Effects of prednisolone on the differentiation of mouse lung adenomas in culture.

Explants of pulmonary adenomas, induced in mice by urethan, were cultured with or without prednisolone for 72 hr. After this time, the cuboidal epithelial cells of the tumor contained many more lamellar bodies than the nonsteroid control cultures. Incorporation of labeled palmitic acid into saturated phosphatidylcholine was not significantly increased in these short-term steroid-treated cultures. The results indicate that steroid enhances the maturation of cultured pulmonary adenoma cells into cells morphologically indistinguishable from type II alveolar epithelial cells of normal lung.

Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury.

The passage of circulating tumor cells across the vascular wall is an important step in the evolution of cancer metastases. Since tumor cells attach preferentially to subendothelial matrix at sites of endothelial injury and retraction in vitro, we have used an established in vivo model of pulmonary endothelial damage to examine the effects of endothelial injury on the localization and metastasis of circulating tumor cells in vivo. C57BL/6 mice were given a single i.v. dose of bleomycin (120 mg/kg) or multiple i.p. injections (10 mg/kg, twice weekly for 6 wk). Five days after the single injection or 4 days after the last i.p. injection, 2 X 10(5) [131I] iododeoxyuridine-labeled fibrosarcoma cells or unlabeled cells were injected i.v. Two to 8 times as many labeled cells were found in the lungs of bleomycin-treated animals after 24 h. Two and 3 wk after injecting unlabeled fibrosarcoma cells, 1.4 to 5 times more metastatic lung colonies were counted in bleomycin-treated animals than in controls. Morphometric analysis of histological sections demonstrated that the percentage of lung area occupied by tumor in bleomycin-treated animals was between 4 and 16 times that of controls. Analysis of bronchoalveolar lavage fluids demonstrated 5-fold increases of total protein content and leakage of previously injected 125l-labeled albumin, indicating increased endothelial permeability. Electron microscopic examination of lungs of bleomycin-treated mice demonstrated endothelial retraction with exposure of the underlying basement membrane. Electron microscopy of [3H]thymidine-labeled tumor cells, located by autoradiography, demonstrated their attachment to exposed basal lamina. Data from these experiments in vivo support the hypothesis that endothelial damage can facilitate the metastasis of circulating tumor cells.

Alveolar macrophage kinetics and multinucleated giant cell formation after lung injury.

Multinucleated giant cells (MGC) are a prominent feature of some chronic inflammatory states in the lung. These cells are formed by macrophage fusion, but how this process relates to the kinetics of alveolar macrophage (AM) production and proliferation is not clear. In this serial study, we compare AM kinetics and MGC formation after instilling carbon, silica, asbestos, bleomycin, and saline into the lungs of mice. Animals were killed up to 16 weeks later with [3H]thymidine injected 1 h before death. Counts of AM and MGC were carried out after bronchoalveolar lavage, and cell labeling was assessed by autoradiography. All test substances induced an inflammatory response with equal AM numbers recovered up to 2 weeks. Subsequently, the number returned to normal after carbon but remained elevated in the other groups. After carbon the lung structure was normal, there was no increase in AM label, and no MGC formed. Bleomycin-injected lungs progressed to fibrosis with only a brief, small increase in AM labeling and no MGC formation. After silica, and particularly asbestos, the lungs showed fibrosis, and many granulomas with large MGC were seen. Lavaged AM from these lungs showed a significant increase in DNA synthesis after 2 weeks, followed by higher numbers of MGC, none of which were labeled. Labeled AM tended to be free of particles, whereas MGC after 4 weeks contained many particles. The results indicate a relationship between AM proliferation and fusion, whereby AM growth appears to be prerequisite for cell infusion and MGC formation as a feature of granulomatous disease.

Enhanced secretion of immunoreactive bombesin by alveolar macrophages exposed to silica.

Bombesin has recently been identified in alveolar macrophages (AM). Since this peptide has been shown to stimulate fibroblast growth in culture, we wished to determine whether AM exposed to the fibrogenic particle silica in vivo were capable of secreting more bombesin than AM recovered after instilling inert carbon particles to the lung. Rats received 10 mg of either carbon or silica by intratracheal injection and were killed at 3 days or 6 weeks. Both particles induced a rapid inflammatory response, and normal levels of immunoreactive bombesin were measured in lung lavage fluid and in freshly recovered macrophages from all rats. However, incubation of normal AM for 4 h in serum free medium produced a significant increase in bombesin levels measured in supernatants. Bombesin in supernatants of AM cultured after recovery from rats exposed to carbon was at the control value, while AM recovered after silica exposure in vivo secreted increased amounts of bombesin when cultured. Cells recovered 6 wk after instilling silica to the lung and cultured for 4 h secreted 50% more bombesin than control AM. At this time, hydroxyproline measured in the silica-injected lungs was also significantly higher than in controls or carbon-injected rats. These results indicate that AM recovered from lungs after exposure to silica secrete increased amounts of bombesin during the development of pulmonary fibrosis.

Quantification of metastatic tumor growth in bleomycin-injured lungs.

The lung is a common target organ in experimental models of tumor metastasis in which quantification usually involves counting labeled tumor cells shortly after injection, or enumeration of grossly visible pleural tumors. In this study, these approaches were used in addition to autoradiographic and morphometric methods to analyse the effect of bleomycin-mediated injury on the development, distribution and quantification of pulmonary metastases. One day after intravenous injection of 2 X 10(5) fibrosarcoma cells, the lungs of C57 bl/6 mice, pretreated with bleomycin (120 mg/kg i.v., 5 days before) contained about nine times as many [131I] iododeoxyuridine-labeled cells as the lungs of control animals given saline injections. At this time, autoradiographic counts of [3H]thymidine-labeled tumor cells in lung sections showed a similar increase in tumor cell localization after bleomycin, with labeled cells distributed equally between parenchymal and pleural areas. However, subsequent tumor growth was demonstrated microscopically to be predominantly in pleural and peribronchial areas, especially at sites of lung injury induced by bleomycin. Counts of grossly visible pleural tumors failed to demonstrate a difference between bleomycin groups and controls at 7 days whereas counts of nodules in lung sections, and quantification of lung area occupied by tumor both showed significantly greater tumor involvement in bleomycin-treated animals. As tumors became confluent, morphometric measurements demonstrated tumor growth in the lung more accurately than did nodule counts. We conclude that bleomycin-induced injury greatly enhances metastatic tumor growth and that morphometric methods are more sensitive than lung colony counts in their ability to quantify pulmonary metastases. Morphometry and autoradiography have also demonstrated that while there is a uniform distribution of arrested tumor cells in the lung initially, there is preferential development of metastatic tumors at sites of pulmonary damage, in particular at the pleura.

Preferential growth of metastatic tumors at the pleural surface of mouse lung.

In an experimental model of lung metastasis we have observed that more metastatic tumors are located on the pleura of the lung than in the parenchyma. To study possible reasons for this differential pattern we have now related the initial distribution of injected tumor cells to the later location and growth rate of metastases in different regions of the lung in C57bl/6 mice. It was found that labeled murine fibrosarcoma cells were evenly distributed throughout the lungs 24 h after intravenous injection into controls and animals previously treated with bleomycin or by exposure to hyperoxia. These treatments, known to induce pulmonary endothelial injury, were associated with increased tumor cell localization in the lung. In all cases, using morphometric methods, we found that after 2 weeks, approximately 75 per cent of metastatic tumors were located at the pleura. By [3H]thymidine labeling in autoradiographs, pleural tumors in all experimental groups had a growth rate 14 times the growth rate of tumors located in the internal regions of the lung. In vitro, the fibrosarcoma cells proliferated more rapidly on connective tissue matrices prepared from normal pleuras than they did on matrices from the remainder of the lung. Protease digestion of these matrices indicated differences in composition with more insoluble collagen, probably type I collagen, present at the pleura. These data suggest that, in spite of the initial random distribution and localization of tumor cells in the lung, there is preferential growth of metastatic tumors at the pleura which may be related to regional differences in the composition of the extracellular matrix.

Retarded development of noenatal rat lung by maternal malnutrition.

Inadequate dietary intake during late pregnancy may have significant effects on the developing fetal lung which undergoes rapid cellular multiplication and differentiation shortly before birth. The morphology, glycogen distribution and acid phosphatase activity in normal and starved neonatal rats have been studied sequentially, by using histochemical and cytochemical methods. It has been shown that the normal pattern of lung growth and enzymatic development is retarded in neonates of malnourished mothers. A slowed rate of cellular division and differentiation in the critical prenatal period resulted in a more immature air-blood barrier at birth, with glycogen retention by some epithelial cells. Delayed Type 2 cell maturation with diminished acid phosphatase activity suggests a decrease in surfactant production in the malnourished newborn. In addition, fewer alveolar macrophages with reduced acid phosphatase activity were observed in the perinatal period of starved rats; this finding might have implications for the handling of inhaled bacteria shortly after birth. These results indicate that nutritional status of the mother has a marked effect on fetal lung growth and development by inhibiting cellular proliferation, differentiation and enzyme development by epithelial and macrophagic cells.

Radiation enhances silica translocation to the pulmonary interstitium and increases fibrosis in mice.

The effects of whole body irradiation (WBR) on particle clearance and the development of pulmonary fibrosis have been investigated. Using carbon, clearance is accomplished by polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM), and only a few particles reach the interstitum. However, in preirradiated mice, the usual eflux of inflammatory cells is much delayed so that more free carbon remains in the alveoli, and by 1 week, many particles cross the epithelium to be phagocytized by interstitial macrophages. Carbon is found in the peribronchiolar interstitium 6 months later with no evidence of fibrosis. In the present study, mice received 1 mg silica intratracheally 2 days after 6.5 Gy WBR when the white blood cell count was low. A much-reduced AM and PMN response was found in the following 2 weeks compared to the reaction to silica alone, and many silica particles reached interstitial macrophages. In this case, macrophage activation by silica was associated with fibroblast proliferation, and by 16 weeks, much more pulmonary fibrosis was produced than after silica or irradiation only. This was measured biochemically and correlated with a large increase in retained silica in the irradiation-silica group. The results indicate that radiation inhibits the inflammatory response to particle instillation, resulting in greater translocation of free particles to the pulmonary interstitium. In the case of silica, the greater, prolonged interaction with interstitial macrophages leads to a much exaggerated fibrotic reaction.

Pulmonary toxicity of bleomycin.

Diffuse pulmonary fibrosis is associated with bleomycin administration to humans. The sequential reactions of lung cells to this drug have now been investigated in mice following injection of 20 mg/kg bleomycin twice per week for 4 to 8 weeks. Cytoplasmic and subendothelial edema was first observed in large vessels and by 4 weeks involved the capillaries. The reaction in many animals did not progress further than endothelial lesions with accumulation of interstitial edema. However, 30% of mice subsequently showed necrosis of type 1 epithelium with a fibrinous exudate in the alveoli. Fibroblastic organization of the fibrin resulted in the deposition of intraalveolar collagen as well as extensive septal fibrosis by 8 weeks. Epithelial repair, normally accomplished by type 2 cell proliferation and transformation to type 1 cells, is characterized in this case by division and metaplasia of type 2 cells. The metaplastic cells were, however, capable of DNA synthesis and probably of further cell division. The results indicate that the pulmonary endothelium is the initial site of injury. Extensive damage to these cells could allow the drug access to interstitial and epithelial cells. Focal necrosis of type 1 epithelium is the critical event that triggers the exudation of fibrin and the subsequent reparative processes.

Drug-induced pulmonary fibrosis.

The lung is a primary target of cell injury in patients receiving cytotoxic drugs, and in many cases the reaction is severe enough to produce diffuse pulmonary fibrosis. Although many different agents may damage the lung, the patterns of cellular injury and repair are relatively constant. Using bleomycin toxicity as an example, it has been shown that, after IV injection, the selective site of lung injury is the vascular endothelium; this is followed by the accumulation of interstitial edema and later by necrosis of Type I epithelial cells. In normal repair, rapid proliferation of Type II cells, followed by differentiation to Type I, restores the epithelial surface without fibrosis. However, after bleomycin, Type II cell proliferation is frequently followed by abnormal differentiation to a metaplastic form of epithelium. Fibroblast proliferation accompanies this abnormal epithelial response which is related either to selective retention of bleomycin by epithelial cells or to the toxic effects of administering more drug at the time of Type II cell division. It is concluded that diffuse interstitial fibrosis results from prolonged disturbance of the normal epithelial-mesenchymal interrelationships at the alveolar wall. Disruption of the fibroblastic control system by extensive epithelial necrosis or by delayed or inappropriate repair may be the key factor in instigating fibroblast proliferation and subsequent deposition of collagen. This mechanism may account for the development of diffuse fibrosis or fibrosing alveolitis in response to a variety of pulmonary toxins.

Early mesothelial cell proliferation after asbestos exposure: in vivo and in vitro studies.

There is some evidence that proliferation of pleural mesothelial cells (MC) occurs soon after deposition of asbestos fibers. To study this effect, we instilled a single dose of 0.1 mg crocidolite into the lungs of mice for 1 and 6 weeks and counted labeled nuclei after 3H-thymidine (3HT) injection. Short fibers (< 1 micron) induced little change in the lung; they were mostly phagocytized and had a minimal effect on MC labeling. Long fibers up to 20 microns damaged the bronchiolar epithelium and were incorporated into connective tissue. Increased 3HT uptake was seen in fibroblasts and epithelial cells and also in MC, which peaked at 2% labeled at 1 week compared to near 0% labeling in controls. No fibers were found in or near labeled MC, which suggested that a cytokine generated in the lung during the early response phase might induce MC proliferation. To look for a cytokine effect in vitro, we instilled asbestos into rat lungs and, after 1 and 6 weeks, bronchoalveolar and pleural lavage fluids as well as macrophages were collected. Alveolar macrophages contained fibers, but pleural macrophages (PM) did not. After short-term culture, macrophage supernatants and the lavage fluids were tested on rat lung MC in culture. At 1 week, PM secreted growth factor(s) for MC, and the mitogenic effect was more pronounced with lavage fluids. No effects on MC were found using material prepared 6 weeks after asbestos. The early MC growth increase was not blocked by antibodies to cytokines, such as platelet-derived growth factor, fibroblast growth factors, or tumor necrosis factor, but was inhibited by anti-keratinocyte growth factor (anti-KGF). The results show that an early growth phase of MC after asbestos exposure appears unrelated to particle translocation to the pleura but is associated with cytokine release, most likely KGF, by lung cells.

Response of mouse lung to carbon deposition during injury and repair.

Increased respiratory disease and daily mortality rates are associated with higher levels of fine particulate air pollutants. We examined the possibility that deposition of even inert particles to previously injured lungs may accentuate pulmonary damage by investigating how the lung handles small carbon particles delivered during acute injury or during fibrotic repair. Mice received 2 mg carbon by intratracheal instillation into lungs already showing acute injury, 3 days after bleomycin (BL), or into lungs with fibrosis, 4 weeks after BL. At 3 days after BL, injury to the type I alveolar epithelium resulted in high protein levels in lavage fluid. Instilling carbon at this time induced a large increase in inflammatory cells, though many particles reached the interstitium, and a high proportion was retained up to 16 weeks later. However, fibrosis in these mice was equal to that found after BL alone. In the mice that received carbon 4 weeks after bleomycin, fibrotic repair had already occurred, and the epithelial surface was restored before particle instillation. After carbon, the subsequent inflammatory reaction cleared most particles, little reached the interstitium, and carbon retained at 16 weeks was not different from that in the carbon-only group. Instilling particles into fibrotic lung did not induce additional fibroblast growth or collagen production. The results indicate that instillation of fine particulates to the alveoli at a time of epithelial injury results in increased translocation to the interstitium. However, deposition of pure carbon into injured lungs does not further stimulate an ongoing fibrotic process, although it alters the patterns of particle deposition and retention in the lung.

Epithelial-fibroblast interactions in bleomycin-induced lung injury and repair.

Intercellular communication between epithelial cells and fibroblasts of the alveolar wall contributes to regulatory control of each cell type. We examined whether lung injury and subsequent fibrosis are associated with disturbance of this mutual control system. Rats received bleomycin intratracheally, and after 10 days, when acute epithelial injury occurs, and at 6 weeks, when repair with fibrosis is found, pure populations of type 2 epithelial cells and lung fibroblasts were prepared to study interactions with respect to growth control. Epithelial cells were cultured alone, on a permeable filter over fibroblasts, and in co-culture with fibroblasts. The results showed that the low growth rate of normal epithelial cells increased when cells were exposed to fibroblast supernatants. This effect was also seen using cells from the 10-day bleomycin group, but it was diminished in the group treated for 6 weeks. However, epithelial cells from exposed or control rats did not show increased DNA synthesis when grown in contact with fibroblasts in co-culture. In contrast, fibroblast growth was inhibited when exposed to epithelial cell secretions in control cultures and when using cells from the 10-day bleomycin group. No inhibition of fibroblast growth by epithelial cells was found using cells from the fibrotic lungs. These results suggest that after lung injury by bleomycin, a fibroblast-secreted factor promotes epithelial growth; however, during repair, regenerating epithelial cells lose the ability to inhibit fibro-blast proliferation. These local changes in cellular control at the alveolar wall may be sufficient to produce pulmonary fibrosis.

Postnatal development of rat lung following retarded fetal lung growth.

Postnatal lung development was examined in rats born with smaller than normal lungs after either prenatal exposure to glucocorticoid or to an inhibitor of collagen synthesis. At birth, treated animals had lower than normal lung weights, lung to body weight ratios, hydroxyproline (HYP) levels, total DNA; and rates of DNA synthesis. Rats exposed to steroid showed a rapid recovery in growth during the normal postnatal cell proliferative phase from 4 to 11 days, though collagen levels did not return to normal until 3 weeks of age. Rats exposed to a prenatal proline analog showed a much slower rise in lung weight and total DNA, and these levels were still much below normal at 2-3 weeks when the cell proliferation phase was completed. Levels of disaturated phosphatidylcholine were significantly below normal up to 11 days, whereas total HYP was significantly reduced and less fibrillar collagen was seen in the lung throughout the study. The results indicate that the smaller but mature lungs at birth after antenatal steroid show a growth rebound and quickly become structurally normal. In contrast, inhibition of fibroblast growth and collagen deposition produces small lungs at birth, which continue to show inhibited growth and development at least up to 3 weeks of age, when the cell division phase is over.

Collagen degradation during postnatal lung growth in rats.

Postnatal lung growth involves remodeling of the structure seen at birth as new alveoli are formed. To determine the role of collagen degradation in this process, in particular of the basement membrane component, we studied the synthesis of total collagen and the degradation of collagen types I or IV in a series of rats from birth to 29 days of age. During the period of rapid cell proliferation to day 11, the collagen level per mg lung did not change though the rate of synthesis increased. Up to 40% of new collagen was rapidly degraded. At the end of the growth phase, the interstitium became thinner and less cellular. Collagen synthesis slowly decreased as the total collagen content increased in the lung, and less than 20% of newly synthesized collagen was degraded. Type I collagenase activity was highest during the cell proliferation phase, though less than 20% was due to active enzyme. In contrast, type IV collagen breakdown, also maximal in the first 11 days, was almost all due to enzymes present in the active form. The results demonstrate that rapid degradation of collagen, particularly the type IV form present in basement membranes, occurs during the early phase of postnatal lung growth.

Effects of dexamethasone on macrophages in fetal and neonatal rat lung.

The effects of injecting dexamethasone to pregnant and newborn rats on the subsequent production of macrophages in the lung and on their phagocytic activity and lysosomal enzyme content were evaluated from late gestation to postnatal day 10 using an organ culture system to collect macrophages. Pieces of lung tissue cultured 6 days on cover glasses produced a halo of macrophages adherent to the glass around the explants. Thymidine labeling showed that the macrophages were derived from dividing precursors in the pulmonary interstitium. DNA synthesis was reduced after steroid treatment, and fewer cells were obtained on cover glasses, particularly just after birth when the macrophage number usually increases. Phagocytic function was also diminished in cells collected after dexamethasone injection, particularly when derived from neonatal animals. In contrast, intracellular levels of non-specific esterase and glucosaminidase were increased, probably indicative of lower phagolysosome formation and lower enzyme secretion. These results demonstrate that steroid administration to fetal or newborn animals subsequently reduces the number and phagocytic activity of macrophages in the lungs. This could reduce the defense mechanisms of the neonate and increase susceptibility to infection.

Relationship of mesenchymal changes to alveolar epithelial cell differentiation in fetal rat lung.

The influence of mesenchymal components on epithelial cell differentiation in fetal lung has mostly been studied in vitro. Here, the relationship of direct epithelial--mesenchymal cell interactions and of matrix changes beneath epithelial cells to the development of Type 2 and Type 1 epithelium is examined in vivo. In late gestation, as epithelial division slows, these cells come in close apposition to fibroblasts. In some places extracellular filaments connect these different cell types, often bridging the basal lamina. In other regions, direct cell-cell contact is made at membrane structures resembling gap junctions which connect fibroblasts to the cuboidal epithelium which develops characteristics of Type 2 cells. After day 20, as endothelial cell proliferation increases, epithelial cells lying directly over the endothelium do not contact fibroblasts. These cells lose lamellar bodies as they flatten out to become Type 1 cells lysing on a fused basal lamina made by epithelium and endothelium. The results provide in vivo evidence that Type 2 cell morphology and function is influenced by direct contact with underlying fibroblasts and collagen fibrils. Differentiation to Type 1 epithelium appears to be modulated by capillary growth, either through loss of epithelial contact with the fibroblast and its products, or through an effect of an endothelial matrix component.

Inhalation toxicology of urban ambient particulate matter: acute cardiovascular effects in rats.

Wistar rats were exposed for 4 hours by nose-only inhalation to clean air, resuspended Ottawa ambient particles (EHC-93*, 48 mg/m3), the water-leached particles (EHC-93L, 49 mg/m3), diesel soot (5 mg/m3), or carbon black (5 mg/m3). Continuous data for physiologic endpoints (heart rate, blood pressure, body temperature, animal's activity) were captured by telemetry before and after exposure. Blood was sampled from jugular cannulas 1 to 3 days before exposure and at 2 and 24 hours after exposure, and by heart puncture on termination at 32 hours (histology group) or 48 hours (telemetry group) after exposure. Lung injury was assessed by 3H-thymidine autoradiography after the rats were killed. We measured endothelins (plasma ET-1, big ET-1, ET-2, ET-3) to assess the vasopressor components; nitric oxide (NO)-related metabolites (blood nitrate, nitrite, nitrosyl compounds, and plasma 3-nitrotyrosine) to assess the vasodilator components; and catecholamines (epinephrine, norepinephrine, L-DOPA, dopamine) and oxidative stressors (m- and o-tyrosine) for additional insight into possible stress components. Lung cell labeling was uniformly low in all treatment groups, which indicates an absence of acute lung injury. Inhalation of EHC-93 caused statistically significant elevations (P < 0.05) of blood pressure on day 2 after exposure, plasma ET-1 at 32 hours after exposure, and ET-3 at 2, 32, and 48 hours after exposure. In contrast, the modified EHC-93L particles, from which soluble components had been extracted, did not affect blood pressure. The EHC-93L particles caused early elevation (P < 0.05) of the plasma levels of ET-1, ET-2, and ET-3 at 2 hours after exposure, but the endothelins returned to basal levels 32 hours after exposure. Exposure to diesel soot, but not carbon black, caused an elevation (P < 0.05) of plasma ET-3 at 36 hours after exposure; blood pressure was not affected by diesel soot. Our results indicate that inhalation of the urban particles EHC-93 can affect blood levels of ET-1 and ET-3 and cause a vasopressor response in Wistar rats without causing acute lung injury. Furthermore, the potency of the particles to influence hemodynamic changes appears to be modified by removing polar organic compounds and soluble elements. Because the pathophysiologic significance of elevated endothelins has been clinically established in humans, our observations suggest a novel mechanism by which inhaled particles may cause cardiovascular effects. These findings in rats contribute to the weight of evidence in favor of a biologically plausible epidemiologic association between ambient particulate matter and cardiovascular morbidity and mortality in human populations.