Dual Functions of Prostaglandin D2 in Murine Contact Hypersensitivity via DP and CRTH2
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.047
ISSN1525-2191
AutoresYoshihiro Yamamoto, Shinobu Otani, Hiroyuki Hirai, Kinya Nagata, Kosuke Aritake, Yoshihiro Urade, Shuh Narumiya, Hiroo Yokozeki, Masataka Nakamura, Takahiro Satoh,
Tópico(s)Vitamin D Research Studies
ResumoProstaglandin D2 (PGD2) exerts its effects through two distinct receptors: the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the D prostanoid (DP) receptor. Our previous study demonstrated that CRTH2 mediates contact hypersensitivity (CHS) in mice. However, the function of DP receptor remains to be fully established. In this study, we examine the pathophysiological roles of PGD2 using DP-deficient (DP−/−) and CRTH2/DP-deficient (CRTH2−/−/DP−/−) mice to elucidate receptor-mediated PGD2 action in CHS. We observed profound exacerbation of CHS in DP−/− mice. CRTH2−/−/DP−/− mice showed similar exacerbation, but to a lesser extent. These symptoms were accompanied by increased production of interferon-γ and IL-17. The increase in IL-17 producing γδ T cells was marked and presumably contributed to the enhanced CHS. DP deficiency promoted the in vivo migration of dendritic cells to regional lymph nodes. A DP agonist added to DCs in vitro was able to inhibit production of IL-12 and IL-1β. Interestingly, production of IL-10 in dendritic cells was elevated via the DP pathway, but it was lowered by the CRTH2 pathway. Collectively, PGD2 signals through CRTH2 to mediate CHS inflammation, and conversely, DP signals to exert inhibitory effects on CHS. Thus, we report opposing functions for PGD2 that depend on receptor usage in allergic reactions. Prostaglandin D2 (PGD2) exerts its effects through two distinct receptors: the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and the D prostanoid (DP) receptor. Our previous study demonstrated that CRTH2 mediates contact hypersensitivity (CHS) in mice. However, the function of DP receptor remains to be fully established. In this study, we examine the pathophysiological roles of PGD2 using DP-deficient (DP−/−) and CRTH2/DP-deficient (CRTH2−/−/DP−/−) mice to elucidate receptor-mediated PGD2 action in CHS. We observed profound exacerbation of CHS in DP−/− mice. CRTH2−/−/DP−/− mice showed similar exacerbation, but to a lesser extent. These symptoms were accompanied by increased production of interferon-γ and IL-17. The increase in IL-17 producing γδ T cells was marked and presumably contributed to the enhanced CHS. DP deficiency promoted the in vivo migration of dendritic cells to regional lymph nodes. A DP agonist added to DCs in vitro was able to inhibit production of IL-12 and IL-1β. Interestingly, production of IL-10 in dendritic cells was elevated via the DP pathway, but it was lowered by the CRTH2 pathway. Collectively, PGD2 signals through CRTH2 to mediate CHS inflammation, and conversely, DP signals to exert inhibitory effects on CHS. Thus, we report opposing functions for PGD2 that depend on receptor usage in allergic reactions. 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Interleukin-4 is a critical cytokine in contact sensitivity.Immunology. 1995; 84: 404-409PubMed Google Scholar In the present study, we examined the respective roles of PGD2 receptors in CHS using genetically modified mice. Our results demonstrate that PGD2 exerts both allergic and anti-allergic roles in inflammation, which are mediated through the reciprocal functions of CRTH2 and DP, respectively. This suggests protective roles for PGD2 under normal physiological and pathogenic conditions. BALB/c mice were purchased from Sankyo Labo Service (Tokyo, Japan). CRTH2−/− mice, DP−/− mice, and H-PGDS−/− mice have been previously described.12Satoh T. Moroi R. Aritake K. Urade Y. Kanai Y. Sumi K. Yokozeki H. Hirai H. Nagata K. Hara T. Utsuyama M. Hirokawa K. Sugamura K. Nishioka K. Nakamura M. Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor.J Immunol. 2006; 177: 2621-2629PubMed Google Scholar, 17Trivedi S.G. Newson J. Rajakariar R. Jacques T.S. Hannon R. Kanaoka Y. Eguchi N. Colville-Nash P. Gilroy D.W. Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity.Proc Natl Acad Sci USA. 2006; 103: 5179-5184Crossref PubMed Scopus (115) Google Scholar, 32Matsuoka T. Hirata M. Tanaka H. Takahashi Y. Murata T. Kabashima K. Sugimoto Y. Kobayashi T. Ushikubi F. Aze Y. Eguchi N. Urade Y. Yoshida N. Kimura K. Mizoguchi A. Honda Y. Nagai H. Narumiya S. Prostaglandin D2 as a mediator of allergic asthma.Science. 2000; 287: 2013-2017Crossref PubMed Scopus (672) Google Scholar Receptor null (CRTH2−/−/DP−/−) mice were generated in Tokyo Medical and Dental University by crossing each of the receptor-deficient mice. Mice lacking PGD2 receptors and those that lack H-PGDS genes are on a BALB/c background. Mice were maintained under specific pathogen-free conditions in our animal facility. The use of animals was in full compliance with the Committee for Animal Experiments of Tokyo Medical and Dental University. Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (CD4-FITC, GK1.5), CD8 (CD8-FITC, 53–6.7), and γδ T cell receptor (TCR) (γδ TCR-FITC, UC7-13D5) antibodies (Abs), phycoerythrin (PE)-conjugated anti-mouse IL-13 (IL-13-PE, eBio 13A), and CD25 (CD25-PE, PC61.5) Abs, biotin-conjugated anti-mouse γδ TCR (γδ TCR-Biotin, GL-3) Ab, PE-Cy5-conjugated anti-mouse Foxp (Foxp3-PE-Cy5, FJK-16s) Ab, and anti-mouse CD28 (clone 37.51) Ab were purchased from eBioscience (San Diego, CA). FITC-conjugated anti-mouse I-Ad (I-Ad –FITC, AMS-32.1) and CD86 (CD86-FITC, GL1) Abs, and PE conjugated anti-mouse CD11c (CD11c-PE, HL3), IFN-γ (IFN-γ-PE, XMG 1.2), and CD16/CD32 (2.4G2) Abs were from BD Biosciences Pharmingen (Franklin Lakes, NJ). PE-conjugated IL-17 (IL-17-PE, TC11-18H10.1), anti-IL-17 (TC11-18H10.1), and IFN-γ (XMG 1.2) Abs were from BioLegend (San Diego, CA) , and anti-IL-13 (eBio 1316H) Ab was from R&D Systems (Minneapolis, MN). Acute CHS was induced by applying 100 μL of 5% 2,4,6-trinitrochlorobenzene (TNCB) (Nacalai Tesque, Kyoto, Japan) in ethanol:acetone (3:1), or 100 μL of 0.5% 2,4-dinitrofluorobenzene (Nacalai Tesque) in acetone:olive oil (4:1). On day 5, each ear lobe was challenged with 20 μL of 1% TNCB in acetone:olive oil (1:4) or 0.2% 2,4-dinitrofluorobenzene in acetone:olive oil (4:1). Ear thickness was determined before and after each challenge. Control ears were challenged with the respective vehicles. Irritation dermatitis was induced by painting 20 μL of 1% croton oil (Nacalai Tesque) in acetone on mouse ear lobes. Ear thickness was measured with a dial thickness gauge (Peacock, Tokyo, Japan) immediately before and after painting and expressed as the mean increment in thickness above basal line control value. Single cell suspensions were prepared in PBS containing 5% fetal calf serum and 0.1% NaN3. After blocking IgG receptors with anti-mouse CD16/CD32 monoclonal Ab, cells were stained with the first Abs against cell surface markers. Intracellular staining was performed by IntraStain kit (Dako Cytomation, Glostrup, Denmark). Spleen cells (SCs) obtained from naive mice were adjusted to 5 × 107 cells/mL and mixed with equal volumes of 10 mmol/L TNBS (2,4,6-trinitrobenzene sulfonic sodium salt) (Nacalai Tesque) in PBS. Cells were then incubated at 37°C for 30 minutes, washed three times and incubated with mytomycin C (Kyowa Hakko Kirin, Tokyo, Japan) at 50 μg/mL, at 37°C for 40 minutes, followed by three further washes with PBS. Axillary and abdominal LNs were collected on day 5 from mice sensitized with TNCB (day 0). Single cell suspensions were prepared by teasing the LN. Immune LN cells (400 μL of 5 × 106 cells/mL) were incubated with 400 μL of 5 × 106 trinitrophenyl (TNP)-SC/mL in a 24-well plate for 24 hours and supernatants were collected and stored at −80°C until use. For the detection of intracellular cytokines, immune LN cells incubated with trinitrophenyl-spleen cells (TNP-SC) for 48 hours were stimulated with 50 ng/mL (phorbol 12-myristate 13-acetate, Sigma Aldrich, St. Louis, MO), 500 ng/mL ionomycin (Calbiochem, San Diego, CA) , and 5 μg/mL Brefeldin A (Sigma Aldrich) for a further 12 hours. They were then analyzed with a flow cytometer. Naive CD4 T cells were isolated using CD4(+)/CD62L (+) T cell isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were stimulated in CD3-coated plates (BD Biosciences Pharmingen) with anti-mouse CD28 Ab (1 μg/mL), and cultured for 4 days in the presence of IL-12 (5 ng/mL), IL-2 (10 ng/mL), and anti-mouse IL-4 Ab (10 μg/mL) for Th 1 cells, IL-4 (5 ng/mL), IL-2 (10 ng/mL), and anti-mouse IFN-γ Ab (10 μg/mL) for Th2 cells, and IL-6 (20 ng/mL), transforming growth factor-β (10 ng/mL), anti-mouse IL-4 Ab (10 μg/mL), and anti-mouse IFN-γ Ab (10 μg/mL) for Th17 cells. γδ T cells were prepared using the TCR γδ (+) T cell isolation kit (Miltenyi Biotec). The γδ T cells were more than 95% pure. Immune LN cells were transferred into each ear lobe (1 × 106 cells/30 μL/ear) into naive mice that immediately received challenge. Inguinal and axillary LN cells were collected from mice sacrificed at 24 hours after application of 1% FITC (Sigma Aldrich) in acetone:dibutyl phthalate (1:1) onto the abdominal skin. Cells were incubated with CD11
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