RESUMO
Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603-607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837-847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-alpha, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.
Assuntos
Autoantígenos/imunologia , Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/fisiologia , RNA/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Receptor 7 Toll-Like/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos de Diferenciação/genética , Autoanticorpos/biossíntese , Autoantígenos/metabolismo , Linfócitos B/metabolismo , Feminino , Hibridomas , Interferon-alfa/fisiologia , Ativação Linfocitária/genética , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide , Receptores de Antígenos de Linfócitos B/genética , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Ribonucleoproteínas/imunologia , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/fisiologiaRESUMO
AM14 B cells are a prototype for those low affinity autoreactive B cells that routinely mature as naïve cells in peripheral lymphoid tissues. These cells express a transgene-encoded receptor specific for IgG2a and can be effectively activated by immune complexes that incorporate either mammalian DNA or mammalian RNA that has been released from dead or dying cells. Activation depends on the ability of the B-cell receptor to deliver antigen to an internal vesicular compartment containing either Toll-like receptor-9 (TLR9) or TLR7. Since TLR9 and TLR7 are thought to recognize microbial DNA and RNA preferentially, it is important to determine under what conditions mammalian DNA and RNA become effective TLR ligands, and whether the determining factor is delivery or structure. This issue has been addressed by using IgG2a mAbs to deliver immune complexes preloaded with defined fragments of DNA or RNA, or by using modified ODNs/ORNs. The data demonstrate that only certain nucleic acid sequences or structures can induce autoreactive B-cell proliferation, even when delivery to the appropriate TLR compartment is facilitated by uptake through the B-cell receptor (BCR).
Assuntos
Autoantígenos/imunologia , Linfócitos B/imunologia , DNA/imunologia , RNA/imunologia , Adjuvantes Imunológicos , Animais , Sequência de Bases , Células da Medula Óssea/fisiologia , Primers do DNA , Imunoglobulina M , Cinética , Ativação Linfocitária , Macrófagos/imunologia , Mamíferos , Camundongos , Camundongos Knockout , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/fisiologiaRESUMO
Synthetic single-stranded oligodeoxynucleotides (15-30 bp) containing CpG motifs and phosphorothioate backbones (CpG s-ODN), immune complexes consisting of anti-nucleosome mAbs and mammalian chromatin (chromatin IC), and immune complexes consisting of anti-hapten mAbs and haptenated-double stranded DNA fragments ( approximately 600 bp) can all effectively stimulate transgenic B cells expressing a rheumatoid factor receptor by a TLR9-dependent process. However, differential sensitivity to both s-ODN and small molecule inhibitors suggests that stimulatory CpG sODN and chromatin IC may either access TLR9 via different routes or depend on discrete activation parameters. These data have important implications regarding the therapeutic application of TLR9 inhibitors to the treatment of systemic autoimmune diseases.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Ilhas de CpG/genética , Proteínas de Ligação a DNA/farmacologia , Fator Reumatoide/fisiologia , Animais , DNA , Inibidores Enzimáticos/farmacologia , Humanos , Macrolídeos/farmacologia , Camundongos , Oligodesoxirribonucleotídeos , Receptores de Superfície Celular , Receptor Toll-Like 9RESUMO
The proliferative response of autoreactive rheumatoid factor (RF) B cells to mammalian chromatin-containing immune complexes (ICs) results from the sequential engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9). We have used ICs constructed from anti-hapten antibodies and defined haptenated dsDNA fragments to determine the form of mammalian DNA that mediates this process. Despite their relatively low abundance in mammalian DNA, we found that inclusion of hypomethylated CpG motifs in these ICs was necessary for effective activation. In the absence of antibody, the same fragments could efficiently stimulate low-affinity hapten-specific and DNA-reactive 3H9 B cells, but not RF B cells. These results extend the BCR/TLR9 coengagement paradigm to a second major class of autoreactive B cells, further confirm the critical role of the BCR in chromatin ligand delivery to TLR9, and implicate hypomethylated CpG motifs as ligand elements necessary for the initiation of systemic autoimmune disease.