Placental macrophages (Hofbauer cells) are located close to trophoblast cells and fetal capillaries, which makes them ideal candidates for involvement in regulatory processes within the villous core. Their production of various cytokines and prostaglandin (PG) synthesizing enzymes has previously been shown immunohistochemically. Hofbauer cells were isolated from human placenta after term deliveries by Ficoll and Percoll gradient centrifugation. Remaining trophoblast cells were removed with anti-epidermal growth factor (EGF)-receptor-coated Dynabeads followed by differential adherence. The identity of isolated cells was investigated by immunohistochemistry with anti-CD68, which showed that >90% cells were positive. After a 36 h recovery period in either 20% O2 or 5% O2, fresh medium was applied and PGE2 and thromboxane (TXA2) production analysed by enzyme immunoassay at 4, 8, and 24 h. PGE2 and TXA2 were both produced by placental macrophages with PGE2 synthesis being predominant. Concentrations of both could be stimulated by lipopolysaccharide with maximum effect after 24 h. Culture in low oxygen caused decreased PGE2 concentrations, whereas TXA2 production remained unchanged. In conclusion, the presented isolation protocol allows further study of Hofbauer cell function. This study also presents novel findings regarding the prostaglandin production of term Hofbauer cells under normal and hypoxic conditions.
Human pregnancy is frequently accompanied by nausea and vomiting that may become severe and life-threatening, as in hyperemesis gravidarum (HG), the cause of which is unknown. Growth Differentiation Factor-15 (GDF15), a hormone known to act on the hindbrain to cause emesis, is highly expressed in the placenta and its levels in maternal blood rise rapidly in pregnancy. Variants in the maternal GDF15 gene are associated with HG. Here we report that fetal production of GDF15, and maternal sensitivity to it, both contribute substantially to the risk of HG. We found that the great majority of GDF15 in maternal circulation is derived from the feto-placental unit and that higher GDF15 levels in maternal blood are associated with vomiting and are further elevated in patients with HG. Conversely, we found that lower levels of GDF15 in the non-pregnant state predispose women to HG. A rare C211G variant in GDF15 which strongly predisposes mothers to HG, particularly when the fetus is wild-type, was found to markedly impair cellular secretion of GDF15 and associate with low circulating levels of GDF15 in the non-pregnant state. Consistent with this, two common GDF15 haplotypes which predispose to HG were associated with lower circulating levels outside pregnancy. The administration of a long-acting form of GDF15 to wild-type mice markedly reduced subsequent responses to an acute dose, establishing that desensitisation is a feature of this system. GDF15 levels are known to be highly and chronically elevated in patients with beta thalassemia. In women with this disorder, reports of symptoms of nausea or vomiting in pregnancy were strikingly diminished. Our findings support a causal role for fetal derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by pre-pregnancy exposure to GDF15, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.
Background The lung undergoes physiologic changes during late gestation in order to prepare for its postnatal environment and function. This comprises up-regulation of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase. Recently we have described perinatal up-regulation of paraoxonase-3 (PON3), an uncharacterized antioxidant gene, in rodent and ovine lungs. In the current study we analysed the functional significance of PON3 expression. Methods We applied RNA interference (RNAi) to reduce gene expression in A549 cells, a human lung epithelial cell line resembling type 2 pneumocytes. Transient and permanent RNAi was achieved by transfection of short interfering or short hairpin RNAs, respectively. To mimic fetal environment cells were cultured in 2% oxygen. Gene expression was analysed by quantitative PCR. IL-8 secretion was analysed by ELISA. Total antioxidant activity was determined in Trolox units. Results siRNAs reduced PON3 expression by 77% (p<0.05) and 69% (p<0.001) in fetal or postnatal (ambient) oxygen concentration, respectively. There was a significant difference in antioxidant capacity between knock-down and control cells. PON3 silencing increased the expression of the gene for heat-shock protein HSP5A by 25% in postnatal oxygen levels; this up-regulation was absent when cells were cultured in low oxygen. Transient or stable silencing did not affect cell viability or the response to H2O2-mediated oxidative stress; however, stable silencing resulted in slower cell proliferation. Conclusion Absence of PON3 causes reduced proliferation and endoplasmic reticulum stress in lung cells. PON3 may be required to protect the lung from consequences of postnatal rise in oxygen concentration.
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