Thomas G. Christensen

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.

Isolation and characterization of an undifferentiated human colon carcinoma cell line (MIP-101).

An undifferentiated human colon carcinoma cell line was established from tumor tissue obtained from metastasis to the liver of colonic adenocarcinoma in a patient with fulminant Dukes D colorectal carcinoma. Histological analysis of the tumor biopsy from the liver confirmed the hospital pathology report of poorly differentiated colonic adenocarcinoma. Explants of this tumor tissue xenografted into a nude mouse were used to establish an epithelioid-like cell culture line, MIP-101. The cell line formed tumors in nude mice that histologically appeared undifferentiated and did not stain for carcinoembryonic antigen (CEA). No CEA was present either by radioimmunoassay (RIA) of the culture supernatant or by immunoperoxidase staining of the tumors or monolayers. MIP-101 appears to be one of the most undifferentiated human colon carcinoma cells lines available. It should prove useful in the search for markers of undifferentiated colonic cancer and in studies of colonic cancer differentiation.

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)

Ultrastructural evidence of dimethylformamide-induced differentiation of cultured human colon carcinoma cells increased expression of desmosomes

N,N‐dimethylformamide (DMF) induces differentiation of human colon carcinoma (DLD‐1) cells in culture and reduces their tumorigenicity in nude mice. The current investigation analyzed DLD‐1 (clone D) cells for ultrastructural evidence of differentiation. Examination of treated and untreated confluent monolayers by transmission electron microscopy revealed an occasional intracytoplasmic lumen indicative of adenocarcinoma. DMF‐treated cells showed no signs of a toxic reaction. Cytoplasmic organelles were essentially unchanged except for an increase in tonofilaments and associated desmosomes. The number of desmosomes per unit length of contiguous cell border increased almost sixfold in treated monolayers. No other type of cell junction was seen. The increased frequency of desmosomes in DMF‐treated cultures is significant because of the direct correlation known to exist between the number of desmosomes and degree of differentiation of some human carcinomas. Desmosomes serve as foci of cell adhesion and are reduced in number in some invasive tumors. Whether the supernumerary desmosomes in DMF‐treated cells contribute to the reduction in malignant behavior of these cells in vivo remains to be determined. Cancer 56: 1559‐1556, 1985.

Effect of immunomodulators on bleomycin-induced lung injury.

Background: The role of lymphocytes and their subpopulations in lung fibrosis is as yet unclear. Objective: To define the role of immunomodulation in bleomycin-induced inflammatory fibrotic lung injury, by testing the effect of two known Th1 inhibitors: linomide and pentoxifylline. Methods: C57BL/6 mice were treated by a single intratracheal instillation of 0.06 mg bleomycin in 0.01 ml saline or saline alone. Treatment groups included: (1) intratracheal bleomycin and daily treatment with linomide or pentoxifylline; (2) intratracheal bleomycin and daily water; (3) intratracheal saline and daily linomide or pentoxifylline; (4) intratracheal saline and daily water. Linomide and pentoxifylline were available per os in the drinking water from 1 day prior to intratracheal instillation. Animals were studied 14 days after intratracheal instillation. Lung injury was evaluated by total and differential cell count in bronchoalveolar lavage fluid, by a semiquantitative morphological index of lung injury and a quantitative image analysis of cellularity, fibrosis fraction and alveolar wall area fraction, and by biochemical analysis of lung hydroxyproline content. Results: Linomide or pentoxifylline did not cause any lung injury in saline-treated control mice. Overt signs of lung injury were apparent in bleomycin-treated mice. These changes were not affected by daily treatment with linomide or pentoxifylline, which were given in the highest tolerable dose. Conclusion: This study does not support the use of linomide or pentoxifylline to prevent or ameliorate lung fibrosis and may suggest that drug-induced differentiation of T lymphocytes into Th1/th2 subpopulations does not affect the evolution of bleomycin-induced lung injury.

Elastase Causes Secretory Discharge in Bronchi of Hamsters with Elastase-Induced Secretory Cell Metaplasia

A single intratracheal instillation of human neutrophil elastase (HNE) into hamsters causes granule discharge from bronchial secretory cells followed by marked accumulation of granules, visible by light microscopy at 21 days and persisting through 18 months. To determine whether persistence of this secretory cell metaplasia (SCM) is due to inability of the metaplastic secretory cells to secrete their granules, hamsters having HNE-induced SCM were challenged with the potent secretagogue HNE. Four groups of 10 hamsters each received 300 micrograms HNE intratracheally. Twenty-one days later, hamsters were intratracheally treated with HNE or saline; the groups were designated HNE-HNE and HNE-SAL, respectively. Hamsters were killed 2 h or 21 days following the second treatment. Using light microscopy, nucleated epithelial cells were counted in plastic sections of the left main intrapulmonary bronchus. Cells were classified as ciliated (C), basal (B), indeterminate (IN), or secretory. Secretory cells were subcategorized as S0 (0 granules), S1 (1-4 granules), S2 (> or = 5 granules with intervening cytoplasm), and S3 (abundant granules completely filling the cytoplasm). At 2 h, S3 cell frequency in the HNE-HNE group was 13.0 +/- 2.2 (% mean +/- SE), significantly lower than in the 2 h HNE-SAL group (31.1 +/- 4.5). Concomitantly, higher cell frequencies were seen in the other secretory categories of the HNE-HNE group compared to the HNE-SAL group; S2 17.1 +/- 1.9 compared to 9.4 +/- 1.9, S1 2.4 +/- 0.4 compared to 1.1 +/- 0.5, and S0 2.4 +/- 0.5 compared to 1.1 +/- 0.5, respectively. The S3 cell frequency of the 21-day HNE-HNE group was 25.4 +/- 4.7, increased significantly compared to the 2 h HNE-HNE group; this change was concomitant with significant decrease in the frequency of the S0 secretory cells. Cell frequencies of C, B, and IN were not affected by treatment or time. It is concluded that metaplastic secretory cells discharge their granules in response to HNE; SCM returns to its original state after HNE rechallenge; persistent SCM is not due to the inability of metaplastic secretory cells to discharge their granules.

Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury.

The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro‐fibrotic effect for lymphocytes. The high‐affinity interleukin‐2 receptor (haIL‐2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL‐2R provides the basis for therapeutic strategies that target IL‐2R‐expressing cells. We hypothesized that elimination of activated lymphocytes by IL‐2R‐targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL‐2‐Bax, a novel apoptosis‐inducing IL‐2R‐targeted chimeric protein, on bleomycin‐induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice‐daily intraperitoneal injections of IL‐2‐Bax; (ii) intratracheal bleomycin and intraperitoneal IL‐2‐PE664Glu, an older‐generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi‐quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL‐2‐Bax and IL‐2‐PE664Glu. However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL‐2‐Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis.

THE EFFECT OF ENOXAPARIN ON BLEOMYCIN-INDUCED LUNG INJURY IN MICE

We have evaluated the effect of enoxaparin, a potent antithrombotic drug, on bleomycin (Bleo)-induced pulmonary inflammation in mice. Pulmonary injury was induced by a single intratracheal (i.t.) instillation of Bleo. Four groups of female C57BL/6 mice, each received one of four treatments: (1) i.t. Bleo and daily intraperitoneal (i.p.) injections of enoxaparin (EN) starting one day before i.t. instillation of Bleo (Bleo-EN); (2) i.t. Bleo and i.p. injections of saline (Bleo-Sal); (3) i.t. saline and i.p. enoxaparin (Sal-EN); (4) i.t. saline and i.p. saline (Sal-Sal). Animals were sacrificed 14 days after i.t. treatment. Lung injury was evaluated by analysis of bronchoalveolar lavage fluid and histologically by an overall semiquantitative index of lung injury and a quantitative image analysis assessing alveolar wall area fraction and fibrosis fraction. Treatment of mice with enoxaparin did not ameliorate Bleo-induced lung injury. Our study does not establish a critical role of procoagulant activity in the evolution of Bleo-induced lung injury and does not support the use of antithrombotic therapy for the prevention of pulmonary fibrosis.