The Effect of Interferon-gamma on Bleomycin Induced Pulmonary Fibrosis in the Rat

Yoon, Hyoung Kyu; Kim, Yong Hyun; Kwon, Soon Seog; Kim, Young Kyoon; Kim, Kwan Hyung; Moon, Hwa Sik; Park, Sung Hak; Song, Jeong Sup

Abstract

OBJECTIVES
The matrix metalloproteinases (MMPs) that participate in the extracellular matrix metabolism play a important role in the progression of pulmonary fibrosis. The effects of the MMPs are regulated by several factors including Th-1 cytokines, interferon-gamma (IFN-gamma). Up to now, IFN-gamma is known to inhibit pulmonary fibrosis, but little is known regarding the exact effect of IFN-gamma on the regulation of the MMPs. This study investigated the effects of interferon-gamma on the pulmonary fibrosis and the expression of the lung MMP-2,-9, TIMP-1,-2, and Th-2 cytokines in aa rat model of bleomycin induced pulmonary fibrosis.
MATERIALS AND METHODS
Male, specific pathogen-free Sprague-Dawley rats were subjected to an intratracheal bleomycin instillation. The rats were randomized to a saline control, a bleomycin treated, and a bleomycin+IFN-gamma treated group. The bleomycin+IFN-gamma treated group was subjected to an intramuscular injection of IFN-gamma for 14 days. At 3, 7, 14, and 28 days after the bleomycin instillation, the rats were sacrificed and the lungs were harvested. In order to evaluate the effects of the IFN-gamma on lung fibrosis and inflammation, the lung hydroxyproline content, inflammation and fibrosis score were measured. Western blotting, zymography and reverse zymography were performed at 3, 7, 14, 28 days after bleomycin instillation in order to evaluate the MMP-2,-9, and TIMP-1,-2 expression level. ELISA was performed to determine the IL-4 and IL-13 level in a lung homogenate.
RESULTS
1.7 days after bleomycin instillation, inflammatory changes were more severe in the bleomycin+IFN-gamma group than the bleomycin group (bleomycin group : bleomycin+IFN-gamma group=2.08 +/- 0.15 : 2.74 +/- 0.29, P<0.05), but 28 days after bleomycin instillation, lung fibrosis was significantly reduced as a result of the IFN-gamma treatment (bleomycin group : bleomycin+IFN-gamma group=3.94 +/- 0.43 : 2.46 +/- 0.13, P<0.05). 2. 28 days after bleomycin instillation, the lung hydroxyproline content was significantly reduced as a result of IFN-gamma treatment (bleomycin group : bleomycin+IFN-gamma group=294.04 +/- 31.73 micro gram/g : 194.92 +/- 15.51 micro gram/g, P<0.05). 3.Western blotting showed that the MMP-2 level was increased as a result of the bleomycin instillation and highest in the 14 days after bleomycin instillation. 4.In zymography, the active forms of MMP-2 were significantly increased as a result of the IFN-gamma treatment 3 days after the bleomycin instillation, bleomycin+IFN-gamma group (bleomycin group : bleomycin+IFN-gamma group=209.63 +/- 7.60% : 407.66 +/- 85.34%, P<0.05), but 14 days after the bleomycin instillation, the active forms of MMP-2 were significantly reduced as a result of the IFN-gamma treatment (bleomycin group : bleomycin+IFN-gamma group=159.36 +/- 20.93% : 97.23 +/- 12.50%, P<0.05). 5.The IL-4 levels were lower in the bleomycin and bleomycin+IFN-gamma groups but this was not significant, and the IL-13 levels showed no difference between the experiment groups.
CONCLUSION
The author found that lung inflammation was increased in the early period but the pulmonary fibrosis was inhibited in the late stage as a result of IFN-gamma. The inhibition of pulmonary fibrosis by IFN-gamma appeared to be associated with the inhibition of MMP-2 activation by IFN-gamma. Further studies on the mechanism of the regulation of MMP-2 activation and the effects of MMP-2 activation on pulmonary fibrosis is warranted in the future.