- Email: [email protected]
Mutation and functional analysis of pregnancy-specific glycoprotein 1 (PSG1)
60
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
(MMP-9), plasminogen activator inhibitor protein (PAI-1), tissue inhibitor of MMP 1 (TIMP-1) and urinary plasminogen avtivator (uPA) were expressed in dKdS1. Except for granulocyte-monocyte colony stimulating factor (GMCSF), which was only expressed in dKdS1 after IL-1 incubation, PAI-1 was the only molecule which disappeared after IL-1 incubation on whole cellular protein level. CONCLUSIONS: The knock-down of Sdc-1 leads to a different whole cellular expression pattern for two important proteins regarding angiogenesis during embryo implantation - GM-CSF and PAI-1. The lack of GM-CSF upon decidualization of KdS1 cells might raise a delayed immune response during implantation. PAI-1 is a key regulator of angiogenesis and its expression seems to be connected to Sdc-1. The knock-down of Sdc-1 might therefore lead to a dysregulated angiogenesis during implantation. Funded by the German Research Foundation (DFG) to AP Hess (HE 3544/2-2) angiogenic factor GM-CSF HGF IGFBP-2 IL-8 MMP-9 PAI-1 TIMP-1 uPA
w/o IL-1 cellular protein + + + + + + +
w IL-1 cellular protein + + + + + + +
doi:10.1016/j.jri.2012.03.347 P 046 Progesterone induced blocking factor expression in decidual stromal cells as a potential marker for successful pregnancy E. Stoyanova 1 , K. Vinketova 1 , T. Oreshkova 1 , R. Karaguozov 2 , W. Chaiwangyen 3 , J. Dimitrov 1 , I. Pastuschek 3 , U. Markert 3 , M. Mourdjeva 1,∗ 1
Institute of Biology and Immunology of Reproduction, Molecular Immunology, Sofia, Bulgaria 2 MHAT “Tokuda Hospital Sofia”, Department of Obstetrics and Gynecology, Sofia, Bulgaria 3 Friedrich-Schiller-University, Placenta-Laboratory, Department of Obstetrics, Jena, Germany PROBLEM: Immunological, endocrine and other factors participate in feto-maternal crosstalk during implantation and pregnancy and contribute for the effectiveness of these processes. Human blastocyst implants in maternal decidua which develops only during pregnancy. Decidual stromal cells (DSC) possess potent immunoregulation ability and induce state of tolerance in maternal immune system accepting the semi-allogeneic fetus. Among other factors and mechanisms involved in immunotolerance induction, progesterone induced blocking factor (PIBF) is known to have significant importance. Here, the expression of PIBF in
DSC was investigated and its correlation to decidualization potential of DSC was evaluated. METHODS: DSC were isolated from human deciduas (7-10 week of gestation) obtained from elective pregnancy terminations. DSC were cultured in standard conditions and after three passages when the cultures are over 90% homogeneous, the samples were evaluated for PIBF protein expression by immunoblot and mRNA by RT-PCR. PIBF expression and localization were studied by immunofluorescent staining with 3A6 anti-PIBF monoclonal antibody and confocal microscopy. Prolactin expression after DSCin vitrodecidualization with progesterone, estradiol and cAMP was evaluated by ELISA and RT-PCR. RESULTS: Thein vitrodecidualization of DSC is sometimes problematic. Cells isolated from different deciduas showing similarity in some parameters like expression of markers of mesenchymal stem cells CD29, CD73 and CD90, differentiate differently under the same decidualization conditions. We hypothesized that differences in PIBF expression can be correlated to the decidualization outcome. Five different DSC cell lines were compared in this study. A comparison of PIBF and prolactin mRNA and protein expression levels from different DSC was used to clarify the significance of PIBF as a marker for biological functions of DSC. Immunofluorescent staining revealed high intensity of PIBF expression in majority of DSC cells but showed heterogeneity in PIBF localization pattern. Most of the cells showed intensive nuclear and cytoplasmic staining; some were only with nuclear PIBF presence; and few were completely PIBF negative. Interestingly, after in vitro decidualization, PIBF seems to be secreted from DSC since the intensity of staining reaction was severely reduced and was restricted to secretory vesicles. CONCLUSIONS: We demonstrate that PIBF is highly expressed in DSC prior to any stimuli and that decidualization triggers PIBF secretion.The possible significance of PIBF as a marker for evaluation of quality and functional abilities of DSC is discussed in this work. The biological function of secreted PIBF remains to be investigated in the context of DSC immunoregulation. This study was supported by the projects DOO250/2008 National Science Fund, Ministry of Education and Science and FP7- REGPOT-2009-1 ReProForce. doi:10.1016/j.jri.2012.03.348 P 047 Mutation and functional analysis of pregnancy-specific glycoprotein 1 (PSG1) R. O‘Riordan 1,∗ , D. Shanley 2 , M. Ball 1 , T. Moore 1 1 2
University College Cork, Biochemistry, Cork, Ireland Trinity College Dublin, Genetics, Dublin, Ireland
PROBLEM: Pregnancy Specific Glycoproteins (PSGs) are highly glycosylated multi-domain proteins that are members of the carcinoembryonic antigen (CEA) family and Immunoglobulin (Ig) superfamily. During pregnancy, PSGs are secreted by the placenta into the maternal bloodstream
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
where they reach levels of between 200-400 g/ml making them, at term, the most abundant fetally derived proteins in the maternal blood (Linet al, 1974). PSGs are widely reported in the literature to be immunomodulatory, inducing IL-6, IL-10, PGE2 and TGF-1 (Wessels et al., 2000, Snyder et al., 2001, Motrán et al., 2002, Ha et al., 2005), and more recently their pro-angiogenic properties have been examined (Wu et al., 2008, Ha et al., 2010). Recent investigations in our group have also identified anti-thrombotic properties for these proteins (Shanleyet al.in preparation). The aforementioned studies have demonstrated that the N domain of human PSGs is sufficient for induction of cytokines from various cell types. However, very little is known about the contributions of the remaining domains or the glycosylation of the proteins to the function of PSGs in humans. METHODS: pTT3 PSG1V5 His was mutated by PCR to generate V5His tagged domain deletion constructs encoding PSG1N, PSG1A1A2, PSG1B2cterm. Constructs were also generated for individual domains PSG1 N, PSG1A1, PSG1A2, PSG1B2cterm, and the PSG1A1A2 dual domain construct. Mutation of a putative glycosylation site at position 111 was carried out to change an asparagine (N) to an aspartic acid (D) yielding PSG1N111D. Proteins were produced using the FreestyleTM mammalian HEK cell expression system and purified by affinity chromatography followed by imidazole fractionation, concentration and dialysis. Protein concentration was spectrophotometrically determined and purity checked by polyacrylymide gel electrophoresis and coomassie staining. HUVEC-2 endothelial cells and THP-1 monocytic cells were treated with the mutant proteins at equimolar concentrations and the levels of TGF-1 in the media measured by ELISA. RESULTS: All domain deletion constructs induced TGF-1 from THP-1 cells using 1 M concentrations. PSG1 N was the only individual domain construct to be expressed successfully in vitroand induced TGF-1 as previously reported. The PSG1A1A2 dual domain protein was expressed and also induced TGF-1 but required double the dose (>2 M). The mutation of the asparagine at position 111 to an aspartic acid in the full length PSG1V5His resulted in a protein that showed reduced glycosylation versus the wildtype when viewed on a comassie stained polyacrylymide gel. This mutant also induced significantly more TGF-1 than the wildtype. CONCLUSIONS: The PSG1 N domain is not required for the induction of TGF-1 in humans. Mutation of the asparagine 111 to remove a putative glycosylation site resulted in a protein that much more efficiently induced TGF-1. The ability of a single point mutation to more efficiently induce TGF-1 raises important questions about the hypothesis of the expansion of the gene family for dosage effects and the role of selective pressure for potentially inhibitory glycosylation sites in this multi-gene family. doi:10.1016/j.jri.2012.03.349
61
P 048 Interleukin (IL) 1 affects the secretory function of the equine endometrial cells in vitro: modulatory effects of ovarian steroids A. Szóstek 1,∗ , M. Majewska 1 , M. Adamowski 1 , G. FerreiraDias 2 , D. Skarzynski 1 1
Institut of Animal Reproduction and Food Research, Department of Reproductive Immunology and Pathology, Olsztyn, Poland 2 Technical University of Lisbon, C.I.I.S.A.,Faculty of Veterinary Medicine, Lisbon, Portugal Interleukins take part in the regulation of many reproductive function while the estrous cycle and early pregnancy. The main function of IL-1 system in periimplantation period are e.g.: regulation of prostaglandin (PG) secretion via modulation of enzymes responsible for arachidonic acid (AA) metabolism (PTGS-2), an induction of leukemia inhibitor factor (LIF) production; endometrial changes and a production of granulocyte-macrophage colony stimulating factor (GM-CSF) and colony stimulating factor 1 (CSF-1). Additionally, PG participate in ovulation, luteolysis and implantation and their secretion change while the estrous cycle and early pregnancy. We suggest that interactions between cytokines, including IL-1 and AA metabolites in the equine uterus are under the control of sex steroids (progesterone - P4 and 17- estradiol - E2). The aims of the study were to determine: [1] effect of IL- on PG secretion and cells proliferation of the equine endometrial epithelial and stromal cells in vitro; [2] modulatory effects of ovarian steroids on the IL-1stimulated PG secretion from equine epithelial and stromal cells in vitro. The endometrial cells were enzymatically obtained at the early luteal stage of estrus cycle (Days 25 after ovulation; n = 5). The phases of the estrous cycle were identified based on P4 analysis in blood plasma and a macroscopic observation of ovaries. The homogeneity of cell culture were evaluated using immunofluorescent staining for specific markers of epithelial cells (cytokeratin) and stromal cells (vimentin). Only cells derived from the endometria which were classified as healthy were used (no endometrial fibrosis, accordingly to Kenney’s classification). The endometrialcells after passage I were stimulated with the ovarian steroids: E2 (10-9 M), P4 (107), and P4 + E2 (10-7/10-9 M) for 24 h. Then the culture medium was changed and the cells were stimulated with IL-1 (10 ng/ml) for the next 24 h. Prostaglandins were determined in the culture medium the by enzyme-linked immunosorbent assay (EIA). IL-1RI and IL-1RII mRNA transcription was determined using Real-time PCR. IL-1 alone had no influence on the epithelial and stromal cells proliferation (P > 0.05). IL-1 increased PGE2 secretion by epithelial cells and stromal cells compared to the control (P > 0.001). Additionally, IL-1 increased PGF2␣ secretion by epithelial cells (P > 0.001) and stromal cells (P > 0.01) compared to the control. The pretreatment of the epithelial cells with E2 - enhanced IL-1-stimulated PGE2 secretion compared to IL-1-stimulated only group (P4 enhanced IL1-stimulated PGF2␣ secretion by stromal cells compared
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
(MMP-9), plasminogen activator inhibitor protein (PAI-1), tissue inhibitor of MMP 1 (TIMP-1) and urinary plasminogen avtivator (uPA) were expressed in dKdS1. Except for granulocyte-monocyte colony stimulating factor (GMCSF), which was only expressed in dKdS1 after IL-1 incubation, PAI-1 was the only molecule which disappeared after IL-1 incubation on whole cellular protein level. CONCLUSIONS: The knock-down of Sdc-1 leads to a different whole cellular expression pattern for two important proteins regarding angiogenesis during embryo implantation - GM-CSF and PAI-1. The lack of GM-CSF upon decidualization of KdS1 cells might raise a delayed immune response during implantation. PAI-1 is a key regulator of angiogenesis and its expression seems to be connected to Sdc-1. The knock-down of Sdc-1 might therefore lead to a dysregulated angiogenesis during implantation. Funded by the German Research Foundation (DFG) to AP Hess (HE 3544/2-2) angiogenic factor GM-CSF HGF IGFBP-2 IL-8 MMP-9 PAI-1 TIMP-1 uPA
w/o IL-1 cellular protein + + + + + + +
w IL-1 cellular protein + + + + + + +
doi:10.1016/j.jri.2012.03.347 P 046 Progesterone induced blocking factor expression in decidual stromal cells as a potential marker for successful pregnancy E. Stoyanova 1 , K. Vinketova 1 , T. Oreshkova 1 , R. Karaguozov 2 , W. Chaiwangyen 3 , J. Dimitrov 1 , I. Pastuschek 3 , U. Markert 3 , M. Mourdjeva 1,∗ 1
Institute of Biology and Immunology of Reproduction, Molecular Immunology, Sofia, Bulgaria 2 MHAT “Tokuda Hospital Sofia”, Department of Obstetrics and Gynecology, Sofia, Bulgaria 3 Friedrich-Schiller-University, Placenta-Laboratory, Department of Obstetrics, Jena, Germany PROBLEM: Immunological, endocrine and other factors participate in feto-maternal crosstalk during implantation and pregnancy and contribute for the effectiveness of these processes. Human blastocyst implants in maternal decidua which develops only during pregnancy. Decidual stromal cells (DSC) possess potent immunoregulation ability and induce state of tolerance in maternal immune system accepting the semi-allogeneic fetus. Among other factors and mechanisms involved in immunotolerance induction, progesterone induced blocking factor (PIBF) is known to have significant importance. Here, the expression of PIBF in
DSC was investigated and its correlation to decidualization potential of DSC was evaluated. METHODS: DSC were isolated from human deciduas (7-10 week of gestation) obtained from elective pregnancy terminations. DSC were cultured in standard conditions and after three passages when the cultures are over 90% homogeneous, the samples were evaluated for PIBF protein expression by immunoblot and mRNA by RT-PCR. PIBF expression and localization were studied by immunofluorescent staining with 3A6 anti-PIBF monoclonal antibody and confocal microscopy. Prolactin expression after DSCin vitrodecidualization with progesterone, estradiol and cAMP was evaluated by ELISA and RT-PCR. RESULTS: Thein vitrodecidualization of DSC is sometimes problematic. Cells isolated from different deciduas showing similarity in some parameters like expression of markers of mesenchymal stem cells CD29, CD73 and CD90, differentiate differently under the same decidualization conditions. We hypothesized that differences in PIBF expression can be correlated to the decidualization outcome. Five different DSC cell lines were compared in this study. A comparison of PIBF and prolactin mRNA and protein expression levels from different DSC was used to clarify the significance of PIBF as a marker for biological functions of DSC. Immunofluorescent staining revealed high intensity of PIBF expression in majority of DSC cells but showed heterogeneity in PIBF localization pattern. Most of the cells showed intensive nuclear and cytoplasmic staining; some were only with nuclear PIBF presence; and few were completely PIBF negative. Interestingly, after in vitro decidualization, PIBF seems to be secreted from DSC since the intensity of staining reaction was severely reduced and was restricted to secretory vesicles. CONCLUSIONS: We demonstrate that PIBF is highly expressed in DSC prior to any stimuli and that decidualization triggers PIBF secretion.The possible significance of PIBF as a marker for evaluation of quality and functional abilities of DSC is discussed in this work. The biological function of secreted PIBF remains to be investigated in the context of DSC immunoregulation. This study was supported by the projects DOO250/2008 National Science Fund, Ministry of Education and Science and FP7- REGPOT-2009-1 ReProForce. doi:10.1016/j.jri.2012.03.348 P 047 Mutation and functional analysis of pregnancy-specific glycoprotein 1 (PSG1) R. O‘Riordan 1,∗ , D. Shanley 2 , M. Ball 1 , T. Moore 1 1 2
University College Cork, Biochemistry, Cork, Ireland Trinity College Dublin, Genetics, Dublin, Ireland
PROBLEM: Pregnancy Specific Glycoproteins (PSGs) are highly glycosylated multi-domain proteins that are members of the carcinoembryonic antigen (CEA) family and Immunoglobulin (Ig) superfamily. During pregnancy, PSGs are secreted by the placenta into the maternal bloodstream
Abstracts / Journal of Reproductive Immunology 94 (2012) 5–130
where they reach levels of between 200-400 g/ml making them, at term, the most abundant fetally derived proteins in the maternal blood (Linet al, 1974). PSGs are widely reported in the literature to be immunomodulatory, inducing IL-6, IL-10, PGE2 and TGF-1 (Wessels et al., 2000, Snyder et al., 2001, Motrán et al., 2002, Ha et al., 2005), and more recently their pro-angiogenic properties have been examined (Wu et al., 2008, Ha et al., 2010). Recent investigations in our group have also identified anti-thrombotic properties for these proteins (Shanleyet al.in preparation). The aforementioned studies have demonstrated that the N domain of human PSGs is sufficient for induction of cytokines from various cell types. However, very little is known about the contributions of the remaining domains or the glycosylation of the proteins to the function of PSGs in humans. METHODS: pTT3 PSG1V5 His was mutated by PCR to generate V5His tagged domain deletion constructs encoding PSG1N, PSG1A1A2, PSG1B2cterm. Constructs were also generated for individual domains PSG1 N, PSG1A1, PSG1A2, PSG1B2cterm, and the PSG1A1A2 dual domain construct. Mutation of a putative glycosylation site at position 111 was carried out to change an asparagine (N) to an aspartic acid (D) yielding PSG1N111D. Proteins were produced using the FreestyleTM mammalian HEK cell expression system and purified by affinity chromatography followed by imidazole fractionation, concentration and dialysis. Protein concentration was spectrophotometrically determined and purity checked by polyacrylymide gel electrophoresis and coomassie staining. HUVEC-2 endothelial cells and THP-1 monocytic cells were treated with the mutant proteins at equimolar concentrations and the levels of TGF-1 in the media measured by ELISA. RESULTS: All domain deletion constructs induced TGF-1 from THP-1 cells using 1 M concentrations. PSG1 N was the only individual domain construct to be expressed successfully in vitroand induced TGF-1 as previously reported. The PSG1A1A2 dual domain protein was expressed and also induced TGF-1 but required double the dose (>2 M). The mutation of the asparagine at position 111 to an aspartic acid in the full length PSG1V5His resulted in a protein that showed reduced glycosylation versus the wildtype when viewed on a comassie stained polyacrylymide gel. This mutant also induced significantly more TGF-1 than the wildtype. CONCLUSIONS: The PSG1 N domain is not required for the induction of TGF-1 in humans. Mutation of the asparagine 111 to remove a putative glycosylation site resulted in a protein that much more efficiently induced TGF-1. The ability of a single point mutation to more efficiently induce TGF-1 raises important questions about the hypothesis of the expansion of the gene family for dosage effects and the role of selective pressure for potentially inhibitory glycosylation sites in this multi-gene family. doi:10.1016/j.jri.2012.03.349
61
P 048 Interleukin (IL) 1 affects the secretory function of the equine endometrial cells in vitro: modulatory effects of ovarian steroids A. Szóstek 1,∗ , M. Majewska 1 , M. Adamowski 1 , G. FerreiraDias 2 , D. Skarzynski 1 1
Institut of Animal Reproduction and Food Research, Department of Reproductive Immunology and Pathology, Olsztyn, Poland 2 Technical University of Lisbon, C.I.I.S.A.,Faculty of Veterinary Medicine, Lisbon, Portugal Interleukins take part in the regulation of many reproductive function while the estrous cycle and early pregnancy. The main function of IL-1 system in periimplantation period are e.g.: regulation of prostaglandin (PG) secretion via modulation of enzymes responsible for arachidonic acid (AA) metabolism (PTGS-2), an induction of leukemia inhibitor factor (LIF) production; endometrial changes and a production of granulocyte-macrophage colony stimulating factor (GM-CSF) and colony stimulating factor 1 (CSF-1). Additionally, PG participate in ovulation, luteolysis and implantation and their secretion change while the estrous cycle and early pregnancy. We suggest that interactions between cytokines, including IL-1 and AA metabolites in the equine uterus are under the control of sex steroids (progesterone - P4 and 17- estradiol - E2). The aims of the study were to determine: [1] effect of IL- on PG secretion and cells proliferation of the equine endometrial epithelial and stromal cells in vitro; [2] modulatory effects of ovarian steroids on the IL-1stimulated PG secretion from equine epithelial and stromal cells in vitro. The endometrial cells were enzymatically obtained at the early luteal stage of estrus cycle (Days 25 after ovulation; n = 5). The phases of the estrous cycle were identified based on P4 analysis in blood plasma and a macroscopic observation of ovaries. The homogeneity of cell culture were evaluated using immunofluorescent staining for specific markers of epithelial cells (cytokeratin) and stromal cells (vimentin). Only cells derived from the endometria which were classified as healthy were used (no endometrial fibrosis, accordingly to Kenney’s classification). The endometrialcells after passage I were stimulated with the ovarian steroids: E2 (10-9 M), P4 (107), and P4 + E2 (10-7/10-9 M) for 24 h. Then the culture medium was changed and the cells were stimulated with IL-1 (10 ng/ml) for the next 24 h. Prostaglandins were determined in the culture medium the by enzyme-linked immunosorbent assay (EIA). IL-1RI and IL-1RII mRNA transcription was determined using Real-time PCR. IL-1 alone had no influence on the epithelial and stromal cells proliferation (P > 0.05). IL-1 increased PGE2 secretion by epithelial cells and stromal cells compared to the control (P > 0.001). Additionally, IL-1 increased PGF2␣ secretion by epithelial cells (P > 0.001) and stromal cells (P > 0.01) compared to the control. The pretreatment of the epithelial cells with E2 - enhanced IL-1-stimulated PGE2 secretion compared to IL-1-stimulated only group (P4 enhanced IL1-stimulated PGF2␣ secretion by stromal cells compared