Osamu Kaminuma

Transcriptional control of the IL-5 gene by human helper T cells: IL-5 synthesis is regulated independently from IL-2 or IL-4 synthesis

BACKGROUND IL-5 is fundamentally involved in eosinophilic inflammation. Control of IL-5 production may be effective for the management of allergic diseases. OBJECTIVE We aimed to find the transcriptional mechanisms that regulate the IL-5 gene to selectively control IL-5 synthesis. METHODS Allergen-specific T-cell clones and T-cell hybridomas were established from the peripheral blood lymphocytes of patients with asthma, and the transcriptional regulation of the IL-5 gene was investigated with transient transfection and electrophoretic mobility shift analysis. RESULTS A human IL-5 promoter/enhancer-luciferase gene construct, pIL-5(-511)Luc, was transcribed on activation of IL-5-producing T-cell clones, but not IL-5-nonproducing clones. pIL-5(-511)Luc was transcribed by T-cell hybridomas derived from fusion between IL-5-producing T-cell clones and an IL-5 gene-nonexpressing T-cell line, but not by hybridomas derived from IL-5-nonproducing T-cell clones. IL-5 synthesis was not only induced by T-cell receptor stimulation but also by IL-2 receptor stimulation. Binding of NF-AT, NF-kappaB, and AP-1 was induced by T-cell receptor (TcR) stimulation, although there was no significant upregulation of binding by IL-2 stimulation. CONCLUSION IL-5 synthesis by human helper T cells is regulated at the transcriptional level. A unique transcriptional mechanism distinct from those regulating the IL-2 or IL-4 genes seems to control the IL-5 gene. Selective regulation of IL-5 gene transcription may be useful for treating eosinophlic inflammation.

Differential Contribution of NFATc2 and NFATc1 to TNF-α Gene Expression in T Cells

The NFAT family transcription factors play crucial roles in immunological and other biological events; however, the functional differences among NFAT members have not been fully elucidated. This study investigated the relative contribution of NFATc2 and NFATc1 to the transactivation of cytokine genes in T cells. Ectopic expression of NFATc2 but not NFATc1, especially its short isoform, enhanced TNF-α synthesis in human T cells at the gene transcription level, whereas both NFATs augmented IL-2 expression. In addition, a reduction of the shortest NFATc1 isoform using RNA interference technology failed to suppress TNF-α expression. The promoter/enhancer activity of the NFAT-binding site in the TNF-α gene was up-regulated by NFATc2 but not by NFATc1, whereas both NFATs associated similarly with this region. A study of mRNA expression using NFATc2/NFATc1 chimeric molecules revealed that the enhancing activity of NFAT on the TNF-α gene was lost by truncation of its C-terminal transactivation domain. In addition, this domain derived from NFATc2 behaved as a dominant negative against the NFAT site in TNF-α promoter-dependent transcriptional activity in T cells. We conclude that the C-terminal transactivation domain in NFAT is crucial for TNF-α gene expression in human T cells.

GATA-3 suppresses IFN-γ promoter activity independently of binding to cis-regulatory elements

The regulatory mechanism by which GATA‐3 suppresses IFN‐γ gene expression was investigated. A reduction of GATA‐3 using RNA interference technology enhanced, whereas overexpression of GATA‐3 suppressed IFN‐γ mRNA expression. IL‐4 expression was reciprocally affected by GATA‐3. GATA‐3‐mediated down‐regulation of IFN‐γ was achieved through the inhibition of its promoter/enhancer activity. Two GATA elements located in the cis‐regulatory elements did not contribute to the suppression of IFN‐γ promoter activity, even though they behaved as binding sites for GATA‐3. The effect of GATA‐3 on IFN‐γ promoter was lost upon removal of the region encompassing −257 to −172. Among several transcription factors putatively interacting with this region, Stat4, which enhanced IFN‐γ promoter activity, was down‐regulated by GATA‐3 at gene transcription level. Although GATA‐3 has the capacity to interact with the cis‐regulatory elements, it suppresses IFN‐γ gene transcription via down‐regulation of Stat4.

Cellular and molecular mechanisms of IL-5 synthesis in atopic diseases: A study with allergen-specific human helper T cells ☆ ☆☆ ★

BACKGROUND Cytokines produced by helper T cells are intimately involved in chronic allergic diseases associated with eosinophilic inflammation. OBJECTIVE We investigated the production of IL-5, a potent growth factor and chemotactic factor for eosinophils, by CD4+ T lymphocytes in patients with asthma. METHODS Allergen-specific T cell clones and T cell hybridomas were established from the peripheral blood lymphocytes of patients with asthma, and the responses to various stimuli were determined. RESULTS After nonspecific stimulation, IL-5 production by CD4+ T cells from both atopic and nonatopic subjects with asthma was significantly enhanced compared with that by cells from healthy controls. Peripheral blood mononuclear cells from atopic asthma patients both proliferated and produced IL-5 after incubation with mite allergen, suggesting that mite-specific helper T cells were involved in the eosinophilic inflammation of atopic asthma. A human IL-5 promoter/enhancer luciferase gene construct transfected into IL-5-producing T cell clones was clearly transcribed after stimulation, indicating that the 515 base pair IL-5 gene segment upstream of the coding region was sufficient to respond to activating signals in human helper T cells. The same gene segment was not transcribed in IL-5-nonproducing T cell clones, suggesting that human T cell IL-5 synthesis is regulated at the transcriptional level. Experiments with T cell hybridomas confirmed these findings and suggested that a unique transcription factor may be essential for human IL-5 gene transcription. CONCLUSION Enhanced IL-5 production by helper T cells seems to cause the eosinophilic inflammation of both atopic and nonatopic asthma. Elucidation of IL-5-specific regulatory mechanisms may facilitate the development of novel treatments for allergic diseases associated with eosinophilic inflammation.

Dynamics of antigen-specific helper T cells at the initiation of airway eosinophilic inflammation

Bronchial asthma is characterized by chronic eosinophilic inflammation of the bronchial mucosa in which Th2 cells play crucial roles. Ovalbumin‐reactive Th2 clones were labeled with a fluorescent‐probe then infused into unprimed mice to elucidate the dynamics of antigen‐specific T cells involved in allergic inflammation. Infiltration of not only labeled antigen‐specific T cells, but alsounlabeled nonspecific CD4+ T cells into the bronchial mucosa following inhaled antigen challenge was detectable under confocal microscopy and flow cytometry. Accordingly, labeled T cells in the spleen were decreased, whereas those in hilar lymph nodes were increased upon antigen challenge. Approximately 45% of antigen‐specific T cells that migrated into the lungs bore CD25, while another early activation marker, CD69, was expressed on 80% of the migrated T cells. Accordingly, antigen challenge to the mice induced in situ proliferation of antigen‐specific T cells as well as bronchial epithelial cells in the lungs. Expression of vascular cell adhesion molecule (VCAM)‐1, but not intercellular adhesion molecule (ICAM)‐1, on the vascular endothelium in the lungs was enhanced following antigen challenge. Nevertheless, treatment with anti‐VCAM‐1 antibody, and also anti‐ICAM‐1 antibody strongly suppressed the accumulation of T cells, suggesting that both VCAM‐1 and ICAM‐1 are essential for antigen‐stimulated T cell mobilization into peripheral tissues. Our current study visualized the kinetics and the mechanism of antigen‐specific T cell migration in response to local challenge with a protein antigen.

T-box 21 transcription factor is responsible for distorted TH2 differentiation in human peripheral CD4+ T cells

BACKGROUND Regardless of T(H)1/T(H)2 theory, CD4(+) T cells of patients with allergic asthma, a typical T(H)2 disease, and those of healthy subjects expressed equivalent levels of IFN-gamma, even though T(H)2 cytokines were significantly upregulated in asthmatic patients. OBJECTIVE The mechanisms underlying distorted T(H)2 cell polarization in human T cells were elucidated. METHODS Cytokine-producing activity and the expression of T(H)1/T(H)2-specific transcription factors in naïve, T(H)1/T(H)2, or both CD4(+) T cells derived from human peripheral and cord blood were comparatively analyzed. The mechanisms of the differential expression of T-box 21 transcription factor (T-bet) in the cells were assessed by determining the chromatin accessibility at the TBX21 gene. The functional roles of T-bet and other transcription factors in human T(H)1/T(H)2 differentiation were further investigated. RESULTS T(H)2 cells derived from naive CD4(+) T cells in peripheral blood but not in cord blood produced IFN-gamma. T-bet was expressed in peripheral, but not cord blood, resting naive T cells. Consistently, the accessibility at the proximal TBX21 gene promoter in peripheral naive T cells was higher than that in cord blood naive T cells. IFN-gamma-producing activity was induced in T(H)2-differentiated cord blood T cells by means of ectopic expression of T-bet. In addition, a reduction of T-bet in peripheral T cells suppressed IFN-gamma production. T-bet not only upregulated IFN-gamma but also downregulated IL-4 and IL-13 gene transcription, independently of the modification of T(H)1/T(H)2 balance. CONCLUSION The expression of T-bet at a naive stage is crucial for the development of IFN-gamma-producing T cells in human peripheral blood, even in T(H)2-related diseases.

Effect of T-440, a Novel Type IV Phosphodiesterase Inhibitor, on Allergen-Induced Immediate and Late Asthmatic Reaction and Leukocyte Infiltration into the Airways of Guinea Pigs

Type IV phosphodiesterase (PDE IV) is expressed in many tissues including airway smooth muscle and inflammatory cells, suggesting that this isozyme has a regulatory role in the pathogenesis of bronchial asthma. We investigated the effect of a novel selective PDE IV inhibitor, T-440, on immediate asthmatic reaction (IAR), late reaction (LAR) and leukocyte infiltration into the airways after allergen challenge in guinea pigs. Inhalation of ovalbumin by sensitized guinea pigs induced an immediate increase in specific airway resistance that peaked at 15 min. LAR was only produced in combination with chronic treatment with metyrapone, an inhibitor of the biosynthesis of adrenocortical steroids, and was suppressed by administration of dexamethasone, suggesting that the expression of LAR is dependent on an intrinsic steroid hormone. These reactions were accompanied by increases in the number of eosinophils and neutrophils recovered in bronchoalveolar lavage fluid. Oral administration of T-440 significantly inhibited IAR, LAR and the infiltration of eosinophils, whereas it suppressed neutrophil accumulation with relative low potency. These findings imply that the inhibition of PDE IV activity is a reasonable target for the management of obstructive airway diseases associated with airway inflammation such as asthma.

CD44 is critical for airway accumulation of antigen-specific Th2, but not Th1, cells induced by antigen challenge in mice.

CD44 is a cell adhesion molecule involved in lymphocyte infiltration of inflamed tissues. We previously demonstrated that CD44 plays an important role in the development of airway inflammation in a murine model of allergic asthma. In this study, we investigated the role of CD44 expressed on CD4+ T cells in the accumulation of T‐helper type 2 (Th2) cells in the airway using CD44‐deficient mice and anti‐CD44 monoclonal antibodies. Antigen‐induced Th2‐mediated airway inflammation and airway hyperresponsiveness (AHR) in sensitized mice were reduced by CD44‐deficiency. These asthmatic responses induced by the transfer of antigen‐sensitized splenic CD4+ T cells from CD44‐deficient mice were weaker than those from WT mice. Lack of CD44 failed to induce AHR by antigen challenge. Expression level and hyaluronic acid receptor activity of CD44, as well as Neu1 sialidase expression on antigen‐specific Th2 cells, were higher than those on antigen‐specific Th1 cells. Anti‐CD44 antibody preferentially suppressed the accumulation of those Th2 cells in the airway induced by antigen challenge. Our findings indicate that CD44 expressed on CD4+ T cells plays a critical role in the accumulation of antigen‐specific Th2 cells, but not Th1 cells, in the airway and in the development of AHR induced by antigen challenge.

Correlation between mRNA expression of Th1/Th2 cytokines and their specific transcription factors in human helper T-cell clones.

The mechanisms that underlie Th1/Th2 differentiation of human T cells are incompletely defined. In the present study, a panel of human T‐cell clones was used to elucidate the relationship between Th1/Th2‐specific transcription factors and cytokine production in human helper T cells. The mRNA expression level of T‐bet, a Th1‐specific transcription factor, was higher in Th1 clones than in Th2 clones. In contrast, inducible expression of Th2‐specific transcription factors (GATA‐3 and c‐Maf) in Th2 clones was higher than that in Th1 clones. The expression level of T‐bet in various T‐cell clones was positively correlated with that of IFN‐γ and negatively correlated with that of Th2 cytokines, particularly IL‐4. Interestingly, the expression of IL‐3 and IL‐13, but not of other Th2 cytokines IL‐4 and IL‐5, was strongly correlated with GATA‐3 mRNA levels. A reduction of GATA‐3 using RNA interference technology suppressed, whereas overexpression of GATA‐3 enhanced, the expression of IL‐3 and IL‐13. In conclusion, the level of T‐bet expression is correlated with Th1/Th2 polarization status, whereas GATA‐3 is a crucial factor in determining the IL‐3 and IL‐13 producing capacity of human T cells.

Selective down‐regulation of Th2 cell‐mediated airway inflammation in mice by pharmacological intervention of CCR4

The chemokine receptor CCR4 has been implicated in Th2 cell‐mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation.