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Expression of the retinoblastoma protein RbAp48 in exocrine glands leads to Sjögren's syndrome-like autoimmune exocrinopathy.

Ishimaru N, Arakaki R, Yoshida S, Yamada A, Noji S, Hayashi Y - J. Exp. Med. (2008)

Bottom Line: Recently, we found that retinoblastoma-associated protein 48 (RbAp48) induces tissue-specific apoptosis in the exocrine glands depending on the level of estrogen deficiency.Surprisingly, we obtained evidence that salivary and lacrimal epithelial cells can produce interferon-gamma (IFN-gamma) in addition to interleukin-18, which activates IFN regulatory factor-1 and class II transactivator.These results indicate a novel immunocompetent role of epithelial cells that can produce IFN-gamma, resulting in loss of local tolerance before developing gender-based autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Molecular Pathology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.

ABSTRACT
Although several autoimmune diseases are known to develop in postmenopausal women, the mechanisms by which estrogen deficiency influences autoimmunity remain unclear. Recently, we found that retinoblastoma-associated protein 48 (RbAp48) induces tissue-specific apoptosis in the exocrine glands depending on the level of estrogen deficiency. In this study, we report that transgenic (Tg) expression of RbAp48 resulted in the development of autoimmune exocrinopathy resembling Sjögren's syndrome. CD4(+) T cell-mediated autoimmune lesions were aggravated with age, in association with autoantibody productions. Surprisingly, we obtained evidence that salivary and lacrimal epithelial cells can produce interferon-gamma (IFN-gamma) in addition to interleukin-18, which activates IFN regulatory factor-1 and class II transactivator. Indeed, autoimmune lesions in Rag2(-/-) mice were induced by the adoptive transfer of lymph node T cells from RbAp48-Tg mice. These results indicate a novel immunocompetent role of epithelial cells that can produce IFN-gamma, resulting in loss of local tolerance before developing gender-based autoimmunity.

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Expressions of IL-18, IFN-γ, and MHC class II (HLA-DR) in HSG cells when treated with Tam and transfected with pCMV-RbAp48. (A) Inhibitory effects of 10−9 M 17β-estradiol (E) or 10 μM caspase 1 inhibitor (Ci) on RbAp48-induced IL-18, IFN-γ, and HLA-DR in HSG cells. IL-18 and IFN-γ of the culture supernatants were detected by ELISA. HLA-DR is shown as mean fluorescence intensity (MFI) by flow cytometric analysis. *, P < 0.05; **, P < 0.005, versus RbAp48-induced. Data are means ± SD of triplicate samples, and representative of two independent experiments. (B) Tam- or RbAp48-induced IL-18 and IFN-γ were detected by confocal microscopic analysis. IL-18, IFN-γ mAbs, and Alexa Fluor 488– or Alexa Fluor 568–conjugated anti–mouse IgG were used. Images are representative of three independent experiments. (C) IFN-γ secretion or production of HSG cells by the addition of recombinant IL-18 was detected by ELISA or intracellular flow cytometric analysis. Data are representative of three independent experiments. (D) IFN-γ expression of IL-18–stimulated HSG cells was detected by confocal microscopic analysis together with cytokeratin and DAPI stainings. Images are representative of three independent experiments. (E) Caspase 1 activity of lacrimal, salivary glands, and spleen from RbAp48-Tg mice at 28 wk of age. Data are shown as means ± SE of four mice in two independent experiments, relative to those of WT mice. *, P < 0.05; **, P < 0.005; WT versus RbAp48-Tg mice. Bars: (B) 20 μm; (D) 50 μm.
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fig6: Expressions of IL-18, IFN-γ, and MHC class II (HLA-DR) in HSG cells when treated with Tam and transfected with pCMV-RbAp48. (A) Inhibitory effects of 10−9 M 17β-estradiol (E) or 10 μM caspase 1 inhibitor (Ci) on RbAp48-induced IL-18, IFN-γ, and HLA-DR in HSG cells. IL-18 and IFN-γ of the culture supernatants were detected by ELISA. HLA-DR is shown as mean fluorescence intensity (MFI) by flow cytometric analysis. *, P < 0.05; **, P < 0.005, versus RbAp48-induced. Data are means ± SD of triplicate samples, and representative of two independent experiments. (B) Tam- or RbAp48-induced IL-18 and IFN-γ were detected by confocal microscopic analysis. IL-18, IFN-γ mAbs, and Alexa Fluor 488– or Alexa Fluor 568–conjugated anti–mouse IgG were used. Images are representative of three independent experiments. (C) IFN-γ secretion or production of HSG cells by the addition of recombinant IL-18 was detected by ELISA or intracellular flow cytometric analysis. Data are representative of three independent experiments. (D) IFN-γ expression of IL-18–stimulated HSG cells was detected by confocal microscopic analysis together with cytokeratin and DAPI stainings. Images are representative of three independent experiments. (E) Caspase 1 activity of lacrimal, salivary glands, and spleen from RbAp48-Tg mice at 28 wk of age. Data are shown as means ± SE of four mice in two independent experiments, relative to those of WT mice. *, P < 0.05; **, P < 0.005; WT versus RbAp48-Tg mice. Bars: (B) 20 μm; (D) 50 μm.

Mentions: The expression of MHC class II molecules is generally regulated at the transcriptional levels, including the transcription factor IFN regulatory factor (IRF)-1 (28, 29) and the class II transactivator (CIITA), which is the master regulator for MHC class II gene expression (30, 31). It has been shown that IRF-1 is a primary responsible gene of the IFN-γ response (32). In in vitro studies using human salivary gland (HSG) cells (33), IFN-γ–induced mRNAs of IRF-1 and CIITA were significantly enhanced by treatment with Tamoxifen (Tam), which is an antagonist of estrogen and can induce RbAp48 (23), or transfection of pCMV-RbAp48 plasmid in the dose-dependent manner (Fig. 5 A), not in MCF-7 cells (human mammary gland cell line; Fig. S5, A and B, available at http://www.jem.org/cgi/content/full/jem.20080174/DC1). In addition, we next analyzed the IRF-1 promoter activity using RbAp48-transfected HSG cells with and without IFN-γ by luciferase assay. We observed significantly enhanced IRF-1 promoter activity in RbAp48-transfected HSG cells with IFN-γ, not in MCF-7 cells (Fig. 5 B). Surprisingly, in RbAp48-Tg mice, a prominent expression of IFN-γ was detected in salivary and lacrimal epithelial cells besides sporadically positive infiltrating cells of RbAp48-Tg mice, not WT mice (Fig. 5 C). These findings were observed mainly in the MHC class II+ ductal epithelium adjacent to lymphoid infiltrates. Epithelial IFN-γ expression in the exocrine glands of RbAp48-Tg mice was up-regulated during the course of autoimmune exocrinopathy. Induction of IFN-γ expression may occur through many different types of stimulation, including cross-linking of cell-surface receptors and stimulation with cytokines, including IL-2, -12, and -18 (34). It has been demonstrated that IFN-γ synthesis is predominantly induced by stimulation with IL-18 (35). Consistent with a previous study (36), IL-18 expression was observed in salivary epithelial cells in RbAp48-Tg mice, not in WT mice (Fig. 5 D). Confocal analysis revealed that differential expression of IL-18 and IFN-γ was clearly observed, i.e., IL-18 mainly in the acinar cells and IFN-γ in the duct cells, within salivary epithelial cells from RbAp48-Tg mice, but not from WT mice (Fig. 5 D). Controls using isotype antibodies were shown in Fig. S4 C. Epithelial IFN-γ and IL-18 productions were confirmed by flow cytometry using MSG cells without immune cells from RbAp48-Tg, not from WT mice, whereas there was no difference in both IFN-γ and IL-18 expressions of cLN cells between WT and RbAp48-Tg mice (Fig. 5 E). Although the production of IL-18 in salivary gland cells was detected in a previous study (36), there has been no proof for IFN-γ production of salivary gland cells in any paper. Therefore, to confirm IFN-γ production of exocrine glands, detection of IFN-γ using tissue homogenates was performed. A high concentration of IFN-γ was detected in the tissue homogenates of lacrimal and salivary glands from RbAp48-Tg mice, compared with that from WT mice, by ELISA (Fig. S6 A). Furthermore, the detection of IFN-γ mRNA of MSG cells was performed by in situ hybridization using the RNA probe of mouse IFN-γ gene. A more intense signal for IFN-γ mRNA in duct cells of salivary glands from RbAp48-Tg mice was observed compared with that from WT mice (Fig. 5 F). As for the expression of BAFF, which is an inducer of IFN-γ in B cells, the expression of epithelial cells was undetectable in both RbAp48-Tg and WT mice (Fig. S7). In vitro studies using HSG cells demonstrated that the expressions of IL-18, IFN-γ, and MHC class II (HLA-DR) were observed when treated with Tam or transfected with pCMV-RbAp48, whereas they were inhibited when treated with 17β-estradiol (E2), caspase 1 inhibitor (Ac-YVAD-CHO; Ci), and siRNA of RbAp48 (si; Fig. 6 A). Confocal analysis confirmed the expression of IL-18 and IFN-γ in HSG cells treated with Tam or transfected with pCMV-RbAp48 (Fig. 6 B). It is important to note that IL-18 is secreted earlier (by 6 h) than IFN-γ production and HLA-DR expression (by 12 h) in Tam-stimulated and RbAp48-transfected HSG cells (Fig. S8). The most prominent function of IL-18 is its capacity to act as a potent costimulus for IFN-γ production (37–39). Indeed, we observed an increase in IFN-γ production in HSG cells treated with recombinant IL-18 in the dose-dependent manner, but not in MCF-7 cells (Fig. S9, available at http://www.jem.org/cgi/content/full/jem.20080174/DC1), by ELISA and flow cytometry (Fig. 6 C). Confocal analysis of IFN-γ production of HSG cells in response to IL-18 together with cytokeratin as an identified marker was shown in Fig. 6 D. Moreover, we found a significant up-regulation of caspase 1 activity in lacrimal and salivary glands from RbAp48-Tg mice relative to that from WT mice (Fig. 6 E). In this regard, we reported previously significantly increased caspase 1 activity in salivary gland tissues from ovariectomized (Ovx) C57BL/6 mice in vivo (22) and Tam-stimulated and RbAp48-transfected HSG cells in vitro (23).


Expression of the retinoblastoma protein RbAp48 in exocrine glands leads to Sjögren's syndrome-like autoimmune exocrinopathy.

Ishimaru N, Arakaki R, Yoshida S, Yamada A, Noji S, Hayashi Y - J. Exp. Med. (2008)

Expressions of IL-18, IFN-γ, and MHC class II (HLA-DR) in HSG cells when treated with Tam and transfected with pCMV-RbAp48. (A) Inhibitory effects of 10−9 M 17β-estradiol (E) or 10 μM caspase 1 inhibitor (Ci) on RbAp48-induced IL-18, IFN-γ, and HLA-DR in HSG cells. IL-18 and IFN-γ of the culture supernatants were detected by ELISA. HLA-DR is shown as mean fluorescence intensity (MFI) by flow cytometric analysis. *, P < 0.05; **, P < 0.005, versus RbAp48-induced. Data are means ± SD of triplicate samples, and representative of two independent experiments. (B) Tam- or RbAp48-induced IL-18 and IFN-γ were detected by confocal microscopic analysis. IL-18, IFN-γ mAbs, and Alexa Fluor 488– or Alexa Fluor 568–conjugated anti–mouse IgG were used. Images are representative of three independent experiments. (C) IFN-γ secretion or production of HSG cells by the addition of recombinant IL-18 was detected by ELISA or intracellular flow cytometric analysis. Data are representative of three independent experiments. (D) IFN-γ expression of IL-18–stimulated HSG cells was detected by confocal microscopic analysis together with cytokeratin and DAPI stainings. Images are representative of three independent experiments. (E) Caspase 1 activity of lacrimal, salivary glands, and spleen from RbAp48-Tg mice at 28 wk of age. Data are shown as means ± SE of four mice in two independent experiments, relative to those of WT mice. *, P < 0.05; **, P < 0.005; WT versus RbAp48-Tg mice. Bars: (B) 20 μm; (D) 50 μm.
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fig6: Expressions of IL-18, IFN-γ, and MHC class II (HLA-DR) in HSG cells when treated with Tam and transfected with pCMV-RbAp48. (A) Inhibitory effects of 10−9 M 17β-estradiol (E) or 10 μM caspase 1 inhibitor (Ci) on RbAp48-induced IL-18, IFN-γ, and HLA-DR in HSG cells. IL-18 and IFN-γ of the culture supernatants were detected by ELISA. HLA-DR is shown as mean fluorescence intensity (MFI) by flow cytometric analysis. *, P < 0.05; **, P < 0.005, versus RbAp48-induced. Data are means ± SD of triplicate samples, and representative of two independent experiments. (B) Tam- or RbAp48-induced IL-18 and IFN-γ were detected by confocal microscopic analysis. IL-18, IFN-γ mAbs, and Alexa Fluor 488– or Alexa Fluor 568–conjugated anti–mouse IgG were used. Images are representative of three independent experiments. (C) IFN-γ secretion or production of HSG cells by the addition of recombinant IL-18 was detected by ELISA or intracellular flow cytometric analysis. Data are representative of three independent experiments. (D) IFN-γ expression of IL-18–stimulated HSG cells was detected by confocal microscopic analysis together with cytokeratin and DAPI stainings. Images are representative of three independent experiments. (E) Caspase 1 activity of lacrimal, salivary glands, and spleen from RbAp48-Tg mice at 28 wk of age. Data are shown as means ± SE of four mice in two independent experiments, relative to those of WT mice. *, P < 0.05; **, P < 0.005; WT versus RbAp48-Tg mice. Bars: (B) 20 μm; (D) 50 μm.
Mentions: The expression of MHC class II molecules is generally regulated at the transcriptional levels, including the transcription factor IFN regulatory factor (IRF)-1 (28, 29) and the class II transactivator (CIITA), which is the master regulator for MHC class II gene expression (30, 31). It has been shown that IRF-1 is a primary responsible gene of the IFN-γ response (32). In in vitro studies using human salivary gland (HSG) cells (33), IFN-γ–induced mRNAs of IRF-1 and CIITA were significantly enhanced by treatment with Tamoxifen (Tam), which is an antagonist of estrogen and can induce RbAp48 (23), or transfection of pCMV-RbAp48 plasmid in the dose-dependent manner (Fig. 5 A), not in MCF-7 cells (human mammary gland cell line; Fig. S5, A and B, available at http://www.jem.org/cgi/content/full/jem.20080174/DC1). In addition, we next analyzed the IRF-1 promoter activity using RbAp48-transfected HSG cells with and without IFN-γ by luciferase assay. We observed significantly enhanced IRF-1 promoter activity in RbAp48-transfected HSG cells with IFN-γ, not in MCF-7 cells (Fig. 5 B). Surprisingly, in RbAp48-Tg mice, a prominent expression of IFN-γ was detected in salivary and lacrimal epithelial cells besides sporadically positive infiltrating cells of RbAp48-Tg mice, not WT mice (Fig. 5 C). These findings were observed mainly in the MHC class II+ ductal epithelium adjacent to lymphoid infiltrates. Epithelial IFN-γ expression in the exocrine glands of RbAp48-Tg mice was up-regulated during the course of autoimmune exocrinopathy. Induction of IFN-γ expression may occur through many different types of stimulation, including cross-linking of cell-surface receptors and stimulation with cytokines, including IL-2, -12, and -18 (34). It has been demonstrated that IFN-γ synthesis is predominantly induced by stimulation with IL-18 (35). Consistent with a previous study (36), IL-18 expression was observed in salivary epithelial cells in RbAp48-Tg mice, not in WT mice (Fig. 5 D). Confocal analysis revealed that differential expression of IL-18 and IFN-γ was clearly observed, i.e., IL-18 mainly in the acinar cells and IFN-γ in the duct cells, within salivary epithelial cells from RbAp48-Tg mice, but not from WT mice (Fig. 5 D). Controls using isotype antibodies were shown in Fig. S4 C. Epithelial IFN-γ and IL-18 productions were confirmed by flow cytometry using MSG cells without immune cells from RbAp48-Tg, not from WT mice, whereas there was no difference in both IFN-γ and IL-18 expressions of cLN cells between WT and RbAp48-Tg mice (Fig. 5 E). Although the production of IL-18 in salivary gland cells was detected in a previous study (36), there has been no proof for IFN-γ production of salivary gland cells in any paper. Therefore, to confirm IFN-γ production of exocrine glands, detection of IFN-γ using tissue homogenates was performed. A high concentration of IFN-γ was detected in the tissue homogenates of lacrimal and salivary glands from RbAp48-Tg mice, compared with that from WT mice, by ELISA (Fig. S6 A). Furthermore, the detection of IFN-γ mRNA of MSG cells was performed by in situ hybridization using the RNA probe of mouse IFN-γ gene. A more intense signal for IFN-γ mRNA in duct cells of salivary glands from RbAp48-Tg mice was observed compared with that from WT mice (Fig. 5 F). As for the expression of BAFF, which is an inducer of IFN-γ in B cells, the expression of epithelial cells was undetectable in both RbAp48-Tg and WT mice (Fig. S7). In vitro studies using HSG cells demonstrated that the expressions of IL-18, IFN-γ, and MHC class II (HLA-DR) were observed when treated with Tam or transfected with pCMV-RbAp48, whereas they were inhibited when treated with 17β-estradiol (E2), caspase 1 inhibitor (Ac-YVAD-CHO; Ci), and siRNA of RbAp48 (si; Fig. 6 A). Confocal analysis confirmed the expression of IL-18 and IFN-γ in HSG cells treated with Tam or transfected with pCMV-RbAp48 (Fig. 6 B). It is important to note that IL-18 is secreted earlier (by 6 h) than IFN-γ production and HLA-DR expression (by 12 h) in Tam-stimulated and RbAp48-transfected HSG cells (Fig. S8). The most prominent function of IL-18 is its capacity to act as a potent costimulus for IFN-γ production (37–39). Indeed, we observed an increase in IFN-γ production in HSG cells treated with recombinant IL-18 in the dose-dependent manner, but not in MCF-7 cells (Fig. S9, available at http://www.jem.org/cgi/content/full/jem.20080174/DC1), by ELISA and flow cytometry (Fig. 6 C). Confocal analysis of IFN-γ production of HSG cells in response to IL-18 together with cytokeratin as an identified marker was shown in Fig. 6 D. Moreover, we found a significant up-regulation of caspase 1 activity in lacrimal and salivary glands from RbAp48-Tg mice relative to that from WT mice (Fig. 6 E). In this regard, we reported previously significantly increased caspase 1 activity in salivary gland tissues from ovariectomized (Ovx) C57BL/6 mice in vivo (22) and Tam-stimulated and RbAp48-transfected HSG cells in vitro (23).

Bottom Line: Recently, we found that retinoblastoma-associated protein 48 (RbAp48) induces tissue-specific apoptosis in the exocrine glands depending on the level of estrogen deficiency.Surprisingly, we obtained evidence that salivary and lacrimal epithelial cells can produce interferon-gamma (IFN-gamma) in addition to interleukin-18, which activates IFN regulatory factor-1 and class II transactivator.These results indicate a novel immunocompetent role of epithelial cells that can produce IFN-gamma, resulting in loss of local tolerance before developing gender-based autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Molecular Pathology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.

ABSTRACT
Although several autoimmune diseases are known to develop in postmenopausal women, the mechanisms by which estrogen deficiency influences autoimmunity remain unclear. Recently, we found that retinoblastoma-associated protein 48 (RbAp48) induces tissue-specific apoptosis in the exocrine glands depending on the level of estrogen deficiency. In this study, we report that transgenic (Tg) expression of RbAp48 resulted in the development of autoimmune exocrinopathy resembling Sjögren's syndrome. CD4(+) T cell-mediated autoimmune lesions were aggravated with age, in association with autoantibody productions. Surprisingly, we obtained evidence that salivary and lacrimal epithelial cells can produce interferon-gamma (IFN-gamma) in addition to interleukin-18, which activates IFN regulatory factor-1 and class II transactivator. Indeed, autoimmune lesions in Rag2(-/-) mice were induced by the adoptive transfer of lymph node T cells from RbAp48-Tg mice. These results indicate a novel immunocompetent role of epithelial cells that can produce IFN-gamma, resulting in loss of local tolerance before developing gender-based autoimmunity.

Show MeSH
Related in: MedlinePlus