Biosynthetic profiles of neutrophil serine proteases in a human bone marrow-derived cellular myeloid differentiation model

Abstract

Background and Objectives. Human leukocyte elastase, proteinase 3 and cathepsin G are neutrophil granule proteins belonging to the hematopoietic serine protease superfamily. In addition to their established roles in inflammation, they have recently been implicated as regulators of granulopoiesis and mediators of apoptosis. We set out to characterize the individual biosynthetic profiles of these proteins in a neutrophil differentiation model. Design and Methods. Bone marrow-derived CD34(+)CD38(+) hematopoietic progenitor cells from 21 healthy human volunteers were cultured in vitro in the presence of recombinant human granulocyte colony-stimulating factor (G-CSF). Biosynthetic radiolabeling was performed in cells from 13 subjects after various periods of differentiation induction. Following protein extraction, the proteins were specifically immunoprecipitated from cell lysates and media and run in gel electrophoresis. Biosynthetic profiles of azurophil granule proteins, in particular members of the neutrophil serine protease family, were examined during myeloid differentiation. Results. The onset of synthesis of myeloperoxidase, lysozyme, leukocyte elastase, and proteinase 3 occurred early after differentiation induction with G-CSF, while synthesis of cathepsin G, azurocidin, and bactericidal/permeability-increasing protein was detected somewhat later. Cathepsin G and proteinase 3 were retained intracellularly relatively efficiently, while leukocyte elastase and lysozyme were secreted to a greater extent. Cell morphology and positive immunocytochemistry for lactoferrin as well as flow cytometric analysis of selected surface antigens confirmed neutrophil-like maturation. Interpretation and Conclusions. We demonstrate that azurophil granule proteins, including proforms of human leukocyte elastase, proteinase 3 and cathepsin G, are constitutively secreted to various degrees during in vitro myeloid differentiation of human hematopoietic progenitor cells, in addition to being stored intracellularly in active forms. These findings suggest protein-specific sorting mechanisms and may have implications for the regulation of granulopoiesis