Activated NK cells in spontaneous abortion cases induce apoptosis on extravillous trophoblast in a granulysin dependent manner



Extravillous trophoblast (EVT) is well known to undergo apoptosis in spontaneous abortion cases although the apoptotic mechanism is still unclear. Granulysin (Gr) is a cytotoxic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) with tumoricidal activities. We have reported that the expression of granulysin, which was mainly produced by decidual NK cells, is significantly higher in the deciduas of spontaneous abortion cases than early normal pregnancy. We then have attempted to investigate the molecular mechanism of apoptosis associated with Gr. We also assessed apoptosis in the HTR8/SV40 cell, extravillous trophoblast (EVT) cell line, which were transfected with GFP-fused Gr. Anti-cleaved cytokeratin 18 antibody and Annexin-V were used for detecting apoptosis by IS and FC. We detected the apoptosis of the EVT in miscarriage cases. In those EVT, we detected nuclear staining of Gr. We next examined the effect of Gr with the target trophoblast cells by using GFP-fused Gr vector. Exogenously expressed GFP-fused Gr preferentially accumulated in nucleus from cytoplasm, resulting in apoptosis. Similar findings were obtained by native granulysin produced by IL-2 stimulated decidual lymphocytes. Our findings suggest that the granulysin-positive NK cells might attack EVT and they release granulysin into the intracellular space, resulting in apoptosis of EVT. So far, other groups have reported that Fas-FasL pathway and and/or TNF-α pathway is associated with apoptosis of EVT. In additiont, we propose granulysin-positive NK cells-induced apoptosis as a new mechanism for EVT cell death.