Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth factor-beta, prevents radiation-induced lung fibrosis

Abstract

Rationale: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta activation is a logical strategy to treat lung fibrosis.

Objectives: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on alpha(v)beta6.

Methods: Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation.

Measurements and main results: Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis.

Conclusions: Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy.

Figures

Figure 1.

αvβ6 Expression in the lung after irradiation. (A) Lungs of irradiated wild-type mice were immunostained with an antibody (ch2A1) specific for the β6 integrin subunit. αvβ6 expression increases at 18 weeks postirradiation. (B) The αvβ6 is highly expressed in fibrotic areas. At 24 weeks postirradiation, immunostaining is also intense in nonfibrotic areas. At 27 weeks postirradiation, immunostaining is less evident in nonfibrotic areas, but remains intense in cells within fibrotic lesions.

Figure 2.

Mice lacking the αvβ6 integrin do not develop radiation-induced lung fibrosis. (A) Representative lungs from Itgb6+/+ and Itgb6−/− mice (27 wk postirradiation) stained with Masson's trichrome. (B) Hydroxyproline content of lungs obtained from Itgb6+/+ and Itgb6−/− mice (27 wk postirradiation). Hydroxyproline content of irradiated Itgb6+/+ lungs is significantly greater than that of unirradiated Itgb6+/+ lungs and of irradiated and unirradiated Itgb6−/− lungs (*P < 0.03 vs. irradiated Itgb6+/+; **P < 0.02 vs. irradiated Itgb6+/+; n = 5–6 for each group). Error bars represent 1 SEM. (C) Kaplan-Meier curves for Itgb6+/+ and Itgb6−/− mice treated with a 14-Gy thoracic radiation.

Figure 3.

Effect of the anti-αvβ6 monoclonal antibody (mAb), 6.3G9, on fibrosis, lung inflammation, and survival after lung irradiation. Mice were irradiated with 14 Gy to the thorax. Weekly intraperitoneal injections with control IgG, control phosphate-buffered saline (PBS), anti-αvβ6 mAb (6.3G9), or recombinant soluble transforming growth factor-β receptor II–Fc fusion protein (rsTGF-βRII–Fc) were started 16 weeks postirradiation (n = 14–27 per group). Doses of 6.3G9 are shown; rsTGF-βRII–Fc doses were 5 mg/kg/week. (A) Pooled results for mice that died between 20 and 26 weeks postirradiation and mice killed at 26 weeks postirradiation. The percent fibrosis area (%FA) is significantly reduced in mice treated with 1 or 10 mg/kg/week 6.3G9 or with rsTGF-βRII–Fc, compared with IgG control (P < 0.001 compared with control IgG). (B) Differential counts of cells in bronchoalveolar lavage (BAL) fluid from mice killed 26 weeks postirradiation. (C) Kaplan-Meier curves for the different treatment groups during the treatment phase. Survival curves do not differ significantly in a composite analysis (P = 0.088 by log-rank test). (D) Representative cytospins of BAL cells. Enlarged macrophages and increases in other inflammatory cell types are evident in BAL fluid from mice treated with 10 mg/kg/week 6.3G9. Error bars represent 1 SEM.

Figure 4.

Analysis of additional treatment doses of anti-αvβ6 monoclonal antibody (mAb). Weekly subcutaneous injections with anti-αvβ6 mAb (6.3G9) or control IgG were started 16 weeks postirradiation (n = 14–27 per group). (A) Representative fibrotic lesions in two control antibody–treated, killed mice. The dashed line indicates the extent of the lesion. The percent fibrosis area (%FA) for the entire lung section is shown to the left. (B) Pooled results for mice that died between 20 and 32 weeks postirradiation and mice killed at either 28 or 32 weeks postirradiation. The %FA is significantly reduced by all doses of 6.3G9. (C) Differential counts of cells in BAL fluid obtained from mice killed at 28 or 32 weeks postirradiation. (D) Kaplan-Meier curves for different treatment groups during the treatment phase. Survival curves differed significantly in a composite analysis (P < 0.005 by log-rank test). (E) Comparison of right ventricle (RV):left ventricle (LV) mass ratio for mice that died between 28 and 32 weeks postirradiation versus mice that survived to be killed at 32 weeks. Results for all mice are shown on left with data from unirradiated controls. On right are data for mice treated with either control IgG or various doses of 6.3G9. For all groupings shown, mice that died had significantly increased RV:LV ratio compared with surviving mice (P < 0.0007) or unirradiated (P < 0.02) mice. (F) Tgfb1+/+ and Tgfb1+/RGE mice were irradiated and killed 26 weeks later (n = 8 and 12, respectively). The %FA was significantly reduced in the mice heterozygous for the Tgfb1 mutation (P < 0.001). Error bars represent 1 SEM.

Figure 5.

Differential effects of high and low doses of 6.3G9 on bronchoalveolar lavage (BAL) fluid protein concentrations. (A) Nonirradiated mice were treated for 4 weeks with control monoclonal antibody (mAb) or the indicated doses of 6.3G9, followed by measurement of concentrations of the indicated proteins in BAL fluid (n = 5–8 per group). (B and C) Irradiated mice were treated for 12 or 16 weeks with control mAb or indicated doses of 6.3G9, beginning 16 weeks after irradiation. The concentrations of the indicated proteins in BAL fluid obtained from these mice were then measured. *P < 0.05, **P < 0.01, compared with control antibody treatment (n = 7–13 per group).

Figure 6.

Microarray analysis of gene expression patterns in irradiated mice treated with 6.3G9, recombinant soluble transforming growth factor-β receptor II–Fc fusion protein (rsTGF-βRII–Fc), control IgG, or PBS. Results are from the experiment shown in Figure 3. Treatments were begun 16 weeks after irradiation, and mice were killed at 26 weeks. The selected genes shown were identified as being differentially expressed in nonirradiated mice and irradiated PBS-treated mice, and in PBS- and 1 mg/kg/week 6.3G9–treated mice. Hierarchical clustering of the gene expression data shows similarity among mice treated with PBS, control IgG, or 0.3 mg/kg/week 6.3G9 (a dose that did not prevent fibrosis). In contrast, the expression pattern of these genes in mice treated with 1 mg/kg/week 6.3G9 or rsTGF-βRII–Fc clustered with that of nonirradiated mice. Increased genes are represented by progressively brighter shades of red, decreased genes are represented by progressively brighter shades of green, and unchanged genes by black.