Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells.

Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating "pro(C)"-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

Hematopoietic secretory granules as vehicles for the local delivery of cytokines and soluble cytokine receptors at sites of inflammation.

Cytokines play an important role in the regulation of homeostasis and inflammation. A de-regulated cytokine function can subsequently promote chronic inflammation. This is supported by clinical evidence showing the beneficial effect of inhibiting TNF-alpha through injection of antibodies and soluble receptor in disorders such as rheumatoid arthritis and Crohn's disease. Systemic anti-TNF-alpha therapy however is associated with infectious complications. We therefore suggest a concept for the local deposition of therapeutically active agents into areas of inflammation or malignancy, based on the use of hematopoietic storage and secretory granules as delivery vehicles. Hematopoietic cells are induced to express the therapeutically active protein and to store it in the secretory lysosomes. The cells migrate into a tumour or site of inflammation, where the cells become activated and release the contents of their secretory lysosomes resulting in the local delivery of the therapeutically active protein. In support of this concept, gene transfer and granule loading can be achieved using the soluble TNF-alpha receptor (sTNFR1) after cDNA expression in hematopoietic cell lines. Endoplasmic reticulum (ER)-export can be facilitated by the addition of a transmembrane domain, and constitutive secretion can be prevented by incorporating a cytosol-sorting signal resulting in secretory lysosome targeting. The sTNFR1 is released from the transmembrane domain by proteolytic cleavage and finally, regulated sTNFR1-secretion can be triggered by a calcium signal. In vivo investigations are currently determining the feasibility of local protein delivery at sites of inflammation.

Sorting of Von Willebrand factor to lysosome-related granules of haematopoietic cells.

The aim of this work was to investigate sorting mechanisms of von Willebrand factor (VWF) when expressed in haematopoietic cells. The processing and sorting of both the wild-type VWF and a multimerization defective propeptide-mutant (VWF(m)) were investigated after expression in the 32D cell line. Normal proteolytic processing was observed for both proteins, however the processing of VWF(m) was much slower and a large portion was unprocessed. Results from subcellular fractionation and immunoelectron microscopy confirmed that a part of VWF, but not VWF(m), was targeted to lysosome-related granules. Partial constitutive secretion was also observed for all forms of VWF and VWF(m). Inhibition of acidification by chloroquine blocked VWF processing but allowed unprocessed pro-VWF targeting to dense organelles. In conclusion, our observations are consistent with VWF multimerization being of importance in cellular retention and targeting to lysosome-related organelles in haematopoietic cells, suggesting a role of protein aggregation for sorting in these cells.

Artificially controlled aggregation of proteins and targeting in hematopoietic cells.

The targeting mechanisms for granule proteins in hematopoietic cells are largely unknown. Aggregation is believed to be important for protein sorting-for-entry and sorting-by-retention in endocrine and neuroendocrine cells. We asked whether artificially induced multimerization/aggregation of chimeric proteins could affect their sorting in hematopoietic cells. A system was used that permits ligand-controlled intracellular oligomerization of hybrid proteins containing the FK506-binding protein (FKBP). The hybrid proteins ELA-(FKBP)3 with neutrophil elastase (ELA) and (FKBP*)4-FCS-hGH with a furin cleavage site (FCS) and human growth hormone (hGH) were expressed in the myeloblastic 32D and the rat basophilic leukemia (RBL-1) hematopoietic cell lines. ELA alone is normally targeted to secretory lysosomes. However, the hybrid proteins and ligand-induced aggregates of them were constitutively secreted and not targeted. The hGH that was released at the FCS in (FKBP*)4-FCS-hGH was also constitutively secreted. We conclude that protein multimerization/aggregation per se is not enough to facilitate sorting-for-entry to secretory lysosomes in hematopoietic cells and that improperly folded proteins may be eliminated from sorting by constitutive secretion.

Sorting of soluble TNF-receptor for granule storage in hematopoietic cells as a principle for targeting of selected proteins to inflamed sites.

Hematopoietic cells have secretory lysosomes that degranulate at the inflammatory site upon stimulation. We asked whether one could target exogenous proteins with a therapeutic potential to secretory lysosomes in hematopoietic cells. For this purpose, we expressed a soluble tumor necrosis factor (TNF) receptor form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting to secretory lysosomes, both endoplasmic reticulum (ER) retention and constitutive secretion have to be prevented. ER export was facilitated by addition of a transmembrane (tm) sequence, and constitutive secretion was overcome by incorporating a cytosolic sorting signal (Y) from CD63. This signal directed the resulting sTNFR1-tm-Y to secretory lysosomes. Confirmation of these results was provided by biosynthetic radiolabeling, subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy. The tm-Y fragment was cleaved by proteolysis, resulting in generation of the membrane-free sTNFR1 in secretory lysosomes. Our results suggest a potential for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.