Possible Involvement of Human Mast Cells in the Establishment of Pregnancy via Killer Cell Ig-Like Receptor 2DL4
2018; Elsevier BV; Volume: 188; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2018.02.012
ISSN1525-2191
AutoresChiyuki Ueshima, Tatsuki R. Kataoka, Masahiro Hirata, Akihiko Sugimoto, Yoshiki Iemura, Sachiko Minamiguchi, Takashi Nomura, Hironori Haga,
Tópico(s)Endometriosis Research and Treatment
ResumoThe involvement of mast cells in the establishment of pregnancy is unclear. Herein, we found that human mast cells are present in the decidual tissues of parous women and expressed a human-specific protein killer cell Ig-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen G expressed on human trophoblasts. In contrast, decreased numbers of decidual mast cells and reduced KIR2DL4 expression were observed in these cells of infertile women who had undergone long-term corticosteroid treatment. Co-culture of the human mast cell line, LAD2, and human trophoblast cell line, HTR-8/SVneo, accelerated the migration and tube formation of HTR-8/SVneo cells in a KIR2DL4-dependent manner. These observations suggest the possible involvement of human mast cells in the establishment of pregnancy via KIR2DL4 and that long-term corticosteroid treatment may cause infertility by influencing the phenotypes of decidual mast cells. The involvement of mast cells in the establishment of pregnancy is unclear. Herein, we found that human mast cells are present in the decidual tissues of parous women and expressed a human-specific protein killer cell Ig-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen G expressed on human trophoblasts. In contrast, decreased numbers of decidual mast cells and reduced KIR2DL4 expression were observed in these cells of infertile women who had undergone long-term corticosteroid treatment. Co-culture of the human mast cell line, LAD2, and human trophoblast cell line, HTR-8/SVneo, accelerated the migration and tube formation of HTR-8/SVneo cells in a KIR2DL4-dependent manner. These observations suggest the possible involvement of human mast cells in the establishment of pregnancy via KIR2DL4 and that long-term corticosteroid treatment may cause infertility by influencing the phenotypes of decidual mast cells. The presence of immunocompetent cells in decidual tissue is essential for the establishment of pregnancy.1Arck P.C. Hecher K. Fetomaternal immune cross-talk and its consequences for maternal and offspring's health.Nat Med. 2013; 19: 548-556Crossref PubMed Scopus (410) Google Scholar, 2Figueiredo A.S. Schumacher A. The T helper type 17/regulatory T cell paradigm in pregnancy.Immunology. 2016; 148: 13-21Crossref PubMed Scopus (191) Google Scholar Decidual natural killer (NK) cells and decidual dendritic cells are involved in angiogenesis in decidual tissues.1Arck P.C. Hecher K. Fetomaternal immune cross-talk and its consequences for maternal and offspring's health.Nat Med. 2013; 19: 548-556Crossref PubMed Scopus (410) Google Scholar Regulatory T cells (Tregs) are essential for implantation.2Figueiredo A.S. Schumacher A. The T helper type 17/regulatory T cell paradigm in pregnancy.Immunology. 2016; 148: 13-21Crossref PubMed Scopus (191) Google Scholar Mast cells, another type of immunocompetent cells,3Gilfillan A.M. Austin S.J. Metcalfe D.D. Mast cell biology: introduction and overview.Adv Exp Med Biol. 2011; 716: 2-12Crossref PubMed Scopus (102) Google Scholar are present in mouse and human decidual tissue.4Horie K. Fujita J. Takakura K. Kanzaki H. Suginami H. Iwai M. Nakayama H. Mori T. The expression of c-kit protein in human adult and fetal tissues.Hum Reprod. 1993; 8: 1955-1962Crossref PubMed Scopus (83) Google Scholar Studies using mast cell–deficient KITW-sh/W-sh mice revealed that mast cells are dispensable for,5Menzies F.M. Higgins C.A. Shepherd M.C. Nibbs R.J. Nelson S.M. Mast cells reside in myometrium and cervix, but are dispensable in mice for successful pregnancy and labor.Immunol Cell Biol. 2012; 90: 321-329Crossref PubMed Scopus (31) Google Scholar but enhance, the establishment of pregnancy.6Woidacki K. Popovic M. Metz M. Schumacher A. Linzke N. Teles A. Poirier F. Fest S. Jensen F. Rabinovich G.A. Maurer M. Zenclussen A.C. Mast cells rescue implantation defects caused by c-kit deficiency.Cell Death Dis. 2013; 4: e462Crossref PubMed Scopus (69) Google Scholar The number of murine mast cells was increased by transfer of Tregs into the decidual tissue,7Woidacki K. Meyer N. Schumacher A. Goldschmidt A. Maurer M. Zenclussen A.C. Transfer of regulatory T cells into abortion-prone mice promotes the expansion of uterine mast cells and normalizes early pregnancy angiogenesis.Sci Rep. 2015; 5: 13938Crossref PubMed Scopus (49) Google Scholar and mast cell chymase was thought to be necessary for decidual vascular remodeling in mice and humans.8Meyer N. Woidacki K. Knöfler M. Meinhardt G. Nowak D. Velicky P. Pollheimer J. Zenclussen A.C. Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth.Sci Rep. 2017; 7: 45106Crossref PubMed Scopus (43) Google Scholar However, the roles of decidual mast cells have not been determined in detail, especially in humans. Human mast cells express killer cell Ig-like receptor 2DL4 (KIR2DL4/CD158d),9Ueshima C. Kataoka T.R. Hirata M. Furuhata A. Suzuki E. Toi M. Tsuruyama T. Okayama Y. Haga H. The killer cell Ig-like receptor 2DL4 expression in human mast cells and its potential role in breast cancer invasion.Cancer Immunol Res. 2015; 3: 871-880Crossref PubMed Scopus (29) Google Scholar a member of the KIRs.10Rajalingam R. Overview of the killer cell immunoglobulin-like receptor system.Methods Mol Biol. 2012; 882: 391-414Crossref PubMed Scopus (49) Google Scholar KIRs are human-specific transmembrane proteins, which are categorized by the presence or absence of the immunoreceptor tyrosine-based inhibitory motif in the cytoplasmic domain.10Rajalingam R. Overview of the killer cell immunoglobulin-like receptor system.Methods Mol Biol. 2012; 882: 391-414Crossref PubMed Scopus (49) Google Scholar KIR2DL4 contains the immunoreceptor tyrosine-based inhibitory motif, which can transduce inhibitory signals to human NK cells.11Faure M. Long E.O. KIR2DL4 (CD158d), an NK cell-activating receptor with inhibitory potential.J Immunol. 2002; 168: 6208-6214Crossref PubMed Scopus (193) Google Scholar The ligand of KIR2DL4 was identified as human leukocyte antigen (HLA)-G, the expression of which is observed in human trophoblasts under physiological conditions.12Rajagopalan S. Long E.O. KIR2DL4 (CD158d): an activation receptor for HLA-G.Front Immunol. 2012; 3: 258Crossref PubMed Scopus (127) Google Scholar, 13Kovats S. Main E.K. Librach C. Stubblebine M. Fisher S.J. DeMars R. A class I antigen, HLA-G, expressed in human trophoblasts.Science. 1990; 248: 220-223Crossref PubMed Scopus (1228) Google Scholar Therefore, KIR2DL4 is thought to suppress the cytotoxic activity of human decidual NK cells against the embryo when interacting with HLA-G derived from trophoblasts at the fetal-maternal interface.14Yan W.H. Lin A. Chen B.G. Zhou M.Y. Dai M.Z. Chen X.J. Gan L.H. Zhu M. Shi W.W. Li B.L. Possible roles of KIR2DL4 expression on uNK cells in human pregnancy.Am J Reprod Immunol. 2007; 57: 233-242Crossref PubMed Scopus (51) Google Scholar To our knowledge, the role of KIR2DL4 on human decidual mast cells in the establishment of pregnancy has not been reported. Herein, we hypothesized that KIR2DL4 of human decidual mast cells plays roles in the establishment of pregnancy. Decidual samples collected at the Kyoto University Hospital (Kyoto, Japan) from 2006 to 2016 were obtained. Thirteen patients who underwent long-term corticosteroid therapy for autoimmune diseases or after liver or kidney transplantation and who were diagnosed as infertile, according to World Health Organization criteria, were included; three patients were excluded from the evaluation because their samples did not include decidual tissues (Table 1). Ten age-matched patients who had given birth to multiple children and 10 other patients diagnosed as infertile because of unknown causes (but who had not undergone corticosteroid therapy) from 2006 to 2016 were selected as normal controls (Table 1). All cases were spontaneously aborted. A total of 6 of 30 decidual tissues were discharged, and 24 of 30 decidual tissues were collected by curettage (Table 1). These patients signed the Kyoto University Hospital Informed Consent Form for the Non-therapeutic Use of Histopathological Materials, and the signed forms were uploaded into each electronic health record.Table 1Clinical Parameters of Study SubjectsCase no.FertilityAge, yearsTPAL∗TPAL shows gravidity and parity, except for pregnancy status before the current samples have been collected.SamplingClinical diagnosisCorticosteroid1Parous223G2PDeliveryIncomplete abortionNone2Parous372G2PDeliveryIncomplete abortionNone3Parous373G3PCurettageMissed abortionNone4Parous312G2PCurettageIncomplete abortionNone5Parous315G2PCurettageIncomplete abortionNone6Parous332G2PCurettageMissed abortionNone7Parous352G2PCurettageMissed abortionNone8Parous304G2PCurettageMissed abortionNone9Parous366G2PCurettageMissed abortionNone10Parous432G2PCurettageMissed abortionNone11Infertile400G0PCurettageMissed abortionNone12Infertile400G0PCurettageMissed abortionNone13Infertile410G0PDeliveryIncomplete abortionNone14Infertile290G0PCurettageMissed abortionNone15Infertile300G0PCurettageMissed abortionNone16Infertile330G0PCurettageMissed abortionNone17Infertile300G0PCurettageIncomplete abortionNone18Infertile320G0PCurettageMissed abortionNone19Infertile330G0PCurettageMissed abortionNone20Infertile230G0PDeliveryIncomplete abortionNone21Infertile421G0PCurettageMissed abortion5–15 mg/day (20 to 33 years old), 25 mg/day (34 to 39 years old), none (40 years old to present) for SLE22Infertile271G0PCurettageMissed abortion15–20 mg/day after liver transplantation (8 years old to present)23Infertile300G0PDeliveryIncomplete abortion5 mg/day from 18 years old for SLE24Infertile350G0PCurettageMissed abortion9 mg/day from 24 years old for SLE25Infertile271G0PCurettageMissed abortion7.5–20 mg/day from 18 years old for SLE26Infertile351G0PCurettageMissed abortion12.5–15 mg/day from 26 years old for MS27Infertile330G0PCurettageMissed abortion10 mg/day from 20 years old for MCTD28Infertile350G0PCurettageMissed abortion4 mg/day from 24 years old for MCTD29Infertile380G0PCurettageMissed abortion5 mg/day after kidney transplantation (27 years old to present)30Infertile371G0PDeliveryIncomplete abortion10 to 20 mg/day from 23 years old for SLEMCTD, mixed connective tissue disease; MS, multiple sclerosis; SLE, systemic lupus erythematosus; TPAL, term births/premature births/abortions/living children.∗ TPAL shows gravidity and parity, except for pregnancy status before the current samples have been collected. Open table in a new tab MCTD, mixed connective tissue disease; MS, multiple sclerosis; SLE, systemic lupus erythematosus; TPAL, term births/premature births/abortions/living children. LAD2 cells (kindly provided by Arnold S. Kirshenbaum, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD), used as a human mast cell model, were cultured in StemPro-34 containing recombinant human stem cell factor (SCF; Peprotech, Rocky Hill, NJ).15Kirshenbaum A.S. Akin C. Wu Y. Rottem M. Goff J.P. Beaven M.A. Rao V.K. Metcalfe D.D. Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia: activation following aggregation of FcεRI or FcγRI.Leuk Res. 2003; 27: 677-682Crossref PubMed Scopus (414) Google Scholar HTR-8/SVneo cells (kindly provided by Dr. Charles H. Graham, Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS).16Graham C.H. Hawley T.S. Hawley R.G. MacDougall J.R. Kerbel R.S. Khoo N. Lala P.K. Establishment and characterization of first trimester human trophoblast cells with extended lifespan.Exp Cell Res. 1993; 206: 204-211Crossref PubMed Scopus (824) Google Scholar Anti-KIR2DL4 agonistic antibody (mouse monoclonal IgG, clone 181703) was purchased from R&D Systems (Minneapolis, MN). Anti-KIR2DL4 antibody (rabbit polyclonal IgG, 2643R), used for immunohistochemistry, immunocytochemistry, and immunoblotting, was purchased from Bioss (Woburn, MA). Anti–mast cell tryptase (MCT) antibody (mouse monoclonal IgG, clone AA1), anti-FOXP3 antibody (mouse monoclonal IgG, clone 236A/E7), and anti–glyceraldehyde 3-phosphate dehydrogenase antibody (mouse monoclonal IgG, clone 6C5) were obtained from Abcam (Cambridge, MA). Anti-CD3 antibody (rabbit monoclonal IgG, clone 2GV6) was purchased from Roche Diagnostics (Mannheim, Germany). Anti-human leukemia inhibitory factor (LIF) antibody (goat polyclonal IgG, P15018) for neutralization assay was purchased from R&D Systems. Anti–phosphorylated STAT3 (Tyr705) antibody (rabbit monoclonal IgG, clone D3A7) was purchased from Cell Signaling Technology (Beverly, MA). Anti–HLA-G antibody (mouse monoclonal IgG, clone 87 G) for blocking the interaction between KIR2DL4 and HLA-G was purchased from Exbio Praha (Praha, Czech Republic). Goat F(ab')2 anti-mouse IgG-H&L (AP) preadsorbed (ab98724), goat anti-mouse IgG H&L (Alexa Fluor 488; ab150113), and goat anti-rabbit IgG H&L (Alexa Fluor 594) preadsorbed (ab150084) were purchased from Abcam. The secondary antibodies for Western blotting and peroxidase-labeled anti-rabbit or anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The fluoroshield mounting medium with DAPI was purchased from Abcam. The corticosteroid dexamethasone was purchased from Sigma-Aldrich (D4902; St. Louis, MO). We used dexamethasone at 1 μmol/L in 100% ethanol.17Da Silva C.A. Kassel O. Mathieu E. Massard G. Gasser B. Frossard N. Inhibition by glucocorticoids of the interleukin-1beta-enhanced expression of the mast cell growth factor SCF.Br J Pharmacol. 2002; 135: 1634-1640Crossref PubMed Scopus (23) Google Scholar KIR2DL4-targeting shRNA lentiviral particles and off-target control particles were purchased from Santa Cruz Biotechnology. Infection and subsequent selection of these viral particles were performed according to the manufacturer's instructions. The matrix metalloproteinase (MMP) inhibitor GM6001 was purchased from Calbiochem (Merck Millipore, Darmstadt, Germany) and was used at a concentration of 0.5 nmol/L in dimethyl sulfoxide. To count the numbers of mast cells and Treg cells in the decidual tissues, tissue sections were deparaffinized with xylene, rehydrated, and pretreated with 0.3% hydrogen peroxide for 5 minutes, followed by blocking of background staining using Protein Block (X0909; DakoCytomation, Glostrup, Denmark). To detect decidual mast cells, anti-MCT antibody was added after steam heating for 30 minutes, and the sections were incubated for 90 minutes at room temperature. Staining was performed using an ENVISION kit (DakoCytomation), according to the manufacturer's instructions. To detect Treg cells, anti-FOXP3 antibody was added after steam heating for 30 minutes, and the sections were incubated for 1 hour at room temperature. Goat F(ab')2 anti-mouse IgG-H&L (AP) preadsorbed was added, and the sections were incubated for 30 minutes at room temperature. Staining was performed using the Vector Blue Alkaline Phosphatase Substrate Kit III (SK-5300; Vector Laboratories, Burlingame, CA), according to the manufacturer's instructions. Anti-CD3 antibody was added, and the sections were incubated for 1 hour at room temperature. Staining was performed using Histofine Simple Stain MAX-PO (R) (Nichirei, Tokyo, Japan), according to the manufacturer's instructions. Stained sections were imaged under a BX45 microscope (Olympus, Tokyo, Japan) equipped with a DP26 digital camera (Olympus). Three fields (×200) showing the largest, second largest, and third largest numbers of MCT-positive or CD3- and FOXP3-positive cells in the decidual tissue were selected, and the total cell numbers in each sample were counted. For double immunofluorescence staining, the tissue sections were deparaffinized with xylene, rehydrated, and pretreated with 0.3% hydrogen peroxide for 5 minutes; they were then steam heated for 30 minutes. Anti-KIR2DL4 antibody (rabbit polyclonal IgG) or control rabbit IgG was added for 90 minutes at room temperature, followed by goat anti-rabbit IgG H&L (Alexa Fluor 594). Anti-MCT antibody or control mouse IgG was added for 30 minutes at room temperature, followed by goat anti-mouse IgG H&L (Alexa Fluor 488). The slides were mounted with fluoroshield mounting medium with DAPI. Sections were imaged with a BX63 microscope (Olympus) equipped with an ORCA Flash 2.8 digital camera (Hamamatsu Photonics, Shizuoka, Japan). LAD2 cells were collected after overnight incubation with ethanol or 1 μmol/L corticosteroid, and cell lysates were prepared. Proteins were separated by gel electrophoresis, and target peptides were detected, as previously described.18Kataoka T.R. Kumanogoh A. Bandara G. Metcalfe D.D. Gilfillan A.M. CD72 negatively regulates KIT-mediated responses in human mast cells.J Immunol. 2010; 184: 2468-2475Crossref PubMed Scopus (35) Google Scholar The immunoblots were scanned using Light-Capture II (ATTO, Tokyo, Japan), and the intensity of protein bands was analyzed using the CS Analyzer (ATTO). LAD2 cells were collected after overnight incubation with ethanol or 1 μmol/L corticosteroid. Cell lysates were prepared, and MCT was detected using the Human MCT Enzyme-Linked Immunosorbent Assay (ELISA) Kit (CSB- E09012h; Cusabio Biotech Co, Ltd, Wuhan, China), according to the manufacturer's protocol. To confirm KIR2DL4 knockdown, immunocytochemistry was performed. Mock or KIR2DL4 knockdown LAD2 cells were collected and centrifuged at 8 × g for 3 minutes, and the cell pellets were resuspended in 20% (v/v) buffered formalin (pH 7.0), followed by centrifugation at 24 × g for 5 minutes. The cell pellets were dehydrated and embedded in paraffin, after which the paraffin blocks were cut into sections (3 to 4 μm thick). The tissue sections were deparaffinized with xylene, rehydrated, and pretreated with 0.3% hydrogen peroxide for 5 minutes. Anti-KIR2DL4 antibody (rabbit polyclonal) was added, and incubation continued for 2 hours at room temperature. Staining was performed using an ENVISION kit (DakoCytomation), according to the manufacturer's instructions. To evaluate STAT3 status in HTR-8/SVneo cells, aliquots of 2 × 105 LAD2 cells in 200 μL medium per well were cultured for 8 hours in two-well chamber slides containing confluent HTR-8/SVneo cells, with or without control IgG or anti-human LIF antibody (10 μg/mL). After three washes with phosphate-buffered saline, the adherent HTR-8/SVneo cells were fixed with 100% ethanol for 1 hour at 4°C. After three washes with phosphate-buffered saline, cells were incubated with anti–phosphorylated STAT3 (Tyr705) antibody for 1 hour at 4°C. Staining was performed using an ENVISION kit (DakoCytomation), according to the manufacturer's instructions. The slides were imaged using the BX45 microscope (Olympus) equipped with a DP26 digital camera (Olympus). Migration of HTR-8/SVneo cells toward LAD2 cells was assessed using Matrigel-coated Transwell polycarbonate membranes (8-μm pores; BD Biosciences, Franklin Lakes, NJ). Aliquots of 400 μL StemPro-34 only, StemPro-34 containing 5 × 104 control LAD2 cells, or StemPro-34 containing 5 × 104 KIR2DL4-knockdown LAD2 cells were incubated in the lower chamber at 37°C, with or without control IgG or anti-LIF neutralizing antibody (10 μg/mL). Then, 5 × 103 HTR-8/SVneo cells (100 μL) were added in cytokine-free medium in the upper chamber. After an 18-hour incubation, the cells migrating to the bottom of the upper wells were fixed with 4% paraformaldehyde, stained with Diff-Quick stain (Sysmex, Kobe, Japan), and counted under a microscope. Aliquots of 1500 μL Corning Matrigel Basement Membrane Matrix Growth Factor Reduced (Corning, Corning, NY) were added to 6-well plates and incubated for 30 minutes at 37°C. Then, 5 × 105 HTR-8/SVneo cells starved of FCS overnight were added to the presolidified Matrigel, in the presence of StemPro-34 only, StemPro-34 and 5 × 103 control LAD2 cells, or StemPro-34 and 5 × 103 KIR2DL4-knockdown LAD2 cells, in the absence or presence of control IgG or anti-LIF neutralizing antibody (10 μg/mL). After a 10-hour incubation, digital images were obtained using a BX45 microscope (Olympus) equipped with a DP26 digital camera (Olympus). Two representative fields (×200) were selected, the total length of the tubes in the field was measured, and the relative value of network formation was calculated (the total length of HTR-8/SVneo only was 100).19Li P. Guo W. Du L. Zhao J. Wang Y. Liu L. Hu Y. Hou Y. microRNA-29b contributes to pre-eclampsia through its effects on apoptosis, invasion and angiogenesis of trophoblast cells.Clin Sci (Lond). 2013; 124: 27-40Crossref PubMed Scopus (120) Google Scholar LAD2 cells were cultured overnight in cytokine-free Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and then resuspended in StemPro-34 containing human SCF. For the assay using the anti-KIR2DL4 agonistic antibody, aliquots of 1 × 105 LAD2 cells in 100 μL of medium per well were cultured for 8 hours with control IgG or anti-KIR2DL4 antibody (1 or 10 μg/mL, respectively). For the co-culture assay, 1 × 105 LAD2 cells in 100 μL of medium per well were cultured for 8 hours in 96-well dishes containing confluent HTR-8/SVneo cells in the presence or absence of control IgG or anti–HLA-G blocking antibody (10 μg/mL). Aliquots of 50 μL of the supernatants were collected and used in ELISA kits [Human LIF ELISA Kit (ab100582), Human MMP9 ELISA Kit (ab100610), and Human VEGF R1 ELISA Kit (FLT1; ab119567); Abcam], according to the manufacturer's protocol. Aliquots of 1 × 105 LAD2 cells were cultured for 8 hours with control IgG or anti-KIR2DL4 antibody (1 or 10 μg/mL, respectively) or in 96-well dishes containing confluent HTR-8/SVneo cells, with or without control IgG or anti–HLA-G blocking antibody (10 μg/mL). The anti-KIR2DL4 antibody-stimulated LAD2 cells or the co-cultured LAD2 and HTR-8/SVneo cells were collected, and their serine protease activity was measured using a FAM-Leu-CMK Green FLISP Assay Kit (Immunochemistry Technologies, Bloomington, MN), according to the manufacturer's protocol. To detect LIF or MMP9 mRNA–producing mast cells, RNA in situ hybridization for LIF or MMP-9 and immunostaining of MCT were performed on the same sections. First, RNA in situ hybridization was performed using RNAscope Target Probe Hs-LIF, Hs-MMP-9, or control scramble probe, and the RNAscope 2.5 HD Detection Reagent-Brown (Advanced Cell Diagnostics, Hayward, CA), according to the manufacturer's instructions, without counterstaining. The sections were incubated with anti-MCT for 30 minutes, followed by incubation with Simple Stain AP (M) (Nichirei Bioscience, Tokyo, Japan) for 30 minutes. Positive signals for MCT were visualized using a Vector Blue alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). Nuclei were stained with methyl green. LAD2 cells were cultured overnight in cytokine-free Iscove's modified Dulbecco's medium supplemented with 10% FCS, then resuspended in StemPro-34 containing human SCF. Aliquots of 1 × 105 LAD2 cells in 100 μL of medium per well were cultured for 8 hours in 96-well dishes containing confluent HTR-8/SVneo cells, with or without dimethyl sulfoxide or GM6001 (0.5 nmol/L). After three washes with phosphate-buffered saline, all of the adherent HTR-8/SVneo cells were collected and lysed. After two washes with cytokine-free Iscove's modified Dulbecco's medium without FCS, the lysates were used for ELISA kits [Human F2/Prothrombin/Thrombin ELISA Kit (LS-F23664-1) and Human F2RL1/PAR2 ELISA Kit (LS-F5365-1); LifeSpan Biosciences, Seattle, WA], according to the manufacturer's protocol. Differences between groups were examined for statistical significance using the U-test (Microsoft Excel 2013; Microsoft Corp., Redmond, WA) or t-test (Microsoft Excel 2013). Data are expressed as means ± SEM. P < 0.05 was considered statistically significant. First, the status of human decidual mast cells in parous women was compared with the status in infertile women, with decidual tissues derived from parous women as controls. The presence of mast cells (MCT positive) was confirmed in the decidual tissue of parous women (33.4 ± 6.92 cells) (Figure 1A). A subpopulation of infertile women who had undergone long-term corticosteroid treatment for autoimmune diseases or after transplantation (Table 1) had significantly reduced numbers of mast cells in the decidua in the first trimester (2.20 ± 0.89 cells) (Figure 1A). No such decrease in the number of decidual mast cells was observed in the women with infertility of unknown cause (23.7 ± 3.51 cells) (Figure 1A). Tregs are thought to induce the migration of mast cells to the decidual tissue in mice.7Woidacki K. Meyer N. Schumacher A. Goldschmidt A. Maurer M. Zenclussen A.C. Transfer of regulatory T cells into abortion-prone mice promotes the expansion of uterine mast cells and normalizes early pregnancy angiogenesis.Sci Rep. 2015; 5: 13938Crossref PubMed Scopus (49) Google Scholar It was examined whether decreased numbers of decidual mast cells were associated with reduced Treg (CD3 and FOXP3 positive) numbers in infertile women who had undergone long-term corticosteroid treatment. There was no significant difference in the number of decidual Tregs among parous women, women with infertility of unknown cause, and infertile women who had undergone long-term corticosteroid treatment (13.3 ± 3.88, 11.3 ± 3.87, and 16.3 ± 4.23 cells, respectively) (Figure 1B). Recently, we reported the expression of KIR2DL4 protein in human mast cells9Ueshima C. Kataoka T.R. Hirata M. Furuhata A. Suzuki E. Toi M. Tsuruyama T. Okayama Y. Haga H. The killer cell Ig-like receptor 2DL4 expression in human mast cells and its potential role in breast cancer invasion.Cancer Immunol Res. 2015; 3: 871-880Crossref PubMed Scopus (29) Google Scholar and evaluated the expression of KIR2DL4 on human decidual mast cells. Herein, immunohistochemical methods were used to evaluate the expression of KIR2DL4 in decidual mast cells. The expression of KIR2DL4 protein was confirmed in MCT-positive cells, a marker of decidual mast cells, from all parous women and infertile women who did not undergo corticosteroid treatment (Figure 2A). However, the expression of KIR2DL4 was undetectable in the decidual mast cells of infertile women who had undergone long-term corticosteroid treatment (Figure 2A). The MCT-negative and KIR2DL4-positive cells were thought to be decidual NK cells.14Yan W.H. Lin A. Chen B.G. Zhou M.Y. Dai M.Z. Chen X.J. Gan L.H. Zhu M. Shi W.W. Li B.L. Possible roles of KIR2DL4 expression on uNK cells in human pregnancy.Am J Reprod Immunol. 2007; 57: 233-242Crossref PubMed Scopus (51) Google Scholar To explore the effect of corticosteroid on mast cells, LAD2 cells were treated with the corticosteroid, dexamethasone. KIR2DL4 protein expression and MCT contents were significantly reduced in corticosteroid-treated LAD2 cells compared with those in control (ethanol-treated) LAD2 cells (Figure 2, B and C). The abnormal phenotypes of decidual mast cells were observed in a subpopulation of infertile women. KIR2DL4 is a receptor for HLA-G, which is physiologically expressed by trophoblasts, and is essential for the establishment of pregnancy.12Rajagopalan S. Long E.O. KIR2DL4 (CD158d): an activation receptor for HLA-G.Front Immunol. 2012; 3: 258Crossref PubMed Scopus (127) Google Scholar We hypothesized that KIR2DL4 on decidual mast cells was involved in the establishment of pregnancy by interacting with HLA-G on trophoblasts and evaluated the effects of co-culture between a human mast cell line LAD2 expressing KIR2DL49Ueshima C. Kataoka T.R. Hirata M. Furuhata A. Suzuki E. Toi M. Tsuruyama T. Okayama Y. Haga H. The killer cell Ig-like receptor 2DL4 expression in human mast cells and its potential role in breast cancer invasion.Cancer Immunol Res. 2015; 3: 871-880Crossref PubMed Scopus (29) Google Scholar and a trophoblast cell line HTR-8/SVneo expressing soluble and membrane-bound forms of HLA-G.16Graham C.H. Hawley T.S. Hawley R.G. MacDougall J.R. Kerbel R.S. Khoo N. Lala P.K. Establishment and characterization of first trimester human trophoblast cells with extended lifespan.Exp Cell Res. 1993; 206: 204-211Crossref PubMed Scopus (824) Google Scholar, 20Guo W. Fang L. Li B. Xiao X. Chen S. Wang J. Yang F. Chen L. Wang X. Decreased human leukocyte antigen-G expression by miR-133a contributes to impairment of proinvasion and proangiogenesis functions of decidual NK cells.Front Immunol. 2017; 8: 741Crossref PubMed Scopus (17) Google Scholar, 21Klitkou L. Dahl M. Hviid T.V. Djurisic S. Piosik Z.M. Skovbo P. Møller A.M. Steffensen R. Christiansen O.B. Human leukocyte antigen (HLA)-G during pregnancy part I: correlations between maternal soluble HLA-G at midterm, at term, and umbilical cord blood soluble HLA-G at term.Hum Immunol. 2015; 76: 254-259Crossref PubMed Scopus (21) Google Scholar Migration and angiogenesis of trophoblasts are essential processes for the establishment of pregnancy; the effects of HTR-8/SVneo cells on these phenotypes were evaluated using the co-culture system. The migration ability of HTR-8/SVneo cells was assessed by two-chamber assay. Herein, the KIR2DL4-knockdown LAD2 cells established previously9Ueshima C. Kataoka T.R. Hirata M. Furuhata A. Suzuki E. Toi M. Tsuruyama T. Okayama Y. Haga H. The killer cell Ig-like receptor 2DL4 expression in human mast cells and its potential role in breast cancer invasion.Cancer Immunol Res. 2015; 3: 871-880Crossref PubMed Scopus (29) Google Scholar and the off-target shRNA-treated LAD2 cells were used as control LAD2 cells (Figure 3A). KIR2DL4 silencing suppresses the proliferation and degranulatio
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