Abstract
Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation, all of which play important roles in inflammation, are themselves induced by various growth factors and cytokines. Less is known about the interaction of these substances on lung fibroblast function in pulmonary fibrosis. The goal of this study was to investigate the effects of PDGF alone and in combination with IL-1beta and TNF-alpha on the production of human lung fibroblast matrix metalloproteinases, proliferation, and the chemotactic response. The assay for MMPs activity against FITC labeled type I and IV collagen was based on the specificity of the enzyme cleavage of collagen. Caseinolytis and gelatinolytic activities of secreted proteinases were analyzed by zymography. Fibronectin in conditioned media was measured using human lung fibronectin enzyme immunoassay. Cell proliferation was measured by 3H-Thymidine incorporation assay. Cell culture supernatants were tested for PGE2 content by ELISA. Chemotactic activity was measured using the modified Boyden chamber. Matrix metalloproteinase assay indicated that IL-1beta, TNF-alpha and PDGF induced intestitial collagenase (MMP-1) production. MMP assay also indicated that IL-1beta and TNF-alpha had inhibitory effects on MMP-2,9(gelatinaseA,B) production. Casein zymography confirmed that IL-1beta stimulated stromlysin (matrix metalloproteinase 3; MMP-3) and gelatin zymography demonstrated that TNF-alpha induced MMP-9 production in human lung fibroblast, whereas PDGF alone did not. PDGF in combination with IL-1beta and TNF-alpha induced MMP-3 and MMP-9 activity, as demonstrated by zymography. PDGF stimulated lung fibroblast proliferation in a concentration-dependent manner, whereas IL-1beta and TNF-alpha alone had no effect. In contrast, the proliferation of human lung fibroblasts by PDGF was inhibited in the presence of IL-1beta and TNF-alpha, and this inhibition was not a consequence of any elevation of PGE2. PDGF stimulated fibroblast chemotaxis in a concentration-dependent manner, and this stimulation was augmented by combining PDGF with IL-1beta and TNF-alpha. These findings suggested that PDGF differentially regulated MMPs production in combination with cytokines, and further that MMP assay and zymography had differential sensitivity for detecting MMPs. The presence of cytokines with PDGF appears to modulate the proliferation and chemotaxis of human lung fibroblasts.
Highlights
Pulmonary fibrosis and other fibrotic lung diseases are characterized by diffuse intestitial inflammation and exaggerated collagen accumulation
The fibrotic process is thought to be initiated by a variety of events following cell migration including extracellular matrix degradation, Table 1
Of particular interest is the role of PDGF in this process given the recently reported finding that PDGF-like proteins are localized in macrophages and epithelial cells of patients with idiopathic pulmonary fibrosis.[17,18,19,20,21]
Summary
Pulmonary fibrosis and other fibrotic lung diseases are characterized by diffuse intestitial inflammation and exaggerated collagen accumulation. This in turn leads to destruction of alveolar structures and remodeling.[1]. Fibroblast migration, proliferation, extracellular matrix protein synthesis and degradation, all of which play important roles in inflammation, are themselves induced by various cytokines, such as IL–1b and TNF-a, and growth factors, such as TGF and PDGF, as well as by the combined use of these agents.[2,3,4,5,6,7,8] Less is known about the interaction of Recent studies have shown that some extracellular matrix components, including collagen, fibronectin and cytokines (e.g. PDGF and TGF) have an effect on fibroblast chemotactic responses.[3,8] The role of PDGF in combination with cytokines in fibroblast collagen production or degradation is unclear. By using a Boyden chamber to assay chemotaxis, the migration of fibroblasts across collagen-coated filters was shown to be a response to proteinase products of fibroblasts as well as to the products of malignant cells.[9,10] Stromelysin (matrix metalloproteinase–3, MMP–3) and the type IV procollagenase (matrix metalloproteinase–9, gelatinase B) are known to degrade a wide range of extracellular matrix molecules, including proteoglycan, gelatin, type IV and V collagen, and fibronectin
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