Inhibition of the αvβ6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis

Abstract

A number of growth factors and signaling pathways regulate matrix deposition and fibroblast proliferation in the lung. The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-β (TGF-β) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGF-α) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGF-α specifically in the lung epithelium develop progressive fibrosis accompanied with cachexia, changes in lung mechanics, and marked pleural thickening. Although recent studies demonstrate that EGFR activation modulates the fibroproliferative effects involved in the pathogenesis of TGF-β induced pulmonary fibrosis, in converse, the direct role of EGFR induction of the TGF-β pathway in the lung is unknown. The αvβ6 integrin is an important in vivo activator of TGF-β activation in the lung. Immunohistochemical analysis of αvβ6 protein expression and bronchoalveolar analysis of TGF-β pathway signaling indicates activation of the αvβ6/TGF-β pathway only at later time points after lung fibrosis was already established in the TGF-α model. To determine the contribution of the αvβ6/TGF-β pathway on the progression of established fibrotic disease, TGF-α transgenic mice were administered Dox for 4 wk, which leads to extensive fibrosis; these mice were then treated with a function-blocking anti-αvβ6 antibody with continued administration of Dox for an additional 4 wk. Compared with TGF-α transgenic mice treated with control antibody, αvβ6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the β6 integrin, TGF-α transgenic mice were mated with β6-null mice and the degree of fibrosis was compared in adult mice following 8 wk of Dox administration. Genetic ablation of the β6 integrin attenuated histological and physiological changes in the lungs of TGF-α transgenic mice although a significant degree of fibrosis still developed. In summary, inhibition of the β6 integrin led to a modest, albeit significant, effect on pleural thickening and lung function decline observed with TGF-α-induced pulmonary fibrosis. These data support activation of the αvβ6/TGF-β pathway as a secondary effect contributing to TGF-α-induced pleural fibrosis and suggest a complex contribution of multiple mediators to the maintenance of progressive fibrosis in the lung.

Keywords: TGF-β; epidermal growth factor receptor; integrin; pleural fibrosis; pulmonary fibrosis.

Fig. 1.

α_vβ₆ expression in the lung following TGF-α overexpression. The lungs of TGF-α mice were administered doxycycline (Dox) to induce TGF-α overexpression in the lung epithelium and immunostained with an antibody (ch.2A1) specific for the β₆ subunit. α_vβ₆ expression is increased from controls after 4 wk of Dox and is highly expressed in the epithelium after 8 wk of Dox. No α_vβ₆ expression is detected in the pleural or adventitial fibrotic regions.

Fig. 2.

Activation of the TGF-β pathway after TGF-α-induced fibrosis is established. A: active TGF-β levels in the bronchoalveolar lavage fluid (BALF) from TGF-α mice are significantly elevated from control mice following 6 wk of Dox (n = 4–7 for each group). B: α_vβ₆/TGF-β pathway specific gene transcripts transforming growth factor β-induced (TGFβi), insulin-like growth factor 1 (IGF1), and matrix metalloproteinase-10 (MMP10) are unchanged in TGF-α transgenic mice 4 days after Dox induction compared with control but are significantly upregulated in the lungs at 6 wk on Dox. Real-time PCR analysis was performed on total lung RNA isolated from the lungs.

Fig. 3.

Therapeutic neutralization of α_vβ₆ integrin attenuates the loss of body weight but does not change total lung collagen production. To assess the efficacy of α_vβ₆ inhibition in established fibrosis, TGF-α transgenic mice were treated with the anti-α_vβ₆ monoclonal antibody 6.3G9 and the isotype control antibody 1E6 after 4 wk of Dox, while remaining on Dox for an additional 4 wk. A: Dox-induced expression of TGF-α for 8 wk caused progressive weight loss in isotype control-treated mice (green line), whereas mice treated with anti-α_vβ₆ 4 wk after TGF-α induction demonstrated an attenuation in the fall in body weight (pink line), but weights remained below CCSP/- controls (blue line). Mice were weighed weekly during treatments and no change in bodyweights of mice is represented as 0%. Data are means ± SE (n = 14–20 for each group). B: total lung hydroxyproline levels were elevated in TGF-α transgenic mice and unchanged in mice treated with anti-α_vβ₆ antibodies.

Fig. 4.

Therapeutic neutralization of α_vβ₆ integrin attenuates progression of lung fibrosis. A: representative photomicrographs of lung tissues for each group stained with Masson's trichrome. Top, pleural regions of the lung; bottom, adventitia. Scale bar, 100 μm. B: TGF-α mice treated with anti-α_vβ₆ antibodies administered 4 wk after the initiation of TGF-α induction did not show a significant attenuation in the lung fibrosis score. C: mean pleural thickness was significantly attenuated in anti-α_vβ₆-treated mice. Data are means ± SE (n = 14–20 for each group). Micrographs in A are representative of sections from 10 mice in each group.

Fig. 5.

Therapeutic neutralization of α_vβ₆ integrin attenuates progression of TGF-α-dependent changes in lung mechanics. TGF-α transgenic mice administered anti-α_vβ₆ antibody 4 wk after treatment with Dox demonstrated attenuated increases in airway resistance and tissue elastance and decreases in compliance compared with isotype control-treated CCSP/TGF-α transgenic mice receiving 8 wk of Dox. Data are means ± SE (n = 14–20 for each group).

Fig. 6.

Genetic loss of β₆ integrin attenuates progression of lung fibrosis. TGF-α transgenic mice were mated with β₆-null mice to test the effects of genetic β₆ integrin inhibition at the initiation of lung fibrosis. Representative photomicrographs of lung tissues stained with Masson's trichrome comparing TGF-α transgenic mice in the presence or absence of the β₆ integrin following 8 wk of Dox. Top, pleural regions of the lung; bottom, adventitia. Scale bar, 100 μm. Presented sections are representative of sections from 6–8 mice in each group.

Fig. 7.

Genetic loss of β₆ integrin attenuates progression of lung fibrosis. A: lung fibrosis score is significantly attenuated in TGF-α transgenic mice in the β₆ knockout mice. B: mean pleural thickness was not significantly attenuated in TGF-α transgenic mice in the β₆-nulls. C: total lung soluble collagen levels were elevated in TGF-α transgenic mice and unchanged in TGF-α transgenic mice in the β₆ knockout mice. Data are means ± SE (n = 4–8 for each group).

Fig. 8.

Genetic loss of β₆ integrin attenuates progression of TGF-α-dependent changes in lung mechanics. Increases in airway resistance and tissue elastance were significantly attenuated in TGF-α transgenic mice in the β₆ knockout mice compared with wild-type TGF-α transgenic mice; there were no differences lung compliance. Data are means ± SE (n = 6–8 for each group).