Abstract:
Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs
including spleen and gut, where they play an important role in the defense against infections. Interaction of NK
cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct
vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts
and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to
correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin
(PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the
purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK
cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK
cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in
staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its
gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in
reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA
reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that
the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to
kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an
additional tool to study activity of cytotoxic lymphocytes in chickens.
