Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not granulocyte colony-stimulating factor (G-CSF) induces plasma membrane expression of proteinase 3 (PR3) on neutrophils in vitro

Abstract

The theoretical risk of triggering vasculitis resulting from administration of G-CSF and GM-CSF to patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), such as Wegener's granulomatosis (WG), who develop agranulocytosis due to cytotoxic therapy, is unknown. Since there is strong evidence that activation of polymorphonuclear neutrophils (PMN) induced by binding of ANCA to PR3 or myeloperoxidase (MPO) expressed on their plasma membrane is involved in the pathogenesis of systemic vasculitides (SV), we studied the surface expression of PR3 and MPO on PMN from healthy donors in response to G-CSF and GM-CSF in vitro by flow cytometric analysis. Increasing doses of G-CSF did not alter PR3 expression on either untreated or tumour necrosis factor-alpha (TNF-alpha)-primed donor PMN significantly. In contrast, GM-CSF significantly increased PR3 membrane expression on both intact PMN and neutrophils primed with TNF-alpha. MPO expression was not significantly altered by either G-CSF or GM-CSF. In summary, these data demonstrate that GM-CSF, but not G-CSF, induces plasma membrane expression of PR3 on PMN in vitro. Since in AAV accessibility of the antigen (PR3 or MPO) to the antibody (ANCA) on the plasma membrane of PMN is thought to be essential for neutrophil activation by ANCA, the results of the present study suggest that administration of GM-CSF to patients with WG with neutropenia implies a definite theoretical risk of deterioration of vasculitis via this mechanism.

Fig. 1

Flow cytometric analysis of the membrane expression of PR3 on polymorphonuclear neutrophils (PMN) derived from healthy donors. Cells were incubated in the presence of increasing doses of tumour necrosis factor-alpha (TNF-α), G-CSF and GM-CSF as described in Materials and Methods. (a) Unstimulated intact PMN. (b) Primed PMN, re-incubated with TNF-α (1 ng/ml) for 10 min. Antigen expression is given as the percentage of total PMN with fluorescence intensities above the threshold fluorescence determined as the upper level of fluorescence intensity of PMN treated with the isotype control antibodies. Results represent means ±s.e.m. of nine individual experiments, by using cells from nine different donors. Statistically significant difference of *P < 0·05 or **P < 0·005 versus control PMN (unstimulated). Statistically significant difference of †P < 0·05 or ††P < 0·001 versus PMN primed with TNF-α (1 ng/ml for 10 min) alone.

Fig. 2

Flow cytometric analysis of the membrane expression of PR3 and myeloperoxidase (MPO) on polymorphonuclear neutrophils (PMN) derived from three healthy donors. Both intact and tumour necrosis factor-alpha (TNF-α)-primed (1 ng/ml for 10 min) PMN were incubated in the presence of increasing doses (0·03–4 ng/ml, respectively) of G-CSF and GM-CSF as described in Materials and Methods. Antigen expression is given as the percentage of total PMN (a,c) with fluorescence intensities above the threshold fluorescence determined as the upper level of fluorescence intensity of PMN treated with the isotype control antibodies, and the median fluorescence intensity (MFI) (b,d). The solid horizontal line represents the basal level of PR3 or MPO expression without any stimulation. Results represent means ±s.e.m. of three individual experiments, by using cells from three different donors.