Cellular Model of Severe Congenital Neutropenia Reveals the Molecular Mechanism of Mutant Elastase-Mediated Agranulocytosis.

Severe congenital neutropenia (SCN) or Kostmann’s syndrome defines an inheritable hematopoietic disorder of an impaired neutrophil production in the bone marrow due to a “maturation arrest” at promyelocytic stage of differentiation in the marrow. SCN patients have recurring severe infections and may evolve to develop leukemia. We reported accelerated apoptosis and cell cycle arrest of bone marrow-derived myeloid progenitor cells in SCN patients with acquired, autosomal dominant, as well as autosomal recessive inheritance. We also reported that approximately 80% of these patients have heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease normally targeted to azurophil granules. Neither knock-in of mutant elastase nor a knock-out of normal neutrophil elastase caused severe neutropenia in mice. Therefore, we established a cellular model of SCN with tetracycline-regulated expression of mutant NE in human promyelocytic tet-off HL-60 cells. Induced expression of mutant elastase in these cells led to a reduced mitochondrial membrane potential and subsequent caspase-independent apoptosis and cell cycle arrest as determined by flow cytometry. Block of differentiation of DMSO-treated cells was also observed in tet-off HL-60 cells with induced expression of mutant NE, similar to that in SCN patients. This cellular model of SCN very closely recapitulates the human severe neutropenia phenotype.

To elucidate the molecular mechanisms of mutant NE-mediated neutropenia, we employed various proteomics methods, including ICAT analysis, two-dimensional gel electrophoresis, immunoprecipitation, LC-MS/MS and identified a number of proteins with altered level of expression including apoptosis and cell cycle regulatory gene products, transcription factor and cytoskeleton proteins. Changes in the protein expression profiles revealed the abnormal molecular events underlying the impaired cell survival and cell cycle arrest and suggested additional pathways implicated in pathogenesis of SCN. Immunostaining of control cells with phalloidin revealed a weak F-actin polymerization in cell periphery, which helps to maintain cell shape flexibility, and intracellular long F-actin filaments supporting the normal cytoskeleton. In contrast, cells expressing mutant NE, exhibited an increased polymerization of F-actin in the periphery, with apparent increase in lamellipodia that may contribute to an increased phosphatydilserine exposure in apoptotic cells expressing mutant elastase. The hyperpolymerization of F-actin, which appears to stem from an elevated level of RhoA, a member of the RAS-superfamily of GTPases, makes the cells more rigid and less flexible thus contributing to impaired cell cycle progression in cells expressing mutant NE. Impaired cell survival in these cells is associated with a significant reduction in the level of phosphorylated PKB/Akt, which in turn appears to be a consequence of a decreased level of PI3kinase in response to expression of mutant NE. Interestingly, G-CSF treatment of mutant NE expressing cells resulted in restoration of the level of phospho-Akt to near normal level comparable to that in control cells with normal NE expression. These data may explain the anti-apoptotic effect of G-CSF in SCN. Thus, these data demonstrate that the cellular model of SCN based on tet-off HL-60/mutNE cells with inducible expression of mutant elastase is useful to unravel the cellular and molecular mechanisms of mutant NE-mediated severe neutropenia.