Mouse Uterine Natural Killer Cell Functions During Early Pregnancy

Early pregnancy is characterized by complex interactions between blood vessels, leukocytes, and conceptus-derived trophoblasts within the gestational uterus. Uterine Natural Killer (uNK) cells become the most abundant leukocyte during decidualization and produce a wide array of angiogenic factors, yet little is known regarding their early pregnancy functions. To characterize the role(s) of uNK cells, whole mount in situ immunohistochemistry of live early implant sites was performed. A timecourse examination of murine early pregnancy (virgin, and gd4.5-9.5) implantation sites was performed. Comparison of Gd6.5, 8.5 and 9.5 implant sites from BALB/c+/+ controls (BALB/c) and BALB/c-Rag2-/-Il2rg-/- (alymphoid) identified anomalies that result from the absence of lymphocytes. In alymphoid decidua basalis, mesometrial angiogenesis was widespread but pruning of nascent vessels within alymphoid decidua basalis was deficient. As early gestation progressed, vessels of alymphoid decidua basalis showed no evidence for remodeling. Alymphoid implantation sites showed ~24h delay in uterine lumen closure and embryonic development. To determine if uNK cells would normalize the anomalies observed in alymphoid implantation sites, adoptive cell transfer of NK+ B- T- marrow to alymphoid mice was performed. All of the above anomalies were reversed by adoptive transfer of NK+B-T- marrow. My results suggest that uNK cells support vascular growth and development which ensures the decidua can support the growing conceptus early in pregnancy prior to formation and function of the placenta. Human decidual NK cells may fill similar roles and be important targets for strategies designed to correct intra-uterine growth restriction.

THE IMPORTANCE OF MHC-INDEPENDENT NATURAL KILLER CELL RECEPTORS IN THE IMMUNOREGULATION OF MURINE PREGNANCY

Numerous leukocyte populations are essential for pregnancy success. Uterine natural killer (uNK) cells are chief amongst these leukocytes and represent a unique lineage with limited cytotoxicity but abundant angiokine production. They possess a distinct phenotype of activating and inhibitory receptors that recognize major histocompatibility complex (MHC) molecules, such as the killer immunoglobulin like receptors (KIRs; mouse Ly49), and MHC-independent activating receptors, including the aryl hydrocarbon receptor (AHR) and natural cytotoxicity receptor 1 (NCR1). While the roles of MHC-dependent receptors are widely addressed in pregnancy, MHC-independent receptors are relatively unstudied. This thesis investigated the roles of MHC-independent receptors in promotion of mouse pregnancy and characterized early leukocyte interactions in the presence and absence of NCR1. It was hypothesized that loss of MHC-independent receptors impairs uNK cell development resulting in aberrations in leukocyte function and decidual vasculature. Implantation sites from Ahr-/- and Ncr1Gfp/Gfp mice were assessed using whole mount in situ immunohistochemistry (WM-IHC) and histochemical techniques. Leukocyte interactions identified during preliminary WM-IHC studies were confirmed as immune synapses. The novel identification of immune synapses in early mouse pregnancy compelled further examination of leukocyte conjugates in wildtype C57BL/6 and Ncr1Gfp/Gfp mice. In Ahr-/- and Ncr1Gfp/Gfp mice, receptor loss resulted in reduced uNK cell diameters, impaired decidual vasculature, and failures in spiral artery remodeling. Ahr-/- mice had severe fertility deficits whereas Ncr1Gfp/Gfp mice had increased fetal resorption indicating differing receptor requirements in pregnancy success. NCR1 loss primarily affected uNK cell maturation and function as identified by alterations in granule ultrastructure, lytic protein expression, and angiokine production. Leukocyte conjugates were frequent in early C57BL/6 decidua basalis and included uNK cells conjugating first with antigen presenting cells and then with T cells. Overall conjugate formation was reduced in the absence of NCR1, but specific uNK cell conjugations were unaffected by receptor loss. While KIR-MHC interactions are associated with numerous pregnancy complications in humans, the role of other uNK cell receptors are not well characterized. These results illustrate the importance of MHC-independent receptors in uNK cell activation during early pregnancy in mice and encourage further studies of pregnancy complications that may occur independently of maternal KIR-MHC contributions.

Role of hypoxia and hypoxia-inducible factor-1 in tumour immune escape

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia.

Role of Nitric Oxide-Signalling in Hypoxia-Induced Immune Escape in Cancer

A key step in malignant progression is the acquired ability of tumour cells to escape immune-mediated lysis. A potential mechanism by which tumour cells avoid immune destruction involves the shedding of MHC Class I Chain-Related Protein A (MICA), a Natural Killer (NK) cell-activating ligand, from the tumour cell membrane. Hypoxia has been shown to cause increased MICA shedding; however, this hypoxia-induced effect can be attenuated by pharmacological activation of the cyclic guanosine monophosphate (cGMP)-dependent nitric oxide (NO)-signalling pathway in cancer cells. The primary objective of the present study was to determine whether treatment of tumour-bearing nude mice with the NO-mimetic glyceryl trinitrate (GTN) attenuates in vivo tumour growth and if so, whether this effect is dependent on the presence of an intact NK cell compartment. Results indicated that continuous transdermal administration of GTN (1.8 µg/h) can significantly attenuate the growth of transplanted human DU-145 prostate tumours but that this effect of GTN is lost in mice whose NK-cells have been depleted. Tumours and serum from the mice in this study were analysed to determine whether GTN treatment had any effect on the expression levels of proteins integral to the proposed MICA shedding mechanism; however, the results of these studies were inconclusive. As phosphodiesterase (PDE) inhibition represents a potential method to enhance NO-signalling, experiments were performed to determine whether treatment with the PDE5/6 inhibitor zaprinast could also attenuate hypoxia-induced MICA shedding and decrease in vivo growth of DU-145 tumours. Results demonstrated that treatment with zaprinast (10 mg/kg) significantly attenuates MICA shedding in DU-145 cancer cells and significantly decreases in vivo tumour growth. Taken together, the results of these experiments indicate that GTN attenuates tumour growth by sensitising tumour cells to innate immunity, likely by increasing membrane-associated tumour cell MICA levels through the reactivation of NO-signalling, and that zaprinast decreases tumour growth likely through a similar mechanism. These findings are important because they indicate that agents capable of reactivating NO-signalling, such as NO-mimetics and PDE inhibitors, can potentially be used as immunosensitisers in the treatment and/or prevention of cancer.

IN SITU AND IN VITRO IMMUNOLOCALIZATION OF OVIDUCTIN BINDING SITES ON HAMSTER UTERINE EPITHELIAL CELLS AND DETECTION OF A HAMSTER OVIDUCTIN HOMOLOGUE IN THE FEMALE RAT REPRODUCTIVE TRACT

Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct.