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Studies of HLA -A, -B, -C and -G expression by amnioepithelial cells in situ and in primary culture
A.58
Placenta (1995), Vol 16
IDENTIFICATION OF ION TRANSPORTER ISOFORMS IN HUMAN PLACENTA. T. L. Powell, M. Dehlin, D.O. Wester~en, K. Ingvarsson and T. Jansson, Dept. of Physiology, G6teborg Umverstty, s-413 90 G6teborg, Sweden Transport studies have demonstrated the presence of a range of ion transport systems in the human placenta. However these tral~sporter proteins have not been characterized as to isoforms and their subcellular dtstribnhon. We used Western blot analysis to study the Na+/H + exchanger moforats NHE 1, 2, and 3, Na+K + ATPase ct 1, 2 and 3 and the HCO3-/CI- exchanger lsoforms AE 1 and 3 m nucrovdlous (MVM) and basal membrane (BM) of term and pre-term human syncyfiotrophoblast. All isotorms of NHE, the ct I isotonn ot Na+K + ATPase and AE 1 were identified. The relative abundance of the transporters between MVM and BM was (MVM arbgrarily = 1, mean + SEM): T r a n s p o r t e r isoform MVM In=4) BM (n=4) J (paired t-test) NHE l 1.0 + 0.22 0 11 + 0.03 0.02 NHE 2 1.0 + 0.17 0 06 + 0.01 0.01 NHE3 1.0 +_ 0.24 0 13 + 0.0l 0.03 Na + K + ATPase ct 1 1.0 + 0.06 0.48 +_ 0.01 0.02 AE 1 1.0 _+ 0.09 0.27 + 0.05 0.01 The relative abundance of NHE tsoforms did not change on the MVM between 16 to 40 weeks of gestation. However, BM NIlE 2 was sigmficantly lower in the 16-22 wk group whtle the density of Na+K+ATPase ctl isotbrm was higher in 16-22 wk on both MVM and BM. In sunmmry we have shown that the human placenta possesses 3 isoforms for the NIlE on MVM and BM where it was previously believed that only the NHEI was found on the MVM and only NHE2 on the BM. The Na+K+ATPase ctl lsoform is in greater abundance on tile MVM compared to BM. MVM exhibits a lower membrane fluidity which has been shown to mhlbtt the function of this protein. Therefore this finding is not contradictory to the role of Na+K + ATPase m transplacental Na + transport. The placental HCO3-/CI" exchanger isoform AEI is located on both tile MVM and BM possibly providing a means of transplacental CI" transport.
STUDIES OF HLA -A, -B, - C AND - G EXPRESSION BY AMNIOEPITHELIAL CELLS IN SITU AND IN PRIM.ARY CULTURE. J. Pr611, A. Blaschitz, M. Hartmann, A. Hammer, I. Ghassempur, S. Haidacher, B. Uchanska-Ziegler 1, A. Ziegler 1, G. Dohr, Department of Histology and Embryology, Karl-Franzens-University of Graz and Institute for Experimental Oncology and Transplantation Medicine, Free University of Berlin, D-14050 Berlin, Germany As the celltype-specific HLA-Ia and HLA-Ib expression during pregnancy is not fully understood we want to contribute some new data concerning their presence in the amnioepithelial cells. The MHC antigen distribution in tissues building up the matemo-fetal interface is of surprising individual diversity and the celltype-specific protein expression pattern displays great heterogeneity. The individual differences of HLA -la, -Ib molecule pattern in amnioepithelia of normally delivered placentae were investigated by immunohistochemistry on cryosections. Since it is of importance that isolated cultured cells, which are usually used for immunopreclpitation, keep their original HLA expression, we studied MHC antigen distributmn after one, three and seven days in culture. Immunohistochemical comparison of staining patterns of each placenta was done simultaneously on cryosections and corresponding cultured cells. For HLA typing we used mab W6/32 (HLA -A, -B, -C, -G), TO 149 (HLA -B, -C, (A)), HC A2 (HLA -A, -G, (B)), HC 10 (HLA -B, -C), LA 45 (HLA -A, -B), TO 48 (HLA -Bw4, (A)), SFRSB6 (HLA -Bw6, (C)), and 152 microglobulin. With some of these antibodies biochemical studies as western blotting and immunoprecipltation were carried out. Additionally we characterized isolated cells with monoclonal antibodies GZ 101 and GZ 112 specific for amniocytes. Further controls were performed with antibodies against cytokeratin, vimentin and the proliferating antigen Ki-67. There is evidence for different downregulation of HLA -A, -B, - C and - G molecules in primary cultures of amniotic cells. Supported by FWF-grant P09922-MED and by EU-grant PL 941593.
Placenta (1995), Vol 16
IDENTIFICATION OF ION TRANSPORTER ISOFORMS IN HUMAN PLACENTA. T. L. Powell, M. Dehlin, D.O. Wester~en, K. Ingvarsson and T. Jansson, Dept. of Physiology, G6teborg Umverstty, s-413 90 G6teborg, Sweden Transport studies have demonstrated the presence of a range of ion transport systems in the human placenta. However these tral~sporter proteins have not been characterized as to isoforms and their subcellular dtstribnhon. We used Western blot analysis to study the Na+/H + exchanger moforats NHE 1, 2, and 3, Na+K + ATPase ct 1, 2 and 3 and the HCO3-/CI- exchanger lsoforms AE 1 and 3 m nucrovdlous (MVM) and basal membrane (BM) of term and pre-term human syncyfiotrophoblast. All isotorms of NHE, the ct I isotonn ot Na+K + ATPase and AE 1 were identified. The relative abundance of the transporters between MVM and BM was (MVM arbgrarily = 1, mean + SEM): T r a n s p o r t e r isoform MVM In=4) BM (n=4) J (paired t-test) NHE l 1.0 + 0.22 0 11 + 0.03 0.02 NHE 2 1.0 + 0.17 0 06 + 0.01 0.01 NHE3 1.0 +_ 0.24 0 13 + 0.0l 0.03 Na + K + ATPase ct 1 1.0 + 0.06 0.48 +_ 0.01 0.02 AE 1 1.0 _+ 0.09 0.27 + 0.05 0.01 The relative abundance of NHE tsoforms did not change on the MVM between 16 to 40 weeks of gestation. However, BM NIlE 2 was sigmficantly lower in the 16-22 wk group whtle the density of Na+K+ATPase ctl isotbrm was higher in 16-22 wk on both MVM and BM. In sunmmry we have shown that the human placenta possesses 3 isoforms for the NIlE on MVM and BM where it was previously believed that only the NHEI was found on the MVM and only NHE2 on the BM. The Na+K+ATPase ctl lsoform is in greater abundance on tile MVM compared to BM. MVM exhibits a lower membrane fluidity which has been shown to mhlbtt the function of this protein. Therefore this finding is not contradictory to the role of Na+K + ATPase m transplacental Na + transport. The placental HCO3-/CI" exchanger isoform AEI is located on both tile MVM and BM possibly providing a means of transplacental CI" transport.
STUDIES OF HLA -A, -B, - C AND - G EXPRESSION BY AMNIOEPITHELIAL CELLS IN SITU AND IN PRIM.ARY CULTURE. J. Pr611, A. Blaschitz, M. Hartmann, A. Hammer, I. Ghassempur, S. Haidacher, B. Uchanska-Ziegler 1, A. Ziegler 1, G. Dohr, Department of Histology and Embryology, Karl-Franzens-University of Graz and Institute for Experimental Oncology and Transplantation Medicine, Free University of Berlin, D-14050 Berlin, Germany As the celltype-specific HLA-Ia and HLA-Ib expression during pregnancy is not fully understood we want to contribute some new data concerning their presence in the amnioepithelial cells. The MHC antigen distribution in tissues building up the matemo-fetal interface is of surprising individual diversity and the celltype-specific protein expression pattern displays great heterogeneity. The individual differences of HLA -la, -Ib molecule pattern in amnioepithelia of normally delivered placentae were investigated by immunohistochemistry on cryosections. Since it is of importance that isolated cultured cells, which are usually used for immunopreclpitation, keep their original HLA expression, we studied MHC antigen distributmn after one, three and seven days in culture. Immunohistochemical comparison of staining patterns of each placenta was done simultaneously on cryosections and corresponding cultured cells. For HLA typing we used mab W6/32 (HLA -A, -B, -C, -G), TO 149 (HLA -B, -C, (A)), HC A2 (HLA -A, -G, (B)), HC 10 (HLA -B, -C), LA 45 (HLA -A, -B), TO 48 (HLA -Bw4, (A)), SFRSB6 (HLA -Bw6, (C)), and 152 microglobulin. With some of these antibodies biochemical studies as western blotting and immunoprecipltation were carried out. Additionally we characterized isolated cells with monoclonal antibodies GZ 101 and GZ 112 specific for amniocytes. Further controls were performed with antibodies against cytokeratin, vimentin and the proliferating antigen Ki-67. There is evidence for different downregulation of HLA -A, -B, - C and - G molecules in primary cultures of amniotic cells. Supported by FWF-grant P09922-MED and by EU-grant PL 941593.