PSGL1 and Siglec-8 distribution on IL-5-activated blood eosinophils and BAL eosinophils were different in that Siglec-8 often had a distribution in several patches per cell while PSGL1 localization was focused at one site per cell (compare Physique 5B and C with Physique 6B and C). Siglec-8, while BAL basophils had lower but detectable Flurizan surface Siglec-8. BAL macrophages, monocytes, neutrophils, and plasmacytoid dendritic cells did not express surface Siglec-8. Microscopy of freshly isolated blood eosinophils exhibited homogeneous Siglec-8 distribution over the cell surface. Upon incubation with IL-5, Siglec-8 on the surface of eosinophils became localized in patches both at the nucleopod tip and at the opposite cell pole. BAL eosinophils also had a patchy Siglec-8 distribution. Conclusions We conclude that 48 h Flurizan after segmental allergen challenge, overall levels of Siglec-8 expression on airway eosinophils resemble those on blood eosinophils, but with a patchier distribution, a pattern consistent with activation. Thus, therapeutic targeting of Siglec-8 has the potential to impact blood as well as lung eosinophils, which may be associated with improved outcome in eosinophilic lung diseases. during allergic inflammation [14]. The presence of an inhibitory motif in the cytoplasmic domain indicates that Siglec-8 should be involved in unfavorable cell signaling [9]. Recent observations indicate that Siglec-8 can, after IL-5 priming, initiate cell signaling leading to eosinophil apoptosis via phosphorylation of intracellular signaling proteins and 2 integrin-dependent adhesion [9,16,17]. Siglec-8-directed therapeutics are currently being developed. A preliminary report involving a phase 1 study in healthy adult volunteers showed that a single intravenous infusion of a humanized afucosylated IgG1 monoclonal antibody (mAb) against Siglec-8 (called AK002) rapidly and selectively depletes blood eosinophils in a sustained manner for at least 84 days [18]. Given the selective expression of Siglec-8 on both eosinophils and mast cells, AK002 is currently undergoing clinical Flurizan trials in eosinophil- and mast cell associated diseases including refractory chronic urticaria (NCT [national clinical trial] 03436797), severe forms of allergic conjunctivitis (“type”:”clinical-trial”,”attrs”:”text”:”NCT03379311″,”term_id”:”NCT03379311″NCT03379311), and indolent systemic mastocytosis (“type”:”clinical-trial”,”attrs”:”text”:”NCT02808793″,”term_id”:”NCT02808793″NCT02808793). IL-5 causes blood eosinophils in suspension to undergo acute shape change and polarize, with granules moving to one pole and the nucleus to the opposite pole into a specialized uropod, the nucleopod [19]. Flurizan The nucleopod is usually enriched in cell surface receptors including P-selectin glycoprotein ligand-1 (PSGL1, CD162); PSGL1 localization on the surface of IL-5-activated eosinophils can be regarded as a reporter of cell activation [19]. However, cell shape and polarization, and localization of cell surface receptors have not been studied on airway eosinophils. In allergic human subjects, segmental bronchoprovocation with allergen is usually a model of allergic airway inflammation that induces intense local recruitment of inflammatory cells, including eosinophils and basophils [20]. We utilized this model to characterize Siglec-8 expression and localization on cells in bronchoalveolar lavage (BAL). 3. Materials and Methods 3.1. Subjects for segmental lung allergen challenge Eight subjects with mild allergic asthma who were allergic to ragweed, house dust mite, or cat dander were screened and enrolled as described [21,22]. At least four weeks before bronchoscopy, subjects underwent a whole-lung inhaled allergen (ragweed, house dust mite, or cat dander) challenge to determine the provocative dose of antigen producing a 20% fall in forced expiratory volume in 1 s (AgPD20) [21,23,24]. These studies were approved CBFA2T1 by the University of Wisconsin-Madison Health Sciences Institutional Review Flurizan Board. Informed written consent was obtained from each subject before participation (protocol No. 2016-0021 for bronchoalveolar lavage study and No. 2013-1570 for eosinophil purification). 3.2. Segmental bronchoprovocation with allergen, bronchoalveolar lavage, and blood draws Allergen was administered by bronchoscopy as previously described [21]. Briefly, two bronchopulmonary segments were lavaged (160 ml sterile 0.9% NaCl) and then a dose of 10% of the subjects AgPD20 was administered into one segment and, when this was well tolerated, a dose of 20% of the AgPD20 was instilled in the second segment [21]. Bronchoscopy with BAL was repeated 48 h later and BAL recovered from the two segments was pooled [21]. Blood was obtained from each subject on the same day as the second bronchoscopy, when BAL was performed. 3.3. Three-color flow cytometry detection of Siglec-8 on airway and blood cell populations Antibodies and reagents used for three-color analysis were the following. Fluorescein isothiocyanate (FITC)-conjugated anti-CD14 mAb clone M5E2 and anti-CD16 clone 3G8 [23]; phycoerythrin (PE)-conjugated.