RESUMO
To elucidate the mechanisms of glomerulonephritis, including Goodpasture's syndrome, mouse models are used that use heterologous Abs against the glomerular basement membrane (GBM) with or without preimmunization with foreign IgG from the same species. These studies have revealed the requirement of either FcgammaR or complement, depending on the experimental model used. In this study, we provide evidence that both FcgammaR and complement are obligatory for a full-blown inflammation in a novel attenuated passive model of anti-GBM disease. We demonstrate that administration of subnephritogenic doses of rabbit anti-GBM Abs followed by a fixed dose of mouse mAbs to rabbit IgG, allowing timing and dosing for the induction of glomerulonephritis, resulted in reproducible complement activation via the classical pathway of complement and albuminuria in wild-type mice. Because albuminuria was absent in FcR-gamma-chain(-/-) mice and reduced in C3(-/-) mice, a role for both FcgammaR and complement is postulated. Because C1q(-/-) and C4(-/-) mice lacking a functional classical and lectin pathway did develop albuminuria, we suggest involvement of the alternative pathway of complement. Anti-GBM glomerulonephritis occurs acutely following the administration of mouse anti-rabbit IgG, and proceeds in a chronic fashion dependent on both FcgammaR and complement. This novel attenuated model allows elucidating the relative contribution of different mediator systems of the immune system to the development of renal injury, and also provides a platform for the assessment of different treatment protocols and evaluation of drugs that ultimately may be beneficial for the treatment of anti-GBM mediated glomerulonephritides.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Nefrite/imunologia , Receptores de Complemento/fisiologia , Receptores de IgG/fisiologia , Albuminúria/etiologia , Animais , Doença Antimembrana Basal Glomerular/etiologia , Doença Antimembrana Basal Glomerular/patologia , Autoanticorpos , Ativação do Complemento , Imunoglobulina G/administração & dosagem , Inflamação , Camundongos , Nefrite/patologiaRESUMO
Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum. Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms. We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.
Assuntos
Via Alternativa do Complemento , Proteínas do Sistema Complemento/fisiologia , Lectina de Ligação a Manose/fisiologia , Anticorpos/sangue , Anticorpos/imunologia , Complemento C1q/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/fisiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Mananas/imunologia , Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/deficiência , Lectina de Ligação a Manose/genética , MutaçãoRESUMO
Neutrophils and complement are key members of innate immunity. The alternative pathway (AP) of complement consists of C3, factor B, factor D and properdin, which amplifies AP activation. AP has been implicated in many neutrophil-mediated diseases, such as anti-neutrophil cytoplasmic antibody-associated vasculitis. The exact mechanism by which the AP and neutrophils interact remains largely unstudied. We investigated the ability of the AP to interact with neutrophil components which can be exposed and released upon activation. Our studies focused on neutrophil enzymes, including myeloperoxidase (MPO), proteinase 3 (PR3), azurocidin, elastase, lysozyme and cathepsin G. All enzymes except for azurocidin were able to bind properdin. However, only MPO could induce C3 activation. MPO mediated AP complement activation in the presence of MgEGTA compared to the EDTA control. This activation resulted in C3 deposition and required properdin to occur. Furthermore, we could show that MPO binds properdin directly, which then serves as a focus for AP activation. In summary, properdin can directly interact with neutrophil components. MPO demonstrates the ability to activate the AP which is dependent on properdin. Finally, MPO is capable of inducing properdin-initiated C3 and C5b-9 deposition in vitro.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Via Alternativa do Complemento , Neutrófilos/imunologia , Peroxidase/metabolismo , Properdina/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Catepsina G/metabolismo , Complemento C3/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Imunidade Inata , Muramidase/metabolismo , Mieloblastina/metabolismo , Elastase Pancreática/metabolismo , Ligação ProteicaRESUMO
IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA1 (pIgA1) and complement. Complement activation via mannose-binding lectin and the lectin pathway is associated with disease progression. Furthermore, recent studies have indicated a possible role for secretory IgA. IgAN is associated with abnormalities in circulating IgA, including aberrant O-linked glycosylation. This study characterized and compared functional properties and N-linked glycosylation of highly purified monomeric IgA (mIgA) and pIgA from patients with IgAN and control subjects. Total serum IgA was affinity-purified from patients (n = 11) and control subjects (n = 11) followed by size separation. pIgA but not mIgA contained secretory IgA, and its concentration was significantly higher in patients with IgAN than in control subjects. Both in patients with IgAN and in control subjects, IgA binding to the GalNAc-specific lectin Helix Aspersa and to mannose-binding lectin was much stronger for pIgA than for mIgA. Furthermore, binding of IgA to mesangial cells largely was restricted to polymeric IgA. Binding of pIgA to mesangial cells resulted in increased production of IL-8, predominantly with IgA from patients with IgAN. Quantitative analysis of N-linked glycosylation of IgA heavy chains showed significant differences in glycan composition between mIgA and pIgA, including the presence of oligomannose exclusively on pIgA. In conclusion, binding and activation of mesangial cells, as well as lectin pathway activation, is a predominant characteristic of pIgA as opposed to mIgA. Furthermore, pIgA has different N-glycans, which may recruit lectins of the inflammatory pathway. These results underscore the role of pIgA in glomerular inflammation in IgAN.
Assuntos
Glomerulonefrite por IGA/imunologia , Imunoglobulina A/química , Lectinas/metabolismo , Células Mesangiais/metabolismo , Adulto , Animais , Quimiocina CCL2/metabolismo , Feminino , Glomerulonefrite por IGA/metabolismo , Glicosilação , Caracois Helix , Humanos , Imunoglobulina A/efeitos dos fármacos , Imunoglobulina A/metabolismo , Interleucina-8/metabolismo , Glomérulos Renais/patologia , Masculino , Lectina de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Neuraminidase/farmacologiaRESUMO
Decorin and biglycan are closely related abundant extracellular matrix proteoglycans that have been shown to bind to C1q. Given the overall structural similarities between C1q and mannose-binding lectin (MBL), the two key recognition molecules of the classical and the lectin complement pathways, respectively, we have examined functional consequences of the interaction of C1q and MBL with decorin and biglycan. Recombinant forms of human decorin and biglycan bound C1q via both collagen and globular domains and inhibited the classical pathway. Decorin also bound C1 without activating complement. Furthermore, decorin and biglycan bound efficiently to MBL, but only biglycan could inhibit activation of the lectin pathway. Other members of the collectin family, including human surfactant protein D, bovine collectin-43, and conglutinin also showed binding to decorin and biglycan. Decorin and biglycan strongly inhibited C1q binding to human endothelial cells and U937 cells, and biglycan suppressed C1q-induced MCP-1 and IL-8 production by human endothelial cells. In conclusion, decorin and biglycan act as inhibitors of activation of the complement cascade, cellular interactions, and proinflammatory cytokine production mediated by C1q. These two proteoglycans are likely to down-regulate proinflammatory effects mediated by C1q, and possibly also the collectins, at the tissue level.
Assuntos
Colectinas/metabolismo , Complemento C1q/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Biglicano , Cálcio/metabolismo , Células Cultivadas , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/biossíntese , Complemento C1q/antagonistas & inibidores , Via Clássica do Complemento/imunologia , Decorina , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Matriz Extracelular/imunologia , Proteínas da Matriz Extracelular , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Lectina de Ligação a Manose/metabolismo , Dados de Sequência Molecular , Células U937RESUMO
Treatment with intravenous immunoglobulin preparations consisting of human IgG (IVIgG) prevents hyperacute rejection of pig xenografts transplanted into primates by inhibition of the classical complement pathway. Recent studies indicate that IVIg preparations mainly consisting of human IgM (IVIgM) have a stronger capacity than IVIgG to inhibit the complement system. IVIg preparations also contain xenoreactive antibodies (XAb) binding to pig cells. In the present study, we compared IVIgG and IVIgM for their capacity to inhibit xenogeneic complement activation, with special reference to the roles of IgG and IgM XAb present in these preparations. Xenogeneic complement activation was studied by exposure of pig cells (PK15) to human serum. For some experiments, IVIgG and IVIgM were depleted from XAb by immune absorption. Exposure of PK15 cells to human serum induced surface deposition of C4 and C3 and cytotoxicity, which could be inhibited in a dose-dependent fashion by both IVIgM and IVIgG. The efficacy of IVIgM was more than 10 times higher than that of IVIgG. IgG XAb were detected IVIgG and IVIgM whereas IgM XAb were only present in IVIgM. Depletion of XAb from the IVIg preparations did not modify the protective properties of IVIgG against cytotoxicity induced by human serum, whereas the IVIgM-mediated protection against xenogeneic cytotoxicity was only slightly improved. IgM-enriched IVIg is a potent inhibitor of xenogeneic complement activation and complement-dependent cytotoxicity of human serum to pig cells, irrespective of the presence of cytotoxic xenoreactive IgM antibodies in this preparation. Therefore, IVIgM has a promising therapeutic significance for the treatment of (hyper)acute xenograft rejection.
Assuntos
Anticorpos Heterófilos/imunologia , Antígenos Heterófilos/imunologia , Ativação do Complemento , Citotoxicidade Imunológica , Rejeição de Enxerto , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Imunoglobulinas Intravenosas/administração & dosagem , Animais , Antígenos Heterófilos/metabolismo , Testes Imunológicos de Citotoxicidade , Dissacarídeos/imunologia , Humanos , Imunoglobulinas Intravenosas/imunologia , Soro/imunologia , SuínosRESUMO
Deficiency of mannose-binding lectin (MBL), a recognition molecule of the lectin pathway of complement, is associated with increased susceptibility to infections. The high frequency of MBL deficiency suggests that defective MBL-mediated innate immunity can be compensated by alternative defense strategies. To examine this hypothesis, complement activation by MBL-binding ligands was studied. The results show that the prototypic MBL ligand mannan can induce complement activation via both the lectin pathway and the classical pathway. Furthermore, antibody binding to mannan restored complement activation in MBL-deficient serum in a C1q-dependent manner. Cooperation between the classical pathway and the lectin pathway was also observed for complement activation by protein 60 from Listeria monocytogenes. MBL pathway analysis at the levels of C4 and C5b-9 in the presence of classical pathway inhibition revealed a large variation of MBL pathway activity, depending on mbl2 gene polymorphisms. MBL pathway dysfunction in variant allele carriers is associated with reduced MBL ligand binding and a relative increase of low-molecular-mass MBL. These findings indicate that antibody-mediated classical pathway activation can compensate for impaired target opsonization via the MBL pathway in MBL-deficient individuals, and imply that MBL deficiency may become clinically relevant in absence of a concomitant adaptive immune response.