Isolation of cells from the feto-maternal interface.
ABSTRACT The mucosal lining of the human uterus is host to a specialized population of leukocytes, which, during pregnancy, interact with invading placental cells (trophoblast) of fetal origin. Of particular interest are uterine natural killer cells, which account for around 70% of the leukocytes at this site during the first trimester of pregnancy, and seem to be specially adapted to recognize invading trophoblast cells. In order to understand the interactions between mucosal immune cells and trophoblast, and those among the immune cells themselves, it is useful to be able to isolate and culture these cells. Here, we describe protocols for the isolation of leukocytes, stromal cells, and trophoblast cells from the feto-maternal interface.
SourceAvailable from: Cheuk-Lun Lee[Show abstract] [Hide abstract]
ABSTRACT: Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05) and increased urokinase (uPA) and matrix metalloproteinases (MMPs) transcripts and their activity (p<0.05) in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.PLoS ONE 10/2013; 8(10):e76023. DOI:10.1371/journal.pone.0076023
[Show abstract] [Hide abstract]
ABSTRACT: Natural killer (NK) cells kill virus-infected and tumor target cells without prior sensitization. Each NK cell expresses a multitude of activating and inhibitory receptors, and the interplay of signals determines the outcome of NK cell activity. NK cell-mediated cytolysis of target cell involves polarized degranulation at effector-target interface. Peripheral blood NK cell constitutes about 10 % of lymphocytes, and approximately 90 % of peripheral blood NK cells are CD56(dim)CD16(+); however, there is a distinct subset of NK cells, CD56(bright)CD16(-), expressed by certain lymphoid organs which are able to produce large amounts of cytokines including interferon-γ, tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor, but the cytotoxicity is attained only on their prolonged activation. In this review, we discuss the accumulated data on distinct phenotypes of NK cells in human uterus, liver, intestine, skin, and lung and also attempt to correlate their phenotype with corresponding activity and functions, with significant stress on the role of NK cells in pathology in the specific organs. Our detailed understanding of altered NK cell activity in different organs and their inherent cytotoxic activity against tumor target cells will help us design better immunotherapeutic strategies in NK cell-mediated cancer therapies.Immunologic Research 12/2013; 58(1). DOI:10.1007/s12026-013-8477-9
[Show abstract] [Hide abstract]
ABSTRACT: Biobanks provide an important repository of samples for research purposes. However, for those samples to reflect the in vivo state, and for experimental reliability and reproducibility, careful attention to collection, processing and storage is essential. This is particularly true for the placenta, which is potentially subjected to stressful conditions during delivery, and sample collection may be delayed owing to routine postpartum inspection by clinical staff. In addition, standardisation of the collection procedure enables samples to be shared among research groups, allowing larger datasets to be established. Here, we provide an evidence-based and experts' review of the factors surrounding collection that may influence data obtained from the human placenta. We outline particular requirements for specific techniques, and propose a protocol for optimal sample collection. We recognise that the relevance of these factors, and of the sample types collected to a particular study will depend on the research questions being addressed. We therefore anticipate that researchers will select from the protocol to meet their needs and resources available. Wherever possible, we encourage researchers to extend their collection to include additional samples that can be shared on an international collaborative basis, with appropriate informed consent, to raise the quality, as well as quantity, of placental research.Placenta 01/2013; 35(1). DOI:10.1016/j.placenta.2013.11.005