Introduction TIMP3 expression in the lung increases with age and in ILD. TIMP3 binds extracellular matrix (ECM) where it influences cellular behaviour both via inhibition of MMPs and unique functions including inhibition of ADAM17 mediated release of membrane-bound TNFa. TIMP3 gene polymorphisms protect against hypersensitivity pneumonitis (HP) and TIMP3−/− mice show spontaneous pulmonary airspace enlargement, increased inflammatory responses in models of hepatic injury and arthritis and increased neoangiogenesis. We hypothesise that TIMP3 and TIMP3 gene polymorphisms contribute to inflammatory processes in ILD. This has been investigated by TIMP3 gene SNP discovery and a case-control SNP association study in sarcoidosis. SNP function has been investigated in primary human cells from sarcoidosis patients and controls of known genotype. Finally the response of TIMP3 -/- mice to lung injury with bleomycin has been examined.
Methods SNP discovery: the TIMP3 gene was PCR amplified and sequenced in 22 subjects. Association studies: 175 UK AfroCarribean sarcoidosis patients and 284 controls were genotyped using TaqMan assays. Functional studies: alveolar macrophages (AM) were isolated from bronchoalveolar lavage (BAL), n=18, and monocyte-derived macrophages (MDM) from venous blood, n=14, with real-time PCR of TIMP3 mRNA via TaqMan assay. Animal studies: TIMP3−/− mice and controls were treated with oropharyngeal bleomycin (2 mg/kg) and lungs assessed at 3, 21 and 28 days for BAL, histology, RNA and protein analysis.
Results The TIMP3 gene is conserved with 2 promoter SNPs and 2 synonymous SNPs in exon 3 identified. Carriage of at least one SNP showed a protective effectagainst sarcoidosis, odds ratio (OR) 0.68 (p=0.019) driven by patients <35 years, OR 0.56 (p=0.016). The protective haplotype associates with increased TIMP3 gene expression in AM and MDM (p=0.021). TIMP3−/− mice show increased lymphocytosis in BAL at days 21 and 28 (p=0.001) (Abstract T3 Figure 1) more diffuse injury and increased neoangiogenesis within lesions on histological assessment.
Conclusions TIMP3 promoter SNPs may increase gene expression and appear protective against granulomatous lung disease. Absence of TIMP3 leads to increased lymphocytic inflammation in the bleomycin model. Mechanisms for this are being investigated. Together these findings suggest that TIMP3 restricts lymphocytic inflammation in the lung.