non-RES organs and tissues. Particles with non-inactivated complement were accumulated in the lung at concentrations of 29% after 30 min, which increased to 71% after 120 min, whereas those coated with inactivated complement reached lung concentrations above 70% already after 30 min. CONCLUSIONS: Particles coated with plasma components are able to avoid uptake by the RES, especially after heat inactivation of the plasma components adsorbed. Adsorption and heat inactivation of complement proteins alone, however, does not have the same result as coating with plasma proteins followed by heat inactivation. Therefore, it is concluded that plasma components other than complement proteins take part in the process of RES activation and phagocytosis of injected nanoparticles.

Animals↗