is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 smallinterfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-␤1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-␤1 and reduced several TGF-␤1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-␤1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-␤1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts. lung; repair; transforming growth factor-␤

Therapies that mitigate the fibrotic process may be able to slow progressive loss of function in many lung diseases. Because cyclic adenosine monophosphate is known to regulate fibroblasts, the current study was designed to evaluate the activity of selective phosphodiesterase (PDE) inhibitors on two in vitro fibroblast responses: chemotaxis and contraction of three-dimensional collagen gels. Selective PDE4 inhibitors, rolipram and cilomilast, each inhibited the chemotaxis of human fetal lung fibroblasts (HFL-1) toward fibronectin in the blindwell assay system (control: 100% versus cilomilast [10 microM]: 40.5 +/- 7.3% versus rolipram: [10 microM] 32.1 +/- 2.7% cells/5 high-power fields; P < 0.05, both comparisons). These PDE4 inhibitors also inhibited contraction of three-dimensional collagen gels (control: 100% versus cilomilast: 167.7 +/- 6.9% versus rolipram: 129.9 +/- 1.9% of initial size; P < 0.05, both comparisons). Amrinone, a PDE3 inhibitor, and zaprinast, a PDE5 inhibitor, had no effect in either system. Prostaglandin E(2) (PGE(2)) inhibited both chemotaxis and gel contraction, and the PDE4 inhibitors shifted the PGE(2) concentration-dependence curve to the left in both systems. The inhibition of endogenous PGE(2) production by indomethacin diminished the effects of the PDE4 inhibitors in both chemotaxis and gel contraction, consistent with the concept that the PDE4 inhibitory effects on fibroblasts are related to the presence of cyclic adenosine monophosphate in the cells. In summary, these in vitro results suggest that PDE4 inhibitors may be able to suppress fibroblast activity and, thus, have the potential to block the development of progressive fibrosis.

Asthma is characterized by chronic inflammation of the airway wall with the presence of activated T helper 2 (Th2) lymphocytes. The current study assessed the ability of Th2 cytokines to modulate fibroblast-mediated contraction of collagen gels to determine if Th2 cytokines could contribute to tissue remodeling by altering mesenchymal cell contraction. Human fetal lung fibroblasts, human adult bronchial fibroblasts and human airway smooth muscle cells were cast into native type I collagen gels and allowed to contract in the presence or absence of IL (interleukin)-4, IL-5, IL-10, or IL-13. IL-4 and IL-13 but not IL-5 and IL-10 augmented collagen gel contraction in a concentration-dependent manner. Neither IL-4 nor IL-13 altered fibroblast production of transforming growth factor-beta or fibronectin. Both, however, decreased fibroblast prostaglandin (PG) E(2) release. Decreased PGE(2) release was associated with a decreased expression of cyclooxygenase 1 and 2 protein and mRNA. Indomethacin completely inhibited PGE(2) release and also augmented contraction. IL-4 and IL-13, however, added together with indomethacin further augmented contraction suggesting both a PGE-dependent and a PGE-independent effect. These findings suggest that IL-4 and IL-13 may modulate airway tissue remodeling and, therefore, could play a role in the altered airway connective tissue which characterizes asthma.

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