All samples immediately after collection were in part snap-frozen and stored at ?80?C and in part, formalin fixed for subsequently analyses. pregnancy3. The levels of MIF in the intervillous space are reportedly higher than in maternal and cord blood and become even higher in consequence of placental insults such as malaria infection15. Altogether, the data on MIF in human pregnancy suggest that MIF is a key molecule in the placental response to endogenous and exogenous harmful stimuli. Despite the number of studies on MIF in pregnancy16,17, only little is known about its role at the maternal-foetal interface. It has been demonstrated that trophoblast MIF reduces the cytotoxicity of human decidual NK cells18. Studies in mice have shown that recombinant MIF, its interaction with CD74 receptor, sustains decidual cell survival by interfering with the fate of these cells when subjected to pro-apoptotic stimuli19. In view of the above evidence, we suggest that MIF exerts its action also in the placenta and, in particular, we hypothesize that MIF protects trophoblast from Ki 20227 apoptosis, a crucial cellular event in the earlier stages of pregnancy that could become harmful if not properly regulated20. To verify the hypothesis, we investigated the role of exogenous and endogenous MIF and that of its receptor the CD74 in human placental explants at first trimester pregnancy, subjected to pro-apoptotic stimuli. Results CD74 expression in first trimester placental tissues and interaction with MIF In order to verify the sensitivity of placenta to MIF action we evaluated the CD74 expression in placental tissues throughout the first trimester of pregnancy. Both CD74 mRNA and protein were detected in first trimester placenta with a peak, although not significant, at 9 weeks of gestation (Fig.?1ACC). Open in a separate window Figure 1 CD74 expression in first trimester placental tissues and interaction with MIF. CD74 mRNA expression assessed by qRT-PCR. (A) CD74 mRNA levels were normalized to those of 18S and expressed as fold increase relative to 8 weeks placental tissue selected as calibrator sample. Representative western blot (B) and densitometric analysis (C) in placental tissues at different weeks of gestation (n?=?5 for each week). (D) Representative immunohistochemical analysis of CD74 in placenta at 9 weeks of gestation. Slides were counterstained with Mayers haematoxylin. Reddish staining represents positive immunoreactivity for CD74. Arrow-head indicates villous Ki 20227 trophoblasts; asterisk marks the mesenchymal cells. Ct: cytotrophoblast; Ki 20227 Sy: syncytiotrophoblast. Bar?=?25?m. (E) Representative immunoprecipitation (IP) of CD74 in placental tissues Ki 20227 from 7 to ?10 weeks of gestation followed by western blot (WB) for CD74 (left panel) and MIF (right panel). IgG: isotype control; rMIF: recombinant MIF. Immunohistochemical analysis showed a dotted staining in trophoblast cells, both in the cytotrophoblast and syncytiotrophoblast layers and to a lesser extent in some mesenchymal cells (Figs?1D and 7S). Immunoprecipitation experiments with anti CD74 antibody, followed by SDS-PAGE and immunoblotting against human MIF, revealed the presence of a positive band at 12?kDa co-migrating with the rMIF, showing an effective interaction between CD74 and MIF (Fig.?1E right panel). As a proof of this, CD74 immunoprecipitated samples, subjected to western blotting for CD74, showed the presence of a positive band at 37?kDa; no bands were obtained when lysates were incubated with normal isotype control IgG (Fig.?1E left panel). Induction of MIF release by hypoxia/re-oxygenation (H/R) in placental explant cultures To evaluate the impact of apoptosis stimulation on placental MIF we pursued two approaches: (1) exposure of explants cultures to FCCP, a well known apoptosis inducer; (2) exposure of the explants Ki 20227 cultures to H/R, a putative pro apoptotic that placenta might encounter in pathological circumstances25. Premature villous exposure to high levels of oxygen as well as hypoxia-reoxygenation injury have been implicated in complications of pregnancy i.e. miscarriage and pre-eclampsia25,26. This study, performed on human placental explants from eight-ten weeks of pregnancy, showed that H/R condition increased release of MIF in the culture medium and that inhibition of MIF, or MIF activity by anti-MIF or anti-CD74, resulted in an induction of apoptosis. On the other hand, treatment with rMIF did not have any PTGFRN influence on apoptosis-induced H/R. The data suggest that an appropriate placental secretion of MIF in response to H/R condition is essential to sensitize the cells against the death-inducing effects. Nevertheless, the addition of rMIF, even at high concentration, to the cultures exposed to H/R did not have any influence on apoptosis resistance. This result,.
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