Regulation of Functional Phenotypes of Cord Blood–Derived Eosinophils by γ-Secretase Inhibitor

Abstract

Eosinophils develop from stem cells in the bone marrow under the influence of hematopoietic cytokines, particularly IL-5. Previously, we have demonstrated that blockage of Notch signaling by a gamma-secretase inhibitor (GSI) promotes the differentiation of umbilical cord blood (UCB)-derived eosinophils. These highly major basic protein (MBP)-positive eosinophils cultured in the presence of the inhibitor lack the migratory response to eotaxin, although their CCR3 levels are similar to those of eosinophils cultured without the inhibitor. We investigated the mechanism underlying the differential responses of differentiating eosinophils and their functionalities in response to eosinophil-active cytokines in the presence and absence of GSI. UCB cells cultured for 4 weeks with hematopoietic cytokines in the presence or absence of GSI were monitored for extracellular signal-regulated kinase (ERK) phosphorylation, MBP expression, and functionality. Eosinophil differentiation from UCB cells was accompanied by activation of the ERK1/2 pathway during the 4-week culture period. In particular, strong ERK1/2 phosphorylation was observed in eosinophils during the final stage of culture when GSI was present. Consistent with this finding, ERK inhibition nullified the effect of GSI on eosinophil differentiation. Eosinophils cultured with GSI resembled airway eosinophils rather than peripheral blood eosinophils based on reduced IL-5Ralpha expression, blunted eosinophil cationic protein (ECP) degranulation, and decreased IL-13 and granulocyte macrophage-colony-stimulating factor production. These results suggest that Notch signaling regulates the terminal differentiation and subsequent effector phenotypes of eosinophils, partly through modulation of the ERK pathway. GSI has therapeutic potential for eosinophilic inflammatory diseases, such as asthma.

Targeting eosinophils in allergy, inflammation and beyond

“…Similar to priming of eosinophils by cytokines, Notch signaling has a role in regulating eosinophil activation state and effector functions 152,153 . The Notch pathway is an evolutionarily conserved signaling pathway that has a key role in deciding the fate of progenitor cells 154 as well as regulating immune cell activation 155 .…”

“…The Notch pathway is an evolutionarily conserved signaling pathway that has a key role in deciding the fate of progenitor cells 154 as well as regulating immune cell activation 155 . In addition to eosinophil priming, Notch signaling regulates eosinophil maturation 152,156 . These findings are relevant as treatment with an inhibitor of Notch signaling (LY450139 dihydrate) in a clinical trial for Alzheimer disease resulted unexpectedly in a statistically significant mean increase (47%) in blood eosinophils in patients 157 .…”

Emerging roles of the γ-secretase-notch axis in inflammation

“…Blocking Notch signaling through γ-secretase inhibitors has been found to promote the differentiation of eosinophils derived from umbilical cord blood (Kang et al, 2005). These eosinophils respond differentially to eosinophil-active cytokines partly through the ERK1/2 pathway in the presence of γ-secretase inhibitors (Kang et al, 2007). The Notch-RBP-J pathway has been shown to regulate macrophage polarization and thus innate immune responses by modulating the transcription factor IRF8.…”

Eosinophil Development, Regulation of Eosinophil-Specific Genes, and Role of Eosinophils in the Pathogenesis of Asthma

“…Notch is an evolutionarily conserved transmembrane protein that regulates a broad spectrum of cell-fate decisions and differentiation 29. Notch signaling promotes eosinophil maturation30 as well as affecting eosinophil functionality 31,32. However, whether Notch signaling modulates the transcription factors or unidentified pathways key for deciding eosinophil fate or is itself affected by these factors remains to be determined.…”