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Fetal loss of adrenomedullin causes vascular pathology typical of preeclampsia
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Abstracts / Journal of Reproductive Immunology 86 (2010) 79–111
IL-33 is a recently described member of the IL-1 family which includes IL-1b and IL-18. Like these cytokines, IL-33 has strong immunomodulatory functions and predominantly induces the production of Th2 cytokines (IL-4, IL-5 and IL-13) in T cells and NK cells and increases serum immunoglobulin. Soluble ST2 is thought to act as a ‘decoy receptor’, competing with the membrane bound receptor (ST2L) for IL-33 binding. Increasing levels of sST2 may therefore negatively regulate IL-33 signalling and diminish the cytokines overall bioactivity. We have shown that IL-33 is produced by the placenta and can be measured in the maternal circulation. Interestingly the expression of ST2L is significantly increased at the time of the pregnancy test on NK cells, T helper cells and T cytotoxic cells in women who have successful implantation, but not in those where implantation failed. Similarly ST2L expression is significantly upregulated in normal pregnancy compared to pre-eclampsia and circulating levels of sST2 are increased. These findings suggest that the IL-33/ST2 axis may play a key role in maternofetal interactions in pregnancy. Funding source: Wellcome Trust, Oxford NIHR Biomedical Research Centre, The Oxford Fertility Unit. doi:10.1016/j.jri.2010.08.043 43 Intrauterine determination of neonatal stress physiology L. Schäffer ∗ , E. Beinder Department of Obstetrics, University Hospital of Zürich, Switzerland Epidemiological data strongly suggest that an unfavorable intrauterine environment may be highly predictive for the risk to develop diseases such as hypertension and the metabolic syndrome. It is hypothesized that intrauterine environmental factors permanently alter gene expression via imprinting mechanisms during sensitive periods of fetal development leading to a permanent alteration of the balance and function of endocrine systems. Two stress regulating endocrine systems involved in the adjustment of the organism towards increased strain, the hypothalamo-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS), are believed to be sensitive towards imprinting during intrauterine life. At the time of their own development, these systems are already challenged if adverse intrauterine conditions are present. Fetuses with intrauterine growth retardation, for example, have increased catecholamine and cortisol levels as stress systems are highly activated. Later in adulthood, the same systems are actively involved in the pathogenesis of hypertension and the metabolic syndrome. Therefore, it is speculated and animal models provide evidence that these systems may undergo developmental alterations during adverse intrauterine conditions leading to permanent changes in their physiological balance and reactivity towards demand. Since the progression towards disease will involve action of primary altered systems and reaction of com-
pensatory activated systems, initial factors for disease development have to be identified if preventive measures or therapeutic strategies are to be developed. Evaluation of neonatal stress physiology provides the opportunity to analyze a period when temporary up-regulated systems normalize in response to a normal postnatal environment, while permanently altered systems may be detectable. Furthermore, at this time compensatory system reactions may not have had the time to develop. Thus, if the HPA axis and the SNS experience permanent alterations during intrauterine development, these alterations should be detectable in the neonate. Considering the fact that these alterations may be mild and clinically not apparent during early life due to a high compensatory reserve being only “exhausted” over many years, we analyzed HPA and SNS balance under resting conditions but also after challenging these systems. Stress physiology in neonates can be assessed noninvasively by measuring cortisol levels in saliva during resting and challenged states providing information on the balance of the HPA axis and saliva amylase levels and heartrate-variability parameters give an insight on the activity of the sympathetic nervous system. Our data provide evidence that the HPA axis functionality is already altered in human neonates after different intrauterine impacts while a normal balance of the sympathetic nervous system seems to be preserved. Furthermore, there is evidence that the HPA axis is subject to functional plasticity during the interval between the initiating event and the end-point as it appears to be a biological substrate for the interaction of prenatal and postnatal events. From the results of our own data in neonates, it appears that the intrauterine HPA axis development is vulnerable towards conditions that alter endogenous glucocorticoid homeostasis and these alterations persist into postnatal live suggesting a permanent imprinting of this system in human infants. Nevertheless, early childhood may still contain a certain plasticity of these systems with putative options for interventions that may restore infant stress physiology. doi:10.1016/j.jri.2010.08.044 44 Fetal loss of adrenomedullin causes vascular pathology typical of preeclampsia N.M.J. Schwerbrock a,∗ , T. Lenhart a , M. Li a , M. Kadmiel a , K. Fritz-Six a , D. Krause c , K. Christine c , C.P. Mack b , K. Caron a a
Department of Cell & Molecular Physiology, The University of North Carolina, Chapel Hill, NC 27599, USA b Department of Pathology and Laboratory Medicine, The University of North Carolina, Chapel Hill, NC 27599, USA c Department of Obstetrics & Gynecology, Duke University Medical Center, Durham, NC 27710, USA Introduction: Adrenomedullin (AM) is a 52-amino acid peptide vasodilator that is highly elevated in nor-
Abstracts / Journal of Reproductive Immunology 86 (2010) 79–111
mal human pregnancies but is often blunted in pregnancy complications such as fetal growth restriction, gestational diabetes, and preeclampsia. Our previous studies showed that genetic reduction of maternal AM leads to poor pregnancy outcomes, but the role of fetal AM remains unknown. Therefore, we examined AM−/− and AM+/+ placentas at mid-gestation for structural, functional, and developmental phenotypes. Methods: Using AM−/− , AM+/+ and AMhi/hi genetargeted mouse models we determined effects of quantitative genetic variation of adrenomedullin in placentation in vivo. Multifaceted techniques were applied to evaluate placental layer differentiation, maternal–fetal interface vessel morphology and placental function. To test the impact of AM on placental immune cells, we isolated uterin natural killer (uNK) cells and evaluated their properties in vitro. Results: Histologic and morphometric analysis revealed normal differentiation and layer formation in the AM−/− placentas. Doppler ultrasound of umbilical cord blood flow showed that placental impedance was unaffected by fetal loss of AM. Although histology and laminin staining showed no difference in the total cross-sectional area of fetal and maternal vessels, the fetal vessels of the AM−/− placentas were large and underbranched compared to those of wild type littermates. Electron microscopy of methyl-methacrylate casts confirmed there were fewer, larger, and less-branched vessels in the AM−/− placentas. Placental alkaline phosphatase staining showed no difference in chorionic trophoblast invasion of the labyrinth indicating that the vascular defects were not caused by reduced trophoblast villi formation. Strikingly, spiral arteries in AM−/− mice failed to remodel and retained their thick smooth muscle cell layer. In conjunction with this phenotype, there was a 40% reduction in uNK cell content in AM−/− placentas compared to wild type (WT) littermate placentas. Placental cytokines profiling revealed that Ccl17, Cxcl9, Cxcl10, Xcl1, TNF, and apelin receptor, which are known to play important roles in the placenta, had opposite regulation in the AMHi/Hi versus AM−/− placentas when normalized to their WT controls, demonstrating that fetal AM levels could regulate the cytokine profile of maternal uNK cells. In vitro studies showed that AM treatment of isolated uNK cells also lead to robust induction of Ccl17, Cxcl10, Cxcl11, GM-CSF and down regulation of IL-23. Cultured smooth muscle cells in the presence of AM-treated uNK cell supernatants had a moderate but significant increase in MMP9 when compared to non-treated controls, supporting the role of AM as an important factor for adapting the maternal vasculature to pregnancy. Conclusions: Taken together, these data indicate that loss of fetal AM causes reduced fetal vascularization and maternal spiral artery remodeling characteristic of preeclampsia, and that these phenotypes can be reversed by overexpressing fetal AM. Keywords: Adrenomedullin; Preeclampsia; Uterin Natural Killer cells. Funding source: KC: March of Dimes Birth Defects Foundation, NIH HD060860, Burroughs Wellcome Fund, doi:10.1016/j.jri.2010.08.045
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45 MIC-1—a novel modulator of dendritic cells in early pregnancy S.E. Segerer ∗ , L. Rieger, M. Kapp, J. Dietl, U. Kämmerer Department of Obstetrics and Gynaecology, University Hospital, Josef-Schneider-Str. 4, D-97080 Würzburg, Germany Introduction: Macrophage inhibitory cytokine-1 (MIC1) is a multifunctional cytokine produced in high levels by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotrophic roles in the establishment and maintenance of pregnancy. So far, only little is known about the decidual cell types producing MIC-1 and the effect of this cytokine on dendritic cells (DC), which are thought to play a distinct role in the development of peripheral tolerance in pregnancy. Material and methods: To identify the cell populations within decidua, which express and secrete MIC-1, immunohistochemistry, RT-PCR experiments, Western blot analysis and ELISA were performed with isolated stoma cells, invasive trophoblasts (CTB), uNK cells and decidual monomyeloic cells. We then generated immature DC (iDC) from peripheral blood-derived monocytes and differentiated them in the presence of MIC-1 or dexamethasone (Dex) as a control. The resulting phenotype of DC was studied using FACS analysis. To test the T-cell stimulatory capacity of pre-incubated DC, mixed lymphocyte reaction was applied. Finally, migratory activity of immature and mature DC after MIC-1 exposure was investigated by migration assay. Results: Immunohistochemical staining, PCR and Western blot experiments ascertained that MIC-1 is mainly expressed by CTB and decidual stromal cells. Analysing the MIC-1 secretion of decidual cells by ELISA, CTB and decidual stromal cells again could be characterized as main producers. Studying the phenotype of DC after MIC-1 exposure by FACS analysis, we observed that MIC-1 decreased the expression of typical maturation molecules like CD25, CD40 and CD83 during cytokine-induced DC maturation similar to Dex. In addition, T-cell stimulatory capacity of DC was significantly reduced after MIC-1 exposure. Studying the migratory activity of iDC and mDC via migration assay, we observed that MIC-1 was able to induce migration of iDC but not of mDC. Conclusion: Herein, we have identified a novel factor of early pregnancy that could promote the increase of iDC in decidua and generate tolerogenic phenotype of this immune cell population. Keywords: MIC-1; Decidua; Dendritic cells; Migration Funding source: This project was in part supported by the Deutsche Forschungsgemeinschaft (DFG, KA1253/2-3) and by the Interdisziplinary Centre for Clinical Research (IZKF, Z-3/5). doi:10.1016/j.jri.2010.08.046
Abstracts / Journal of Reproductive Immunology 86 (2010) 79–111
IL-33 is a recently described member of the IL-1 family which includes IL-1b and IL-18. Like these cytokines, IL-33 has strong immunomodulatory functions and predominantly induces the production of Th2 cytokines (IL-4, IL-5 and IL-13) in T cells and NK cells and increases serum immunoglobulin. Soluble ST2 is thought to act as a ‘decoy receptor’, competing with the membrane bound receptor (ST2L) for IL-33 binding. Increasing levels of sST2 may therefore negatively regulate IL-33 signalling and diminish the cytokines overall bioactivity. We have shown that IL-33 is produced by the placenta and can be measured in the maternal circulation. Interestingly the expression of ST2L is significantly increased at the time of the pregnancy test on NK cells, T helper cells and T cytotoxic cells in women who have successful implantation, but not in those where implantation failed. Similarly ST2L expression is significantly upregulated in normal pregnancy compared to pre-eclampsia and circulating levels of sST2 are increased. These findings suggest that the IL-33/ST2 axis may play a key role in maternofetal interactions in pregnancy. Funding source: Wellcome Trust, Oxford NIHR Biomedical Research Centre, The Oxford Fertility Unit. doi:10.1016/j.jri.2010.08.043 43 Intrauterine determination of neonatal stress physiology L. Schäffer ∗ , E. Beinder Department of Obstetrics, University Hospital of Zürich, Switzerland Epidemiological data strongly suggest that an unfavorable intrauterine environment may be highly predictive for the risk to develop diseases such as hypertension and the metabolic syndrome. It is hypothesized that intrauterine environmental factors permanently alter gene expression via imprinting mechanisms during sensitive periods of fetal development leading to a permanent alteration of the balance and function of endocrine systems. Two stress regulating endocrine systems involved in the adjustment of the organism towards increased strain, the hypothalamo-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS), are believed to be sensitive towards imprinting during intrauterine life. At the time of their own development, these systems are already challenged if adverse intrauterine conditions are present. Fetuses with intrauterine growth retardation, for example, have increased catecholamine and cortisol levels as stress systems are highly activated. Later in adulthood, the same systems are actively involved in the pathogenesis of hypertension and the metabolic syndrome. Therefore, it is speculated and animal models provide evidence that these systems may undergo developmental alterations during adverse intrauterine conditions leading to permanent changes in their physiological balance and reactivity towards demand. Since the progression towards disease will involve action of primary altered systems and reaction of com-
pensatory activated systems, initial factors for disease development have to be identified if preventive measures or therapeutic strategies are to be developed. Evaluation of neonatal stress physiology provides the opportunity to analyze a period when temporary up-regulated systems normalize in response to a normal postnatal environment, while permanently altered systems may be detectable. Furthermore, at this time compensatory system reactions may not have had the time to develop. Thus, if the HPA axis and the SNS experience permanent alterations during intrauterine development, these alterations should be detectable in the neonate. Considering the fact that these alterations may be mild and clinically not apparent during early life due to a high compensatory reserve being only “exhausted” over many years, we analyzed HPA and SNS balance under resting conditions but also after challenging these systems. Stress physiology in neonates can be assessed noninvasively by measuring cortisol levels in saliva during resting and challenged states providing information on the balance of the HPA axis and saliva amylase levels and heartrate-variability parameters give an insight on the activity of the sympathetic nervous system. Our data provide evidence that the HPA axis functionality is already altered in human neonates after different intrauterine impacts while a normal balance of the sympathetic nervous system seems to be preserved. Furthermore, there is evidence that the HPA axis is subject to functional plasticity during the interval between the initiating event and the end-point as it appears to be a biological substrate for the interaction of prenatal and postnatal events. From the results of our own data in neonates, it appears that the intrauterine HPA axis development is vulnerable towards conditions that alter endogenous glucocorticoid homeostasis and these alterations persist into postnatal live suggesting a permanent imprinting of this system in human infants. Nevertheless, early childhood may still contain a certain plasticity of these systems with putative options for interventions that may restore infant stress physiology. doi:10.1016/j.jri.2010.08.044 44 Fetal loss of adrenomedullin causes vascular pathology typical of preeclampsia N.M.J. Schwerbrock a,∗ , T. Lenhart a , M. Li a , M. Kadmiel a , K. Fritz-Six a , D. Krause c , K. Christine c , C.P. Mack b , K. Caron a a
Department of Cell & Molecular Physiology, The University of North Carolina, Chapel Hill, NC 27599, USA b Department of Pathology and Laboratory Medicine, The University of North Carolina, Chapel Hill, NC 27599, USA c Department of Obstetrics & Gynecology, Duke University Medical Center, Durham, NC 27710, USA Introduction: Adrenomedullin (AM) is a 52-amino acid peptide vasodilator that is highly elevated in nor-
Abstracts / Journal of Reproductive Immunology 86 (2010) 79–111
mal human pregnancies but is often blunted in pregnancy complications such as fetal growth restriction, gestational diabetes, and preeclampsia. Our previous studies showed that genetic reduction of maternal AM leads to poor pregnancy outcomes, but the role of fetal AM remains unknown. Therefore, we examined AM−/− and AM+/+ placentas at mid-gestation for structural, functional, and developmental phenotypes. Methods: Using AM−/− , AM+/+ and AMhi/hi genetargeted mouse models we determined effects of quantitative genetic variation of adrenomedullin in placentation in vivo. Multifaceted techniques were applied to evaluate placental layer differentiation, maternal–fetal interface vessel morphology and placental function. To test the impact of AM on placental immune cells, we isolated uterin natural killer (uNK) cells and evaluated their properties in vitro. Results: Histologic and morphometric analysis revealed normal differentiation and layer formation in the AM−/− placentas. Doppler ultrasound of umbilical cord blood flow showed that placental impedance was unaffected by fetal loss of AM. Although histology and laminin staining showed no difference in the total cross-sectional area of fetal and maternal vessels, the fetal vessels of the AM−/− placentas were large and underbranched compared to those of wild type littermates. Electron microscopy of methyl-methacrylate casts confirmed there were fewer, larger, and less-branched vessels in the AM−/− placentas. Placental alkaline phosphatase staining showed no difference in chorionic trophoblast invasion of the labyrinth indicating that the vascular defects were not caused by reduced trophoblast villi formation. Strikingly, spiral arteries in AM−/− mice failed to remodel and retained their thick smooth muscle cell layer. In conjunction with this phenotype, there was a 40% reduction in uNK cell content in AM−/− placentas compared to wild type (WT) littermate placentas. Placental cytokines profiling revealed that Ccl17, Cxcl9, Cxcl10, Xcl1, TNF, and apelin receptor, which are known to play important roles in the placenta, had opposite regulation in the AMHi/Hi versus AM−/− placentas when normalized to their WT controls, demonstrating that fetal AM levels could regulate the cytokine profile of maternal uNK cells. In vitro studies showed that AM treatment of isolated uNK cells also lead to robust induction of Ccl17, Cxcl10, Cxcl11, GM-CSF and down regulation of IL-23. Cultured smooth muscle cells in the presence of AM-treated uNK cell supernatants had a moderate but significant increase in MMP9 when compared to non-treated controls, supporting the role of AM as an important factor for adapting the maternal vasculature to pregnancy. Conclusions: Taken together, these data indicate that loss of fetal AM causes reduced fetal vascularization and maternal spiral artery remodeling characteristic of preeclampsia, and that these phenotypes can be reversed by overexpressing fetal AM. Keywords: Adrenomedullin; Preeclampsia; Uterin Natural Killer cells. Funding source: KC: March of Dimes Birth Defects Foundation, NIH HD060860, Burroughs Wellcome Fund, doi:10.1016/j.jri.2010.08.045
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45 MIC-1—a novel modulator of dendritic cells in early pregnancy S.E. Segerer ∗ , L. Rieger, M. Kapp, J. Dietl, U. Kämmerer Department of Obstetrics and Gynaecology, University Hospital, Josef-Schneider-Str. 4, D-97080 Würzburg, Germany Introduction: Macrophage inhibitory cytokine-1 (MIC1) is a multifunctional cytokine produced in high levels by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotrophic roles in the establishment and maintenance of pregnancy. So far, only little is known about the decidual cell types producing MIC-1 and the effect of this cytokine on dendritic cells (DC), which are thought to play a distinct role in the development of peripheral tolerance in pregnancy. Material and methods: To identify the cell populations within decidua, which express and secrete MIC-1, immunohistochemistry, RT-PCR experiments, Western blot analysis and ELISA were performed with isolated stoma cells, invasive trophoblasts (CTB), uNK cells and decidual monomyeloic cells. We then generated immature DC (iDC) from peripheral blood-derived monocytes and differentiated them in the presence of MIC-1 or dexamethasone (Dex) as a control. The resulting phenotype of DC was studied using FACS analysis. To test the T-cell stimulatory capacity of pre-incubated DC, mixed lymphocyte reaction was applied. Finally, migratory activity of immature and mature DC after MIC-1 exposure was investigated by migration assay. Results: Immunohistochemical staining, PCR and Western blot experiments ascertained that MIC-1 is mainly expressed by CTB and decidual stromal cells. Analysing the MIC-1 secretion of decidual cells by ELISA, CTB and decidual stromal cells again could be characterized as main producers. Studying the phenotype of DC after MIC-1 exposure by FACS analysis, we observed that MIC-1 decreased the expression of typical maturation molecules like CD25, CD40 and CD83 during cytokine-induced DC maturation similar to Dex. In addition, T-cell stimulatory capacity of DC was significantly reduced after MIC-1 exposure. Studying the migratory activity of iDC and mDC via migration assay, we observed that MIC-1 was able to induce migration of iDC but not of mDC. Conclusion: Herein, we have identified a novel factor of early pregnancy that could promote the increase of iDC in decidua and generate tolerogenic phenotype of this immune cell population. Keywords: MIC-1; Decidua; Dendritic cells; Migration Funding source: This project was in part supported by the Deutsche Forschungsgemeinschaft (DFG, KA1253/2-3) and by the Interdisziplinary Centre for Clinical Research (IZKF, Z-3/5). doi:10.1016/j.jri.2010.08.046