Noriko Kitamura

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.

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.

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.

Eosinophils Are Required for the Induction of Bronchial Hyperresponsiveness in a Th Transfer Model of BALB/c Background

Background: Helper T (Th) cells are deeply involved in the pathophysiology of bronchial asthma, such as eosinophilic inflammation, bronchial hyperresponsiveness (BHR), airflow limitation and remodeling. It is still unclear whether Th cells contribute to BHR independently of eosinophilic inflammation. The double GATA (dblGATA) site is a high-affinity GATA-binding site in the GATA-1 promoter. dblGATA site-deficient (ΔdblGATA) mice lack eosinophils. Method: Ovalbumin (OVA)-reactive Th clones were transferred into ΔdblGATA and wild-type (WT) mice of BALB/c background. The number of eosinophils in the bronchoalveolar lavage fluid (BALF) and bronchial responsiveness to methacholine were examined after OVA challenge. Results: The number of BALF eosinophils was significantly increased in WT mice, but not detectable in ΔdblGATA mice. BHR was also induced in WT mice, but significantly attenuated in ΔdblGATA mice. Conclusion: Eosinophils are involved in T-cell-mediated BHR.

A contraction assay system using established human bronchial smooth muscle cells.

Background: To further understand the mechanisms of airway obstruction in asthma, it is crucial to investigate contractile responses of human airway smooth muscle. An in vitro assay system employing collagen gels embedded with well-established bronchial smooth muscle cells of human origin was explored in the present study. Methods: Commercially available cultured human bronchial smooth muscle cells were embedded into a collagen gel. Well-known constrictors, histamine and methacholine, were added to the gel. The gel images were captured by an image analyzer, and contractile responses were evaluated. Results: Histamine and methacholine induced contraction of the gels in a dose-dependent manner. Pyrilamine, an H1 receptor antagonist, inhibited gel contraction in an agonist-specific manner. Conclusion: Our contraction assay system, employing widely distributed cultured cells, was highly reproducible and precise. It may go a long way toward understanding mechanisms of asthmatic responses and evaluation of antiasthma drugs.

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.

The expression of granulysin in systemic anaplastic large cell lymphoma in childhood

The expression of granulysin, a cytolytic protein produced by activated T and NK cells, has been revealed to be correlated with the prognosis of some adult cancer patients. By examination on various childhood lymphoma tissues, we found that granulysin level was especially high in systemic anaplastic large cell lymphoma (ALCL) cases, whereas no close correlation with the expression of CD96, a marker for activated T and NK cells, was observed. We further demonstrated that both ALCL cells in biopsy specimens and cell lines established from ALCL express granulysin, indicating some correlation of granulysin with biological features of ALCL.

Role of GATA-3 in IL-5 Gene Transcription by CD4+ T Cells of Asthmatic Patients

Background: Helper T cells and T cell cytokines play central roles in allergic disorders including bronchial asthma. We reported enhanced IL-5 production by peripheral blood T cells of asthmatic patients. A transcription factor, GATA-3, has been implicated in IL-5 gene expression. This study was undertaken to clarify the role of GATA-3 in the upregulation of IL-5 synthesis in asthmatic patients. Method: Peripheral CD4+ T cells were transfected with an IL-5 promoter reporter construct as well as its mutants in the presence or absence of a GATA-3 expression vector. Messenger RNA expression level of GATA-3 in CD4+ T cells of asthmatic subjects was compared to that of healthy donors. Results: IL-5 promoter activity in CD4+ T cells was enhanced by overexpression of GATA-3, whereas it was diminished by the introduction of mutations in the putative GATA-3 binding sites. The GATA-3 expression level in CD4+ T cells of asthmatic patients was equivalent to that of healthy controls. Conclusion: The expression level of GATA-3 may not be an essential factor to cause IL-5 hyperproduction in bronchial asthma, though GATA-3 is crucially involved in IL-5 gene transcription in human peripheral CD4+ T cells.