Neutrophil Elastase Modulates Granulopoiesis through the Interaction of G-CSF and G-CSF Receptor.

Primary granule proteases provide negative feedback on granulopoiesis. Recent study showed that neutrophil elastase potentially inhibits granulopoiesis by enzymatic digestion of G-CSF. We studied the effects of neutrophil elastase on granulopoiesis in the interaction of G-CSF and G-CSF receptor (G-CSFR). Primitive myeloid cells of the bone marrow in normal subjects were purified based on the expression of CD34 and G-CSFR by FACS Vantage. The cells were incubated with or without human neutrophil elastase for 2 hours in serum-deprived media. Cells were washed once and then cultured in various serum-deprived conditions. The proliferation and colony formation of CD34⁺/G-CSFR⁺ cells in the presence of G-CSF were significantly reduced by the pretreatment of elastase. Furthermore, those of CD34⁺/G-CSFR⁺ cells in response to hematopoietic factors without G-CSF were similarly inhibited by elastase. On the other hand, the responses of CD34⁺/G-CSFR⁻ cells were not affected by the treatment of elastase in the stimulation of any hematopoietic factors. When CD34⁺ cells were incubated with or without elastase, further G-CSFR expression was analysed by flow cytometry. The treatment of elastase did not affect the expression of surface expression of G-CSFR on CD34⁺ cells. Next, the binding of G-CSF to G-CSFR was examined by flow cytometry using antibody to G-CSF. The mean channel of fluorescence of G-CSF binding to G-CSFR-positive cells was reduced by the treatment of elastase, suggesting the loss of the binding of G-CSF to G-CSFR. When CD34⁺/G-CSFR⁺ cells were treated with elastase, the observation by confocal immunofluorescence microscopy revealed neither accumulation of elastase on the surface of cell membrane nor in intracellular compartment. No evidences that cells treated with elastase directly cleave the G-CSF leading to inactivation of G-CSF. Collectively, these results suggest that G-CSFR-positive cells are selectively affected by neutrophil elastase, and that neutrophil elastase as negative regulator may play a role in modulating granulopoiesis through G-CSF-G-CSFR interaction.