Eosinophils are major effector cells in diseases including asthma, rhinitis, certain gastrointestinal disorders and atopic dermatitis. Current treatments include mediator antagonists and anti-inflammatory drugs that reduce allergic cell numbers and inhibit mediator release, but they are not fully effective or curative. On their surface, eosinophils selectively express Siglec-8 (sialic acid-binding immunoglobulin-like lectin-8), a CD33 subfamily member. Engagement of Siglec-8 has previously been shown to cause eosinophil apoptosis, but exact signaling mechanisms are unknown. Using a monoclonal antibody (mAb) or a multimeric synthetic sulfated sialoglycan ligand selectively recognizing Siglec-8, I examined the signaling mechanism of Siglec-8 on eosinophils in the presence or absence of IL-5 priming. First, I showed that Siglec-8 engagement on IL-5-primed eosinophils promoted rapid β2-integrin-dependent eosinophil spreading and adhesion, and that β2-integrin-dependent adhesion was necessary for subsequent reactive oxygen species (ROS) generation and apoptosis. Furthermore, analysis of the eosinophil phosphoproteome using liquid chromatography – mass spectrometry (LC-MS/MS) identified 237 proteins that were differentially phosphorylated following Siglec-8 engagement and some of these proteins were linked to pathways associated with cell adhesion and cytoskeleton organization. Additional experiments demonstrated that Siglec-8-mediated ROS was generated via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and engagement of Siglec-8 on IL-5-primed eosinophils resulted in increased phosphorylation of Akt, p38 and c-Jun N-terminal kinases (JNK)-1 that was also β2-integrin dependent. Additional proteins that were phosphorylated following Siglec-8 engagement on IL-5-primed eosinophils include Src530, Csk and c-Abl. Lastly, co-immunoprecipitation data shows that Siglec-8 associates with Src-homology 2 domain-containing phosphatase (SHP)-2, a protein tyrosine phosphatase. In addition to the effects seen in peripheral blood human eosinophils, I explored the function of Siglec-8 in a novel mouse strain made in our lab (in collaboration with the Zhu lab at Yale University) that expresses Siglec-8 exclusively on the surface of mouse eosinophils. Using eosinophils from the spleen of IL-5 transgenic mice crossed with Rosa26-STOP-SIGLEC8 eoCre mice, I showed that Siglec-8 engagement with mAb induces eosinophil apoptosis, but unlike in human eosinophils, has no effect on adhesion, CD11b upregulation or ROS generation. Similar results were observed following Siglec-F engagement, suggesting that while Siglec-8 and Siglec-F on mouse eosinophils can mediate apoptosis, the mechanisms responsible for this are different compared to Siglec-8 on human eosinophils. In conclusion, we demonstrate for the first time that Siglec-8 functions as an activating receptor on cytokine-primed human eosinophils via regulation of SHP-2, β2-integrins, NADPH oxidase and a subset of protein kinases.

Last modified

• 01/30/2019

Creator

• Daniela Janevska Carroll

DOI

• https://doi.org/10.21985/N22475

Subject

• Driskill Graduate Training Program in Life Sciences

Keyword

• Siglec-8
• Eosinophils
• Apoptosis
• Signaling

Date created

• 2017-01-01

Resource type

• Dissertation

Rights statement

• In Copyright