Karen H. Oppenheimer

Modulation of monocyte chemotactic protein-1 expression during lipopolysaccharide-induced preterm delivery in the pregnant mouse.

Preterm delivery is often associated with increased cytokine and chemokine production. These studies characterize the expression of the chemokine monocyte chemotactic protein-1 (MCP-1) in mice during lipopolysaccharide (LPS)—induced preterm delivery. Uterine and other tissues were harvested from CD-1 mice on gestational day 15 after intrauterine LPS injection. Quantitative real-time reverse-transcriptase polymerase chain reactions determined MCP-1 and toll-like receptor 4 (TLR4) mRNA expression during the 24 hours after LPS. MCP-1 protein expression was determined using a cytokine/chemokine protein array, enzyme-linked immunosorbant assay, and immunohistochemistry. Intrauterine LPS injection caused preterm delivery in CD-1 mice between 12 and 24 hours. Expression of MCP-1 mRNA significantly increased at 2 and 6 hours, while TLR4 expression did not significantly change over 24 hours. The MCP-1 protein levels peaked by 2 to 6 hours in maternal serum, liver, lung, kidney, and uterus. Immunohistochemistry confirmed MCP-1 in the myometrium and endometrium. These studies provide evidence suggesting that MCP-1 potentially plays an important role during the proinflammatory immune response, leading to preterm labor in the mouse.

Evidence that CD8 T-cell homeostasis and function remain intact during murine pregnancy

Evolving models of immune tolerance have challenged the view that the response of the maternal immune system to environmental or fetal antigens must be suppressed or deviated. CD8 T cells play a central role in the immune response to viruses and intracellular pathogens so the maintenance of both the number and function of these cells is critical to protect both the mother and fetus. We show that the numbers of maternal CD8 T cells in both the spleen and the uterine draining lymph nodes are transiently increased at mid‐gestation and this correlates with enhanced CD8 T‐cell proliferation and an increased relative expression of both pro‐survival and pro‐apoptotic molecules. In transgenic mice bearing T‐cell antigen receptors specific for the male HY or allo‐antigens, the transgenic CD8 T cells retain the ability to proliferate and function during pregnancy. Moreover, anti‐HY T‐cell receptor transgenic mice have normal numbers of male pups despite the presence of CD8 T cells at the maternal–fetal interface. These data suggest that pregnancy is a dynamic state in which CD8 T‐cell turnover is increased while the function and ending size of the CD8 T‐cell compartment are maintained.

Selective Accumulation of Th2-skewing Immature Erythroid Cells in Developing Neonatal Mouse Spleen

Environmental factors likely regulate neonatal immunity and self-tolerance. However, evidence that the neonatal immune system is suppressed or deviated is varied depending on the antigen and the timing of antigen exposure relative to birth. These disparate findings may be related to the availability of the appropriate antigen presenting cells but also point to the possibility of homeostatic changes in non-lymphoid cells in the relevant lymphoid tissues. Here we show that, while leukocytes are the most abundant cell population present in spleen during the first 4-5 days after birth, a massive accumulation of nucleated immature erythroid population in the spleen takes places on day 6 after birth. Although the relative frequency of these immature erythorid cells slowly decreases during the development of neonates, they remain one of the most predominant populations up to three weeks of age. Importantly, we show that the immature erythroid cells from neonate spleen have the capacity to modulate the differentiation of CD4 T cells into effector cells and provide a bias towards a Th2 type instead of Th1 type. These nucleated erythroid cells can produce cytokines that participate in the Th2/Th1 balance, an important one being IL-6. Thus, the selective accumulation of immature erythroid cells in the spleen during a specific period of neonatal development may explain the apparent differences observed in the type(s) of immune responses generated in infants and neonates. These findings are potentially relevant to the better management of immune deficiency in and to the design of vaccination strategies for the young.

Expression of coagulation-related protein genes during LPS-induced preterm delivery in the pregnant mouse.

Preterm delivery (PTD) has been associated with inflammation along with activation of the coagulation pathway. These studies sought to characterize the expression of several coagulation pathway genes including plasminogen activator inhibitor 1 (PAI-1), tissue factor (TF), protease-activated receptor 1 (Par1), protease-activated receptor 2 (Par2), fibrinogen-like protein 2 (Fgl2), and thrombomodulin (TM) during lipopolysaccharide (LPS)-induced PTD in day 15 pregnant CD-1 mice. Western blot studies confirmed protein expression for PAI-1, Par1, Par2, Fgl2, and TM in the mouse uterus. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) confirmed increased PAI-1 messenger RNA (mRNA) in the uteri, lung, kidney, and liver tissues at 2 to 6 hours after LPS injection. In contrast, TF expression significantly decreased by 12 hours in uterine tissue; whereas, its expression was unchanged in the other maternal tissues. The uterine mRNA for Par1, Par2, Fgl2, and TM remained stable. In summary, these studies have confirmed expression of coagulation pathway genes within the pregnant uterus; some of which are modulated during LPS-induced PTD.

Impairment of IKCa Channels Contributes to Uteroplacental Endothelial Dysfunction in Rat Diabetic Pregnancy

Diabetes in rat pregnancy is associated with impaired vasodilation of the maternal uteroplacental vasculature. In the present study, we explored the role of endothelial cell (EC) Ca(2+)-activated K(+) channels of small conductance (SKCa channels) and intermediate conductance (IKCa channels) in diabetes-induced uterine vascular dysfunction. Diabetes was induced by injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with citrate buffer. Changes in smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and vasodilation induced by SKCa/IKCa channel activators were studied in uteroplacental arteries of control and diabetic rats. The impact of diabetes on SKCa- and IKCa-mediated currents was explored in freshly dissociated ECs. NS309 evoked a potent vasodilation that was effectively inhibited by TRAM-34 but not by apamin. NS309-induced smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and dilator responses were significantly diminished by diabetes; N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA)-evoked responses were not affected. Ca(2+)-activated ion currents in ECs were insensitive to paxilline, markedly inhibited by charybdotoxin (ChTX), and diminished by apamin. NS309-induced EC currents were generated mostly due to activation of ChTX-sensitive channels. Maternal diabetes resulted in a significant reduction in ChTX-sensitive currents with no effect on apamin-sensitive or CyPPA-induced currents. We concluded that IKCa channels play a prevalent role over SKCa channels in the generation of endothelial K(+) currents and vasodilation of uteroplacental arteries. Impaired function of IKCa channels importantly contributes to diabetes-induced uterine endothelial dysfunction. Therapeutic restoration of IKCa channel function may be a novel strategy for improvement of maternal uteroplacental blood flow in pregnancies complicated by diabetes.

Phospholipid Scramblase Isoform Expression in Pregnant Rat Uterus

Objective: Phospholipid scramblases (PLSCRs), a family of novel membrane proteins, facilitate the translocation of aminophospholipids from the inner to the exterior leaf of the cell membrane. Four isoforms of PLSCR (PLSCR1-4) have been reported in mouse and human. The studies described in this report sought to characterize the uterine expression of the PLSCR isoforms in the near-term pregnant rat. Methods: Uterine tissue was obtained from timed-pregnant Sprague-Dawley rats. Total RNA was isolated, treated with DNase, and used in qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) studies utilizing PLCSR isoform PCR primers. A rat spleen cDNA bacteriophage library was used as a template for PCR-based sequencing to determine the cDNA and translated amino acid sequences for the PLSCR3 and PLSCR4 homologs expressed in rats. The 5′ and 3′ untranslated regions were obtained using 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) techniques. Results: RT-PCR studies confirmed expression of PLSCR3 and PLSCR4 in the endometrial and myometrial layers of the pregnant rat uterus; in contrast, PLSCR1 and PLSCR2 were not found in uterine tissues. The cDNA sequence for the rat PLSCR3 homolog was found to be 1642 nucleotides, having 92% identity with mouse and 80% with human PLSCR3. The rat PLSCR4 homolog has a cDNA sequence of 1879 nucleotides, having an 89% identity with mouse and 72% identity with human PLSCR4 homologs. Conclusion: The intrauterine expression of PLSCR3 and PLSCR4 provides a dynamic mechanism by which aminophospholipid translocation can be regulated, thereby modulating the activity of various membrane proteins that are involved in inflammation and coagulation-related events.

Inhibition of PPARγ during rat pregnancy causes intrauterine growth restriction and attenuation of uterine vasodilation

Decreased peroxisome proliferator-activated receptor gamma (PPARγ) activity is thought to have a major role in preeclampsia through abnormal placental development. However, the role of PPARγ in adaptation of the uteroplacental vasculature that may lead to placental hypoperfusion and fetal growth restriction during pregnancy is not known. Here, pregnant Sprague–Dawley rats (n = 11/group) were treated during the second half of pregnancy with the PPARγ inhibitor GW9662 (10 mg/kg/day in food) or vehicle. Pregnancy outcome and PPARγ mRNA, vasodilation and structural remodeling were determined in maternal uterine and mesenteric arteries. PPARγ was expressed in uterine vascular tissue of both non-pregnant and pregnant rats with ~2-fold greater expression in radial vs. main uterine arteries. PPARγ mRNA levels were significantly higher in uterine compared to mesenteric arteries. GW9662 treatment during pregnancy did not affect maternal physiology (body weight, glucose, blood pressure), mesenteric artery vasodilation or structural remodeling of uterine and mesenteric vessels. Inhibition of PPARγ for the last 10 days of gestation caused decreased fetal weights on both day 20 and 21 of gestation that was associated with impaired vasodilation of radial uterine arteries in response to acetylcholine and sodium nitroprusside. These results define an essential role of PPARγ in the control of uteroplacental vasodilatory function during pregnancy, an important determinant of blood flow to the placenta and fetus. Strategies that target PPARγ activation in the uterine circulation could have important therapeutic potential in treatment of pregnancies complicated by hypertension, diabetes or preeclampsia.

Estradiol and progesterone influence on influenza infection and immune response in a mouse model

Influenza infection severity may be mediated by estradiol and/or progesterone.