engleski

Effect of Heat shock protein 70 on granulysin and perforin expression in normal human first trimester decidual NK cells

Introduction: Granulysin (GNLY) is an apoptotic molecule stored in cytoplasmic granules of early human decidual NK cells dominantly with a cytotoxic, calcium-dependent pore forming protein, perforin. Perforin is indispensable for GNLY entrance in the cells. GNLY access cytoplasm of infected or aberrant eukaryotic cells by the perforin pore, and such interplay of these two cytotoxic mediators seem to provide protection of the maternal-fetal unit from infection, malignant alteration, and uncontrolled trophoblast growth. Both cytotoxic mediators are tightly regulated by numerous cellular and soluble factors occurring at the maternal-fetal interface during an extensive tissue remodeling implantation process. We have previously shown members of heat shock protein 70 (HSP70) and progesterone induced blocking factor (PIBF) to be expressed at maternal-fetal interface. Significant increase of HSP70, in the intracellular space, due to the leak from dead or damaged cells, acts through binding to surface receptors on antigen presenting cells and stimulate proinflammatory immune response. It possibly increases cytotoxic potential of NK cells and counteract the immunosuppressive role of specific progesterone mediator PIBF. Aim of the study was to investigate the effect of HSP70 on GNLY and perforin protein expression in decidual NK cells untreated or PIBF pretreated in vitro. Methods of study: Decidual mononuclear cells were obtained by enzymatic digestion of normal early pregnancy decidual tissue and density gradient centrifugation. They were untreated or pretreated with 2 μg/ml of PIBF for 2 hours and subsequently cultured in the medium only or with HSP 70 at 100 ng/ml or 1000 ng/ml without washing for further 18 hours. Simultaneous labeling of surface (CD3 and CD56) and intracellular (GNYL or perforin) antigens was performed by immunofluorescence. Cells were acquired by flow cytometer (Becton Dickinson FACSCalibur) using CellQuestPro software (BD Biosciences, San Jose, California, USA). Data were analyzed using WinMdi 1.9 program. Statistical and graphical processing was performed with the data analysis software system Statistica 7.0 (StatSoft, Inc., Tulsa, OK, USA). Results: HSP 70 at concentration of 100 ng/ml increased MFI for perforin in CD56 bright+ subset of decidual NK cells. PIBF prevents this increase and decreased simultaneously the percentage of perforin expressing CD56 dim+ cells after 18 hrs stimulation with HSP 70. We did not observe significant changes in GNLY expression after HSP 70 and/or PIBF stimulation. Conclusion: HSP 70 mediated increase in perforin expression in decidual NK cells in vitro was abolished with PIBF, suggesting that HSP 70/PIBF interplay could be important in perforin regulation at maternal-fetal interface in vivo. Although GNLY expression did not change in our experiments, the access of GNLY in target eukaryotic cells might be reduced in presence of HSP 70 and PIBF.

granulysin; perforin; PIBF; decudua; first trimester pregnancy

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