PERSPECTIVE
Stressing Fibrogenesis in Cell Culture

Gilbert F. Morris and Arnold R. Brody

Lung Biology Program, Department of Pathology and Laboratory Medicine, Tulane/Xavier Center for Bioenvironmental Research, Tulane University Medical Center, New Orleans, Louisiana

Inhaled fibrous particles deposited in the lung periphery elicit a chronic inflammatory response that leads to production of fibrogenic mediators (1, 2). The fibrogenesis observed in asbestosis or silicosis, despite differences in histopathologic appearance, leads to similar disruptions in lung architecture, with deposition of extracellular matrix and cell proliferation characteristic of interstitial pulmonary fibrosis (IPF) (1). The mechanisms of fibrogenesis induced by inhaled particles are not completely understood but appear to involve both physical and chemical properties of the particles. Pulmonary retention of particles correlates with length, whereas the surfaces promote bioreactivity through adsorptive and catalytic properties (1).

To understand the complexities of IPF, experimental animal models of fiber-induced lung diseases have been developed to serve as a paradigm for human diseases. Fiber-induced lung diseases develop in animal models in patterns similar to those observed in humans (3). Consequently, our understanding of the initial response of the lung to inhaled particles comes primarily from animals exposed to particles at high concentrations for short time periods (4). Observations in rodent models indicate that particle-induced injury of the pulmonary epithelium initiates a cascade of events leading to fibrogenesis (1, 2, 4). Reactive oxygen species (ROS) generated by activated inflammatory cells, by catalysis on the particle surface and by particle-cell interactions, are probably contributors to injury of the epithelium (1, 2). The injured epithelium releases cytokines that initiate macrophage accumulation and begin the process of wound healing. The accumulated alveolar macrophages also secrete inflammatory and fibrogenic mediators, and alveolar type II cells proliferate to regenerate the disrupted epithelium (1, 2, 5). Concurrent proliferation of mesenchymal cells and deposition of extracellular matrix lead to development of the fibrotic scar. The complexity of the fibrogenic process in animal models and cellular heterogeneity of the lung hinder identification of a specific role for any particular mediator, and interactions among these factors further confound the problem.

To reduce the complexity of the analyses of fibrogenesis, some of the events in animal models of pneumoconiosis have been recapitulated in cell culture. As early as the 1960s, cell culture experiments suggested a central role for macrophages in silicosis. Silica was toxic to macrophages in culture, and an extract from silica-exposed macrophages stimulated collagen accumulation by fibroblasts (6). These early findings prompted a model of particle-induced fibrogenesis, in which macrophage death caused by particle phagocytosis released destructive intracellular enzymes, and reuptake of released particles perpetuated the process leading to chronic tissue injury. Many years later, alveolar macrophages from patients with interstitial lung disorders were shown to express elevated levels of transforming growth factor- (TGF-) (7), a potent inducer of extracellular matrix deposition (8). Alveolar macrophages from patients with lung fibrosis were also found to secrete growth factors for lung fibroblasts (9), and subsequent cell culture experiments demonstrated release of platelet-derived growth factor by particle-exposed macrophages (10). These and many more recent findings suggest the currently accepted model wherein particle-activated macrophages and epithelial cells recruit fibroblasts to the sites of injury, stimulate their proliferation, and promote synthesis of extracellular matrix.

Much of the analyses of the response of fibers in cell culture have focused on alveolar macrophages, fibroblasts, and mesothelial cells, with lung epithelial cells receiving less attention because they were more difficult to maintain in vitro. Ideally, cell culture models of fibrosis would assess the effect of fibrogenic agents upon replication, differentiation, and accumulation of connective tissue for each type of parenchymal cell. In the current issue of the AJRCMB, Tsuda and colleagues describe the response of A549 cells, an alveolar epithelial cell line, to fibrous particle exposures in cell culture (11). A novel finding in this report is that cyclic stretching of the particle-exposed cells enhances the fiber pathogenesis, as measured by secretion of interleukin-8 (IL-8), a potent neutrophil chemotactic factor (12). Secretion of IL-8 by fiber-exposed lung epithelial cells in culture is consistent with previous findings (13) and suggests a mechanism by which the proinflammatory response to fibers can be initiated. Coating the fibers with fibronectin, an abundant protein in the extracellular matrix and in serum, increased the pathogenesis of asbestos but not of silica fibers. The authors clarify this disparate result by demonstrating that fibronectin remains tightly bound to asbestos but not to silica. Fibronectin binds to cells by interacting with integrin receptors on the cell surface (14), thus facilitating the interaction between asbestos fibers and the cellular surface. The addition of peptides to block interactions between the fibronectin-coated asbestos fibers and integrins on the cell surface ameliorates the pathogenesis of the fibers (11). The authors conclude from these findings that fiber-induced physical insults on cells are enhanced under dynamic conditions, and therefore, the cyclic motion of the lung increases the physical stress imparted by deposited fibers.

In addition to questions regarding fiber toxicity, the effect of stretching upon pulmonary cell proliferation and differentiation has been explored to understand the potential of fetal breathing movements in influencing lung development. Thus, cyclic mechanical strain of pulmonary epithelial cells in culture enhances surfactant expression (15), increases the proliferative response to fibroblast conditioned medium (16), and influences epithelial cell differentiation (17). These and other findings have led to the postulate that fetal breathing movements trigger cellular proliferation and lung development (18). However, wound healing in the adult may respond differently because mechanical strain appears to inhibit wound repair by airway epithelial cells in culture (19).

It is clear that cell culture assays must be viewed as model systems that do not necessarily reflect just what occurs in vivo. This problem can be ameliorated by a wise choice of cell type. Whether or not primary cells respond in the same manner as cell lines generally needs to be determined. Eventually, cell culture approaches like the one reviewed here (11) may coalesce to broaden our comprehension of fibrogenesis. Understanding the biology of particle-induced fibrogenesis enhances the possibility of therapeutic interventions that may have applications in the treatment of pulmonary fibrosis with a wide range of etiologies.

	Footnotes

(Received in original form June 26, 1999).

Acknowledgments: Work in the authors' laboratories is supported, in part, by research grants from the Louisiana Cancer and Lung Trust Fund Board, the Department of Defense, and Tulane/Xavier Center for Bioenvironmental Research, and NIEHS grant R29 ES07856 (G.F.M.) and NHLBI RO1 HL60532 and NIEHS RO1 ES06766 (A.R.B.).

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