Novel findings on cellular trafficking and targeting for granule storage of neutrophil elastase, a multifunctional effector molecule of innate immunity

Neutrophil elastase (NE) has important roles in innate immunity, killing pathogens and controlling the immune response; but how NE is targeted to developing granules is not understood. Therefore, the aim of this thesis was to investigate the sorting of NE.

Transfection experiments in a leukemic cell line, which were confirmed in normal hematopoietic cells, showed that a population of proNE was targeted to the plasma membrane and endocytosed. This targeting required an intact carboxy-terminal propeptide. Furthermore, modified proNE was not endocytosed, indicating structural requirements for endocytosis.

An association was demonstrated between the tetraspanin CD63 and proNE upon coexpression in COS cells. Furthermore, depletion of CD63 in a promyelocytic cell line (achieved by RNA interference or a CD63 mutant) caused reduced processing of proNE into mature NE and reduced constitutive secretion of proNE. We therefore propose that CD63 may be a transmembrane linker that facilitates granule targeting of proNE.

Results from a monocytic cell line indicated that the sorting of proNE was a multistage process including trafficking to the cell surface, endocytosis through coated vesicles and possibly lipid rafts, and possible conversion to mature NE in late endosomes. The inhibition of proNE’s activation into mature NE was accompanied by the accumulation of proNE, suggesting a requirement for activation before granule targeting.

This research provided new perspectives on the cellular trafficking of NE. The thesis proposes that granule sorting of proNE is facilitated by a tetraspanin protein serving as a transmembrane linker and transporter. The hypothesis needs further testing in primary cells to acquire additional evidence of the interactions involved.