Raphael Breuer

Time course of bleomycin-induced lung fibrosis.

Intratracheal instillation (IT) of bleomycin is a widely used experimental model for lung fibrosis. In this study we describe the time‐course of bleomycin‐induced lung fibrosis in mice using computer‐assisted morphometry. C57Bl/6J mice were treated with a single IT dose of bleomycin or control saline. Animals were killed 3, 6, 14 and 21 days post‐IT. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, hydroxyproline concentration in the lung, routine light microscopic examination resulting in a semiquantitative morphological index (SMI) of lung injury, and quantitative morphological measurements (fibrosis fraction and alveolar wall area fraction) aided by optimas image analysis software. Changes in BAL fluid attributed to bleomycin treatment include increased total cell count (days 14 and 21), and increased percentage of neutrophils (days 3 and 6) followed by a sustained increase in lymphocytes (days 6, 14 and 21). Hydroxyproline levels increased in bleomycin‐treated mice on days 14 and 21. Median SMI grades were significantly elevated on days 3, 14 and 21. Computer‐assisted morphometry demonstrated a 3‐fold increase in fibrosis fraction and a 1.3‐fold increase in wall area fraction in bleomycin‐treated mice on day 14, with no further increase on day 21. These data also demonstrate that the most suitable time point for assessing lung fibrosis in this model is 14 days after IT instillation of bleomycin, based on the observation that at 14 days the animals developed extensive fibrosis, but had less variability in the fibrotic response and lower mortality than later at 21 days. Computer‐assisted morphometry provides objective and quantitative measurements that are a useful tool for the evaluation of bleomycin‐induced lung injury.

Pulmonary involvement in lymphoma

Intrathoracic involvement is common in both Hodgkins disease (HD) and non-Hodgkins lymphoma (NHL). The most common manifestation is mediastinal lymphadenopathy. In HD, nodal involvement is by contiguity and usually involves the superior mediastinum, while the findings in NHL are more variable. Pulmonary parenchymal disease occurs in 38% of HD and 24% of NHL. In untreated HD, parenchymal involvement is invariably associated with mediastinal lymphadenopathy and often with widespread disease. Three distinct radiological patterns of pulmonary lymphoma are recognised: nodular, bronchovascular-lymphangitic and pneumonic-alveolar. Rarely lymphoma may be endobronchial. Pleural effusion occurs in 16% of lymphoma patients and is usually associated with disease elsewhere. It is frequently caused by lymphatic obstruction but may be due to direct pleural involvement by tumour. Chylothorax may occur in NHL but is unusual in HD. Diagnosis of intrathoracic lymphoma is by transbronchial or transthoracic biopsy or by needle aspiration of tissue or pleural fluid. The addition of immunostaining improves the diagnostic yield in equivocal cases. Treatment and prognosis vary depending on cell-type, location and extent of disease.

Retinoic acid does not affect alveolar septation in adult FVB mice with elastase-induced emphysema.

Background: Administration of all-trans retinoic acid (ATRA) to adult Sprague-Dawley rats with emphysema induced by porcine pancreatic elastase (PPE) reversed the emphysema perhaps by inducing new alveolar formation. Objective: A study was conducted to determine whether ATRA can induce new alveolar septa and reverse the airspace enlargement caused in adult mice by PPE treatment. Methods: 48 FVB mice were divided into 6 groups. Three groups received 15 µg of PPE in 0.1 ml of 0.9% saline and 3 groups received 0.1 ml of saline, intratracheally. Starting at day 22, the mice received 12 daily intraperitoneal injections of cottonseed oil, with or without ATRA (12.5 µg or 50 µg). The mice were killed for study 1 day after the last injection. Results: Measurements of plasma and lung tissue ATRA levels showed statistically significant elevated levels after the 50-µg but not after the 12.5-µg doses of ATRA. In situ hybridization studies of elastin and α1(I) collagen mRNA expression in pulmonary parenchyma as well as in airways and blood vessels showed no effect of ATRA. Airspace size was determined by the mean linear intercept (Lm) method. The Lm of the groups receiving PPE and ATRA (46.2 ± 4.1 µm, mean ± SD) was not significantly different from the group receiving PPE and oil (47.8 ± 6.0 µm). The Lm for groups receiving saline and ATRA (40.6 ± 2.5 µm) were not significantly different from the group receiving saline and oil (41.0 ± 2.7 µm). Comparison of the fixed lung volume data and calculated internal surface area also showed no differences between the control and ATRA-treated groups. Conclusion: ATRA treatment does not affect airspace size or expression of elastin or α1(I) collagen mRNA in adult FVB mice with PPE-induced emphysema.

Osteopontin Induces Airway Remodeling and Lung Fibroblast Activation in a Murine Model of Asthma

Airway remodeling is a central feature of asthma; however, the mechanisms underlying its development have not been fully elucidated. We have demonstrated that osteopontin, an inflammatory cytokine and an extracellular matrix glycoprotein with profibrotic properties, is up-regulated in a murine model of allergen-induced airway remodeling. In the present study, we determined whether osteopontin plays a functional role in airway remodeling. Osteopontin (OPN)-deficient (OPN(-/-)) and wild-type mice were sensitized and exposed to inhaled ovalbumin (OVA) or saline for 5 weeks. Collagen production, peribronchial smooth muscle area, mucus-producing cell number, and bronchoalveolar cell counts were assessed. The functional behavior and phenotype of lung fibroblasts from OVA-treated OPN(-/-) and from wild-type mice were studied using ex vivo cultures. OVA-treated OPN(-/-) mice exhibited reduced lung collagen content, smooth muscle area, mucus-producing cells, and inflammatory cell accumulation as compared with wild-type mice. Reduced matrix metalloproteinase-2 activity and expression of transforming growth factor-beta1 and vascular endothelial growth factor were observed in OVA-treated OPN(-/-) mice. Lung fibroblasts from OVA-treated OPN(-/-) mice showed reduced proliferation, migration, collagen deposition, and alpha-smooth muscle actin expression in comparison with OVA-treated wild-type lung fibroblasts. Thus, OPN is key for the development of allergen-induced airway remodeling in mice. In response to allergen, OPN induces the switching of lung fibroblasts to a pro-fibrogenic myofibroblast phenotype.

Evasion of myofibroblasts from immune surveillance: A mechanism for tissue fibrosis

Tissue fibrosis evolving from impaired tissue remodeling after injury is characterized by myofibroblast accumulation. We propose that during the development of fibrosis myofibroblasts acquire an immune-privileged cell phenotype, allowing their uninterrupted accumulation. Using the murine model of bleomycin-induced lung fibrosis in mice, we show that myofibroblasts that accumulate in lungs with fibrosis, but not in normal lungs, kill Fas+ lymphocytes, resist Fas-induced apoptosis, and survive longer when grafted into allogeneic mice. In contrast, bleomycin-treated FasLigand (FasL)-deficient (gld) chimeric mice did not accumulate myofibroblasts or collagen in their lungs, and their FasL− myofibroblasts did not survive after alloengraftment. This finding indicates that myofibroblasts possess Fas/FasL-pathway-dependent characteristics that allow them to escape from immune surveillance and resulting organ fibrosis.

Use of Fiberoptic Bronchoscopy in Bone Marrow Transplant Recipients

Bone marrow transplantation (BMT) has become the therapy of choice for a number of malignant and nonmalignant hematologic and nonhematologic disorders. A frequent complication after BMT is pulmonary disease which is associated with a high mortality rate. We examined the results of 79 bronchoscopies performed between May 1991 and May 1995 in 62 patients for the evaluation of pulmonary complications after BMT. In all cases bronchoalveolar lavage (BAL) was performed, in 10% transbronchial biopsy (TBB) was also carried out and in 13% bronchoscopy was followed by open lung biopsy. Positive results were found in 67% of bronchoscopies. Fungal infection (Candida and Aspergillus species) was the most common finding (18%), bacterial infection was found in 13%, mixed (fungal and bacterial) infection in 6%, cytomegalovirus in 11% and Pneumocystis carinii pneumonia in 4%. Diffuse alveolar hemorrhage was detected in 11% of cases. Idiopathic pneumonia syndrome (IPS) was diagnosed by TBB in 3% of procedures. We conclude that BAL is a safe and accurate procedure for the evaluation of pulmonary complications after BMT. TBB should be considered in the absence of thrombocytopenia for the diagnosis of IPS. If bronchoscopy findings are negative, open lung biopsy should be considered.

Enhanced osteopontin expression in a murine model of allergen-induced airway remodelling.

Background Airway remodelling is a central pathophysiological feature of chronic asthma. A wide variety of cytokines and growth factors are likely to be involved in the development of airway remodelling. Osteopontin (OPN) is a cytokine with pro‐fibrotic properties; however, its role in airway remodelling in asthma has not been explored.

An 18-month study of the effects on hamster lungs of intratracheally administered human neutrophil elastase.

A study was made of the evolution of emphysema and airway injury induced in the lungs of male golden Syrian hamsters by a single intratracheal injection of 350 micrograms human neutrophil elastase (HNE). Saline control and HNE-treated groups of 8 animals were studied 1, 3, 6, 12, and 18 months posttreatment. HNE treatment caused a significant increase in all lung volumes and a significant decrease in maximum expiratory flows at all study times. The mean linear intercept (MLI) values of the left lung were significantly increased over control values. There was no progression with time in MLI values, lung volumes, or lung compliance. Secretory-cell metaplasia was present at 1 month and persisted throughout the study. The HNE-treated lungs showed clusters of ferric iron-containing macrophages in the terminal airspaces. The amount of iron in the lungs, determined morphometrically, was greatest at 1 month, was decreased by 6 months, and then did not change further to 18 months. At 18 months the amount of iron was still significantly above control amounts. We conclude that the airway and parenchymal lesions induced by HNE persist without progression for 18 months. Clearance of ferric iron, which was probably a result of the hemorrhage induced by HNE treatment, continued for 6 months with no evident subsequent clearance.

An Ultrastructural Study of the Response of Hamster Bronchial Epithelium to Human Neutrophil Elastase

The central intrapulmonary bronchi of hamsters were examined by transmission electron microscopy at varying times following intratracheal instillation of human neutrophil elastase (HNE) or its vehicle, saline. Two hours after HNE treatment, there was a marked irregularity of the surfaces of many nonciliated epithelial cells; a differential count of transepithelial cells (those with both a basal lamina and luminal border) demonstrated a significant decrease in the proportion of granule-containing (granulated) secretory cells and a corresponding increase in nongranulated secretory cells. By 3 days after HNE injection, the differential count had returned to control levels and cell surface alterations were less evident. By 8 days, the proportion of granulated secretory cells had significantly increased, while that of nongranulated secretory cells had decreased. Many Clara cells developed the characteristics of mucous cells so that mucous cells constituted 57% of the secretory cells compared to 14% for the saline controls. The mucous cells contained an increased number of mucous granules including bizarre forms never seen in controls. By day 16, the average mucous cell proportion had increased to 75%; the mucous cells were larger and contained many more secretory granules than at day 8. At no time was there evidence of overt cell injury or alteration of extracellular connective tissue due to HNE. Basal and pseudobasal cells, distinguished by the presence or absence of hemidesmosomes, did not change as a percentage of total nucleated epithelial cells. Saline had no effect on the differential cell count compared to untreated values. Our results indicate a strong likelihood that HNE causes early discharge of secretory granules and alters the phenotypic expression of Clara cells so that they produce abundant, often abnormal mucous granules. The mechanism of HNE-induced disturbance of epithelial homeostasis is unknown, but the early irregularity of nonciliated epithelial cell surfaces may signify an important event in the evolution of the resultant lesion.

Proteolytic Activity of Human Neutrophil Elastase and Porcine Pancreatic Trypsin Causes Bronchial Secretory Cell Metaplasia in Hamsters

The authors wished to determine whether secretory cell metaplasia (SCM) induced in the bronchi of hamsters by human neutrophil elastase (HNE) was enzymatically mediated. We also wished to determine whether SCM could be induced by a proteolytic enzyme devoid of elastolytic activity. Accordingly, groups of weight-matched hamsters were given a single intratracheal instillation of 0.5 ml of saline solution containing one of the following: 300 micrograms of HNE purified from blood neutrophils, n = 14; 300 micrograms of HNE inactivated with Suc-Ala-Ala-Pro-Val chloromethyl ketone (CMK), n = 10; 500 micrograms of porcine pancreatic trypsin treated with CMK to eliminate residual active elastase, n = 10; 500 micrograms of trypsin inactivated by tosyl lysine chloromethyl ketone, n = 10; 2 micrograms CMK, n = 10; and saline alone, n = 10. Seven untreated animals served as additional controls. Twenty-one days post treatment, 5-6 micron paraffin-embedded sections, from the left lung hilar region, stained by Alcian blue and periodic acid-Schiff reaction were graded on a five-point scale for determination of the secretory cell index, which reflects SCM. Both elastase and trypsin produced severe SCM: mean +/- SEM secretory cell indices were 2.96 +/- 0.11 and 2.72 +/- 0.19, respectively, compared with values of 0.90 +/- 0.35 for the untreated group and 0.93 +/- 0.46 for the saline group (p less than .05). The secretory cell indices of the groups treated with inactivated elastase or trypsin were comparable to those of the saline-treated and untreated groups.(ABSTRACT TRUNCATED AT 250 WORDS)