Cross-Talk between ICAM-1 and GM-CSF Receptor Signaling Modulates Eosinophil Survival and Activation

Abstract

Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor β-chain (GMRβ) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480–488) showed binding to Shp2 phosphatase and GMRβ. However, the interaction of GMRβ with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRβ with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRβ. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-α. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.

Priming of Eosinophils by GM-CSF Is Mediated by Protein Kinase CβII-Phosphorylated L-Plastin

“…7 B ). We previously showed that prolonged survival of GM-CSF-stimulated eosinophils was critically dependent on signals from both the GM-CSF receptor and the ICAM-1 adhesion molecule (48). …”

Toward the proteome of the human peripheral blood eosinophil

“…Blood donors for eosinophil isolation included three female and five male non-smoking donors (ages 18–50 y) who showed neither asthmatic nor allergic symptoms [5]. Briefly, 1.5 ml of 15% Dextran and 1.5 ml of 0.25 M EDTA was immediately added to 60 ml of collected blood in two 50 ml polypropylene conical centrifuge tubes and allowed to sediment for 30 min at RT.…”

“…Most current established methods of preparing eosinophils from peripheral blood utilizing anti-CD16 immunomagnetic beads to remove neutrophils yield a population of cells which is relatively homogenous typically greater than 98% [5]. However, density gradient centrifugation can further distinguish two populations of eosinophils termed “normodense” (specific gravity > 1.085 g/l) and “hypodense” (specific gravity < 1.085 g/l) [6].…”

Lipoxin A4 Counterregulates GM-CSF Signaling in Eosinophilic Granulocytes

“…Both ERK1/2 and SHP-2 are known to acquire increased phosphorylation after GM-CSF treatment of human eosinophils and play important roles in signal transduction (23). Thus, we investigated whether LXA 4 had any effect on their level of phosphorylation.…”

“…Cell culture media, FBS, and antibiotics (penicillin and streptomycin) were obtained from Invitrogen. Human peripheral blood eosinophils were isolated and purified as we previously described (23). FITC-phalloidin was a product of Sigma-Aldrich.…”