Attenuation of bleomycin-induced lung fibrosis by oral sulfhydryl containing antioxidants in rats: erdosteine and N-acetylcysteine

Antioxidant therapy may be useful in diseases with impaired oxidant antioxidant balance such as lung fibrosis. The effects of sulfhydryl-containing antioxidant agents N-acetylcysteine (NAC) and erdosteine on the bleomycin-induced lung fibrosis were compared in rats. The animals were divided into four groups: Vehicle + vehicle, vehicle + bleomycin (2.5 U/kg), bleomycin + (10 mg/kg), and bleomycin + NAC (3 mmol/kg). Bleomycin administration resulted in prominent lung fibrosis as measured by lung hydroxyproline content and lung histology which is almost completely prevented by erdosteine and NAC. Hydroxyproline content was 18.7 +/- 3.5 and 11.2 +/- 0.6 mg/g dried tissue in bleomycin and saline treated rats, respectively (P < 0.001), and this level was 11.3 +/- 1.2 and 13.8 +/- 1.2 mg/g dried tissue in erdosteine and NAC pretreated, respectively. Erdosteine and NAC significantly reduced depletion of glutathione peroxidase, and prevented increases in myeloperoxidase activities, nitric oxide, and malondialdehyde levels in lung tissue produced by bleomycin. Data presented here indicate that erdosteine and NAC similarly prevented bleomycin-induced lung fibrosis and their antioxidant effects were also similar in this experiment.     

Oral N-acetylcysteine reduces bleomycin-induced lung damage and mucin Muc5ac expression in rats.

Oxidative stress is involved in the pathogenesis of pulmonary fibrosis, therefore antioxidants may be of therapeutic value. Clinical work indicates that N-acetylcysteine (NAC) may be beneficial in this disease. The activity of this antioxidant was examined on bleomycin-induced lung damage, mucus secretory cells hyperplasia and mucin Muc5ac gene expression in rats. NAC (3 mmol x kg(-1) x day(-1)) or saline was given orally to Sprague-Dawley rats for 1 week prior to a single intratracheal instillation of bleomycin (2.5 U x kg(-1)) and for 14 days postinstillation. NAC decreased collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,257+/-323 and 3,200+/-192 microg x lung(-1) in vehicle- and NAC-treated rats, respectively) and lessened the fibrotic area assessed by morphometric analysis. The bleomycin-induced increases in lung tumour necrosis factor-alpha and myeloperoxidase activity were reduced by NAC treatment. The numbers of mucus secretory cells in airway epithelium, and the Muc5ac messenger ribonucleic acid and protein expression, were markedly augmented in rats exposed to bleomycin. These changes were significantly reduced in NAC-treated rats. These results indicate that bleomycin increases the number of airway secretory cells and their mucin production, and that oral N-acetylcysteine improved pulmonary lesions and reduced the mucus hypersecretion in the bleomycin rat model.     

Pediatric lung disease: from proteinases to pulmonary fibrosis

One distinctive outcome of interstitial lung diseases in childhood is the abnormal accumulation of pulmonary extracellular matrix. The clinical consequence of such excessive connective tissue accumulation is known as pulmonary fibrosis. While numerous aspects of its pathogenesis have become familiar, many key events involved in its inception and progression still remain unclear. There is now compelling evidence that lung damage due to uncontrolled proteolysis may help drive critical processes that regulate fibrotic matrix remodeling. In this regard, a number of proteinases have been implicated in promoting both the initial lung injury and the fibroproliferative repair that follows. This review summarizes the knowledge of how different matrix-targeting enzymes may act to influence the development of pediatric pulmonary fibrosis. Understanding the scientific basis of this complex process may highlight opportunities to limit unwanted proteolysis and the intensity of its fibrotic sequelae.     

Progressive pulmonary fibrosis in hamsters

The concomitant treatment of hamsters with bleomycin and hyperoxia results in a synergistic development of pulmonary injury. We exposed hamsters for 72 hr to 70% oxygen following a single intratracheal instillation of bleomycin (0.16 U/100 g body weight). Groups of 10 animals were killed at 3, 6, 10, 30, 60, 90, and 120 days after instillation for histopathologic and morphometric assessment. Diffuse alveolar damage developed acutely. At 30 days, the intense acute cellular infiltrate had subsided, leaving a focal interstitial pneumonitis. Morphometric quantitation at 10 days revealed that 33.5 +/- 5.3% (x +/- SE) of the lung was diseased; there was apparent healing by 30 days, when 10.5 +/- 2.0% of the lung was diseased. However, progression to diffuse pneumonitis with fibrosis was seen at 60, 90, and 120 days, when 30.2 +/- 4.9%, 38.5 +/- 5.8%, and 38.8 +/- 4.5% of the lung was diseased, respectively. In vivo pulmonary function studies on treated animals at 25 and 55 days showed decreasing dynamic compliance and increased minute ventilation, which corroborates the presence of interstitial fibrosis. We conclude that simultaneous treatment of hamsters with bleomycin and hyperoxia results in interstitial fibrosis with a distribution and progression that mimics human pulmonary fibrosis. This model appears ideally suited for the study of progressive fibrosis and will be useful when development of a widely distributed lesion is crucial.     

趋化因子CXC受体3在博莱霉素诱导的肺纤维化中的作用

目的　采用趋化因子受体即CXCR3基因敲除小鼠 ,探讨CXCR3在博莱霉素诱导的肺损伤及纤维化中的作用。方法　采用基因打靶技术得到无CXCR3基因小鼠 6 2只 ,同时选择性别、年龄和体重配对的野生型小鼠 4 8只 ,随机分入不同的实验组及对照组。大鼠气管内注入博莱霉素0 0 2 5U。实验动物按要求处死后 ,取无血的肺组织切片 ,经 10 %甲醛固定后 ,用苏木精 伊红 (HE)和Massontrichrome染色 ,分别观察实验动物肺炎症和纤维化的程度 ;用浓盐酸消化肺组织提取羟脯氨酸并定量来表示肺胶原的含量 ;用磷酸盐缓冲液 (PBS)灌洗肺组织 ,酶联免疫吸附 (ELISA)法测定灌洗液中肿瘤坏死因子α(TNF α)、白细胞介素 5 (IL 5 )及转化生长因子 β(TGF β)的浓度 ;用流式细胞仪检测T细胞亚群。结果　气管内注入博莱霉素后 14d ,CXCR3基因敲除小鼠肺组织炎症程度及纤维化程度较野生型小鼠明显减轻。肺组织炎症程度评分和羟脯氨酸含量 (左肺 )在CXCR3基因敲除小鼠分别为 3 92± 0 37和 (6 7 0± 2 4 2 ) μg ,在野生型小鼠则分别为 5 33± 0 34和 (2 11 5± 2 4 2 )μg ,两组比较差异有统计学意义 (P分别 <0 0 5 ,<0 0 1) ;TGF β水平在CXCR3基因敲除小鼠为(2 2 11± 2 89) μg/L ,在野生型小鼠为 (5 388± 10 71) μg/L ,     

肺间质纤维化大鼠肺组织基质金属蛋白酶的动态改变

进一步评价基质金属蛋白酶在肺间质纤维化形成过程中的作用。方法Ｗｉｓｔａｒ大鼠２１只随机分为模型组１４只,对照组７只,模型组在额镜直视下插管,注入０．４％博莱霉素注射液５ｍｇ／ｋｇ,制成动物模型。观察注射药物后１周和４周肺泡巨噬细胞和纤维细胞ＭＭＰ的活性及表达。利用空斑扩散法,测定胶原酶活性。     

Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans

Pulmonary fibrosis is a progressive and largely untreatable group of disorders that affects up to 100,000 people on any given day in the United States. To elucidate the molecular mechanisms that lead to end-stage human pulmonary fibrosis we analyzed samples from patients with histologically proven pulmonary fibrosis (usual interstitial pneumonia) by using oligonucleotide microarrays. Gene expression patterns clearly distinguished normal from fibrotic lungs. Many of the genes that were significantly increased in fibrotic lungs encoded proteins associated with extracellular matrix formation and degradation and proteins expressed in smooth muscle. Using a combined set of scoring systems we determined that matrilysin (matrix metalloproteinase 7), a metalloprotease not previously associated with pulmonary fibrosis, was the most informative increased gene in our data set. Immunohistochemisry demonstrated increased expression of matrilysin protein in fibrotic lungs. Furthermore, matrilysin knockout mice were dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin. Our results identify matrilysin as a mediator of pulmonary fibrosis and a potential therapeutic target. They also illustrate the power of global gene expression analysis of human tissue samples to identify molecular pathways involved in clinical disease.     

Genetic deficiency of alpha1-PI in mice influences lung responses to bleomycin.

It has recently been suggested that proteinase inhibitors modulate the fibrotic response in the lung. This study investigated the development of bleomycin-induced pulmonary changes in pallid mice, deficient in serum alpha1-proteinase inhibitor, and with a lower elastase inhibitory capacity, and in congenic C57Bl/6J mice. Male pallid and C57Bl/6J mice received a single intratracheal instillation of either saline or bleomycin. The investigation was carried out by means of biochemical, morphological and morphometrical methods. In both strains, 21 and 72 h after bleomycin, the lungs showed foci of inflammatory cell infiltration associated with emphysema. Fibrosis developed with time after bleomycin. At 14 days fibrosis affected 23.46+/-9.48% (mean +/- SD) and 40.62+/-13.34% (p < 0.01) of the lungs of C57Bl/6J and pallid mice, respectively. Emphysema affected 3.68+/-3.11% and 12.57+/-4.13% (p<0.01) of lung in C57Bl/6J and pallid mice, respectively. In C57Bl/6J mice bleomycin increased lung hydroxyproline content by 34% and desmosine content by 44% (p < 0.01 for both). In pallid mice these increases were only 21% (p < 0.01) and 6% which may reflect parenchymal loss. Thus, the lung destructive response (emphysema) and the subsequent proliferative reaction (fibrosis) to bleomycin are potentiated in alpha1-proteinase inhibitor deficiency.     

New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a serious and progressive chronic lung disease that is characterised by altered cellular composition and homoeostasis in the peripheral lung, leading to excessive accumulation of extracellular matrix and, ultimately, loss of lung function. It is the interstitial pneumonia with the worst prognosis--mortality 3-5 years after diagnosis is 50%. During the past decade, researchers have described several novel cellular and molecular mechanisms and signalling pathways implicated in the pathogenesis of idiopathic pulmonary fibrosis, resulting in the identification of new therapeutic targets. These advances will hopefully result in increased survival rates and improved quality of life for patients with this disorder in future.     

骨髓间充质干细胞对博来霉素致大鼠肺纤维化形成的影响

目的通过观察骨髓间充质干细胞（MSC）对博来霉素致大鼠肺纤维化动物模型的影响，探讨治疗肺纤维化的新方法。方法体外分离、培养雄性6周龄SD大鼠的骨髓MSC。将48只雌性SD大鼠随机分为6组，第1—5组经气管注入5．0mg／kg博来霉素0．3Tnl，第1和第3组分别于博来霉素注射第1天和第7天经尾静脉注入雄性大鼠MSC液0．2ml（细胞数为2．5×10^6个）；第2和第4组分别于博来霉素注射第1天和第7天经尾静脉注入等量的磷酸缓冲液0．2ml；第5组作为模型阳性对照，注射博来霉素后未给予其他处理；第6组作为模型阴性对照，经气管注入等量生理盐水0．3ml代替博来霉素，未给予其他处理。于实验第28天统一处死大鼠，留取肺组织行病理学检查、羟脯氨酸含量测定；提取肺组织的DNA，通过聚合酶链反应（PCR）-琼脂糖电泳法检测雄鼠的性别决定基因（sry基因），以判断外源给予的MSC是否在雌性大鼠肺组织中存在。结果在博来霉素致肺损伤第1天和第7天给予MSC干预治疗后的大鼠肺脏病理改变均较对照组减轻，肺纤维化程度评分分别为（1．0±0．2）分、（2．5±0．5）分和（1．6±0．5）分、（2．3±0．8）分；肺组织羟脯氨酸含量分别为（83±17）μg／mg、（96±20）μg／mg和（123±32）μg／mg、（127±34）μg／mg，在博来霉素致肺损伤第1天较第7天给予MSC其作用更明显。PCR检测结果显示，在博来霉素致肺损伤第1天给予MSC组的大鼠肺组织可以检测到sry基因。结论外源给予的MSC可以减轻肺纤维化的形成，在肺损伤早期给予MSC干预的疗效更好。     

Characterization of fibroblast-specific protein 1 in pulmonary fibrosis

Because fibroblasts produce collagen and other extracellular matrix components that are deposited during tissue fibrosis, defining the behavior of these cells is critical to understanding the pathogenesis of fibrotic diseases. We investigated the utility of fibroblast-specific protein 1 (FSP1), a member of the calmodulin S100 troponin C superfamily, for identifying lung fibroblasts in a murine model of pulmonary fibrosis induced by intratracheal administration of bleomycin. Protein and mRNA expression of FSP1 was minimal in untreated lungs, but increased by 1 week after bleomycin administration and remained increased at 2 and 3 weeks after treatment. By immunohistochemistry, the number of FSP1(+) cells increased in a dose-dependent manner in the lungs after bleomycin treatment. Colocalization of alpha1 procollagen and FSP1 in interstitial cells demonstrated that FSP1(+) fibroblasts contribute to the deposition of collagen after bleomycin administration. In primary lung cell cultures, lung fibroblasts, but not macrophages or type II alveolar epithelial cells, expressed FSP1. FSP1 also identified fibroblasts in lung biopsy specimens from patients with documented usual interstitial pneumonitis. Therefore, FSP1 is an improved marker for lung fibroblasts that could be useful for investigating the pathogenesis of pulmonary fibrosis.     

Attenuation by oral N-acetylcysteine of bleomycin-induced lung injury in rats.

Antioxidant therapy may be useful in diseases with impaired oxidant-antioxidant balance such as pulmonary fibrosis. This study examines the effect of N-acetylcysteine (NAC) on bleomycin-induced lung fibrosis in rats. NAC (3 mmol x kg(-1); oral) was given daily from 1 week prior to a single intratracheal instillation of bleomycin (2.5 U x kg(-1)) or saline, until 14 days postinstillation. NAC partially decreased the augmented collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,354+/-386 and 3,416+/-326 microg x lung(-1) in vehicle-treated and NAC-treated rats, respectively; p < 0.05). The histological assessment using a semiquantitative score showed less collagen deposition and inflammatory cells in NAC-treated rats compared to those receiving bleomycin alone. NAC failed to inhibit the bleomycin-induced increases in lung wet weight and in cell counts and protein levels of bronchoalveolar lavage fluid, but significantly increased total glutathione and taurine levels in bronchoalveolar lavage fluid. These results indicate that oral N-acetylcysteine improves the pulmonary antioxidant protection and may be useful in reducing lung damage produced by bleomycin.     

Losartan attenuates bleomycin-induced pulmonary fibrosis in rats

BACKGROUND: In addition to regulating blood pressure and body fluid homeostasis, the renin-angiotensin system is also involved in lung fibrogenesis. OBJECTIVE: To study the effect of losartan, an angiotensin II antagonist, on bleomycin-induced pulmonary fibrosis in rats and its possible mechanism. METHODS: Pulmonary fibrosis was induced in SD rats by intratracheal instillation of bleomycin (5 mg x kg(-1)). Subsequently, the rats received daily losartan (3, 9 and 27 mg x kg(-1)) or prednisone (20 mg x kg(-1)) orally. Six rats in each group were sacrificed 14 and 21 days after intratracheal instillation. Hydroxyproline, superoxide dismutase (SOD), and malondialdehyde (MDA) levels in lung tissues were determined by spectroscopy. The levels of TGF-beta1 in serum were measured by ELISA. Histological changes in the lungs were evaluated by hematoxylin-eosin stain, and scored. RESULTS: Rat body weight evidently decreased while the indices of lung and hydroxyproline contents in lung tissue were significantly increased 14 and 21 days after intratracheal bleomycin instillation. Inflammatory cell infiltration and fibrotic scores were more prominent in the model group compared to the sham group. Losartan (3, 9 and 27 mg.kg(-1), i.g.) apparently attenuated the degree of pulmonary fibrosis. Further study showed that losartan significantly increased SOD levels while it decreased MDA contents in lung homogenates. Serum TGF-beta1 levels of pulmonary fibrosis rats were also decreased by losartan. CONCLUSIONS: Losartan had an inhibitory effect on bleomycin-induced pulmonary fibrosis, and its effect may be associated with its anti-free radicals and the reduction in TGF-beta1.     

γ干扰素转基因表达对博莱霉素致小鼠肺纤维化的作用

目的　探讨小鼠γ干扰素 (mIFN γ)在肺内局部转基因表达对博莱霉素致小鼠肺纤维化的作用。方法　 6～ 8周龄雄性ICR小鼠 ,模型组第 0天经鼻滴入BLM 3mg/kg建立肺间质纤维化模型 ,预防治疗组于滴入BLM前 4 8h经鼻给予重组复制缺陷型mIFN γ腺病毒 (AdCMVmIFN γ) 5×10 8空斑形成单位 (pfu) /鼠 ,治疗组于滴入BLM后 72h经鼻给予等量AdCMVmIFN γ ,同时设立无外源活性基因的复制缺陷型腺病毒 (AdCMVNull)对照组和生理盐水阴性对照组。第 0、7、14天分别测小鼠体重 ,第 14天收获其支气管肺泡灌洗液 (BALF)和肺组织 ,右肺行苏木精 伊红 (HE)和Masson三色染色 ,左肺经酸解法检测肺组织羟脯氨酸 (HYP)含量。用酶联免疫吸附法 (ELISA)检测BALF中mIFN γ的浓度。结果　AdCMVmIFN γ转基因预防治疗组和治疗组BALF中mIFN γ含量明显增高[(2 12 5 0± 6 4 97)pg/ml,(2 10 0 0± 5 4 5 1)pg/ml],其它组BALF中mIFN γ浓度为接近零水平。上述两组与其余各组比较 ,小鼠体重明显减轻 (P <0 .0 5 ) ,肺泡炎、纤维化程度均有加重倾向 (P =0 .0 7) ,肺HYP含量增高 (P <0 .0 5 )。结论　 (1)AdCMVmIFN γ经鼻滴入可在小鼠肺内实现mIFN γ转基因表达 ;(2 )在BLM模型中 ,早期mIFN γ转基因表达可在某种程度上加重BLM诱导的肺     

Idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a devastating, age-related lung disease of unknown cause that has few treatment options. This disease was once thought to be a chronic inflammatory process, but current evidence indicates that the fibrotic response is driven by abnormally activated alveolar epithelial cells (AECs). These cells produce mediators that induce the formation of fibroblast and myofibroblast foci through the proliferation of resident mesenchymal cells, attraction of circulating fibrocytes, and stimulation of the epithelial to mesenchymal transition. The fibroblast and myofibroblast foci secrete excessive amounts of extracellular matrix, mainly collagens, resulting in scarring and destruction of the lung architecture. The mechanisms that link idiopathic pulmonary fibrosis with ageing and aberrant epithelial activation are unknown; evidence suggests that the abnormal recapitulation of developmental pathways and epigenetic changes have a role. In this Seminar, we review recent data on the clinical course, therapeutic options, and underlying mechanisms thought to be involved in the pathogenesis of idiopathic pulmonary fibrosis.     

Mechanical aspects of lung fibrosis: a spotlight on the myofibroblast

Contractile myofibroblasts are responsible for the irreversible alterations of the lung parenchyma that hallmark pulmonary fibrosis. In response to lung injury, a variety of different precursor cells can become activated to develop myofibroblast features, most notably formation of stress fibers and expression of α-smooth muscle actin. Starting as an acute and beneficial repair process, myofibroblast secretion of collagen and contraction frequently becomes excessive and persists. The result is accumulation of stiff scar tissue that obstructs and ultimately destroys lung function. In addition to being a consequence of myofibroblast activities, the stiffened tissue is also a major promoter of the myofibroblast. The mechanical properties of scarred lung and fibrotic foci promote myofibroblast contraction and differentiation. One essential element in this detrimental feed-forward loop is the mechanical activation of the profibrotic growth factor transforming growth factor-β1 from stores in the extracellular matrix. Interfering with myofibroblast contraction and integrin-mediated force transmission to latent transforming growth factor-β1 and matrix proteins are here presented as possible therapeutic strategies to halt fibrosis.     

The vitronectin-binding function of PAI-1 exacerbates lung fibrosis in mice

Plasminogen activator inhibitor-1 (PAI-1) is increased in the lungs of patients with pulmonary fibrosis, and animal studies have shown that experimental manipulations of PAI-1 levels directly influence the extent of scarring that follows lung injury. PAI-1 has 2 known properties that could potentiate fibrosis, namely an antiprotease activity that inhibits the generation of plasmin, and a vitronectin-binding function that interferes with cell adhesion to this extracellular matrix protein. To determine the relative importance of each PAI-1 function in lung fibrogenesis, we administered mutant PAI-1 proteins that possessed either intact antiprotease or vitronectin-binding activity to bleomycin-injured mice genetically deficient in PAI-1. We found that the vitronectin-binding capacity of PAI-1 was the primary determinant required for its ability to exacerbate lung scarring induced by intratracheal bleomycin administration. The critical role of the vitronectin-binding function of PAI-1 in fibrosis was confirmed in the bleomycin model using mice genetically modified to express the mutant PAI-1 proteins. We conclude that the vitronectin-binding function of PAI-1 is necessary and sufficient in its ability to exacerbate fibrotic processes in the lung.     

An increase in hyaluronan by lung fibroblasts: A biomarker for intensity and activity of interstitial pulmonary fibrosis?

The purpose of the present study was to clarify the roles of hyaluronan (HA) production of lung fibroblasts in the pathogenesis of pulmonary fibrosis. Quantitative and comparative assessments of the HA levels in bronchoalveolar lavage fluid (BALF) and lung fibroblast-conditioned media (F-CM) were made at various stages during the development of bleomycin-induced pulmonary fibrosis in rats. In bleomycin-treated animals, the HA levels in F-CM increased significantly (P < 0.01) on day 1 after bleomycin treatment, peaked on day 3, and then gradually declined and returned to control values on days 14-28. The HA concentrations of BALF in the bleomycin group were significantly increased (P < 0.01) on day 3, were maximal on day 7, and thereafter gradually decreased, remaining significantly above normal values (P < 0.01) on day 14, but returning to control values by day 28. In the bleomycin group, the HA levels both in BALF and in F-CM were significantly correlated with the cell components in BALF and there was a significant correlation between the HA concentration in BALF and in the F-CM. Lung fibroblasts were activated and produced increased HA which resulted in excessive accumulation of HA in the lung in the early stage of pulmonary fibrosis; the increased HA synthesis of lung fibroblasts and enhanced HA concentrations of BALF might reflect the intensity of alveolitis and the disease activity.     

Oral N-acetylcysteine reduces bleomycin-induced collagen deposition in the lungs of mice.

N-acetylcysteine (NAC) has been employed in the treatment of acute lung injury but its therapeutic value is as yet unproven. In the present study we examined the effect of both L- and D-forms of NAC as inhibitors of bleomycin-induced fibrosis in mice. We hypothesized that, because of the D-form is not metabolized, it may be more effective than the L-form in ameliorating lung injury and fibrosis. Both drugs were given daily in the drinking water at a dose of approximately 400 mg.kg-1 body weight commencing one week prior to a single intratracheal instillation of bleomycin at a dose of 6 mg.kg-1 body weight. Lung injury was assessed 35 days later by measuring total lung collagen content, lung wet weight and examination of tissues by light and electron microscopy. Total collagen content and lung wet weight of animals receiving bleomycin together with L-NAC were 2.90 +/- 0.03 mg and 0.23 +/- 0.01 g, respectively. The values for collagen content, but not wet weight, were significantly less (p less than 0.05) than those given for bleomycin alone (3.90 +/- 0.02 mg and 0.260 +/- 0.005 g, respectively), but greater than (p less than 0.05) controls (2.10 +/- 0.01 mg and 0.160 +/- 0.002 g, respectively). Values for collagen content and wet weight of animals given bleomycin together with D-NAC (3.10 +/- 0.02 mg and 0.21 +/- 0.01 mg, respectively) were both significantly greater than values for control animals but lower than animals given bleomycin alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bleomycin-induced pulmonary fibrosis is attenuated in gamma-glutamyl transpeptidase-deficient mice

To investigate repair mechanisms in bleomycin-induced pulmonary fibrosis, we used mice deficient in gamma-glutamyl transpeptidase (GGT-/-), a key enzyme in glutathione (GSH) and cysteine metabolism. Seventy-two hours after bleomycin (0.03 U/g), GGT-/- mice displayed a different inflammatory response to wild-type mice as judged by a near absence of neutrophils in lung tissue and bronchoalveolar lavage and a less pronounced rise in matrix metalloproteinase-9. Inflammation in GGT-/- mice consisted mainly of lymphocytes and macrophages. At 1 month, lungs from bleomycin-treated GGT-/- mice exhibited minimal areas of fibrosis compared with wild-type mice(light microscopy fibrosis index: 510 +/- 756 versus 1975 +/- 817, p < 0.01). Lung collagen content revealed a significant increase in bleomycin-treated wild-type (15.1 +/- 3.8 versus 8.5 +/- 0.7 microg hydroxy(OH)-proline/mg dry weight, p < 0.01) but not in GGT-/- (10.4 +/- 1.7 versus 8.8 +/- 0.8). Control lungs from GGT-/- showed a significant reduction of cysteine (0.03 +/- 0.005 versus 0.055 +/- 0.001, p < 0.02) and GSH levels (1.24 +/- 0.055 versus 1.79 +/- 0.065, p < 0.002). These values decreased after 72 hours of bleomycin in both GGT-/- and wild-type but reached their respective control values after 1 month. Supplementation with N-acetyl cysteine partially ameliorated the effects of GGT deficiency. These findings suggest that increased neutrophils and matrix metalloproteinase-9 during the early inflammatory response and adequate thiol reserves are key elements in the fibrotic response after bleomycin-induced pulmonary injury.