IL‐3 up‐regulates and activates human eosinophil CD32 and αMβ2 integrin causing degranulation

Abstract

Background Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. Objective We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. Methods Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation, which was then compared to IgG-mediated degranulation of freshly isolated (unstimulated) airway eosinophils activated in vivo by segmental allergen challenge. Results When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. Conclusions We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.

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

“…In those studies, the discharge of mitochondrial DNA was followed by the release of reactive oxygen species (ROS), which was required for vacuolization, loss of membrane integrity, and release of granule proteins (21) . Based on our prior finding that IL-3 primes EOS for release of granule proteins upon exposure to aggregated IgG (8) , we speculate that alterations in mitochondria may be part of the priming process.…”

“…The present studies were motivated by immuno-blotting and qPCR analyses demonstrating that SEMA7A and FCGR2B/C are regulated at the translational level in EOS by IL-3, but not by the other ß-chain cytokines, IL-5 or GM-CSF (6–8) . SEMA7A and two splice variants (isoforms 2/5 and 1/3/4) of FCGR2B were among the top 30 proteins up-regulated by IL-3 as defined by Log2 >0.5 fold increase and q values <0.01 (Figure 1 left and Supplemental Spreadsheet 1).…”

“…Subjects with prescriptions for low doses of inhaled corticosteroids did not use their corticosteroids the day of the blood draw. Eosinophils were purified by negative selection as previously described (8) . Briefly, heparinized blood was diluted 1:1 in HBSS and was overlaid above Percoll (1.090 g/ml).…”

“…In addition, IL-3 is elevated in serum in poorly controlled asthmatic patients, and airway IL-3-positive cells are increased with asthma severity (12, 13) . Importantly we found that EOS chronically activated in vitro with IL-3 react vigorously to aggregated immunoglobulin-G (IgG) via activated FCGR2 and ITGAM/ITGB2 integrin receptor (8) .…”

“…We have demonstrated that among the three IL-5-family cytokines, IL-3 is uniquely effective in prolonging intracellular signaling, maintaining a polarized shape, and up-regulation of the abundance of FCGR2B and SEMA7A as assessed by immuno-blotting and flow cytometry (6–8) . IL-3 is relevant in allergy inasmuch as it is released by activated Th-2 lymphocytes, mast cells and basophils following IgE cross-linking (9, 10) , and elevated in the airways to as much as 10 ng/ml after a segmental allergen challenge (11) .…”