Coordinated Events During Eosinophil Activation: The First Sixty Minutes.

摘要

By flow cytometry, IL-5 (>1 ng/ml) increased forward scatter of suspended eosinophils, enhanced CD11bCD18 affinity, and upregulated CD11bCD18 within 5 min. These changes were characterized cytologically and by immunofluorescence. The rapid change in forward scatter correlates with polarization of eosinophils into a granular compartment and an agranular protrusion. The agranular protrusion was characterized as a uropod because of the enrichment of activated CD11bCD18, P-selectin glycoprotein ligand-1 (PSGL-1), and CD44, and less of microfilaments. Remarkably, the eosinophil uropod contains the nucleus. IL-3, granulocyte-monocyte colony stimulating factor, and eotaxin polarized eosinophils similar to IL-5. Phorbol myristate acetate activated CD11bCD18 but did not polarize eosinophils. Upon IL-5 stimulation, cortical microfilaments were replaced by denser networks in the granular compartment. Microtubules in the uropod were reorientated and distributed around the nucleus. The centrosome was located between the nucleus and the granular compartment of polarized eosinophils in suspension. Vimentin intermediate filaments gathered in the uropod, especially in the tip region. Activated ezrin-radixin-moesin proteins and phosphorylated myosin light chain were associated with the cell membrane at the uropod. Nocodazole and colchicine induced CD44 or PSGL-1 capping and cell shape change, but not nucleus translocation. Cytochalasin B, calyculin A, withaferin A, and ML-7 blocked IL-5-induced polarization and PSGL-1 capping. Y27632 partially inhibited uropod protrusion but did not block PSGL-1 capping. IL-5 receptor alpha and common beta subunits were centrally located in resting eosinophils and transported to the uropod of IL-5-treated eosinophils with some located in the nucleus. The uropod tip stained positively for pJAK2, pSTAT1, pSTAT5, and pERK and the nucleus stained positively for pSTAT1, pSTAT5, and pCREB. fMLF induced ERK phosphorylation in more IL-5-primed eosinophils than unprimed cells. In summary, IL-5 family cytokines and eotaxin polarize suspended eosinophils into a granular compartment and the unique uropod containing the nucleus, the nucleopod. The nucleopod is enriched with adhesion receptors on the cell membrane, various signaling molecules in the cytoplasm, and IL-5 family cytokine receptors upon stimulation of cognate cytokines. The IL-5-induced polarization of eosinophils enhances ERK phosphorylation in response to fMLF.