ABSTRACT
The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk-/- mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk-/- mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk-/- mice. A transgene expressing a low level of Btk in B cells (Btk(lo)) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk(lo) B cells. anti-DNA IgG was not observed in 56R.Btk(lo) mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.
Subject(s)
Antigens, Nuclear/immunology , B-Lymphocytes/immunology , Gene Rearrangement, B-Lymphocyte/immunology , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation , Protein-Tyrosine Kinases/immunology , Agammaglobulinaemia Tyrosine Kinase , Animals , Antibodies, Antinuclear/genetics , Antibodies, Antinuclear/immunology , Antibodies, Antinuclear/metabolism , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , B-Lymphocytes/enzymology , Cell Proliferation , CpG Islands/immunology , Gene Knock-In Techniques , Gene Rearrangement, B-Lymphocyte/genetics , Germinal Center/enzymology , Germinal Center/immunology , Immune Tolerance , Immunoglobulin M/genetics , Immunoglobulin M/immunology , Immunoglobulin M/metabolism , Lupus Erythematosus, Systemic/enzymology , Lupus Erythematosus, Systemic/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Mice , Mice, Knockout , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/immunology , Receptors, Antigen, B-Cell/metabolism , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/immunology , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/metabolismABSTRACT
The pre-BCR and the BCR regulate B cell development via a signalosome nucleated by the adaptor protein B cell linker protein (BLNK). Formation of this complex facilitates activation of phospholipase C (PLC) gamma2 by Bruton's tyrosine kinase (Btk). To determine whether Btk and PLCgamma2 also have separate functions, we generated Btk(-/-)PLCgamma2(-/-) mice. They demonstrated a block in development at the pre-B stage and increased pre-BCR surface expression. This phenotype was more severe than that of Btk(-/-) or PLCgamma2(-/-) mice. Although both Btk and PLCgamma2 were required for proliferation of splenic B cells in response to BCR cross-linking, they contributed differently to anti-IgM-induced phosphorylation of ERK. Btk(-/-) and PLCgamma2(-/-) mice each had a reduced frequency of Iglambda-expressing B cells and impaired migration of pre-B cells towards stromal cell-derived factor 1. However, the increase in pre-B cell malignancy that occurs in BLNK(-/-) mice in the absence of Btk was not observed in the absence of PLCgamma2. Thus, Btk and PLCgamma2 act both in concert and independently throughout B cell development.
