Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia

Abstract

Cyclic neutropenia (CN) is a congenital hematopoietic disorder characterized by remarkably regular oscillations of blood neutrophils from near normal to extremely low levels at 21-day intervals. Recurring episodes of severe neutropenia lead to repetitive and sometimes life-threatening infections. To investigate the cellular mechanism of CN, the ultrastructure and the proliferative and survival characteristics of bone marrow-derived CD34(+) early progenitors, CD33(+)/CD34(-) myeloid progenitors, and CD15(+) neutrophil precursors from CN patients and healthy volunteers were studied. The ultrastructural studies showed profound apoptotic features in bone marrow progenitor cells in CN. Colony-forming assays demonstrated a 75% decrease in the number of early myeloid-committed colonies compared with controls. Long-term culture-initiating cell assays demonstrated a 6-fold increase in production of primitive progenitor cells in CN. To determine whether accelerated apoptosis might account for the underproduction of myeloid progenitors, the hematopoietic subpopulations were labeled with fluorescein isothiocyanate-annexin V and analyzed by flow cytometry. Short-term culture of CN cells resulted in apoptosis of approximately 65% of CD34(+) cells, 80% of CD33(+)/CD34(-) cells, and more than 70% of CD15(+) cells, as compared with 20%, 7%, and 15% apoptosis in respective control subpopulations. Evidence of accelerated apoptosis of bone marrow progenitor cells was observed in all 8 patients participating in the study, regardless of the stage in the CN cycle in which bone marrow aspirations were obtained. Granulocyte colony-stimulating factor therapy of CN patients significantly improved survival of bone marrow progenitor cells. These data indicate that ineffective production of neutrophils is due to accelerated apoptosis of bone marrow myeloid progenitor cells in CN.

Observations on the Pathophysiology and Mechanisms for Cyclic Neutropenia

“…This marrow abnormality is due to defective neutrophil formation with interruption of cell production at the promyelocyte-myelocyte stage, the stage in neutrophil development when the enzyme neutrophil elastase is normally synthesized and packaged in the neutrophil granules [18]. Several recent studies now link the finding of "maturation arrest" and the mutations in ELA2 gene.…”

“…Examination of marrow samples by electron microscopy show cytoplasmic blebbing and autophagy of debris of degenerating neutrophils, features that are not observed in normal marrow samples [18]. Annexin 5 staining and fluorescence activated cell sorting (FACS analysis) show that patients have degenerating cells with exposure of phospholipids on the surface of cells which are usually maintained on the cells' internal surfaces [18]. Transfection studies utilizing several cell lines, as well as normal human CD34 cells, showed that expression of mutant ELA2 leads to the unfolded protein response with up-regulation of the master endoplasmic reticulum chaperone referred to as BiP, a molecular correlate of accelerated apoptosis.…”

“…These studies all point to accelerated apoptosis of myeloid cells at the promyelocyte to myelocyte transition being the primary cellular event leading to the interruption of cell production in cyclic neutropenia. Examination of marrow samples by electron microscopy show cytoplasmic blebbing and autophagy of debris of degenerating neutrophils, features that are not observed in normal marrow samples [18]. Annexin 5 staining and fluorescence activated cell sorting (FACS analysis) show that patients have degenerating cells with exposure of phospholipids on the surface of cells which are usually maintained on the cells' internal surfaces [18].…”

“…It is significant that the break in cell development is at the pro-myelocyte to myelocyte transition, the stage at which the enzyme neutrophil elastase is normally synthesized and packaged in the primary granules. At the cellular and molecular level, mutant neutrophil elastase triggers accelerated apoptosis, either as a direct effect of the mutant protein or secondary to the unfolded protein response with induction of caspase-independent apoptosis [18,21].…”

Characterization of Effector Cells with Anti‐Candida Activity Obtained from Murine Bone Marrow Cells Cultured in the Presence of rhG‐CSF: Comparison between Normal and CY‐Treated Mice

“…Assay of growth inhibition of C. albicans by BMC was performed on the basis of a crystal violet (CV) staining method, as described previously (13). BMC (4ϫ10 4 or 8ϫ10 4 cells) were seeded in the wells of a 96-well flatbottomed microplate with 100 µl of complete medium. After pre-incubation with a designated concentration of rhG-CSF at 37 C for 24 hr, C. albicans suspension (5ϫ10 2 cells) in 50 µl of complete medium was added to each well.…”