title: Identification of surface molecules associated with physiologic activation of eosinophils. Application of whole-blood flow cytometry to eosinophils.
abstract: Activation is central to the eosinophil's functional role as an immune responder cell. To evaluate such activation in cells freshly isolated from peripheral blood, a method for whole-blood immunostaining and flow cytometry-based eosinophil selection was developed. Simultaneous comparison of purified eosinophils and whole-blood cells revealed significant differences in the levels of expression of various surface molecules, which suggested that the purification process activated the eosinophils. Subsequent analyses were conducted with the whole-blood assay. When eosinophils from helminth-infected persons (n = 18) were compared with those from normal individuals (n = 10), the early activation marker CD69 was found to be significantly increased (geometric mean (GM) = 4.3 vs. 1.0%, p = 0.04). The granulocyte activation marker CD66 was also up-regulated on eosinphils from helminth patients (GM = 53.3 vs. 31.0%, p = 0.044), as was the tetraspan family molecule CD81 (TAPA-1; GM = 79.4 vs. 48.2%, p = 0.02). Conversely, in vivo CD23 (FcepsilonRII) expression on eosinophils was decreased in the presence of parasitic infection (GM = 0.9 vs. 5.7%, p = 0.02). Expression of the eosinophil surface molecules CD69, CD81, and CD23 was significantly enhanced after cytokine stimulation in vitro with IL-3 or GM-CSF. In vivo, specific anthelmintic therapy resulted in decreased CD66 and CD25 expression (p < 0.05 compared with pretreatment) to levels approaching those seen in uninfected normal individuals. These findings indicate the dynamic nature of eosinophil surface molecules and demonstrate an important role for whole-blood staining in developing an understanding of the nature of eosinophil activation and of their role in inflammatory reactions to helminth parasites.
authors.affiliation: Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Mawhorter
authors.forename: D A
authors.initials: DA
authors.lastname: Stephany
authors.forename: E A
authors.initials: EA
authors.lastname: Ottesen
authors.forename: T B
authors.initials: TB
authors.lastname: Nutman
mesh_terms: D000871:Anthelmintics / Q000627:therapeutic use; D000954:Antigens, Surface / Q000032:analysis; D002469:Cell Separation; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004804:Eosinophils / Q000737:chemistry*; D005260:Female; D005434:Flow Cytometry; D006373:Helminthiasis / Q000097:blood* / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008297:Male
chemical_list: D000871:Anthelmintics; D000954:Antigens, Surface; D016207:Cytokines