RESUMO
A hallmark of autoimmunity in murine models of lupus is the formation of germinal centers (GCs) in lymphoid tissues where self-reactive B cells expand and differentiate. In the host response to foreign antigens, follicular dendritic cells (FDCs) maintain GCs through the uptake and cycling of complement-opsonized immune complexes. Here, we examined whether FDCs retain self-antigens and the impact of this process in autoantibody secretion in lupus. We found that FDCs took up and retained self-immune complexes composed of ribonucleotide proteins, autoantibody, and complement. This uptake, mediated through CD21, triggered endosomal TLR7 and led to the secretion of interferon (IFN) α via an IRF5-dependent pathway. Blocking of FDC secretion of IFN-α restored B cell tolerance and reduced the amount of GCs and pathogenic autoantibody. Thus, FDCs are a critical source of the IFN-α driving autoimmunity in this lupus model. This pathway is conserved in humans, suggesting that it may be a viable therapeutic target in systemic lupus erythematosus.
Assuntos
Autoimunidade/imunologia , Linfócitos B/imunologia , Células Dendríticas Foliculares/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Autoantígenos/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Interferon-alfa/biossíntese , Interferon-alfa/imunologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Reação em Cadeia da Polimerase , Receptor 7 Toll-Like/imunologia , TranscriptomaRESUMO
Properdin is the only known positive regulator of complement activation by stabilizing the alternative pathway convertase through C3 binding, thus prolonging its half-life. Recent in vitro studies suggest that properdin may act as a specific pattern recognition molecule. To better understand the role of properdin in vivo, we used an experimental model of acute anti-glomerular basement membrane disease with wild-type, C3- and properdin knockout mice. The model exhibited severe proteinuria, acute neutrophil infiltration and activation, classical and alternative pathway activation, and progressive glomerular deposition of properdin, C3 and C9. Although the acute renal injury was likely due to acute neutrophil activation, we found properdin deposition in C3-knockout mice that was not associated with IgG. Thus, properdin may deposit in injured tissues in vivo independent of its main ligand C3.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Ativação do Complemento/imunologia , Complemento C3/imunologia , Properdina/imunologia , Animais , Doença Antimembrana Basal Glomerular/patologia , Complemento C3/genética , Complemento C3/metabolismo , Modelos Animais de Doenças , Feminino , Membrana Basal Glomerular/citologia , Membrana Basal Glomerular/imunologia , Membrana Basal Glomerular/patologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Properdina/genética , Properdina/metabolismo , Ligação Proteica/imunologiaRESUMO
Dendritic cells (DCs) and complement are both key members of the innate and adaptive immune response. Recent experimental mouse models have shown that production of alternative pathway (AP) components by DCs strongly affects their ability to activate and regulate T-cell responses. In this study we investigated the production and regulation of properdin (fP) and factor H (fH) both integral regulators of the AP, by DCs and tolerogenic DCs (tolDCs). Both fP and fH were produced by DCs, with significantly higher levels of both AP components produced by tolDCs. Upon activation with IFN-γ both cells increased fH production, while simultaneously decreasing production of fP. IL-27, a member of the IL-12 family, increased fH, but production of fP remained unaffected. The functional capacity of fP and fH produced by DCs and tolDCs was confirmed by their ability to bind C3b. Inhibition of fH production by DCs resulted in a greater ability to induce allogenic CD4+ T-cell proliferation. In contrast, inhibition of fP production led to a significantly reduced allostimulatory capacity. In summary, this study shows that production of fP and fH by DCs, differentially regulates their immunogenicity, and that the local cytokine environment can profoundly affect the production of fP and fH.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fator H do Complemento/metabolismo , Via Alternativa do Complemento , Células Dendríticas/fisiologia , Properdina/metabolismo , Proliferação de Células , Células Cultivadas , Complemento C3b/metabolismo , Fator H do Complemento/genética , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Interleucina-27/metabolismo , Isoantígenos/imunologia , Ativação Linfocitária , Properdina/genética , RNA Interferente Pequeno/genéticaRESUMO
Previous studies have shown that complement activation on renal tubular cells is involved in the induction of interstitial fibrosis and cellular injury. Evidence suggests that the tubular cell damage is initiated by the alternative pathway (AP) of complement with properdin having an instrumental role. Properdin is a positive regulator of the AP, which can bind necrotic cells as well as viable proximal tubular epithelial cells (PTECs), inducing complement activation. Various studies have indicated that in the circulation there is an unidentified inhibitor of properdin. We investigated the ability of C-reactive protein (CRP), both in its monomeric (mCRP) and pentameric (pCRP) form, to inhibit AP activation and injury in vitro on renal tubular cells by fluorescent microscopy, ELISA, and flow cytometry. We demonstrated that preincubation of properdin with normal human serum inhibits properdin binding to viable PTECs. We identified mCRP as a factor able to bind to properdin in solution, thereby inhibiting its binding to PTECs. In contrast, pCRP exhibited no such binding and inhibitory effect. Furthermore, mCRP was able to inhibit properdin-directed C3 and C5b-9 deposition on viable PTECs. The inhibitory ability of mCRP was not unique for viable cells but also demonstrated for binding to necrotic Jurkat cells, a target for properdin binding and complement activation. In summary, mCRP is an inhibitor of properdin in both binding to necrotic cells and viable renal cells, regulating complement activation on the cell surface. We propose that mCRP limits amplification of tissue injury by controlling properdin-directed complement activation by damaged tissue and cells.
Assuntos
Proteína C-Reativa/metabolismo , Ativação do Complemento/efeitos dos fármacos , Complemento C3/metabolismo , Complemento C5/metabolismo , Rim/metabolismo , Properdina/farmacologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Rim/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacosRESUMO
Lipoproteins can induce complement activation resulting in opsonization and binding of these complexes to complement receptors. We investigated the binding of opsonized native LDL and acetylated LDL (acLDL) to the complement receptor 1 (CR1). Binding of complement factors C3b, IgM, C1q, mannose-binding lectin (MBL), and properdin to LDL and acLDL were investigated by ELISA. Subsequent binding of opsonized LDL and acLDL to CR1 on CR1-transfected Chinese Hamster Ovarian cells (CHO-CR1) was tested by flow cytometry. Both native LDL and acLDL induced complement activation with subsequent C3b opsonization upon incubation with normal human serum. Opsonized LDL and acLDL bound to CR1. Binding to CHO-CR1 was reduced by EDTA, whereas MgEGTA only reduced the binding of opsonized LDL, but not of acLDL suggesting involvement of the alternative pathway in the binding of acLDL to CR1. In vitro incubations showed that LDL bound C1q, whereas acLDL bound to C1q, IgM, and properdin. MBL did neither bind to LDL nor to acLDL. The relevance of these findings was demonstrated by the fact that ex vivo up-regulation of CR1 on leukocytes was accompanied by a concomitant increased binding of apolipoprotein B-containing lipoproteins to leukocytes without changes in LDL-receptor expression. In conclusion, CR1 is able to bind opsonized native LDL and acLDL. Binding of LDL to CR1 is mediated via the classical pathway, whereas binding of acLDL is mediated via both the classical and alternative pathways. Binding of lipoproteins to CR1 may be of clinical relevance due to the ubiquitous cellular distribution of CR1.
Assuntos
Ativação do Complemento , Complemento C3b/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de Complemento 3b/metabolismo , Animais , Apolipoproteínas B/metabolismo , Células CHO , Células Cultivadas , Complemento C1q/metabolismo , Via Alternativa do Complemento , Via Clássica do Complemento , Cricetinae , Cricetulus , Ácido Edético/farmacologia , Citometria de Fluxo , Humanos , Imunoglobulina M/metabolismo , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas Opsonizantes/metabolismo , Properdina/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Complemento 3b/genéticaRESUMO
The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo.
Assuntos
Ativação do Complemento/imunologia , Complemento C3b/imunologia , Complemento C3c/imunologia , Rim/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Ativação do Complemento/genética , Complemento C3b/genética , Complemento C3b/metabolismo , Complemento C3c/genética , Complemento C3c/metabolismo , Complemento C9/imunologia , Complemento C9/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Estudos de Viabilidade , Rim/irrigação sanguínea , Rim/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Uptake of apoptotic cells by DCs is considered to contribute to induction and maintenance of immunological tolerance. TolDCs are sought after as cellular therapy in transplantation and autoimmunity and can be generated in vitro using GCs. In this study, we investigated how uptake of dead cells affects the production and expression of different members of the IL-12 family by immature DCs or TolDCs. We show that compared to regular immature DCs, TolDCs display elevated levels of PS-recognizing bridge molecule receptors αvß5 and CD36, and have enhanced phagocytic abilities with accelerated uptake of apoptotic cells. We confirm that apoptotic cell uptake results in diminished production of IL-12p40 and IL-12p70 by DCs. We now show that this also results in increased expression of IL-12p35 and Ebi3. TolDCs completely lack expression of IL-12p40 yet have enhanced levels of Ebi3 and IL-12p35. Uptake by TolDCs of apoptotic or necrotic cells does not affect the expression of Ebi3/IL-12p35 and also does not increase IL-12p40. This is distinct from the culture of immature DCs with necrotic cells, which is sufficient to induce IL-12p40 secretion. Conversely, ingestion of apoptotic cells by DCs leads to increased expression of IL-12p35 and Ebi3 without affecting IL-12p40. In conclusion, we have shown that uptake of apoptotic versus necrotic cells by DCs differentially regulates members of the IL-12 family. Apoptotic cells favor expression of Ebi3 and IL-12p35, and we propose that differential regulation of the IL-12 family is an additional mechanism in determining the immune response to dying cells.
Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-12/imunologia , Fagocitose/imunologia , Células Cultivadas , Humanos , Necrose/imunologiaRESUMO
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
Properdin is a member of the alternative pathway of complement. It is unique in that it is the only known positive regulator of the complement system. Properdin can stabilize and promote complement activation, but in addition is also capable of initiating the alternative pathway, making it a unique protein. This chapter focuses on the methods required for purifying human properdin and testing its functionality.
Assuntos
Properdina/isolamento & purificação , Properdina/metabolismo , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Complemento C3b/metabolismo , Via Alternativa do Complemento , Humanos , Ligação ProteicaRESUMO
Various studies indicate a role for both anti-nucleosome and anti-C1q autoantibodies in glomerulonephritis in patients with systemic lupus erythematosus. However, a causal relationship between these autoantibodies and the development of lupus nephritis has not been fully established. Since injury of the endothelium is a major target in lupus nephritis we assessed the interaction of C1q and nucleosomes with glomerular endothelial cells in vitro in the presence or absence of autoantibodies against these antigens. We demonstrate a direct and dose-dependent binding of both nucleosomes and C1q to immortalized human glomerular endothelial cells (GEnC) in vitro, which in part is mediated by cell surface heparan sulfate. We demonstrate that nucleosomes and C1q serve as targets for monoclonal and polyclonal antibodies as well as for anti-nuclear autoantibodies from patients with systemic lupus erythematosus. An additive effect of anti-C1q autoantibodies on anti-nucleosome mediated complement activation was observed. Furthermore, we showed that the activation of complement on glomerular endothelial cells is mediated by the classical pathway since the deposition of C3 on GEnC is abrogated by MgEGTA and does not occur in C1q-depleted serum. Taken together, our studies demonstrate a direct binding of both nucleosomes and C1q to glomerular endothelial cells in vitro. The subsequent binding of autoantibodies against nucleosomes in patients with systemic lupus erythematosus is potentially pathogenic and autoantibodies against C1q seem to have an additional effect.