In mouse and human, precursors of NK cell lineage home to decidualizing uteri. To assess the requirement for IL-15, an essential cytokine for NK differentiation in lymphoid tissue, on uterine NK (uNK) cell differentiation, implantation sites from IL-15−/− mice were analyzed histologically. IL-15−/− implantation sites had no uNK cells, no spiral-artery modification, and lacked the decidual integrity found in normal mice. IL-15−/− recipients of C57BL/6 marrow displayed similar pathology. However, implantation sites from recombination-activating gene-2−/−γc−/− (alymphoid) recipients of IL-15−/− marrow showed normal uNK cells, modified spiral arteries, and well-developed decidua basalis. Deletion of the IFN-regulatory factor (IRF)-1, but not IRF-2 (factors important in peripheral NK cell differentiation) limited but did not prevent uNK cell development. In situ hybridization localized IRF-1 largely to placental trophoblast cells. IRF-1−/− marrow transplanted into recombination-activating gene-2−/−γc−/− displayed competence for full uNK cell differentiation. IL-15 mRNA expression at implantation sites of IRF-1−/− and C57BL/6 was similar, suggesting that, unlike in bone marrow and spleen, IRF-1 does not regulate IL-15 in the pregnant uterus. Terminal differentiation of uNK cells was not promoted in pregnant IRF-1−/− mice by 5-day infusion of murine rIL-15, suggesting that IRF-1 deficiency rather than IL-15 deficiency limits uNK cell differentiation in these mice. Further, IRF-1 regulates placental growth, birth weight, and postnatal growth of offspring. These studies indicate that uNK cell development and maturation share some aspects with NK cell development in other tissues, but also display distinctive tissue-specific regulation.

Prenatal mortality reaching 30% occurs during the first weeks of gestation in commercial swine. Mechanisms for this are unknown although poor uterine blood supply has been postulated. In other species, vascular endothelial growth factor, hypoxia-inducible factor 1-α, and IFN-γ regulate gestational endometrial angiogenesis. Vascular endothelial growth factor and hypoxia-inducible factor 1-α are also important for placental angiogenesis while trophoblastic expression of Fas ligand is thought to protect conceptuses against immune-mediated pregnancy loss. In this study, we document dynamic, peri-implantation differences in transcription of genes for angiogenesis, cytokine production, and apoptosis regulation in the endometrium, and laser capture microdissected endometrial lymphocytes and trophoblasts associated with healthy or viable but arresting porcine fetuses. In healthy implantation sites, endometrial gene expression levels differed between anatomic subregions and endometrial lymphocytes showed much greater transcription of angiogenic genes than trophoblasts. In arresting fetal sites, uterine lymphocytes had no angiogenic gene transcription and showed rapid elevation in transcription of proinflammatory cytokines Fas and Fas ligand while trophoblasts showed elevated transcription of IFN-γ and Fas. This model of experimentally accessible spontaneous fetal loss, involving blocked maternal angiogenesis, should prove valuable for further investigations of peri-implantation failure of normally conceived and surgically transferred embryos in many species, including the human.

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During human and rodent uterine decidualization, transient but abundant numbers of uterine natural killer (uNK) cells appear, proliferate, and differentiate. uNK cells share features with peripheral NK cells but are specialized to promote interferon-gamma (IFN-gamma)-mediated, pregnancy-associated, structural changes in maternal placental arteries. In CD8+ T cells and NK cells, the transcription factors T-bet and eomesodermin (Eomes) regulate maturation and effector functions, including IFN-gamma production. No studies are reported for uNK cells. Implantation sites in T-bet null mice, which have a defect in NK cell maturation, had uNK cells normal in morphology and number and normally modified spiral arteries. As Eomes null mice are not viable, real-time polymerase chain reaction comparisons between C57Bl/6J (B6) and alymphoid (Rag2(0/0)gammac0/0) mice were used to assess uNK cell expression of T-bet, Eomes, and the target genes IFN-gamma, granzyme A, and perforin. Gestation dated (gd) uterine tissues (mixed cell composition) and 200 morphologically homogeneous, laser-capture, microdissected uNK cells of different maturation stages were used. In uterus, Eomes transcripts greatly outnumbered those of T-bet, whether donors were nonpregnant or pregnant, and increased to gd10. In uNK cells, transcripts for T-bet, Eomes, and IFN-gamma were most abundant in mature stage cells, and transcripts for granzyme A and perforin were lower at this stage than in immature or senescent cells. Thus, Eomes dominance to T-bet discriminates regulation of the uNK cell subset from that observed for peripheral NK cells.

ABSTRACT:Vinyl carbamate (VC) is derived from ethyl carbamate, a carcinogen formed in fermentation of food and alcoholic products. We have undertaken studies to test the hypothesis that an epoxide generated from VC oxidation leads to formation of 1,N 6 -ethenodeoxyadenosine (dAS). We have developed approaches using liquid chromatography-mass spectrometry and liquid chromatographytandem mass spectrometry for identification and quantitation of dAS. Peak rates were produced by lung microsomes and recombinant CYP2E1 at 3.0 and 2.5 mM VC, respectively. In inhibitory studies, incubations of VC were performed using lung microsomes from mice treated with the CYP2E1 inhibitor diallyl sulfone (100 mg/kg, p.o.). Results from these studies showed significantly decreased dAS formation in microsomes incubated with VC, with an inhibition of 70% at 3.0 mM. These findings suggested that CYP2E1 is a major enzyme mediating VC oxidation, leading to the formation of a metabolite that alkylates DNA to form the dAS adduct. Scanning and fragment ion analyses confirmed the identity of dAS based on the molecular ion [M ؉ H]