Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes, and, Expression and function of the transient receptor potential 2 (TRPM2) i

Part I. Severe congenital neutropenia (SCN) is a bone marrow failure disorder usually diagnosed in the first year of life and characterized by extremely low numbers of peripheral blood neutrophils, a myeloid maturation arrest in the bone marrow, and recurrent infections. Despite dramatic improvements in survival and quality of life with granulocyte colony-stimulating factor (G-CSF) therapy, patients with SCN have a life-long increased risk of developing leukemia. Mutations in the ELA2 gene encoding neutrophil elastase (NE) are present in most patients with SCN. However, the mechanisms by which these mutations cause neutropenia remain unknown. To investigate the effects of mutant NE expression on granulopoiesis, we used the HL-60 promyelocytic cell line retrovirally transduced with the G185R NE mutant that is associated with a severe SCN phenotype. We show that the mutant enzyme accelerates apoptosis of differentiating but not of proliferating cells. Using metabolic labeling, confocal immunofluorescence microscopy, and immunoblot analysis of subcellular fractions, we also demonstrate that the G185R mutant is abnormally processed and localizes predominantly to the nuclear and plasma membranes rather than to the cytoplasmic compartment observed with the wild-type (WT) enzyme. Expression of the G185R mutant appeared to alter the subcellular distribution and expression of adaptor protein 3 (AP3), which traffics proteins from the trans-Golgi apparatus to the endosome. These observations provide further insight into potential mechanisms by which NE mutations cause neutropenia and suggest that abnormal protein trafficking and accelerated apoptosis of differentiating myeloid cells contribute to the severe SCN phenotype resulting from the G185R mutation.; In the subset of patients with SCN transforming to acute myeloid leukemia (AML), mutations that truncate the cytoplasmic tail of the G-CSF receptor (G-CSFR) have been detected. We identified a novel mutation in the extracellular portion of the G-CSFR within the WSXWS motif in a patient with SCN without AML who was refractory to G-CSF treatment. The mutation affected a single allele and introduced a premature stop codon that deletes the distal extracellular region and the entire transmembrane and cytoplasmic portions of the G-CSFR. Subsequent reports have demonstrated that this mutant decreases the surface expression of the wild-type receptor and thereby inhibits proliferative signaling by the wild-type G-CSFR, suggesting a common mechanism underlying G-CSF refractoriness in SCN patients.; NE is a serine protease stored in the primary granules of neutrophils that proteolytically cleaves multiple cytokines and cell surface proteins on release from activated neutrophils. Recent reports of mutations in the gene encoding this enzyme in some patients with neutropenic syndromes prompted us to investigate whether G-CSF or its receptor G-CSFR were also substrates for NE. Previous research in the laboratory demonstrated that NE enzymatically degrades both G-CSF and the G-CSFR, strongly arguing in favor of a catalytic mechanism. We show that NE abrogates proliferative signals generated by the G-CSFR in myeloid progenitor cells, as indicated by the decreased numbers and size of CFU-GM arising from marrow progenitors pre-treated with NE. These findings provide additional insights into mechanisms by which G-CSF/G-CSFR interactions may be modulated.; Collectively, our data indicate that the G185R NE mutant that is associated with the most severe phenotype in SCN is missorted to the plasma membrane and that the normal or WT NE can degrade and inactivate both G-CSF and the G-CSFR. This suggests that aberrant interaction of NE mutants with membrane proteins critical for the survival of maturating myeloid cells is the pathophysiologic mechanism leading to neutropenia.; Part II. Dendritic cells (DCs) orchestrate immunity by amplifying innate and initiating adaptive immune responses. DCs traffic in respons...