Aureo T. Yamada

DBA-lectin reactivity defines natural killer cells that have homed to mouse decidua.

During normal mouse pregnancy, abundant numbers of uterine natural killer (uNK) cells differentiate at implantation sites and contribute to early, post-implantation endometrial angiogenesis and to spiral arterial modification. Mouse uNK cells are confidently recognized by light microscopy in tissue sections stained with special protocols. Classically, mouse uNK cells were identified as lymphocytes containing Periodic Acid Schiffs (PAS) reactive cytoplasmic granules. More recently, Dolichos biflorus lectin (DBA) reactions which stain not only the cytoplasmic granules but also the uNK cell membranes have been widely adopted. No lymphocytes in any tissues of virgin mice or external to the uterus of pregnant mice have DBA lectin reactivity equivalent to that of uNK cells; however, some uNK cells are now recognized as DBA-. Here, we describe a PAS/DBA lectin double staining protocol and assess the coincident staining of C57Bl/6J uNK cells from gestation day (gd)6, the first day of uNK cell abundance, to gd12, the day when Tunel+ nuclear senecence appears widely in uNK cells before their numerical decline. For these gd, PAS+ DBA- and PAS+DBA+ cells but not PAS-DBA+ cells were identified. Dual positive cells increased from 47% at gd6 to 85% at gd12. Transplantation of normal bone marrow into alymphoid mice who were subsequently mated revealed the uterus repopulated by doubly reactive PAS+DBA+ uNK cells (>95%). Thus, in normal pregnancies, most uNK cells appear to arise from progenitor cells that have homed to the uterus.

DBA-Lectin Reactivity Defines Mouse Uterine Natural Killer Cell Subsets with Biased Gene Expression

ABSTRACT Endometrial decidualization, a process essential for blastocyst implantation in species with hemochorial placentation, is accompanied by an enormous but transient influx of natural killer (NK) cells. Mouse uterine NK (uNK) cell subsets have been defined by diameter and cytoplasmic granule number, reflecting stage of maturity, and by histochemical reactivity with Periodic Acid Schiff (PAS) reagent with or without co-reactivity with Dolichos biflorus agglutinin (DBA) lectin. We asked whether DBA− and DBA+ mouse uNK cells were equivalent using quantitative RT-PCR analyses of flow-separated, midpregnancy (Gestation Day [gd] 10) cells and immunohistochemistry. CD3E (CD3)−IL2RB (CD122)+DBA cells were identified as the dominant Ifng transcript source. Skewed IFNG production by uNK cell subsets was confirmed by analysis of uNK cells from eYFP-tagged IFNG-reporter mice. In contrast, CD3E−IL2RB+DBA+ uNK cells expressed genes compatible with significantly greater potential for IL22 synthesis, angiogenesis, and participation in regulation mediated by the renin-angiotensin system (RAS). CD3E−IL2RB+DBA+ cells were further divided into VEGFA+ and VEGFA− subsets. CD3E−IL2RB+DBA+ uNK cells but not CD3E−IL2RB+DBA− uNK cells arose from circulating, bone marrow-derived progenitor cells by gd6. These findings indicate the heterogeneous nature of mouse uNK cells and suggest that studies using only DBA+ uNK cells will give biased data that does not fully represent the uNK cell population.

SPIRAL ARTERIAL REMODELING IS NOT ESSENTIAL FOR NORMAL BLOOD PRESSURE REGULATION IN PREGNANT MICE

Maternal cardiovascular adaptations occur in normal pregnancy, systemically, and within the uterus. In humans, gestational control of blood pressure is clinically important. Transient structural remodeling of endometrial spiral arteries normally occurs in human and mouse pregnancies. In mice, this depends on uterine natural killer cell function. Using normal and immune-deficient mice, we asked whether spiral artery remodeling critically regulates gestational mean arterial pressure and/or placental growth. Radiotelemetric transmitters were implanted in females and hemodynamic profiles to a dietary salt challenge and to pregnancy were assessed. Implantation sites from noninstrumented females were used for histological morphometry. Both normal and immune-deficient mice had normal sensitivity to salt and showed similar 5-phase gestational patterns of mean arterial pressure correlating with stages of placental development, regardless of spiral artery modification. After implantation, mean arterial pressure declined during the preplacental phase to reach a midgestation nadir. With gestation day 9 opening of placental circulation, pressure rose, reaching baseline before parturition, whereas heart rate dropped. Heart rate stabilized before parturition. Placental sizes deviated during late gestation when growth stopped in normal mice but continued in immune-deficient mice. As an indication of the potential for abnormal hemodynamics, 2 pregnant females delivering dead offspring developed late gestational hypertension. This study characterizes a dynamic pattern of blood pressure over mouse pregnancy that parallels human gestation. Unexpectedly, these data reveal that spiral artery remodeling is not required for normal gestational control of blood pressure or for normal placental growth.

Biological characteristics and pathogenicity of avian Escherichia coli strains.

Fifty avian (chicken) pathogenic Escherichia coli strains (APEC) isolated from individuals suffering from omphalitis, septicaemia and swollen head syndrome, and 30 strains isolated from healthy chickens were studied regarding their biological characteristics such as serogroups, haemolysin, colicin, cytotoxin, toxin and siderophore production, adhesion capacity to in vitro cultivated cells, and absorption of Congo red dye. Serotyping demonstrated that most of the omphalitis and normal strains were untypable, whereas most of the septicaemic strains were either untypable or rough. There was no prevalent serogroup among the pathogenic strains studied. The capacity for adhesion and invasion of in vitro cultured cells (HeLa, HEp-2, KPCC), as well as the agglutination of different types of red blood cells and the LD50 of each strain were also evaluated. No correlation was observed between the biological characteristics and pathogenicity, except that colicin was characteristically produced by swollen head syndrome E. coli strains. No correlation was found between adhesion or haemagglutination patterns and pathogenicity. Only six of the 50 strains revealed invasive capacity and the strain that best invaded the cell lines was the one with the lowest LD50.

Analysis of uterine natural killer cells in mice.

The term uterine natural killer (uNK) cell is applied in mice to an abundant but transient NK cell population that undergoes unique, terminal differentiation within embryo implantation sites during endometrial decidualization and pregnancy. In mice, decidualization is induced by attachment and implantation of hatched, blastocyst-stage embryos. Within each implantation site, uNK cells proliferate and rapidly differentiate into highly restricted regions called decidua basalis and the mesometrial lymphoid aggregate of pregnancy (MLAp). uNK cells begin to die within healthy decidua basalis by day 8 of the 19-20 day pregnancy of mice. By gestation day 12, uNK cell numbers have peaked and most uNK cells show in situ nuclear fragmentation indicative of disintegration. Morphological studies (standard histology, ultrastructure, immunohistochemistry, in situ hybridization, and RNA analyses from laser capture microdissected uNK cells) have provided most of the current understanding regarding this cell lineage. These approaches identified the special angiogenic properties of uNK cells and their regulatory relationships with normal physiological changes to the uterine (endometrial) arterial tree that accompany successful pregnancy. This chapter highlights key information needed for successful dissection of the dynamically changing decidua basalis that is enriched in uNK cells and special morphological procedures used for uNK cell study. Preparation of viable mouse uNK cell suspensions is difficult but can be achieved. This chapter includes techniques for isolation of uterine leukocyte suspensions and their enrichment for uNK cells that permit immediate downstream applications such as culture, isolation of high quality RNA, or flow cytometry.

Heterogeneity in composition of mouse uterine natural killer cell granules

uNK cells differ from cNK cells, as they produce angiogenic molecules critical for normal implantation site development. We evaluated heterogeneity among DBA+uNK cells for Prf, Gzma, and Vegfa. Ctsd and Srgn expression was used to assign intracellular sorting of these molecules on gd7, ‐9, and ‐14. Vegfa was present in small, granule‐free DBA+uNK cells at gd7 and in large, granule‐rich DBA+uNK cells at gd9 and ‐14. Prf and Gzma were only found in granulated DBA+uNK cells (gd9 and ‐14). All granule‐rich Prf+DBA+uNK cells appeared to coexpress Vegfa. Thus, all DBA+uNK cells were Vegfa‐producing cells. PC analysis and immunogold ultrastructure confirmed colocalization of Prf/Ctsd in secretory‐lysosome granules (PC>0.5). Surprisingly, Gzma and Prf+Ctsd+ were not colocalized (PC<0.5). Rather, Gzma colocalized with Srgn (PC>0.5) in small granules in cells with Vegfa expression (PC<0.5). NK1.1+sNK cells and DBA+uNK cells expressed genes regulating vesicular traffic (rab11, rab27a, snap23, vamp7), but uNK cells also expressed rab34 and vamp8, molecules associated with constitutive secretion. SEE activated the regulated secretory pathway of DBA+uNK cells in vivo, mobilizing Prf and Gzma but not Vegfa. Thus, DBA+uNK cells display constitutive and regulated secretion. Further, these results demonstrate that granule‐free DBA+uNK cells are not quiescent immature cells, but they are cells with potentially significant angiogenic roles before and in addition to their initiation of spiral arterial remodeling.

Delta-Like Ligand (DLL)1 Expression in Early Mouse Decidua and Its Localization to Uterine Natural Killer Cells

Uterine vascular changes, critical for pregnancy success, occur at each implant site during endometrial decidualization. Mesometrial decidualization recruits high numbers of angiogenic, uterine Natural Killer (uNK) cells that trigger midpregnancy spiral arterial remodeling. We postulated that uNK cells contribute to early decidual angiogenesis as endothelial-cell extrinsic sources of Delta-like ligand 1 (DLL1), a molecule that induces endothelial tip cell differentiation and orthogonal vascular growth in other tissues. Virgin uteri expressed Dll1 mesometrially and anti-mesometrially and relative expression increased in both anatomic regions as pregnancy progressed. Analyses of transcripts from gd10.5 uNK cells flow sorted on the basis of expression of Dolichos biflorus agglutinin (DBA) lectin revealed that DBA+ but not DBA- uNK cells expressed Dll1. Immunostaining at gd4.5 found DLL1-expressing cells rare. At gd6.5, DBA+ uNK cells at all stages of maturation expressed DLL1. By gd10.5, DLL1 immunoreactivity was strongly expressed by some but not all DBA+ uNK cells and more weakly by DBA- cells. DLL1+ cells were mesometrially stratified and concentrated within central decidua basalis. Our data suggest that uNK cells have the potential to induce endothelial tip cell differentiation and to promote non-planar vascular growth within early decidua basalis.

Identification of the primary outcomes that result from deficient spiral arterial modification in pregnant mice

Pre-eclampsia, an acute complication of human pregnancy, is associated within complete physiological modification of decidual spiral arteries. This is thought to promote oxidative stress from perfusion/reperfusion of the placenta and to restrict placental and fetal growth. Alymphoid (genotype Rag2(-/-)/Il2rg(-/-)) mice, sufficient in dendritic and myeloid cell functions, lack spiral arterial modification with individual spiral arteries having ~1.7x the vascular resistance and 0.66x the blood velocity of +/+ mice. Their placentae are measurably hypoxic yet neither placental growth nor fetal survival is impaired and gestational hypertension is not seen. Thus, lymphocytes rather than vascular adaptations appear to be the pivotal contributors to the clinical complications of pre-eclampsia.

Fetal–Placental Hypoxia Does Not Result from Failure of Spiral Arterial Modification in Mice

OBJECTIVES To determine if fetal-placental hypoxia is a primary outcome of defective spiral artery remodeling. STUDY DESIGN Pregnancies in Rag2(-/-)Il2rg(-/-) double knock-out mice, which fail to undergo normal physiological spiral arterial remodeling, were compared to syngeneic BALB/c control pregnancies. Mice at gestation day (gd)6, 8, 10, 12 and 18 were infused with Hypoxyprobe-1 before euthanasia to enable detection of cellular hypoxia by immunohistochemistry. RESULTS In implantation sites of both phenotypes, trophoblast cells were reactive to Hypoxyprobe-1. No major differences were observed between the phenotypes in decidua or placenta at any gd or in gd18 fetal brain, lung, heart, liver or intestine or in maternal heart, brain, liver or spleen. Maternal kidneys from BALB/c were significantly hypoxic to Rag2(-/-)Il2rg(-/-) kidneys. CONCLUSIONS In mice, lack of pregnancy-associated spiral artery remodeling does not impair oxygen delivery to the conceptus, challenging the concept that deficient spiral arterial remodeling leads to fetal hypoxia in human gestational complications such as preeclampsia and fetal growth restriction. The isolated hypoxic response of normal kidney has revealed that renal lymphocytes may have unique, tissue-specific regulatory actions on vasoconstriction that are pregnancy independent.

Ribonucleic acid and protein synthesis in the uterus of pregnant mouse during activation of implantation window

Ribonucleic acid (RNA) and protein synthesis in the mouse endometrium during activation of the implantation window was studied. Ovulations of BALB/c mice were controlled by pregnant mare serum gonadtropin (PMSG) and human chorionic gonadotropin (HCG), and pregnant females were ovariectomized on the 4th day of pregnancy. The delay implantation state was maintained for 48 hr. After 0 to 18 hr of estrogen supply, a 1-hr pulse of3H-uridine or3H-leucine was injected intraperitoneally. The uterine tissues were processed for light and electron microscope radioautography, and analyzed at three distinct regions as follows: interimplantation site (IN), antimesometrial side (AI) and mesometrial side (MI) of the implantation site. The RNA and protein synthesis showed a peak at 6 hr after estrogen induction both in the AI stromal and epithelial cells, whereas in the cells of IN sites it increased only slightly. The presence of the blastocyst in the uterine lumen induced selective changes in the behavior of endometrial cells after the nidatory estradiol effect. The peak of RNA and protein synthesis seen in the AI cells reflected a commitment to restrict cell population during activation of the implantation window.