Involvement of Lipid Rafts in Impaired fMLP-Stimulated ROS Production of GM-CSF-Primed Neutrophils from Patients with Myelodysplasia.

Abstract

Neutrophils from MDS patients are generally dysfunctional, resulting in increased susceptibility to bacterial infections. An important bactericidal activity of neutrophils is their production of reactive oxygen species (ROS) upon stimulation with the bacterial product fMLP. ROS production by fMLP can be enhanced by pre-treatment of neutrophils with pro-inflammatory cytokines such as GM-CSF. In MDS patients, this GM-CSF priming of ROS production is severely impaired. Furthermore, activation of the extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) signal transduction pathways, involved in ROS production, are impaired in MDS (Fuhler et al, Blood , 101, 2003). In the current study we investigated the involvement of lipid rafts in neutrophil ROS production, and show that treatment of healthy neutrophils with the lipid-raft disrupting agent methyl-b-cyclodextrin (CD) abrogates fMLP-induced ROS production in unprimed cells (mean fluorescence intensity [MFI] of 16±2 vs 4±0.3, n=5, P=0.03) and GM-CSF-primed neutrophils (MFI 50±14 vs 9±1, P=0.04). We further demonstrate that activation of ERK and PKB by fMLP was abolished by CD treatment of unprimed or GM-CSF-primed neutrophils. In contrast, GM-CSF-stimulated phosphorylation of STAT5 was not affected by CD pre-treatment of healthy neutrophils. These results indicate the importance of lipid rafts in fMLP-stimulated ROS production and in activation of signal transduction pathways involved in this process. The expression of the ganglioside GM1 at the plasma membrane of cells has been described to be a marker for the presence of lipid rafts. We analyzed the expression of GM1 on healthy neutrophils by FACS analysis and show that stimulation of cells with fMLP increased the expression of GM1 at the plasma membrane (MFI 102±20 vs 161±38, n=6, P=0.03). Furthermore, pretreatment of neutrophils with GM-CSF further increased the fMLP-induced GM1 expression (MFI 161 ±38 vs 193±36, P=0.03). We next assessed the expression of GM1 at the plasma membrane, in conjunction with ROS production, in 8 MDS patients compared to healthy donors. As described previously, ROS production in response to fMLP was normal in unprimed MDS neutrophils. In contrast, in 6 patients, fMLP-induced ROS production in GM-CSF primed neutrophils was significantly impaired when compared to their healthy controls (mean MFI 128±54 vs 191±72, P=0.03). When investigating the GM1 expression in these patients, we found that although fMLP-triggered GM1 expression was normal in unprimed neutrophils from MDS patients, fMLP-induced GM1 expression on GM-CSF-primed cells was significantly lower on MDS neutrophils compared to their healthy counterpart (mean MFI 174±76 vs 214±78, P=0.03). GM-CSF primed neutrophils from one patient with normal ROS production also exhibited normal GM1 expression. Taken together, these data indicate an involvement of lipid rafts in ROS production, and suggest that in MDS neutrophils, an impaired lipid raft formation in GM-CSF primed cells correlates with impaired fMLP-induced ROS production.