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Characterization of human decidual mast cells and establishment of culture system
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Abstracts / Journal of Reproductive Immunology 118 (2016) 109–141
70 Characterization of human decidual mast cells and establishment of culture system Takayuki Matsuno 1,∗ , Yoshimichi Okayama 2 , Motomi Yamazaki 1 , Jun-ichi Kashiwakura 2 , Tomomi Sakamoto 2 , Shota Toyoshima 2 , Hiromitsu Azuma 1 , Takayuki Murase 1 , Fumihisa Chishima 1 , Tatsuo Yamamoto 3 1
Department of Obstetrics and Gynecology, Nihon University School of Medicine, Japan 2 Allergy and Immunology Project Team, Division of Medical Education Planning and Development, Nihon University School of Medicine, Japan 3 Department of Obstetrics and Gynecology, Kasukabe Medical Center, Japan The rodent uterine mast cells (MCs) have been reported to play a role in implantation, placenta formation and uterine contraction. However, the roles of human uterine MCs during pregnancy have been unknown well. The aims to the study are to investigate distribution of MCs in human decidua and to establish culture system of decidual MCs. Materials and methods: Decidual tissues were obtained from patients who underwent a legal elective abortion (6-10 weeks). The decidual tissues were stained by immunohistochemistry using anti-tryptase mAb. Decidual MCs were enzymatically dispersed and semi-purified with percoll gradient. Expression of MC markers such as CD117 (KIT), FcRI␣, tryptase and chymase, was analyzed using FACS. Cultured deciduea-derived MCs were generated by culturing semi-purified MCs with stem cell factor and IL-6. Histamine released from MCs were measured using EIA. Results: Decidual tissues (4.9 ± 2.7 g/one donor, mean ± SD) were obtained from 14 patients. Using imminohistochemical analysis, the number of MCs in decidual tissues was ∼150 cells/mm2 . After semi-purification, 14.8 × 105 ± 18.3 × 105 MCs were obtained from 1 g decidual tissue (purity; 16.9 ± 8.0%). Using FACS analysis, 0.3 ± 0.2% of total decidual cell was CD117+ FcRI␣+ cells, which were tryptasehigh chymaselow cells. After 12 week-culture, the percentage of MCs increased to ∼100%. The MCs released 20.7 ± 0.05% of net histamine following aggregation of FcRI. Conclusion: Human decidua were rich in MCs. We established dedcidual MC culture system. The cultured dedcidual MC may become a good tool to evaluate the roles of decidual MCs. Conflicts of interest: The authors have no conflict of interest to declare. http://dx.doi.org/10.1016/j.jri.2016.10.079 71 Is HLA-G present in body fluids including maternal plasma and supernatant of in vitro fertilization? Noriko Ouji-Sageshima 1,∗ , Akiko Ishitani 2 , Daniel E. Geraghty 3 , Toshihiro Ito 1 1 Department of Immunology, Nara Medical University, Japan 2 Department of Legal Medicine, Nara Medical University, Japan 3 Fred Hutchinson Cancer Research Center, Seattle, WA, USA
HLA-G has limited expression on placental fetal-derived trophoblast and is a ligand for the inhibitory receptor, leukocyte
immunoglobulin like receptor (LILR)-B1/B2. It has been considered that HLA-G protects the fetus by inhibition of maternal immune cells through LILR-B1/B2, expressed on maternal immune cells. Furthermore, HLA-G produces several isoforms by alternative splicing. The detection of HLA-G protein in body fluids including supernatant of in vitro fertilization (SF), follicular fluids (FF) and plasma from pregnant women (PP) has been reported, but the detection of HLA-G in body fluids has been contradicted in some studies. In order to help resolve this controversy, we established an improved ELISA system that allows for the reliable detection of HLA-G levels in body fluids. Denatured proteins were analyzed in this improved ELISA system in order to inhibit non-specific reaction of materials in body fluids with anti-HLA-G antibodies. The ELISA utilized anti HLA-G antibodies, MEM-G/1 and 4H84 in combination for binding and detection respectively. This test was used on 49 PP, 75 SF, 16 FF, 75 amniotic fluids (AF) and 24 placental fluids (PF) to measure HLA-G expression. To confirm the ELISA results, samples were also tested immunoprecipitation (IP) and western blotting (WB). HLA-G was present in PF and AF at 0.89–24.1 ng/ml and 4.67–440.2 ng/ml, respectively. However, HLA-G was under the detection limit of the ELISA (<1.5 ng/ml) in PP, SF and FFs. These results were confirmed by IP and WB. These results indicated that the immunoregulatory function of HLA-G was focused on placental decidua after implantation. Conflicts of interest: The authors have no conflict of interest to declare. http://dx.doi.org/10.1016/j.jri.2016.10.080 72 A case of postpartum-onset severe preeclamsia Mineo Yamasaki ∗ , Kahori Shimizu, Hiromi Suzuki, Hiromi Suzuki, Nobuya Tatsumi Palmore Hospital, Kobe, Japan A case of postpartum preeclampsia is presented. A 31-yearold primigravida delivered a healthy 3150 g female baby under epidural anesthesia at 40 weeks 3 days of gestation. Her pregnant course was uneventful and without hypertension or proteinuria. Her previous medical history and family history were not particular except hypertension of her grandparents. She did not show significant increase in blood pressure during labor. The postpartum course was also uneventful. She discharged after 5 days of postpartum stay. The 15th day of puerperium severe leg edema was noticed. Three days later her blood pressure was 154/98 at the postpartum clinic. Edema was also noted, while proteinuria was negative. No medications were prescribed. At night on the following day she was presented in the clinic with high blood pressure (159/92) at home associated with headache, visual disturbance and epigastric pain. She was given nifedipine, and went back home because of disappearance of the symptoms and reductioon of blood pressure. However, she returned back to the hospital, because an attack of loss of consciousness developed 5 h after the medication. The blood analysis revealed platelet: 190,000/mm3 , hematocrit: 51.4%, total bilirubin: 2.0 mg/dL, LDH: 767U/L, AST: 59U/L, suggesting severe preeclamptic state with partial HELLP syndrome. Immediate administration of antihypertensive agents and magnesium sulfate and anti-DIC medication was started and made the patient recovered soon. She was discharged at the 5th hospital day. Although postpartum onset of preeclampsia is not common, stressful situations surrounding puerperal women may possibly cause
Abstracts / Journal of Reproductive Immunology 118 (2016) 109–141
70 Characterization of human decidual mast cells and establishment of culture system Takayuki Matsuno 1,∗ , Yoshimichi Okayama 2 , Motomi Yamazaki 1 , Jun-ichi Kashiwakura 2 , Tomomi Sakamoto 2 , Shota Toyoshima 2 , Hiromitsu Azuma 1 , Takayuki Murase 1 , Fumihisa Chishima 1 , Tatsuo Yamamoto 3 1
Department of Obstetrics and Gynecology, Nihon University School of Medicine, Japan 2 Allergy and Immunology Project Team, Division of Medical Education Planning and Development, Nihon University School of Medicine, Japan 3 Department of Obstetrics and Gynecology, Kasukabe Medical Center, Japan The rodent uterine mast cells (MCs) have been reported to play a role in implantation, placenta formation and uterine contraction. However, the roles of human uterine MCs during pregnancy have been unknown well. The aims to the study are to investigate distribution of MCs in human decidua and to establish culture system of decidual MCs. Materials and methods: Decidual tissues were obtained from patients who underwent a legal elective abortion (6-10 weeks). The decidual tissues were stained by immunohistochemistry using anti-tryptase mAb. Decidual MCs were enzymatically dispersed and semi-purified with percoll gradient. Expression of MC markers such as CD117 (KIT), FcRI␣, tryptase and chymase, was analyzed using FACS. Cultured deciduea-derived MCs were generated by culturing semi-purified MCs with stem cell factor and IL-6. Histamine released from MCs were measured using EIA. Results: Decidual tissues (4.9 ± 2.7 g/one donor, mean ± SD) were obtained from 14 patients. Using imminohistochemical analysis, the number of MCs in decidual tissues was ∼150 cells/mm2 . After semi-purification, 14.8 × 105 ± 18.3 × 105 MCs were obtained from 1 g decidual tissue (purity; 16.9 ± 8.0%). Using FACS analysis, 0.3 ± 0.2% of total decidual cell was CD117+ FcRI␣+ cells, which were tryptasehigh chymaselow cells. After 12 week-culture, the percentage of MCs increased to ∼100%. The MCs released 20.7 ± 0.05% of net histamine following aggregation of FcRI. Conclusion: Human decidua were rich in MCs. We established dedcidual MC culture system. The cultured dedcidual MC may become a good tool to evaluate the roles of decidual MCs. Conflicts of interest: The authors have no conflict of interest to declare. http://dx.doi.org/10.1016/j.jri.2016.10.079 71 Is HLA-G present in body fluids including maternal plasma and supernatant of in vitro fertilization? Noriko Ouji-Sageshima 1,∗ , Akiko Ishitani 2 , Daniel E. Geraghty 3 , Toshihiro Ito 1 1 Department of Immunology, Nara Medical University, Japan 2 Department of Legal Medicine, Nara Medical University, Japan 3 Fred Hutchinson Cancer Research Center, Seattle, WA, USA
HLA-G has limited expression on placental fetal-derived trophoblast and is a ligand for the inhibitory receptor, leukocyte
immunoglobulin like receptor (LILR)-B1/B2. It has been considered that HLA-G protects the fetus by inhibition of maternal immune cells through LILR-B1/B2, expressed on maternal immune cells. Furthermore, HLA-G produces several isoforms by alternative splicing. The detection of HLA-G protein in body fluids including supernatant of in vitro fertilization (SF), follicular fluids (FF) and plasma from pregnant women (PP) has been reported, but the detection of HLA-G in body fluids has been contradicted in some studies. In order to help resolve this controversy, we established an improved ELISA system that allows for the reliable detection of HLA-G levels in body fluids. Denatured proteins were analyzed in this improved ELISA system in order to inhibit non-specific reaction of materials in body fluids with anti-HLA-G antibodies. The ELISA utilized anti HLA-G antibodies, MEM-G/1 and 4H84 in combination for binding and detection respectively. This test was used on 49 PP, 75 SF, 16 FF, 75 amniotic fluids (AF) and 24 placental fluids (PF) to measure HLA-G expression. To confirm the ELISA results, samples were also tested immunoprecipitation (IP) and western blotting (WB). HLA-G was present in PF and AF at 0.89–24.1 ng/ml and 4.67–440.2 ng/ml, respectively. However, HLA-G was under the detection limit of the ELISA (<1.5 ng/ml) in PP, SF and FFs. These results were confirmed by IP and WB. These results indicated that the immunoregulatory function of HLA-G was focused on placental decidua after implantation. Conflicts of interest: The authors have no conflict of interest to declare. http://dx.doi.org/10.1016/j.jri.2016.10.080 72 A case of postpartum-onset severe preeclamsia Mineo Yamasaki ∗ , Kahori Shimizu, Hiromi Suzuki, Hiromi Suzuki, Nobuya Tatsumi Palmore Hospital, Kobe, Japan A case of postpartum preeclampsia is presented. A 31-yearold primigravida delivered a healthy 3150 g female baby under epidural anesthesia at 40 weeks 3 days of gestation. Her pregnant course was uneventful and without hypertension or proteinuria. Her previous medical history and family history were not particular except hypertension of her grandparents. She did not show significant increase in blood pressure during labor. The postpartum course was also uneventful. She discharged after 5 days of postpartum stay. The 15th day of puerperium severe leg edema was noticed. Three days later her blood pressure was 154/98 at the postpartum clinic. Edema was also noted, while proteinuria was negative. No medications were prescribed. At night on the following day she was presented in the clinic with high blood pressure (159/92) at home associated with headache, visual disturbance and epigastric pain. She was given nifedipine, and went back home because of disappearance of the symptoms and reductioon of blood pressure. However, she returned back to the hospital, because an attack of loss of consciousness developed 5 h after the medication. The blood analysis revealed platelet: 190,000/mm3 , hematocrit: 51.4%, total bilirubin: 2.0 mg/dL, LDH: 767U/L, AST: 59U/L, suggesting severe preeclamptic state with partial HELLP syndrome. Immediate administration of antihypertensive agents and magnesium sulfate and anti-DIC medication was started and made the patient recovered soon. She was discharged at the 5th hospital day. Although postpartum onset of preeclampsia is not common, stressful situations surrounding puerperal women may possibly cause