Outcomes of Fibronectin and Procollagen 3 Levels in Bronchoalveolar Lavage of Asbestos-Exposed Human Subjects and Sheep

Sheep

Sheep

Bronchoalveolar Lavage. The results of analysis of bronchoalveolar lavage fluid are presented in Figure 2. In the saline-exposed sheep, total cells from lavage averaged 250,000/ml and did not vary significantly in the course of the experiment. Similarly, the levels of macrophages, neutrophils, fibronectin, and procollagen 3 did not change. In the latex-exposed sheep, we have previously reported a clear increase in total cells from lavage at month 1 to eight times the control value, and at month 12, this had returned to the level of saline-exposed sheep. From that point and after, there was no significant difference in levels of macrophages, neutrophils, fibronectin, and procollagen 3 between latex-exposed and saline-exposed sheep. In the asbestos-exposed sheep, total cells were clearly increased through the experiment; and one year after exposure, total cells in bronchoalveolar lavage fluid remained at 150 percent of controls, macrophages at 150 percent of controls, and neutrophils at 285 percent of controls. Fibronectin in bronchoalveolar lavage fluid was significantly elevated only in the asbestos-exposed sheep, and the levels increased throughout the experiment. Procollagen 3 in lavage fluid increased significantly early and returned to control levels at month 4 after exposure (Fig 2). Albumin was increased significantly only after month 4, and it paralleled the second increase in fibronectin ordered via My Canadian Pharmacy (data not shown).

Pulmonary Histopathology. In the saline-exposed sheep, the histopathology of the tracheal lobe remained normal during the experiment. In the latex-exposed sheep at month 1, there was a pneumonia-like infiltrate of the tracheal lobe; the infiltrate was predominantly alveolar and was largely composed of macrophages without distortion of the pulmonary architecture. At month 4, the infiltrate had regressed, and areas of residual cell accumulation constituted less than 5 percent of the total lobe (Fig 3). After month 4, the histopathology of the latex-exposed tracheal lobes had returned to normal (Fig 4). In the asbestos-exposed sheep at month 1, there was also a pneumonialike infiltrate composed of macrophages and neutrophils, with considerable distortion of the architecture of the lung and airway; at month 4, the infiltrate had considerably regressed to some 20 percent of the initial extent; it was located predominantly in the peribronchiolar and endobronchiolar areas, with persistent severe distortion of the small airways (Fig 5). At month 8, the infiltrate continued to regress but peribronchiolar and endobronchiolar fibrosis could be detected; at month 12, the infiltrate was at 5 to 10 percent of the initial extent, but peribronchiolar and endobronchiolar fibrosis were severely distorting the pulmonary architecture (Fig 6).

Human Subjects

Clinical Data. On the basis of clinical, radiologic, and functional data, 19 of the 34 subjects were recognized as having asbestosis. In comparison with the other 15 workers and the controls, this group had identical age and smoking habits. They had a significantly higher rale score, higher score of radiographic pulmonary opacities, lower vital capacity, lower total lung capacity (TLC), lower total lung capacitydiffusing capacity for carbon monoxide, higher Ga pulmonary uptake, and more rigid pulmonary pressure-volume curve (Fig 1) (p < 0.05 in all). The workers of group B differed (by definition) from group A only in terms of rigidity of the pulmonary pressure-volume curve and increased Ga pulmonary uptake. Eight had a roentgenogram scored at 0/0 or 0/1; none of them had bilateral rales. The clinical data in the asbestos workers of groups A, B, and C are essentially in keeping with our previous work.

Bronchoalveolar Lavage. In Figure 7, we present the results of analysis of fibronectin and procollagen in bronchoalveolar lavage fluid. In the controls, the ratio of fibronectin/albumin in bronchoalveolar lavage fluid was 33.7 μg/mg±6.4 μg/mg, and the ratio of procollagen 3/albumin was 1.22 ±0.22 ng/mg. In the asbestos workers without asbestosis and with normal pulmonary pressure-volume curve and normal Ga pulmonary uptake (group A), the fibronectin/albumin ratio was 33.1 μg/mg ± 6.0μ/mg, and the procollagen 3/ albumin ratio was 1.03 ±0.24 μg/mg. In group B, the fibronectin albumin ratio was 45.7 μg/mg±34 μg/mg (not significant), and the procollagen 3/albumin ratio was 2.3 ±0.8 ng/mg (p<0.05 vs controls and group A). In the workers with asbestosis, the fibronectin/ albumin level was at 71.4μg/mg±32μg/mg and was significantly higher than controls and group A (p<0.05); the procollagen 3/albumin ratio was at 2.0±0.8ng/mg and was comparable to levels in group B (p<0.05 vs controls and group A). Analysis of the data excluding smokers did not significantly change the results.

Figure 2. Analyses of bronchoalveolar lavage fluid in sheep model. Sheep were exposed once at one to seven days after control lavage (month 0) either to 100 ml of PBS (solid squares), to 100 mg of 0. l|t latex beads in 100 ml of PBS (open squares), or to 100 mg of U.I.C.C. chrysotile in 100 ml of PBS (triangles). Star indicates p<0.05 between asbestos-exposed sheep and latex-exposed or PBS-exposed sheep.

Figure 2. Analyses of bronchoalveolar lavage fluid in sheep model. Sheep were exposed once at one to seven days after control lavage (month 0) either to 100 ml of PBS (solid squares), to 100 mg of 0. l|t latex beads in 100 ml of PBS (open squares), or to 100 mg of U.I.C.C. chrysotile in 100 ml of PBS (triangles). Star indicates p<0.05 between asbestos-exposed sheep and latex-exposed or PBS-exposed sheep.

Figure 3. Latex-exposed tracheal lobe at month 4, showing early pneumonia-like reaction with preservation of airway structures (hematoxylin-eosin, original magnification x 160).

Figure 3. Latex-exposed tracheal lobe at month 4, showing early pneumonia-like reaction with preservation of airway structures (hematoxylin-eosin, original magnification x 160).

Figure 4. Latex-exposed tracheal lobe at month 12, showing nearly complete resolution of process seen in Figure 3 (hematoxylin-eosin, original magnification x 63).

Figure 4. Latex-exposed tracheal lobe at month 12, showing nearly complete resolution of process seen in Figure 3 (hematoxylin-eosin, original magnification x 63).

Figure 5. Representative histopathologic findings in asbestos-exposed tracheal lobes at month 4. Early pneumonia-like reaction is associated with severe structural distortion of most small airways (hematoxylin-eosin, original magnification x 160).

Figure 5. Representative histopathologic findings in asbestos-exposed tracheal lobes at month 4. Early pneumonia-like reaction is associated with severe structural distortion of most small airways (hematoxylin-eosin, original magnification x 160).

Figure 6. Histopathologic findings in asbestos-exposed tracheal lobe at month 12, demonstrating less intense inflammatory process than at month 4 (Fig 5), but lesions of small airways did not regress and became fibrotic (hematoxylin-eosin, original magnification x  63).

Figure 6. Histopathologic findings in asbestos-exposed tracheal lobe at month 12, demonstrating less intense inflammatory process than at month 4 (Fig 5), but lesions of small airways did not regress and became fibrotic (hematoxylin-eosin, original magnification x 63).

Figure 7. Levels of fibronectin and procollagen 3 in human bronchoalveolar lavage fluid reported as ratios to albumin (A). Groups N, A, B, and C are as in Figure 1.

Figure 7. Levels of fibronectin and procollagen 3 in human bronchoalveolar lavage fluid reported as ratios to albumin (A). Groups N, A, B, and C are as in Figure 1.