Three-dimensional reconstruction of leukocyte internalisation in the luminal uterine epithelium following mating
Introduction
The observation that whole cells can be internalised into the cytoplasm of other cells has been reported for over a century, but recent evidence has identified a unique method of formation of cell-in-cell structures that is distinct from phagocytosis and termed “entosis”. Entosis occurs when the host cell is not necessarily phagocytic, and the internalised cells are living and otherwise viable [1,2]. The result is the formation of “cell-in-cell structures” between cells of the same origin (homotypic) or cells of different origin (heterotypic). Although the target cells are almost always of haematopoietic origin such as neutrophils and lymphocytes, these structures have been reported in a wide range of normal and neoplastic host cell types both in vitro and in vivo, including epithelial cells [[3], [4], [5]]. Once formed, a range of fates have been observed for both the host and target cells. Most commonly, the internalised cells will undergo an atypical form of cell death, lacking the normal molecular and ultrastructural features of apoptosis. When studied in vitro approximately 20% of cell-in-cell structures result in the target cell being expelled from the host without apparent consequence to either host or target, and transient migration of lymphocytes through endothelium, thyroid and thymus cells have been reported in vivo [[1], [4], [43]].
The physiological significance of heterotypic non-neoplastic cell-in-cell structures is not well understood, with many factors such as the cell type of host and target playing a significant role. One of the more documented examples occurring in normal physiology is during extravasation from circulating blood into surrounding connective tissue, where lymphocytes can complete an intracellular migration and pass through the cytoplasm of an endothelial cell [[6], [7], [8]]. Neutrophils have previously been observed to occasionally transit through the cytoplasm of megakaryocytes within the bone marrow, but this is correlated with malignancies and is increased in pathological conditions [9,10]. However, there have not previously been any reports of cell-in-cell structures being formed in the uterine epithelium in either normal or pathological conditions.
The leukocytic inflammatory response of the endometrium to mating is common to many mammalian species. In humans, pigs and horses this is primarily caused by the spermatozoa, while in rodents it is elicited by transforming growth factor-β and other seminal plasma factors [11,12]. These factors stimulate the synthesis of pro-inflammatory cytokines in the endometrium, including secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) by the endometrial epithelium. The leukocytic response recruits neutrophils and macrophages from the bloodstream into the uterus, cervix and vagina and is primarily responsible for the rapid processing and elimination of the majority of the spermatozoa and induction of immune tolerance to seminal material.
In addition to clearance of excess spermatozoa, the leukocytic response to mating is implicated in priming the endometrium for successful pregnancy through mediation of the maternal immune system to ‘tolerate’ the implanting blastocyst. This is achieved via recognition of paternal antigens shared by semen and the blastocyst which induces an immunosuppressive function of regulatory T-lymphocytes in the endometrium at the site of implantation. However, the precise mechanism by which the paternal antigens found in seminal plasma are presented to the maternal immune system is yet to be elucidated [13].
Inflammatory neutrophils activated by the innate immune response express the anti-apoptotic protein Mcl-1 and have an increased longevity with a half-life of up to 36 h compared to a 7–12 h half-life in the inactive circulating form [14,15]. In humans, mature neutrophils are responsible for the majority of leukocyte glycogen content and this is elevated during leukocytosis, and stored glycogen in neutrophils is readily used during phagocytosis [[16], [17], [18]]. Following phagocytosis of invading microorganisms, neutrophils undergo programmed cell death or are themselves phagocytosed by long-lived recruited macrophages in order to protect the surrounding tissues from histotoxic enzymes [14,19].
In the present study we used array tomography to examine the cellular relationships between leukocytes and uterine epithelial cells (UECs) approximately 12 h post-mating. This technique involves production of serial arrays of ultrathin tissue sections which were imaged sequentially and then aligned to form high resolution 3-dimensional reconstructions, delivering large volume ultrastructural information down to the nanometre scale [20,21]. Array Tomography reconstruction confirmed that inflammatory neutrophils that are recruited to the endometrium post-mating can become fully contained within a membrane-bound vacuole in the cytoplasm of luminal uterine epithelial cells, thereby forming cell-in-cell structures.
Section snippets
Tissue collection
Adult, virgin Wistar rats were housed in plastic cages in a temperature-controlled environment at 21 °C. Rat pellets and tap water were provided ad libitum. Animals were selected for mating using vaginal smearing at 4:30 p.m. for pro-oestrus stage of the oestrous cycle and caged overnight with males of proven fertility. The presence of spermatozoa in the vaginal smear at 8:30 a.m. the next morning confirmed mating and was designated as Day 1 of pregnancy. On the chosen day of pregnancy animals
Leukocytes are internalised within uterine epithelial cells during the maternal inflammatory response to mating
On Day 1 of pregnancy approximately 12 h post mating, we report the first documented appearance of leukocyte internalisation within the luminal uterine epithelium of the rat in all four animals used in this study (Fig. 1). There were no internalised leukocytes observed on Day 6 of pregnancy (n = 4, data not shown) which corresponds to the time of blastocyst implantation in rats. The presence of internalised leukocytes was confirmed by imaging serial ultrathin sections and creating a z-stack
Discussion
Array tomography has been applied to reconstruct 3-dimensional models that conclusively demonstrate for the first time the internalisation of leukocytes within uterine epithelial cells on Day 1 of pregnancy in the rat. Through large-volume serial sectioning and manually tracing structures of interest, it was shown that following mating, inflammatory neutrophils and macrophages recruited to the endometrium are internalised by luminal uterine epithelial cells. The internalised cells described in
Acknowledgements
The authors acknowledge the facilities and expert technical assistance of Microscopy Australia (MA) at Sydney Microscopy & Microanalysis (SMM) at the University of Sydney and the Advanced Microscopy Facility of The Bosch Institute, The University of Sydney. This work was funded by the NWG Macintosh Memorial Fund Grant.
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