engleski

Molecular mechanisms involved in short term cytolytic activity of early pregnancy decidual NK cells

NK cells of CD3-CD16-CD56 bright+ phenotype are predominant leukocyte population in the first trimester normal human pregnancy decidua. These cells are activated and express high average number of perforin molecule in their granules. Perforin expression is strongly regulated by cytokine network operating in decidua. Interleukin-15, secreted by decidual macrophages and IL-18, produced by extra villous trophoblast cells increase perforin expression and cytolytic activity of decidual lymphocytes against K-562 cells in vitro. Fas ligand (FasL), the member of TNF super family, is expressed on extra villous cytotrophoblast cells. This molecule induces apoptosis of Fas+ decidual targets and seems to be involved in trophoblast invasion. A little is known about Fas L expression on decidual NK (dNK) cells and its involment in dNK cell cytotoxicity. The aim of this study is to analyze molecular mechanism of short-term cytolytic activity of dNK cells. We have used decidual tissue of first trimester pregnancy decidua obtained following elective termination of 6-10 week old pregnancy. Tissue was enzymatic digested by collagenase IV and decidual mononuclear cells were obtained by Ficoll density gradient centrifugation. Mononuclear cell suspension was cultured in the presence of Th1 cytokines (IL-2, IL-12, IL-15, IL-18) or medium only with antimetalloproteinase KB 8301 at 37oC with 5% CO2. Following 18 hrs, the cells were simultaneously labeled for detection of Fas L (using cell permeabilization method) and cell surface CD56 molecule. Decidual CD56+ NK cells were isolated from decidual mononuclear cell suspension immediately after centrifugation (fresh dNK cells) or following 18hr culture at 37o C (cultured dNK cells) by positive magnetic cell separation method (anti-CD56 MACS Beads). Cytolytic activity of these cells was tested in 2hr PKH-26 red (Sigma) cyotoxicity assay in the presence of inhibitor of perforin egzocytosis concanamycin A, concanamycin A and anti-Fas L antibody or in the medium only and analyzed by flow cytometer. Cultured dNK cells lysed NK sensitive K-562 cells line mostly by perforin mechanism and with equal efficiency as fresh dNK cells. Contrary to K-562 targets, cultured dNK cells lyse NK resistant P815 cells more efficiently than fresh dNK cells. Concanamycin A partially, but Concanamycin A and anti-FasL antibody completely abrogate higher cytolytic activity of cultured dNK cells against P815 targets. Intracellular detection of FasL showed that IL-2, IL-12, IL-15 and IL-18 following 18 hr culture in vitro, could increase negligible expression of FasL in fresh dNK cells. Although dNK cells constitutively express perforin, Th1 cytokines can up regulate expression of FasL protein. We have shown that dNK efficiently use both: perforin and FasL mediated cytotoxicity pathways. Supported by Ministry of Science and Technology, Republic of Croatia (Grant No.0062029).

decidual NK cells; perforin; early pregnancy; fas ligand protein

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