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1.
Exp Biol Med (Maywood) ; 229(6): 494-502, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15169968

ABSTRACT

The suppression of the B cell population during senescence has been considered to be due to the suppression of interleukin-7 (IL-7) production and responsiveness to IL-7; however, the upregulation of transforming growth factor-beta (TGF-beta) was found to contribute to B cell suppression. To investigate the mechanism of this suppression based on the interrelationship between IL-7 and TGF-beta during senescence, senescence-accelerated mice (SAMs), the mouse model of aging, were used in this study to elucidate the mechanisms of B lymphopoietic suppression during aging. Similar to regular senescent mice, SAMs showed a decrease in the number of IL-7-responding B cell progenitors (i.e., colony-forming unit pre-B [CFU-pre-B] cells in the femoral bone marrow [BM]). A co-culture system of B lymphocytes and stromal cells that the authors established showed a significantly lower number of CFU-pre-B cells harvested when BM cells were co-cultured with senescent stromal cells than when they were co-cultured with young stromal cells. Interestingly, cells harvested from a senescent stroma and those from the control culture without stromal cells were higher in number than those harvested from a young stroma, thereby implying that an altered senescent stromal cell is unable to maintain self-renewal of the stem cell compartment. Because TGF-beta is supposed to suppress the proliferative capacity of pro-B/pre-B cells, we added a neutralizing anti-TGF-beta antibody to the co-culture system with a pro-B/pre-B cell-rich population to determine whether such suppression may be rescued. However, unexpectedly, any rescue was not observed and the number of CFU-pre-B cells remained unchanged when BM cells were co-cultured with senescent stromal cells compared with the co-culture with young stromal cells, which essentially showed an increase in the number of CFU-pre-B cells (P < 0.001 in 5 microg/ml). Furthermore, TGF-beta protein level in the supernatant of cultured senescent stroma cells was evaluated by enzyme-linked immunoabsorbent assay, but surprisingly, it was found that TGF-beta concentration was significantly lower than that of cultured young stromal cells. Thus, TGF-beta activity was assumed to decline particularly in a senescent stroma, which means a distinct difference between the senescent suppression of B lymphopoiesis and secondary B lymphocytopenia. Concerning proliferative signaling, on the other hand, the level of IL-7 gene expression in cells from freshly isolated BM decreased significantly with age. Therefore, the acceleration of proliferative signaling and the deceleration of suppressive signaling may both be altered and weakened in a senescent stroma (i.e., homeosuppression).


Subject(s)
B-Lymphocytes/cytology , Cellular Senescence/physiology , Interleukin-7/biosynthesis , Lymphopoiesis/physiology , Stromal Cells/metabolism , Transforming Growth Factor beta/metabolism , Animals , Antibodies, Monoclonal/pharmacology , B-Lymphocytes/metabolism , Cell Differentiation , Cell Division , Colony-Forming Units Assay , Femur/cytology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Homeostasis , Mice , Mice, Inbred AKR , RNA, Messenger/biosynthesis , Stromal Cells/immunology , Time Factors , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/immunology
2.
J Exp Med ; 198(9): 1427-37, 2003 Nov 03.
Article in English | MEDLINE | ID: mdl-14581609

ABSTRACT

Pax5 activity is enhanced in activated B cells and is essential for class switch recombination (CSR). We show that inhibitor of differentiation (Id)2 suppresses CSR by repressing the gene expression of activation-induced cytidine deaminase (AID), which has been shown to be indispensable for CSR. Furthermore, a putative regulatory region of AID contains E2A- and Pax5-binding sites, and the latter site is indispensable for AID gene expression. Moreover, the DNA-binding activity of Pax5 is decreased in Id2-overexpressing B cells and enhanced in Id2(-/-) B cells. The kinetics of Pax5, but not E2A, occupancy to AID locus is the same as AID expression in primary B cells. Finally, enforced expression of Pax5 induces AID transcription in pro-B cell lines. Our results provide evidence that the balance between Pax5 and Id2 activities has a key role in AID gene expression.


Subject(s)
Cytidine Deaminase/genetics , DNA-Binding Proteins/physiology , Gene Expression Regulation, Enzymologic/physiology , Repressor Proteins , Transcription Factors/physiology , Animals , Base Sequence , DNA , DNA Primers , Inhibitor of Differentiation Protein 2 , Mice , Mice, Knockout , Molecular Sequence Data , PAX5 Transcription Factor , Promoter Regions, Genetic , Sequence Homology, Nucleic Acid
3.
J Biol Chem ; 277(23): 20234-42, 2002 Jun 07.
Article in English | MEDLINE | ID: mdl-11925429

ABSTRACT

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca(2+), and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 microm tubulin. The Ca(2+) dependence mainly resulted from Ca(2+) binding to the Ca(2+) ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca(2+). These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca(2+) concentrations.


Subject(s)
Calcium-Binding Proteins , Calcium/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Tubulin/metabolism , Animals , Chromatography, Affinity , Protein Binding , Recombinant Proteins/metabolism , Swine , Synaptotagmin I , Synaptotagmins
4.
Int Immunol ; 14(2): 167-75, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11809736

ABSTRACT

T(h)1 cells but not T(h)2 cells accumulate at the inflamed gastric mucosa (GM), while both subsets co-exist in the regional lymph node (RLN) in a murine experimental model for autoimmune gastritis (AIG). To understand the relationship between the immuno-microenvironment and effector localization in GM versus RLN of AIG-bearing mice, cells or tissue sections were stained with several mAb against adhesion molecules. The expression of RNA of various cytokines at these contrasting sites was also assessed. IFN-gamma-producing memory CD4(+) (T(h)1) and CD8(+) T cells as well as IL-12-producing mature macrophages which express P-selectin ligand and/or alpha(4)beta(7)-integrin selectively accumulated in the inflamed GM. Vessel endothelium at the site of infiltration expressed those counter-receptors, P-selectin and mucosal adressin cell adhesion molecule-1. Therefore, the tissue destruction of target tissue in autoimmune diseases might be promoted by a vicious circle between the selective accumulation of type 1 effectors mediated by multiple adhesion molecules and following an unusual type 1-biased microenvironment away from the type 2 response.


Subject(s)
Autoimmune Diseases/immunology , Gastritis/immunology , Integrins/analysis , Membrane Glycoproteins/analysis , Th1 Cells/physiology , Animals , Cell Adhesion Molecules , Immunoglobulins/analysis , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Macrophages/physiology , Mice , Mice, Inbred BALB C , Mucoproteins/analysis
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