Interleukin-33 and alveolar macrophages contribute to the mechanisms underlying the exacerbation of IgE-mediated airway inflammation and remodelling in mice

Abstract

Allergen-specific IgE has long been regarded as a major molecular component of allergic asthma. Additionally, there is increasing evidence of the important roles of interleukin-33 (IL-33) in the disease. Here, we show that IL-33 and alveolar macrophages play essential roles in the exacerbation of IgE-mediated airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were challenged with OVA seven times intratracheally. The seventh challenge exacerbated airway inflammation and remodelling compared with the fourth challenge; furthermore, markedly increased expression of IL-33 in the lungs was observed at the fourth and seventh challenges. When anti-IL-33 or anti-ST2 antibody was administered during the fourth to seventh challenge, airway inflammation and remodelling were significantly inhibited at the seventh challenge. Because increases of IL-33(+) and ST2(+) alveolar macrophages and ST2(+) CD4(+) T cells in the lungs were observed at the fourth challenge, the roles of macrophages and CD4(+) cells were investigated. Depletion of macrophages by 2-chloroadenosine during the fourth to seventh challenge suppressed airway inflammation and remodelling, and IL-33 production in the lung at the seventh challenge; additionally, anti-CD4 mAb inhibited airway inflammation, but not airway remodelling and IL-33 production. Meanwhile, treatment with 2-chloroadenosine or anti-CD4 mAb decreased IL-33-induced airway inflammation in normal mice; airway remodelling was repressed only by 2-chloroadenosine. These results illustrate that macrophage-derived IL-33 contributes to the exacerbation of IgE-mediated airway inflammation by mechanisms associated with macrophages and CD4(+) cells, and airway remodelling through the activation of macrophages.

Figure 1

Changes in interleukin-33 (IL-33) production, airway inflammation and airway remodelling after repeated antigen challenges in IgE-sensitized mice. (a) Experimental protocol for sensitization with OE-1 and challenge with antigen in BALB/c mice. (b) Levels of IL-33 in lung homogenate supernatants 24 hr after the seventh challenge in non-sensitized-challenged [NS-C (7th)] and before the fourth [OE-1 (4th before)] or 24 hr after the fourth [OE-1 (4th)] and seventh [OE-1 (7th)] challenges in mice sensitized with OE-1. (c) Changes in inflammatory cell numbers in bronchoalveolar lavage fluid in NS-C (7th), OE-1 (4th) and OE-1 (7th) groups. (d–g) Changes in airway inflammation (d), goblet cell hyperplasia (e), sub-epithelial fibrosis (f) and smooth muscle cell hypertrophy (g) in NS-C (7th) (i) and OE-1 (7th) (ii) groups. Bar = 100 µm. (h) Changes in airway inflammation (i), epithelial area (ii), PAS⁺ area (iii), fibrosis area (iv), total collagen (v) and α-SMA⁺ area (vi) in NS-C (7th), OE-1 (4th) and OE-1 (7th) groups. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of six or seven animals. *P < 0·05 and **P < 0·01 compared with the NS-C (7th) group. P < 0·05 and P < 0·01 compared with the S-C (4th) group. &&P < 0·01 compared with the OE-1 (4th before) group. Total, all cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.

Figure 2

Effect of anti-interleukin-33 monoclonal antibody (IL-33 mAb) on airway inflammation and remodelling after the seventh antigen challenge in IgE-sensitized mice. (a) Experimental protocol for treatment with anti-IL-33 mAb. Anti-IL-33 mAb was intratracheally administered on days 8, 9, 10 and 15 [OE-1 (7th) + anti-IL-33]. Negative and positive controls were non-sensitized-challenged [NS-C (7th)] and OE-1-sensitized-challenged, control rat IgG2a mAb-treated [OE-1 (7th) + rat IgG2a] mice, respectively. (b) Effect of treatment with anti-IL-33 mAb on inflammatory cell number in bronchoalveolar lavage fluid 24 hr after the seventh challenge. (c) Effect of treatment with anti-IL-33 mAb on increased levels of IL-4 (i), IL-5 (ii) and IL-13 (iii) in the lung tissue supernatant 24 hr after the seventh challenge. (d) Effect of treatment with anti-IL-33 mAb on airway inflammation (i), epithelial area (ii), PAS⁺ area (iii), fibrosis area (iv), total collagen (v) and α-SMA⁺ area (vi) 24 hr after the seventh challenge. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of six to eight animals. **P < 0·01 compared with the NS-C (7th) group. #P < 0·05 and ##P < 0·01 compared with the OE-1 (7th) + rat IgG2a group. Total, all cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.

Figure 3

Effect of anti-ST2 polyclnoal antibody (pAb) on airway inflammation and remodelling after the seventh antigen challenge in IgE-sensitized mice. (a) Experimental protocol for treatment with anti-ST2 pAb. Anti-ST2 pAb was intratracheally administered on days 8, 9, 10 and 15 [OE-1 (7th) + anti-ST2]. Negative and positive controls were non-sensitized-challenged [NS-C (7th)] and OE-1-sensitized-challenged, control goat IgG-treated [OE-1 (7th) + goat IgG] mice, respectively. (b) Effect of treatment with anti-ST2 pAb on inflammatory cell number in bronchoalveolar lavage fluid 24 hr after the seventh challenge. (c) Effect of treatment with anti-ST2 pAb on increased levels of interleukin-4 (IL-4) (i), IL-5 (ii) and IL-13 (iii) in the lung tissue supernatant 24 hr after the seventh challenge. (d) Effect of treatment with anti-ST2 pAb on airway inflammation (i), epithelial area (ii), PAS⁺ area (iii), fibrosis area (iv), total collagen (v) and α-SMA⁺ area (vi) 24 hr after the seventh challenge. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of five to eight animals. #P < 0·05 and ##P < 0·01 compared with the OE-1 (7th) + goat IgG group. Total, all cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.

Figure 4

Effect of 2-chloroadenosine (2-CA) on interleukin-33 (IL-33) production in the lungs after the seventh antigen challenge in IgE-sensitized mice. (a) (A) Representative dot plots depicting the percentages of alveolar macrophages (F4/80⁺ CD11c⁺) and IL-33⁺ alveolar macrophages (F4/80⁺ CD11c⁺ IL-33⁺) in the lungs 24 hr after the fourth challenge in non-sensitized [NS-C (4th)] (i) and IgE-sensitized mice [OE-1 (4th)] (ii). (B) Changes in the number of alveolar macrophages (F4/80⁺ CD11c⁺ cells) (i) and IL-33⁺ alveolar macrophages (ii) in the lungs. (b) Representative dot plots depicting the percentage of F4/80⁺ CD11c⁺ ST2⁺ in the lungs 24 hr after the fourth challenge in NS-C (4th) (i) and OE-1 (4th) (ii) groups. Changes in the number of F4/80⁺ CD11c⁺ ST2⁺ in the lungs (iii). (c) Representative dot plots depicting the percentage of F4/80⁺ CD11c⁺ CD206⁺ in the lungs 24 hr after the fourth challenge in NS-C (4th) (i) and OE-1 (4th) (ii) groups. Changes in the number of F4/80⁺ CD11c⁺ CD206⁺ in the lungs (iii). (d) Representative dot plots depicting the percentage of CD3⁺ CD4⁺ ST2⁺ in the lungs 24 hr after the fourth challenge in NS-C (4th) (i) and OE-1 (4th) (ii) groups. Changes in the number of CD3⁺ CD4⁺ ST2⁺ in the lungs (iii). (e) Experimental protocol for treatment with 2-CA or anti-CD4 mAb. 2-CA was intratracheally administered on days 7, 8, 9 and 14 [OE-1 (7th) + 2-CA]. Anti-CD4 monoclonal antibody (mAb) was intraperitoneally administered on day 7 [OE-1 (7th) + anti-CD4]. Negative and positive controls were non-sensitized-challenged [NS-C (7th)] and OE-1-sensitized-challenged, vehicle or control rat IgG2b-treated [OE-1 (7th)] mice, respectively. (f) Effects of 2-CA (i) and anti-CD4 mAb (ii) on IL-33 production in lung homogenate supernatants 24 hr after the seventh challenge. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of four to seven animals. *P < 0·05 and **P < 0·01 compared with the NS-C (4th) group. #P < 0·05 and ##P < 0·01 compared with the OE-1 (7th) group.

Figure 5

Effect of 2-chloroadenosine (2-CA) on airway inflammation and remodelling after the seventh antigen challenge in IgE-sensitized mice. (a) Effect of treatment with 2-CA on inflammatory cell number in bronchoalveolar lavage fluid 24 hr after the seventh challenge. (b) Effect of treatment with 2-CA on increased levels of interleukin-4 (IL-4) (i), IL-5 (ii) and IL-13 (iii) in the lung tissue supernatant 24 hr after the seventh challenge. (c) Effect of treatment with 2-CA on airway inflammation (i), epithelial area (ii), PAS⁺ area (iii), fibrosis area (iv), total collagen (v) and α-SMA⁺ area (vi) 24 hr after the seventh challenge. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of six or seven animals. #P < 0·05 and ##P < 0·01 compared with the OE-1 (7th) + vehicle group. Total, all cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.

Figure 6

Effect of anti-CD4 monoclonal antibody (mAb) on airway inflammation and remodelling after the seventh antigen challenge in IgE-sensitized mice. (a) Effect of treatment with anti-CD4 mAb on inflammatory cell number in bronchoalveolar lavage fluid 24 hr after the seventh challenge. (b) Effect of treatment with anti-CD4 mAb on increased levels of interleukin-4 (IL-4) (i), IL-5 (ii) and IL-13 (iii) in the lung tissue supernatant 24 hr after the seventh challenge. (c) Effect of treatment with anti-CD4 mAb on airway inflammation (i), epithelial area (ii), PAS⁺ area (iii), fibrosis area (iv), total collagen (v) and α-SMA⁺ area (vi) 24 hr after the seventh challenge. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of five to seven animals. #P < 0·05 and ##P < 0·01 compared with the OE-1 (7th) + rat IgG2b group. Total, all cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.

Figure 7

Effect of 2-chloroadenosine (2-CA) on interleukin-33 (IL-33) -induced airway inflammation and remodelling in mice. (a) Experimental protocol for treatment with 2-chloroadenosine (2CA). The 2-CA was intratracheally administered on days –1, 0, 1 and 6 (IL-33 + 2-CA). Negative and positive controls were vehicle-administered (vehicle) and IL-33-administered, vehicle-treated (IL-33 + vehicle) mice, respectively. (b) Effect of treatment with 2-CA on inflammatory cell number in bronchoalveolar lavage fluid 24 hr after the last administration of IL-33. (c) Effect of treatment with 2-CA on increased levels of IL-4 (i), IL-5 (ii) and IL-13 (iii) in the lung tissue supernatant 24 hr after the last administration. (d) Effect of treatment with 2-CA on airway inflammation (i), epithelial area (ii), PAS⁺ area (iii), fibrosis area (iv), total collagen (v) and α-SMA⁺ area (vi) 24 hr after the last administration of IL-33. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of six animals. **P < 0·01 compared with the vehicle group. #P < 0·05 and ##P < 0·01 compared with the IL-33 + vehicle group. Total, all cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.

Figure 8

Effect of anti-CD4 monoclonal antibody (mAb) on interleukin-33 (IL-33) -induced airway inflammation and remodelling in mice. (a) Experimental protocol for treatment with anti-CD4 mAb. Anti-CD4 mAb was intraperitoneally administered on day -1 (IL-33 + anti-CD4). Negative and positive controls were vehicle-administered (vehicle) and IL-33-administered, control rat IgG2b-treated (IL-33 + rat IgG2b) mice, respectively. (b) Effect of treatment with anti-CD4 mAb on inflammatory cell number in bronchoalveolar lavage fluid 24 hr after the last administration of IL-33. (c) Effect of treatment with anti-CD4 mAb on increased levels of IL-4 (i), IL-5 (ii) and IL-13 (iii) in the lung tissue supernatant 24 hr after the last administration. (d) Effect of treatment with anti-CD4 mAb on airway inflammation (i), epithelial area (ii), PAS⁺ area (iii), fibrosis area (iv), total collagen (v) and α-SMA⁺ area (vi) 24 hr after the last administration of IL-33. Results shown are from one experiment representative of two independent trials. Each value is the mean ± SEM of six animals. #P < 0·05 and ##P < 0·01 compared with the IL-33 + rat IgG2b group. Total, all cells; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils.