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.

Rhinovirus (RV) infections can alter lower airway physiology and inflammation, yet the characteristics of RV replication in lower airway cells are incompletely understood. An RV serotype 16 (RV16)-specific monoclonal antibody was identified. Immunohistochemistry and an infectious center assay were used to quantitate the infectivity of RV16 in primary bronchial and adenoidal epithelial cells. The proportion of infectible epithelial cells increased with the inoculum but did not exceed 10%. Analysis of bronchial tissue samples infected ex vivo demonstrated a small subset of RV-infected cells in the epithelial layer. These data confirm previous reports that RV infects only a small subset of epithelial cells in upper airway tissues and indicate that lower airway epithelial cells have a similar susceptibility to RV infection. In confirming that RV can infect cells in the lower airway, these results suggest that lower airway dysfunction occurs through this mechanism in susceptible persons.

Segmental antigen bronchoprovocation was used to define the nature of the inflammatory process in allergic airway disease. Bronchoalveolar lavage fluid obtained from allergic rhinitis patients 12 min after segmental antigen instillation (immediate response) revealed a significant increase in histamine and tryptase, but no cellular response. Repeat segmental lavage 48 h later (late response) showed marked and significant increases in both low and normal density eosinophils as well as striking elevations of eosinophil granular protein levels (major basic protein, eosinophil-derived neurotoxin, eosinophil cationic protein, and eosinophil peroxidase). Leukotriene C4, but not tryptase, concentrations were also consistently elevated in late lavage samples. Further, the late lavage samples showed a significant increase in interleukin-5 concentrations that correlated with the presence of eosinophils and eosinophil granular proteins. Neither eosinophils nor soluble mediators of eosinophils increased when normal subjects were similarly challenged with antigen. These data suggest that eosinophils are attracted to the airway during the late-phase allergic reaction and that IL-5 may produce changes in airway eosinophil density and promote the release of granular proteins to cause airway injury.

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.

The mechanism of airway eosinophilia during antigen-induced inflammation was investigated by measurement of eosinophil-active cytokines utilizing an eosinophil survival assay. In the first study, 4 patients with allergic rhinitis underwent segmental bronchoprovocation (SBP) with low, medium, and high doses of ragweed extract instilled into different bronchial subsegments; bronchoalveolar lavage (BAL) fluids were collected from each segment 12 min and 48 h after challenge. Eosinophil granule proteins and eosinophil survival activity were significantly elevated in the 48-h (late-phase) BAL fluids from these segments. Correlations were observed between the concentrations of eosinophil granule proteins and eosinophil survival activity (rs = 0.717 to 0.880, p < 0.001) in BAL fluids. Eosinophil survival activity was completely neutralized by anti-IL-5 monoclonal antibody in five of the seven 48-h samples tested representing three of the 4 patients. In the two remaining samples, eosinophil survival activity was only partially neutralized by either anti-IL-5 antibody or anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) but was completely neutralized by anti-IL-5 and anti-GM-CSF in combination. Subsequently, in the second study, 10 patients with allergic rhinitis were challenged by SBP with ragweed extract. Eosinophil survival activity was significantly elevated in the 48-h BAL fluids; this activity was partially neutralized by anti-IL-5 antibody about (48%) and completely neutralized by the combination of anti-IL-5 and anti-GM-CSF antibodies. These findings suggest that the eosinophil survival activity in the late inflammatory lesions following SBP with allergen is mainly associated with IL-5, with small contributions from GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

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 microM)-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-beta2-integrin antibody. These results suggest that ICAM-1 and VCAM-1 are important in determining the eventual function of airway EOS.

Our data demonstrate that SEB-induced generation of Th1 and Th2 cytokines is increased in the presence of human blood and airway eosinophils. Thus, eosinophils can have an immunoregulatory function in pathogen-associated allergic diseases such as atopic dermatitis, chronic sinusitis, and asthma exacerbations.

No abstract