Comparative proteomic analysis of peripheral blood eosinophils from healthy donors and atopic dermatitis patients with eosinophilia

Abstract

Atopic dermatitis (AD) is an allergic disease that has recently shown a dramatic increase of incidence in developed countries. Eosinophilia, the accumulation of eosinophils, occurs in AD patients through an anti-apoptotic mechanism. To understand the target proteins involved in the anti-apoptotic signaling of eosinophilia, we used a proteomic approach to analyze eosinophil proteins from AD patients with eosinophilia and healthy donors. Protein spots in two-dimensional electrophoresis (2-DE) gels were identified with peptide mass fingerprinting (PMF) based on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and data-base searching. More spots were observed in the 2-DE proteome map from AD patient samples (1310 ± 58 spots) than in those from healthy donors (1121 ± 40 spots). We identified 51 proteins affected by eosinophilia: 19 related to signaling, 8 involved in regulation of metabolism, 4 related to apoptosis, and 3 involved in inflammation. The other identified proteins were associated with transcription, RNA processing, translation, the cytoskeleton, and unknown functions. Among the identified proteins, we observed prominent increases in the expressions of cyclinA2, voltage-dependent anion channel protein 2, and 38 kDa FK506 binding protein 8 in eosinophils from AD patients in comparison to healthy donors. PMF and immunoblotting of a single spot that was expressed in eosinophils from healthy individuals but not in AD patients identified the protein as phosphorylated growth receptor binding 7 (Grb7) adaptor protein. Increased phosphorylation of Grb7 and its upstream signaling protein, focal adhesion kinase (FAK), was detected in low viability eosinophils such as those from healthy donors or in cultured eosinophils (AML14.3D10 cells) treated with dexamethasone. These results suggest that phosphorylation of Grb7 and the expressions of cydinA2, voltage-dependent anion channel protein 2, and 38 kDa FK506 binding protein 8 may be related with the anti-apoptosis mechanism of eosinophilia.