Selective induction of tissue inhibitor of metalloproteinase-1 in bleomycin-induced pulmonary fibrosis

Tissue inhibitors of metalloproteinases (TIMPs) are multifunctional proteins that have the capacity to modify cellular activities and to modulate matrix turnover. We demonstrate that TIMP-1 messenger RNA (mRNA) and protein expression are selectively and markedly increased in a murine model of bleomycin-induced pulmonary fibrosis. Northern analysis showed that lung steady-state TIMP-1 mRNA levels increased 14-fold after bleomycin administration compared with control mice. Expression of the genes for TIMP-2, TIMP-3, and interstitial collagenase (matrix metalloproteinase-13) was unaltered in the injured lung. In situ hybridization demonstrated that TIMP-1 gene induction was spatially restricted to areas of lung injury. Metalloproteinase inhibitory activity of relative molecular mass of similar to 21 to 28 kD, corresponding to the molecular weights for TIMP-1 and TIMP-2, was identified in lung extracts of bleomycin-injured mice by reverse zymography. Western analysis demonstrated that TIMP-1 protein levels in bronchoalveolar lavage fluid (BALF) of bleomycin-treated mice increased 220- and 151-fold at Days 4 and 28, respectively, compared with control mice. TIMP-2 immunoreactive protein in the BALF increased 20- and 103-fold relative to controls at Days 4 and 28, respectively. These results demonstrate that TIMP-1 gene expression is selectively increased, and that the expression of TIMP-1 and TIMP-2 is differentially regulated in bleomycin-induced pulmonary fibrosis. The profound and durable increase in TIMP-1 and TIMP-2 proteins suggests an important regulatory role for these antiproteases in the inflammatory and fibrotic responses to bleomycin-induced lung injury.