Transforming growth factor-beta (TGF-beta) can regulate cell growth and differentiation as well as production of extracellular matrix proteins. Elevated production of TGF-beta has been associated with human and rodent chronic inflammatory and fibrotic diseases. Using immunohistochemical staining, we have examined lung sections of patients with advanced idiopathic pulmonary fibrosis (IPF), a disease characterized by chronic inflammation and fibrosis and demonstrated a marked and consistent increase in TGF-beta production in epithelial cells and macrophages when compared to patients with nonspecific inflammation and those with no inflammation or fibrosis. In patients with advanced IPF, intracellular staining with anti-LC (1-30) TGF-beta antibody was seen prominently in bronchiolar epithelial cells. In addition, epithelial cells of honeycomb cysts and hyperplastic type II pneumocytes stained intensely. Anti-CC (1-30) TGF-beta antibody, which reacts with extracellular TGF-beta, was localized in the lamina propria of bronchioles and in subepithelial regions of honeycomb cysts in areas of dense fibroconnective tissue deposition. The close association of subepithelial TGF-beta to the intracellular form in advanced IPF suggests that TGF-beta was produced and secreted primarily by epithelial cells. Because of the well-known effects of TGF-beta on extracellular matrix formation and on epithelial cell differentiation, the increased production of TGF-beta in advanced IPF may be pathogenic to the pulmonary fibrotic and regenerative responses seen in this disease.

Although it is recognized that three isoforms of transforming growth factor-beta (TGF-beta) exist in mammals, their expression, distribution, and function in injury and repair are not well characterized. Using immunohistochemistry and antibodies to synthetic peptides of TGF-beta 1, TGF-beta 2, and TGF-beta 3, we determined the distribution of TGF-beta isoforms in lung sections with acute and chronic lesions of idiopathic pulmonary fibrosis (IPF), chronic asbestosis and hypersensitivity pneumonitis, as well as non-specific pneumonitis. In lung sections with advanced pulmonary fibrosis and honeycombing, irrespective of the diagnosis, TGF-beta 1 was prominently expressed in epithelial cells and macrophages and was found to be associated with the extracellular matrix. In lungs with early lesions of IPF and only inflammatory changes, TGF-beta 1 was present in alveolar macrophages but TGF-beta 1 was not present in epithelial cells. Small amounts of matrix-associated TGF-beta 1 were present subepithelially in areas of lung sections from patients with IPF with minimal inflammation and no fibrosis. In normal lungs with no evidence of inflammation or fibrosis TGF-beta 1 was not seen in alveolar macrophages, epithelial cells, or extracellularly. TGF-beta 2 and TGF-beta 3 were expressed in alveolar macrophages, epithelial cells, and smooth muscle cells of vessels and bronchi of normal lungs and lungs with both inflammatory and fibrotic changes. Our findings suggest that while TGF-beta 2 and TGF-beta 3 are ubiquitously expressed in the lung, TGF-beta 1 is expressed in epithelial cells of fibrotic lungs where the presence of TGF-beta 1 is not disease-specific but an indication of the chronicity of the injury.

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal fibrotic lung disease. Transforming growth factor (TGF)-beta1 is present in a biologically active conformation in the epithelial cells lining lesions with advanced IPF. To determine the role of aberrant expression of biologically active TGF-beta1 by alveolar epithelial cells (AECs), the AECs of explanted normal rat lungs were transfected with the TGF-beta1 gene using the retrovirus pMX-L-s223,225-TGF-beta1. In situ hybridization using a digoxigenin-labeled cDNA of the puromycin resistance gene contained in the pMX demonstrated that pMX-L-s233,225-TGF-beta1 was selectively transfected into AECs of the explants. Conditioned media overlying explants obtained 7 days after being treated with pMX-L-s223,225-TGF-beta1 contained 14.5 +/- 3.15 pg/ml of active TGF-beta1. With the use of Masson's trichrome staining of explant sections obtained 14 days after transfection, there were lesions similar to those in IPF, characterized by type II AEC hyperplasia, interstitial thickening, extensive increase in interstitial and subepithelial collagen, an increase in the number of fibroblasts, and areas resembling fibroblast buds. Collagens I, III, IV, and V and fibronectin were increased in explants treated with pMX-L-s223,225-TGF-beta1. The findings in the current study suggest that IPF may be a disorder of epithelial cells and not inflammatory cells.

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Transforming growth factor-beta-1 (TGF-beta1) is secreted by cells in a latent form (L-TGF-beta1) noncovalently bound to a latency-associated peptide. Activated alveolar macrophages obtained from rat lungs after bleomycin-induced pulmonary injury released increased amounts of active TGF-beta1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- beta1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- beta1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-beta1, indicating that TSP-1/L-TGF-beta1 complexes are present on alveolar macrophages. However, in the presence of plasmin both latency-associated peptide and TGF-beta1 were decreased in the same cell lysates, indicating that L-TGF-beta1 associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-beta1 and CD36, a receptor for TSP-1, there was colocalization of TGF-beta1 with CD36. Because TSP-1 but not TGF-beta1 is a natural ligand for CD36, these findings suggest that the L-TGF-beta1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-beta1 complex with CD36 is necessary to the activation of L-TGF-beta1 because antibodies to CD36 prevent the colocalization of TGF-beta1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-beta1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-beta1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction.