RESUMEN
Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood.
Asunto(s)
Anopheles/inmunología , Activación de Complemento/inmunología , Factor H de Complemento/inmunología , Evasión Inmune/inmunología , Mucosa Intestinal/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Convertasas de Complemento C3-C5/biosíntesis , Convertasas de Complemento C3-C5/inmunología , Complemento C3b/inmunología , Sistema Digestivo/inmunología , Humanos , Insectos Vectores/inmunología , Mucosa Intestinal/parasitología , Estadios del Ciclo de Vida , Receptores de Complemento/inmunologíaRESUMEN
Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.
Asunto(s)
Convertasas de Complemento C3-C5/biosíntesis , Complemento C3a/genética , Complemento C3a/metabolismo , Complemento C3b/genética , Complemento C3b/metabolismo , Síndrome Hemolítico-Urémico/inmunología , Animales , Síndrome Hemolítico Urémico Atípico , Células COS , Chlorocebus aethiops , Activación de Complemento , Convertasas de Complemento C3-C5/genética , Convertasas de Complemento C3-C5/metabolismo , Complemento C3a/biosíntesis , Complemento C3b/biosíntesis , Complemento C9/biosíntesis , Complemento C9/metabolismo , Factor B del Complemento/metabolismo , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento/genética , Glomerulonefritis/genética , Síndrome Hemolítico-Urémico/genética , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Several recent studies have shown that interacting antigen presenting cells and/or T cells produced complement activation products C5a and C3a, are integrally involved in T-cell activation, and promote the generation of myelin oligodendrocyte glycoprotein (MOG(35-55))-specific interferon-gamma and interleukin-17-producing T cells in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. In this study, we tested whether FUT-175, a clinical pharmaceutical that has been shown to inhibit the formation of C3/C5 convertases, can attenuate myelin-specific T-cell responses, as well as disease severity in experimental autoimmune encephalomyelitis. In vitro, FUT-175 inhibited local C5a/C3a production by antigen presenting cell-T-cell complexes and attenuated MOG(35-55)-specific Th1 and Th17 responses with little nonspecific cytotoxicity. In vivo administration of FUT-175 delayed experimental autoimmune encephalomyelitis disease onset, lowered clinical scores, decreased central nervous system inflammation, and reduced demyelination. The FUT-175-treated mice exhibited decreased numbers of MOG(35-55)-specific interferon-gamma- and interleukin-17-producing T cells. In addition, results from the FUT-175 treatment of naive recipients of adoptively transferred splenocytes from MOG(35-55)-immunized mice suggested that the effect of FUT-175 was on MOG-specific cellular responses and not on anti-MOG antibodies. These results argue that complement regulators, which inhibit C5a/C3a production, may have therapeutic efficacy in multiple sclerosis and in other clinical conditions in which T cells drive disease pathogenesis.
Asunto(s)
Convertasas de Complemento C3-C5/antagonistas & inhibidores , Encefalomielitis Autoinmune Experimental/inmunología , Guanidinas/administración & dosificación , Inhibidores de Serina Proteinasa/farmacología , Linfocitos T/efectos de los fármacos , Animales , Benzamidinas , Convertasas de Complemento C3-C5/biosíntesis , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Guanidinas/farmacología , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunologíaRESUMEN
C3 is a central protein of the complement system, which is important to immune defense and provides a link between innate and adaptive immunity. Three pathways of complement activation converge at the activation of C3 yielding a diverse set of biological responses. This versatile and flexible molecule interacts with various proteins to fulfill its functions. Here we review recent insights gained from the crystal structure determinations of human, native C3 and its physiological down-regulation product C3c. The data provided, for the first time, a complete and detailed view of the composition and arrangement of the domains in C3. Comparison of C3 with C3c indicates marked flexibility of the molecule, particularly in the alpha-chain. We discuss the observed domain rearrangements, conformational changes and the location of various protein binding sites. These detailed, and structural, insights are important for developing models of the molecular mechanisms underlying the diverse biological activities of this large and complex molecule.
Asunto(s)
Complemento C3/química , Sitios de Unión , Complemento C3/metabolismo , Convertasas de Complemento C3-C5/biosíntesis , Complemento C3b/química , Humanos , Estructura Terciaria de ProteínaRESUMEN
The complement system is an important part of the innate immune system. Three pathways, the classical, the alternative and the lectin pathway, lead to the cleavage of complement factor C3, a central event in the activation of the complement system. We investigated the deposition of C3b (solid-phase C3 activation product) on a mannan-coated surface at high concentration of human serum (17%). At these conditions, mannan-binding lectin (MBL) promoted the activation of C3 through the combined action of MBL-associated serine protease (MASP)-1 and MASP-2 without appreciable involvement of the alternative pathway. In serum depleted of MASP-1, MASP-2 and MASP-3, we observed synergetic effect of reconstitution with MASP-1 and MASP-2. This was inhibited by MASP-3. No C3b deposition was observed with C2- or C4-depleted serum. Depletion of factor B had no effect on the MBL-MASP-promoted C3b deposition. Our results demonstrate a function of the orphan protease MASP-1 by providing evidence that this enzyme collaborates with MASP-2 in the generation of C3 convertase, a process observable at high serum concentration, but not at low serum concentration.
Asunto(s)
Activación de Complemento/fisiología , Convertasas de Complemento C3-C5/biosíntesis , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Animales , Complemento C3b/metabolismo , Humanos , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Normal human IgG contains naturally occurring anti-C3 antibodies (anti-C3 NAbs) that have been proposed to regulate complement amplification. Here, we report a novel procedure for anti-C3 NAb purification. Pooled human IgG was fractionated on a DEAE column prior to affinity chromatography on IgG and then on C3. Anti-C3 NAbs co-purified with anti-F(ab')2 NAbs. In a refined protocol, IgG fractions were absorbed on Fc, F(ab')2, and C3, which allowed to isolate the directly accessible NAbs and to remove IgG hinge-region-specific NAbs. Since a substantial fraction of total anti-C3 NAbs in whole IgG pre-existed as complexes, IgG that did not bind to the three affinity columns was treated with urea and the affinity chromatography repeated to collect the dissociated NAbs. The urea-accessible anti-F(ab')2 NAbs were rather pure but anti-C3 NAbs yet contained substantial amounts of anti-F(ab')2 NAbs. Anti-C3 NAbs showed up to 400-fold and anti-F(ab')2 NAbs, up to 30-fold enrichment as compared to pooled normal human IgG. Anti-C3 NAb preparations exhibited nephritic factor activity that was up to 60 times stronger than that of total IgG from a patient with membranoproliferative glomerulonephritis type 2. In addition, anti-C3 NAbs promoted C3 convertase generation, when added to the convertase precursor or during convertase assembly, suggesting a non-nephritic-factor mechanism. Factors H and I reduced the overall level of activity but had no influence on the NAb dose-response curve meaning that NAbs did not interfere with factor H binding. Convertase promoting activity during assembly correlated with the content of anti-C3 NAbs in NAb complexes. In conclusion, anti-C3 NAbs associated with framework-specific anti-idiotypic NAbs stabilize C3 convertase and promote its generation but their activity is compensated for in whole IgG.
Asunto(s)
Convertasas de Complemento C3-C5/metabolismo , Inmunoglobulina G/metabolismo , Anticuerpos Antiidiotipos/aislamiento & purificación , Anticuerpos Antiidiotipos/metabolismo , Complemento C3/antagonistas & inhibidores , Complemento C3/inmunología , Complemento C3/metabolismo , Convertasas de Complemento C3-C5/biosíntesis , Factor H de Complemento/metabolismo , Factor H de Complemento/farmacología , Vía Alternativa del Complemento , Estabilidad de Enzimas , Fibrinógeno/metabolismo , Fibrinógeno/farmacología , Humanos , Inmunidad Innata , Inmunoglobulina G/aislamiento & purificación , Técnicas In VitroRESUMEN
Diamond-like carbon (DLC) and silicon carbide (SiC) coatings are attractive because of low friction coefficient, high hardness, chemical inertness and smooth finish, which they provide to biomedical devices. Silicon wafers (Si(waf)) and silicone rubber (Si(rub)) plates were coated using plasma-enhanced chemical vapour deposition (PE-CVD) techniques. This article describes: 1- the characterization of modified surfaces using attenuated total reflection-Fourier transform infrared spectroscopy (ATR/FTIR) and contact angle measurements, 2- the results of three in-vitro haemocompatibility assays. Coated surfaces were compared to uncoated materials and various substrates such as polymethylmethacrylate (PMMA), polyethylene (LDPE), polydimethylsiloxane (PDMS) and medical steel (MS). Thrombin generation, blood platelet adhesion and complement convertase activity tests revealed the following classification, from the most to the least heamocompatible surface: Si(rub)/ DLC-Si(rub)/ DLC-Si(waf)/ LDPE/ PDMS/ SiC-Si(waf)/ Si(waf)/ PMMA/ MS. The DLC coating surfaces delayed the clotting time, tended to inhibit the platelet and complement convertase activation, whereas SiC-coated silicon wafer can be considered as thrombogenic. This study has taken into account three events of the blood activation: coagulation, platelet activation and inflammation. The response to those events is an indicator of the in vitro haemocompatibility of the different surfaces and it allows us to select biomaterials for further in vivo blood contacting investigations.
Asunto(s)
Compuestos Inorgánicos de Carbono , Carbono , Materiales Biocompatibles Revestidos , Diamante , Compuestos de Silicona , Materiales Biocompatibles/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Carbono/sangre , Carbono/farmacología , Compuestos Inorgánicos de Carbono/sangre , Compuestos Inorgánicos de Carbono/farmacología , Materiales Biocompatibles Revestidos/farmacología , Convertasas de Complemento C3-C5/biosíntesis , Diamante/sangre , Diamante/farmacología , Inducción Enzimática/efectos de los fármacos , Humanos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Compuestos de Silicona/sangre , Compuestos de Silicona/farmacología , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie/efectos de los fármacos , Trombina/biosíntesisRESUMEN
Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.
Asunto(s)
Convertasas de Complemento C3-C5/biosíntesis , Proteínas Inactivadoras del Complemento C3b , Complemento C4b , Proteínas Inactivadoras de Complemento , Eritrocitos/metabolismo , Fibrinógeno/metabolismo , Glicoproteínas/metabolismo , Zinc/farmacología , Animales , Proteínas Sanguíneas , Células Cultivadas , Complemento C3b/química , Complemento C3b/metabolismo , Factor H de Complemento , Vía Clásica del Complemento/efectos de los fármacos , Vía Clásica del Complemento/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Eritrocitos/efectos de los fármacos , Fibrinógeno/química , Glicoproteínas/química , Ovinos , Zinc/químicaRESUMEN
Complement convertases are bimolecular complexes expressing protease activity only against C3 and C5. Their action is necessary for production of the biological activities of the complement system. Formation of these complexes proceeds through sequential protein-protein interactions and proteolytic cleavages of high specificity. Recent structural, mutational and functional data on factors D and B have significantly enhanced our understanding of the assembly, action, and regulation of the alternative pathway convertase. These processes were shown to depend critically on conformational changes, only some of which are reversible. The need for such changes is dictated by the zymogen-like configurations of the active centers of these unique serine proteases. The structural determinants of some of these changes have been defined from structural and mutational analyses of the two enzymes. Transition of factor D from the zymogen-like to the catalytically active conformation is completely reversible, while the active conformation of the catalytic center of the Bb fragment of factor B is irreversibly attenuated to a great extent on dissociation of the convertase complex. Both mechanisms contribute to the regulation of the proteolytic activity of these enzymes. Additional studies are necessary for a complete description of the elegant mechanisms mediating these processes.
Asunto(s)
Convertasas de Complemento C3-C5/biosíntesis , Convertasas de Complemento C3-C5/química , Factor D del Complemento/química , Vía Alternativa del Complemento , Animales , Sitios de Unión , Dominio Catalítico , C3 Convertasa de la Vía Alternativa del Complemento , Convertasas de Complemento C3-C5/fisiología , Complemento C3b/inmunología , Complemento C3b/metabolismo , Factor B del Complemento/metabolismo , Factor D del Complemento/fisiología , Activación Enzimática , Precursores Enzimáticos/química , Ratones , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Serina Endopeptidasas/química , Especificidad por Sustrato , Factor de von Willebrand/químicaRESUMEN
Mannan-binding lectin (MBL) is an acute phase protein which activates the classical complement pathway at the level of C4 and C2 via two novel serine proteases homologous to C1r and C1s. We recently reported that haemolysis via this lectin pathway requires alternative pathway amplification. The present experiments sought to establish the basis for this requirement, and hence focused on the activity and regulation of the C3 convertases. Complement activation was normalized between the lectin and classical pathways such that identical amounts of bound C4 and of haemolytically active C4,2 sites were present on the indicator cells. Under these conditions, there was markedly less haemolysis, associated with markedly less C3 and C5 deposited, via the lectin pathway than via the classical pathway, particularly when alternative pathway recruitment was blocked by depletion of factor D. Lectin pathway activation was associated with enhanced binding in the presence of MBL of complement control proteins C4bp and factor H to C4b and C3b, respectively, with decreased stability of the C3-converting enzyme C4b,2a attributable to C4bp. Immunodepletion of C4bp and/or factor H increased lectin pathway haemolysis and allowed lysis to occur in absence of the alternative pathway. Thus, the lectin pathway of humans is particularly susceptible to the regulatory effects of C4bp and factor H, due at least in part to MBL enhancement of C4bp binding to C4b and factor H binding to C3b.
Asunto(s)
Proteínas Portadoras/inmunología , Factor H de Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Antígenos de Histocompatibilidad/inmunología , Lectinas/inmunología , Mananos/inmunología , Animales , Colectinas , Convertasas de Complemento C3-C5/biosíntesis , Complemento C3b/inmunología , Complemento C4/inmunología , Proteína de Unión al Complemento C4b , Complemento C5/inmunología , Complemento C5b , Inducción Enzimática , Eritrocitos/inmunología , Hemólisis , Humanos , Properdina/inmunología , OvinosRESUMEN
Complement-mediated mode of action of bisbenzylisoquinoline alkaloid fangchinoline was investigated in vivo and in vitro. The application of fangchinoline intraperitoneally (i.p.) to complement normal mice, strain ICR, inhibited the complement activity in serum and peritoneal exudate. The substance activated serum complement of C5-deficient DBA/2 mice. Fangchinoline was able to provoke local inflammatory reaction in both strains after subcutaneous (s.c.) injection. The alkaloid suppressed paw swelling induced by live Candida albicans in ICR and DBA/2 mice. Its effect depended on the dose and time of injection prior to inflammatory reaction. The in vitro experiments proved the interference of fangchinoline action with post-C5 reactions. The substance augmented C5-convertase formation and functional activity. These results are in correspondence with our previous investigations, proving the complement-mediated action of fangchinoline. The antiinflammatory effect could be a consequence of the caused complement exhaustion.
Asunto(s)
Alcaloides/farmacología , Bencilisoquinolinas , Proteínas del Sistema Complemento/inmunología , Medicamentos Herbarios Chinos/farmacología , Alcaloides/administración & dosificación , Alcaloides/uso terapéutico , Animales , Candida albicans/fisiología , Convertasas de Complemento C3-C5/biosíntesis , Convertasas de Complemento C3-C5/deficiencia , Convertasas de Complemento C3-C5/metabolismo , Proteínas del Sistema Complemento/metabolismo , Medicamentos Herbarios Chinos/administración & dosificación , Medicamentos Herbarios Chinos/uso terapéutico , Ácido Edético/farmacología , Hemólisis/efectos de los fármacos , Miembro Posterior/efectos de los fármacos , Miembro Posterior/microbiología , Miembro Posterior/patología , Técnicas In Vitro , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/microbiología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Fitoterapia , Plantas MedicinalesAsunto(s)
Humanos , Complejo Antígeno-Anticuerpo , Proteínas del Sistema Complemento/inmunología , Activación de Complemento/fisiología , Complemento C1s/ultraestructura , Convertasas de Complemento C3-C5/biosíntesis , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Proteínas Opsoninas/inmunología , Properdina/fisiologíaRESUMEN
Thermal injury quantitatively and qualitatively alters hematopoiesis, including monocyte-macrophage lineage changes, resulting in altered mononuclear cell function. These bone marrow cells (BMCs) ultimately become fixed tissue macrophages (e.g., Kupffer cells). To study the effects of thermal injury on macrophage-hepatocyte interactions, rat BMCs were isolated 24 hours after burn injury, and myelopoiesis was induced by 7-day culture in granulocyte-macrophage colony-stimulating factor. Separate cultures included inflammatory mediators with growth factor function (IL-6 or PGE2). Cultured cells were incubated up to 96 hours with isolated normal hepatocytes (+/- lipopolysaccharide stimulation). The 96-hour exposure to postburn BMCs produced less of the acute phase proteins (APPs), C3 and transferrin, but more cytotoxicity as measured by 1-lactate dehydrogenase release. Sham BMCs cultured with added IL-6 caused higher APP release and minimal cytotoxicity, whereas burn BMCs stimulated lower APP release and retained cytotoxicity. In conclusion, myeloid cells regulate APP synthesis differently after thermal injury and may become more cytotoxic to hepatocytes.
Asunto(s)
Células de la Médula Ósea/fisiología , Quemaduras/inmunología , Citotoxicidad Inmunológica/fisiología , Hematopoyesis/fisiología , Macrófagos del Hígado/fisiología , Proteínas de Fase Aguda/biosíntesis , Animales , Quemaduras/fisiopatología , Comunicación Celular , Diferenciación Celular , Convertasas de Complemento C3-C5/biosíntesis , Técnicas In Vitro , Hígado/citología , Hígado/fisiopatología , Masculino , Ratas , Ratas Endogámicas ACI , Transferrina/biosíntesisRESUMEN
Recent identification of a C3-like gene in sea urchins revealed the presence of a complement system in invertebrates. To elucidate further the components and function of the pre-vertebrate complement system, we attempted to isolate an ascidian (urochordata) C3 convertase. After identification of C3 cDNA from Halocynthia roretzi, a Japanese ascidian, reverse transcriptase-PCR amplification of hepatopancreas RNA was performed using primers encoding highly conserved amino acid sequences of the vertebrate Bf and C2 serine protease domain. Two candidate sequences were identified, and the corresponding cDNA clones were isolated from a hepatopancreas library. Surprisingly, neither clone is related to Bf/C2 but rather share the same domain structure of mammalian C1r/C1s/MASP (mannan binding protein-associated serine protease), and are more related evolutionarily to mammalian MASP than to mammalian C1r or C1s. The identification of the tunicate MASP clones, amplified with primers designed to amplify Bf or C2, suggests that the lectin pathway antedated the classical and alternative pathways of complement activation.
Asunto(s)
Evolución Biológica , Proteínas Portadoras/biosíntesis , Convertasas de Complemento C3-C5/biosíntesis , Serina Endopeptidasas/biosíntesis , Urocordados/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Clonación Molecular , Colectinas , Complemento C3/química , Convertasas de Complemento C3-C5/química , Convertasas de Complemento C3-C5/inmunología , Cartilla de ADN , ADN Complementario , Evolución Molecular , Mamíferos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Serina Endopeptidasas/inmunología , Transcripción Genética , Urocordados/genética , Urocordados/inmunologíaRESUMEN
The ability to form a covalent dimer of human C4b was investigated with purified isotypes C4A and C4B, and antibody-sensitized liposomes supplemented with C1. In this system, no C4A or C4B formed a complex with the antibody or C1. Whereas both C4A and C4B isotypes formed dimers to a similar extent, C4B formed an ester-linked dimer and C4A an amide-linked dimer. Both of these dimers served as a subunit for the C3-bypass pathway C5 convertase, since liposomes bearing Ab, C1 and a dimer of C4A or C4B, allowed the formation of C5 convertase by the addition of C2. The degree of complement-mediated liposome lysis however, was observed to be 2-3 times higher in the C4B-bearing particles than in those bearing C4A. These results indicate that the second C4b-binding site on the first C4b is different between C4A and C4B, and that in the C3-bypass pathway, C4B has a higher degree of hemolytic activity than C4A, as in the conventional classical complement pathway.
Asunto(s)
Convertasas de Complemento C3-C5/biosíntesis , Complemento C4a/fisiología , Complemento C4b/fisiología , Ensayo de Actividad Hemolítica de Complemento/métodos , Biopolímeros/fisiología , Complemento C3/fisiología , Complemento C4a/metabolismo , Complemento C4b/metabolismo , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Humanos , LiposomasRESUMEN
Antibody independent activation of complement C1s was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster C1s L chain. Approx. 50% of C1s secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active C1s was found in a culture medium of hamster embryo fibroblasts (HEF). Active C1s was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of C1s was also observed in rat cell lines but not in secondary rat embryo fibroblasts. C1s in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that C1s was activated after secretion. The activation of C1s was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that C1s is activated either by a serine proteinase(s) other than those reported to cleave C1s or by an activator which directly stimulates autoactivation of C1s.
Asunto(s)
Activación de Complemento , Complemento C1s/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Línea Celular , Activación de Complemento/efectos de los fármacos , Complemento C1s/inmunología , Convertasas de Complemento C3-C5/biosíntesis , Cricetinae , Medio de Cultivo Libre de Suero , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Fibroblastos/metabolismo , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacologíaRESUMEN
It has been accepted that decay-accelerating factor (DAF) and membrane cofactor protein (MCP) on human cells block C3 deposition, thereby preventing homologous complement attack. In this study, we discovered that a human myeloid cell line, P39, was a target for human C3 even though it expressed normal DAF and MCP. This homologous C3 deposition was induced by serum containing Mg2+ and EGTA (Mg(2+)-EGTA serum) selectively on a P39 subline [P39(+)cells] having the capacity to form cell aggregates. Another P39 subline [P39(-)cells] growing as a separated form did not induce homologous C3 deposition. Multiple C3 fragments, C3b and C3bi, were fixed on P39(+) cells and a significant amount of C5a was released. Several distinct C3 fragment-membrane acceptor molecule complexes were immunoprecipitated with anti-C3c antibody from surface-labeled P39(+) cells treated with Mg(2+)-EGTA serum and from unlabeled cells incubated with 125I-labeled C3 and Mg(2+)-EGTA serum. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 90, 60 and < or = 40 kDa C3 acceptors on P39(+) cells. On these cells, some of the C3 bound to these acceptors remained in the form of C3b, which can form C3/C5 convertases. P39(+) cells differed phenotypically from P39(-) cells in that P39(+) cells expressed intercellular adhesion molecule-1 (ICAM-1), whereas P39(-) cells did not. Both cells were lymphocyte function-associated antigen 1 (LFA-1)+/CR3- counter-receptors for ICAM-1. However, homotypic cell adhesion was not completely inhibited by antibodies against ICAM-1 and LFA-1, suggesting that the homotypic cell aggregation of P39(+) cells is due only in part to ICAM-1 and LFA-1. In addition, C3 deposition, the expression of ICAM-1, and cell aggregation were enhanced by both tumor necrosis factor-alpha and interferon-gamma. Although the principal causative mechanisms remain obscure, C3 deposition and cell adhesion appear in parallel in this cell line and may be involved in the modulation of cell-mediated immune reactions.
Asunto(s)
Adhesión Celular , Complemento C3/metabolismo , Moléculas de Adhesión Celular/análisis , Agregación Celular , Línea Celular , Convertasas de Complemento C3-C5/biosíntesis , Electroforesis en Gel Bidimensional , Humanos , Molécula 1 de Adhesión Intercelular , Antígeno-1 Asociado a Función de Linfocito/análisis , Proteínas de la Membrana/análisisRESUMEN
C3 nephritic factor (NEF) is an IgG autoantibody directed against neoantigenic determinants of the alternative C3 convertase (C3b.Bb). Structural and functional studies require important amounts of this antibody, which are difficult to obtain from patients' sera. We have developed a method for increasing NEF production in vitro. Epstein-Barr virus is a herpes virus which transforms B lymphocytes. Some authors were able to induce the production of NEF in vitro after infection with Epstein-Barr virus (EBV). These works were preformed without any previous cellular selection of B cells. We have performed a method of preselecting antigen-binding cells prior to EBV transformation. Non-preselected cells yielded 0.16 U/million cells in culture (U/M) of NEF antibody, whereas enriched cells for NEF antibody in eliminated 8 U/M (sheep erythrocytes coated with anti-IgG, A, M). Specific NEF synthesis can be increased, in peripheral blood mononuclear cells (PBMC) from patients by in vitro stimulation with the antigens recognized by NEF [C3b.Bb, 21,000 MW protein from patients' E membranes and 26,000 MW protein from sheep E membranes (ShE)]. The highest stimulation is induced by the C3b.Bb and by 26,000 MW protein, 21,000 MW protein had lowest stimulatory effect. In this work also we have shown that patients having NEF antibody in sera have an increase of the CD5-CD19 subset, when compared with the controls.
Asunto(s)
Factor Nefrítico del Complemento 3/biosíntesis , Glomerulonefritis/inmunología , Antígenos CD/análisis , Linfocitos B/inmunología , Transformación Celular Viral , Convertasas de Complemento C3-C5/biosíntesis , Convertasas de Complemento C3-C5/inmunología , Herpesvirus Humano 4 , HumanosRESUMEN
A C intermediate, LAC14, was prepared from TNP-aminocaproyl liposomes sensitized with anti-TNP antibody (Ab) and purified human C1 and C4. LAC14, containing radiolabeled C4, was analyzed by SDS-PAGE followed by autoradiography, and yielded a 210-kDa band and a predominant 400-kDa band. The 210-kDa band consisted of monomeric C4b bound to low molecular mass acceptors. The 400-kDa band was comprised of a 200-kDa moiety, as well as beta- and gamma-chains of C4. The 200-kDa moiety contained neither C1 nor sensitizing Ab, but it was largely decreased by treatment with NH2OH to the 90-kDa moiety with the mobility corresponding to the alpha'-chain of C4b. A covalent dimer of C4b, therefore, is the predominant form of C4b deposited on liposomes sensitized with antibody. The C4b-C4b dimer formed rapidly (within 5 min) followed by slow dissociation into monomers. The LAC14 bearing the C4b dimer but not the monomer was lysed, although with relatively low efficiency, by the addition of oxyC2 and EDTA-supplemented C3-deficient serum (C3DS), and, furthermore, LAC142 possessed the ability to convert C5 into C5a and C5b. Moreover, lysis was inhibited not by anti-C3 Ab but by anti-C4 Ab. In other experiments, the dimer served as an element of C3 convertase, as well. These findings imply that the C4b dimer, when complexed with C2, expresses C3/C5 convertase activity without participation of C3, and may provide a molecular mechanism whereby sera from patients with complete C3 deficiency retain the ability to induce C-mediated cytolysis.
Asunto(s)
Convertasas de Complemento C3-C5/biosíntesis , Complemento C4b/metabolismo , Autorradiografía , Complemento C3/metabolismo , Convertasas de Complemento C3-C5/fisiología , Vía Alternativa del Complemento , Vía Clásica del Complemento , Electroforesis en Gel Bidimensional , Humanos , Liposomas/inmunologíaRESUMEN
The assembly of the classical-pathway C3 convertase from C4 and I2-treated C2 by the action of C1s is an Mg2(+)-dependent reaction. The Mg2+ concentration necessary for the assembly of C3 convertase in the fluid phase was found to be dependent on NaCl concentration. In the absence of NaCl more than 5 mM-MgCl2 was found to be required, whereas 0.5 mM-MgCl2 was adequate for the assembly of C3 convertase in the presence of 150 mM-NaCl. The C3 convertase assembled in a low-ionic-strength buffer was extremely labile compared with that assembled in buffer of physiological ionic strength, and the stability of C3 convertase was improved with the increase in NaCl concentration. It was found that the stabilizing effect of NaCl on C3 convertase was due to inhibition of the dissociating activity of C2b, which was formed during the assembly of C3 convertase. In addition to the dissociation-accelerating effect, C2b inhibited the assembly of C3 convertase in low-ionic-strength buffer, and this effect also was diminished with increase in NaCl concentration. An increase in NaCl concentration to more than 200 mM resulted in a decrease in the assembly of C3 convertase. This effect was not due to the lability of the assembled C3 convertase but due rather to the inhibition of C2 cleavage by C1s. Purified C3 convertase itself is stable in dilute medium or high-ionic-strength medium such as 500 mM-NaCl, suggesting that the interactions between C4b and C2a are hydrophobic. In these respects C2b seemed to be functionally similar to C4bp, but C2b failed to act as a cofactor for the Factor I-catalysed C4b cleavage.