Sedgwick, Julie B.

Abstract In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Rα expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Rα is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Rα mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Rα, which in turn contributes to the presence of sIL-5Rα. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Rα was accompanied by an increase in sIL-5Rα in the supernatant. IL-5 had no ligand-specific effect on mIL-5Rα or sIL-5Rα mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Rα, suggesting that sIL-5Rα may be produced by proteolytic cleavage of mIL-5Rα. IL-5 transiently reduced surface expression of β-chain, but had no effect on the expression of GM-CSFRα. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Rα rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Rα and release sIL-5Rα in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.

Abstract IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Rα on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Rα in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Rα on airway eosinophils compared with circulating cells. Furthermore, sIL-5Rα concentrations were elevated in BAL fluid, but steady state levels of sIL-5Rα mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Rα on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Rα, the presence of sIL-5Rα, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.

Abstract Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are members of the immunoglobulin superfamily adhesion molecules on vascular endothelium and important in the development of eosinophil (EOS) accumulation in allergic inflammation. To define the role of these adhesion proteins in EOS inflammation, peripheral blood EOS from allergic donors were incubated in either buffer (control)-, recombinant human (rh)-VCAM-1-, or rh-ICAM-1-coated plates, and the effects of these adhesion proteins on EOS effector functions were determined. VCAM-1 induced spontaneous EOS adhesion whereas EOS adhesion to ICAM-1 required a second signal, such as granulocyte macrophage colony-stimulating factor (GM-CSF). Although only VCAM-1 stimulated EOS superoxide anion (O2−) generation, the addition of GM-CSF (100 pM) to the reactions resulted in a greater and equivalent production of O2− with VCAM-1 and ICAM-1. In the presence of GM-CSF, ICAM-1 and VCAM-1 caused significant release of EOS-derived neurotoxin (EDN). Moreover, only ICAM-1 (no GM-CSF) promoted calcium ionophore A23187 (0.2 μM)-induced EOS leukotriene C4 (LTC4). Enhanced O2− generation, EDN release, and LTC4 generation observed with ICAM-1 and VCAM-1 were significantly inhibited by anti-β2-integrin antibody. These results suggest that ICAM-1 and VCAM-1 are important in determining the eventual function of airway EOS.

Abstract Human blood eosinophils exposed ex vivo to hematopoietic cytokines (e.g., IL-5 or GM-CSF) subsequently display enhanced responsiveness to numerous chemoattractants, such as chemokines, platelet-activating factor, or FMLP, through a process known as priming. Airway eosinophils, obtained by bronchoalveolar lavage after segmental Ag challenge, also exhibit enhanced responsiveness to selected chemoattractants, suggesting that they are primed during cell trafficking from the blood to the airway. Earlier work has shown that chemoattractants stimulate greater activation of ERK1 and ERK2 following IL-5 priming in vitro, thus revealing that ERK1/ERK2 activity can be a molecular readout of priming under these circumstances. Because few studies have examined the intracellular mechanisms regulating priming as it relates to human airway eosinophils, we evaluated the responsiveness of blood and airway eosinophils to chemoattractants (FMLP, platelet-activating factor, CCL11, CCL5, CXCL8) with respect to degranulation, adherence to fibronectin, or Ras–ERK signaling cascade activation. When compared with blood eosinophils, airway eosinophils exhibited greater FMLP-stimulated eosinophil-derived neurotoxin release as well as augmented FMLP- and CCL11-stimulated adherence to fibronectin. In airway eosinophils, FMLP, CCL11, and CCL5 stimulated greater activation of Ras or ERK1/ERK2 when compared with baseline. Ras activation by FMLP in blood eosinophils was also enhanced following IL-5 priming. These studies are consistent with a model of in vivo priming of eosinophils by IL-5 or related cytokines following allergen challenge, and further demonstrate the key role of priming in the chemoattractant-stimulated responses of eosinophils. These data also demonstrate the importance of the Ras–ERK signaling pathway in the regulation of eosinophil responses to chemoattractants in the airway. Human airway eosinophils respond to several chemoattractants with increased activation of the Ras–ERK cascade, eosinophil-derived neurotoxin release, and adherence to fibronectin relative to blood eosinophils.

Abstract Oxidized low-density lipoprotein (oxLDL) has been reported as a major participant in the pathogenesis of atherosclerosis. We hypothesized that oxLDL can also interact with granulocytes during inflammatory airway diseases, such as asthma. To test the chemotactic effect of oxLDL, isolated human peripheral granulocytes were added to the upper chambers of Transwell filters and migration in response to oxLDL was determined. Cu+2-oxidized LDL stimulated neutrophil (23.4 ± 3.2% for 100 μg/ml oxLDL versus 2.9 ± 1.1% for buffer, P < 0.05) and eosinophil (19.3 ± 3.5% versus 0.6 ± 0.02% for buffer, P < 0.05) chemotaxis in a concentration-dependent manner. The magnitude of chemotaxis was dependent on the degree of LDL oxidation. Granulocyte transmigration across IL-1β–activated human pulmonary microvascular endothelial cell monolayers was similarly stimulated by oxLDL. OxLDL activated significant degranulation of both neutrophils (100.9 ± 9.8 versus 49.6 ± 8.4 ng lactoferrin released/5 × 105 neutrophils for buffer, P < 0.05) and eosinophils (342 ± 115.4 versus 85.8 ± 30.4 ng eosinophil-derived neurotoxin/1 × 106 eosinophils for buffer, P < 0.05). Therefore, in vivo influx and oxidation of LDL may be an important mediator for the initiation of bronchial inflammation where granulocytes are recruited to the lung.