Tissue inhibitor of metalloproteinase-1 deficiency amplifies acute lung injury in bleomycin-exposed mice

Abstract

Bleomycin-induced lung injury triggers a profound and durable increase in tissue inhibitor of metalloproteinase (TIMP)-1 expression, suggesting a potential role for this antiproteinase in the regulation of lung inflammation and fibrosis. TIMP-1 protein induction is spatially restricted to areas of lung injury as determined by immunohistochemistry. Using TIMP-1 null mutation mice, we demonstrate that TIMP-1 deficiency amplifies acute lung injury as determined by exaggerated pulmonary neutrophilia, hemorrhage, and vascular permeability compared with wild-type littermates after bleomycin exposure. The augmented pulmonary neutrophilia observed in TIMP-1-deficient animals was not found in similarly treated TIMP-2-deficient mice. Using TIMP-1 bone marrow (BM) chimeric mice, we observed that the TIMP-1-deficient phenotype was abolished in wild-type recipients of TIMP-1-deficient BM but not in TIMP-1-deficient recipients of wild-type BM. Acute lung injury in TIMP-1-deficient mice was accompanied by exaggerated gelatinase-B activity in the alveolar compartment. TIMP-1 deficiency did not alter neutrophil chemotactic factor accumulation in the injured lung nor neutrophil migration in response to chemotactic stimuli in vivo or in vitro. Moreover, TIMP-1 deficiency did not modify collagen accumulation after bleomycin injury. Our results provide direct evidence that TIMP-1 contributes significantly to the regulation of acute lung injury, functioning to limit inflammation and lung permeability.

Figure 1.

Temporal changes in neutrophil percent and concentration in the BAL recovered from wild-type and TIMP-1^−/− mice after bleomycin administration. Mean values (± SE) for neutrophil percent (A) and neutrophil concentration (B) in the BAL of TIMP-1^+/+ (open squares) and TIMP-1^−/− (filled diamonds) mice before or after a single intratracheal dose of bleomycin. Six TIMP-1^+/+ and six TIMP-1^−/− mice were analyzed at each time point. *P < 0.05, **P < 0.01. Saline-instilled mice had < 6,000 PMN/ml recovered in the BAL at Day 7 for each genotype.

Figure 2.

Neutrophil percent and concentration in the BAL of wild-type, TIMP-1^−/−, and TIMP-2^−/− mice at Day 7 after bleomycin administration. Mean values (± SE) for neutrophil percent (A) and neutrophil concentration (B) in the BAL of wild-type (white column), TIMP-1^−/− (black column), and TIMP-2^−/− (striped column) mice at Day 7 after a single intratracheal dose of bleomycin. Six mice were analyzed for each genotype. *P < 0.05, comparison of TIMP-1^+/+ with TIMP-1^−/−; **P < 0.001 comparison of TIMP-1^−/− with TIMP-2^−/−.

Figure 3.

Lung histology for TIMP-1^−/− and wild-type mice at Day 7 after saline or bleomycin administration. (A) TIMP-1^−/− mouse lung at Day 7 after saline instillation shows little evidence of inflammation. a, airway; v, blood vessel (H&E, magnification: ×400; bar = 50 μm). (B) TIMP-1^−/− mouse lung at Day 7 after bleomycin instillation shows extensive inflammatory cell accumulation in the peri-airway regions (H&E, magnification: ×400; bar = 50 μm). (C) Higher magnification of image in B stained with chloroacetate ester shows extensive neutrophil accumulation (arrowheads) in the alveolar compartment at Day 7 after bleomycin administration to TIMP-1^−/− mice. (Leder stain, magnification: ×1,000; bar = 10 μm). (D) Wild-type mouse lung at Day 7 after bleomycin instillation shows predominantly mononuclear cell infiltration (arrows) rather than neutrophil accumulation (arrowheads) in the alveolar compartment. (Leder stain, magnification: ×1,000; bar = 10 μm).

Figure 4.

Morphometric analysis of leukocyte and erythrocyte concentrations in the lungs of TIMP-1^−/− and wild-type mice at Day 7 after bleomycin instillation. Mean values (± SE) for neutrophils, mononuclear inflammatory cells, and erythocytes within 40,000 μm² areas centered on medium-sized bronchovascular bundles (airway diameter ∼ 100 μm) for TIMP-1^+/+ (white columns) and TIMP-1^−/− (black columns) at Day 7 after bleomycin instillation. Three 40,000 μm² areas were analyzed for each of five TIMP-1^+/+ and five TIMP-1^−/− mice. *P < 0.001, **P < 0.01.

Figure 5.

Localization of TIMP-1 protein by immunohistochemistry in bleomycin-injured and control mouse lung. (A) Wild-type mouse lung at Day 5 after bleomycin instillation incubated with antibody to murine TIMP-1 demonstrates strong TIMP-1 immunostaining of airway epithelial cells, alveolar macrophages (arrows), and lung interstitial cells (arrowheads) within a focus of injury in the peri-airway region. a, airway; v, blood vessel (magnification: ×400; bar = 50 μm). (B) Higher magnification of A shows that TIMP-1 protein localizes to alveolar macrophages (arrows) and lung interstitial cells (arrowheads) within the focus of lung injury (magnification: ×1,000; bar = 10 μm). (C) Wild-type mouse lung at Day 5 after bleomycin instillation incubated with murine TIMP-1 antibody demonstrates only occasional immunostaining of macrophages (arrow) in an area of lung remote from damage (magnification: ×400; bar = 50 μm). (D) Wild-type mouse lung at Day 5 after bleomycin administration incubated with isotype control antibody shows no background staining within a focus of injury (magnification: ×400; bar = 50 μm).

Figure 6.

Neutrophil concentration in the BAL of TIMP-1 BM chimeric mice at Day 7 after bleomycin administration. Mean values (± SE) for neutrophil concentration in the BAL of TIMP-1^+/+ recipients of TIMP-1^+/+ BM (white column), TIMP-1^−/− recipients of TIMP-1^−/− BM (black column), TIMP-1^−/− recipients of TIMP-1 ^+/+ BM (striped column), and TIMP-1^+/+ recipients of TIMP-1^−/− BM (checked column) at Day 7 after a single intratracheal dose of bleomycin. At least seven mice were analyzed per group. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 7.

Temporal changes in albumin concentration in the BAL recovered from wild-type and TIMP-1^−/− mice after bleomycin administration. Mean values (± SE) for albumin concentration in the BAL of TIMP-1^+/+ (open squares) and TIMP-1^−/− (solid diamonds) mice before or after a single intratracheal dose of bleomycin. Six TIMP-1^+/+ and six TIMP-1^−/− mice were analyzed at each time point. *P < 0.03.