Search

Tetrathiomolybdate is an anticopper drug with a unique mechanism of action. Tetrathiomolybdate complexes copper to protein and itself, rendering the copper unavailable for cellular uptake. It was originally developed for Wilson's disease, and is now being developed as an antiangiogenic agent for the treatment of cancer. Many angiogenic cytokines require normal levels of copper, and lowered copper levels reduce cytokine signaling while cellular copper requirements are met. Cytokines of fibrosis and inflammation may be similarly copper dependent, since tetrathiomolybdate inhibits bleomycin induced pulmonary inflammation and fibrosis. The basis for this inhibition was evaluated here by examination of tetrathiomolybdate effects on cytokines in lung pathophysiologically important in the bleomycin mouse model of pulmonary damage. Results in mice injected endotracheally with bleomycin confirmed that tetrathiomolybdate therapy was effective in reducing fibrosis. This effect was associated with significant inhibition of bleomycin-induced tumor necrosis factor alpha and transforming growth factor beta expression in lung homogenates. These effects were shown to be independent of one another. This indicates that tetrathiomolybdate therapy can be effective even when fibrosis is at a more chronic stage, wherein inflammatory cytokines are playing a diminishing role. The inhibition of tumor necrosis factor alpha suggests that diseases of tumor necrosis factor alpha overexpression are also potential targets of tetrathiomolybdate therapy.

Cytokines are critical to a myriad of fundamental homeostatic and pathophysiological processes such as fever, wound healing, inflammation, tissue repair and fibrosis. They play important roles in regulating cell function such as proliferation, migration, and matrix synthesis. It is the balance or the net effect of the complex interplay between these mediators, which appears to play a major role in regulating the initiation, progression and resolution of wounds. Wound healing involves a complex process including induction of acute inflammation by the initial injury, followed by parenchymal and mesenchymal cell proliferation, migration, and activation with production and deposition of extracellular matrix. Failure to resolve or abnormal wound healing results in fibrosis. The latter process involves similar cellular interactions via complex cytokine networks, which result in extensive remodeling with heightened extracellular matrix production and their abnormal deposition in the tissue. Various cytokines, both promoting and inhibiting fibrogenesis, have been implicated in the pathogenesis of fibrosis and wound healing. Recent progress in understanding the mechanisms underlying the pathogenesis of fibrosis leads us to expect that inhibitors of pro-fibrogenic cytokines and growth factors may be useful as novel therapeutic agents in controlling undesirable fibrosis. In this review, the role of cytokines in wound healing and fibrosis will be summarized and highlighted with more detailed discussion reserved for the possible points of therapeutic attack in pulmonary fibrosis. In this review, the major cytokines that are in current clinical use will be also discussed. In addition, advances in the application of novel cytokines and anti-cytokines for accelerating wound healing and attenuating fibrosis both at the experimental and the clinical trial levels will be discussed.

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.

Despite abundant evidence documenting the importance of TNF-alpha in the pathogenesis of pulmonary fibrosis, its actual role has not been fully elucidated. Recent observations also indicate that eosinophils found in fibrotic lung express elevated levels of cytokines known to be important in lung fibrosis. These findings suggest a possible role for TNF-alpha in eosinophil recruitment and cytokine expression in this disease. To examine this hypothesis, pulmonary fibrosis was induced in mice by endotracheal bleomycin treatment, and separate groups of animals were also treated with either anti-TNF-alpha Ab or control serum. On days 7 and 14 post-bleomycin treatment, lungs were harvested and analyzed for fibrosis, cytokine expression, and eosinophil influx. Anti-TNF-alpha caused a significant reduction in lung fibrosis, as indicated by a reduction in hydroxyproline content, which was accompanied by suppression of lung TGF-beta1, IL-5, and JE mRNA expression. Examination of tissue sections revealed a significant reduction in lung eosinophils and overall cellularity by anti-TNF-alpha treatment without a significant effect on the number of lung macrophages. The number of IL-5-expressing cells was also significantly reduced by anti-TNF-alpha treatment. Since IL-5 is important in eosinophil differentiation, activation, and recruitment, these findings suggest a novel mechanism by which TNF-alpha could mediate pulmonary fibrosis via induction of IL-5-mediated eosinophil recruitment and fibrogenic cytokine production. Since these eosinophil-derived cytokines include JE/monocyte chemotactic factor-1 and TGF-beta1, this cytokine networking orchestrated by TNF-alpha could, in turn, amplify the inflammatory response and drive the progression to fibrosis and end-stage lung disease.

In the past several years, significant progress in many aspects of pulmonary fibrosis research has been made. Among them, the finding that a variety of cytokines play important roles in the complex process appears most intriguing. These cytokines include at least transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), platelet-derived growth factor, fibroblast growth factors, (TGF-alpha), interleukin-1, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 alpha. These cytokines have been demonstrated to be produced at the sites of active fibrosis where they appear to be expressed by activated inflammatory cells, such as macrophages and eosinophils. More interestingly, other noninflammatory lung cells including mesenchymal cells, such as myofibroblasts, and epithelial cells, have been found to be significant sources as well, albeit in most instances at somewhat different time points than those by inflammatory cells. Study of the individual cytokines in vitro has revealed a variety of potential roles for these cytokines in the regulation of the fibrotic process in vivo, including chemoattractant, mitogenic activities for fibroblasts, stimulation of extracellular matrix and alpha-smooth muscle actin gene expression, alteration of the contractile phenotype of fibroblasts and regulation of diverse functions of lung inflammatory and epithelial cells which can further impact on the fibrotic process by autocrine and paracrine mechanisms. Of these cytokines, it appears that TGF-beta is probably the most important cytokine in terms of the direct stimulation of lung matrix expression which typifies fibrosis. Recently however, there is accumulating evidence to indicate that the situation is much more complex than any one single cytokine being solely responsible for the fibrotic response. The concept of complex lung cytokine networks, orchestrated by a few key cytokines, such as TNF-alpha, being responsible for this response has received strong support from recent studies. This means that it is the balance of positive (profibrogenic) and negative (antifibrogenic) forces generated from interaction among the various cytokines constituting these networks, which may finally determine the outcome of lung injury and inflammation. The importance of these cytokines also suggests new potential targets for designing new therapies for progressive pulmonary fibrosis, and perhaps their utility in prognostication as well.

Pulmonary fibrosis is the end point of a chronic inflammatory process characterized by leukocyte recruitment and activation, fibroblast proliferation, and increased extracellular matrix production. Previous studies of models of pulmonary fibrosis have investigated the role of cytokines in the evolution of the fibrotic response. The involvement of tumor necrosis factor and interleukin-1 in bleomycin-induced lung injury, a model of idiopathic pulmonary fibrosis, has been well established, suggesting that cytokines mediate the initiation and maintenance of chronic inflammatory lesions. However, the aforementioned cytokines alone cannot account for the recruitment and activation of specific leukocyte populations found in the bleomycin model. Recently, a family of novel proinflammatory cytokines (chemokines) was cloned and characterized, yielding many putative mediators of leukocyte functions. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemoattractant protein-1 (MCP-1) belong to the C-C chemotactic cytokine family, a group of low-molecular-weight peptides. These molecules modulate chemotaxis, proliferation, and cytokine expression in leukocyte subsets. Our group has investigated the roles of MCP-1 and MIP-1 alpha in the bleomycin model. Both MCP-1 and MIP-1 alpha are expressed in a time-dependent manner after bleomycin challenge, and passive immunization of these animals with either anti-MIP-1 alpha or anti-MCP-1 antibodies attenuated leukocyte accumulation. In addition, we have identified specific cell types expressing MCP-1 or MIP-1 alpha by in situ hybridization and immunohistochemical localization, respectively. Furthermore, our results indicate that MIP-1 alpha expression is mediated by alveolar macrophage-derived tumor necrosis factor, identifying an important cytokine pathway in the initiation of pulmonary fibrosis. Finally, anti-MIP-1 alpha therapy attenuated fibrosis, providing direct evidence for its involvement in fibrotic pathology. Our work has clearly established that the C-C chemokines MCP-1 and MIP-1 alpha are expressed and contribute to the initiation and maintenance of the bleomycin-induced pulmonary lesion.

We investigated the role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in bleomycin-induced lung injury, a model of interstitial lung disease. Bleomycin stimulates a T cell-dependent pulmonary inflammatory response characterized by an increase in leukocyte infiltration, fibroblast proliferation, and collagen synthesis. Intratracheal challenge of CBA/J mice with bleomycin resulted in a significant time-dependent increase in MIP-1 alpha protein levels both in whole-lung homogenates and bronchoalveolar lavage fluid. The kinetics of MIP-1 alpha expression were biphasic, with the first peak occurring at 2 days postinstillation and the second peak at 16 days. These levels of Ag expression temporally correlated with the accumulation of granulocytes, lymphocytes, and mononuclear phagocytes in the lung. In addition, immunohistochemical staining identified alveolar macrophages and bronchial epithelial cells as the primary cellular sources of MIP-1 alpha production. Interestingly, passive immunization of bleomycin-challenged mice with anti-MIP-1 alpha Abs significantly reduced pulmonary mononuclear phagocyte accumulation and fibrosis. These experiments establish that MIP-1 alpha protein is expressed in the lungs of bleomycin-treated mice and provide evidence that MIP-1 alpha promotes leukocyte accumulation and activation. Furthermore, these findings support the notion that leukocyte accumulation and activation are linked to fibrosis.

Recent studies indicate that monocyte chemoattractant protein-1 (MCP-1) may play an important role in pulmonary inflammation. In vitro studies show that a number of cell types are capable of producing MCP-1. In this study, MCP-1 expression in lungs of rats with bleomycin (BLM)-induced pulmonary fibrosis is examined to evaluate its cellular origin and potential role in pathogenesis. Lung fibrosis was induced in male Fisher 344 rats by endotracheal injection on day 0. On selected days after injection, lungs were harvested for in situ and Northern hybridization analyses for MCP-1 mRNA expression, immunochemical and histochemical analyses for MCP-1 protein expression, and identification of cell type. Northern analysis revealed significant elevation in lung MCP-1 mRNA expression beginning on day 3 post-BLM treatment, increasing to a peak on day 7, and then decreasing toward control levels after day 21. In situ hybridization combined with histochemical staining with chromotrope 2R indicate that most of the cells expressing MCP-1 mRNA at these time points are primarily eosinophils. A few scattered reactive fibroblasts, some mononuclear cells, epithelial cells, and cells of certain blood vessel walls also express this mRNA. Increased MCP-1 protein expression also was found to be predominantly within and adjacent to eosinophils. The eosinophils expressing this mRNA were found predominantly within areas of active fibrosis. The kinetics of increase in the number of cells expressing significant MCP-1 mRNA in lung sections paralleled that for MCP-1 mRNA expression, as assessed by Northern analysis. These results, for the first time, demonstrate that MCP-1 is up-regulated significantly in this rat animal model, and that infiltrating eosinophils represent the major cellular source for this increased MCP-1 expression.

The alteration of normal dermal fibroblast function that leads to the development of hypertrophic scar after thermal injury is unknown. To determine functional differences that might explain this process, fibroblasts were cultured from biopsies of post-thermal injury mature hypertrophic scars and patient-matched normal skin. The mitogenic responses of scar cells to fetal bovine serum, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and tumor necrosis factor alpha (TNF alpha) were determined and compared to normal skin cells. Collagen synthetic rate was also compared in the presence and absence of transforming growth factor beta 1 (TGF beta 1). Whereas both scar and normal cells responded with increased thymidine uptake to serum and cytokines, the stimulation to EGF and serum was significantly lower in scar cells. In contrast, synthesis of collagen, but not of non-collagenous proteins, was increased in scar relative to normal cells, both basally and when stimulated with low doses of TGF beta 1. Additionally, the fraction of protein synthesized as collagen was significantly higher in scar fibroblasts. These results suggest that fibroblasts from hypertrophic scars demonstrate stable phenotypic differences in cytokine responsiveness in comparison to cells from unaffected skin. The increased rate of collagen synthesis and decreased responsiveness to mitogens are consistent with the increased extracellular matrix content and decreased cellularity of hypertrophic scars.

Using a well-characterized model of bleomycin-induced pulmonary fibrosis in the rat, we determined that there was a time-dependent elaboration of monocyte chemotactic activity in bronchoalveolar lavage fluid. Northern hybridization analysis revealed markedly increased expression of rat monocyte chemoattractant protein-1 (MCP-1) mRNA in alveolar macrophages (AMs) from rats following induction of pulmonary fibrosis. Monocyte chemotactic activity was also significantly increased in conditioned media from AMs retrieved from injured rat lungs. These data suggest that one important role of AMs in the pathogenesis of chronic inflammatory lung injury and pulmonary fibrosis is the regulation of monocyte recruitment and activation within the lung secondary to secretion of monocyte chemoattractants including MCP-1.

In bleomycin-induced pulmonary fibrosis, lung injury is accompanied with inflammation and subsequent fibrosis. In this study, lung mRNA for several cytokines was measured in bleomycin-treated mice to evaluate their roles in lung fibrosis. Significant increases in tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) mRNA were found in lungs of bleomycin-treated responder CBA mice but not in nonresponder BALB/c mice. Increases in responder animals peaked on day 7 after bleomycin administration, and subsequently returned toward control levels. This time course paralleled that for the increase in beta-actin mRNA, but preceded the peak increase in mRNA for collagens I and III. When lung macrophages were analyzed for cytokine secretion, differences were observed between alveolar macrophages and interstitial cells, and between cells from bleomycin-responsive CBA and nonresponsive BALB/c mice. Only alveolar macrophages from CBA mice secreted increased amounts of IL-1. TNF-alpha activity was increased in conditioned media of alveolar and interstitial cells of CBA mice, while only alveolar macrophages of nonresponder BALB/c mice secreted any activity. The kinetics of the increased secretion of TNF-alpha was dissimilar for these different cells. These results are consistent with the conclusion that increased production of TNF-alpha and TGF-beta is an important component of the fibrotic process.

Many acute and chronic lung diseases are characterized by the presence of increased numbers of activated macrophages. These macrophages are derived predominantly from newly recruited peripheral blood monocytes and may play a role in the amplification and perpetuation of an initial lung insult. The process of inflammatory cell recruitment is poorly understood, although the expression of inflammatory cell-specific chemoattractants and subsequent generation of chemotactic gradients is likely involved. Although immune cells such as macrophages and lymphocytes are known to generate several inflammatory cell chemoattractants, parenchymal cells can also synthesize and secrete a number of bioactive factors. We now demonstrate the generation of significant monocyte chemotactic activity from tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta-treated pulmonary type II-like epithelial cells (A549). The predominant inducible monocyte chemotaxin had an estimated molecular mass of approximately 14-15 kDa and was neutralized by specific antibody to human monocyte chemotactic protein-1 (MCP-1). Induction of activity was accompanied by increases in steady-state mRNA level for MCP-1. These data are consistent with the induction of MCP-1 expression from A549 cells by TNF and IL-1. MCP-1 production from A549 cells could be induced by lipopolysaccharide (LPS)-stimulated alveolar macrophage (AM)-conditioned media, but not by LPS alone. The inducing activity in AM-conditioned media was neutralized with specific antibodies to IL-1 beta, but not TNF-alpha. Our findings suggest that the alveolar epithelium can participate in inflammatory cell recruitment via the production of MCP-1 and that cytokine networking between contiguous alveolar macrophages and the pulmonary epithelium may be essential for parenchymal cell MCP-1 expression.