Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast.

Idiopathic Pulmonary Fibrosis (IPF) is characterized by injury and loss of lung epithelial cells, accumulation of fibroblasts/myofibroblasts and abnormal remodeling of the lung parenchyma. The prognosis for IPF patients is poor and current therapies are largely ineffective in preventing respiratory failure. Current therapeutic approaches target epithelial cell replacement, manipulation of fibroblasts/myofibroblasts, modulation of procoagulant/fibrinolytic activities, cytokine and growth factor production, angiogenesis, and reduction of oxidative stress. Myofibroblasts are the primary effector cells in fibrosis. These cells may be derived by the activation and proliferation of resident lung fibroblasts, from epithelial-mesenchymal transition (EMT), or through recruitment of circulating fibrocytes. Transforming growth factor beta (TGFbeta) is a profibrotic factor that increases fibroblast proliferation, stimulates the synthesis and deposition of connective tissue, and inhibits connective tissue breakdown. TGFbeta acts through the promoter of the type 1 collagen gene causing increased collagen synthesis. In addition, TGFbeta induces EMT in alveolar epithelial cells (AECs) in vitro and in vivo. AECs exhibit substantial plasticity and may serve as a source of fibroblasts and/or myofibroblasts in lung fibrosis. Therapeutic interventions interfering with the pathways that lead to myofibroblast expansion and AEC apoptosis should be of considerable benefit in the treatment of IPF. This review will focus on the critical role of TGFbeta on AECs EMT and myofibroblasts in the development of fibrosis.

An essential role for CCAAT/enhancer binding protein beta in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is characterized by inflammation, genesis of myofibroblasts, and abnormal tissue repair. Despite extensive research, its pathogenesis remains incompletely understood. Previously, the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) was found to be a key regulator of myofibroblast differentiation in vitro, and to be involved in the acute phase and inflammatory responses. In an attempt to test the role of C/EBPbeta in the development of pulmonary fibrosis, experiments using C/EBPbeta null mice and their wild-type littermates were conducted. Our findings indicated that, compared to wild-type mice, animals deficient in C/EBPbeta showed significantly reduced fibrotic lesions and collagen deposition in the lung upon endotracheal injection of bleomycin. Further studies on the mechanisms by which C/EBPbeta regulates fibrosis indicated that knockout of C/EBPbeta attenuates inflammatory cytokine expression in bleomycin-treated mice. The reduced alpha-smooth muscle actin gene expression in either isolated lung fibroblasts or lung tissue from bleomycin or saline-treated C/EBPbeta deficient mice suggests that C/EBPbeta regulates myofibroblast differentiation during fibrosis. Consistent with this finding, cells from C/EBPbeta deficient mice exhibited higher proliferative rates than those from wild-type mice. These data suggest that C/EBPbeta plays an essential role in pulmonary fibrosis and that this role appears to be multifactorial with respect to cytokine expression, cell differentiation, and proliferation.

Role of Eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycin-induced lung injury and fibrosis.

Eotaxin-1/CCL11 and its receptor CCR3 are involved in recruitment of eosinophils to diverse tissues, but their role in eosinophil recruitment in pulmonary fibrosis is unclear. The present study examined the pulmonary expression of CCL11 and CCR3 during bleomycin (blm)-induced lung injury and determined their importance in the recruitment of inflammatory cells and the development of lung fibrosis. In mice, blm induced a marked pulmonary expression of CCL11 and CCR3. Immunostaining for CCR3 revealed that this receptor was not only expressed by eosinophils but also by neutrophils. CCL11-deficient (CCL11(-/-)) mice developed significantly reduced pulmonary fibrosis. Expression of profibrotic cytokines such as transforming growth factor-beta1 was diminished in the absence of CCL11. Furthermore, increased lung expression of CCL11 significantly enhanced blm-induced lung fibrosis and production of profibrotic cytokines. These effects were also associated with an increase of eosinophil and neutrophil pulmonary infiltration. In contrast, mice treated with neutralizing CCR3 antibodies developed significantly reduced pulmonary fibrosis, eosinophilia, neutrophilia, and expression of profibrotic cytokines. Together, these data suggest that CCL11 and CCR3 are important in the pulmonary recruitment of granulocytes and play significant pathogenic roles in blm-induced lung fibrosis.

Molecular mechanisms of and possible treatment strategies for idiopathic pulmonary fibrosis.

Pulmonary fibrosis is characterized by lung inflammation and abnormal tissue repair, resulting in the replacement of normal functional tissue with an abnormal accumulation of fibroblasts and deposition of collagen in the lung. This process involves cellular interactions via a complex cytokine-signaling mechanism and heightened collagen gene expression, ultimately resulting in its abnormal collagen deposition in the lung. Our current understanding of the pathogenesis of pulmonary fibrosis suggests that in addition to inflammatory cells, the fibroblast and signaling events that mediate fibroblast proliferation and myofibroblasts, play important roles in the diverse processes that constitute fibrosis. Increasing knowledge of cytokine biology, cytokine-signaling and cell matrix interactions have shed some light on the genesis of pulmonary fibrosis; however, the importance of inflammation in pulmonary fibrosis remains controversial. This remains true because the inflammatory component is variable at the time of diagnosis, and the most potent anti-inflammatory drugs that have been widely used in the treatment of pulmonary fibrosis do not seem to interfere with the fibrotic disease progression. Pulmonary fibrosis is a highly lethal disorder, which continues to pose major clinical challenges because an effective therapeutic regimen is yet to be determined. This review summarizes recent progress in understanding the molecular mechanisms of pulmonary fibrosis, and includes a more detailed discussion of the potential points of therapeutic attack in pulmonary fibrosis. In addition, a detailed discussion is presented regarding each of the potential therapies which have emerged from the animal models of pulmonary fibrosis, and which have been developed through advances in cellular and molecular biology.

Lung interleukin-4 gene expression in a murine model of bleomycin-induced pulmonary fibrosis.

Interleukin-4 (IL-4) is known to activate mononuclear cells as well as fibroblasts, all of which are important in the pathogenesis of pulmonary fibrosis. To investigate the potential role of this cytokine, lung IL-4 expression was examined in a murine model of bleomycin-induced pulmonary fibrosis. Lung fibrosis was induced in CBA/J mice by endotracheal injection of bleomycin on day 0. On selected days after treatment, lungs were harvested for reverse transcriptase polymerase chain reaction (RT-PCR), Northern, in-situ hybridization and immunohistochemical analyses. RT-PCR and Northern analyses revealed significant increases in lung IL-4 mRNA content between days 3 and 14 after induction of lung injury, which decreased toward control level after day 21. Both in-situ hybridization and immunohistochemistry showed low or undetectable IL-4 expression in control lungs and in injured lungs before day 3 after bleomycin injection. There was however elevated expression in mononuclear cells and macrophages between days 3 and 14, localized to areas of active fibrosis. These results demonstrate that IL-4 is upregulated significantly in this model. They suggest a potential role for this cytokine in pulmonary fibrosis, perhaps via its ability to stimulate and amplify the inflammatory response, stimulate collagen synthesis in fibroblasts, and thus promote the progression to fibrosis and end stage lung disease.

Role of cytokines and cytokine therapy in wound healing and fibrotic diseases.

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.

Induction of telomerase activity in fibroblasts from bleomycin-injured lungs.

Bleomycin-induced lung injury causes increased fibroblast numbers in the lung and pulmonary fibrosis. Studies of fibroblasts isolated from such injured lungs have revealed evidence of increased intrinsic proliferative capacity, but the mechanism is unknown. Telomerase catalyzes the addition of telomeric DNA repeats onto chromosomal ends, which is associated with increased cellular life span or immortality. To examine whether telomerase might play a role in regulating fibroblast proliferative capacity in pulmonary fibrosis, lung fibroblasts were isolated from rats treated with endotracheal injections of phosphate-buffered saline or bleomycin. At selected time points, the rats were killed and lung fibroblasts isolated. The isolated cells and lung tissue were then used in experiments for measurement of telomerase activity. The results show undetectable telomerase activity in fibroblasts isolated from control uninjured lungs, or in the control lung tissue extracts. Similar results were obtained in cells and lung tissue from Days 1, 3, and 28 bleomycin-injured lungs. However, significant telomerase activity was detected in fibroblasts and tissue extracts isolated from Days 7, 14, and 21 bleomycin-treated rat lungs, with maximal activity observed in the Day 14 samples. Analysis of the isolated cells for telomerase messenger RNA or reverse transcriptase expression, combined with alpha-smooth-muscle actin expression by immunohistochemistry, revealed that telomerase expression localized primarily to nonmyofibroblasts. These findings suggest that in addition to elevated growth factor expression, the injured lung fibroblast population may contain cells with increased life span, which could contribute to the observed overall increase in lung fibroblast numbers.

Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.

Damage to human skin due to ultraviolet light from the sun (photoaging) and damage occurring as a consequence of the passage of time (chronologic or natural aging) are considered to be distinct entities. Photoaging is caused in part by damage to skin connective tissue by increased elaboration of collagen-degrading matrix metalloproteinases, and by reduced collagen synthesis. As matrix metalloproteinase levels are known to rise in fibroblasts as a function of age, and as oxidant stress is believed to underlie changes associated with both photoaging and natural aging, we determined whether natural skin aging, like photoaging, gives rise to increased matrix metalloproteinases and reduced collagen synthesis. In addition, we determined whether topical vitamin A (retinol) could stimulate new collagen deposition in sun-protected aged skin, as it does in photoaged skin. Sun-protected skin samples were obtained from 72 individuals in four age groups: 18-29 y, 30-59 y, 60-79 y, and 80+ y. Histologic and cellular markers of connective tissue abnormalities were significantly elevated in the 60-79 y and 80+ y groups, compared with the two younger age groups. Increased matrix metalloproteinase levels and decreased collagen synthesis/expression were associated with this connective tissue damage. In a separate group of 53 individuals (80+ y of age), topical application of 1% vitamin A for 7 d increased fibroblast growth and collagen synthesis, and concomitantly reduced the levels of matrix-degrading matrix metalloproteinases. Our findings indicate that naturally aged, sun-protected skin and photoaged skin share important molecular features including connective tissue damage, elevated matrix metalloproteinase levels, and reduced collagen production. In addition, vitamin A treatment reduces matrix metalloproteinase expression and stimulates collagen synthesis in naturally aged, sun-protected skin, as it does in photoaged skin.

Immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (RSV) for human vaccine.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in infants, young children, and the elderly. Efforts to develop satisfactory live or inactivated vaccines have not yet been proven successful. Our research focuses on the development of four purified live attenuated RSV sub-type A human vaccine clones. Temperature sensitive (ts) and attenuated purified clones of either cold-adapted (ca) RSV or high-passage (hp) RSV were administered intra-nasally (i.n.) to BALB/c mice and tested for immunogenicity. All four clones produced significant anti-RSV F IgG2a and IgG1 titres in the sera of mice, RSV-specific neutralizing titres higher than those produced by their wild-type progenitor viruses, cytotoxic T-lymphocyte (CTL) activity, and total protection against wild-type (wt) viral challenge. These purified vaccine candidates await testing in humans to determine which contain the required balance between immunogenicity and attenuation.

The role of eosinophils in pulmonary fibrosis (Review).

Pulmonary fibrosis is commonly characterized by inflammation of the alveolar wall, leading to derangement of normal alveolar architecture, and interstitial as well as intra-alveolar fibrosis. The process involves cellular interactions via a complex cytokine network and heightened collagen gene expression with abnormal deposition in the lung. Recent studies have identified a myriad of cytokines with potential roles in pulmonary fibrosis. Based on in vivo antibody neutralization studies, important roles for tumor necrosis á (TNFá), macrophage inflammatory protein 1á (MIP-1á) and transforming growth factor (TGF), have been established. The recent demonstration that the eosinophil is a major source for several of these key pro-fibrogenic cytokines during the early stages of fibrosis, strongly suggest a role for the eosinophil in pulmonary fibrosis. In vitro, eosinophils can elaborate factors capable of stimulating fibroblast proliferation, and their presence in lungs undergoing many forms of pulmonary fibrosis has been well documented. Further support for a role for eosinophils in pulmonary fibrosis are suggested by clinical data showing a correlation between lung eosinophil count and a poor prognosis and decreased responsiveness to therapy. This review will focus on the recent findings, which suggest novel potential roles for the eosinophil in the pathogenesis of pulmonary fibrosis.

The role of IL-5 in bleomycin-induced pulmonary fibrosis.

Eosinophils are known to express cytokines capable of promoting fibrosis. Interleukin-5 (IL-5) is important in regulating eosinophilopoiesis, eosinophil recruitment and activation. Lung IL-5 expression is elevated in pulmonary fibrosis, wherein the eosinophil is a primary source of fibrogenic cytokines. To determine the role of IL-5 in pulmonary fibrosis, the effects of anti-IL-5 antibody were investigated in a model of bleomycin-induced pulmonary fibrosis. Fibrosis was induced in mice by endotracheal bleomycin treatment. Animals were also treated with either anti-IL-5 antibody or control IgG. Lungs were then analyzed for fibrosis, eosinophil influx, chemotactic activity, and cytokine expression. The results show that a primary chemotactic activity at the height of eosinophil recruitment is IL-5. Furthermore, anti-IL-5 antibody caused significant reduction in lung eosinophilia, cytokine expression, and fibrosis. These findings taken together suggest an important role for IL-5 in pulmonary fibrosis via its ability to regulate eosinophilic inflammation, and thus eosinophil-dependent fibrogenic cytokine production.

Lung interleukin-5 expression in murine bleomycin-induced pulmonary fibrosis.

Eosinophils are primary sources of fibrogenic cytokines in lung fibrosis, and interleukin (IL)-5 is important in their differentiation, proliferation, recruitment and activation. To investigate the potential role of this cytokine, lung IL-5 expression was examined in a murine model of bleomycin-induced pulmonary fibrosis. Analysis of lung RNA showed significant increases in lung IL-5 mRNA content between days 3 and 14 after induction of lung injury, which decreased toward control levels after day 21. In situ hybridization revealed essentially no detectable IL-5 mRNA expression before day 3, but showed elevated expression in mononuclear cells and eosinophils between days 3 and 14, localized within areas of active fibrosis. After 21 days, the intensity and number of IL-5-expressing cells significantly declined. Immunostaining with anti-IL-5 antibodies confirmed the predominant IL-5 expression by mononuclear cells and eosinophils in areas of active fibrosis. The kinetics of increase in the number of cells expressing significant IL-5 mRNA in lung sections paralleled that for IL-5 mRNA expression in whole-lung homogenates. These results demonstrate for the first time that IL-5 is upregulated in this murine model and suggest a novel role for this cytokine in pulmonary fibrosis via its ability to recruit and activate eosinophils.

Transforming growth factor-beta 1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells.

PURPOSE: This investigation was designed to test the hypothesis that transforming growth factor-beta 1 (TGF-beta 1) regulates lysyl oxidase secretion from vascular smooth muscle cells. Lysyl oxidase is an enzyme that catalyzes an essential step in collagen and elastin cross-linking in the extracellular matrix, and TGF-beta 1 has been implicated in the pathogenesis of restenosis after vascular injury. The effect of TGF-beta 1 on lysyl oxidase in vascular smooth muscle cells has not been previously defined. METHODS: Rat aortic smooth muscle cells were grown in culture to confluence. Cells in passage 2 to 6 were incubated for 24 hours in media containing 0.1, 0.5, 1.0, or 10.0 ng/ml of TGF-beta 1. Lysyl oxidase activity in the media was quantitated with a tritium-release bioassay against an insoluble 3H-labeled aortic clastin substrate. Northern blot analyses were performed to determine steady-state levels of lysyl oxidase mRNA in the smooth muscle cells. RESULTS: Lysyl oxidase activity in the media increased 1.5-fold above control levels after exposure to 10 ng/ml of TGF-beta 1 (p < 0.01). This increase in lysyl oxidase activity was associated with a concentration-dependent increase in steady-state levels of lysyl oxidase mRNA, being 4.3- and 6.2-fold above control levels after exposure to 1 and 10 ng/ml TGF-beta 1, respectively (p < 0.01). The observed increase in steady-state lysyl oxidase mRNA after exposure to TGF-beta 1 was also time-dependent over the 24-hour experimental period. CONCLUSIONS: TGF-beta 1 appears to regulate lysyl oxidase in cultured rat aortic smooth muscle cells. Increases in lysyl oxidase activity may be one of the mechanisms by which TGF-beta 1 contributes to arterial restenosis after vascular injury.

Regulation of lung fibroblast alpha-smooth muscle actin expression, contractile phenotype, and apoptosis by IL-1beta.

IL-1 is important in regulating lung inflammation and potentially fibrosis as well. To clarify the role of this cytokine vis-à-vis changes in lung fibroblast phenotype in pulmonary fibrosis, the effects of IL-1beta on isolated lung fibroblasts were examined. Rat lung fibroblasts were treated with increasing doses of IL-1beta and examined for effects on cell number, alpha-smooth muscle actin expression, apoptosis, nitric oxide (NO) production, and contractility in collagen gels. The results show that IL-1beta caused dose-dependent down-regulation of alpha-smooth muscle actin protein and mRNA expression. The kinetics of mRNA inhibition was rapid and preceded the effects on protein expression. This IL-1beta-induced decrease in actin expression was associated with inhibition of contractility evaluated using fibroblast-populated collagen gels. Since IL-1beta inhibition of actin expression was accompanied by reduction in cell number, the effect on apoptosis was examined. Significant increase in the number of apoptotic nuclei and DNA fragmentation was observed upon IL-1beta treatment, with a dose-response curve that mirrored that for the decline in actin-positive cells. More than one-half of the apoptotic cells were actin positive at high IL-1beta doses, suggesting that the actin-expressing cells may be more susceptible to IL-1beta-induced apoptosis. IL-1beta also induced NO production in these cells, which was inhibited by NG-monomethyl-L-arginine. Similarly, IL-1beta-induced apoptosis and inhibition of actin expression were inhibited by this arginine analogue. Hence, induction of apoptosis by IL-1beta via NO production may be an important mechanism for regulating lung fibroblast alpha-smooth muscle actin expression, and consequently its contractile phenotype as well.

TNF-alpha-mediated lung cytokine networking and eosinophil recruitment in pulmonary fibrosis.

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.

Costimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte chemoattractant protein-1 via specific receptors.

Recent studies indicate potential roles of monocyte chemotactic protein-1 (MCP-1) in recruitment of monocytes to sites of inflammation. However, their increased expression does not always correlate with monocyte influx, suggesting other possible biological activities for this member of the C-C chemokine family. In view of its potential role in regulating extracellular matrix expression in fibrotic disorders, the effects of MCP-1 on lung fibroblast collagen expression were evaluated. Isolated rat lung fibroblasts were treated with increasing doses of MCP-1 for variable periods of time and examined for effects on collagen synthesis and expression of procollagen alpha1(I) mRNA expression. The results show that MCP-1 was able to stimulate collagen expression in these cells in a dose-dependent manner but required over 24 h for significant elevation to occur. In view of this delayed time course, the possibility of mediation via endogenous transforming growth factor beta (TGFbeta) was tested by the ability of anti-TGFbeta antibody to inhibit this MCP-1 stimulation of collagen expression. Significant but incomplete inhibition by this antibody was observed. Pretreatment of the cells with antisense but not by sense or missense TGFbeta1 oligodeoxyribonucleotides caused essentially complete inhibition of this MCP-1 stimulatory effect. Furthermore, MCP-1 treatment was found to also stimulate TGFbeta secretion and mRNA expression, which was also abolished by pretreatment with antisense TGFbeta1 oligodeoxyribonucleotides. The kinetics of TGFbeta expression indicates that significant increase preceded that for collagen expression. Binding studies using 125I-labeled MCP-1 indicated the presence of specific and saturable binding sites with a dissociation constant consistent with the dose response curves for stimulation of fibroblast collagen synthesis and TGFbeta activity by MCP-1. These results taken together suggest that MCP-1 stimulates fibroblast collagen expression via specific receptors and endogenous up-regulation of TGFbeta expression. The latter then results in autocrine and/or juxtacrine stimulation of collagen gene expression.

Fibronectin is the major fibroblast chemoattractant in rabbit anti-glomerular basement membrane disease.

The mechanism for fibroblast recruitment in renal fibrosis due to anti-glomerular basement membrane (anti-GBM) disease is unknown. Since fibroblast recruitment can occur via chemotaxis, assessment of the possible production of fibroblast chemotactic activity by affected renal tissue and its identification could provide important clues. Anti-GBM disease was induced by injection of guinea pig anti-rabbit GBM immunoglobulin G into rabbits previously sensitized to guinea pig immunoglobulin G. On days 4, 7, and 14 after induction, renal tissue was harvested and glomeruli isolated. Overnight serum-free conditioned media from whole cortex and glomeruli were prepared and assayed for fibroblast chemotactic activity. The results show low level activity in both conditioned media from control animals. In contrast, conditioned media from anti-GBM-treated animals at all time points showed significantly elevated fibroblast chemotactic activity peaking on day 4 with subsequent reduction thereafter. The magnitude of increase in cortical conditioned media was significantly higher than that for glomerular conditioned media, suggesting that most of the activity was derived from extraglomerular sources. Gel filtration analysis revealed the activity to be heterogeneous, consisting of at least four major species with estimated molecular weights ranging from 10 to > 100 kd. Acidification of conditioned media failed to increase chemotactic activity significantly, whereas protease digestion abolished it. Treatment of conditioned media with antifibronectin inhibited > 85% of the chemotactic activity, whereas antibodies to platelet-derived growth factor and transforming growth factor-beta did not have a significant effect. These findings taken together suggest that fibronectin-derived peptides represent the predominant fibroblast chemoattractant produced by renal cortex in anti-GBM disease.

Lung fibroblast alpha-smooth muscle actin expression and contractile phenotype in bleomycin-induced pulmonary fibrosis.

The emergence of the myofibroblast phenotype (characterized by alpha-smooth muscle actin expression) in wound healing and in tissues undergoing fibrosis is thought to be responsible for the increased contractility of the affected tissues. In bleomycin-induced pulmonary fibrosis, the myofibroblast is also responsible for the observed increase in collagen gene expression. To evaluate further these phenotypic changes in lung fibroblasts, contractile and other phenotypic properties of fibroblasts isolated from lungs of rats with bleomycin-induced fibrosis were compared with those of normal rats using in vitro models. Pulmonary fibrosis was induced in rats by endotracheal injection on day 0, and 7 and 14 days later the animals were sacrificed and lung fibroblasts isolated. Using immunofluorescence, < 10% of fibroblasts from control animals express alpha-smooth muscle actin when cultured as a monolayer. In contrast, 19% and 21% of cells from day 7 and day 14 bleomycin-treated animals, respectively, expressed this actin and with greater intensity than in control lung cells. This increase in actin expression was associated with enhanced contractility when evaluated using a three-dimensional cell culture model consisting of fibroblast-populated collagen gels. This enhanced contractility was abolished by treatment with antibody to transforming growth factor-beta (TGF-beta), whereas exogenous TGF-beta 1 and serum-stimulated contraction of control lung fibroblasts. TGF-beta 1 gene expression was greater in cells from bleomycin-treated animals than those from control lungs. These results show that cells with the myofibroblast phenotype are more abundant in fibrotic lung, and that these cells possess greater contractile capacity in vitro at least partly by virtue of their enhanced endogenous TGF-beta 1 gene expression.

Lung monocyte chemoattractant protein-1 gene expression in bleomycin-induced pulmonary 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.