Increased Unfolded Protein Response in Hematopoietic Cells from Cyclic Hematopoietic Dogs.

Canine cyclic hematopoiesis, a model of human cyclic neutropenia/congenital neutropenia (CN), is caused by a mutation in the AP3b1 gene. Human CN is caused by mutations in the Ela2 gene. The exact mechanism(s) whereby mutations in the Ela2 and AP3b1 cause CN or CH is not well understood. Elastase is directed to the primary granules by AP3 in myeloid progenitor cells, suggesting abnormal trafficking of elastase as a cause for CN/CH. Discontinuous percoll gradients on PMN cells from normal and CH dogs were performed. Fractions corresponding to the cytosol, plasma membrane, nuclei, primary, secondary and tertiary granules were collected and assayed for elastase and myeloperoxidase (MPO) enzyme activity and protein levels by Western blot using canine specific antibodies. Percoll density gradient fractionation results indicated that MPO, a primary granule protein that is not an AP3 cargo protein, is present in the primary granules in approximately equivalent amounts in both normal and CH dog PMN’s. Elastase was also localized in the primary granule fraction from both normal and CH dog PMN, but with a lower amount in CH dogs. Elastase was not present in the plasma membrane fraction in either CH or normal dog PMN’s. Quantitatively the CH dog primary granules had 10–20% of normal dog primary granule elastase. These results suggest PMN’s from CH dogs correctly sort elastase to the primary granules but at a lower level compared to PMNs from dogs without AP3b1 defect. Glucose-regulated protein (GRP78/BiP) is an indicator of ER stress. ER stress leads to activition of the unfolded protein response (UPR) and is cytoprotective. However, prolonged UPR leads to apoptosis. Analysis of GRP78 expression in PMN’s and bone marrow cell cultures from normal and CH dogs stimulated with SCF and G-CSF demonstrated 3–4 fold increase of GRP78 in CH cells compared to normal dog cells. These results indicate mistrafficking or accumulation of misfolded elastase induces the UPR in myeloid precursor cells and disrupts normal PMN production in CH dogs. These results are consistent with in vitro studies in which over-expression of mutant but not normal human elastase induces the UPR in myeloid cells and cell death. These results suggest that induction of the UPR is a common event, in myeloid progenitor cells from patients and animal models with either Ela2 or AP3b1 mutations, which ultimately results in congenital and/or cyclic neutropenia.