B.A. Croy

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.

A Review of Trafficking and Activation of Uterine Natural Killer Cells

Enrichment of uterine natural killer (uNK) cells occurs during pregnancy in many species. However, functions of uNK cells and regulation of their uterine homing are not fully defined. In mice and women, uNK cells contribute to angiogenesis, a role reviewed here and now addressed in a mammal with an alternative placental type.

Uterine NK cells in murine pregnancy

Murine uterine natural killer (uNK) cells are transient, short-lived, terminally differentiated lymphocytes found in decidualized endometrium. Cells expressing natural killer cell surface markers are present in uteri of infant mice. Terminal uNK cell differentiation coincides with mesometrial decidual development subsequent to blastocyst implantation and begins about gestation day 5. uNK cells proliferate rapidly and, within 3 days, senescent uNK cells appear in normal implantation sites. Mid-gestation, senescent cells become dominant and uNK cell numbers decline until term when remaining cells are shed with the placenta. Transplantable uNK cell progenitors occur outside the uterus, suggesting that blood cell homing augments any in-utero progenitors. Early in healthy pregnancies, uNK cells produce cytokines and angiogenic molecules. Their lytic capacity in normal gestation and in pregnancy failure is incompletely defined. A significant shift recently occurred in thinking about major uNK cell functions. Activated uNK cells are now considered critical for appropriate endometrial angiogenesis in early implantation site development and in non-gestational endometrium. Because analogous cells appear in the endometria of women during each menstrual cycle and become abundant in early pregnancy, studies involving experimental pregnancy termination in genetically manipulated mice continue to have great importance for understanding regulation at the human maternal-fetal interface.

Uterine NK cells, spiral artery modification and the regulation of blood pressure during mouse pregnancy.

Citation Burke SD, Barrette VF, Gravel J, Carter ALI, Hatta K, Zhang J, Chen Z, Leno‐Durán E, Bianco J, Leonard S, Murrant C, Adams MA, Anne Croy B. Uterine NK cells, spiral artery modification and the regulation of blood pressure during mouse pregnancy. Am J Reprod Immunol 2010

A Map of Relationships Between Uterine Natural Killer Cells and Progesterone Receptor Expressing Cells During Mouse Pregnancy

Uterine natural killer (uNK) cells appear in implantation sites with decidualization. Initially, they are rare, highly proliferative cells that become abundant by midgestation and then die abruptly. Steps regulating the cyclic appearance of uNK cells are incompletely defined. Although progesterone (P4) has an essential role, mature uNK cells of women and mice lack progesterone receptors (PR). Immunohistochemical studies suggest that mouse PR(-) uNK cells may co-localize with PR(+) stromal cells while human PR(-) uNK cells co-localize with immature, DC-SIGN(+) dendritic cells (DCs). DCs have the potential to produce progesterone-regulated interleukin (IL)-15, a growth factor essential for uNK cells. Since the high affinity IL-15Ralpha is presented to differentiating NK cells in trans, requiring cell contact, histologically-detected interactions may be of central importance for uNK cell differentiation. Thus, a pregnancy time course, histological study of uNK cell differentiation and localization was undertaken in PR(lacZ) transgenic mice. PR(+) cells and uNK cells were co-localized using LacZ histochemistry and Dolichos biflorus (DBA) lectin staining, respectively. uNK cells appeared mesometrially, where PR(+) cells were rare, at gestation day 5.5. uNK cells had limited, apparently random contact with PR(+) cells throughout pregnancy and never themselves expressed PR. Thus, uNK cell differentiation does not appear to require contact with PR(+) cells.

Functions of alpha 2 macroglobulins in pregnancy

The alpha 2 macroglobulins (A2M) are a family of abundant plasma proteins produced predominantly by the mammalian liver. Pregnancy zone proteins (PZP) of humans and rats are A2M family members that bind a wide variety of macromolecules including the important pregnancy-associated molecules such as vascular endothelial growth factor, placenta growth factor and glycodelin (also called PP14). Recently, a mouse gene analogous to PZP (A2M of pregnancy or A2Mp) was cloned. A2Mp has a unique pattern of expression in reproductive and cardiovascular tissues and, unexpectedly, is not expressed by liver. Since changes in heart function and remodeling of renal and uterine vasculature are amongst the earliest maternal responses to pregnancy, the product of the A2Mp gene has been postulated to systemically regulate these changes. A2Ms with and without non-covalently bound ligands also down regulate immune cell activation but promote immune cell migration, additional features associated with gestational success. Here, we review the A2M gene families of mice and humans, the predicted structural relationships between A2M and its pregnancy induced forms and the postulated roles for this gene family in normal pregnancy.

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.

Expression of chemokine decoy receptors and their ligands at the porcine maternal-fetal interface

Successful pregnancy requires coordinated maternal–fetal cross‐talk to establish vascular connections that support conceptus growth. In pigs, two waves of spontaneous fetal loss occur and 30–40% of conceptuses are lost before parturition. Previous studies associated these losses with decreased angiogenic and increased inflammatory cytokines. Chemokines, a sub‐category of cytokines, and decoy receptors control leukocyte trafficking, angiogenesis and development. The availability of chemokines is regulated by three non‐signalling decoy receptors: chemokine decoy receptor (D6), Duffy antigen receptor for chemokines (DARC) and Chemocentryx decoy receptor (CCX CKR). We hypothesized that the expression of these receptors and their chemokine ligands regulate the porcine pregnancy success or failure. Here, we describe for the first time the transcription and translation of all three decoy receptors and several chemokine ligands in endometrium and trophoblast associated with healthy and arresting conceptuses at gestation day (gd) 20 and gd50. Among decoy receptors, transcripts for DARC were significantly reduced in endometrium, whereas that for CCX CKR were significantly increased in endometrium and trophoblast at gd50 arresting compared with healthy sites. However, western blot analysis revealed no differences in decoy receptor expression between healthy and arresting tissues. Transcripts for decoy receptor ligands CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL21, CXCL2 and CXCL8 were stable between healthy and arresting littermates. Quantification by SearchLight chemiluminescent protein array confirmed ligand expression at the protein level. These data indicate that decoy receptors and ligands are expressed at the porcine maternal–fetal interface and dysregulation of decoy receptor (DARC and CCX CKR) transcripts occurs at sites of fetal arrest.

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.

Brain Structural and Vascular Anatomy Is Altered in Offspring of Pre-Eclamptic Pregnancies: A Pilot Study

The authors assessed the brain structural and vascular anatomy in 7- to 10-year-old offspring of pre-eclamptic pregnancies compared with matched controls (n=10 per group). TOF-MRA and a high-resolution anatomic T1-weighted MPRAGE sequence were acquired for each participant. Offspring of pre-eclamptic pregnancies exhibited enlarged brain regional volumes of the cerebellum, temporal lobe, brain stem, and right and left amygdalae. These offspring displayed reduced cerebral vessel radii in the occipital and parietal lobes. The authors conclude that these structural and vascular anomalies may underlie the cognitive deficits reported in the pre-eclamptic offspring population. BACKGROUND AND PURPOSE: Pre-eclampsia is a serious clinical gestational disorder occurring in 3%–5% of all human pregnancies and characterized by endothelial dysfunction and vascular complications. Offspring born of pre-eclamptic pregnancies are reported to exhibit deficits in cognitive function, higher incidence of depression, and increased susceptibility to stroke. However, no brain imaging reports exist on these offspring. We aimed to assess brain structural and vascular anatomy in 7- to 10-year-old offspring of pre-eclamptic pregnancies compared with matched controls. MATERIALS AND METHODS: Offspring of pre-eclamptic pregnancies and matched controls (n = 10 per group) were recruited from an established longitudinal cohort examining the effects of pre-eclampsia. Children underwent MR imaging to identify brain structural and vascular anatomic differences. Maternal plasma samples collected at birth were assayed for angiogenic factors by enzyme-linked immunosorbent assay. RESULTS: Offspring of pre-eclamptic pregnancies exhibited enlarged brain regional volumes of the cerebellum, temporal lobe, brain stem, and right and left amygdalae. These offspring displayed reduced cerebral vessel radii in the occipital and parietal lobes. Enzyme-linked immunosorbent assay analysis revealed underexpression of the placental growth factor among the maternal plasma samples from women who experienced pre-eclampsia. CONCLUSIONS: This study is the first to report brain structural and vascular anatomic alterations in the population of offspring of pre-eclamptic pregnancies. Brain structural alterations shared similarities with those seen in autism. Vascular alterations may have preceded these structural alterations. This pilot study requires further validation with a larger population to provide stronger estimates of brain structural and vascular outcomes among the offspring of pre-eclamptic pregnancies.