engleski

Regulation of granulysin protein expression in decidual NK cells by Th1 cytokines and antigen presenting cells

Problem: The aim of our study was to analyze the expression of a cytolytic protein granulysin in decidual CD56 positive cells after the stimulation with Th1 cytokines and decidual antigen presenting cells. Methods of study: Decidual mononuclear cells (DMC) were obtained by enzymatic digestion of decidual tissue and density gradient centrifugation. CD56+ cells and subsequently CD83+ cells were purified from the suspension of a non-adherent DMC fraction using positive magnetic separation after 18-hour culture at 37°C. The immature CD1a+ and subsequently CD14+ cells were isolated from the adherent DMC fraction. Granulysin expression was analyzed using cell permeabilisation method, flow cytometry, immunohistochemistry and confocal microscopy. Results: Approximately 90% of decidual CD56+CD3- NK cells express granulysin in their cytoplasmatic granules near the cell membrane. This percentage did not significantly change after 18-hour culture of NK cells in DMC suspension, nor after CD56 positive magnetic separation. The addition of anti-PIBF monoclonal antibody in the suspension of freshly isolated DMC decreased the percentage of granulysin expressing CD56+CD3- cells after 18-hour culture. The culture of purified CD56+ cells in the medium only for 18 hours, decreased the percentage of granulysin in their cytoplasm. This decrease is prevented by stimulation with IL-15, IL-2, and IL-18 in a dose-dependent manner. The co-culture of purified CD56+ cells with immature CD1a+ dendritic cells or CD14+ macrophages also upregulated the percentage of granulysin expressing NK cells, whereas CD83+ cells had no effect. Conclusion: Decidual tissue specific environment, comprising PIBF, cytokines and antigen presenting cells, favorizes granulysin expression in CD56+ NK cells. The experiments are partially financed by Croatian Ministry of Science (Grants No. 0620402-0376 and 0379) and EMBIC project, European FP6, NoE, LSHM-CT-2004-512040.

pregnancy; granulysin; decidual NK cells; Th1 cytokines

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