Neuropeptide S increases fMLP-stimulated CD11b levels in eosinophils from patients with elevated IgE

P Ilmarinen-Salo1, E Moilanen1, V Pulkkinen2, S Saarelainen3, T Laitinen4, J Kere2,5, H Kankaanranta1,6. 1The Immunopharmacology Research Group, University of Tampere School of Medicine and Tampere University Hospital, Tampere, Finland, 2Department of Medical Genetics, University of Helsinki and Folkhälsan Institute of Genetics, Helsinki, Finland, 3Department of Respiratory Medicine, Tampere University Hospital, Tampere, Finland, 4Department of Pulmonary Diseases and Clinical Allergology, Turku University Hospital and University of Turku, Turku, Finland, 5Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden, 6Department of Respiratory Medicine, Seinäjoki Central Hospital, Seinäjoki, Finland

Objectives: Eosinophils are important inflammatory cells in asthma contributing especially to asthma exacerbations. Neuropeptide S receptor 1 (NPSR1) was identified in a search for asthma susceptibility genes, in which the risk haplotypes of NPSR1 gene were found to associate with high serum IgE levels and asthma. The aim of the present study was to investigate the effects of neuropeptide S (NPS), the natural ligand of NPSR1, on functions of human peripheral blood eosinophils and to compare its effects on eosinophils derived from patients with IgE above and below 100 (high IgE and low IgE eosinophils, respectively). Methods: Expression of NPSR1 was studied by western blotting and real-time RT-PCR in freshly purified eosinophils. For cell surface CD11b expression analysis eosinophils were pretreated with 2 µM NPS or solvent, after which 10 pM granulocyte macrophage-colony stimulating factor (GM-CSF) or solvent was added and incubation continued for 18 h. Finally, 0.1 µM formyl-methionyl-leucyl-phenylalanine (fMLP) or solvent was added for 10 min and CD11b expression determined by flow cytometry. Intracellular cyclic AMP (cAMP) levels were assessed by ELISA after stimulation of cells with 2-20 µM NPS or solvent for 30 sec and cell lysis. Results: We found expression of NPSR1 in human peripheral blood eosinophils. Interestingly, NPS (2 µM) increased fMLP- but not GM-CSF-stimulated CD11b integrin levels by 12.7 ± 5.9 % (p<0.05, n=8) in high but not in low IgE eosinophils and there was a positive correlation between serum IgE level and the magnitude of the effect of NPS on fMLP-stimulated CD11b expression (r=0.5253, p=0.05, n=14). NPS increased intracellular cAMP levels by 57.0 ± 27 % (p<0.05, n=8) but only at 20 µM concentration suggesting that the effects of NPS on CD11b levels are not mediated via cAMP. Conclusions: Our data indicates that NPSR1 is expressed in human eosinophils and may have a pathologic role in augmenting eosinophil adhesion molecule expression in individuals with elevated serum IgE-levels.