B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.
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Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. We show that selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells were identified as precursors for mature B cells. Transitional B cells of type 1 (T1) are recent immigrants from the bone marrow. They develop into the transitional B cells of type 2 (T2), which are cycling and found exclusively in the primary follicles of the spleen. Mature B cells can be generated from T1 or T2 B cells. Mice with genetic deletions of elements participating in the B cell receptor signaling cascade display developmental arrest at the T1 or T2 stage. The analysis of these defects showed that the development of T2 and mature B cells from T1 precursors requires defined qualitative and quantitative signals derived from the B cell receptor and that the induction of longevity and maturation requires different signals. (+info)
Effects on the ciliated epithelium of protein D-producing and -nonproducing nontypeable Haemophilus influenzae in nasopharyngeal tissue cultures.
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A pair of isogenic, nontypeable Haemophilus influenzae strains, one expressing protein D and the other protein D-negative, was compared in their ability to cause damage in a human nasopharyngeal tissue culture model. Damage was assessed by measuring the ciliary beat frequency (CBF) of tissue specimens at 12 h intervals. Cultures inoculated with H. influenzae manifested a decrease in CBF beginning after 12 h, with a maximum decrease after 36 h. The impairment of ciliary function by the protein D-expressing strain was significantly greater than that caused by the protein D-negative mutant (P<.01). Tissue specimens examined by scanning and transmission electron microscopy after 24 h appeared normal. After 48 h of incubation, the protein D-expressing strain caused a significant loss of cilia. These findings suggest that protein D is involved in the pathogenesis of upper respiratory tract infections due to nontypeable H. influenzae, probably by enhancing functional and morphological damage to cilia. (+info)
Distinctive roles of Fyn and Lyn in IgD- and IgM-mediated signaling.
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Src family kinases Fyn and Lyn associate with the B cell antigen receptor (BCR). Accumulating data show that Lyn plays important roles in BCR-mediated signaling, while the role of Fyn remains obscure. Here we dissected the role of Fyn and Lyn in BCR signaling using B cells from fyn(-/-), lyn(-/-) and fyn/lyn double-deficient (fyn(-/-)lyn(-/-)) mice. In contrast to previous reports, fyn(-/-) B cells were slightly hyporeactive to both anti-IgM and anti-IgD-dextran. Although lyn(-/-) B cells were hyper-reactive to anti-IgM, anti-IgD-induced proliferation was impaired in lyn(-/-) B cells. Most of the other phenotypes of fyn(-/-)lyn(-/-) mice were similar to that of lyn(-/-) mice, except that proliferative responses of B cells to various stimuli, such as BCR cross-linking and lipopolysaccharide, were significantly lower in fyn(-/-)lyn(-/-) mice than in lyn(-/-) mice. Finally, immune responses to thymus-independent type 2 antigen were affected in these mutant mice. These observations suggest that Fyn and Lyn are involved in B cell functions, and play similar, but partly distinct, roles in BCR signaling. (+info)
RAG2:GFP knockin mice reveal novel aspects of RAG2 expression in primary and peripheral lymphoid tissues.
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We generated mice in which a functional RAG2:GFP fusion gene is knocked in to the endogenous RAG2 locus. In bone marrow and thymus, RAG2:GFP expression occurs in appropriate stages of developing B and T cells as well as in immature bone marrow IgM+ B cells. RAG2:GFP also is expressed in IgD+ B cells following cross-linking of IgM on immature IgM+ IgD+ B cells generated in vitro. RAG2:GFP expression is undetectable in most immature splenic B cells; however, in young RAG2:GFP mice, there are substantial numbers of splenic RAG2:GFP+ cells that mostly resemble pre-B cells. The latter population decreases in size with age but reappears following immunization of older RAG2:GFP mice. We discuss the implications of these findings for current models of receptor assembly and diversification. (+info)
Multivalent cross-linking of membrane Ig sensitizes murine B cells to a broader spectrum of CpG-containing oligodeoxynucleotide motifs, including their methylated counterparts, for stimulation of proliferation and Ig secretion.
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We have previously reported that B cells that are activated by multivalent but not bivalent membrane Ig cross-linking ligands synergize with various B cell activators culminating in enhanced B cell proliferation. In this study we asked whether B cells that are activated by a multivalent mIg cross-linking agonist could respond to oligodeoxynucleotides (ODN) containing non-stimulatory motifs. Earlier reports have shown that ODN containing a CpG motif in which the cytosine is unmethylated and is flanked by two 5' purines and two 3' pyrimidines induce high levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other than the optimal two 5' purines and two 3' pyrimidines were non-stimulatory. In this manuscript we show that when B cells are stimulated in vitro with dextran-conjugated anti-IgD antibodies (anti-IgD-dex), as the multivalent mIg ligand, their proliferation is enhanced and they can be induced to secrete Ig in response to ODN containing various non-optimal motifs, both methylated and non-methylated. Furthermore we could induce synergistic levels of proliferation with concentrations of anti-IgD-dex that were in the picomolar concentration range and with concentrations of ODN that were 10- to 100-fold less than previously reported to be necessary for mitogenic activity. These data provided a model to explain how low concentrations of a multi-epitope-expressing microorganism in the context of mammalian (methylated) or microorganism (non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion. (+info)
T(h)2 cytokine dependence of IgD production by normal human B cells.
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IgD is a minor component of serum Ig and the control of IgD secretion is virtually unknown. We measured concentrations of IgD (and IgE and IgM as controls) in culture supernatants of peripheral blood mononuclear cells (PBMC) from 60 normal donors as well as mononuclear cells from 10 tonsils following culture in the absence or presence of CD40 mAb and cytokines. Low levels of IgD were measured in cultures of PBMC, either unstimulated or stimulated by anti-CD40 antibodies. IL-4 and IL-10 significantly increased IgD production by CD40 mAb-stimulated cells in the majority of normal subjects studied, whereas in a limited number of individuals, spontaneous IgD production was either low or high, but with no increase upon stimulation. Spontaneous IgD production by tonsil-derived mononuclear cells was higher than by PBMC and increased after CD40 stimulation and even more in the presence of IL-10, but not IL-4. IL-2 and IFN-gamma exerted a dose-dependent inhibition on spontaneous as well as CD40- and cytokine-induced IgD production by PBMC, but not by tonsil mononuclear cells. Activation by IL-4 of CD40-stimulated purified B cells from tonsil and PBMC, and by IL-10 of tonsil B cells increased IgD production, whereas IL-2 and IFN-gamma had no detectable inhibitory effect. This suggests that accessory cells indirectly regulate IgD synthesis. IgD production induced in PBMC by IL-4 or IL-10 appeared to result from an active synthesis, and correlated with an increase in the number of IgD-containing plasma cells as demonstrated by immunofluorescence and increased expression of secreted IgD transcripts. These findings suggest that IgD production by normal peripheral blood human B cells is regulated positively by T(h)2 cytokines and negatively by T(h)1 cytokines. (+info)
Induction of autoimmunity in a transgenic model of B cell receptor peripheral tolerance: changes in coreceptors and B cell receptor-induced tyrosine-phosphoproteins.
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Abrogation of peripheral tolerance in transgenic mice that express a uniform B-cell receptor may create a powerful tool to examine the molecular mechanisms that underlie the autoimmune response in B cells. Here we report that processes that induce a systemic lupus erythematosus-like syndrome in normal mice, namely chronic graft vs host reaction, trigger systemic autoimmunity in a well-established transgenic mice model of B cell receptor peripheral tolerance. The induction of graft vs host reaction in mice that carry both a rearranged B cell Ag receptors specific for hen egg lysozyme and expressing chronically circulating hen egg lysozyme Ag resulted in induction of high and sustained levels of circulating anti-hen egg lysozyme autoantibodies and glomerulonephritis with proteinuria. This was associated with marked changes in expression of cell-surface proteins, such as CD23 and complement receptor 2. B cells from the graft vs host-induced mice could proliferate in vitro in response to self-Ag, and upon stimulation with anti-IgD demonstrated rapid phosphotyrosine phosphorylation of specific proteins, which could not be induced in the anergic double transgenic B cells. Conversely, loss of tolerance was not associated with a higher induction in the level of Syk kinase phosphorylation following stimulation with anti-IgD. Taken collectively, these data establish that 1) processes that induce a systemic lupus erythematosus-like syndrome in normal mice can abrogate peripheral tolerance in transgenic mice expressing self-tolerized B cells, and that 2) loss of tolerance in this model is associated with marked changes in surface expression of B cell coreceptors as well as with selective changes in IgD-induced signaling by discrete tyrosine-phosphoproteins, but not Syk kinase. (+info)
E2A activity is induced during B-cell activation to promote immunoglobulin class switch recombination.
(16/605)
The basic helix-loop-helix protein, E2A, is required for proper early B lymphopoiesis. Specifically, in E2A-deficient mice, B-cell development is blocked at the progenitor stage prior to the onset of immunoglobulin (Ig) V(D)J recombination. Here, we demonstrate that E2A plays an additional role during peripheral B lymphopoiesis. Upon activation of primary mature B lymphocytes, both E2A protein levels and DNA-binding activity are induced. Furthermore, we show that mature B cells, expressing a dominant-negative E2A antagonist, proliferate normally in response to mitogenic signaling and appropriately express the early and late activation markers CD69, CD44, IgD and B220. However, in the absence of E2A activity, B lymphocytes are blocked in their ability to express secondary Ig isotypes. We demonstrate that the defect lies at the level of DNA rearrangements between the Ig switch regions. These data suggest that E2A is an essential target during B-cell activation and its induction is required to promote Ig class switch recombination. (+info)