GM-CSF and TNFalpha modulate protein expression of human neutrophils visualized by fluorescence two-dimensional difference gel electrophoresis

Abstract

Increased serum levels of TNFalpha and GM-CSF are found in various chronic inflammatory diseases and these cytokines affect the function of circulating and tissue neutrophils. TNFalpha- and GM-CSF-induced protein expression profiles could, therefore, serve as biomarker for the action of these cytokines in vivo. We stimulated human peripheral neutrophils with TNFalpha and GM-CSF in vitro and analyzed changes in their proteome by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). We report the differential expression of 3 and 18 protein spots following TNFalpha and GM-CSF stimulation, respectively. Differences in protein expression induced by TNFalpha were limited and did not show discriminatory power in a principal component analysis, whereas the profile induced by GM-CSF did. TNFalpha- and GM-CSF-induced both de novo IL-1beta and sIL-1Ra protein expression as detected by Western blot analysis, which confirmed proper neutrophil activation by these cytokines in vitro. Mass spectrometry analysis of cytokine-regulated protein spots resulted in the identification of 8 proteins. Among the identified proteins, enolase 1 and annexin A1 might function as markers for peripheral neutrophil activation. In conclusion, a proteomic analysis of neutrophils by 2D-DIGE provides proof-of-principle that cytokine-induced protein profiles can serve as biomarkers for the action of individual cytokines in vivo.