Tissue inhibitor of metalloproteinase-1 deficiency abrogates obliterative airway disease after heterotopic tracheal transplantation

Abstract

Obliterative bronchiolitis (OB) is a major cause of allograft dysfunction after lung transplantation and is thought to result from immunologically mediated airway epithelial destruction and luminal fibrosis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the regulation of lung inflammation, airway epithelial repair, and extracellular matrix remodeling and therefore may participate in the pathogenesis of OB. The goals of this study were to determine the expression profiles of MMPs and TIMPs and the role of TIMP-1 in the development of airway obliteration using the murine heterotopic tracheal transplant model of OB. We demonstrate the selective induction of MMP-3, MMP-9, MMP-12, and TIMP-1 in a temporally restricted manner in tracheal allografts compared with isografts. In contrast, the expression of MMP-7, TIMP-2, and TIMP-3 was decreased in allografts relative to isografts during the period of graft rejection. TIMP-1 protein localized to epithelial, mesenchymal, and inflammatory cells in the tracheal grafts in a temporally and spatially restricted manner. Using TIMP-1-deficient mice, we demonstrate that the absence of TIMP-1 in the donor trachea or the allograft recipient reduced luminal obliteration and increased re-epithelialization in the allograft compared with wild-type control at 28 d after transplantation. Our findings provide direct evidence that TIMP-1 contributes to the development of airway fibrosis in the heterotopic tracheal transplant model, and suggest a potential role for this proteinase inhibitor in the pathogenesis of OB in patients with lung transplant.

Figure 1.

Tracheal histology at Day 28 after transplantation. (A) Isografts had patent lumens and were fully re-epithelialized with a ciliated pseudo-stratified, columnar epithelium (arrow) (H&E; bar = 50 μm; original magnification: ×200; insets ×20). (B) Allografts develop OAD with inflammatory and mesenchymal cell infiltration into the obliterated lumen. An epithelium is not present, and the basement membrane (arrowhead) is pulled away from the cartilage (*) (H&E; bar = 50 μm; original magnification: ×200; insets ×20). (C) Morphometric analysis of luminal obliteration. Mean values (± SE) for percentage of luminal obliteration of isografts (white column) and allografts (black column) after tracheal transplantation. Five transplanted tracheas were analyzed at each time point for each group. *P < 0.05; ^†P < 0.0005. (D) Morphometric analysis of re-epithelialization. Mean values (± SE) for percentage of the tracheal lumen covered with epithelial cells for isografts (white column) and allografts (black column) after tracheal transplantation. Five transplanted tracheas were analyzed at each time point for each group. *P < 0.005; ^†P < 0.001.

Figure 2.

Temporal changes in MMP-3, MMP-9, MMP-12, and MMP-7 steady-state mRNA levels after tracheal transplantation. Mean values (± SE) for the signal intensity for MMP-3 (A), MMP-12 (B), MMP-9 (C), and MMP-7 (D) of normal tracheas (white column), isografts (gray column), and allografts (black column) expressed as arbitrary units (A.U.) after normalization to β-actin. Six normal tracheas, five isografts, and five allografts were analyzed at each time point. *P < 0.05; ^†P < 0.01 of allografts compared with isografts.

Figure 3.

Temporal changes in TIMP-1, TIMP-2, and TIMP-3 steady-state mRNA levels after tracheal transplantation. Mean values (± SE) for the signal intensity for TIMP-1 (A), TIMP-3 (B), and TIMP-2 (C) of normal tracheas (white column), isografts (gray column), and allografts (black column) expressed as arbitrary units (A.U.) after normalization to β-actin. Six normal tracheas, five isografts, and five allografts were analyzed at each time point. *P < 0.05; ^†P < 0.01 of allografts compared with isografts.

Figure 4.

Localization of TIMP-1 protein by immunohistochemistry in isografts, allografts, and normal trachea. (A) Tracheal allograft at Day 7 incubated with TIMP-1 antibody shows prominent TIMP-1 immunostaining of the flattened epithelium (arrow) along with fibroblasts in the submucosa and chondrocytes (bar = 50 μm; original magnification: ×400). (B) Allograft at Day 14 incubated with TIMP-1 antibody demonstrates strong TIMP-1 positivity in the metaplastic epithelium (arrow) (bar = 50 μm; original magnification: ×400). (C) Allograft at Day 28 stained with TIMP-1 antibody shows TIMP-1 immunostaining of inflammatory cells and fibroblasts in the submucosa and tracheal lumen (arrowheads) (bar = 50 μm; original magnification: ×400). (D) Tracheal isograft at Day 7 after transplantation incubated with antibody to murine TIMP-1 demonstrates faint TIMP-1 immunostaining (brown) of airway epithelial cells and infrequent staining of chondrocytes and fibroblasts in the submucosa (bar = 50 μm; original magnification: ×400). (E) Isograft at Day 14 after transplantation in- cubated with TIMP-1 antibody shows TIMP-1 immunostaining of fully differentiated ciliated epithelium (dashed arrow) (bar = 50 μm; original magnification: ×400). (F) Isograft at Day 28 after transplantation incubated with TIMP-1 antibody shows strong TIMP-1 immunostaining of the restored tracheal epithelium (dashed arrow) (bar = 50 μm; original magnification: ×400). (G) Normal trachea incubated with TIMP-1 antibody shows TIMP-1 protein localized to the intact tracheal epithelium (dashed arrow) (bar = 50 μm; original magnification: ×400). (H) Allograft at Day 14 incubated with isotype control antibody shows no background staining (bar = 50 μm; original magnification: ×400).

Figure 5.

Tracheal allografts recovered from TIMP-1–deficient recipients have decreased luminal obliteration and increased re-epithelialization at Day 28 after transplantation. (A) Wild-type allograft recovered from a TIMP-1 −/− recipient after 28 d shows epithelial cells covering large portions of the basement membrane (arrowhead) and minimal inflammation in the submucosa between the epithelium and cartilage (*) (H&E; bar = 50 μm; original magnification: ×200; insets ×20). (B) Wild-type allograft recovered from a wild-type recipient after 28 d demonstrates no identifiable epithelium, prominent inflammatory cells, and fibroblasts in the submucosa and luminal obliteration. The basement membrane (arrowhead) is pulled away from the cartilage (*) (H&E; bar = 50 μm; original magnification: ×200; insets ×20). (C) Morphometric analysis of luminal obliteration. Mean values (± SE) for percentage of luminal obliteration of wild-type allografts recovered from TIMP-1−/− (white column) and wild-type (black column) recipients at Day 28 after tracheal transplantation. Allografts from four TIMP-1 −/− and five wild-type recipients were analyzed. *P < 0.05. (D) Morphometric analysis of re-epithelialization. Mean values (± SE) for percentage of the tracheal lumen covered with epithelial cells for wild-type allografts recovered from TIMP-1−/− (white column) and wild-type (black column) recipients at Day 28 after tracheal transplantation. Allografts from four TIMP-1 −/− and five wild-type recipients were analyzed. *P < 0.05.

Figure 6.

TIMP-1 deficiency in donor tracheas decreased luminal obliteration and increased re-epithelialization of allografts at Day 28 after transplantation. (A) TIMP-1 −/− trachea recovered from wild-type recipient 28 d after allograft transplantation shows squamous epithelial cells covering the basement membrane (arrowhead) with minimal inflammation seen in the submucosa between the epithelium and cartilage (*) (H&E; bar = 50 μm; original magnification: ×200; insets ×20). (B) Wild-type trachea recovered from wild-type recipient 28 d after allograft transplantation shows no identifiable epithelium and inflammatory cell and fibroblast accumulation in the submucosa and lumen. The basement membrane (arrowhead) is pulled away from the cartilage (*) (H&E; bar = 50 μm; original magnification: ×200; insets ×20). (C) Morphometric analysis of luminal obliteration. Mean values (± SE) for percentage of luminal obliteration of TIMP-1−/− (white column) and wild-type (black column) tracheas recovered from wild-type recipients at Day 28 after transplantation. Four TIMP-1 −/− and five wild-type allografts were analyzed. *P < 0.05. (D) Morphometric analysis of re-epithelialization. Mean values (± SE) for percentage of the lumen covered with epithelial cells for TIMP-1−/− (white column) and wild-type (black column) tracheas recovered from wild-type recipients at Day 28 after transplantation. Four TIMP-1 −/− and five wild-type allografts were analyzed. *P < 0.05.