 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
BASIC SCIENCE |
,,#,
,
*Institute of Clinical Medicine, Institute of Basic Medical Sciences, and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan; Laboratory for Immune Receptor, RIKEN Research Center for Allergy and Immunology, Ibaraki, Japan; ||Institute for Basic Medicine, Ehime University, Ehime, Japan; ¶Department of Pathology, University of Geneva, Geneva, Switzerland; #PRESTO, Japan Science and Technology Corporation, Saitama, Japan; and **Department of Biochemistry, Molecular Biology, and Cellular Biology, Northwestern University, Evanston, Illinois.
Correspondence to Dr. Satoru Takahashi, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Phone: 81-29-853-7516; Fax: 81-29-853-6965;
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
levels. Although IL-4 production remained unchanged, intracellular cytokine analyses demonstrated that IL-5 was induced and IFN- was suppressed in stimulated T cells from the GATA-3-transgenic Yaa mice. These results indicated that abundant GATA-3 was unable to stimulate complete differentiation of Th2 cells but did counteract the dominance of Th1 cells and alleviated the disease severity in Yaa mice. These data suggest that transcriptional regulation therapy may have potential as an effective strategy for treating autoimmune glomerulonephritis. E-mail: satoruta@md.tsukuba.ac.jp |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13. The differentiation of the Th subsets requires the activity of distinct transcription factors (11). The transcription factors T-bet and GATA-3 are thought to be key regulators of Th1/Th2 differentiation. T-bet expression is strongly correlated with IFN- expression and is specifically upregulated in primary Th cells that differentiate along the Th1 but not the Th2 pathway (12). In contrast, GATA-3 plays an indispensable role during Th2 cell differentiation. GATA-3 is a member of the GATA family of zinc-finger transcription factors that bind the GATA consensus motif (13,14). GATA-3 is proposed to be important for early T cell development and to be predominantly responsible for late Th2 cellular differentiation (15–20). A number of studies have attempted to control cellular pathways that contribute to autoimmune responses and the subsequent inflammatory cascade that leads to progressive autoimmune disease (1). One hypothesis suggested that correction of the Th1/Th2 imbalance might represent an effective treatment for autoimmune disease. A number of reports have described approaches to improve the imbalance of the Th1 and Th2 cell populations. These approaches include the administration of cytokines or anticytokine agents that counteract the effects of cytokines secreted by Th1 or Th2 cells (1,2,5,21,22), the targeting of autoantigens to the surface of B cells for antigen presentation (23), and the administration of autoantigens (24,25). However, whether Th1/Th2 imbalances can be corrected with internal metabolic alterations, through exploitation of the activity of specific transcription factors that are known to affect T cell differentiation, has not been clearly demonstrated.
BXSB/MpJ-Yaa (Y chromosome-linked autoimmune acceleration) mice, which display a dominant Th1 background, represent a murine model for autoimmune glomerulonephritis; the Th1 imbalance is thought to be the factor responsible for the observed pathogenesis (26,27). The Yaa gene apparently modifies the type of IgG autoantibody response, so that the production of IgG2a and IgG3 antibodies is increased and the production of IgG1 antibody is decreased (26). Th1 cells support macrophage activation, delayed-typed hypersensitivity responses, and Ig isotype switching to IgG2a and IgG3. In contrast, Th2 cells provide efficient help for B cell activation and class switching to IgG1 and IgE antibodies (5,28). The Yaa gene upregulates Th1 responses over Th2 responses, thereby enhancing IgG2a and IgG3 antibody production and downregulating IgG1 antibody production (26,27).
In this study, we established GATA-3-overexpressing C57BL/6 x BXSB/MpJ-Yaa F1 (Yaa) transgenic mice, with the goal of determining whether T cell-mediated transcriptional responses might alter the pathogenesis in genetically defined Yaa animals. The data demonstrate that the Th1-dominant condition of these mice can be alleviated with alterations of the transcriptional cascades within Th cells, decreasing the severity of glomerulonephritis and extending the life span of mice with a BXSB/MpJ-Yaa genetic background.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Southern Hybridization Analysis of Genomic DNA
High-molecular weight DNA was prepared from the tail of each mouse, and 10 µg of DNA was digested with EcoRI and ClaI and then subjected to electrophoresis on 0.8% agarose gels. After electrophoresis, the DNA was transferred to a nylon membrane (Zeta-probe; Bio-Rad, Richmond, CA). Southern hybridization was performed by using a 32P-labeled SacII/ClaI fragment (0.1 kb) of the GATA-3 gene as a probe. Transgene copy number was determined from the blot with a BAS 1500 Mac image analyzer.
Expression of GATA-3 Protein from the Transgene
Thymocyte nuclear extracts were prepared from 10-wk-old GATA-3-transgenic mice or transgene-negative (non-Tg) littermates. The extracts were size-fractionated on a 15% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (FluoroTrans), and reacted with primary and secondary antibodies. For detection of the GATA-3 protein, a mAb (HG3-31; Santa Cruz Biochemicals, Santa Cruz, CA) was used as the primary antibody and peroxidase-conjugated goat anti-rat IgG (Zymed Laboratories, South San Francisco, CA) was used as the secondary antibody. For normalization with respect to the amount of protein in each sample, anti-laminin B antibody (Santa Cruz Biochemicals) was used as a control antibody.
Urinary Protein Excretion and Serum Creatinine Concentration Measurements
The urine of 30-wk-old mice was collected in individual metabolic cages during a 24-h period. The methods for urinary protein determination and measurement of serum creatinine levels were described previously (30). Protein values of >2 mg/24 h were considered abnormal. Serum creatinine values of >0.5 mg/dl were considered abnormal.
Histopathologic Analyses of Renal Tissues
Organs were fixed with 10% formalin in 0.01 M phosphate buffer (pH 7.2) and embedded in paraffin. Sections (3 µm) were stained with hematoxylin and eosin for histopathologic examinations with light microscopy. For semiquantitative histologic analyses, >20 glomeruli from each kidney section were examined. The severity of the glomerular changes was graded on a scale of 0 to 3, as previously reported (30) (Figure 1). Sections frozen for immunofluorescence analyses were stained with FITC-labeled anti-mouse Ig (ICN Pharmaceuticals, Cleveland, OH).
|
(Genzyme-Techne, Cambridge, MA).
Serologic and Anti-Double-Stranded DNA Antibody Assays
Serum levels of Ig and anti-double-stranded DNA (dsDNA) antibody were determined with ELISA, as described previously (30). To measure IgG3 levels, single-radial immunodiffusion assays were performed. Agarose (1%) gel in 0.1 M PBS (pH 7.2) containing anti-Ig isotype serum (rabbit anti-mouse IgG3; Miles Laboratories, Elkhart, IN) was prepared and spread on a glass plate. Samples (4 ml each) were applied to punched holes (diameter, 1.5 mm), and the slides were incubated at room temperature for 48 h. Precipitation rings were measured after staining with Aminoblock 10B.
Culture Medium, Cytokines, and Antibodies
RPMI 1640 medium supplemented with 10% FCS, 2-mercaptoethanol (0.05 mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), HEPES buffer (10 mM), and sodium pyruvate (1 mM) was used as culture medium. Recombinant mouse cytokines were IL-2 (Genzyme-Techne), IL-4 (BD PharMingen, San Diego, CA), and IL-12 (BD PharMingen). Purified rat anti-mouse IL-4 (11B11), IL-12 (C17.8), CD3
(145-2C11), and CD28 (37.51) mAb, phycoerythrin-conjugated anti-mouse IL-4 (11B11), allophycocyanin-conjugated anti-mouse IL-5 (TRFK5), and FITC-conjugated anti-mouse IFN- (XMG1.2) were purchased from BD PharMingen.
Preparation of T Cells
CD4+ T cells were prepared from each mouse spleen and were enriched by positive selection, using a magnetically activated cell-sorting system with anti-CD4 mAb. CD4+ T cells were enriched >98%, as analyzed by flow cytometry.
Stimulation of Transgenic CD4+ T Cells for Cytokine Production
Primary stimulations of CD4+ T cells (2.5 x 105 cells/well) were performed with crosslinked anti-CD3 (1 µg/ml) and anti-CD28 (10 µg/ml) plus IL-2 (10 ng/ml), in a total volume of 2 ml, in 24-well plates. In addition, some cultures received cytokines (10 ng/ml IL-4 or 10 ng/ml IL-12) or mAb to block endogenous cytokines (10 µg/ml anti-IL-4 or 10 µg/ml anti-IL-12). T cells were expanded and maintained under constant culture conditions for 1 wk.
Flow Cytometric Analysis of Intracellular IL-4, IL-5, and IFN- Synthesis
Cells were resuspended at 105 to 106 cells/ml and were stimulated with PMA (50 ng/ml) plus ionomycin (500 ng/ml). Two hours before cell harvesting, brefeldin A was added at 10 µg/ml, using a stock solution of 1 mg/ml in ethanol (100%). Cells were harvested, washed, and resuspended in PBS with brefeldin A before the addition of an equal volume of 4% formaldehyde fixative (final concentration, 2%). After fixation for 20 min at room temperature, cells were stained for cytokines. For intracellular staining, all reagents and washes contained 1% BSA and 0.5% saponin (Sigma Chemical Co., St. Louis, MO), and all incubations were performed at room temperature. Cells were washed and preincubated for 10 min in PBS/BSA/saponin and were then incubated with phycoerythrin-conjugated anti-mouse IL-4 (5 µg/ml), allophycocyanin-conjugated anti-mouse IL-5 (5 µg/ml), and anti-mouse IFN- (5 µg/ml), or isotype-matched control antibodies (10 µg/ml), for 30 min. After 20 min, cells were washed twice with PBS/BSA/saponin and were then washed with PBS/BSA without saponin, to allow membrane closure. Samples were analyzed with a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA). Results were analyzed by using Cellquest software.
Statistical Analyses
All data are expressed as means ± SEM. Multiple data comparisons were performed by using one-way ANOVA. P values of <0.05 were considered statistically significant. Comparisons of survival rates were performed with the Kaplan-Meier method, with differences in survival curves being evaluated with log-rank sum testing.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Longer Life Span of Yaa Mice Overexpressing GATA-3
During an 80-wk period of observation, we observed that both non-Tg and transgenic mice developed renal failure and died. Further analysis of the transgenic mouse cohort demonstrated that GATA-3-overexpressing Yaa transgenic mice exhibited longer life spans than did non-Tg mice (Figure 3). Non-Tg, Tg 379, and Tg 720 mice began to die at 17.6, 15.0, and 24.1 wk, respectively. Fifty percent mortality incidence occurred at 30.9 wk for non-Tg mice but at 35.4 and 41.6 wk for Tg 379 and Tg 720 mice, respectively. Ninety percent mortality incidence occurred at 47.7 wk for non-Tg mice but at 73.7 wk for Tg 379 mice. Ninety percent mortality incidence did not occur for Tg 720 mice within the 80-wk observation period. Yaa mice began to die within 20 wk and demonstrated 50% and 90% mortality incidences at 30.9 and 47.7 wk, respectively, yielding a survival curve comparable to that for BXSB/MpJ-Yaa mice, as previously reported (27,31–34). More specifically, in earlier reports we demonstrated that 50% mortality incidence occurred at approximately 8 mo and 90% mortality incidence occurred within 12 mo for BXSB/MpJ-Yaa mice. Although both non-Tg and transgenic mice exhibited progression to renal failure, the times to 50% and 90% mortality incidences for transgenic mice were longer than those for non-Tg mice, and Tg 720 mice displayed significantly longer life spans in the survival analysis than did non-Tg mice, presumably because of elevated GATA-3 expression.
|
|
|
Lower Serum IFN- Levels in GATA-3-Transgenic Yaa Mice
To extend our observations to the cytokine level, we measured the serum levels of IL-4 and IFN- (Table 1). The IL-4 levels in the GATA-3-transgenic Yaa mice were higher than those in non-Tg mice but not significantly. In contrast, the serum IFN- levels in the transgenic mice were significantly lower than those in non-Tg mice (P < 0.01).
Diminished Synthesis of IFN- in GATA-3-Transgenic Yaa Mice
To confirm the observed differences in cytokine production at the single-cell level, we studied the intracellular synthesis of cytokines in GATA-3-transgenic Yaa mice (Tg 720 mice) with flow cytometry. CD4+ T cells from non-Tg mice demonstrated higher levels of IFN- than did those from Tg 720 mice under conditions with medium alone (Figure 5). In contrast, IL-4 levels in non-Tg mice were similar to those in Tg 720 mice. CD4+ T cells from Tg 720 mice demonstrated lower levels of IFN- in the presence of anti-IL-4 antibody and IL-12 (Th1 differentiation conditions). However, the production of IL-4 in cells from Tg 720 mice was similar to that of non-Tg mice in the presence of anti-IL-12 antibody and IL-4 (Th2 differentiation conditions). The production of IL-5 in cells from Tg 720 mice was significantly greater than that of non-Tg mice with culture either in medium alone or under Th2 differentiation conditions. These results demonstrated that T cells from Yaa mice exhibited a Th1-dominant differentiation pattern and overexpression of GATA-3 prevented Th1 differentiation. However, prevention of Th1 differentiation was not sufficient to promote full Th2 differentiation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production. The results suggest that GATA-3 overexpression can modulate Th1 cytokine production in vivo.
It is well established that a specific genetic background is necessary for the full development of autoimmune disease induced by the Yaa gene (27). In this experiment, it could be argued that some loci in the BDF1 or C56BL/6 mouse backgrounds might act as resistance genes for autoimmune disease. However, two independent transgenic lines displayed suppression of disease development and those two lines expressed different levels of GATA-3, as indicated in Table 1 and Figures 2 and 3
. Furthermore, the level of suppression in each line was correlated with the transgene copy number and the protein expression level, and non-Tg mice within each intercross developed autoimmune disease at rates virtually identical to those for the original BXSB/MpJ-Yaa mice. Fifty percent and 90% mortality incidences for non-Tg mice were consistent with those for BXSB/MpJ-Yaa mice (27,31–34). Therefore, the development of glomerulonephritis by non-Tg mice was attributable to the genetic BXSB/MpJ-Yaa background. Also, our results strongly indicated that the effects of suppression on autoimmune disease were attributable solely to expression of the GATA-3 transgene.
In a transgenic mouse system, Zheng and Flavell (19) used reverse transcription-PCR to demonstrate that elevated GATA-3 levels in CD4+ T cells activated Th2 cytokine gene expression in developing Th1 cells. With a retrovirus system, Ferber et al. (35) and Ouyang et al. (36) reported that GATA-3 significantly downregulated IFN- production during in vitro Th1 differentiation of naive CD4+ T cells, through downregulation of IL-12 receptor 
2 and IFN- production, independent of IL-4 production. GATA-3 has been reported to trans-activate the IL-5 promoter but to have only limited effects on IL-4 gene transcription (19,37,38). It has also been reported that GATA-3 regulates the locus accessibility of the IL-4 and IL-13 genes with chromatin remodeling (39,40). On the basis of those reports, it might be concluded that GATA-3 is an important transcription factor for Th2 cytokine secretion. In this study, we observed higher serum IL-4 levels in transgenic mice, although levels were not significantly different from those of non-Tg mice. However, IFN- levels were significantly lower in transgenic mice, compared with non-Tg mice. FACS analysis revealed stimulation of IL-5 production and suppression of IFN- production in individual T cells from transgenic mice. We also demonstrated, in intracellular cytokine analyses, that low levels of IL-4 production in transgenic mice were similar to those in non-Tg mice, although under Th2 cell differentiation conditions. Intracellular cytokine analyses also demonstrated that non-Tg mice could secrete IFN- under Th2 differentiation conditions. These results suggest that Yaa mice have a strong Th1 background. Therefore, the high level of IL-5 secretion and the suppression of IFN- production might be independent of IL-4. Furthermore, overexpression of GATA-3 might have prevented Th1 differentiation but not supported full Th2 differentiation in the Yaa mice. Such a possibility might explain why overexpression of GATA-3 could extend the survival times of Yaa mice and reduce the severity of glomerulonephritis but not completely reverse the disease phenotype.
The Th1/Th2 paradigm is a useful description of autoimmune disease. Inhibition of GATA-3 activity in T cells with overexpression of a dominant-negative mutant of GATA-3 in transgenic mice improved asthma (41), which is a Th2 model disease (42). In contrast, T-bet is reported to be specifically upregulated in primary Th cells differentiated along the Th1 pathway (12). Finotto et al. (43) reported that T-bet deficiency, in the absence of allergen exposure, induced a murine phenotype reminiscent of both acute and chronic human asthma. It would be interesting to determine whether T-bet deficiency or T-bet overexpression in Yaa mice could result in synergistic effects. There are currently >600 gene therapy trials throughout the world, as documented on the Gene Therapy Clinical Trials web page maintained by the Journal of Gene Medicine (www.wiley.co.uk/genemd). However, the potential use of gene therapy for the treatment of autoimmune disease has not been fully explored. In the area of autoimmune disease, gene therapy strategies can be divided into three approaches (44). The first is targeting of a known genetic defect. The second is delivery of immune-modulating molecules, such as IL-4, IL-10, cytokine antagonists, and soluble cytokine receptors, all of which antagonize proinflammatory autoimmune reactions. The third is interference with signaling processes involved in autoimmune reactions, for example, T cell receptor signaling and costimulation or apoptosis pathways. Furthermore, one group described the use of Th2-like transfected T cells that produced transgenic IL-10 in an antigen-inducible manner, and those authors reported that such a strategy was effective in ameliorating ongoing experimental autoimmune encephalomyelitis (45). As an alternative to these strategies, Th1/Th2 cell regulation by transcription factors is a novel approach for gene therapy. With transcription factor regulation, it should be possible not only to control Th cell differentiation but also to modulate cytokine and Ig production. Although transcription factor regulation therapy cannot currently be used for the treatment of autoimmune disease among human patients, future studies may open possibilities for this novel approach for therapeutic intervention. This study suggests that analyses of transcriptional regulation and transcription factor function may provide new therapies for the treatment of autoimmune disease.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene enhancer. Mol Cell Biol 11: 2778–2784, 1991, interleukin 4, and prostaglandin E expression in the brain. J Exp Med 176: 1355–1364, 1992
chain. J Exp Med 178: 1189–1197, 1993 production from developing Th1 cells in addition to inducing IL-4 and IL-5 levels. Clin Immunol 91: 134–144, 1999[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  M. Stojakovic, L. I. Salazar-Fontana, Z. Tatari-Calderone, V. P. Badovinac, F. R. Santori, D. Kovalovsky, D. Sant'Angelo, J. T. Harty, and S. Vukmanovic Adaptable TCR Avidity Thresholds for Negative Selection J. Immunol., November 15, 2008; 181(10): 6770 - 6778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Matsuno, Y. Ishii, K. Yoh, Y. Morishima, N. Haraguchi, N. Kikuchi, T. Iizuka, T. Kiwamoto, S. Homma, A. Nomura, et al. Overexpression of GATA-3 Protects against the Development of Hypersensitivity Pneumonitis Am. J. Respir. Crit. Care Med., November 15, 2007; 176(10): 1015 - 1025. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rotondi, L. Chiovato, S. Romagnani, M. Serio, and P. Romagnani Role of Chemokines in Endocrine Autoimmune Diseases Endocr. Rev., August 1, 2007; 28(5): 492 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ishizaki, A. Yamada, K. Yoh, T. Nakano, H. Shimohata, A. Maeda, Y. Fujioka, N. Morito, Y. Kawachi, K. Shibuya, et al. Th1 and Type 1 Cytotoxic T Cells Dominate Responses in T-bet Overexpression Transgenic Mice That Develop Contact Dermatitis J. Immunol., January 1, 2007; 178(1): 605 - 612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Genton, Y. Wang, S. Izui, B. Malissen, G. Delsol, G. J. Fournie, M. Malissen, and H. Acha-Orbea The Th2 Lymphoproliferation Developing in LatY136F Mutant Mice Triggers Polyclonal B Cell Activation and Systemic Autoimmunity J. Immunol., August 15, 2006; 177(4): 2285 - 2293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kiwamoto, Y. Ishii, Y. Morishima, K. Yoh, A. Maeda, K. Ishizaki, T. Iizuka, A. E. Hegab, Y. Matsuno, S. Homma, et al. Transcription Factors T-bet and GATA-3 Regulate Development of Airway Remodeling Am. J. Respir. Crit. Care Med., July 15, 2006; 174(2): 142 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kimura, Y. Ishii, K. Yoh, Y. Morishima, T. Iizuka, T. Kiwamoto, Y. Matsuno, S. Homma, A. Nomura, T. Sakamoto, et al. Overexpression of the Transcription Factor GATA-3 Enhances the Development of Pulmonary Fibrosis Am. J. Pathol., July 1, 2006; 169(1): 96 - 104. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
n (with 
