T helper 1 cells stimulated with ovalbumin and IL-18 induce airway hyperresponsiveness and lung fibrosis by IFN-gamma and IL-13 production

Abstract

We previously reported that ovalbumin (OVA) and IL-18 nasally administered act on memory type T helper (Th)1 cells to induce airway hyperresponsiveness (AHR) and inflammation, which is characterized by peribronchial infiltration with neutrophils and eosinophils. Here, we report this administration also induces lung fibrosis in an IL-13-dependent manner. Th1 cells secrete several cytokines, including IFN-gamma and bronchogenic cytokine IL-13, when stimulated with antigen (Ag) and IL-18. However, IL-13 blockade failed to attenuate AHR, although this treatment inhibited eosinophilic infiltration. To understand the mechanism by which Th1 cells induce AHR after Ag plus IL-18 challenge, we established "passive" and "active" Th1 mice by transferring OVA-specific Th1 cells into naïve BALB/c mice or by immunizing naïve BALB/c mice with OVA/complete Freund's adjuvant, respectively. Administration of Ag and IL-18 induced both types of Th1 mice to develop AHR, airway inflammation, and lung fibrosis. Furthermore, this treatment induced deposition of periostin, a novel component of lung fibrosis. Neutralization of IL-13 or IFN-gamma during Ag plus IL-18 challenges inhibited the combination of eosinophilic infiltration, lung fibrosis, and periostin deposition or the combination of neutrophilic infiltration and AHR, respectively. We also found that coadministration of OVA and LPS into Th1 mice induced AHR and airway inflammation via endogenous IL-18. Thus, IL-18 becomes a key target molecule for the development of a therapeutic regimen for the treatment of Th1-cell-induced bronchial asthma.

Fig. 1.

Anti-IFN-γ Ab treatment protected against Ag- plus IL-18-induced AHR and accumulation of neutrophils in Th1 mice. Passive (A and C) and active (B and D) Th1 mice were exposed to daily intranasally administered PBS (50 μl) or OVA (100 μg per 50 μl of PBS) plus IL-18 (0.5 μg per 50 μl of PBS) for 3 days. A total of 20 μg of sIL-13Rα₂-Fc chimera (sIL13Rα2) was used for IL-13 blockade in vivo. For the blockade of IFN-γ in vivo, 50 μg of anti-IFN-γ Ab was intranasally coadministered with OVA and IL-18 for 3 days. (A and B) At 24 h after the final exposure to OVA plus IL-18, AHR in response to increased concentrations of inhaled β-methacholine and inflammatory cell composition of BALF was examined. (C and D) Cell differential percentages were determined by light microscopic evaluation of cytospin preparation. Representative results of five to seven animals per group are shown.

Fig. 2.

Noninvasive or invasive measurement of Ag- plus IL-18-induced AHR. Active Th1 mice were exposed daily to intranasal administration of PBS (50 μl) or OVA (100 μg per 50 μl of PBS) plus IL-18 (0.5 μg per 50 μl of PBS) for 3 days. At 24 h after the final exposure to OVA plus IL-18, AHR in response to increased concentrations of inhaled β-methacholine was determined by noninvasive [specific airway resistance (sRaw)] (A) and invasive [pulmonary resistance (RLung) and Cdyn] (B) measurement. Baseline values for pulmonary resistance (cmH₂O/ml·sec⁻¹) and Cdyn (ml/cmH₂O) were 4.58 for PBS, 4.65 for OVA plus IL-18, 0.0119 for PBS, and 0.0133 for OVA plus IL-18. Representative results of five to seven animals per group are shown.

Fig. 3.

Histological examination of the lung tissues from Th1 mice exposed to OVA and IL-18. Both passive and active Th1 mice were exposed daily to intranasally administered PBS or OVA plus IL-18 with anti-IFN-γ or sIL-13Rα2 as described in the legend of Fig. 1. At 24 h after final exposure, lungs from each group of mice were prepared for histological examination by perfusing the animal via the right ventricle with 10 ml of PBS; the lungs were then fixed in formalin, cut into 3-μm sections, and stained with H&E as described in Material and Methods. [Original magnification, ×40 (Insets, ×200).]

Fig. 4.

IL-4Rα^−/− mice often developed Th1-cell-induced AHR. BALB/c WT or BALB/c background IL-4Rα^−/− mice, which were immunized with OVA in CFA 2 weeks previously, were exposed to daily intranasal administration of PBS or OVA plus IL-18 for 3 days. At 24 h after final exposure, the mice were analyzed for β-methacholine-induced AHR (A) and BALF (B) and their histological changes (C), as described for in the legends of Figs. 1 and 2. White arrowhead, neutrophil; black arrowhead, eosinophil. Representative results obtained from five to seven animals per group are shown. (Magnification, ×200.)

Fig. 5.

Neutralization of IFN-γ or IL-18 abolished OVA plus LPS-induced AHR and neutrophilic infiltration in active Th1 mice. BALB/c WT mice or BALB/c background IFN-γ^−/− or IL-18^−/− mice immunized with OVA/CFA 2 weeks previously were challenged intranasally with 5 μg of LPS and 100 μg of OVA. To block endogenous IL-18 or IFN-γ, Ab against IL-18 or IFN-γ mixed with OVA and LPS was coadministered daily. At 24 h after final administration, β-methacholine-induced AHR (A and C) and cell population in BALF (B) were analyzed as described in the legend of Fig. 1. Representative results of five to seven animals per group are shown. The results shown in A Left and C Left were obtained from the same experiment.

Fig. 6.

IL-13-dependent lung fibrosis in Ag- plus IL-18-administered Th1 mice or Ag-administered Th2 mice. Active Th1 mice or Th2 were induced by immunization with OVA/CFA (A) or OVA/alum (B), respectively. Immunized mice were exposed daily to intranasally administered OVA or OVA plus IL-18 with or without sIL-13Rα2 as described in the legend of Fig. 1. At 24 h after final exposure, lungs from each group of mice were prepared, stained with H&E (a, b, e, and f) or Azan–Mallory (c, d, g, and h), and used for histological examination. (Original magnification, ×40.)

Fig. 7.

Immunohistochemical staining of periostin in the lungs of Th1 mice after exposure to OVA and IL-18. Active Th1 mice were exposed daily to intranasally administered OVA plus IL-18 or OVA plus IL-18 plus sIL-13Rα2 as described in the legend of Fig. 1. At 24 h after final exposure, lungs from each group of mice were fixed and stained with H&E or Azan–Mallory. Immunohistochemical staining for periostin was as described in Material and Methods. (Original magnification, ×40.)