[Rapid biosynthesis and release of 35 kD granzyme B by NK92 cells bypassing secretory lysosomes]

To investigate the possibility of the biosynthesis and release of granzyme B (GZB) by NK92 cells bypassing the way of secretory lysosomes (SLs) and the possible mechanism.

As cell models, NK92 cells were activated by the phorbol myristate acetate (PMA) and ionomycin (ION). Within 4 hours following the activation, immuno- fluorescence and electron microscopy were used to detect the content and distribution of 35 000 (Mr) and 32 000 (Mr) GZB in the cytoplasm of NK92 before and after the protein synthesis was inhibited; Western blotting was performed to detect GZB inside and outside the SLs. After blocking the release of 32 000 (Mr) GZB by inhibiting the exocytosis of SLs with EDTA, we tested the content of Mr 35 000 GZB in activated NK92 supernatant. Activated NK92 cells were co-cultured with K562 cells to observe whether the Mr 35 000 GZB could enter the K562 cells. Activated NK92 cell death rate was determined and the enzyme activity of secreted Mr 35 000 GZB was examined.

Four hours after stimulated by PMA/ION, NK92 cells generated large amount of Mr 35 000 GZB in the cytoplasm outside SLs where Mr 32 000 GZB was located. Immunoelectron microscope and immunofluorescence further approved that Mr 35 000 GZB outside SLs was located in vesicles. In addition, Mr 35 000 GZB could be secreted outside NK92 cells. Further investigation found that GZB/Serpinb9 composite and Mr 35 000 GZB could simultaneously emerge in the cytoplasm outside SLs. However, activated NK92 cell death rate did not rise. Mr 32 000 GZB inside SLs had enzyme activity in contrast with the Mr 35 000 GZB in zymogen form outside SLs, which suggested that Mr 35 000 GZB was not originated from the SLs.

The activated human NK cell lines could secreted rapidly inactive Mr 35 000 GZB outside SLs, and the GZB could enter the extracellular matrix or target cells bypassing SLs, which provides a part of the extracellular GZB.