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

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 multiplexed
isobaric labeling combined with high-resolution mass spectrometry.
Purified blood eosinophils of five individuals were treated with IL-3
or no cytokine for 20 h, 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,
pairwise t test with Benjamini–Hochberg 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.