Effects of cigarette smoke and asbestos on airway, vascular and mesothelial cell proliferation

Int. J. Exp. Path. (1995), 76, 411-418 Effects of cigarette smoke and asbestos on airway, vascular and mesothelial cell proliferation H. SEKHON, J. WRIGHT AND A. CHURG Departments of Pathology, University of British Columbia and the Vancouver Hospital and Health Sciences Centre, Vancouver, BC, Canada Received for publication 2 March 1995 Accepted for publication 14 September 1995 Summary. In order to determine whether exposure to both cigarette smoke and asbestos leads to enhanced cell proliferation, and whether pleura cell proliferation reflects the presence of fibres at or near the pleura, rats were exposed to air (control), daily cigarette smoke, a single intratracheal instillation of amosite asbestos, or a combination of smoke and asbestos. Dividing cells were labelled with bromodeoxyuridine (BrdU) and animals were sacrificed at 1, 2, 7 or 14 days. Both cigarette smoke and asbestos produced increases in the labelling index of small airway wall, epithelial cells and pulmonary artery cells. In the small airways there was a brief marked positive synergistic interaction between these two agents, but synergism was not seen in the vessels. Cigarette smoke did not increase the labelling of mesothelial or submesothelial cells, whereas asbestos caused a persisting increase in mesothelial cell labelling. There was no correlation between the number of BrdU labelled mesothelial or submesothelial cells and the number of fibres touching the pleura, or located within 180pIm of the pleura. We conclude that the combination of cigarette smoke and asbestos exposure produces a complex set of interactions and has the potential to markedly increase cell proliferation in the parenchyma, an effect that may be important in both fibrogenesis and carcinogenesis. In contrast to the diminishing effects over time of a single dose of asbestos on cell proliferation in the small airways and vessels, the same dose of asbestos leads to sustained mesothelial cell proliferation. However, the latter process does not correlate with local accumulation of asbestos fibres. Keywords: asbestos, cigarette smoke, tracheobronchial epithelium, mesothel ium There is considerable evidence that cigarette smoking Jones et al. 1989; Kilburn et a/. 1986). In contrast, increases the incidence of asbestos-induced lung can- cigarette smoking has no effect on the incidence of cers and of asbestosis (Hammond et a/. 1979; McDonald asbestos-induced pleural mesothelioma (McDonald & & McDonald 1986a; Weiss 1984; Barnhart et a/. 1990; McDonald 1986b). The mechanism(s) of these combined Correspondence: Dr Andrew Churg, Department of Pathology, effects (or lack of effects) is unclear (Oberdorster 1989). University of British Columbia, 2211 Westbrook Mall, Vancou- Animal models and very limited human data suggest ver, BC, Canada V6T 2B5. that cigarette smoke increases the retention of asbestos 1995 Blackwell Science Ltd 411

412 H. Sekhon et a/. fibres in the lung and also increases their uptake by pulmonary epithelial cells (McFadden et a/. 1986a,b; Churg & Stevens 1995), thus leading to effectively greater doses of fibres in cigarette smokers. Cell proliferation is a process that appears to be closely associated with asbestos induced fibrogenesis and carcinogenesis. Heintz et a/. (1993) have recently demonstrated that exposure of cultured mesothelial and bronchial epithelial cells to asbestos is associated with prolonged upregulation of expression of the protooncogenes c-fos and c-jun, suggesting that one effect of asbestos may be chronic stimulation of cell proliferation. Several groups (Brody & McGavran 1989; McGavran et a/. 1990; Gardner & Brody 1995; Bowden & Adamson 1985; Adamson et a/. 1994) have shown that asbestos produces cell proliferation in both the pulmonary epithelium and underlying mesenchymal cells and in the associated branches of the pulmonary artery. Asbestos also produces pleural cell proliferation, even when administered by intratracheal instillation (Adamson et a/. 1993, 1994). We have recently reported that cigarette smoke causes cell proliferation in the small airways and associated small pulmonary arteries, but not in the mesothelial cells (Sekhon et a/. 1994), raising the question of whether additive or synergistic increases in cell proliferation play a role in smoke-asbestos interactions. In this paper we examine cell proliferation in the lungs of rats exposed to both agents. Materials and methods Bromodeoxyuriudine (BrdU) administration Bromodeoxyuridine was used to label cells synthesizing DNA prior to cell division. Three hours before sacrifice, each animal was given an intravenous injection of 5 mg/ loog body weight BrdU (Boeringher-Mannheim) in phosphate buffered saline. The animals were then sacrificed by urethane overdose and the lungs removed and fixed for 24 hours by intratracheal inflation with paraformaldehyde to 10cm of water pressure. Representative sections of the left lung at the level of the hilus were selected, dehydrated, embedded in paraffin, and cut at 5ptm thickness. Bromodeoxyuridine (BrdU) staining To visualize nuclei that had incorporated BrdU, deparaffinized sections were stained with mouse anti-brdu (Boehringher-Mannheim) diluted 1 :60 overnight at 40C. The sections were then washed in PBS containing 0.005% Tween 20 in order to remove excess primary antibody, and incubated for 1 hour at room temperature with biotinylated horse anti-mouse serum (Vector Labs). The sections were again washed and then incubated with 3-amino-9-ethylcarbazole for 10 minutes to produce a visible product. Counting of BrdU stained cells Proliferating cells were counted in the small airways, small vessels and pleura. Each section was examined Exposure groups Male Sprague-Dawley rats weighing 250 g were divided into 4 treatment groups: (1) air exposure (control); (2) amosite asbestos exposure; (3) cigarette smoke exposure; and (4) both asbestos and cigarette smoke exposure. Treatment periods were 1, 2, 7 or 14 days. Five or 6 animals were sacrificed in each group at each time point. UICC Amosite asbestos was administered as a single intratracheal dose of 2.5mg in 0.5 ml physiologic saline using light halothane anaesthesia. The animals were allowed to recover for one hour and cigarette smoking was then commenced for the smoke exposure groups. Smoke exposure was carried out in a nose-only smoking system as described (Sekhon et at. 1994). Each rat was exposed daily to the whole smoke of 7 commercial non-filter cigarettes. All sacrifices were performed 24 hours after the last smoke exposure. Figure 1. Light micrograph of the pleura showing mesothelial cells (arrows) and submesothelial cells (arrowheads); the submesothelial cells are labelled with BrdU. The mesothelial and submesothelial cells are separated by the pleural connective tissue and elastica which do not stain but appear as a gap in this preparation. Immunoperoxidase method for BrdU, very lightly counterstained with haematoxylin. x800.

Asbestos, smoke and cell proliferation 413 using a microscope stage and every membranous bronchiole (MB) and associated pulmonary artery, or respiratory bronchiole (RB) and associated pulmonary artery, was identified. The fraction of staining nuclei was determined by counting stained and unstained nuclei in the arterial endothelium, airway epithelium, arterial wall and airway wall at a magnification of x1000. Similar counts were made of arteries at the alveolar duct (AD) level. In the pleura, mesothelial cells and submesothelial cells, defined as those cells located external to or directly internal to the pleural elastica (Figure 1), were counted around the entire circumference of the section. The average number of nuclei counted per animal was approximately 4000. Pleural accumulation of fibres To determine whether cell proliferation in the pleura is related to direct contact with or proximity to asbestos fibres, we selected 3 animals from each asbestos exposed or asbestos plus smoke exposed group and from the histologic sections of these animals we randomly selected 20 fields of the pleura using a magnification of x1000. Each field had a diameter of 180,tm. We initially counted the number of staining pleural cells per field. The number of fibres in contact with the pleura was then counted; if fibres were present in a macrophage that touched the pleura, these fibres were counted as being in contact. The pleura was then moved to one edge of the field so that the subpleural alveolar parenchyma was visible, and this was divided in half with a microscope eyepiece graticule. A count was made of fibres in the half of the field near the pleura and a separate count of fibres in the half of the field distant from the pleura. Statistics Statistical analyses were performed using SYSTAT (Wilkinson 1991). We have discussed elsewhere the problems associated with statistical analysis of labelling indices in experiments of this type (Sekhon et a/.1994). Because labelling indices are very low, at most a few per cent and in some sites frequently zero, use of statistical methods that depend on normal distributions can be misleading. In a previous study with a similar design in which we compared smokers with controls (Sekhon et a/. 1994), we attempted to avoid this problem by grouping all data from any time and treatment together and using the nonparametric Kruskal-Wallis test to look for differences among treatments.however, in the present study we wished specifically to examine the questions of interactions between smoke and asbestos, and we were not able to find any non-parametric tests suitable for this purpose. We chose, therefore, to employ a traditional two-way ANOVA to analyse the data using the mean value from each animal in each treatment group at each time point. Synergistic (i.e. greater or less than additive) interactions were considered to be present when the significance value for the interaction term in the regression equation was 0.05 or less. Initial examination showed that distributions were normal (or could be normalized by log transformation) for counts in the walls and epithelium of both membranous bronchioles and respiratory bronchioles, the walls of arteries associated with membranous bronchioles, and the endothelium and wall of arteries at the alveolar duct level. Reasonably close to normal distributions were also found for most treatment groups when examining mesothelial and submesothelial cell labelling indices. Distributions of counts for the endothelium of arteries at the membranous bronchiole and respiratory bronchiole level, and for the arterial walls at the respiratory bronchiole level, could not be normalized because of the presence in some treatment groups of many zero counts. For these groups no attempt was made to examine the data with ANOVA; rather, the Kruskal- Wallis test was used to look for differences among treatment groups. This test does not permit testing for interactions. As a precaution, the Kruskal-Wallis test was also used to examine the mesothelial and submesothelial cell labelling indices. Because the counts of BrdU staining pleural cells and counts of fibres in contact with, near, or distant from the pleura contained many zero values, Spearman rank correlations and Kruskal-Wallis tests were used for analysis (see Results). Results Membranous and respiratory bronchioles Tables 1 and 2 show labelling indices by time and treatment group for membranous bronchioles and respiratory bronchioles. Results in the two sites were very similar. Both asbestos and cigarette smoke produced increases in labelling indices in the epithelium and walls of the airways themselves within 24 hours. In the epithelium a synergistic increase in labelling was present at day 1, but had disappeared by day 2, although an additive effect on cell proliferation was still present, and in the walls an additive effect was seen at 1 and 2

414 H. Sekhon et al. Table 1. Labelling indices in Artery Artery Airway membranous bronchioles and Endothelium* Wall Epithelium Wall associated arteries (values as mean ± s.d.) One day Control 0.22 ± 0.31 Asbestos 0.18 ± 0.29 Smoke 0.19+0.26 Asbestos + smoke 0.22 ± 0.37 Two days Control 0.18 ± 0.25 Asbestos 1.35 ± 0.70 Smoke 0.53 ± 0.49 Asbestos + smoke 0.39 ± 0.48 Seven days Control 0.26 ± 0.37 Asbestos 0.63 ± 0.65 Smoke 0.54±1.06 Asbestos+ smoke 0.46 ± 0.27 Fourteen days Control 0.45 ± 0.31 Asbestos 0.07 + 0.17 Smoke 0.23 ± 0.47 Asbestos + smoke 0.21 ± 0.34 0.15±0.14 0.38±0.06 0.24+0.07 0.67 0.38 4.39 ± 0.75 3.63 ± 1.24 0.21 ± 0.20 3.01 ± 1.47 1.77 + 0.64 0.51 ± 0.28 9.38 ± 1.97 t 6.25 ± 1.32 0.29 ± 0.33 0.34± 0.07 0.35± 0.13 1.86 ± 0.55 4.31 ± 1.57 4.63 ± 1.30 0.70 ± 0.26 2.28 ± 0.33 1.64 ± 0.28 1.40 ± 0.40 t 5.62 ± 0.84 5.03 ± 1.40 0.06±0.14 0.27±0.09 0.40+0.14 0.96 ± 0.24 1.28 ± 0.61 2.00 ± 1.15 0.54 + 0.40 0.81 ± 0.64 1.38 ± 0.49 1.16 ± 0.39 t 1.28 ± 0.89 1.75±1.03 0.14±0.19 0.27 ± 0.10 0.33 0.14 0.77 ± 0.73 0.43 ± 0.20 0.80 + 0.21 0.59 + 0.44 0.70 ± 0.29 1.35 ± 0.63 0.29 ± 0.16t 0.67 ± 0.36 0.96 ± 0.68 Significance values are comparison to control at level P < 0.05 or less; P < 0.01 or less. *Statistical analysis by Kruskal-Wallis test. t Indicates synergistic (positive or negative) interaction P < 0.05 or less by two-way ANOVA. Artery Airway Table 2. Labelling indices in respiratory bronchioles and associated arteries Treatment Endothelium* Wall* Epithelium Wall (values as mean± s.d.) One day Control 0.15±0.34 Asbestos 0.90 ±1.03 Smoke 0.00 ± 0.00 Asbestos + smoke 0.45 ± 0.70 0.11 ±0.23 0.41 ±0.14 0.26± 0.15 1.11 ± 0.88 4.58 ± 0.96 4.12 ± 0.96 0.55 ± 0.81 2.99 ± 0.98 2.45 ± 1.33 0.91 ± 0.79 12.45 ± 2.04 t 7.96 ± 1.78 Two days Control 0.00±0.00 0.19±0.43 0.40±0.10 0.36±0.20 Asbestos 0.77 ± 1.20C 1.82 ± 1.01 4.60 ± 2.08 4.80 ± 1.64 Smoke 0.17 ± 0.39 0.79 ± 0.87 2.13 ± 0.22T 1.64 ± 0.37 Asbestos + smoke 0.50 ± 0.64 1.25 ± 0.82 7.82 ± 0.45 5.37 ± 0.78 Seven days Control 0.45 ± 0.62 Asbestos 1.53 ± 1.11 Smoke 0.69 ± 0.70 Asbestos + smoke 0.71 ± 0.78 Fourteen days Control 0.18 ± 0.41 Asbestos 0.54 ± 0.60 Smoke 0.85 ±1.01 Asbestos + smoke 0.42 ± 0.74 0.00±0.00 0.38±0.13 0.55± 0.28 1.07 ± 0.94 2.19 ± 0.94 2.20 ± 1.24 1.01 ± 0.26 1.34 ± 1.04 1.98±1.47 1.36 ± 1.13 1.64 ± 0.651t 1.96±1.09 0.41 ± 0.38 0.87 ± 0.81 0.96±0.79 0.54±0.65 0.26±0.11 0.44±0.23 0.84 ± 0.38 0.80 ± 0.19 0.70 ± 0.20C 0.65 ± 0.37 0.86 ± 0.32 1.73 ± 1.00 Significance values are comparison to control at level P < 0.05 or less; P < 0.01 or less. *Statistical analysis by Kruskal-Wallis test. t Indicates synergistic (positive or negative) interaction P < 0.05 or less by two-way ANOVA. Blackwell Science Ltd, International Journal of Experimental Pathology, 76, 411-418

Asbestos, smoke and cell proliferation 415 Table 3. Labelling indices in arteries associated with alveolar ducts (values as mean ± s.d.) Table 4. Labelling indices in mesothelial and submesothelial cells (values as mean ± s.d.) Endothelium Wall Mesothelial Submesothelial One day Control Asbestos Smoke Asbestos + smoke Two days Control Asbestos Smoke Asbestos + smoke Seven days Control Asbestos Smoke Asbestos + smoke Fourteen days Control Asbestos Smoke Asbestos + smoke 0.24 ± 0.15 0.64 ± 0.34 0.61 ± 0.27 0.38± 0.33 0.25±0.14 1.02 ± 0.35 0.62 ± 0.36 1.16±0.96 0.23 ± 0.20 1.23 ± 0.84 0.87 ± 0.28T 1.08 ± 0.35 t 0.18 ± 0.24 0.53 ± 0.28T 0.87 ±0.43 0.43 ± 0.13 t 0.18 ± 0.16 0.91 ± 0.28 0.54± 0.29 0.82 ±0.44 0.28 ±0.20 1.47 ± 0.40 0.87 ± 0.12 1.21 ± 0.46 t 0.12 ± 0.16 0.69 ± 0.39 1.38 ±0.27 1.36±0.19 t 0.14 ± 0.17 0.71 ± 0.42 0.93 ± 0.37 0.92 ± 0.41 One day Control 0.22 ± 0.32 0.69 ± 0.37 Asbestos 0.18 ± 0.24 0.70 ± 0.35 Smoke 0.58 + 0.38 1.38± 0.88 Asbestos + smoke 1.64 ± 0.76 t 3.52 ± 2.20 t Two days Control 0.17 ± 0.36 0.95 ± 0.26 Asbestos 1.76 ± 1.28 3.10 ± 1.34 Smoke 0.73 ± 0.86 1.64 ± 0.67 Asbestos + smoke 1.36± 1.23 3.61 ± 2.32 Seven days Control 0.23 ± 0.32 0.79 ± 0.50 Asbestos 1.58 ± 1.07 3.81 ± 1.11 Smoke 0.74 ± 0.84 2.01 ± 0.86 Asbestos + smoke 0.86 ± 0.34 2.93 ± 0.94 t Fourteen days Control 0.49 ± 0.36 0.44 ± 0.52 Asbestos 1.30 ± 0.39T 2.64 ± 1.08 Smoke 1.25 ± 0.87 2.09 ±1.19 Asbestos + smoke 0.76 ± 0.86t 2.46 ± 1.29 Significance values are comparison to control at level TP < 0.05 or less; P < 0.01 or less. t Indicates synergistic (positive or negative) interaction P < 0.05 or less by two-way ANOVA. Significance values are comparison to control at level P < 0.05 or less; P < 0.01. or less t Indicates synergistic (positive or negative) interaction P < 0.05 or less by two-way ANOVA. days. There was a suggestion that at days 7 and 14 the combined effects of smoke and asbestos were less than additive, but for the most part this could not be confirmed statistically. In the airway epithelium the proliferative effects of both smoke and asbestos declined over time; this was also true for asbestos in the airway wall. However, the smoke induced increase in nuclear labelling in the airway wall was relatively stable over the period of the experiment. Airway-associated pulmonary arteries Tables 1-3 present data on labelling indices for arteries associated with membranous bronchioles, respiratory bronchioles, and alveolar ducts respectively. Both smoke and asbestos produced increases in endothelial labelling, mostly within 24 hours, although smoke effects were largely confined to the endothelium of alveolar ducts, and were generally less marked than the effects of asbestos. Smoke effects were more clearly apparent in the walls of vessels at all three anatomic levels, again at a level less marked than the effects of asbestos. There was no evidence of a positive synergistic interaction or even an additive interaction in any vessels; rather significantly negative interactions were present at a number of time points. Asbestos effects declined over time, while smoke effects tended to persist over the experimental period. Pleural cells Table 4 shows data for mesothelial and submesothelial cells analysed by two-way ANOVA. Except for submesothelial cells at day 7, smoke did not increase the labelling index for either cell type. In contrast, asbestos produced a significant increase in labelling in both cell types starting at day 2 and this did not decrease over time. Analysis using the Kruskal-Wallis test produced similar results comparing the effects of smoke or asbestos to control. The combination of smoke and asbestos produced synergistic increases in labelling at day 1, followed by a trend (largely non-significant) toward less than additive interactions. Pleural accumulation of fibres Spearman rank correlations were used to examine the relation between the number of BrdU stained mesothelial or submesothelial cells and the number of fibres in contact with the pleura, near the pleura, or distant from the pleura as defined in Materials and methods. There were no significant correlations between these variables

416 H. Sekhon et al. either overall (correlation coefficients: -0.02 for pleural staining cells and fibres in contact with the pleura; -0.02 for pleural staining cells and fibres near the pleura; and 0.07 for pleural staining cells and fibres distant from the pleura), or when specific time and treatment subsets were examined. There was, overall, a correlation between the number of pleural fibres and the number of fibres in the field near the pleura (correlation coefficient 0.31, P < 0.001); and between the numbers of near and distant fibres (correlation coefficient 0.25, P < 0.001). To determine whether there was any evidence of movement of fibres toward the pleura with time, Kruskal-Wallis tests were used to look for differences over time. There was no statistical evidence of a trend toward movement of fibres to the pleura over time with either asbestos or asbestos plus smoke treatment, although the number of BrdU labelled pleural cells increased between day 1 and later time points (P0.01) in both groups, findings similar to those obtained by formal counting of the entire pleural surface (Table 4). Discussion In this study we have shown that smoke and asbestos induce different patterns of cell proliferation in different pleuropulmonary tissues, along with a complex set of interactions. Both asbestos instillation and cigarette smoke exposure cause proliferation of small airway epithelial cells and airway wall (fibroblast and smooth muscle) cells which is quite marked by 24 hours, persists through 48 hours, and then slowly declines toward control values over the next 2 weeks. A fairly similar, but less marked, pattern of response is seen in the endothelial cells and smooth muscle cells of the juxta-airway pulmonary arteries, although smoke effects, when present, appear to be more persistent in the vessels. Evidence of an initial positive (greater than additive) synergistic effect or an additive effect was seen in the epithelium and walls of the airways in both the membranous and respiratory bronchioles. This effect disappeared by 7 days, at which point there was a suggestion (not statistically provable) of a less than additive effect. Neither positive synergism nor even an additive effect was present in the vessels, and here the evidence for a significantly less than additive effect (negative synergistic effect) was clearer. Whether these results are generalizable to all types of asbestos is unclear. We have used amosite because it is easy to administer by intratracheal instillation and is also easy to count by light microscopy. Also, amosite asbestos has been clearly linked to mesothelioma (thus by implication to mesothelial cell proliferation), whereas the association of chrysotile and mesothelioma is less certain (McDonald & McDonald, 1986b). Chrysotile asbestos might behave differently, particularly in conjunction with cigarette smoke. However, increases in cell proliferation in the epithelium and walls of the small conducting airways have been described after both chrysotile and amphibole asbestos exposure (Brody & McGavran 1989; McGavran et a/. 1990; Bowden & Adamson 1985; Adamson et a/. 1994; Gardner & Brody 1995), so that it is likely that the fundamental reactions we observe, at least in the airways and surrounding vessels, apply to chrysotile as well as amosite. The exact location of the proliferative response probably depends somewhat on the method of dust exposure. In rats, inhalation produces distinct increases in labelling at the first alveolar duct bifurcations, the major site of fibre impaction, as well as in the membranous and respiratory bronchioles (Brody & McGavran 1989; McGavran et a/. 1990; Gardner & Brody 1995), whereas intratracheal instillation results in marked fibre deposition in and around the membranous and respiratory bronchioles but not at the alveolar duct bifurcations (Bowden & Adamson 1985; Tron et a/. 1987). However, the underlying response is similar with both methods of dust administration and produces, over a greater or lesser time period, increases in the number of mesenchymal cells and in the amounts of interstitial connective tissue (Bowden & Adamson 1985; Chang et at. 1988). The exact intrapulmonary distribution of cigarette smoke is uncertain, although deposition models for small particles suggest that the membranous and respiratory bronchioles should receive very high concentrations of smoke (Oberdorster 1989; Muller et at. 1990). This conclusion is consistent with our finding of increased labelling in both the small airway walls and epithelium after smoke exposure. Intratracheal instillation is a highly artificial system and must be interpreted with great caution, but what is of interest in our present study is that the combination of smoke and asbestos administered in such a way as to target the same site does cause an initial synergistic or additive increase in cell proliferation in the small airways. Given that both agents independently cause cell division, this observation makes intuitive sense and could help explain the increased disease incidence seen in humans exposed to smoke and asbestos. What is difficult to explain is the tendency towards a less than additive effect as the experiment progresses. Asbestos generates active oxygen species, known mitogenic agents (Murrell et a/. 1990; Burdon 1995), and

Asbestos, smoke and cell proliferation 417 additionally causes release of growth factors such as PDGF from evoked macrophages (Schapira et a/. 1991). Smoke also contains active oxygen species and other oxidants (Church & Pryor 1985) and produces marked, albeit poorly defined, interference with normal lung cytokine regulation (Brown et a/. 1989; Nagai et a/. 1988; Dubar et a/. 1993; McCrea et a/. 1994). Both active oxygen species and cytokines might be involved in driving cell proliferation in these experimental cells, and both might also produce substances that interfere with cell proliferation. For the present experiments these comments are entirely speculative. However, since we have shown in previous animal studies that the combination of smoke and asbestos exposure given in exactly the fashion employed here produces longterm increases compared to asbestos alone in fibrosis in and around the small airways (Tron et a/. 1987), the initial positive synergistic/additive reaction is probably the most significant. Much less attention has been directed to asbestos or smoke induced cell proliferation in small vessels. McGavran et a/. (1990) pointed out that the pulmonary artery branches near alveolar duct bifurcations, but not those distant from the bifurcations, showed increased endothelial and muscular wall labelling after chrysotile inhalation in rats, and Adamson et a/. (1994) reported increases in endothelial labelling after intratracheal administration of crocidolite. We have previously shown that cigarette smoke by itself also causes cell proliferation in the branches of the small pulmonary arteries that are adjacent to the small airways (Sekhon et al. 1994). This phenomenon is not limited to smoke or asbestos, but has also been observed in the lungs of animals exposed to hyperoxia and to substances producing marked local airway inflammation (Coflesky et a/. 1988; Herget et a/. 1980). All these agents evoke marked inflammatory responses and/or cause production of active oxygen species in the small airways, and these findings, along with the very localized nature of the vascular response, suggest that vascular cell proliferation is a direct result of mitogenic factors (cytokines, active oxygen species, or both) diffusing from the airways to the vessels. Again, we have no explanation for the observation that the combined effect of smoke and asbestos in the small vessels is generally less than additive. Perhaps most intriguing in our study is the very different pattern of response in the pleura. We have previously reported (Sekhon et a/. 1994) that cigarette smoke by itself fails to produce an increase in cell labelling in either the mesothelial or submesothelial cells, and this observation was confirmed in the present experiments. Asbestos does cause cell proliferation in both sites, but with a 2-day lag period, as opposed to the much more rapid response in the airways and vessels, and this reaction, in contrast to the airway and vessel effects, shows no evidence of a statistically significant decrease by day 14. Thus, during a period in which cell proliferation in the airways and vessels is rapidly declining to control levels, mesothelial proliferation persists. This process does not appear to represent a direct effect of fibres in contact with or near the mesothelial cells, since there was no correlation at all between the presence of BrdU labelled mesothelial or submesothelial cells and the number of fibres touching the pleura or the number of fibres within 180,um of the pleura, nor was there evidence of an increase in the number of fibres touching or near the pleura over time, even though the numbers of labelled cells increased. Somewhat similar findings in regard to the effects of asbestos and other pleural mitogenic agents have been reported by Adamson et a/. (1994) who found that intrapulmonary labelling of epithelial cells, endothelial cells, and fibroblasts, considered in aggregate, decreased over 12 weeks after a single instillation of crocidolite asbestos, whereas the labelling of submesothelial (but not mesothelial) cells persisted at an elevated level over the same time period. Adamson et a/. (1993) noted that asbestos fibres were readily observed in and around the small airways, where they evoked a marked inflammatory and fibroblastic response, but could not be found in or under the pleura, findings slightly different from ours in that we did observe small numbers of fibres in contact with the pleura. These same authors observed that silica and bleomycin, both fibrogenic agents, also caused a long-lasting stimulation of pleural cell proliferation, whereas hyperoxia, which was non-fibrogenic, produced only a relatively brief burst of proliferation. They suggested that a diffusible growth factor released from asbestos-evoked inflammatory cells, particularly a factor related to intrapulmonary fibrogenesis (?PDGF) was driving the longterm proliferation of pleural cells. Our present observations are consistent with this hypothesis, but suggest that the source of the growth factor is physically fairly remote from the pleura itself. Acknowledgements H. Sekhon is a Fellow of the Canadian Lung Association. Supported by grant MA8051 from the Medical Research Council of Canada.

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