Granulysin expression and colocalization with perforin in decidual and peripheral blood lymphocytes of the first trimester pregnancy

Abstract

Shorter 9 kDa granulysin isoform often colocalizes with perforin in the same granules of cytotoxic NK and T cells. Granulysin rapidly enter into the target cells via perforin pores inducing apoptotic death. The longer, 15 kDa form does not colocalize with perforin in the same granules and it exerts regulatory functions after secretion by activated cells. We aimed to analyze the expression of granulysin by different antibodies within the early pregnancy decidual lymphocytes (DL) and peripheral blood lymphocytes (PBL) in vitro and colocalization of granulysin and perforin. MATERIAL AND METHODS: DL were isolated by enzymatic digestion and gradient density centrifugation. PBL were obtained by gradient density centrifugation. Intracellular granulysin was labelled by flow cytometry, using rabbit polyclonal anti-granulysin antibodies (provided by Prof. Clayberger, Chicago, USA) and RF10 anti-granulysin monoclonal antibody - mAb (MBL, Japan) in CD3 and CD56 labelled lymphocytes. Granulysin and perforin were visualized by immunofluorescence, and analyzed by confocal microscopy using ImageJ 1.44p quantification software. RESULTS: There is a difference between colocalization of granulysin and perforin in DL and PBL, with much more colocalization in PBL. Upon the cell activation the colocalization pattern of DL is much more similar to the one observed in PBL. Significant difference granulysin labelling by polyclonal and monoclonal RF10 antibodies in decidual NK cells is observed as well. In the decidual NK cells there is significant difference in the granulysin labelling by polyclonal and monoclonal RF10 antibodies, which can be interpreted as a difference in the granuylsin isoform expression. CONCLUSION: Investigation of the dynamics of colocalization following the cell activation showed significant increase of colocalization in DL, pointing to the functional maturation of the cytolytic potential of these cells.