engleski

Cytolytic molecule trio of granulysin, perforin and perforin-2 in CD56+ and CD14+ cells of peripheral blood

INTRODUCTION: NK cells and T cells highly express cytolytic molecules of granulysin and perforin. Granulysin is member of SAPLIP family and it has two isoforms - longer of 15 kDa and shorter of 9 kDa. Recent studies suggest two distinctive roles of these isoforms. Shorter isoform has strictly cytolytic property while longer is an immune alarmin. To enter in procariotic cell granulysin forms scissor-like shape and enter by friction between its molecules while for eucariotic cell it needs to collaborate with perforin. Perforin is a pore forming protein, highly expressed in human pregnancy decidua than in any physiological and pathological state known. Perforin-2 is a novel cytolytic molecule and one of the key mediators of innate immunity in the battle against intracellular bacteria. Perforin-2 is a transmembrane protein with role to form the pores in outer wall of intracellular bacteria of the eucariotic host cells. Aim of this study is to elucidate the mechanisms of expression and interplay of this cytolytic molecule trio. MATERIAL AND METHODS: Peripheral blood lymphocytes were obtained by density gradient centrifugation of heparinized blood specimens. Lymphocyte CD56+ and CD14+ subpopulations were purified by positive magnetic separation by using LS columns of VarioMacs separator. The purity of isolation was always above 90%. Isolated CD56+ and CD14+ cells were incubated with 300 IU/106 of IFN-γ and IFN-α + IFN-β + IFN-γ respectively for 24 hours at 37°C with 5% CO2. Total RNA was isolated by Trizol/chloroform extraction. The concentration of RNA was determined by NanoVue (Thermo Scientific). Real-time RT-PCR analysis was performed with the ABI PRISM 7300 SDS (Applied Biosystems) and the commercially available TaqMan assay reagents (Applied Biosystems) for the granulysin gene (Hs00246266_m1), the perforin gene (Hs00169473_m1), the perforin-2 gene (Hs00909102_s1) and for the human eukaryotic 18S rRNA (4352930E). RESULTS: IFN-γ induce the expression fold change of cytolytic molecules granulysin, perforin and perforin-2 in both CD56+ and CD14+ peripheral blood subpopulations. The combination of interferons: IFN-α + IFN-β + IFN-γ was much stronger stimulator of cytolytic molecules mRNA expression. Extremely high is the fold change of granulysin mRNA in CD14+ subpopulation upon stimulation with interferons combination. CONCLUSION: The increase of cytolytic molecule expression on gene level in both CD56+ and CD14+ cells of peripheral blood upon the stimulation with interferons points to the importance of these molecules in fight against intracellular pathogens and infected or changed cells. Granulysin is molecule of dual nature that can also act as an immune alarmin capable of recruiting antigen-presenting cells via TLR-4 receptor and to direct towards the innate or adaptive immune responses. Extremely high fold change of granulysin in CD14+ cells upon stimulation with IFN-α + IFN-β + IFN-γ suggests the important immunomodulatory role of granulysin in inflammatory conditions. Acknowledgement: The experiments were financed by Croatian Ministry of Science, Education and Sports Grants No. 062- 0620402-0376.

perforin; granulysin; pregnancy

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