immunohistochemical localization of insulin-like growth factor-i (igf-i), its receptor and...

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rlbstracts: 4th IFPA Conference, Poster Session II.55 EICOSANOID PRODUCTION PA’ITERN OF MACROPHAGES FROM HUMAN TERM PLACENTA AND DECIDUA. B.Wetzka, W.R.Schafer, K.Wemer, F.Darlau, H.P.Zahradnik, Dept. Obstetrics and Gynaecology, Endocrinology and Reproductive Medicine, University of Freiburg. Germany. At the end of pregnancy, intrauterine tissue macmphages constitute a considerable proportion of cells. i.e. approximately 15%-20% of cells in the placenta and the decidua. However, there is little concrete knowledge about their importance for the maintenance of the special immwologicul balance between the mother and the fetus or for the induction of labour. The effects of hormones and cytokiies involved in these mechanisms are often mediated by autacoids like eicosanoids and nitric oxide. Tissue explants of placenta and decidua were shown to produce large quantities of eicosanoids.’ Their synthesizing enzymes, especially cyclooxygenases 1 and 2 and tbromboxane synthase, were expressed mainly in tissue macmphages.z’ We therefore prepared primary cell cultures from human placenta and decidua derived fmm term Caesarean sections. Placental macmphageswere isolated by Ficoll gradient centlifugation and depletion of tmphoblsst by anti-EGF-weptor- coated magnetic beads. Decidual cell suspensions were subjected to Percoll gradient centrifugation. The cell cultures contained at least 50% macrophages as shown by immunocytochemistry and FACS analysis using anti-CD&?. Their eicossnoid production was investigated with and without addition of inflammatory mediators(IL-1, LPS. TNFa ). PGE, PGF,. and TXA, synthesis from endogenous arachidonic acid&Q stores was analysed by enzyme immnoassay. The spectrum od AA metabolites was studied after addition of ‘H-AA for 24 h and analysis of radioactive metabolites by revcrsephase HPLC. The macmphage-enriched cell populations produced PGE,. PGF,, as well as TXAa but the main AA metabolites were hydmxyeicosatetraenoic acids(HETE’s) and epoxyeicmatrienoic acids(EEI’s). The pattern of AA metabolites was influenced by addition of inflammatory mediators. In conclusion. cytokie effects seem to be mediated by local eicosanoid synthesis. These results together with previous reports point to a very important role of tissue macrophages for immune-endocrine interactions within placenta and decidua. ‘Placenta 17:231-8,19%. *Placenta 17573.81,1996.‘HumsnRepmduction 12:2313-20.1997. IMMUNOHISTOCHEMICAL LOCALIZATION OF INSULIN- LIKE GROWTH FACTOR-l (Iff-I), ITS RECEPTOR AND IGF- BINDING PROTEIN-1 (IGFBP-1) IN THE HUMAN PLACENTAL MEMBRANE. S.Funavama and M. Iwashita. Department of Obstetrics & Gynecology, Tokyo Women’s Medical University, Tokyo,Japan The placental membrane is composed of amnion, chorion laevc and decidua. IGF-I involved in the proliferation and differentiation of trophoblasts in the placenta. IGFBP- 1, one of IGF-binding proteins is produced by decidua and modifys I(% action. We investigated the localization of IGF-I, its receptor and IGFBP-1 in the placental membrane to elucidate the physiological significance of IGF system in the placental membrane. w Fresh frozen blocks were made from term placental membrane. The stain was performed by strept-avidin-biotin method using monoconal antibodies against IGF-I, its receptor and IGFBP-1. Anti- vimentin and anti-cytokeratin antibodies were also used to identify cell types. m IGF-I wasstained in decidua and chorion laeve faintly. IGF-I receptor was stained in chorion laeve. Staining for IGFBP-1 was laid scatterd all over the decidua. Conclusien Since IGF-I and IGFBP-1 were localized in decidua and IGF-I receptor was localized in chorion laeve, it is suggested that there is the interaction between decidua and chorion faeve through IGF system. Endothelin-activated calcium signaling and human trophoblast dlflerentiation. L. Cronier, A. Dubut, J. Guibourdenche* and A. MaIassinC. CNRS UMR 6558, 86022 Poitiers, *INSERM U 427, Paris,France. Regarding their endocrine and paracrine activities, endothelins (ET) are considered as peptide hormones and growth factors. Endothelins receptors are expressed on the trophoblast and the biosynthesis of ET by syncytiotrophoblast (ST) was previously demonstrated. Here we investigated the effects of ET on the in vitro trophoblast differentiation and on the cytosolic f?ee Cd’ activity ([Ca”]i). The presence of ET, (1V7 and 1V’M) for 3 days partially inhibited ST formation as conlirmed by desmoplakin immunostaining. In parallel hCG release and hCS expression were significantlyreduced (33% and 40% respectively at 1o”M ETI). ET1 and ET3 (lV’M, 1V8M) perifbsion induced a dose dependent transient increase of [Ca”]i in ST as measured by means of a ratiometric fluorescence method with Indo-1. Moreover [Ca”]i response to ET were inhibited by BQ 123 and BQ 788 (ETA and ETa receptors antagonists). The absence of extracellular Cd’ has no effect on the endothelin induced [Ca++]i. We conclude that ET inhibits human trophoblast differentiation and behaves as a typical Ca” mobilizing peptide in trophoblast. KERATINOCYTE GROWTH FACTOR STIMULATES THE HUMAN CHORIONIC GONADOTROPIN SECRETION IN THE BEWO, CHORIOCARCINOMA CELL LINE. H. Matsui, K.Suyama, K.Kurogi, M.Taga and H.Minaguchi, Department of Obstetrics and Gynecology, Yokohama City University School of Medicine, Yokohama, Japan. In order to investigate the significance of keratinocyte growth factor (KGF) in the function of trophoblastic cells, we examined the gene expression of KGF and KGF receptor (KGF-R) and the effect of KGF on human chorionic gonadotropin (hCG) secretion as well as on DNA synthesis in the BeWo, human choriocarcinoma cell line. BeWo cells obtained from ATCC were grown in F12k modification of Ham’s medium supplemented with 15% FBS at 37c in a humidified atmosphere of 95% air 5% CO,. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with specific primers for KGF and KGF-R on the RNA extracted from BeWo cells. hCG concentrations in the medium were determined by enzyme immunoassay. DNA synthesis in BeWo cells was determined by counting the amount of [3H]- thymidine incorporated into trichloroacetic acid (TCA)-precipitable material. RT-PCR revealed that KGF and KGF-R genes were expressedin BeWo cells. KGF significantly stimulated hCG secretion from BeWo cells, but did not affect DNA synthesis. We therefore assumethat BeWo cells, which have KGF-R, produce KGF and that KGF plays an important role in the cell function by influencing hCG secretion in an autocrine fashion.

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rlbstracts: 4th IFPA Conference, Poster Session II.55

EICOSANOID PRODUCTION PA’ITERN OF MACROPHAGES FROM HUMAN TERM PLACENTA AND DECIDUA. B.Wetzka, W.R.Schafer, K.Wemer, F.Darlau, H.P.Zahradnik, Dept. Obstetrics and Gynaecology, Endocrinology and Reproductive Medicine, University of Freiburg. Germany.

At the end of pregnancy, intrauterine tissue macmphages constitute a considerable proportion of cells. i.e. approximately 15%-20% of cells in the placenta and the decidua. However, there is little concrete knowledge about their importance for the maintenance of the special immwologicul balance between the mother and the fetus or for the induction of labour. The effects of hormones and cytokiies involved in these mechanisms are often mediated by autacoids like eicosanoids and nitric oxide. Tissue explants of placenta and decidua were shown to produce large quantities of eicosanoids.’ Their synthesizing enzymes, especially cyclooxygenases 1 and 2 and tbromboxane synthase, were expressed mainly in tissue macmphages.z’ We therefore prepared primary cell cultures from human placenta and decidua derived fmm term Caesarean sections. Placental macmphageswere isolated by Ficoll gradient centlifugation and depletion of tmphoblsst by anti-EGF-weptor- coated magnetic beads. Decidual cell suspensions were subjected to Percoll gradient centrifugation. The cell cultures contained at least 50% macrophages as shown by immunocytochemistry and FACS analysis using anti-CD&?. Their eicossnoid production was investigated with and without addition of inflammatory mediators(IL-1, LPS. TNFa ). PGE, PGF,. and TXA, synthesis from endogenous arachidonic acid&Q stores was analysed by enzyme immnoassay. The spectrum od AA metabolites was studied after addition of ‘H-AA for 24 h and analysis of radioactive metabolites by revcrsephase HPLC. The macmphage-enriched cell populations produced PGE,. PGF,, as well as TXAa but the main AA metabolites were hydmxyeicosatetraenoic acids(HETE’s) and epoxyeicmatrienoic acids(EEI’s). The pattern of AA metabolites was influenced by addition of inflammatory mediators. In conclusion. cytokie effects seem to be mediated by local eicosanoid synthesis. These results together with previous reports point to a very important role of tissue macrophages for immune-endocrine interactions within placenta and decidua. ‘Placenta 17:231-8,19%. *Placenta 17573.81,1996.‘HumsnRepmduction 12:2313-20.1997.

IMMUNOHISTOCHEMICAL LOCALIZATION OF INSULIN- LIKE GROWTH FACTOR-l (Iff-I), ITS RECEPTOR AND IGF- BINDING PROTEIN-1 (IGFBP-1) IN THE HUMAN PLACENTAL MEMBRANE. S.Funavama and M. Iwashita. Department of Obstetrics & Gynecology, Tokyo Women’s Medical University, Tokyo,Japan

The placental membrane is composed of amnion, chorion laevc and decidua. IGF-I involved in the proliferation and differentiation of trophoblasts in the placenta. IGFBP- 1, one of IGF-binding proteins is produced by decidua and modifys I(% action. We investigated the localization of IGF-I, its receptor and IGFBP-1 in the placental membrane to elucidate the physiological significance of IGF system in the placental membrane. w Fresh frozen blocks were made from term placental membrane. The stain was performed by strept-avidin-biotin method using monoconal antibodies against IGF-I, its receptor and IGFBP-1. Anti- vimentin and anti-cytokeratin antibodies were also used to identify cell types. m IGF-I wasstained in decidua and chorion laeve faintly. IGF-I receptor was stained in chorion laeve. Staining for IGFBP-1 was laid scatterd all over the decidua. Conclusien Since IGF-I and IGFBP-1 were localized in decidua and IGF-I receptor was localized in chorion laeve, it is suggested that there is the interaction between decidua and chorion faeve through IGF system.

Endothelin-activated calcium signaling and human trophoblast dlflerentiation. L. Cronier, A. Dubut, J. Guibourdenche* and A. MaIassinC. CNRS UMR 6558, 86022 Poitiers, *INSERM U 427, Paris, France.

Regarding their endocrine and paracrine activities, endothelins (ET) are considered as peptide hormones and growth factors. Endothelins receptors are expressed on the trophoblast and the biosynthesis of ET by syncytiotrophoblast (ST) was previously demonstrated. Here we investigated the effects of ET on the in vitro trophoblast differentiation and on the cytosolic f?ee Cd’ activity ([Ca”]i). The presence of ET, (1V7 and 1V’M) for 3 days partially inhibited ST formation as conlirmed by desmoplakin immunostaining. In parallel hCG release and hCS expression were significantly reduced (33% and 40% respectively at 1o”M ETI). ET1 and ET3 (lV’M, 1V8M) perifbsion induced a dose dependent transient increase of [Ca”]i in ST as measured by means of a ratiometric fluorescence method with Indo-1. Moreover [Ca”]i response to ET were inhibited by BQ 123 and BQ 788 (ETA and ETa receptors antagonists). The absence of extracellular Cd’ has no effect on the endothelin induced [Ca++]i. We conclude that ET inhibits human trophoblast differentiation and behaves as a typical Ca” mobilizing peptide in trophoblast.

KERATINOCYTE GROWTH FACTOR STIMULATES THE HUMAN CHORIONIC GONADOTROPIN SECRETION IN THE BEWO, CHORIOCARCINOMA CELL LINE. H. Matsui, K.Suyama, K.Kurogi, M.Taga and H.Minaguchi, Department of Obstetrics and Gynecology, Yokohama City University School of Medicine, Yokohama, Japan.

In order to investigate the significance of keratinocyte growth factor (KGF) in the function of trophoblastic cells, we examined the gene expression of KGF and KGF receptor (KGF-R) and the effect of KGF on human chorionic gonadotropin (hCG) secretion as well as on DNA synthesis in the BeWo, human choriocarcinoma cell line. BeWo cells obtained from ATCC were grown in F12k modification of Ham’s medium supplemented with 15% FBS at 37c in a humidified atmosphere of 95% air 5% CO,. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with specific primers for KGF and KGF-R on the RNA extracted from BeWo cells. hCG concentrations in the medium were determined by enzyme immunoassay. DNA synthesis in BeWo cells was determined by counting the amount of [3H]- thymidine incorporated into trichloroacetic acid (TCA)-precipitable material. RT-PCR revealed that KGF and KGF-R genes were expressed in BeWo cells. KGF significantly stimulated hCG secretion from BeWo cells, but did not affect DNA synthesis. We therefore assume that BeWo cells, which have KGF-R, produce KGF and that KGF plays an important role in the cell function by influencing hCG secretion in an autocrine fashion.