ABSTRACT
Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin, and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here, we show that anti-PF4/heparin antibodies are readily generated in wild-type mice on challenge with PF4/heparin complexes, and that antibody production is severely impaired in B-cell-specific Notch2-deficient mice that lack marginal zone (MZ) B cells. As expected, Notch2-deficient mice responded normally to challenge with T-cell-dependent antigen nitrophenyl-chicken γ globulin but not to the T-cell-independent antigen trinitrophenyl-Ficoll. In addition, wild-type, but not Notch2-deficient, B cells plus B-cell-depleted wild-type splenocytes adoptively transferred into B-cell-deficient µMT mice responded to PF4/heparin complex challenge. PF4/heparin-specific antibodies produced by wild-type mice were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular, B cells adoptively transferred into B-cell-deficient µMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells are critical for PF4/heparin-specific antibody production.
Subject(s)
Antibody Formation , Autoantibodies/immunology , B-Lymphocytes/immunology , Heparin/immunology , Platelet Factor 4/immunology , Thrombocytopenia/immunology , Adoptive Transfer , Animals , Anticoagulants/adverse effects , Anticoagulants/immunology , Antigen-Presenting Cells/immunology , Autoantibodies/blood , B-Lymphocytes/chemistry , Coagulants/adverse effects , Coagulants/immunology , Flow Cytometry , Heparin/adverse effects , Immunization , Immunoglobulin Class Switching/immunology , Immunoglobulin G/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Factor 4/adverse effects , Receptor, Notch2/physiology , Thrombocytopenia/chemically induced , Thrombocytopenia/diagnosisABSTRACT
Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase ß, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-x(L), a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , B-Cell Activating Factor/immunology , Cell Survival/immunology , NF-kappa B/immunology , Signal Transduction , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis , B-Cell Activating Factor/genetics , B-Cell CLL-Lymphoma 10 Protein , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Survival/genetics , Clonal Anergy , Gene Expression Regulation/immunology , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , Mice , Mice, Transgenic , Muramidase/immunology , NF-kappa B/genetics , NF-kappa B p52 Subunit/genetics , NF-kappa B p52 Subunit/immunology , Phosphorylation , Proto-Oncogene Proteins c-rel/genetics , Proto-Oncogene Proteins c-rel/immunology , Spleen/cytology , Spleen/immunology , Spleen/metabolism , Transcription Factor RelB/genetics , Transcription Factor RelB/immunology , bcl-X Protein/genetics , bcl-X Protein/immunologyABSTRACT
Recent work has identified a new subset of CD4(+) T cells named as Tfh cells that are localized in germinal centers and critical in germinal center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and B cell lymphoma 6 (Bcl6) is specifically expressed in Tfh cells and required for their lineage specification. In the current study, we characterized the role of STAT5 in Tfh cell development. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors (CXC motif) receptor 5 (CXCR5), musculoaponeurotic fibrosarcoma (c-Maf), Bcl6, basic leucine zipper transcription factor ATF-like (Batf), and IL-21, and STAT5 deficiency greatly enhanced Tfh gene expression. Importantly, STAT5 regulated the expression of Tfh cell suppressor factor B lymphocyte-induced maturation protein 1 (Blimp-1); STAT5 deficiency impaired Blimp-1 expression and resulted in elevated expression of Tfh-specific genes. Similarly, inhibition of IL-2 potentiated Tfh generation, associated with dampened Blimp-1 expression; Blimp-1 overexpression inhibited Tfh gene expression in Stat5-deficient T cells, suggesting that the IL-2/STAT5 axis functions to regulate Blimp-1 expression. In vivo, deletion of STAT5 in CD4(+) T cells resulted in enhanced development of Tfh cells and germinal center B cells and led to an impairment of B cell tolerance in a well defined mouse tolerance model. Taken together, this study demonstrates that STAT5 controls Tfh differentiation.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation , STAT5 Transcription Factor/metabolism , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Mice , Positive Regulatory Domain I-Binding Factor 1 , STAT5 Transcription Factor/deficiency , Transcription Factors/genetics , Up-RegulationABSTRACT
The N-terminal nuclear export sequence (NES) of inhibitor of nuclear factor kappa B (NF-κB) alpha (IκBα) promotes NF-κB export from the cell nucleus to the cytoplasm, but the physiological role of this export regulation remains unknown. Here we report the derivation and analysis of genetically targeted mice harboring a germline mutation in IκBα NES. Mature B cells in the mutant mice displayed nuclear accumulation of inactive IκBα complexes containing a NF-κB family member, cRel, causing their spatial separation from the cytoplasmic IκB kinase. This resulted in severe reductions in constitutive and canonical NF-κB activities, synthesis of p100 and RelB NF-κB members, noncanonical NF-κB activity, NF-κB target gene induction, and proliferation and survival responses in B cells. Consequently, mice displayed defective B cell maturation, antibody production, and formation of secondary lymphoid organs and tissues. Thus, IκBα nuclear export is essential to maintain constitutive, canonical, and noncanonical NF-κB activation potentials in mature B cells in vivo.
Subject(s)
B-Lymphocytes/pathology , I-kappa B Proteins/metabolism , Immunologic Deficiency Syndromes/genetics , Lymphoid Tissue/pathology , Nuclear Export Signals/physiology , Active Transport, Cell Nucleus , Animals , B-Lymphocytes/metabolism , Cell Death , Cell Division , Gene Expression Regulation/genetics , Germ-Line Mutation , I-kappa B Kinase/metabolism , I-kappa B Proteins/genetics , Immunologic Deficiency Syndromes/immunology , Immunologic Deficiency Syndromes/pathology , Lymph Nodes/pathology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Nuclear Export Signals/genetics , Organ Size , Peyer's Patches/pathology , Proto-Oncogene Proteins c-rel/metabolism , Spleen/pathology , Transcription, GeneticABSTRACT
The kinase TAK1 is essential for T-cell receptor (TCR)-mediated nuclear factor kappaB (NF-kappaB) activation and T-cell development. However, the role of TAK1 in B-cell receptor (BCR)-mediated NF-kappaB activation and B-cell development is not clear. Here we show that B-cell-specific deletion of TAK1 impaired the transition from transitional type 2 to mature follicular (FO) B cells and caused a marked decrease of marginal zone (MZ) B cells. TAK1-deficient B cells exhibited an increase of BCR-induced apoptosis and impaired proliferation in response to BCR ligation. Importantly, TAK1-deficient B cells failed to activate NF-kappaB after BCR stimulation. Thus, TAK1 is critical for B-cell maturation and BCR-induced NF-kappaB activation.
Subject(s)
B-Lymphocytes/metabolism , MAP Kinase Kinase Kinases/physiology , NF-kappa B/metabolism , Receptors, Antigen, B-Cell/metabolism , Animals , Apoptosis/physiology , Blotting, Western , Cell Nucleus/metabolism , Electrophoretic Mobility Shift Assay , Flow Cytometry , Fluorescent Antibody Technique , Mice , Mice, Knockout , Signal TransductionABSTRACT
B-cell development is orchestrated by complex signaling networks. Rap1 is a member of the Ras superfamily of small GTP-binding proteins and has 2 isoforms, Rap1a and Rap1b. Although Rap1 has been suggested to have an important role in a variety of cellular processes, no direct evidence demonstrates a role for Rap1 in B-cell biology. In this study, we found that Rap1b was the dominant isoform of Rap1 in B cells. We discovered that Rap1b deficiency in mice barely affected early development of B cells but markedly reduced marginal zone (MZ) B cells in the spleen and mature B cells in peripheral and mucosal lymph nodes. Rap1b-deficient B cells displayed normal survival and proliferation in vivo and in vitro. However, Rap1b-deficient B cells had impaired adhesion and reduced chemotaxis in vitro, and lessened homing to lymph nodes in vivo. Furthermore, we found that Rap1b deficiency had no marked effect on LPS-, BCR-, or SDF-1-induced activation of mitogen-activated protein kinases and AKT but clearly impaired SDF-1-mediated activation of Pyk-2, a key regulator of SDF-1-mediated B-cell migration. Thus, we have discovered a critical and distinct role of Rap1b in mature B-cell trafficking and development of MZ B cells.
Subject(s)
B-Lymphocytes/physiology , Chemotaxis, Leukocyte , rap GTP-Binding Proteins/physiology , rap1 GTP-Binding Proteins/physiology , Animals , B-Lymphocytes/cytology , Chemokine CXCL12/metabolism , Focal Adhesion Kinase 2/metabolism , Lymph Nodes/cytology , Mice , rap GTP-Binding Proteins/deficiency , rap1 GTP-Binding Proteins/deficiencyABSTRACT
The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B(-/-) fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3(-/-) mice. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B(-/-) fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.