RESUMO
OBJECTIVE: Lupus-associated IgG anti-double-stranded DNA antibodies are thought to be pathogenic in the kidney due to cross-reaction with glomerular antigens, leading subsequently to immune complex formation in situ and complement activation. We undertook this study to determine if pathogenic anti-DNA antibodies may also contribute to renal damage by directly influencing mesangial gene expression. METHODS: Complementary DNA microarray gene profiling was performed in primary mesangial cells (derived from lupus-prone MRL/lpr mice) treated with pathogenic, noncomplexed anti-DNA antibodies. Significant gene up-regulation induced by anti-DNA antibodies as determined by microarray analysis was further investigated by real-time polymerase chain reaction and methods to detect the relevant proteins. Induction of proinflammatory genes by pathogenic antibodies was confirmed by comparing gene expression in glomeruli of old versus young MRL/lpr mice, and by antibody injection in vivo. RESULTS: Pathogenic, but not nonpathogenic, antibodies significantly induced a number of transcripts, including CXCL1/KC, LCN2, iNOS, CX3CL1/fractalkine, SERPINA3G, and IkappaBalpha ("marker genes"). Blocking of Fcgamma receptors or using Fcgamma chain-knockout mesangial cells had no effect on the gene regulation effect of the pathogenic antibody R4A, indicating a non-Fc-dependent mechanism. The glomerular expression of these marker genes increased over time with the development of glomerular antibody deposition and active nephritis in MRL/lpr mice. Moreover, injection of R4A into SCID mice in vivo significantly up-regulated glomerular marker gene expression. CONCLUSION: These findings indicate that the renal pathogenicity of anti-DNA antibodies may be attributed in part to their ability to directly modulate gene expression in kidney mesangial cells through both Fc-dependent and non-Fc-dependent mechanisms.
Assuntos
Anticorpos Antinucleares/efeitos adversos , Anticorpos Antinucleares/farmacologia , Vasculite Associada ao Lúpus do Sistema Nervoso Central/genética , Células Mesangiais/metabolismo , Camundongos Endogâmicos MRL lpr/genética , Regulação para Cima/efeitos dos fármacos , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animais , Células Cultivadas , Quimiocina CX3CL1 , Quimiocina CXCL1 , Quimiocinas CX3C/genética , Quimiocinas CX3C/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Feminino , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/fisiopatologia , Lipocalina-2 , Lipocalinas , Vasculite Associada ao Lúpus do Sistema Nervoso Central/metabolismo , Vasculite Associada ao Lúpus do Sistema Nervoso Central/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Inibidor de NF-kappaB alfa , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serpinas/genética , Serpinas/metabolismo , Regulação para Cima/fisiologiaRESUMO
The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety concerns. Since it has been suggested that FcgammaR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcgammaR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcgammaRIII and inhibitory FcgammaRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD+ RBCs and a potent FcgammaRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoglobulina G/metabolismo , Receptores de IgG/fisiologia , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Eritrócitos/imunologia , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Receptores de IgG/metabolismoRESUMO
Binding of intact Ag is a hallmark of Ag-specific B cells. Apart from B cells, a small number of non-B cells can bind Ag with comparable efficacy as B cells and are found in the peripheral blood, spleen, and bone marrow of mice. This population has been observed for a long time and recently named "Ag-capturing cells." Their identity remained enigmatic. In this study, we show that these cells are basophilic granulocytes. Their ability to capture Ags is dependent on surface IgE receptors and on Ag-specific plasma IgE molecules appearing after immunization. Several surface markers including surface bound IgE, IL-3R, CD45, CD16/32, and the chemokine receptor CCR2 were used to clearly identify these cells. Cross-linkage of surface Igs results in the release of large amounts of IL-4 and IL-6. The data identify basophils as Ag-capturing cells and support the concept of basophils as important regulators of humoral immune responses.