Nazeeh Hanna

Polybrominated Diphenyl Ethers Enhance the Production of Proinflammatory Cytokines by the Placenta

Polybrominated diphenyl ether(s) (PBDE) are ubiquitous environmental contaminants that bind and cross the placenta but their effects on pregnancy outcome are unclear. It is possible that environmental contaminants increase the risk of inflammation-mediated pregnancy complications such as preterm birth by promoting a proinflammatory environment at the maternal-fetal interface. We hypothesized that PBDE would reduce IL-10 production and enhance the production of proinflammatory cytokines associated with preterm labor/birth by placental explants. Second-trimester placental explants were cultured in either vehicle (control) or 2 μM PBDE mixture of congers 47, 99 and 100 for 72 h. Cultures were then stimulated with 10(6) CFU/ml heat-killed Escherichia coli for a final 24 h incubation and conditioned medium was harvested for quantification of cytokines and PGE(2). COX-2 content and viability of the treated tissues were then quantified by tissue ELISA and MTT reduction activity, respectively. PBDE pre-treatment reduced E. coli-stimulated IL-10 production and significantly increased E. coli-stimulated IL-1β secretion. PBDE exposure also increased basal and bacteria-stimulated COX-2 expression. Basal, but not bacteria-stimulated PGE(2), was also enhanced by PBDE exposure. No effect of PBDE on viability of the explants cultures was detected. In summary, pre-exposure of placental explants to congers 47, 99, and 100 enhanced the placental proinflammatory response to infection. This may increase the risk of infection-mediated preterm birth by lowering the threshold for bacteria to stimulate a proinflammatory response(s).

Gestational Age-Dependent Expression of IL-10 and Its Receptor in Human Placental Tissues and Isolated Cytotrophoblasts

Control of antifetal immune responses is thought to be regulated locally by the placenta. Because the physiologic programming of the placenta across gestation is likely to influence the local immunity, we hypothesize that a potent anti-inflammatory cytokine such as IL-10 may be produced in a gestational age-dependent manner. In the present study, we examined the expression of IL-10 and its receptor in placental explants or freshly isolated cytotrophoblasts from different gestational ages and compared it with the expression profiles of other cytokines. First and second trimester placental tissues from normal pregnancies predominantly expressed IL-10, whereas the levels of IL-2, IL-4, and IFN-γ were mostly below detection throughout pregnancy. The expression of IL-10, but not its receptor, diminished significantly in term placental tissues collected “before” the onset of labor and did not change appreciably “after” labor. On the other hand, TNF-α and IL-1β were significantly up-regulated in response to labor-associated conditions. IL-10 expression was transcriptionally attenuated at term as observed in cytotrophoblasts. In contrast to the placental cytokine milieu, autologous PBMCs, when activated with PHA, secreted significant amounts of IL-2, IL-4, IL-10, and IFN-γ, albeit with a statistically significantly enhanced IL-10 production in first trimester compared with age-matched nonpregnant women. These data suggest that IL-10 is expressed in the placenta in a gestational age-dependent manner and that its down-regulation at term may be an important mechanism underlying the subtle changes associated with parturition.

Uterine NK Cells Mediate Inflammation-Induced Fetal Demise in IL-10-Null Mice

Specialized NK cells are recruited in high numbers to the mammalian embryo implantation sites, yet remain pregnancy compatible. It is not well understood whether uterine NK (uNK) cells become adversely activated and mediate fetal demise, a common complication of early pregnancy. In this study we show that mating of IL-10−/− mice resulted in fetal resorption or intrauterine growth restriction in response to very low doses of LPS. Pregnancy in congenic wild-type mice was normal even at 10-fold higher LPS doses. Fetal resorption in IL-10−/− mice was associated with a significant increase in uNK cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells, TNF-α neutralization, or IL-10 administration rescued pregnancy in LPS-treated IL-10−/− animals. Our results identify an immune mechanism of fetal demise involving IL-10 deficiency, NK cells, and inflammation. These results may provide insight into adverse pregnancy outcomes in humans.

Evidence for Interleukin-10-Mediated Inhibition of Cyclo- oxygenase-2 Expression and Prostaglandin Production in Preterm Human Placenta

Problem  Interleukin‐10 (IL‐10) is thought to be a key cytokine for the maintenance of pregnancy. Here we examined the expression profiles of IL‐10 and cyclo‐oxygenase‐2 (COX‐2), and the effect of IL‐10 on COX‐2 expression and prostaglandin release in the human placenta from preterm labor deliveries associated with chorioamnionitis.

Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery

OBJECTIVE The purpose of this study was to determine in a mouse model whether uterine natural killer (uNK) cell cytotoxic activation induces infection/inflammation-associated preterm labor and delivery. STUDY DESIGN Wild type or interleukin (IL)-10(-/-) mice were injected intraperitoneally with lipopolysaccharide on gestational day 14. Mice were either killed for collection of uteroplacental tissue, spleen, and serum or allowed to deliver. Uteroplacental tissue was used for histology and characterization of uNK cells. RESULTS Low-dose lipopolysaccharide treatment triggered preterm labor and delivery in IL-10(-/-), but not wild type mice, in a manner independent of progesterone levels. Preterm labor and delivery in IL-10(-/-) mice was associated with an increased number and placental infiltration of cytotoxic uNK cells and placental cell death. Depletion of NK cells or tumor necrosis factor (TNF)-alpha neutralization in these mice restored term delivery. Furthermore, TNF-alpha neutralization prevented uNK cell infiltration and placental cell apoptosis. CONCLUSION The uNK cell-TNF-alpha-IL-10 axis plays an important role in the genesis of infection/inflammation-induced preterm labor/delivery.

Deficiency of decidual IL-10 in first trimester missed abortion: a lack of correlation with the decidual immune cell profile.

PROBLEM: To determine if first trimester missed abortion decidua is characterized by an altered immune cell profile and/or a modified interleukin (IL)‐10 and interferon (IFN)‐γ production pattern compared with decidua from elective termination.

Labor-Associated Changes in Fas Ligand Expression and Function in Human Placenta

Fas ligand (FasL)-dependent apoptosis has been implicated in the control of tissue-damaging inflammatory responses in several immune privileged sites. Here, we present data demonstrating that FasL is expressed on human trophoblast cells throughout pregnancy and transduces growth inhibitory/death signals in cells bearing its receptor, Fas (CD95). Immunohistochemical analysis detected FasL-positive staining in the trophoblast layer of villi of first- and second-trimester and term (no labor) placental tissues, as well as in freshly isolated cytotrophoblasts representing these gestational ages. In contrast, term placental tissues and cytotrophoblasts from labor-associated deliveries exhibited significantly reduced FasL expression, suggesting that parturition altered the characteristics of trophoblast cells. FasL-specific immunoblotting of cytotrophoblast cell lysates further confirmed these results. To assess the functionality of FasL expressed on cytotrophoblasts, we co-cultured these cells with Fas-bearing Jurkat T cells. Cytotrophoblasts from early pregnancy, or term with no labor, significantly inhibited growth in Jurkat cells, evident even at a 1:1 effector:target cell ratio, as determined by the incorporation of [3H]thymidine. Similar results were obtained when a FasL-positive colon carcinoma cell line, SW620, was used in place of cytotrophoblasts. In contrast, term cytotrophoblasts from labor deliveries exhibited poor FasL expression and were quantitatively much less proficient in inhibiting [3H]thymidine incorporation in Jurkat cells. These data indicate that FasL could participate in modulating the inflammatory responses associated with labor and suggest intrinsic molecular differences in the placental microenvironment before and after labor.

Mechanisms underlying reduced apoptosis in neonatal neutrophils.

Apoptosis, which leads to phagocytosis by mononuclear cells, represents the primary mechanism for removing neutrophils from inflamed tissues and minimizing injury. The present studies show that membrane phosphatidylserine turnover and permeability, as well as DNA fragmentation, were reduced in neutrophils from neonates when compared with adults. The activity of caspase 3 and expression of the proapoptotic proteins Bax, Bad, and Bak were also decreased in neonatal relative to adult neutrophils. These findings are consistent with impaired apoptosis in neonatal cells, which may contribute to prolonged inflammation in infants after oxidative stress or infection. Neutrophil apoptosis is induced by endogenous ligands such as Fas (FasL), which engage death receptors of the tumor necrosis factor/nerve growth factor superfamily, including Fas receptor (FasR). We found that expression of FasR was decreased in neonatal when compared with adult cells. Moreover, neonatal neutrophils did not undergo apoptosis in response to anti-FasR antibody and exhibited impaired chemotaxis to soluble FasL. However, in both adult and neonatal cells, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase inhibitors blocked Fas-induced activity. These data suggest that prolonged survival of neonatal neutrophils at injured sites is due, in part, to reduced responsiveness to FasL. This may be related to decreased expression of both FasR and Bcl-2–family proteins that mediate neutrophil apoptosis.

IFN‐γ‐Mediated Inhibition of COX‐2 Expression in the Placenta from Term and Preterm Labor Pregnancies

Problem:  The inflammatory‐anti‐inflammatory cytokine network is thought to play a critical role in regulated progression and termination of pregnancy. The aim of this study was to evaluate the effects of interferon (IFN)‐γ on the expression of Cyclooxygenase (COX)‐2 and production of prostaglandin E2 (PGE2) in the human placenta from term and preterm labor deliveries.

Comprehensive amniotic fluid cytokine profile evaluation in women with a short cervix: which cytokine(s) correlates best with outcome?

OBJECTIVE The objective of this study was to determine whether an expanded amniotic fluid cytokine profile predicts spontaneous preterm birth in patients with short cervix in the midtrimester. STUDY DESIGN Amniocentesis was performed on singleton gestations between 16-24 weeks with a cervical length <or=25 mm. Amniotic fluid from patients who received no surgical or hormonal treatment was assayed for 25 cytokines. Univariate analysis identified cytokine(s) that correlated with the interval between amniocentesis to delivery. Stepwise regression identified which cytokine(s) was most predictive of delivery, followed by the generation of receiver-operator characteristic curves. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated. RESULTS Forty-four amniotic fluid samples were analyzed. After stepwise regression, only monocyte chemotactic protein-1 remained significant and was the most predictive of early delivery. With a cutoff of 1320 pg/mL, monocyte chemotactic protein-1 had a 69% sensitivity, 83% specificity, 36% positive predictive value, and 87% negative predictive value to predict spontaneous preterm birth within 1 week of amniocentesis (P = .015). CONCLUSION Among 25 cytokines, monocyte chemotactic protein-1 was most predictive of spontaneous preterm birth.