RESUMEN
BACKGROUND: The granulocyte colony-stimulating factor receptor (G-CSFR) plays a critical role in maintaining homeostatic levels of circulating neutrophils (PMN). The mechanisms modulating G-CSFR surface expression to prevent chronic neutrophilia are poorly understood. Here, we report that neutrophil elastase (NE) proteolytically cleaves the G-CSFR on human PMN and blocks G-CSFR-mediated granulopoiesis in vitro. METHODS: Human peripheral blood PMN isolated from healthy donors were incubated with NE. Expression of the G-CSFR was analyzed by flow cytometry and western blot analyses. Detection of G-CSFR cleavage products from the culture supernatants was also performed. Human bone marrow mononuclear cells were also cultured in the presence or absence of NE to determine its effects on the proliferation of granulocyte-macrophage colony forming units (CFU-GM). RESULTS: Treatment of PMN with NE induced a time-dependent decrease in G-CSFR expression that correlated with its degradation and the appearance of proteolytic cleavage fragments in conditioned media. Immunoblot analysis confirmed the G-CSFR was cleaved at its amino-terminus. Treatment of progenitor cells with NE prior to culture inhibited the growth of granulocyte-macrophage colony forming units. CONCLUSIONS: These findings indicate that in addition to transcriptional controls and ligand-induced internalization, direct proteolytic cleavage of the G-CSFR by NE also downregulates G-CSFR expression and inhibits G-CSFR-mediated granulopoiesis in vitro. Our results suggest that NE negatively regulates granulopoiesis through a novel negative feedback loop.
RESUMEN
Neutrophil elastase (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 granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) are also substrates for NE. To further address this, we examined the effect of NE on G-CSF and the G-CSFR both in solution and on intact cells. Incubation of recombinant G-CSF or a G-CSFR form corresponding to its extracellular domain with purified NE resulted in rapid proteolytic cleavage of both proteins. Addition of NE to tissue culture medium or pretreatment of G-CSF with NE before its addition to media suppressed the growth of G-CSF-responsive cells. NE also cleaved the G-CSFR on the surface of intact cells resulting in a time-dependent reduction in cell surface expression of the G-CSFR. Notably, decreased G-CSFR surface expression resulting from treatment of cells with NE was also associated with a reduction in cell viability and proliferation in response to G-CSF. These results are the first to demonstrate that G-CSF and G-CSFR are proteolytically cleaved by NE and that NE-induced degradation of these proteins correlates with a reduction in the biologic activity of the cytokine and a decrease in the signaling function of the receptor because of decreased G-CSFR surface expression. These findings provide additional insights into mechanisms by which G-CSF/G-CSFR interactions may be modulated.