Induced sputum-retrieved matrix metalloproteinase 9 and tissue metalloproteinase inhibitor 1 in granulomatous diseases

Abstract

Matrix metalloproteinases (MMPs) capable of degrading various components of connective tissue matrices, and tissue inhibitor metalloproteinases (TIMPs) are considered important in lung parenchymal remodeling and repair processes in pulmonary diseases. Induced sputum (IS) is a reliable noninvasive method to investigate pathogenesis, pathophysiology and treatment of lung disease. This study was designed to determine whether IS-MMP9/TIMP1 levels demonstrate lung parenchymal remodeling in sarcoidosis (SA) and Crohn's disease (CRD) patients. Sputum was induced and processed conventionally in 13 SA patients, 18 CRD patients and 9 controls. Two-hundred cells were counted on Giemsa-stained cytopreps, and T lymphocytes subsets (CD4 = T helper and CD8 = T suppressor cytotoxic cells) were analysed by FACS using monoclonal antibodies.MMP-9 and TIMP-1 were measured using commercial ELISA kits. MMP-9 concentrations, but not those of TIMP-1, were significantly greater in the sputum supernatant in SA and CRD patients compared to controls (P = 0.018 and P = 0.0019, respectively). The molar ratio, MMP-9/TIMP-1, was significantly higher in SA and CRD patients compared to controls (P = 0.008 and P = 0.024, respectively). Gelatinase species having a molecular weight similar to that of MMP-9 were demonstrated by zymographic analysis. MMP-9 levels were highly correlated with the CD4/CD8 ratio and DLCO capacity in SA but less in CRD patients. MMP-9 levels in IS provide a sensitive marker for pulmonary damage.

Fig. 1

Differential cell counts. Results are expressed as percentage of 200 cells counted in a Giemsa-stained cytoprep as described in Materials and methods. ▪ Crohn's disease; □ Sarcoidosis; Controls. Eos, eosinophils; Mast, metachromatic cells; Lymph, lymphocytes; Mac, macrophages; neut, neutrophils.

Fig. 2

MMP-9 (a) and TIMP-1 (b) concentrations in sputum samples. Supernatants were recovered and mediators expressed in ng/ml were measured as described in Materials and methods. ▪ Crohn's disease; ▴ Sarcoidosis; • Controls.

Fig. 3

MMP-9/TIMP-1 molar ratio in sputum samples. ▪ Crohn's disease; ▴ Sarcoidosis; • Controls.

Fig. 4

Correlation between cellular and physiological parameters with MMP-9 and TIMP-1: (a) CD4/CD8 ratio and TIMP-1 (ng/ml), r = 0.44, P = 0.024; (b) CD4/CD8 ratio and MMP-9 (ng/ml), r = 0.78, P = 0.002; (c) TIMP-1 (ng/ml) and DLCO capacity, r = 0.56, P = 0.025; (d) MMP-9 (ng/ml) and DLCO capacity, r = 0.64, P = 0.009. Statistical analysis by Spearman's rank test.

Fig. 5

Analysis of MMPs in the induced sputum by zymography. This is a representative zymographic analysis of sputum supernatant samples obtained from one control (lane 1), one patient with Crohn's disease (lane 2) and one patient with Sarcoidosis (lane 3). Gelatin zymography revealed a major band at 92 kD in all lanes. Lane 1 shows the standard.

Fig. 6

Levels of concentrations (pg/ml) by ELISA were plotted against OD (nm) values of the protein performed by zymography.