Differential Expression of Granzymes A and K in Subsets of Human T-Cells and NK-Cells.

Abstract Granzymes are highly specific proteases stored in secretory granule in T-cells and NK-cells. Five different granzymes are known in human (A, B, H, K and M). A and B are the best studied and both can induce apotosis when delivered to target cells. The role for granzyme H, K and M are principally unknown. We have studied the expression of granzymes in subsets of T-cells and NK-cells both in fresh circulating cells and cultured cells stimulated with IL-2, IL-15 and OKT3/antiCD28. Protein expression (biosynthesis/immunoprecipitation) and mRNA level (RT-PCR) was studied in parallel. This report focus on Granzyme A and K which are close relatives, both tryptases located 70 kb apart on chromosome 5. Granzyme A have the highest expression in NK-cells were granzyme K are barely detected. Granzyme K on the other hand have a particular high expression in CD8+ T cells. Stimulation with IL-2 or IL-15 give a strong upregulation of both enzymes in NK-cells. Stimulation of CD8+ T cells gives mainly upregulation of granzyme A. Fresh CD4+ T cells show very weak synthesis of granzymes but treatment with IL-2 or IL-15 give a clear upregulation of both granzyme A and K. T-cells (both CD4+ and CD8+) are highly responsive to stimulation with OKT3/anti CD28 producing granzyme B but not A and K. This rather discordant pattern of expression for granzyme A and K talks in favour of different modes of regulation and most likely different functions. A striking difference between granzyme A and K concern the propensity for granzyme A to be secreted into the culture medium after stimulation while this is not the case for granzyme K. The secreted form of granzyme A has a molecular weight slightly higher than the intracellular form indicating the presence of a proform. Secretion of granzymes to the extracellular medium is probably not only an in vitro phenomena since both granzyme A and B can be detected in serum from healthy individuals in picomolar concentrations. Moreover, significant elevation of granzyme levels in serum can bee seen in different diseases with activation of cellular immunity and in T- and NK-cells malignancies. We have previously reported that proforms of certain serine proteases including the five human granzymes have a downregulating activity on myeloid proliferation, a mechanism probably implicated in certain forms of neutropenia. We propose that secreted granzymes (especially A, B and H) constitute a part of T regulatory function working both locally at the site of inflammation and distantly via the circulation.