TGF-beta1 is well known to induce Ig germ-line alpha (GLalpha) transcription and subsequent IgA isotype class switching recombination (CSR). Homeodomain protein TG-interacting factor (TGIF) and E3-ubiquitin ligases TGIF interacting ubiquitin ligase 1 (Tiul1) are implicated in the negative regulation of TGF-beta signaling. In the present study, we investigated the roles of Tiul1 and TGIF in TGFbeta1-induced IgA CSR. We found that over-expression of Tiul1 decreased TGFbeta1-induced GLalpha promoter activity and strengthened the inhibitory effect of Smad7 on the promoter activity. Likewise, overexpression of TGIF also diminished GLalpha promoter activity and further strengthened the inhibitory effect of Tiul1, suggesting that Tiul1 and TGIF can down-regulate TGFbeta1-induced GLalpha expression. In parallel, overexpression of Tiul1 decreased the expression of endogenous IgA CSR-predicitive transcripts (GLT(alpha), PST(alpha), and CT(alpha)) and TGFbeta1-induced IgA secretion, but not GLT(gamma3) and IgG3 secretion. Here, over-expressed TGIF further strengthened the inhibitory effect of Tiul1. These results suggest that Tiul1 and TGIF act as negatively regulators in TGFbeta1-induced IgA isotype expression.
Down-Regulation ; Immunoglobulin A ; Immunoglobulin Class Switching ; Immunoglobulin G ; Ligases ; Recombination, Genetic ; Transforming Growth Factor beta ; Transforming Growth Factor beta1 ; Ubiquitin

BACKGROUND: Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. METHODS: In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. RESULTS: First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-beta1-induced germ line transcript alpha (GLTalpha) and IL-4-induced GLTgamma1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. CONCLUSION: Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.
B-Lymphocytes ; Cell Proliferation ; Germ Cells ; Immunoglobulin A ; Immunoglobulin Class Switching ; Immunoglobulin G ; Oligodeoxyribonucleotides ; Recombination, Genetic ; Toll-Like Receptors

It is well established that TGF-beta1 and retinoic acid (RA) cause IgA isotype switching in mice. We recently found that lactoferrin (LF) also has an activity of IgA isotype switching in spleen B cells. The present study explored the effect of LF on the Ig production by mouse peritoneal B cells. LF, like TGF-beta1, substantially increased IgA production in peritoneal B1 cells but little in peritoneal B2 cells. In contrast, LF increased IgG2b production in peritoneal B2 cells much more strongly than in peritoneal B1 cells. LF in combination with RA further enhanced the IgA production and, interestingly, this enhancement was restricted to IgA isotype and B1 cells. Similarly, the combination of the two molecules also led to expression of gut homing molecules alpha4beta7 and CCR9 on peritoneal B1 cells, but not on peritoneal B2 cells. Thus, these results indicate that LF and RA can contribute to gut IgA response through stimulating IgA isotype switching and expression of gut-homing molecules in peritoneal B1 cells.
Animals ; B-Lymphocytes ; Immunoglobulin A* ; Immunoglobulin Class Switching ; Immunoglobulin G ; Lactoferrin* ; Mice ; Spleen ; Transforming Growth Factor beta1 ; Tretinoin*

Dectin-1 is a major receptor that recognizes fungal cell wall β-glucan. We previously reported that heat-killed Saccharomyces cerevisiae (HKSC), a Dectin-1 agonist, selectively induces IgG1 class switching in mouse B cells. Dectin-1 is also expressed on human B cells; however, Dectin-1 function in human B cells remains unknown. This study aimed to investigate the direct effect of in vitro stimulation using HKSC on Ig class switching in human B cells. HKSC selectively induced the expression of germline γ4 transcripts (GLTγ4) by human B cell line 2E2, and HKSC significantly augmented GLTγ4 promoter activity. Moreover, HKSC selectively enhanced GLTγ4 expression and IgG4 production by anti-CD40-activated human tonsillar resting B cells. Thus, these results suggest that Dectin-1 maybe involved in selective IgG4 class switching by human B cells.
Animals ; B-Lymphocytes* ; Cell Line ; Cell Wall ; Humans* ; Immunoglobulin Class Switching ; Immunoglobulin G* ; In Vitro Techniques ; Mice ; Saccharomyces cerevisiae* ; Saccharomyces*

Lymph nodes, one of peripheral lymphoid organs, are the sites, where the lymphocytes receive their initial instructions for producing effector functioning resulting in humoral or cell-mediated immunity. Each lymph node consists of an outer cortex in which there are aggregates of cells constituting the follicles, B-cell areas. Some follicles have central areas called germinal centers, which stain lightly. Germinal centers are B lymphoblast cell areas arising eccentrically in primary lymphoid follicles in response to T-cell dependent antigenic stimulation and are the generally accepted sites of generation of memory B cells and undergoing isotype switching and somatic mutation. We observed the morphologic, cellular, protein and molecular events arising in mouse popliteal lymph nodes in response to T-cell dependent antigenic stimulation. In this study mice were immunized into footpads with TNP-chicken ovalbumin. The germinal center formation in primary follicles of popliteal lymph nodes was first observed 6 days after immunization and germinal centers persisted until 24 days of immunization. Lymph node cells were stained with PE-labeled anti-B220 antibody and/or FITC labeled PNA and analyzed by using FACScan. B cells (B220(+) cell) in lymph node increased after 3 days and peaked between 6 and 18 days after immunization. The proportion of germinal center B cells (B220, PNA(high) cells) among lymph node B cells was low (2%) before immunization but increased at day 6 (9%) and reached the peak (30%) at day 18. The expression of IgG1 productive mRNAs and germline transcripts were observed by using RT-PCR. The expression of IgG1 productive mRNA was detected at day 10 and continued until 24 days after immunization. The expression of IgG1 germline transcripts was observed 10 days after immunization and rapidly declined over the next one week. IgG1 anti-TNP antibody, main isotype of anti-TNP antibodies, was first detected at day 14 and reached the peak level 24 days after immunization. Taken these data together, we can conclude that the first immunological event observed from mouse popliteal lymph node in response to T-cell dependent antigenic stimulation is the increase in the number of B cells, and this event is followed by appearance of germinal center B cells and at the same time by the formation of germinal center in primary lymphoid follicles. Once the germinal center is formed, the process of isotype switching to IgG1 occurs in lymph node and antigen-specific IgG1 antibody is produced.
Animals ; Antibodies ; B-Lymphocytes ; Fluorescein-5-isothiocyanate ; Germinal Center ; Immunity, Cellular ; Immunization ; Immunoglobulin Class Switching ; Immunoglobulin G ; Immunoglobulins* ; Lymph Nodes* ; Lymphocytes ; Memory ; Mice ; Ovalbumin ; RNA, Messenger ; T-Lymphocytes

Vitamin C, one of essential micronutrients, has been reported to modulate the humoral immune responses in some mammals. We investigated whether vitamin C might modulate this response in mice by directly affecting B cells. Splenic B cells were isolated and activated by CD40- and B cell receptor-ligation in vitro. The cells were cultured with a pretreatment of vitamin C from 0 to 1 mM of concentrations. Vitamin C slightly increased apoptosis of B cells dose-dependently and behaved as an antioxidant. We found that in vivo administration of vitamin C by intraperitoneal injection affected isotype switching as previously reported: the titer of antigen-specific IgG1 antibody was decreased, while that of IgG2a was unaffected. Somewhat different from those observed in vivo, in vitro exposure to vitamin C slightly decreased isotype switching to IgG1 and increased isotype switching to IgG2a. Pretreatment with vitamin C in the safe range did not affect either proliferation of cultured B cells or the expression of CD80 and CD86 in those cells. Taken together, in vivo results suggest that vitamin C acts to modulate isotype switching in the mouse. However, because of our in vitro results, we suggest that the modulation exerted by vitamin C in vivo is by indirectly affecting B cells, perhaps by directly influencing other immune cells such as dendritic cells.
Animals ; Apoptosis ; Ascorbic Acid ; B-Lymphocytes ; Dendritic Cells ; Immunity, Humoral ; Immunoglobulin Class Switching ; Immunoglobulin G ; Injections, Intraperitoneal ; Mammals ; Mice ; Micronutrients ; Reactive Oxygen Species ; Vitamins

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTgamma1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.
Alum Compounds ; Aluminum Hydroxide ; Animals ; B-Lymphocytes ; Cell Count ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Humans ; Hydroxides ; Immunoglobulin Class Switching ; Immunoglobulin G ; Mice ; Spleen ; Vaccines

BACKGROUND: It is well known that IgA isotype switching is induced by TGF-beta1. LPS-activated mouse normal B cells well differentiate into IgA secreting plasma cells under the influence of TGF-beta1. Nevertheless, there are lots of difficulties in studying normal B cells in detail because it is not simple to obtain highly purified B cells, showing low reproducibility and transfection efficacy, moreover impossible to keep continuous culture. To overcome these obstacles, it is desperately needed to develop B cell line which acts like normal B cells. In the present study, we investigated whether CH12F3-2A lymphoma cells are appropriate for studying IgA isotype switching event. METHODS: CH12F3-2A B cell line was treated with LPS and TGF-beta1, then levels of germ-line (GL) transcripts were measured by RT-PCR, and GLalpha promoter activity was measured by luciferase assay. In addition, membrane IgA (mIgA) expression and IgA secretion were determined by FACS and ELISA, respectively. RESULTS: TGF-beta1, regardless of the presence of LPS, increased level of GLalpha transcripts but not GLgamma2b transcripts. However, IgA secretion was increased dramatically by co-stimulation of LPS and TGF-beta1. Both mIgA and IgA secretion in the presence of TGF-beta1 were further increased by over-expression of Smad3/4. Finally, GLalpha promoter activity was increased by TGF-beta1. CONCLUSION: CH12F3-2A cell line acts quite similarly to the normal B cells which have been previously reported regarding IgA expression. Thus, CH12F3-2A lymphoma cell line appears to be adequate for the investigation of the mechanism(s) of IgA isotype switching at the cellular and molecular levels.
Animals ; B-Lymphocytes ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulin A* ; Immunoglobulin Class Switching* ; Luciferases ; Lymphoma* ; Membranes ; Mice* ; Plasma Cells ; Transfection ; Transforming Growth Factor beta1

BACKGROUND AND OBJECTIVES: High total serum IgE level is one of the characteristics seen in allergic rhinitis. IL-13 provides impetus to immunoglobulin class switching to IgE. The IL-13 promoter single nucleotide polymorphism has been shown to be associated with allergic diseases and abnormal IL-13 production. We tested whether a polymorphism in the coding region of IL-13 gene is associated with allergic rhinitis, blood eosinophil counts and total serum IgE levels in Korean. SUBJECTS AND METHOD: Blood samples for genetic analysis were obtained from 307 individuals with allergic rhinitis and from 268 healthy subjects without atopic diseases. Polymerase chain reaction-based assay for IL-13 exon 4 G2044A was used for genotyping. Serum total IgE levels were determined by using the immunoassay. Eosinophil values were determined by eosinophil numbers per total cell numbers per microl. RESULTS: There were no differences in the frequencies of the genotypes of IL-13 in the controls and patients (p>0.05). The frequencies of the IL-13 exon 4 2044A allele were statistically different between controls and patients (p<0.05). Blood eosinophil count and total serum IgE levels were not statistically different in the genotypes of IL-13 exon 4 G2044A in allergic rhinitis (p>0.05). CONCLUSION: Our result suggests that the IL-13 exon 4 G2044A polymorphism might give susceptibility to the development of allergic rhinitis in Koreans.
Alleles ; Cell Count ; Clinical Coding ; Eosinophils ; Exons* ; Genotype ; Humans ; Immunoassay ; Immunoglobulin Class Switching ; Immunoglobulin E ; Interleukin-13* ; Polymorphism, Single Nucleotide ; Rhinitis*

Production of thymus-dependent antibodies by autoreactive B cells requires help from T cells. Follicular helper T (Tfh) cells are a unique lineage of CD4+ T subsets present in the follicles of peripheral lymphoid tissues which functions primarily to provide help to cognate B cells. Within germinal centers Tfh cells stimulate germinal center B cells to undergo affinity maturation, Ig class switching, and differentiation to memory B cells and plasma cells. Proposals that activity of Tfh cells is crucial for long-lived humoral autoimmunity are supported by the correlation of numbers and/or functions of Tfh cells with disease activity in many autoimmune disorders. In this review, we discuss recent findings regarding Tfh cell development and function. In addition, we discuss putative roles of Tfh cells in the pathogenesis and highlight the potential of Tfh cells as therapeutic targets in autoimmune diseases.
Antibodies ; Autoimmune Diseases ; Autoimmunity ; B-Lymphocytes ; Germinal Center ; Immunity, Humoral ; Immunoglobulin Class Switching ; Lymphoid Tissue ; Memory ; Plasma Cells ; T-Lymphocytes ; T-Lymphocytes, Helper-Inducer