ABSTRACT
One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.
Subject(s)
Antibodies/immunology , B-Lymphocytes/immunology , Germ Cells/immunology , Germinal Center/immunology , Immunologic Memory/immunology , Signal Transduction/immunology , Animals , Antibodies/genetics , Antibodies/metabolism , B-Lymphocytes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Female , Flow Cytometry , Gene Expression Profiling , Germ Cells/metabolism , Germinal Center/metabolism , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Immunoglobulin Variable Region/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-bcl-6 , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Signal Transduction/geneticsABSTRACT
Finding a means to ameliorate and prevent bone destruction is one of the urgent issues in the treatment of rheumatoid arthritis. Recent studies revealed bone-resorbing osteoclasts to be essential for arthritic bone destruction, but to date there has been scarce experimental evidence for the underlying mechanism of the bone-protective effect of antirheumatic drugs. Here we examined the effects of one or a combination of disease-modifying antirheumatic drugs (DMARDs) on osteoclast differentiation to provide a cellular and molecular basis for their efficacy against bone destruction. The effects on osteoclast precursor cells and osteoclastogenesis-supporting cells were distinguished by two in vitro osteoclast culture systems. Methotrexate (MTX), bucillamine (Buc) and salazosulphapyridine (SASP) inhibited osteoclastogenesis by acting on osteoclast precursor cells and interfering with receptor activator of NF-kappaB ligand (RANKL)-mediated induction of the nuclear factor of activated T cells (NFAT) c1. MTX and SASP also suppressed RANKL expression on osteoclastogenesis-supporting mesenchymal cells. Interestingly, the combination of three antirheumatic drugs exerted a marked inhibitory effect on osteoclastogenesis even at a low dose at which there was much less of an effect when administered individually. These results are consistent with the reported efficacy of combined DMARDs therapy in humans and suggest that osteoclast culture systems are useful tools to provide an experimental basis for the bone-protective effects of antirheumatic drugs.
Subject(s)
Antirheumatic Agents/pharmacology , Arthritis, Rheumatoid/complications , Arthritis, Rheumatoid/drug therapy , Bone Resorption/drug therapy , Bone Resorption/etiology , Cell Differentiation/drug effects , Animals , Bone Resorption/immunology , Cells, Cultured , Coculture Techniques , Cysteine/analogs & derivatives , Cysteine/pharmacology , Dose-Response Relationship, Drug , Drug Therapy, Combination , Methotrexate/pharmacology , Mice , Mice, Inbred C57BL , NFATC Transcription Factors/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects , RANK Ligand/drug effects , RANK Ligand/metabolism , Sulfasalazine/pharmacologyABSTRACT
Our previous studies showed that expression of the GalNAcbeta1-->4GlcNAc group on N-linked oligosaccharides is associated with functional differentiation of the bovine mammary gland. In the present study, the occurrence of the GalNAcbeta1-->4GlcNAc group was established in human milk proteins and membrane glycoproteins from a human breast cancer cell line, MRK-nu-1, by structural analysis of oligosaccharides released by hydrazinolysis. Whether the expression level of the disaccharide group is affected upon malignant transformation was examined in human breast cancer specimens using Wistaria floribunda agglutinin (WFA) which interacts with oligosaccharides with N-acetylgalactosamine at their termini. Lectin blot analysis of membrane glycoprotein samples from human breast cancer specimens showed that the number of protein bands reacting with WFA, as well as their intensities, are lower in samples from primary carcinoma lesions compared with samples from surrounding normal tissues. No lectin binding was observed when the blots were treated with jack bean beta-N-acetylhexosaminidase or N-glycanase, indicating that WFA-reactive oligosaccharides are N-linked. A histochemical study of tissue specimens from 92 patients with breast cancer revealed that the reduced WFA staining levels in primary carcinoma lesions correlate with advancing clinical stages and prognostic status (i.e., 58% of patients in a group showing reduced/negative staining died of disease recurrence, whereas more than 90% of those in the positive staining group survived for 5 years after surgery). These results indicate that reduced expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides on primary carcinoma lesions predicts a poor prognosis for patients with breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Disaccharides/metabolism , Female , Histocytochemistry , Humans , Membrane Glycoproteins/chemistry , Plant Lectins/metabolism , Receptors, N-Acetylglucosamine , Tumor Cells, CulturedABSTRACT
The role of phosphatidylinositol (PI) 3-kinase in the regulation of pancreatic beta-cell function was investigated. PI 3-kinase activity in p85 alpha regulatory subunit-deficient (p85 alpha(-/-)) islets was decreased to approximately 20% of that in wild-type controls. Insulin content and mass of rough endoplasmic reticula were decreased in beta-cells from p85 alpha(-/-) mice with increased insulin sensitivity. However, p85 alpha(-/-) beta-cells exhibited a marked increase in the insulin secretory response to higher concentrations of glucose. When PI 3-kinase in wild-type islets was suppressed by wortmannin or LY294002, the secretion was also substantially potentiated. Wortmannin's potentiating effect was not due to augmentation in glucose metabolism or cytosolic [Ca(2+)] elevation. Results of p85 alpha(-/-) islets and wortmannin-treated wild-type islets stimulated with diazoxide and KCl showed that inhibition of PI 3-kinase activity exerted its effect on secretion, at least in part, distal to a cytosolic [Ca(2+)] elevation. These results suggest that PI 3-kinase activity normally plays a crucial role in the suppression of glucose-stimulated insulin secretion.