engleski

Intragranular co-localization of granulysin and perforin during decidual lymphocyte activation

Introduction: Granulysin (GNLY) is a cytolytic protein of NK cells, NKT cells and activated but not resting T lymphocytes (Pena et al., 1997, Gansert et al., 2003). It has two forms: shorter of 9 KDa and longer of 15 kD. (Pena et al., 1997). Important function of GNLY is lysis and apoptosis induction of target cells: tumors cells, cells infected by intracellular pathogens or viruses (Stenger et al., 1998, Saini et al., 2011, Hata et al., 2001). Perforin is calcium-dependent pore-forming protein which penetrate the eucariotic target cell and enable GNLY to enter the cell via formed pores (Lowin et al., 1995, Saini et al., 2011). At the maternal-fetal interface, in decidual lymphocytes (DL) GNLY expression is two times higher compared to peripheral blood (PBL) of pregnant and non-pregnant women (Veljkovic Vujaklija et al., 2011). More than 85% of uterine CD56-positive (NK) cells express GNLY. Beside NK cells, GNLY is highly expressed in other decidual subopopulation as well, including dendritic cells and macrophages, thus we assume GNLY could be a molecule of great importance for the successful outcome of the pregnancy (Veljkovic Vujaklija et al., 2011). The role of GNLY at the maternal-fetal interface is dual, to protect mother and fetus from pathogens like bacteria, viruses, fungi or parasites and to protect from the hyperactivation of semiallograft trophoblast (Stenger et al., 1998, Hata et al., 2001., Nakashima et al., 2008) At the maternal-fetal interface perforin is expressed higher than in any known physiological and pathological state. (Rukavina and Podack, 2000). GNLY and perforin often co-localize on cytolytic granules of NK cells and T lymphocytes and have great synergetic cytolytic potential (Pena and Krensky 1997, Stenger et al., 1998). Most of the freshly isolated decidual CD56-positive and CD3-positive cells express both GNLY and perforin, while less than 10% express only GNLY or only perforin. Extravillous trophoblast cells of maternal-fetal interface have unique HLA molecule expression - HLA-E, HLA-C and HLA-G molecules. This kind of molecule expression could mediate or inhibit decidual NK cell cytotoxicity (Tabiasco et al., 2006). Aim of study: Our goal is to investigate the amount of GNLY-perforin intragranular co-localization in DL and PBL prior and during lymphocyte activation. These findings could enable the better understanding of lymphocyte action specificities at the maternal-fetal interface. Methods of study: Decidual mononuclear cells (DMC) and peripheral blood lymphocytes (PBL) were obtained by enzymatic digestion of decidual tissue and density gradient centrifugation. Granulysin and perforin protein expression were analyzed using confocal microscopy. All images were acquired by JACoP plugin (http://rsb.info.nih.gov/ij/plugins/track/jacop.html) of ImageJ 1.44p program (http://imagej.nih.gov/ij) (Bolte, S., Cordelieres, F.P., 2006). Results: In DL 10% of total GNLY is co-localized in the same granules with perforin, thus makes significant difference compared to PBL (approximately 50% of co-localization). In DL around 20% of total perforin is co-localized with GNLY, which is also significantly lower compared to PBL. After 2 hours co-cultivation with K562 and its HLA-G transfectant this percentage of co-localized GNLY in DL is significantly increased to around 45%. In the case of PBL there is no significant change after co-cultivation with target cell line and its values are around 50%. After 2 hours co-cultivation with K562 and its HLA-C and HLA-G transfectants the percentage of DL with co--localized perforin significantly increases to around 50%, while there was no change in the percentage of co-localized perforin in PBL following the two hours co-cultivation with HLA-C and HLA-G transfectant compared to PBL cultivated in the medium only. Conclusion: The profile of DL after 2 hrs co-cultivation becomes more alike the profile of PBL since the initial significant difference of the co-localized GNLY and co-localized perforin percentage disappears after 2 hours co-cultivation, which points to the potential “functional maturation”. Our results suggest DL are now activated but possibly still inhibited to carry out cytotoxicity against extravillous trophoblast due to interaction with its HLA molecules thus directed to attack intracelluler pathogens. Acknowledgement: The investigation was financed by Croatian Ministry of Science, grant No. 0620402-0376 to Dr. D. Rukavina.

Granulysin; NkT cells; maternal-fetal interface; perforin

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