Characterization of Trophoblast Cells and Decidual Lymphocytes Isolated from Fetal- Maternal Interface

Aim. The aim of our study was to describe the leukocyte phenotype at the fetalmaternal interface after separation of the trophoblast cells and uterine leukocytes from endometrial tissue samples. Materials and methods. The tissues were processed and submitted to 3 cycles of enzymatic digestion then the cells were separated by centrifugation over a Percoll gradient. We investigated by flow-cytometry the lymphocyte subpopulations in peripheral blood or endometrial tissue samples obtained after legal abortion in 10 normal first trimester pregnancies. Leukocyte tissue distribution and trophoblast invasion in the endometrium were investigated by immunohistochemistry. Results. Our data show that NK cells are the predominant lymphocytes in pregnant uterus, that they express a higher level of CD56 compared to their circulatory equivalent, lack CD16 and partially coexpress CD3. One third of the uterine lymphocytes are T cells, with a CD4/CD8 ratio of 1. Immunohistochemistry investigation showed that leukocytes aggregate around spiral arteries and endometrial glands and also disseminate in the stroma. Conclusion. We devised a feasible method for isolation of trophoblast cells and uterine leukocytes from fetal-maternal interface that could be useful in further investigations in order to outline some useful immune tests for detecting and monitoring pregnancies with high obstetrical risk. Key-words: fetal-maternal interface, trophoblast, lymphocytes, NK cells Rezumat. Scop. Scopul acestei lucrări a fost acela de a descrie fenotipul populaţiilor leucocitare de la nivelul interfeţei materno-fetale după separarea trofoblastului şi a leucocitelor din probe de ţesut endometrial. Material şi metodă. Fragmentele tisulare au fost prelucrate şi supuse la trei cicluri de digestie enzimatică apoi celulele au fost separate prin centrifugare pe gradient de Percoll. Am investigat flow-citometric subpopulaţiile limfocitare din sânge periferic şi ţesut endometrial obţinut prin chiuretaj solicitat de 10 gravide cu sarcină normală în primul trimestru. Distribuţia tisulară a leucocitelor ţi invazia trofoblastului în endometru au fost investigate prin imunohistochimie. Rezultate. Rezultatele noastre arată faptul că celulele NK reprezintă populaţia limfocitară predominanta în uterul gravid, ele exprimând un nivel crescut de CD56 în comparaţie cu echivalentul lor circulant, un nivel redus de CD16 şi coexpresie parţială de CD3. O treime din limfocitele uterine sunt celule T cu un raport CD4/CD8 de 1. Imunohistochimia a relevat faptul ca leucocitele sunt fie agregate în jurul arterelor spiralate şi a glandelor endometriale, fie diseminate în stroma. Concluzie. În această lucrare descriem optimizarea unei tehnici fezabile de separare a trofoblastului şi a leucocitelor uterine de la nivelul interfeţei materno-fetale, metodă ce va servi investigaţiilor ulterioare care TROPHOBLAST AND LYMPHOCYTES AT FETAL-MATERNAL INTERFACE 89 vizează descrierea unor parametri imuni utili pentru depistarea si monitorizarea sarcinilor cu risc obstetrical crescut. Cuvinte-cheie: interfata materno-fetala, trofoblast, limfocite, celule NK INTRODUCTION Leukocytes are an important component of the fetal-maternal interface during the first trimester of pregnancy, representing 40 – 45% of the decidual cells (1, 2). Decidua, the pregnancy-modified endometrium, is infiltrated at the implantation site by NK cells and T lymphocytes being in close active contact with the invading fetal trophoblast cells (3). There are two populations of trophoblast cells: the villous trophoblast that covers the placental villi and mediates the nutritive changes between the mother and the fetus. The extra-villous trophoblast is responsible with the invasive behavior, it migrates in the maternal decidua, surrounds and replaces the endothelium of the maternal spiral arteries changing them to low-resistance vessels which are not any more responsive to maternal stimuli and offer larger amounts of blood to the fetus (4, 5). The immune cells and their products at the maternal-fetal interface contribute to limiting the invasiveness of the trophoblast in a way that allows the fetus to develop and mature to term and the mother to survive to the increasing demands of the new being. From the immunological point of view pregnancy is a puzzling issue; it is still unclear how the immune cells of the mother tolerate the fetus which, due to the paternal genes, is half non-self. For a normal evolution of pregnancy to term, a well-balanced equilibrium between invasive trophoblast and maternal immune cells is required, in order to support the complex endocrine and cytokine network which regulate the activity at the fetal-maternal interface. This paper presents our adaptation of a method of separation of trophoblast and immune cells from first trimester placental tissue aimed to explore the ratios between different lymphocyte subpopulations in the normal first trimester-endometrium. MATERIAL AND METHODS Preparation of trophoblast cells and uterine leukocyte suspensions We processed placental tissue obtained after legal abortions in 10 normal first trimester-pregnancies (6-14 weeks). The tissues were transported to the laboratory in ice cold heparinised saline 0.9% NaCl, during the first 30 minutes after the curettage, due to the protease-rich nature of these tissues. Processing of the samples was performed in the laminar-flow cabinet, preserving the sterility oaf the products. After removing the blood clots and fragments of the fetal membranes, the tissue fragments were rinsed several times in phosphate buffered saline (PBS) (pH 7.4) to remove residual blood and then mechanically disaggregated with scissors, to fragments of approximately 3-10 mm volume. Minced tissue was transferred to the digestion bottle Daniela Constantinescu, Carmen Cozmei, Laurette Graziella Cozma, et al 90 containing 20 U/ml DNAse I type IV (Sigma), 0,125 % trypsin (Sigma), 50 μl CaCl2 (Reactivul Bucureşti) 100mM, 50 μl MgSO4 (Reactivul Bucureşti) 800 mM and RPMI 1640 (Sigma) up to 50 ml. The presence of divalent ions is necessary for trypsin activation. The enzymatic tissue digestion was performed at 37°C, in the shaking water bath. After incubation, tissue pieces were allowed to settle for 5 min then half of the supernatant containing the syncytiotrophoblast cells was discarded. Two more cycles of enzymatic digestion were performed, in similar conditions except that the second and third digestion mixtures did not contain DNAse. After the last incubation trypsin activity was stopped by adding heat inactivated fetal calf serum (FCS) up to 10% of the final volume (6). Undigested tissue was removed by filtration through a dense mesh and filtrate was washed by centrifugation (620xg, 20°C, 10 min). After discarding the supernatant, the cell pellet was resuspended in RPMI 1640 containing 4.5‰ glucose and layered on the Percoll gradient (7). As reported by Kliman (1986) (7) when centrifuged over a Percoll gradient cytotrophoblast cells migrate in layers ranging from 1.062 to 1.048 densities. We adapted the protocol described by Nagamatsu et al (2004) and used three gradients of Percoll as table 1 shows (8). Table 1. The composition of the three gradients used in Percoll separation of trophoblast and uterine mononuclear cells. Percoll gradient Corresponding density Percoll (ml) PBS (ml)