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72: Longitudinal modulation of immune system cytokine profile during pregnancy
SMFM Abstracts
www.AJOG.org 71
72
NICOTINE SUPPRESSES SOLUBLE FMS-LIKE TYROSINE KINASE-1 (SFLT-1) EXPRESSION IN TROPHOBLASTS VIA ALPHA 7 NICOTINIC ACETYLCHOLINE RECEPTOR BUT NOT IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) JA YOUNG KWON1, YOUNG HAN KIM1, MUONG HWA KANG1, YONG SUN MAENG2, YOUNG GUEN KWON2, YONG WON PARK1, 1Yonsei University College of Medicine, Department of Obstetrics and Gynecology, Seoul, South Korea, 2Yonsei University, Department of Biochemistry, Seoul, South Korea OBJECTIVE: Cigarette smoking is a unique factor known to reduce the risk of preeclampsia and serum sFlt-1 level is known to be decreased in smokers. Present study was to evaluate the effect of nicotine on production in trophoblasts and HUVEC STUDY DESIGN: Commercially available term placenta trophoblasts and HUVEC extracted from umbilical cord of normal pregnancy were treated with nicotine of 10-7 and 10-6 M. Gene expression of sFlt-1 mRNA expressions were evaluated by RT-PCR and protein level in supernatants were assayed by ELISA following 0, 3, 6, and 12 h of nicotine treatment. Subsequently, alphabungarotoxin (BGT), an á7 nicotinic acetylcholine receptor (nAchR) antagonist was pretreated to evaluate this receptor=s potential role in sFlt-1 production modulation by nicotine. The presence of á7 nAchR in both cell types were confirmed by immunostaining. RESULTS: After 3, 6, 12 h of incubation of trophoblasts and HUVECs with nicotine, sFlt-1 mRNA expression significantly decreased in trophoblasts but not in HUVECs. Concordantly, sFlt-1 protein level in the supernatants of trophoblast following 3, 6, and 12 h of nicotine treatment significantly decreased (19.0 ⫾ 0.6, 23.7 ⫾ 2.0, 32.1 ⫾ 1.9 pg/mL vs. 29.0 ⫾ 1.0, 41.5 ⫾ 3.1, 57.4 ⫾ 5.6 pg/mL, p⬍0.001). To evaluate whether this effect on trophoblast was mediated via á7 nAchR, áBGT was pretreated for 2 h prior to nicotine treatment and as a result, previous nicotinic effect of reduced sFlt-1 protein level was reversed in the áBGTtreated group (28.8 ⫾ 0.8, 40.7 ⫾ 1.3, 58.9 ⫾ 2.4 pg/mL vs. 19.0 ⫾ 0.6, 23.7 ⫾ 2.0, 32.1 ⫾ 1.9 pg/mL, p⬍0.05). CONCLUSION: Nicotine downregulated sFlt-1 production in trophoblast but not in HUVEC. And this effect was mediated by á7 nAchR. This mechanism may be involved in preeclampsia risk reduction in smokers and thus may provide a potential therapeutic target for preventing or treating preeclampsia.
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0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.080
74
LONGITUDINAL MODULATION OF IMMUNE SYSTEM CYTOKINE PROFILE DURING PREGNANCY JEFF DENNEY1, THADDEUS WATERS1, EDWARD NELSON2, LENY MATHEW1, ROBERT GOLDENBERG1, JENNIFER CULHANE1, 1Drexel University College of Medicine, Obstetrics & Gynecology, Center for Perinatal Research, Philadelphia, Pennsylvania, 2University of California, Irvine, Department of Medicine, Division of Hematology/Oncology, Orange, California OBJECTIVE: To characterize normal modulation of the immune system as expressed by whole blood stimulation and cytokine assay at three time-points in human pregnancy. STUDY DESIGN: This is a prospective longitudinal study conducted over the course of pregnancy. Eligibility criteria included having singleton IUP ⬍ 15 weeks gestation and ⱖ 17 years old. Women were excluded for antibiotic or systemic steroid use in the prior two weeks, autoimmunity, or immunocompromised states. Data were obtained at 8-14 weeks, 18-22 weeks, and 28-32 weeks gestation. Six cytokines were measured in supernatants obtained from whole blood ex vivo 24 hour stimulations with 5g/ml PHA and 1g/ml LPS. Samples were processed by Luminex-100 MAP® methods using commercially available kits. Since the data on cytokines are highly skewed and constrained at a lower limit of zero, it is not appropriate to use regression models with normality assumptions. We used Generalized Linear Models (GLM) with gamma family distributions and log link to evaluate cytokine trajectories. We used Huber White sandwich estimator to adjust for effect of within subject correlation in cytokine measures. RESULTS: Complete data were obtained for 80 women. We evaluated the following cytokines: IL-1, IL-6, TNF-, IL-10, IL-4, and IFN-. We observed enhanced counter-regulatory cytokine expression (e.g., increased IL-10). Lymphocytes and monocytes demonstrated dampened responses to global stimuli (i.e., IL-6, IL-4, and IFN-; IL-1 and TNF-, respectively). In contrast to the widely held perception of shift to the TH2 profile, we observed a generalized decrease in inflammatory response over gestation. CONCLUSION: This is the first longitudinal study of a broad panel of cytokines in pregnancy. The overall decrease in pro-inflammatory cytokine trajectory in both the innate and adaptive arms of the immune system and increase in counter-regulatory cytokines as gestation progresses supports the role of immune modulation to permit maintenance of a viable pregnancy. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.081
EFFECTS OF TOBACCO EXPOSURE DURING PREGNANCY ON THE FETO PLACENTAL CIRCULATION CELINE BROCHOT1, MARIE HELENE FLINE1, ESTELLE AUBRY1, PHILIPPE DERUELLE1, LAURENT STORME1, VERONIQUE HOUFFLIN-DEBARGE1, 1JE 24 90 Universite de Lille 2, Lille, France OBJECTIVE: Cigarette smoking during pregnancy is associated with a higher risk of intra uterine growth restriction (IUGR), whereas the incidence of preeclampsia is reduced in smoking women. To explain this paradoxical effect, we studied the effect of maternal passive smoking on the feto-placental circulation in chronically prepared late gestation fetal lambs. STUDY DESIGN: Surgery was performed at 128 days gestation (term, 147 days). Catheters were inserted in the fetal and placental circulations. An ultrasonic flow transducer was placed around the umbilical common artery to measure umbilical flow (Qp). The umbilical vascular resistance(UVR) were calculated. . Ewes were exposed for 2 hours to passive smoking. To examine mechanisms of the placental vascular response to tobacco smoke, L-Nitro-Arginine (LNA, a NOsynthase inhibitor) and tetra-ethyl-ammonium (TEA, a K⫹ channels inhibitor,) were infused to the fetus during cigarette exposure. Maternal and fetal blood gas tension were obtained before and after each experiments. RESULTS: Passive smoking increased Qp by 17% (p⬍0.05) without change in aortic pressure, resulting in a 15% decrease in UVR (p⬍0,05). Fetal PaO2 decreased from 17 to 11 mmHg (p⫽0.05) without change in maternal PaO2. Fetal HbCO increased from 5 to 15% (p⬍0.05). Smoking induced-vasodilation was not attenuated by L-NA. In contrast, TEA completely abolished the vasodilatory effects of passive smoking. CONCLUSION: We conclude that passive cigarette smoking during pregnancy causes feto-placental vasodilation mediated by K⫹ channels activation, despite fetal hypoxemia. We speculate that these effects may explain that cigarette smoke may cause IUGR and prevent from preeclampsia. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.082
ADVERSE UTERINE ENVIRONMENT LEADS TO FAILURE TO DOWNREGULATE ENDOGENOUS CHOLESTEROL SYNTHESIS IN A MOUSE MODEL. NIMA GOHARKHAY1, ESTHER TAMAYO1, FANGXIAN LU1, HUAIZHI YIN1, GARY D.V. HANKINS1, GEORGE R. SAADE1, MONICA LONGO1, 1The University of Texas Medical Branch, Obstetrics and Gynecology, Galveston, Texas OBJECTIVE: We have previously shown evidence of atherosclerosis, dyslipidemia and changes in mediators of lipid homeostasis in mice born to hypercholesterolemic mothers. Sterol regulatory-binding proteins (SREBPs) are transcription factors that control sterol synthesis in mammals. Our aim was to determine hepatic expression rates of SREBP-1a, ⫺1c and ⫺2, and to correlate them with other mediators of cholesterol metabolism in this model. STUDY DESIGN: We crossbred apoE knockout (apoE⫺/⫺) and wild-type C57BL/6J (apoE⫹/⫹) mice to obtain: heterozygous born to hypercholesterolemic apoE⫺/⫺ mothers (apoE⫹/⫺mat), heterozygous born to wild-type mothers (apoE⫹/⫺pat), homozygous knockout (apoE⫺/⫺), and homozygous wild-type (apoE⫹/⫹) offspring. All litters were maintained on regular chow and sacrificed at eight months. Real-time RT-PCR was performed from liver samples to detect mRNA expression levels for SREBP-1a, ⫺1c and ⫺2. RESULTS: We found a significant variation in SREBP-1a levels among the four study groups (p⫽0.025), with the highest levels in apoE⫹/⫺mat offspring (figure). Levels of SREBP-2 expression were also increased in apoE⫹/⫺mat mice (figure). SREBP-1c mRNA was not detectable in our samples. We observed significant positive correlations of mRNA expression levels of SREBP-1a and SREBP-2 with that of HMG-CoA reductase (HMGR) and SREBP cleavage activator protein (SCAP), respectively.
CONCLUSION: Offspring born to hypercholesterolemic mothers fail to downregulate endogenous cholesterol synthesis in adulthood. The data underscore our hypothesis that uninhibited SCAP expression is an early event underlying in-utero programming of hypercholesterolemia. Interventions focused on this mechanism can have important long-term implications. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.083
Supplement to DECEMBER 2007 American Journal of Obstetrics & Gynecology
S33
www.AJOG.org 71
72
NICOTINE SUPPRESSES SOLUBLE FMS-LIKE TYROSINE KINASE-1 (SFLT-1) EXPRESSION IN TROPHOBLASTS VIA ALPHA 7 NICOTINIC ACETYLCHOLINE RECEPTOR BUT NOT IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) JA YOUNG KWON1, YOUNG HAN KIM1, MUONG HWA KANG1, YONG SUN MAENG2, YOUNG GUEN KWON2, YONG WON PARK1, 1Yonsei University College of Medicine, Department of Obstetrics and Gynecology, Seoul, South Korea, 2Yonsei University, Department of Biochemistry, Seoul, South Korea OBJECTIVE: Cigarette smoking is a unique factor known to reduce the risk of preeclampsia and serum sFlt-1 level is known to be decreased in smokers. Present study was to evaluate the effect of nicotine on production in trophoblasts and HUVEC STUDY DESIGN: Commercially available term placenta trophoblasts and HUVEC extracted from umbilical cord of normal pregnancy were treated with nicotine of 10-7 and 10-6 M. Gene expression of sFlt-1 mRNA expressions were evaluated by RT-PCR and protein level in supernatants were assayed by ELISA following 0, 3, 6, and 12 h of nicotine treatment. Subsequently, alphabungarotoxin (BGT), an á7 nicotinic acetylcholine receptor (nAchR) antagonist was pretreated to evaluate this receptor=s potential role in sFlt-1 production modulation by nicotine. The presence of á7 nAchR in both cell types were confirmed by immunostaining. RESULTS: After 3, 6, 12 h of incubation of trophoblasts and HUVECs with nicotine, sFlt-1 mRNA expression significantly decreased in trophoblasts but not in HUVECs. Concordantly, sFlt-1 protein level in the supernatants of trophoblast following 3, 6, and 12 h of nicotine treatment significantly decreased (19.0 ⫾ 0.6, 23.7 ⫾ 2.0, 32.1 ⫾ 1.9 pg/mL vs. 29.0 ⫾ 1.0, 41.5 ⫾ 3.1, 57.4 ⫾ 5.6 pg/mL, p⬍0.001). To evaluate whether this effect on trophoblast was mediated via á7 nAchR, áBGT was pretreated for 2 h prior to nicotine treatment and as a result, previous nicotinic effect of reduced sFlt-1 protein level was reversed in the áBGTtreated group (28.8 ⫾ 0.8, 40.7 ⫾ 1.3, 58.9 ⫾ 2.4 pg/mL vs. 19.0 ⫾ 0.6, 23.7 ⫾ 2.0, 32.1 ⫾ 1.9 pg/mL, p⬍0.05). CONCLUSION: Nicotine downregulated sFlt-1 production in trophoblast but not in HUVEC. And this effect was mediated by á7 nAchR. This mechanism may be involved in preeclampsia risk reduction in smokers and thus may provide a potential therapeutic target for preventing or treating preeclampsia.
73
0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.080
74
LONGITUDINAL MODULATION OF IMMUNE SYSTEM CYTOKINE PROFILE DURING PREGNANCY JEFF DENNEY1, THADDEUS WATERS1, EDWARD NELSON2, LENY MATHEW1, ROBERT GOLDENBERG1, JENNIFER CULHANE1, 1Drexel University College of Medicine, Obstetrics & Gynecology, Center for Perinatal Research, Philadelphia, Pennsylvania, 2University of California, Irvine, Department of Medicine, Division of Hematology/Oncology, Orange, California OBJECTIVE: To characterize normal modulation of the immune system as expressed by whole blood stimulation and cytokine assay at three time-points in human pregnancy. STUDY DESIGN: This is a prospective longitudinal study conducted over the course of pregnancy. Eligibility criteria included having singleton IUP ⬍ 15 weeks gestation and ⱖ 17 years old. Women were excluded for antibiotic or systemic steroid use in the prior two weeks, autoimmunity, or immunocompromised states. Data were obtained at 8-14 weeks, 18-22 weeks, and 28-32 weeks gestation. Six cytokines were measured in supernatants obtained from whole blood ex vivo 24 hour stimulations with 5g/ml PHA and 1g/ml LPS. Samples were processed by Luminex-100 MAP® methods using commercially available kits. Since the data on cytokines are highly skewed and constrained at a lower limit of zero, it is not appropriate to use regression models with normality assumptions. We used Generalized Linear Models (GLM) with gamma family distributions and log link to evaluate cytokine trajectories. We used Huber White sandwich estimator to adjust for effect of within subject correlation in cytokine measures. RESULTS: Complete data were obtained for 80 women. We evaluated the following cytokines: IL-1, IL-6, TNF-, IL-10, IL-4, and IFN-. We observed enhanced counter-regulatory cytokine expression (e.g., increased IL-10). Lymphocytes and monocytes demonstrated dampened responses to global stimuli (i.e., IL-6, IL-4, and IFN-; IL-1 and TNF-, respectively). In contrast to the widely held perception of shift to the TH2 profile, we observed a generalized decrease in inflammatory response over gestation. CONCLUSION: This is the first longitudinal study of a broad panel of cytokines in pregnancy. The overall decrease in pro-inflammatory cytokine trajectory in both the innate and adaptive arms of the immune system and increase in counter-regulatory cytokines as gestation progresses supports the role of immune modulation to permit maintenance of a viable pregnancy. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.081
EFFECTS OF TOBACCO EXPOSURE DURING PREGNANCY ON THE FETO PLACENTAL CIRCULATION CELINE BROCHOT1, MARIE HELENE FLINE1, ESTELLE AUBRY1, PHILIPPE DERUELLE1, LAURENT STORME1, VERONIQUE HOUFFLIN-DEBARGE1, 1JE 24 90 Universite de Lille 2, Lille, France OBJECTIVE: Cigarette smoking during pregnancy is associated with a higher risk of intra uterine growth restriction (IUGR), whereas the incidence of preeclampsia is reduced in smoking women. To explain this paradoxical effect, we studied the effect of maternal passive smoking on the feto-placental circulation in chronically prepared late gestation fetal lambs. STUDY DESIGN: Surgery was performed at 128 days gestation (term, 147 days). Catheters were inserted in the fetal and placental circulations. An ultrasonic flow transducer was placed around the umbilical common artery to measure umbilical flow (Qp). The umbilical vascular resistance(UVR) were calculated. . Ewes were exposed for 2 hours to passive smoking. To examine mechanisms of the placental vascular response to tobacco smoke, L-Nitro-Arginine (LNA, a NOsynthase inhibitor) and tetra-ethyl-ammonium (TEA, a K⫹ channels inhibitor,) were infused to the fetus during cigarette exposure. Maternal and fetal blood gas tension were obtained before and after each experiments. RESULTS: Passive smoking increased Qp by 17% (p⬍0.05) without change in aortic pressure, resulting in a 15% decrease in UVR (p⬍0,05). Fetal PaO2 decreased from 17 to 11 mmHg (p⫽0.05) without change in maternal PaO2. Fetal HbCO increased from 5 to 15% (p⬍0.05). Smoking induced-vasodilation was not attenuated by L-NA. In contrast, TEA completely abolished the vasodilatory effects of passive smoking. CONCLUSION: We conclude that passive cigarette smoking during pregnancy causes feto-placental vasodilation mediated by K⫹ channels activation, despite fetal hypoxemia. We speculate that these effects may explain that cigarette smoke may cause IUGR and prevent from preeclampsia. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.082
ADVERSE UTERINE ENVIRONMENT LEADS TO FAILURE TO DOWNREGULATE ENDOGENOUS CHOLESTEROL SYNTHESIS IN A MOUSE MODEL. NIMA GOHARKHAY1, ESTHER TAMAYO1, FANGXIAN LU1, HUAIZHI YIN1, GARY D.V. HANKINS1, GEORGE R. SAADE1, MONICA LONGO1, 1The University of Texas Medical Branch, Obstetrics and Gynecology, Galveston, Texas OBJECTIVE: We have previously shown evidence of atherosclerosis, dyslipidemia and changes in mediators of lipid homeostasis in mice born to hypercholesterolemic mothers. Sterol regulatory-binding proteins (SREBPs) are transcription factors that control sterol synthesis in mammals. Our aim was to determine hepatic expression rates of SREBP-1a, ⫺1c and ⫺2, and to correlate them with other mediators of cholesterol metabolism in this model. STUDY DESIGN: We crossbred apoE knockout (apoE⫺/⫺) and wild-type C57BL/6J (apoE⫹/⫹) mice to obtain: heterozygous born to hypercholesterolemic apoE⫺/⫺ mothers (apoE⫹/⫺mat), heterozygous born to wild-type mothers (apoE⫹/⫺pat), homozygous knockout (apoE⫺/⫺), and homozygous wild-type (apoE⫹/⫹) offspring. All litters were maintained on regular chow and sacrificed at eight months. Real-time RT-PCR was performed from liver samples to detect mRNA expression levels for SREBP-1a, ⫺1c and ⫺2. RESULTS: We found a significant variation in SREBP-1a levels among the four study groups (p⫽0.025), with the highest levels in apoE⫹/⫺mat offspring (figure). Levels of SREBP-2 expression were also increased in apoE⫹/⫺mat mice (figure). SREBP-1c mRNA was not detectable in our samples. We observed significant positive correlations of mRNA expression levels of SREBP-1a and SREBP-2 with that of HMG-CoA reductase (HMGR) and SREBP cleavage activator protein (SCAP), respectively.
CONCLUSION: Offspring born to hypercholesterolemic mothers fail to downregulate endogenous cholesterol synthesis in adulthood. The data underscore our hypothesis that uninhibited SCAP expression is an early event underlying in-utero programming of hypercholesterolemia. Interventions focused on this mechanism can have important long-term implications. 0002-9378/$ - see front matter doi:10.1016/j.ajog.2007.10.083
Supplement to DECEMBER 2007 American Journal of Obstetrics & Gynecology
S33