RESUMO
Factor H-related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.
Assuntos
Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , Ativação do Complemento , Proteínas Inativadoras do Complemento C3b/imunologia , Proteínas Inativadoras do Complemento C3b/metabolismo , Sítios de Ligação , Proteína C-Reativa/química , Proteína C-Reativa/farmacologia , Convertases de Complemento C3-C5 , Complemento C3b/imunologia , Complemento C3b/farmacologia , Proteínas Inativadoras do Complemento C3b/farmacologia , Fator H do Complemento , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Ligantes , Degeneração Macular/imunologia , Ligação Proteica , Componente Amiloide P Sérico/imunologia , Componente Amiloide P Sérico/metabolismoRESUMO
Purified C4-binding protein (C4-bp) was shown to bind to cell-bound C4b by radioactive tracer techniques. With EAC4 bearing greater than 3,000 C4b-molecules/cell, the number of C4-bp molecules bound was directly proportional to the number of C4b molecule on the cell surface; EAC4 bearing less than 3,000 C4b-molecules/cell bound a very small amount of C4-bp. Scatchard analysis of binding of C4-bp indicated an equilibrium constant of 4.6 X 10(8) L/M and a maximum of 0.43 C4-bp molecules bound per C4b molecule, equivalent to an average of one molecule of C4-bp per two or three molecules of C4b. Fluid-phase C4b inhibited the binding of C4-bp to cell-bound C4b in a dose-dependent manner, whereas native C4 had little effect. C2 inhibited this binding and also released C4-bp from EAC4,C4-bp. However, C2 was 27 times less effective than unlabeled C4-bp on a molar basis and a considerable amount of C4-bp remained bound to C4b on the cell surface even in the presence of a large excess of C2. We also examined the cofactor activity of C4-bp in the cleavage of cell-bound C4b by C3b/C4b inactivator (I). Cleavage of the alpha' chain of C4b on the cell surface by I alone was incomplete and an intermediate cleavage product, alpha-75, was observed. When C4-bp bound to C4b on the cell surface, the alpha' chain of the C4b cleaved into three fragments, alpha 2, alpha 3, and alpha 4. The alpha 3, alpha 4, beta, and gamma peptides (C4c) were released into the fluid phase, and the alpha 2 fragment (C4d) remained linked covalently to the cell membrane via an ester bond. In some situations, therefore, C4-bp enhances the proteolytic activity of I on cell-bound C4b.
Assuntos
Proteínas de Transporte/metabolismo , Complemento C4/metabolismo , Animais , Ligação Competitiva , Proteínas de Transporte/análise , Complemento C2/fisiologia , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C4b , Relação Dose-Resposta Imunológica , Eritrócitos/metabolismo , Cobaias , Humanos , Hidrólise , Integrina alfaXbeta2 , Peso MolecularRESUMO
Antibody to the binding site (idiotype) of anti-factor H was shown to have specificity for both B lymphocyte membrane H receptors and C3b. Goat F(ab')(2) anti-human H was purified by absorption and elution from H agarose and used for rabbit immunization to produce anti-anti-H (aaH). After absorption with nonimmune goat IgG, (125)I-labeled aaH bound to B lymphocytes and to sheep erythrocytes coated with C3b (EC3b) but did not bind to T lymphocytes or to EC3d. All B cell- and C3b-specific activities of the aaH were removed and subsequently recovered by absorption and elution of the antibody from either C3-agarose or goat-anti-H-agarose. This indicated that the aaH probably recognized a single common antigenic structure that was shared by anti-H, C3b, and the membranes of B cells. Affinity-purified aaH resembled H in that it bound to B cells, blocked the uptake of H onto B cell H receptors, and triggered B cells to release endogenous factor I (C3b inactivator). In addition, aaH functioned with factor I as either a cofactor for cleavage of fluid-phase C3b or a potentiator for cleavage of bound C3b. This same spectrum of C3 binding functions could not be demonstrated with either sheep anti-C3b or rabbit-anti-C3c. Analysis by sodium dodecyl sulfate- polyacrylamide get electrophoresis of the [(3)H]leucine intrinsically labeled B cell proteins reactive with the purified aaH revealed proteins of 100,000 M(r) and 50,000 M(r) without reduction, and after complete reduction of disulfide bonds, a single protein band of 50,000 M(r). This same protein molecular weight profile was also demonstrated with labeled B cell proteins that were absorbed and eluted from H-agarose. Thus, aaH is apparently specific for both B cell H receptors and C3b. However, because parallel analysis of C3b confirmed its known 115,000- and 75,000-M(r) polypeptide chain structure, the H receptor is probably not C3b and shares only the structure of the H binding site with C3b.
Assuntos
Sítios de Ligação de Anticorpos , Proteínas Inativadoras do Complemento C3b/farmacologia , Idiótipos de Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Anticorpos Anti-Idiotípicos/imunologia , Especificidade de Anticorpos , Ligação Competitiva , Linhagem Celular , Complemento C3b/imunologia , Proteínas Inativadoras do Complemento C3b/imunologia , Fator H do Complemento , Cabras , Humanos , Proteínas de Membrana , Peso Molecular , CoelhosRESUMO
The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.
Assuntos
Doenças Autoimunes/imunologia , Proteínas Inativadoras do Complemento/farmacologia , Traumatismo por Reperfusão Miocárdica/imunologia , Miocárdio/patologia , Receptores de Complemento/farmacologia , Animais , Doenças Autoimunes/patologia , Ativação do Complemento , Complemento C3/antagonistas & inibidores , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C4b/antagonistas & inibidores , Complemento C5/antagonistas & inibidores , Proteínas Inativadoras do Complemento/ultraestrutura , Modelos Animais de Doenças , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Necrose , Ratos , Receptores de Complemento/ultraestrutura , Proteínas Recombinantes/farmacologiaRESUMO
Cigarette smoking is associated with significant increases in the number of pulmonary mononuclear phagocytes and neutrophils. A potent chemoattractant for these cells is C5a, a peptide generated during complement (C) activation. We, therefore, investigated the possibility that cigarette smoke could activate the complement system in vitro. Our results show that factor(s) (mol wt less than 1,000) present in an aqueous solution of whole, unfiltered cigarette smoke can deplete the hemolytic capacity of whole human serum in a dose-dependent manner. The particle-free, filtered gas phase of cigarette smoke is inactive. The smoke factor(s) do not activate serum C1, but do deplete serum C4 activity. Treatment of purified human C3 with whole smoke solution modifies the molecule such that its subsequent addition to serum (containing Mg/EGTA to block the classical pathway) results in consumption of hemolytic complement by activation of the alternative pathway. Smoke-modified C3 shows increased anodal migration in agarose electrophoresis, but this is not due to proteolytic cleavage of the molecule as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to methylamine-treated C3, C3 treated with smoke is only partially susceptible to the action of the complement regulatory proteins Factors H and I. In addition, smoke-modified C3 has diminished binding to Factor H as compared with methylamine-treated C3. Finally, smoke-modified C3 incorporates [14C]methylamine which suggests that the thiolester bond may be intact. These data indicate that aqueous whole cigarette smoke solution can modify C3 and activate the alternative pathway of complement, perhaps by a previously unrecognized mechanism. Should this occur in vivo, complement activation might partly account for the extensive pulmonary leukocyte recruitment observed in smokers.
Assuntos
Ativação do Complemento , Complemento C3/metabolismo , Via Alternativa do Complemento , Fumar , Complemento C1/metabolismo , Complemento C3/imunologia , Proteínas Inativadoras do Complemento C3b/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C4/metabolismo , Fator H do Complemento , Fator I do Complemento , Endopeptidases/farmacologia , Hemólise , Humanos , Técnicas In Vitro , Iodoacetamida/metabolismo , Metilaminas/metabolismo , Ligação Proteica , SoluçõesRESUMO
The molecular mechanism of complement resistance of the human SK-MEL-170 melanoma cell line was investigated. The cells have been shown to express the C3b-cleaving membrane protease p65. To delineate the molecular consequences of the C3b-cleaving activity for the complement cytotoxicity, the molecular events during the initiation (R24 monoclonal antibody, C1), amplification (C4, C3), and membrane attack (C5, C9) phases of complement were studied in comparison to a complement-susceptible human melanoma line (SK-MEL-93-2). No cleavage of C4b and C5b, 2 molecules structurally similar to C3b, was observed on the cells during classical pathway activation indicating the specificity of the p65 protease for the C3b molecule. The rapid degradation of C3b by p65 on the surface of complement-resistant SK-MEL-170 cells generates a M(r) 30,000 C3 alpha'-chain-fragment detectable as early as 1 min after complement activation, whereas no such fragment was present in detectable amounts on complement-susceptible cells. As a result of the rapid C3b proteolysis by p65 on resistant SK-MEL-170 cells, less C5 convertases are formed, which in turn results in the formation of a lower number of terminal complement components and membrane attack complexes. R24 antibody and C1q binding to the resistant cells was slightly lower as to susceptible cells. C4 binding studies, however, revealed that the observed difference in antibody and C1q binding has no influence on the complement resistance of SK-MEL-170 cells: significantly more C4b was bound to complement-resistant (1565 +/- 92 fg/cell) as compared to susceptible cells (715 +/- 31 fg/cell). On extraction of the molecular forms of C4 bound to the cell membranes, an additional high molecular weight C4 species--apparently a C4b-C4b homodimer--appeared only on the resistant SK-MEL-170 cells that may function as a residual back-up C5 convertase. Collectively, these results show that SK-MEL-170 human melanoma cells evade complement-mediated cytolysis despite sufficient activation of early components of the classical complement pathway by p65-mediated rapid degradation of surface-bound C3b, leading to a significant reduction in membrane attack complex formation. Thus, rapid cleavage of surface deposited C3b was established as a powerful mechanism of complement resistance.
Assuntos
Ativação do Complemento/fisiologia , Complemento C3b/metabolismo , Proteínas do Sistema Complemento/imunologia , Endopeptidases/fisiologia , Melanoma/enzimologia , Animais , Anticorpos Monoclonais/metabolismo , Membrana Celular/enzimologia , Complemento C1q/metabolismo , Complemento C3/metabolismo , Complemento C3/fisiologia , Complemento C3b/fisiologia , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C4/metabolismo , Complemento C4/fisiologia , Complemento C5/metabolismo , Complemento C5/fisiologia , Complemento C9/metabolismo , Complemento C9/fisiologia , Complexo de Ataque à Membrana do Sistema Complemento/fisiologia , Humanos , Melanoma/metabolismo , Melanoma/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais CultivadasRESUMO
The macrophage system in man plays a significant role in the detection of foreign cells. The mechanisms by which macrophages recognize malignant cells, however, are not well understood. We used human monocytes and four lymphoblastoid cell (LC) lines derived from human acute lymphocytic leukemia to investigate the initial recognition of tumor cells by monocytes. IgM antibody mediated the binding of these cells to monocytes only in the presence of complement. The stepwise addition of complement components to IgM-coated LC indicated that C3 was necessary to monocyte binding. Similarly, monocyte recognition of IgM-coated LC was maximal in the presence of sera from patients with congenital C5 or C6 deficiency, but absent in the presence of sera deficient C4 or from a patient with congenital C2 deficiency. Complement activation was associated with C3 consumption and the deposition of substantial amounts of C3 on to LC. Although 3H-C3 bound to LC appeared stable for 2 hours, approximately 4.0 +/- 2 X 10(5) 3H-C3 per LC was necessary for monocyte recognition, compared to approximately 2.7 +/- 0.5 X 10(3) 3H-C3 per RBC. The data indicate that LC can be recognized by monocytes through complement by mechanisms similar to nonmalignant target cells. However, substantial amounts of C3 are necessary to induce monocyte recognition of IgM-coated LC and, thus, such complement mediated recognition may be inefficient.
Assuntos
Proteínas do Sistema Complemento/fisiologia , Linfócitos/fisiologia , Monócitos/fisiologia , Comunicação Celular , Linhagem Celular , Colchicina/farmacologia , Complemento C3/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Proteínas do Sistema Complemento/deficiência , Proteínas do Sistema Complemento/metabolismo , Humanos , Imunoglobulina M/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Antígeno de Macrófago 1 , Monócitos/imunologia , Monócitos/metabolismo , Receptores de Complemento/análise , Receptores de Complemento/efeitos dos fármacos , Linfócitos T/metabolismo , Azul Tripano/farmacologiaRESUMO
C3f, a heptadeca-peptide having the amino acid sequence of NH2-Ser-Ser-Lys-Ile-Thr-His-Arg-Ile-His-Trp-Glu-Ser-Ala-Ser-Leu-Leu-Arg- COOH, is liberated during the catabolic degradation of C3b in serum. The amino acid sequence of C3f is known both from the cDNA-derived structure of C3 and from protein analysis after isolation of the natural factor. C3f was synthesized by solid phase peptide synthesis. Both natural and synthetic C3f had identical retention times by RP-18 high performance liquid chromatography (HPLC) analysis and the respective amino acid compositions agreed with the expected theoretical values. C3f, but not des-Arg-C3f, was weakly spasmogenic inducing contraction of guinea pig ileum at a level of 5-10 x 10(-6) M. Since C3f and C3a were cross-tachyphylactic, it was concluded that these two spasmogens compete for the same receptors. Both C3f and des-Arg-C3f at concns of 1-4 x 10(-4) M enhanced vascular permeability in guinea pig skin. These observations further suggest that C3f functionally resembles C3a anaphylatoxin. Formation of C3f in human serum following CVF activation of C3 could be demonstrated by radioimmunoassay (RIA). Digestion of C3f with purified human serum carboxypeptidase N produced C3f-desArg. These observations suggest that when serum complement protein C3 undergoes conversion to C3b, further degradation by Factors H and I readily generates C3f. C3f is a weak spasmogen that functions like C3a anaphylatoxin and C3f-desArg is a major metabolite in serum.
Assuntos
Complemento C3/fisiologia , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C3b/metabolismo , Serina Endopeptidases/farmacologia , Sequência de Aminoácidos , Animais , Permeabilidade Capilar , Cromatografia Líquida de Alta Pressão , Complemento C3/isolamento & purificação , Complemento C3/metabolismo , Fator H do Complemento , Fator I do Complemento , Venenos Elapídicos/farmacologia , Cobaias , Humanos , Dados de Sequência Molecular , Contração Muscular , RadioimunoensaioRESUMO
Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.
Assuntos
Ativação do Complemento/fisiologia , Complemento C3b/metabolismo , Endopeptidases , Catepsina B/metabolismo , Catepsina D/metabolismo , Catepsina L , Catepsinas/metabolismo , Ativação do Complemento/efeitos dos fármacos , Complemento C3b/efeitos dos fármacos , Proteínas Inativadoras do Complemento C3b/farmacologia , Cisteína Endopeptidases , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Immunoblotting , Lisossomos/metabolismo , Microesferas , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The accumulation of polymorphonuclear leukocytes (PMN) beneath the stratum corneum is a characteristic histopathologic finding in various aseptic pustular dermatoses. To elucidate the pathomechanism involved in this phenomenon, we investigated whether PMN also attach to a sheet of corneocytes in vitro. A 1-cm2 corneocyte sheet was attached to a sterile glass slide with double adhesive tape used for skin graft surgery before incubating with human serum. The PMN suspension then was applied to the sheet. Attached cells were stained with May-Grunwald-Giemsa and counted with a computer image analyzer. We quantitatively assessed PMN adhesion to the serum-treated corneocyte sheets, which was mediated by activation of the alternative complement pathway. Addition of either anti-CD18 or anti-CD11b antibody to the assay system resulted in a marked reduction of PMN adhesion. We also demonstrated immunohistochemically that iC3b was formed on the serum-treated corneocytes. These findings suggest that PMN attach to serum-treated corneocytes through an interaction of CR3 expressed on PMN with iC3b-coated corneocytes. In addition, we found that this adhesion was enhanced by activation of PMN with phorbol myristate acetate. From these results, we speculate that complement activation by corneocytes occurs in the cutaneous lesions of aseptic pustular dermatoses and that PMN can be stimulated by the interaction with iC3b-opsonized corneocytes as well as by chemotaxins, leading to damage of the surrounding epidermal keratinocytes.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Neutrófilos/citologia , Pele/citologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Ativação do Complemento , Proteínas Inativadoras do Complemento C3b/farmacologia , Humanos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Applying 2 M KBr, membranes of Ehu were solubilized. By C3-affinity chromatography an activity could be isolated that inhibited the immune adherence reaction and C3b-dependent rosette formation. Since this material did not agglutinate EAC14oxy23b we termed it monovalent C3b receptor (mC3bR). PAGE and SDS-PAGE and staining with Coomassie brilliant blue and PAS reagent revealed a single glycoprotein band with a mol. wt. of 55,000-60,000 daltons and an electrophoretic mobility comparable to ovalbumin. This mC3bR proved to be antigenetically related to gp 205 [17]. The potential of mC3bR to react with C3b-carrying particles was not destroyed by heat and trypsin treatment but by neuraminidase or periodic acid treatment suggesting that mC3bR reacted by its carbohydrate moiety with C3b. As by mC3bR, immune adherence could be inhibited by D-glucose and D-galactose but not by their optical antipodes, L-glucose and L-galactose.
Assuntos
Complemento C3b/isolamento & purificação , Eritrócitos/análise , Receptores de Complemento/isolamento & purificação , Animais , Antígenos/imunologia , Fenômenos Químicos , Química , Complemento C3b/imunologia , Proteínas Inativadoras do Complemento C3b/farmacologia , Eritrócitos/imunologia , Cobaias , Humanos , Reação de Imunoaderência , Monossacarídeos/farmacologia , Coelhos , Formação de RosetaRESUMO
An intramolecular thioester bond in complement protein C3 is vital for covalent attachment of C3b (the proteolytically activated form of C3) to biological surfaces and for activation of the complement system. Proteolytic removal of C3a from C3 activates the thioester in the C3b fragment. Activated C3b primarily forms ester bonds with hydroxyl groups of carbohydrates on complement activating surfaces, but it has also been shown to react with the hydroxyl group of tyrosine and with specific Ser and Thr residues on IgG and on complement protein C4b. To examine the reactivity of the thioester, several families of hydroxylated compounds were examined. Reactivity of a series of substituted phenols varied over two orders of magnitude and demonstrated a linear correlation between reactivity and the Hammett substituent constants. Hydroxylated drugs including members of the L-DOPA/epinephrine family and hydroxamic acids also were examined. Compounds were identified that were 20,000 times more reactive than carbohydrates. These compounds were found to inhibit both the classical and alternative pathways of complement activation. Although the specificity of the thioester for its natural biological targets appears to be determined by many structural features, the data presented here demonstrate that increasing the nucleophilic character of the target hydroxyl group can increase the potency of a synthetic inhibitor many orders of magnitude.
Assuntos
Complemento C3/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C3b/metabolismo , Humanos , Hidroxilação , Fenóis/metabolismo , Fenóis/farmacologia , Tirosina/análogos & derivados , Tirosina/farmacologiaRESUMO
Rosmarinic acid has been reported to inhibit complement activation in vivo as well as in vitro. Previous studies suggested that the inhibitory effect was due to inhibition of C3/C5 convertases, but inhibition of C3b attachment would yield the same results. Recent work in our laboratory demonstrated that compounds with polyhydroxylated phenyl rings are highly reactive with the thioester bond in nascent C3b. These compounds block complement activation by preventing attachment of C3b to the activating surface. Because rosmarinic acid contains two 3,4-dihydroxyphenyl groups, the current study was undertaken to re-examine the mechanism of inhibition by analyzing the effect of rosmarinic acid on C3b attachment. In assays using purified complement proteins, rosmarinic acid inhibited covalent attachment of C3b to cells with an 1C50 = 34 microM. Inhibition of C5 convertase activity required 1500 microM rosmarinic acid, and no significant inhibition of the C3 convertase enzyme, which produces C3b from C3, was observed at 10,000 microM. In hemolytic assays using human serum, rosmarinic acid was shown to inhibit activation of both the classical (IC50 = 180 microM) and the alternative (IC50 = 160 microM) pathways of complement. Rosmarinic acid concentrations up to 10,000 microM did not cause direct inactivation of C3. Radioiodination of rosmarinic acid was used to demonstrate covalent activation-dependent incorporation of rosmarinic acid specifically into the thioester-containing alpha'-chain of nascent C3b. These findings indicate that inhibition of complement activation by rosmarinic acid is due to the reaction of rosmarinic acid with the activated thioester of metastable C3b, resulting in covalent attachment of the inhibitor to the protein.
Assuntos
Cinamatos/farmacologia , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C3b/efeitos dos fármacos , Animais , Cinamatos/química , Ativação do Complemento , Complemento C3/antagonistas & inibidores , Convertases de Complemento C3-C5/antagonistas & inibidores , Convertases de Complemento C3-C5/metabolismo , Depsídeos , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Ovinos , Ácido RosmarínicoRESUMO
The role of cell membrane-associated human factor H for the binding of cell-bound C3b to complement receptor-carrying (CR+) cells was investigated. Pretreatment of CR+ cells with antibodies to factor H inhibited the adherence of C3b-coated red cells to human tonsil lymphocytes (TL) and peripheral blood monocytes (M phi). The C3b receptor reactivity of human polymorphonuclear leucocytes (PMN) was not influenced and the one of Raji lymphoblastoid cells only slightly influenced; iC3b and C3d receptor reactivity was in no case affected. When diisopropylfluorophosphate (DFP) in a concentration of 0.1 mM was present during pretreatment of the CR+ cells with anti H, the antibodies gained the capacity to inhibit the adherence of C3b-coated erythrocytes to Raji cells; this effect was dose-dependent with respect to DFP. In contrast, there was no influence of DFP on the inhibition pattern of anti H in the case of TL and M phi. The adherence of C3b-coated erythrocytes to PMN remained unaffected by anti-H antibodies in the presence of DFP. Polyclonal as well as monoclonal antibodies directed against human factor I inhibited the binding of C3b cells to Raji cells but not to TL. Additionally, when anti I and anti H antibodies were both present, C3b receptor reactivity of Raji cells was inhibited to a larger extent than with either antibody alone; again, TL remained unaffected. Results obtained by washing the Raji cells before and after treatment with anti H and anti I suggest that the respective antibodies act on factor H primarily on the level of the cell membrane and on factor I in the fluid phase.
Assuntos
Ativação do Complemento , Proteínas Inativadoras do Complemento C3b/farmacologia , Proteínas Inativadoras do Complemento C3b/fisiologia , Via Alternativa do Complemento , Eritrócitos/metabolismo , Receptores de Complemento/análise , Animais , Proteínas Inativadoras do Complemento C3b/imunologia , Fator H do Complemento , Humanos , Fragmentos Fab das Imunoglobulinas , Isoflurofato/farmacologia , Linfócitos/metabolismo , Monócitos/metabolismo , Coelhos , Receptores de Complemento/efeitos dos fármacos , Receptores de Complemento/fisiologia , Receptores de Complemento 3b , Formação de RosetaRESUMO
The physicochemical nature of the human glomerular complement receptor was studied. Receptor activity was measured by determining the avidity of glomeruli of normal human renal tissue for fluorescein-labeled bacteria (S.typhi) coated with C3b. Maximal binding of C3b-coated bacteria to normal human glomeruli took place in phosphate-saline buffers of pH 6.5 and 0.08 to 0.15 mu ionic strength. Pretreatment of renal tissue with neuraminidase enhanced receptor activity. On the other hand, binding of C3b-coated bacteria to the glomeruli was diminished by pretreatment of the tissue with proteolytic enzymes, phospholipase C and certain lipid solvents. The binding of C3b-coated bacteria to the glomeruli was also diminished by pretreatment of the tissue with fluid-phase C3b, or by pretreatment of the bacteria with C3b inactivator. Normal human serum and purified fluid-phase C3 or the absence of magnesium and calcium ions had little effect on glomerular complement receptor activity.
Assuntos
Sítios de Ligação de Anticorpos , Complemento C3b , Glomérulos Renais/imunologia , Sítios de Ligação de Anticorpos/efeitos dos fármacos , Criança , Proteínas Inativadoras do Complemento C3b/farmacologia , Técnicas de Cultura , Humanos , Neuraminidase/farmacologia , Papaína/farmacologia , Fosfolipases/farmacologia , Salmonella typhi/imunologia , Solventes/farmacologia , Tripsina/farmacologiaAssuntos
Enzimas Ativadoras do Complemento/antagonistas & inibidores , Convertases de Complemento C3-C5/antagonistas & inibidores , Properdina/farmacologia , Ligação Competitiva , Convertases de Complemento C3-C5/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Fator B do Complemento/farmacologia , Fator H do Complemento , Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Humanos , Técnicas In Vitro , Properdina/metabolismoRESUMO
PURPOSE: Studies implicate activation of complement among the processes involved in the pathogenesis of age-related macular degeneration (AMD). Questions pertain to the trigger(s) responsible for the complement-associated events. The authors previously reported that photooxidation products of A2E can activate complement. Here they have further explored these events. METHODS: In vitro assays using human serum as a source of complement were used, and the C3 split product iC3b was measured by enzyme immunoassay. Serum was placed in contact with ARPE-19 cells and polarized human fetal retinal pigment epithelium that had accumulated A2E and were irradiated (430 nm). Serum was also incubated in wells precoated with bisretinoid pigments of lipofuscin and their oxidized forms. iC3b generation in normal human serum (NHS) was compared with that in factor B-depleted and C1q-depleted human serum. RESULTS: iC3b levels were elevated in NHS placed in contact with A2E-laden retinal pigment epithelium that were irradiated to generate A2E photooxidation products. iC3b was also increased in serum incubated in wells precoated with peroxy-A2E, the lipofuscin pigment all-trans-retinal dimer, and oxidized forms of all-trans-retinal dimer. Substitution of NHS with factor B-depleted sera abrogated these increases in iC3b. Complement activation was also suppressed by the addition of C-reactive protein and by a C3 cleavage inhibitor. CONCLUSIONS: The authors suggest that bisretinoid pigments of retinal pigment epithelial lipofuscin, subsequent to photoactivation and cleavage, serve to activate complement. Complement activation by this mechanism is dependent on the alternative pathway and can be modulated by an inhibitor of C3 cleavage. These events in the setting of complement dysregulation could contribute to the chronic inflammation that underlies AMD pathogenesis.
Assuntos
Ativação do Complemento/fisiologia , Complemento C3b/biossíntese , Lipofuscina/metabolismo , Compostos de Piridínio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Retinoides/metabolismo , Apoptose/efeitos dos fármacos , Proteína C-Reativa/farmacologia , Células Cultivadas , Complemento C3b/antagonistas & inibidores , Proteínas Inativadoras do Complemento C3b/farmacologia , Via Alternativa do Complemento/fisiologia , Etoposídeo/farmacologia , Humanos , Técnicas Imunoenzimáticas , Lipofuscina/química , Oxirredução , Peptídeos Cíclicos/farmacologia , Compostos de Piridínio/química , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/embriologia , Retinoides/químicaRESUMO
Shortly after grafting of Ehrlich ascites carcinoma cells, the serum of tumour-bearing mice loses the capacity to mediate immunoadherence phenomena, because of a sharp decrease in the concentration of C3b and C3d, while the cellular receptors for such factors are unaffected by tumour growth. It is suggested that complement is consumed through the alternative pathway which is activated during the inflammatory responses accompanying tumour growth.
Assuntos
Carcinoma de Ehrlich/imunologia , Proteínas do Sistema Complemento , Formação de Roseta , Animais , Complemento C3/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Camundongos , Fatores de TempoRESUMO
IgG mouse monoclonal antibody (mAb) was prepared by fusion of spleen cells from mice immunized with human C3d (mAb:C3d) using syngeneic thymocytes as feeder cells. mAb:C3d was assessed for its effect on the stabilization of the cell-bound alternative pathway C3 convertase EAC3bBb. It bound to cell-bound C3b and stabilized C3bBb at 30 degrees in the presence of EDTA-GVB. The plasma protein H reduced the stabilization effect of the stabilized C3 convertase. These results suggest that binding of antibody to C3d may stabilize C3bBb. It seems likely that such antibody induces in C3b conformational change, which increases the C3bBb complex stability.