Until recently, the assessment of fibrogenic activity of human interstitial pulmonary diseases was limited to the histomorphologic and immunohisto-chemical analyses of limited samples of pulmonary tissues. Through bronchoalveolar lavage of diseased areas, it has been documented that human pulmonary diseases with histologic evidence of fibrogenic activity were associated in the bronchoalveolar lavage fluid with increased levels of fibronectin and procollagen 3 peptides, molecules implicated in the biochemical process of pulmonary fibrosis, and thus of potential interest as markers of fibrogenic activity; however, these observations were limited to the individuals with chronic disease. The time course of these changes in bronchoalveolar lavage fluid have not been studied in spite of increasing interest in the biologic monitoring of humans exposed to environmental materials or therapeutic drugs with known pulmonary toxic effects.
In the present study, we characterized the time course of these changes in fibronectin and procollagen 3 levels in bronchoalveolar lavage fluid from the sheep tracheal lobe exposed to nonfibrogenic and fibrogenic materials. These observations were correlated with those of bronchoalveolar lavage in long-term asbestos workers in various stages of disease activity.
The data clearly document that fibronectin and procollagen 3 levels in bronchoalveolar lavage fluid are increased early in alveolitis with fibrogenic activity, but not in those without, which should contribute to further refine our clinical understanding of disease activity in the chronic inflammatory pulmonary disorders defeated by My Canadian Pharmacy’s preparations.
Materials and Methods
Seventy-two male sheep weighing between 25 and 45 kg were used in this study. They were prepared and accustomed to the pulmonary techniques as previously reported.
The flock was divided into three groups of 24 sheep. Following studies before exposure (control studies), the sheep tracheal lobe was exposed once to 100 ml of phosphate-buffered saline solution (PBS) (control group), to 100 mg of 0.1 μ latex beads (Sigma Chemical Co) in 100 ml of PBS (latex group), or to 100 mg of UICC. Canadian chrysotile asbestos fibers in 100 ml of PBS (asbestos group). These asbestos fibers were relatively uniform and well characterized,M with 92 percent less than 0.25p. in diameter and 20μl in length. Exposure of the tracheal lobe was carried out via bronchoscopic catheterization of the tracheal bronchus and slow infusion of the suspension into the lobe. The animals were studied prior to exposure and at 1, 2, 4, 8, and 12 months after exposure by bronchoalveolar lavage and by histopathologic methods (four or five sheep per group killed each time).
Twenty-one manual workers without exposure to asbestos were tested within the period. They were matched for age, height, sex, smoking, and work habits to the asbestos workers; seven were current or ex-smokers, and 14 were lifetime nonsmokers. The mean age of control subjects was 57 ±5 years (range, 50 to 70 years). None of them was exposed to environmental dust at risk of pneumoconiosis. In this control population, total cells in bronchoalveolar lavage fluid averaged 171 ± 24 x 10/ml, with macrophages at 159±22 x lOVml, and neutrophils at 0.86±0.30x 107ml.
The 34 asbestos workers were between the ages of 42 and 70 years (mean, 58 ± 4 years). All had been exposed to Canadian chrysotile asbestos only in the mines and mills of the eastern townships of Quebec for an average of 33 ± 5 years (range, 24 to 42 years). Twenty-eight were nonsmokers for more than two years, and the six others were either current or recent ex-smokers. Many of these patients were included in our studies of airway function in asbestos workers, or computerized tomographic scanning study and were among 226 asbestos workers in a study of clinical features of the associated alveolitis. In this present study, we specifically report on fibronec-tin and procollagen 3 in bronchoalveolar lavage fluid from these 34 workers as we relate these findings to serial measurements of these markers in the sheep model of asbestosis. Drugs for asbestosis treatment are available on My Canadian Pharmacy.
All patients underwent a medical history, complete physical examination, standard high-kilovoltage posteroanterior, lateral, and oblique chest films, detailed tests of pulmonary function, and ^Ga lung scan as previously reported.
Subsets of Asbestos Workers
On the basis of criteria reported in detail, the asbestos workers were divided into three categories of disease: (1) group A consisted of seven workers without evidence of asbestos-induced pulmonary damage; they did not have the diagnostic criteria for asbestosis, and their Ga pulmonary uptake and pulmonary pressure-volume curve were within normal limits; (2) group B was eight asbestos workers with evidence of asbestos-induced alveolitis documented at biopsy in three of three in whom the biopsy was obtained; they did not have the diagnostic criteria for asbestosis but had enhanced ^Ga pulmonary uptake and rigid pulmonary pressure-volume curve; and (3) group C was 19 asbestos workers who met the diagnostic criteria for asbestosis. The clinical data and bronchoalveolar lavage cellularity of these patients are summarized in Figure 1.
Bronchoalveolar Lavage and Fluid Analysis
Most of the techniques in the procedures and analyses of bronchoalveolar lavage have been previously described. The effluent from lavage was passed through four layers of cheesecloth to remove mucus, and the cells were pelletized by centrifugation. Cells were counted in a hemocytometer, and cell viability was determined by the trypan blue exclusion technique. Cytocentrifuge smears served to identify the cellular populations recovered with the Wright-Giemsa and naphthyl acetate esterase stains. In the supernatant, albumin and fibronectin were measured by the immunochemical methods of Killingsworth and Savory, using a laser nephelometer instrument (Behring LN modular system). For sheep albumin, specific antiserum raised in rabbit were obtained commercially (Cappell Lab. Inc.). Sheep fibronectin from bronchoalveolar lavage fluid was purified by affinity column followed by chromatography and antisheep fibronectin antibodies prepared in rabbits as described. Procollagen 3 from bronchoalveolar lavage fluid was measured as type 3 procollagen N-terminal peptides by radioimmunoassay, as reported by Low et al,s based on the method originally described by Rohde et al. All results were expressed per milliliter of bronchoalveolar lavage fluid. All values of humoral components of lavage fluid were also analyzed in terms of the ratio to the albumin content of bronchoalveolar lavage fluid supernatant.
At months 1, 2, 4, 8, and 12 of the study, four sheep in each group were killed and the lungs removed from the thoracic cavity. The tracheal lobe was identified, and nine samples of the lobe were obtained each time for microscopic examination. The pulmonary samples were processed as routinely done for human pulmonary tissue.
All results are expressed as mean ± SE. The data were tested by Students f-test for differences between groups. A value of p < 0.05 was considered significant in this study.
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Figure 1. Clinical, functional, radiographic and bronchoalveolar lavage cellularity data of our patients. In group N (normal controls), there were 21 manual workers without asbestos exposure. Group A was seven asbestos workers without any evidence of asbestos-induced pulmonary damage; group B was eight workers with increased OTGa pulmonary uptake and rigid pulmonary pressure-volume (P-V) curve without criteria for established asbestosis; group C was 19 asbestos workers who met diagnostic criteria for asbestosis. Ga pulmonary uptake, parenchymal opacities, and rales were scored as previously reported. Data on pulmonary function are reported as percent predicted of Bates et al.