Research

Neutrophil elastase downmodulates native G-CSFR expression and granulocyte-macrophage colony formation

Melissa G Piper^1,2,8 , Pam R Massullo³ , Megan Loveland⁴ , Lawrence J Druhan^2,5 , Tamila L Kindwall-Keller⁶ , Jing Ai^2,4 , Alexander Copelan⁷ and Belinda R Avalos^2,4,8

¹  Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, The Ohio State University, Columbus, OH, 43210, USA

²  The Davis Heart and Lung Research Institute, The Ohio State University, Columbus, 43210, OH, USA

³  Northeastern Ohio University College of Pharmacy, Rootstown, OH, 44272, USA

⁴  The Division of Hematology and Oncology, The Ohio State University, Columbus, OH, 43210, USA

⁵  Division of Cardiovascular Medicine, The Ohio State University, Columbus, OH, 43210, USA

⁶  Department of Medicine, Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH, 44106, USA

⁷  Loyola University Chicago Stritch School of Medicine, Chicago, IL, 60660, USA

⁸  Department of Internal Medicine, The Ohio State University, Columbus, OH, 43210, USA

author email corresponding author email

Journal of Inflammation 2010, 7:5doi:10.1186/1476-9255-7-5

Published:	21 January 2010

Abstract

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.