RESUMO
Defects in apoptosis can cause autoimmune disease. Loss-of-function mutations in the 'death receptor' FAS impair the deletion of autoreactive lymphocytes in the periphery, leading to progressive lymphadenopathy and systemic lupus erythematosus-like autoimmune disease in mice (Fas(lpr/lpr) (mice homozygous for the lymphoproliferation inducing spontaneous mutation)) and humans. The REL/nuclear factor-κB (NF-κB) transcription factors regulate a broad range of immune effector functions and are also implicated in various autoimmune diseases. We generated compound mutant mice to investigate the individual functions of the NF-κB family members NF-κB1, NF-κB2 and c-REL in the various autoimmune pathologies of Fas(lpr/lpr) mutant mice. We show that loss of each of these transcription factors resulted in amelioration of many classical features of autoimmune disease, including hypergammaglobulinaemia, anti-nuclear autoantibodies and autoantibodies against tissue-specific antigens. Remarkably, only c-REL deficiency substantially reduced immune complex-mediated glomerulonephritis and extended the lifespan of Fas(lpr/lpr) mice. Interestingly, compared with the Fas(lpr/lpr) animals, Fas(lpr/lpr)nfkb2(-/-) mice presented with a dramatic acceleration and augmentation of lymphadenopathy that was accompanied by severe lung pathology due to extensive lymphocytic infiltration. The Fas(lpr/lpr)nfkb1(-/-) mice exhibited the combined pathologies caused by defects in FAS-mediated apoptosis and premature ageing due to loss of NF-κB1. These findings demonstrate that different NF-κB family members exert distinct roles in the development of the diverse autoimmune and lymphoproliferative pathologies that arise in Fas(lpr/lpr) mice, and suggest that pharmacological targeting of c-REL should be considered as a strategy for therapeutic intervention in autoimmune diseases.
Assuntos
Lúpus Eritematoso Sistêmico/complicações , Doenças Linfáticas/complicações , Subunidade p50 de NF-kappa B/deficiência , Subunidade p52 de NF-kappa B/deficiência , Proteínas Proto-Oncogênicas c-rel/metabolismo , Receptor fas/metabolismo , Animais , Autoanticorpos/sangue , Quimiocinas/sangue , Quimiocinas/metabolismo , Dermatite/sangue , Dermatite/complicações , Dermatite/imunologia , Fatores de Transcrição Forkhead/metabolismo , Genótipo , Hipergamaglobulinemia/sangue , Hipergamaglobulinemia/complicações , Tolerância Imunológica/imunologia , Leucócitos/patologia , Longevidade , Lúpus Eritematoso Sistêmico/sangue , Doenças Linfáticas/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Subunidade p50 de NF-kappa B/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Especificidade de Órgãos , Esplenomegalia/sangue , Fatores de Transcrição/metabolismo , Proteína AIRERESUMO
FASL/FAS signaling imposes a critical barrier against autoimmune disease and lymphadenopathy. Mutant mice unable to produce membrane-bound FASL (FasL(Δm/Δm)), a prerequisite for FAS-induced apoptosis, develop lymphadenopathy and systemic autoimmune disease with immune complex-mediated glomerulonephritis. Prior to disease onset, FasL(Δm/Δm) mice contain abnormally high numbers of leukocytes displaying activated and elevated NF-κB-regulated cytokine levels, indicating that NF-κB-dependent inflammation may be a key pathological driver in this multifaceted autoimmune disease. We tested this hypothesis by genetically impairing canonical or non-canonical NF-κB signaling in FasL(Δm/Δm) mice by deleting the c-Rel or NF-κB2 genes, respectively. Although the loss of NF-κB2 reduced the levels of inflammatory cytokines and autoantibodies, the impact on animal survival was minor due to substantially accelerated and exacerbated lymphoproliferative disease. In contrast, a marked increase in lifespan resulting from the loss of c-REL coincided with a striking reduction in classical parameters of autoimmune pathology, including the levels of cytokines and antinuclear autoantibodies. Notably, the decrease in regulatory T-cell numbers associated with loss of c-REL did not exacerbate autoimmunity in FasL(Δm/Δm)c-rel(-/-) mice. These findings indicate that selective inhibition of c-REL may be an attractive strategy for the treatment of autoimmune pathologies driven by defects in FASL/FAS signaling that would be expected to circumvent many of the complications caused by pan-NF-κB inhibition.
Assuntos
Doenças Autoimunes/metabolismo , Proteína Ligante Fas/metabolismo , Mutação , Subunidade p52 de NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Receptor fas/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Doenças Autoimunes/prevenção & controle , Proteína Ligante Fas/genética , Camundongos , Camundongos Knockout , Subunidade p52 de NF-kappa B/genética , Proteínas Proto-Oncogênicas c-rel/genética , Transdução de Sinais/genética , Receptor fas/genéticaRESUMO
The complexity of the lymphostromal interplay that is essential to alphabetaT-cell development is reflected by the heterogeneity of both lymphocytes and thymic stromal cells. While panels of monoclonal antibodies have described many of the cellular components of these microenvironments, the means to quantify stromal cell subsets using flow cytometry remains poorly defined. This study refines and compares various stromal cell isolation procedures and determines the effects of various digestion enzymes on important surface molecules. Three- and four-color flow cytometry is used to correlate established and novel stromal cell markers to define thymic fibroblasts, epithelium and a unique subset of thymic endothelium that express MHC class II. This work provides a basis for the purification of thymic stromal cells for further phenotypic, functional and genetic analysis.
Assuntos
Células Estromais/citologia , Timo/citologia , Animais , Separação Celular/métodos , Citometria de Fluxo/métodos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células Estromais/imunologia , Timo/imunologiaRESUMO
T cells developing in the thymus undergo rigorous positive and negative selection to ensure that those exported to peripheral lymphoid organs bear T-cell receptors (TCRs) capable of reacting with foreign antigens but tolerant of self. At each checkpoint, whether a thymocyte survives or dies is determined by antiapoptotic and proapoptotic Bcl-2 family members. We used Mcl-1 transgenic (tg) mice to investigate the impact of elevated expression of antiapoptotic Mcl-1 on thymocyte apoptosis and selection, making a side-by-side comparison with thymocytes from BCL-2tg mice. Mcl-1 was as effective as Bcl-2 at protecting thymocytes against spontaneous cell death, diverse cytotoxic insults and TCR-CD3 stimulation-driven apoptosis. In three different TCR tg models, Mcl-1 markedly enhanced positive selection of thymocytes, as did Bcl-2. In H-Y TCR tg mice, elevated Mcl-1 and Bcl-2 were equally effective at inhibiting deletion of autoreactive thymocytes. However, in the OT-1tg model where deletion is mediated by a peripheral antigen whose expression is regulated by Aire, Mcl-1 was less effective than Bcl-2. Thus, the capacity of Mcl-1 overexpression to inhibit apoptosis triggered by TCR stimulation apparently depends on the thymocyte subset subject to deletion, presumably due to differences in the profiles of proapoptotic Bcl-2 family members mediating the deletion.