Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3

Abstract

Purified human eosinophils treated for 18-24 h with IL-3 adopt a unique activated phenotype marked by increased reactivity to aggregated immunoglobulin-G (IgG). To characterize this phenotype, we quantified protein abundance and phosphorylation by multi-plexed isobaric labeling combined with high-resolution mass spectrometry. Purified blood eosinophils of five individuals were treated with IL-3 or no cytokine for 20 hours, and comparative data were obtained on abundance of 5385 proteins and phosphorylation at 7330 sites. The 1150 proteins that were significantly up-regulated (q<0.05, pair-wise t test with Benjamini-Hoachberg correction) by IL-3 included the IL3RA and CSF2RB subunits of the IL-3 receptor, the low-affinity receptor for IgG (FCGR2B), 96 proteins involved in protein translation, and 55 proteins involved in cytoskeleton organization. Among the 703 proteins that decreased were 78 mitochondrial proteins. Dynamic regulation of protein phosphorylation was detected at 4218 sites. These included multiple serines in CSF2RB; Y694 of STAT5, a key site of activating phosphorylation downstream of IL3RA/CSF2RB; and multiple sites in RPS6KA1, RPS6, and EIF4B, which are responsible for translational initiation. We conclude that IL-3 up-regulates overall protein synthesis and targets specific proteins for up-regulation, including its own receptor.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho‐associated protein kinase signalling

“…However, we believe that a period of priming in vitro is appropriate because it recapitulates in vivo events (ie after exposure to a relevant allergen), the change in cell phenotype from blood to airway eosinophil and the activation of αMß2 integrin as the cells move through the airway tissue. To demonstrate the importance of priming, we have recently shown, using a proteomic approach, that IL3 priming led to differential amounts of 1853 proteins and changes in phosphorylation at 7330 sites compared with unactivated eosinophils . Furthermore, measuring adhesion along with degranulation (ie EDN release) and cytolysis allowed us to attribute a role for PI3K in adhesion rather than in cytolysis per se as proposed in the Radonjic‐Hoesli et al study, where PI3K was considered a messenger linking necroptosis to p38 phosphorylation upstream of ROS production .…”

“…To demonstrate changes in events linked to MT growth and rescue frequency, we analysed the phosphorylation status of two regulators of MT polymerization/depolymerization and catastrophe/rescue events, stathmin and MAP4 . The functions of these two MT‐associated proteins (MAPs) have not been studied in eosinophils, although we had previously shown, using a proteomic approach, that stathmin and MAP4 were present in resting and IL3‐activated eosinophils . We thus analysed the phosphorylation status of Ser38 in stathmin and Ser696 in MAP4 in IL3‐primed eosinophils on HA‐IgG compared with no HA‐IgG.…”

“…The pathway responsible for stathmin phosphorylation in eosinophils on IgG remains unknown. It is interesting to note that we recently observed in a proteomic analysis that IL3 priming of eosinophils for 20 hours was associated with decreased phosphorylation of MAP4 at Ser696 and increased phosphorylation of stathmin at Ser38, compared with resting eosinophils (supplemental spreadsheet 2 in). These proteomic data suggest that IL3 priming already changes or conditions the phosphorylation states of MAP4 and stathmin before contact with IgG and ligand(s) for integrins.…”

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin

“…This is in line with the observation that IL-3 upregulates the cell-surface expression of and induces the high-activity conformation of α M β 2 integrin at 20 h to a significantly greater degree than does IL-5 [ 21 ]. The differential effect between the cytokines may in turn be a function of the fact that IL-3 upregulates the cell-surface level of its cognate IL-3 receptor α subunit (IL3RA), whereas IL-5 downregulates IL-5 receptor α (IL5RA) at 4–24 h [ 20 , 23 – 27 ]. Overall, the greater effect by IL-3 at 20 h is consistent with the scenario that IL-3 induces stronger and prolonged signaling and translation in eosinophils than does IL-5 or GM-CSF [ 21 , 25 , 28 – 30 ].…”

Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B′δ holoenzyme

“…100 µg of the mixed TMT sample was fractionated into six 'global proteome' fractions using high pH reverse phase spin columns (Pierce). The remaining mixed TMT sample (2.3 mg) was enriched for phosphopeptides using MagReSyn Ti-MAC beads (ReSyn Biosciences), as previously described by Esnaut et al (44). The phosphopeptide-enriched samples were fractionated into three 'phosphoproteome' fractions using high pH reverse phase spin columns (Pierce).…”