Abstract
BackgroundGranulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein.ResultsIn this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation.Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells.ConclusionThis first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.
Highlights
Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes
This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing
Transcriptional regulation of granulysin in lymphokine activated killer (LAK) cells Transcriptional analysis of the granulysin expression has been assessed in cell lines but not in primary cultured lymphocytes
Summary
Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. A crucial role in destroying tumor cells, virus infected cells and intracellular pathogens is played by natural killer cells (NK) cells and cytotoxic T lymphocytes (CTL) [1,2,3]. They are armed with cytotoxic granules containing various effector molecules such as granulysin, perforin, and several granzymes [4,5]. Manning et al [8] investigated the structure and transcription of the granulysin gene in the functional T cell line AJY and found the alternative spliced transcripts 519, 520, and 522 but no NKG5. NKG5, which is very similar to 520, but is absent in the AJY, would have the same function in NK cells
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