Reduction of bleomycin induced lung fibrosis by transforming growth factor β soluble receptor in hamsters

BACKGROUND: Transforming growth factor beta (TGF-beta) is a key mediator of collagen synthesis in the development of lung fibrosis. It has previously been shown that the administration of TGF-beta antibody and TGF-beta binding proteoglycan, decorin, reduced bleomycin (BL) induced lung fibrosis in animals. The present study was carried out to investigate whether intratracheal instillation of TGF-beta soluble receptor (TR) would minimise the BL induced lung fibrosis in hamsters. METHODS: The effect of a recombinant TR (TGFbetaRII) on the lung collagen accumulation was evaluated in a BL hamster model of pulmonary fibrosis. Animals were divided into four groups and intratracheally injected with saline or BL at 6.5 U/4 ml/kg followed by intratracheal instillation of phosphate buffered saline (PBS) or 4 nmol TR in 0.3 ml twice a week. Twenty days after the first intratracheal instillation the hamsters were killed for bronchoalveolar lavage (BAL) fluid, biochemical, and histopathological analyses. RESULTS: Treatment of hamsters with TR after intratracheal instillation of BL significantly reduced BL induced lung fibrosis as shown by decreases in the lung hydroxyproline level and prolyl hydroxylase activity, although they were still significantly higher than those of the saline control. Histopathological examination showed a considerable decrease in BL induced fibrotic lesions by TR treatment. However, TR did not prevent the BL induced increases in total cells and protein in the BAL fluid. CONCLUSIONS: These results suggest that TR has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where increased TGF-beta is associated with excess collagen accumulation.     

Reduction of bleomycin induced lung fibrosis by candesartan cilexetil, an angiotensin II type 1 receptor antagonist

BACKGROUND: Signalling of angiotensin II via angiotensin II type 1 receptor (AT1) promotes cardiac and renal fibrosis, but its role in lung fibrosis is little understood. Using a rat bleomycin (BLM) induced model of pulmonary fibrosis, we examined the expression of AT1 in the lung and the effect of an AT1 antagonist on pulmonary fibrosis. METHODS: Adult male Sprague-Dawley rats were given 0.3 mg/kg BLM intratracheally. Two days earlier they had received 10 mg/kg/day of the AT1 antagonist candesartan cilexetil mixed in the drinking water. AT1 expression in the lungs was examined by immunohistochemistry and immunoblot methods. The effect of the AT1 antagonist on pulmonary fibrosis was studied by analysis of bronchoalveolar lavage (BAL) fluid, histopathology, and hydroxyproline assay. RESULTS: Immunohistochemical studies showed overexpression of AT1 in inflammatory immune cells, alveolar type II cells, and fibroblasts. A quantitative assay for AT1 showed that AT1 expression was significantly upregulated in cells from BAL fluid after day 3 and in the lung homogenates after day 21. Candesartan cilexetil significantly inhibited the increase in total protein and albumin, as well as the increase in total cells and neutrophils in BAL fluid. On day 21 candesartan cilexetil also ameliorated morphological changes and an increased amount of hydroxyproline in lung homogenates. In addition, BLM increased the expression of transforming growth factor (TGF)-beta1 in BAL fluid on day 7; this increase was significantly reduced by candesartan cilexetil. CONCLUSION: AT1 expression is upregulated in fibrotic lungs. Angiotensin II promotes lung fibrosis via AT1 and, presumably, in part via TGF-beta1.     

T lymphocytes and silica-induced pulmonary inflammation and fibrosis in mice.

BACKGROUND: Silica-induced pulmonary inflammation and fibrosis in animals provides a good model for chronic pulmonary inflammation and fibrosis. Although lymphocytes are implicated in the pathogenesis of pulmonary fibrosis, experimental models using silica-treated athymic nude mice have not been successful in showing the fibrogenic mechanism regulated by T cells. The aim of this study was to re-evaluate the role of T lymphocytes in the development of silicosis by comparing the response to silica administration of nude athymic mutants with that of euthymic animals. METHODS: Suspensions of silica particles were transnasally administered to nude athymic mice (Balb/c nu/nu) as well as to their euthymic littermates (Balb/c nu/+). The degree of pulmonary inflammation and fibrosis was assessed on days 14, 28, and 56 based upon histological observation, analysis of collagen deposition in the lungs, and analysis of the cellular constituent, protein, and phospholipid content in the bronchoalveolar lavage fluid. RESULTS: Histologically, athymic mice developed less severe interstitial pneumonitis than euthymic mice. In euthymic mice the lung hydroxyproline content increased with time after silica administration from 6.48 (0.38) micrograms hydroxyproline/mg dry lung weight on day 0 to 8.87 (0.41) micrograms/mg on day 56. A gradual increase in lung hydroxyproline content was also observed in athymic mice but the increase was significantly smaller than in euthymic mice (6.63 (0.43) micrograms/mg on day 0, 7.90 (0.19) micrograms/mg on day 56). Administration of silica resulted in an increase in the number of macrophages and neutrophils and in the total protein and phospholipid content of the bronchoalveolar lavage (BAL) fluid in both mouse strains. No significant difference was detected between athymic and euthymic mice in the numbers of macrophages, but the increase in neutrophils in the BAL fluid of athymic mice was significantly smaller than in euthymic mice on days 14 and 56. The total protein and phospholipid content of the BAL fluid from athymic mice was lower than that from euthymic mice. CONCLUSIONS: T lymphocytes appear to be involved in the pathogenesis of silica-induced pneumonitis. Since pulmonary fibrosis develops even in nude athymic mice, T cells do not seem to play a primary part in fibrogenic response but they regulate, at least to some extent, the response of inflammatory cells and fibrogenesis of the lung.     

Attenuation of bleomycin induced pulmonary fibrosis in mice using the heme oxygenase inhibitor Zn-deuteroporphyrin IX-2,4-bisethylene glycol

BACKGROUND: Pulmonary fibrosis is associated with a poor prognosis. The pathogenesis of fibrotic lung disorders remains unclear, but the extent of tissue damage due to the persistent presence of oxidants or proteases is believed to be important. The heme degrading enzyme heme oxygenase (HO) has been found to be expressed in experimental fibrosis, and generation of free iron and carbon monoxide (CO) by HO has been implicated in oxidant induced lung damage. A study was undertaken to examine the effects of the HO inhibitor Zn-deuteroporphyrin-IX-2,4-bisethylene glycol (Zndtp) on the development of pulmonary fibrosis in the bleomycin model of lung injury and repair. METHODS: Zndtp (10 micro mol/kg) was administered subcutaneously twice daily to mice 1 week following the intratracheal instillation of 0.025 U bleomycin. Animals were killed 10 or 21 days after bleomycin instillation and indices of lung damage and fibrosis were evaluated. RESULTS: Bleomycin treatment induced pulmonary cytotoxicity, increased levels of active transforming growth factor beta (TGF-beta), enhanced lung collagen accumulation, and decreased glutathione content. Zndtp administration significantly attenuated these indices. CONCLUSIONS: Administration of Zndtp in the bleomycin model resulted in appreciable alveolar cytoprotection and amelioration of pulmonary fibrosis. This molecule and its analogues may warrant further consideration in the treatment of acute lung injury and fibrotic lung disorders.     

Enhanced type III collagen gene expression during bleomycin induced lung fibrosis.

BACKGROUND--Intratracheal instillation of bleomycin into mice leads to deposition of collagen in the lung and fibrosis, but the mechanism for this is poorly understood. Enhanced collagen gene expression, increased collagen synthesis, decreased collagen degradation, and proliferation of fibroblasts have all been proposed as possible contributors. To obtain information on the activity of collagen producing cells at an early stage in the development of pulmonary fibrosis in situ hybridisation was used to detect and localise products of the type III procollagen gene. In addition, assay of type III procollagen gene expression was performed using dot-blot analysis of lung RNA extracts. METHODS--Lung fibrosis was induced in mice by intratracheal instillation of bleomycin sulphate (6 mg/kg body weight) and tissues were examined after three, 10, 21 and 35 days. RNA-RNA hybridisation was accomplished with riboprobes labelled with sulphur-35 which were generated from a 1.7 kb mouse procollagen a1(III) cDNA. In situ hybridisation was performed on sections fixed in paraformaldehyde and embedded in paraffin wax and steady state values of type III procollagen mRNA were assayed by dot-blot analysis of total lung RNA extracted by guanidium isothiocyanate. RESULTS--Data obtained using both techniques suggest that type III procollagen gene expression was enhanced in bleomycin induced fibrosis and that expression was maximal between 10 and 35 days after a single dose of bleomycin. The most active cells were located in interstitial areas around the conducting airways, although these cells were usually seen in areas with no histological evidence of fibrosis. Regions with the most advanced fibrosis, as assessed by histological methods, rarely contained cells with activity above the threshold detectable by this technique. CONCLUSIONS--These results suggest that activation of interstitial fibroblasts, with enhanced type III collagen gene expression, forms at least part of the mechanism leading to increased collagen deposition in bleomycin induced fibrosis and that this occurs before fibrosis is detected by conventional histological staining.     

Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice.

BACKGROUND--Increased production of transforming growth factor beta (TGF-beta) seems to have an important role in the pathophysiology of bleomycin induced lung fibrosis. This is attributed to the ability of TGF-beta to stimulate infiltration of inflammatory cells and promote synthesis of connective tissue, leading to collagen deposition. METHODS--The study was designed to evaluate the antifibrotic potential of TGF-beta antibody in mice treated with bleomycin, which is a model of lung fibrosis. Under methoxyflurane anaesthesia, each mouse received intratracheally either 50 microliters sterile isotonic saline or 0.125 units bleomycin in 50 microliters. Within five minutes after the instillation, mice received into the tail vein 100 microliters non-immune rabbit IgG, TGF-beta 2 antibody, or a combination of TGF-beta 2 and TGF-beta 1 antibodies at various dose regimens. Mice were killed 14 days after the instillation and their lungs processed for morphological and biochemical studies. RESULTS--Administration of 250 micrograms of TGF-beta 2 antibody after instillation of bleomycin followed by 100 micrograms on day 5 and 100 micrograms on day 9 significantly reduced the bleomycin induced increases in the accumulation of lung collagen from 445.8 (42.3) micrograms/lung to 336.7 (56.6) micrograms/lung at 14 days. Similarly, the combined treatment with 250 micrograms TGF-beta 2 antibody and 250 micrograms TGF-beta 1 antibody after bleomycin instillation followed by 100 micrograms of each antibody on day 5 also caused a significant reduction in bleomycin induced increases in lung collagen accumulation and myeloperoxidase activity at 14 days. CONCLUSIONS--These results suggest that TGF-beta has an important role in the aetiology of bleomycin induced lung fibrosis; the neutralisation of TGF-beta by systemic treatment with its antibodies offers a new mode of pharmacological intervention which may be useful in treating lung fibrosis.     

Losartan attenuates bleomycin induced lung fibrosis by increasing prostaglandin E2 synthesis

BACKGROUND: The angiotensin system has a role in the pathogenesis of pulmonary fibrosis. This study examines the antifibrotic effect of losartan, an angiotensin II type 1 receptor antagonist, in bleomycin induced lung fibrosis and its possible implication in the regulation of prostaglandin E(2) (PGE(2)) synthesis and cyclooxygenase-2 (COX-2) expression. METHODS: Rats were given a single intratracheal instillation of bleomycin (2.5 U/kg). Losartan (50 mg/kg/day) was administrated orally starting one day before induction of lung fibrosis and continuing to the conclusion of each experiment. RESULTS: Losartan reduced the inflammation induced by bleomycin, as indicated by lower myeloperoxidase activity and protein content in the bronchoalveolar lavage fluid. Collagen deposition induced by bleomycin was inhibited by losartan, as shown by a reduction in the hydroxyproline content and the amelioration of morphological changes. PGE(2) levels were lower in fibrotic lungs than in normal lungs. Losartan significantly increased PGE(2) levels at both 3 and 15 days. A reduction in COX-2 expression by bleomycin was seen at 3 days which was relieved by losartan. CONCLUSIONS: The antifibrotic effect of losartan appears to be mediated by its ability to stimulate the production of PGE(2). Losartan, which is already widely used clinically, could be assessed as a new treatment in lung fibrosis.     

Transforming growth factor-β: crossroad of glucocorticoid and bleomycin regulation of collagen synthesis in lung fibroblasts

Fibrosis is a consequence of injury which is characterized by accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of normal tissue architecture and function. Transforming growth factor-beta, a potent wound healing agent, has also been shown to be an agent that can produce fibrosis because it is a potent stimulator of collagen synthesis. Both glucocorticoids and bleomycin have recently been shown to affect collagen synthesis in opposite directions, by utilizing a common pathway of involving transforming growth factor-beta activator protein binding to the transforming growth factor-beta element. This article presents a mechanistic overview of collagen synthesis regulation by glucocorticoids and bleomycin through the transforming growth factor-beta pathway.     

Development of acute lung injury after the combination of intravenous bleomycin and exposure to hyperoxia in rats.

Pulmonary toxicity is an important adverse effect of bleomycin treatment. Very little is known of the mechanisms underlying the development of lung injury, especially after intravenous administration, or how it can be modulated. In this study acute lung injury induced by bleomycin has been examined in rats by assessment of alveolar lavage cell profiles, histological examination, and measurement of the total pulmonary extravascular albumin space. Intratracheal instillation of bleomycin 1.5 mg resulted in a severe pneumonitis with influx of inflammatory cells into the alveoli as assessed by alveolar lavage, oedema of the alveolar walls, and up to an eight fold increase in the total pulmonary extravascular albumin space, maximal at 72 hours. Intravenous bleomycin 0.15-5 mg produced no detectable injury when assessed in these ways. Exposure to hyperoxia (40-90%) after intravenous bleomycin, however, induced lung injury similar to that produced by intratracheal bleomycin. A much more severe injury followed administration of intravenous bleomycin after an exposure to hyperoxia, which itself resulted in lung injury; but lung injury was still detectable after bleomycin when the exposure to hyperoxia was insufficient to induce changes in control animals. Lung injury was not observed when the exposure to hyperoxia preceded bleomycin treatment. These results indicate the importance of oxygen in the pathways leading to acute lung injury following intravenous bleomycin. We conclude that exposure to oxygen might induce lung injury during and after bleomycin treatment, and suggest that in these circumstances oxygen therapy should be kept to a minimum.     

Collagen breakdown during acute lung injury.

Injury to the capillary endothelium and to alveolar epithelial cells of the lung may result in damage to the underlying collagen of the extracellular matrix. To examine this possibility, whole body irradiation, bleomycin injections, and exposure to hyperoxia were used to induce various types of lung damage in mice. The morphology of the lung and the cellular and protein content of bronchoalveolar lavage fluid were used to assess injury. Collagen breakdown was assessed from the hydroxyproline concentrations in bronchoalveolar lavage fluid. When lung cell injury was observed, protein leaked in to alveoli and hydroxyproline was detected in bronchoalveolar lavage fluid. An increase in hydroxyproline followed endothelial damage by irradiation and was greatly increased when type 1 epithelial cell necrosis also occurred after bleomycin injection or hyperoxia. Maximal concentrations of hydroxyproline occurred in mice showing respiratory distress after six days of hyperoxia. Concentrations returned to zero during the subsequent phases of cell regeneration and fibrosis seen after bleomycin injection and irradiation. There was little change in the cellular components of bronchoalveolar lavage fluid at any time. The results indicate that collagen breakdown occurs during acute lung injury and can be quantified in terms of the hydroxyproline concentration in lavage fluid. Such a change in the extracellular matrix might influence the subsequent division and differentiation of regenerating cells during repair.     

Therapeutic effects of bone marrow-derived mesenchymal stem cells engraftment on bleomycin-induced lung injury in rats

Previous studies have demonstrated that bone marrow-derived mesenchymal stem cell (MSC) engraftment attenuated lung injury in a model induced by bleomycin in mice. However, the mechanisms are not completely understood. The primary objective of the present study was to determine whether MSC engraftment can also protect lungs against bleomycin-induced injury in rats and to observe any beneficial effects of cytokines. Twelve hours after bleomycin (5 mg/kg) or phosphate-buffered saline was perfused into the trachea, 5x10(6) DAPI-labeled MSCs or DMEM-F12 were injected into the tail vein of rats. Two weeks later, MSCs labeled with DAPI were detected by pan-cytokeratin staining. The level of laminin and hyaluronan in bronchoalveolar lavage fluid was measured by radioimmunoassay. Collagen content in lung tissue was calculated by the hydroxyproline assay. TGF-beta1, PDGF-A, B, and IGF-I were measured by real-time PCR. It was observed that some MSCs positive for pan-cytokeratin staining, an indicator of alveolar epithelial cells, were present in injured lung tissue. Bleomycin injection increased the content of hydroxyproline in lung tissue, as well as laminin and hyaluronan in bronchoalveolar lavage fluid, markers for lung injury and fibrosis. However, these effects were attenuated by MSC treatment. Furthermore, the increased mRNA levels of TGF-beta1, PDGF-A, PDGF-B, and IGF-I following bleomycin injection were also significantly decreased by MSC treatment. These observations provided evidence that MSCs are still present in the lung 2 weeks after the initial MSC treatment in rats, as well as documented the beneficial effects of MSC engraftment against bleomycin-induced lung injury associated with changes in TGF-beta1, PDGF-A, PDGF-B, and IGF-I. These results may provide an experimental base for clinical therapy of pulmonary fibrosis in the future.     

Abrogation of the fibrotic effect of transforming growth factor-β in dermal wound healing

The growth factor, transforming growth factor-beta1, which under normal circumstances promotes wound healing by stimulating local fibroblasts to produce collagen and other extracellular matrix proteins, has also been implicated as the primary causative agent of fibrosis. Because transforming growth factor-beta1 is capable of stimulating its own production by fibroblasts, its normally beneficial effects may become amplified to the point where excess extracellular matrix accumulation occurs, thereby causing abnormal scarring. Therefore, strategies that block or counter the effects of transforming growth factor-beta1 may be useful in preventing or decreasing fibrosis. One such strategy is the use of glucocorticoid steroids such as dexamethasone, which normally have the opposite effect of transforming growth factor-beta1, namely the impairment of wound healing. When used in conjunction with transforming growth factor-beta1, glucocorticoid steroids may normalize the effect of transforming growth factor-beta1 on collagen synthesis, thereby reducing excessive collagen deposition and fibrosis.     

Fibroblast chemotactic response elicited by native bronchoalveolar lavage fluid from patients with fibrosing alveolitis.

BACKGROUND--In fibrosing alveolitis activation of lung fibroblasts is the decisive event in the pathogenetic sequence leading to pulmonary fibrosis. Fibroblast stimulating activity was measured in bronchoalveolar lavage (BAL) fluid to assess its relationship to the activity of fibrosing alveolitis. METHODS--Nine control subjects and 40 patients with fibrosing alveolitis caused by idiopathic pulmonary fibrosis (n = 22) or pulmonary involvement in systemic sclerosis (n = 18) were studied. All patients were followed up by lung function testing for a minimum of six months (mean (SE) 13.3 (1.4) months). Twenty five patients received immunosuppressive therapy and 15 refused. At the beginning of follow up BAL was performed and, as a possible indicator of fibroblast stimulating mediators within the lungs, chemotactic migration of cultured human fibroblasts elicited by native BAL fluid was measured in Boyden-type chambers and expressed as a percentage of the chemoattractant effect of 25 ng/ml platelet derived growth factor. The procollagen III peptide level in BAL fluid served as a marker for collagen synthesis. RESULTS--Chemoattractant activity was elevated in the patients with idiopathic pulmonary fibrosis and systemic sclerosis compared with the control group, (mean (SE) 56.4% (8.5%)) and 72.3% (16.3%) v 12.6% (4.0%). Chemoattractant activity was inversely correlated with total lung capacity (TLC) (r = -0.45) and with vital capacity (VC) (r = -0.33). Procollagen III peptide concentrations in BAL fluid and chemoattractant activity were not significantly correlated. For further evaluation chemoattractant activity of 36% (mean value of controls +2 SD) was used to separate normal (< 36%) from elevated (> or = 36%) activity. At the end of follow up, untreated patients with high chemoattractant activity (> or = 36%) showed a significant reduction of VC, TLC, and exercise arterial oxygen tension (PaO2) and a small decrease in carbon monoxide transfer factor (TLCO), whereas a significant improvement in VC, TLC, and TLCO and a small increase of exercise PaO2 occurred in treated patients with high chemoattractant activity. Patients with low chemoattractant activity (< 36%) showed no consistent change in lung function measurements, irrespective of treatment. In contrast, lung function results and differential cell counts in BAL fluid failed to identify progressive disease. CONCLUSIONS--In patients with fibrosing alveolitis the chemoattractant activity of BAL fluid seems to be an independent indicator of lung fibroblast stimulating activity providing relevant information about disease activity, and may help to improve the clinical management of these patients.     

Type I and II collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells.

Chondrogenic differentiation by mesenchymal progenitor cells (MPCs) is associated with cytokines such as transforming growth factor-beta 1 (TGF-beta1) and dexamethasone. Extracellular matrix (ECM) also regulates the differentiation by MPCs. To define whether ECM plays a functional role in regulation of the chondrogenic differentiation by MPCs, an in vitro model was used. That model exposed to dexamethasone, recombinant human TGF-beta1(rhTGF-beta1) and collagens. The results showed that MPCs incorporated with dexamethasone and rhTGF-beta1 increased proliferation and expression of glycosaminoglycan (GAG) after 14 days. Type II collagen enhanced the GAG synthesis, but did not increase alkaline phosphatase (ALP) activity. When adding dexamethasone and rhTGF-beta1 MPCs increased mRNA expression of Sox9. Incorporation with type II collagen, dexamethasone and rhTGF-beta1, MPCs induced mRNA expression of aggrecan and enhanced levels of type II collagen, and Sox9 mRNA. In contrast, incorporation with type I collagen, dexamethasone and rhTGF-beta1 MPCs reduced levels of aggrecan, and Sox9 mRNA, and showed no type II collagen mRNA. Altogether, these results indicate that type I and II collagen, in addition to the cytokine effect, may play a functional role in regulating of chondrogenic differentiation by MPCs.     

Pirfenidone Inhibits Lung Allograft Fibrosis through L-Arginine–Arginase Pathway

Transplant-related lung fibrosis is characterized by excessive fibro-collagenous deposition. Induction of arginase, an enzyme that metabolizes L-arginine to urea and L-ornithine, is vital for collagen synthesis. Pirfenidone is an investigational anti-fibrotic agent shown to be effective in blocking pulmonary fibrosis. The purpose of this study was to determine if pirfenidone was protective against the development of fibro-collagenous injury in rat lung orthotopic transplants through altering L-arginine-arginase metabolic pathways. Lung transplants were performed using Lewis donors and Sprague-Dawley recipients (allografts) or the same strain (isografts). Recipients were given pirfenidone (0.5% chow) 1-21-day post-transplantation. A significantly increased peak airway pressure (PawP) with excessive collagen deposition was found in untreated lung allografts. Pirfenidone treatment decreased PawP and collagen content in lung allografts. The beneficial effects were associated with downregulation of arginase protein expression and activity. In addition, pirfenidone decreased endogenous transforming growth factor (TGF)-beta level in lung allografts, and TGF-beta stimulated arginase activity in a dose-dependent manner in both lung tissue and fibroblasts. These results suggest that pirfenidone inhibits local arginase activity possibly through suppression of endogenous TGF-beta, hence, limiting the development of fibrosis in lung allografts.     

Effects of gabexate mesilate (FOY) on ischemia-reperfusion-induced acute lung injury in dogs

BACKGROUND: To assess the effects of gabexate mesilate (FOY), a protease inhibitor, on a canine model of pulmonary ischemia-reperfusion injury. FOY has been applied clinically to treat acute pancreatitis and disseminated intravascular coagulation (DIC) and has been found to suppress some leukocyte-mediated tissue injuries in both in vitro and in vivo studies. MATERIALS AND METHODS: DESIGN: Comparison of four experimental groups: group 1 (untreated control, n = 8), unilateral (left) pulmonary ischemia due to perfusion and ventilation obstruction followed by reperfusion, without receiving any specific treatment; group 2 (negative control, sham operation, n = 8), left pulmonary hilar dissection without ischemia; group 3 (FOY posttreatment, n = 8), FOY treatment during the reperfusion stage only; and group 4 (FOY pretreatment, n = 8), FOY treatment before ischemia and then continued during reperfusion. SETTING: University animal laboratory. SUBJECTS: Heart-worm-free mongrel dogs (12 to 15 kg body wt) were anesthetized with pentobarbital and mechanically ventilated. INVESTIGATIONS: Lung ischemia was made by snaring the left pulmonary artery and veins and clamping the bronchus with peribronchial tissue for 90 min followed by reperfusion for 18 h. Animals of the two treatment groups received a 1 mg/kg bolus of FOY at the beginning of reperfusion, with infusion of 2 mg/kg/h of FOY continuously starting 30 min before ischemia (group 4) or after reperfusion (group 3). During this study the following were measured: hemodynamics and aerodynamics, blood gas, bronchoalveolar lavage (BAL) fluid neutrophil percentage and protein concentration, lung wet to dry weight ratio (W/D ratio), myeloperoxidase (MPO) activity of the lung tissue, alveolar neutrophil infiltration, and degree of injury. RESULTS: This model of lung ischemia-reperfusion induced significant pulmonary hypertension, increased pulmonary vascular resistance, decreased pulmonary dynamic compliance and arterial hypoxemia, increased BAL fluid total protein amount and neutrophil percentage, and increased alveolar neutrophil infiltration, histological injury score, and lung tissue MPO assay (group 1). Animals of the sham operation (negative control, group 2) showed only minimal changes in the above parameters. Treatment with FOY significantly attenuated the injury by decreasing the lung W/D ratio, alveolar neutrophil infiltration, histological injury score, lung tissue MPO assay, BAL fluid neutrophil percentage, and protein amount. Pretreatment with FOY (group 4) attenuated the injury to a significantly greater degree than it did when administered at the reperfusion stage only (group 3), which was reflected by the above-mentioned parameters, and as well significantly improved gas exchange function. FOY treatment was found to have little effect in altering hemodynamics and aerodynamics at most time points in this model of lung injury. CONCLUSIONS: FOY can attenuate the ischemia-reperfusion-induced acute lung injury in dogs by ameliorating the degree of alveolar membrane permeability change, neutrophil aggregation, and activation. FOY treatment starting before ischemia attenuated this injury to a significantly higher degree than its use after ischemia. However, the effect of FOY may be partial because it cannot alter the hemodynamics or aerodynamics as prominently as other parameters in this type of lung injury. Concomitant use of FOY with other agents will have additive or synergic effects in preventing lung ischemia-reperfusion injury.     

Tumor necrosis factor-alpha neutralization reduces lung injury after experimental allogeneic bone marrow transplantation

BACKGROUND: Idiopathic pneumonia syndrome (IPS) is a frequent and potentially fatal complication of bone marrow transplantation (BMT). We have previously shown that experimental IPS is associated with increased levels of lipopolysaccaride (LPS) and tumor necrosis factor-alpha (TNFalpha) in the bronchoalveolar lavage (BAL) fluid, and that administration of LPS to animals with extensive graft versus host exacerbated underlying lung injury (Blood 1996; 88: 3230). METHODS: Lethally irradiated CBA mice received BMT from allogeneic (B10.BR) or syngeneic (CBA) donors. The role of TNFalpha in the exacerbation of pulmonary toxicity caused by LPS injection and in the evolution of IPS after allogeneic BMT was examined by neutralizing TNFalpha after BMT using a soluble binding protein (rhTNFR:Fc). RESULTS: Five weeks after BMT, administration of rhTNFR:Fc dramatically reduced mortality and prevented the exacerbation of lung injury caused by LPS administration. This protective effect was associated with preservation of pulmonary function and with marked reductions of cells, neutrophils, and LPS in the BAL fluid of treated animals. TNFalpha neutralization from week 4 to 6 after allogeneic BMT effectively halted the progression of systemic GVHD and significantly reduced, but did not prevent lung injury that developed during the treatment period. CONCLUSIONS: We conclude that TNFalpha is central to early LPS induced toxicity in this model and is a significant, but not the exclusive contributor to the development of IPS after allogeneic BMT.     

Kupffer cell inactivation delays repair in a rat model of reversible biliary obstruction

BACKGROUND: During cholestatic liver injury, Kupffer cells (KC) and activated macrophages modulate cell proliferation and subsequent matrix deposition. The role of KC in the restoration of cell architecture and matrix metabolism during repair following chronic cholestatic liver injury is unknown. MATERIALS AND METHODS: To determine the effect of KC inactivation, adult male Sprague-Dawley rats underwent bile duct suspension (BDS) for 5 days followed by reversal of the obstruction. Saline (control) and gadolinium chloride (10 mg/kg) were administered 1 day prior to BDS and 1 day prior to reversal, to inactivate KC during both injury and repair. Serum bilirubin and quantitative cell morphometry were compared to verify the reversibility of the model. Collagen content of the liver was measured in trichrome-stained paraffin sections using NIH imaging software. RESULTS: Reversibility of the obstruction was verified by normalization of direct serum bilirubin, which peaked at 8.42 +/- 0.76 mg/dL following 5 days of BDS and returned to sham-operated levels 2 days after reversal, 0.36 +/- 0.15 mg/dL. Hematoxylin and eosin (H&E)-stained paraffin-embedded liver sections from gadolinium-treated animals at 4 and 7 days after reversal exhibited persistent bile duct proliferation, matrix deposition, and inflammation. Gadolinium-treated animals had altered collagen metabolism compared to saline controls. Whereas the collagen content in the saline group slowly returned to sham-operated levels over time, the treatment group demonstrated progressive accumulation of collagen during repair which was statistically significant at 7 days following reversal (8.79%/mm(2) +/- 2.17 in gadolinium group vs 2. 33%/mm(2) +/- 0.34 in saline group, P = 0.0003). CONCLUSIONS: These results demonstrate that inactivation of resident hepatic macrophages during liver repair impairs collagen metabolism, inhibits the resolution of fibrosis, and allows the persistence of inflammatory cell infiltrates in the portal areas. This is the first evidence of profibrogenic responses in the absence of an intact KC compartment during repair after cholestatic injury.     

Effect of lung T lymphocytes on fibroblasts in idiopathic pulmonary fibrosis and extrinsic allergic alveolitis.

Increased fibroblast replication and interstitial collagen accumulation occur commonly in the interstitial lung disease that progress to fibrosis. The processes controlling lung fibrogenesis are not completely understood, however. This study was designed to analyse the influence of T lymphocytes from lung tissue obtained at open lung biopsy from four patients with idiopathic pulmonary fibrosis and four patients with extrinsic allergic alveolitis on fibroblast proliferation and collagen synthesis in vitro. Lung T cell supernatants from patients with both diseases induced a moderate but significant inhibition of human lung fibroblast cell line growth. In contrast, there was a clear difference in the effect of T cells from the two groups of patients in relation to collagen production. Lung T lymphocytes from all four patients with idiopathic pulmonary fibrosis produced a substantial increase in collagen synthesis (from 371% to 514% of control values), whereas T cells from three of the four patients with extrinsic allergic alveolitis induced a significant decrease in collagen production (to 35%, 36%, and 43% of control values); in the fourth case there was an increase in collagen synthesis but this was lower than that seen with T cells from any of the patients with idiopathic pulmonary fibrosis. Peripheral T cells from six patients and control subjects caused a small increase in fibroblast proliferation and no change in collagen synthesis. The findings suggest that at least two types of interaction occur between lung T cells and fibroblasts in these disorders. A variable degree of inhibition of cell proliferation is observed in response to lung T cell supernatants from patients with both idiopathic pulmonary fibrosis and extrinsic allergic alveolitis; a substantial increase in collagen synthesis is triggered by lymphokines from patients with idiopathic pulmonary fibrosis.     

Human amnion epithelial cells prevent bleomycin-induced lung injury and preserve lung function

Human amnion epithelial cells (hAECs) have attracted recent attention as a promising source of cells for regenerative therapies, with reports that cells derived from human term amnion possess multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties. Specifically, in animal models of lung disease characterized by significant loss of lung tissue secondary to chronic inflammation and fibrosis, the transplantation of hAECs has been shown to reduce both inflammation and subsequent fibrosis. To further explore the mechanisms by which hAECs reduce pulmonary fibrosis and enhance lung regeneration, we utilized a bleomycin-induced model of pulmonary fibrosis and investigated the ability of hAECs to reduce fibrosis and thereby improve pulmonary function. We aimed to determine if hAECs, injected into the peritoneal cavity could migrate to the lung, engraft, and form functional lung epithelium, and whether hAECs could modulate the inflammatory environment in the bleomycin-injured lung. We demonstrated that, compared to bleomycin alone, IP administration of hAECs 24 h after bleomcyin, decreased gene expression of the proinflammatory cytokines TNF-α, TGF-β, IFN-γ, and IL-6 and decreased subsequent pulmonary fibrosis with less pulmonary collagen deposition, reduced levels of α-smooth muscle actin and decreased inflammatory cell infiltrate. We also showed that hAECs are able to prevent a decline in pulmonary function associated with bleomycin-induced lung damage. We were unable to detect any significant engraftment of hAECs in injured, or uninjured, lung after administration. The findings from this study support the further investigation of hAECs as a potential cell therapy for inflammatory and fibrogenic diseases.