Gwenny M. Fuhler

Impaired interleukin-8- and GROα-induced phosphorylation of extracellular signal-regulated kinase result in decreased migration of neutrophils from patients with myelodysplasia

Patients with myelodysplasia suffer from recurrent bacterial infections as a result of differentiation defects of the myeloid lineage and a disturbed functioning of neutrophilic granulocytes. Important physiological activators of neutrophils are the cytokines interleukin‐8/CXC chemokine ligand 8 (IL‐8/CXCL8), which activates CXC chemokine receptor 1 and 2 (CXCR1 and CXCR2), and growth‐related oncogene (GROα)/CXCL1, which stimulates only CXCR2. In this study, we show that migration toward IL‐8/GROα gradients is decreased in myelodysplastic syndrome (MDS) neutrophils compared with healthy donors. We investigated the signal transduction pathways involved in IL‐8/GROα‐induced migration and showed that specific inhibitors for extracellular signal‐regulated kinase (ERK)1/2 and phosphatidylinositol‐3 kinase (PI‐3K) abrogated neutrophil migration toward IL‐8/GROα. In accordance with these results, we subsequently showed that IL‐8/GROα‐stimulated activation of ERK1/2 was substantially diminished in MDS neutrophils. Activation of the PI‐3K downstream target protein kinase B/Akt was disturbed in MDS neutrophils when cells were activated with IL‐8 but normal upon GROα stimulation. IL‐8 stimulation resulted in higher migratory behavior and ERK1/2 activation than GROα stimulation, suggesting a greater importance of CXCR1. We then investigated IL‐8‐induced activation of the small GTPase Rac implicated in ERK1/2‐dependent migration and found that it was less efficient in neutrophils from MDS patients compared with healthy donors. In contrast, IL‐8 triggered a normal activation of the GTPases Ras and Ral, indicating that the observed defects were not a result of a general disturbance in CXCR1/2 signaling. In conclusion, our results demonstrate a disturbed CXCR1‐ and CXCR2‐induced neutrophil chemotaxis in MDS patients, which might be the consequence of decreased Rac‐ERK1/2 and PI‐3K activation within these cells.

The reduced GM-CSF priming of ROS production in granulocytes from patients with myelodysplasia is associated with an impaired lipid raft formation

Patients with myelodysplasia (MDS) show an impaired reactive oxygen species (ROS) production in response to fMLP stimulation of GM‐CSF‐primed neutrophils. In this study, we investigated the involvement of lipid rafts in this process and showed that treatment of neutrophils with the lipid raft‐disrupting agent methyl‐β‐cyclodextrin abrogates fMLP‐induced ROS production and activation of ERK1/2 and protein kinase B/Akt, two signal transduction pathways involved in ROS production in unprimed and GM‐CSF‐primed neutrophils. We subsequently showed that there was a decreased presence of Lyn, gp91phox, and p22phox in lipid raft fractions from neutrophils of MDS. Furthermore, the plasma membrane expression of the lipid raft marker GM1, which increases upon stimulation of GM‐CSF‐primed cells with fMLP, was reduced significantly in MDS patients. By electron microscopy, we showed that the fMLP‐induced increase in GM1 expression in GM‐CSF‐primed cells was a result of de novo synthesis, which was less efficient in MDS neutrophils. Taken together, these data indicate an involvement of lipid rafts in activation of signal transduction pathways leading to ROS production and show that in MDS neutrophils, an impaired lipid raft formation in GM‐CSF‐primed cells results in an impaired ROS production.

Distinct roles of the mTOR components Rictor and Raptor in MO7e megakaryocytic cells

Objective:  During megakaryopoiesis, hematopoietic progenitor cells in the bone marrow proliferate and ultimately differentiate in mature megakaryocytes (MK). We and others have recently described a role for the mammalian target of Rapamycin (mTOR) in proliferation and differentiation of MK cells. Two non‐redundant complexes of mTOR have been described; mTORC1 containing rapamycin‐associated TOR protein (Raptor) and mTORC2 containing Rapamycin‐insensitive companion of mTOR (Rictor). The individual roles of these complexes in MK development have so far not been elucidated, and were investigated in this study.

Insulin-like growth factor binding protein-1 activates integrin-mediated intracellular signaling and migration in oligodendrocytes

J. Neurochem. (2010) 113, 1319–1330.

Disturbed granulocyte macrophage-colony stimulating factor priming of phosphatidylinositol 3,4,5-trisphosphate accumulation and Rac activation in fMLP-stimulated neutrophils from patients with myelodysplasia

The production of reactive oxygen species (ROS) by human neutrophils is imperative for their bactericidal activity. Proinflammatory agents such as granulocyte macrophage‐colony stimulating factor (GM‐CSF) can prime ROS production in response to chemoattractants such as N‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine (fMLP). In neutrophils from patients suffering from Myelodysplastic syndromes (MDS), a clonal, hematological disorder characterized by recurrent bacterial infections, this GM‐CSF priming is severely impaired. In this study, we set out to further delineate the defects in neutrophils from MDS patients. We examined the effect of GM‐CSF priming on fMLP‐triggered activation of Rac, a small GTPase implicated in neutrophil ROS production. In contrast to healthy neutrophils, activation of Rac in response to fMLP was not enhanced by GM‐CSF pretreatment in MDS neutrophils. Furthermore, activation of Rac was attenuated by pretreatment of neutrophils with the phosphatidylinositol 3‐kinase (PI‐3K) inhibitor LY294002. Unlike healthy neutrophils, fMLP‐induced accumulation of the PI‐3K lipid product PI(3,4,5)trisphosphate was not increased by GM‐CSF pretreatment in MDS neutrophils. The disturbed Rac and PI‐3K activation observed in MDS neutrophils did not appear to reflect a general GM‐CSF or fMLP receptor‐signaling defect, as fMLP‐triggered Ras activation could be primed by GM‐CSF in MDS and healthy neutrophils. Moreover, fMLP‐induced activation of the GTPase Ral was also normal in neutrophils from MDS patients. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI‐3K–Rac signaling pathway, located at the level of PI‐3K, results in a decreased GM‐CSF priming of ROS production.

Reduced expression of flavocytochrome b558, a component of the NADPH oxidase complex, in neutrophils from patients with myelodysplasia

OBJECTIVE Patients with myelodysplasia (MDS) show a disturbed production of ROS in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils. Because generation of ROS is mediated by the NADPH oxidase complex, a component of which is flavocytochrome b558, we investigated whether the expression of flavocytochrome b558 in neutrophils from MDS patients is affected. MATERIAL AND METHODS Neutrophils were stimulated with fMLP and GM-CSF, and plasma membrane expression of flavocytochrome b558 and specific granule markers were assessed by fluorescence-activated cell sorting analysis. Protein levels of the flavocytochrome b558 subunits gp91phox and p22phox in whole neutrophil lysates were detected by Western blotting. RESULTS Stimulation of neutrophils with GM-CSF and fMLP increased the flavocytochrome b558 plasma membrane expression. The fMLP-induced translocation of flavocytochrome b558 was reduced in neutrophils from MDS patients (140%+/-9% vs 180%+/-13%, p<0.05). Analysis of cell surface expression of markers of flavocytochrome b558 containing granules (CD35 and CD66b) indicated that exocytosis of these granules in response to fMLP stimulation was not affected in MDS patients. Western blot analysis demonstrated a decreased protein expression level of the flavocytochrome b558 subunits gp91phox and p22phox in neutrophils from MDS patients. CONCLUSION Our results indicate both a lower basal protein level and a disturbed fMLP-induced increase in plasma membrane expression of flavocytochrome b558 in neutrophils from MDS patients, which together might play a role in decreased ROS production.