We tested a novel hypothesis that elevated levels of proteases in the maternal circulation of preeclamptic women activate neutrophils due to their pregnancy-specific expression of proteaseactivated receptor 1 (PAR-1). Plasma was collected longitudinally from normal pregnant and preeclamptic women and analyzed for MMP-1 and neutrophil elastase. Neutrophils were isolated for culture and confocal microscopy. Omental fat was collected for immunohistochemistry. Circulating proteases were significantly elevated in preeclampsia. Confocal microscopy revealed that tet methylcytosine dioxygenase 2 (TET2), a DNA de-methylase, and p65 subunit of NF-κB were strongly localized to the nucleus of untreated neutrophils of preeclamptic women, but in untreated neutrophils of normal pregnant women they were restricted to the cytosol. Treatment of normal pregnancy neutrophils with proteases activated PAR-1, leading to activation of RhoA kinase (ROCK), which triggered translocation of TET2 and p65 from the cytosol into the nucleus, mimicking the nuclear localization in neutrophils of preeclamptic women. IL-8, an NF-κB gene, increased in association with TET2 and p65 nuclear localization. Co-treatment with inhibitors of Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. http://www.springer.com/gb/openaccess/authors-rights/aam-terms-v1
The objective of this study was to determine: 1) if neutrophils of preeclamptic women express cyclooxygenase-2 (COX-2), and 2) if COX-2 mediates neutrophil interleukin-8 (IL-8) production. Neutrophils were isolated from heparinized blood collected from women with preeclampsia and normal pregnancy, and from normal nonpregnant women. COX-2 protein was measured in the neutrophils of the three groups by specific ELISA. To study the role of COX-2 in IL-8 production, neutrophils of normal pregnant women were activated with arachidonic acid and incubated overnight in Medium 199 with and without NS398, a specific COX-2 inhibitor. We found that neutrophils from preeclamptic women expressed significantly more COX-2 than neutrophils from normal pregnant or normal nonpregnant women. Arachidonic acid activated neutrophils to produce IL-8 which was completely inhibited by NS398. These data demonstrate that COX-2 is expressed in neutrophils of preeclamptic women and that COX-2 mediates IL-8 production by activated neutrophils.
Gestational choriocarcinomas are derived from placental trophoblast cells, with HLA-C being the only class I polymorphic molecule expressed. However, choriocarcinomas have not been profiled for endoplasmic reticulum aminopeptidase 2 (ERAP2) expression. ERAP2 trims peptides presented by human leukocyte antigens (HLA) that have shown to modulate immune response. Over 50% of choriocarcinomas we screened lack ERAP2 expression, which suggests that the absence of ERAP2 expression allows immune evasion of choriocarcinoma cells. We demonstrate that the ability of choriocarcinoma cells to activate lymphocytes was lowest with cells lacking ERAP2 (JEG-3) or HLA-C (JAr). This observation suggests that activation is dependent on expression of both ERAP2 and HLA-C molecules. In addition, an ERAP2 variant in which lysine is changed to asparagine (K392N) results in increased trimming activity (165-fold) for hydrophobic peptides and biologically never been detected. We hypothesize that homozygosity for the N392 ERAP2 variant is prohibited because it modulates the immune recognition of placental trophoblasts. We demonstrate that NK-cell activation and killing were significantly dependent on forced expression of the N392 ERAP2 isoform in JEG-3 cells. Cytotoxicity was confirmed by 7AAD killing assays showing that N392 ERAP2-isoform expressing JEG-3 cells had the highest percentage of apoptotic cells independent of the expression level of CD11a on lymphocytes. This is the first report showing that N392 ERAP2 promotes an immune clearance pathway for choriocarcinoma cells, and provides an explanation for why embryonic homozygosity for the N392 ERAP2 variant is not detected in any population.
The cause of spontaneous preterm labor (sPTL) is not known, but it could be due to epigenetic alterations that increase the sensitivity of decidual tissue to inflammatory stimuli. We collected decidual tissue from women at term not in labor (TNL), women at term in labor (TL), and women with sPTL. Illumina Infinium HumanMethylation450 BeadChip analysis revealed significantly reduced DNA methylation for TLR-2 and TLR-9 in sPTL as compared to TL. Immunohistochemical staining documented significantly increased expression of TLR-2 and TLR-9 in decidual tissue of women with sPTL as compared to TL or TNL. TLR expression was not present in decidual cells, but localized to tissue leukocytes as revealed by staining for CD14, a macrophage antigen, and neutrophil elastase. Microarray analysis of inflammatory genes to assess innate immune response demonstrated marked increases in expression of inflammatory cytokines and chemokines in women with TL as compared to TNL. However, when sPTL was compared to TL, there was a further increase in inflammatory cytokines, and a remarkable increase in neutrophil chemokines. These results suggest that epigenetic mechanisms could play a role in increasing leukocyte infiltration, and increasing the sensitivity of decidual tissue to inflammatory stimuli that could precipitate labor prematurely.
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