Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma

Abstract

The primary function of IL-7 is to promote maturation and survival of T cells. Through microarray expression analysis, we previously observed that human blood eosinophils express mRNA for IL-7R alpha (CD127) and its common gamma chain (CD132). The purpose of this study was to determine whether eosinophils have functional IL-7 receptors and to assess the potential contribution of IL-7 to eosinophilic airway inflammation by evaluating its presence in bronchoalveolar lavage (BAL) fluid of subjects with atopic asthma before and after segmental bronchoprovocation with allergen. Immunoblot analysis revealed that CD127 is present in highly purified human blood eosinophils. Furthermore, eosinophils responded to IL-7 with phosphorylation of STAT5, up-regulation of the activation marker CD69, and prolonged survival. Neutralization of GM-CSF but not IL-5 significantly blunted these functional responses, suggesting that IL-7 mediates its effects by promoting eosinophil release of autologous GM-CSF. Notably, the suppressive effect of anti-GM-CSF on STAT5 phosphorylation occurred within 10 min of eosinophil exposure to IL-7. Thus, IL-7 likely activates eosinophil release of preformed rather than newly synthesized GM-CSF. The biological relevance of IL-7 to eosinophilia in vivo was implicated in a study of airway allergen challenge in patients with allergic asthma. IL-7 concentrations in BAL fluid increased significantly 48 h after segmental allergen challenge and were highly correlated with BAL eosinophils (r = 0.7, p < 0.001). In conclusion, the airway response to allergen is associated with the generation of IL-7, which may contribute to airway inflammation by promoting enhanced eosinophil activation and survival. Activation of eosinophils is a novel function for IL-7.

Figure 1

Evidence of IL-7 receptor expression on human eosinophils. A, Representative immunoblot analysis for CD127 in cell lysates from highly purified (>99%) eosinophils (Eos) or CD4+ T lymphocytes (CD4) immunoblotted with either a monoclonal antibody specific to CD127 (left panel) or an isotype control IgG₁ (right panel). B, CD127 expression was analyzed by immunoblotting of 30 µg of cell lysate protein from 7 separate eosinophil donors and quantified by densitometry. For each donor, the immunoblot density is summarized as the % of CD127 signal from 30 µg of the positive control T lymphocyte cell lysate run on the same gel and immunoblot.

Figure 2

Effect of IL-7 on survival of human eosinophils. A, Eosinophil survival in the presence of increasing concentrations of IL-7 or 300 pM GM-CSF. The number of viable eosinophils at 48 h was determined by trypan blue exclusion and expressed as a percentage of the number of cells at 0 h (*p<0.05, n=7). B, Effect of GM-CSF neutralization on IL-7-induced eosinophil survival (n=3). Eosinophils were preincubated for 1 h in the presence (black bars) or absence (white bars) of anti-GM-CSF (20 µg/ml) and then cultured for 48 h with medium, IL-7 (50 nM), or GM-CSF (300 pM).

Figure 3

Induction and kinetics of CD69 expression on eosinophils cultured with IL-7. Eosinophils were preincubated for 1 h with anti-GM-CSF (20 µg/ml, triangles) or an isotype control antibody (circles) and then cultured with medium (white squares), IL-7 (50 nM, black) or GM-CSF (300 pM, gray). Expression of CD69 was determined by flow cytometric analysis of eosinophils at 0, 0.5, 1, 3, 20, and 48 h. (n = 4, *p<0.05 compared to 0 h, ^†p< 0.05 compared to treatment plus anti-GM-CSF). B, Effect GM-CSF neutralization on eosinophil morphology 48 h after culture with IL-7. Eosinophils were treated with IL-7 (50nM), GM-CSF (300 pM), or medium in the presence of control antibody, or neutralizing antibody to GM-CSF (20 µg/ml).

Figure 4

Effect of IL-7 on eosinophil STAT5 phosphorylation. A, Intracellular flow cytometric analysis for detection of phosphotyrosine STAT5 in eosinophils cultured for 15 min in medium (dotted line) or IL-7 (50 nM, filled histogram). Isotype control is represented by solid line. Data are representative of eosinophils from 3 subjects. B. Eosinophils were preincubated with (upper labels) medium (lanes 1 and 8), isotype control antibody (20 µg/ml; lanes 2, 4, and 6), or a GM-CSF neutralizing antibody (20 µg/ml; lanes 3, 5, and 7) before stimulation with (bottom labels) medium (lane 1), 50 nM IL-7 (lanes 2–5 and 8), or 300 pM GM-CSF (lanes 6 and 7) for the indicated times. To ensure equal protein loading between samples, 60 µg of total protein was loaded and the immunoblots were reprobed for total ERK1/2 as a loading control (lower illustration). These data are representative of experiments from 3 eosinophil donors.

Figure 5

Effect of neutralization of GM-CSF or IL-5 on IL-7-induced eosinophils expression of CD69. A-D. Representative histogram with solid lines depicting stimulant and hatched lines representing stimulant plus neutralizing Ab. Purified eosinophils were stimulated (3 h) with IL-7 in the presence of neutralizing Ab to GM-CSF (A) or IL-5 (B). The efficacy of the Abs was confirmed by blocking CD69 expression induced by GM-CSF (C) or IL-5 (D) using the respective neutralizing Ab. E. Summary data of three experiments from different blood donors.

Figure 6

IL-7 concentrations in BAL fluid before and 48 h after SBP-Ag. A, Concentrations of IL-7 were determined by ELISA analysis of 10 × concentrated BAL fluid obtained immediately before (baseline) and 48 h after SBP-Ag (n=18) B, Correlation between levels of IL-7 and percentage of eosinophils in BAL fluid 48 h after SBP-Ag (R_s=0.71, p<0.001).