Ian Y. R. Adamson

Acute Effects of Inhaled Urban Particles and Ozone : Lung Morphology, Macrophage Activity, and Plasma Endothelin-1

We studied acute responses of rat lungs to inhalation of urban particulate matter and ozone. Exposure to particles (40 mg/m3 for 4 hours; mass median aerodynamic diameter, 4 to 5 microm; Ottawa urban dust, EHC-93), followed by 20 hours in clean air, did not result in acute lung injury. Nevertheless, inhalation of particles resulted in decreased production of nitric oxide (nitrite) and elevated secretion of macrophage inflammatory protein-2 from lung lavage cells. Inhalation of ozone (0.8 parts per million for 4 hours) resulted in increased neutrophils and protein in lung lavage fluid. Ozone alone also decreased phagocytosis and nitric oxide production and stimulated endothelin-1 secretion by lung lavage cells but did not modify secretion of macrophage inflammatory protein-2. Co-exposure to particles potentiated the ozone-induced septal cellularity in the central acinus but without measurable exacerbation of the ozone-related alveolar neutrophilia and permeability to protein detected by lung lavage. The enhanced septal thickening was associated with elevated production of both macrophage inflammatory protein-2 and endothelin-1 by lung lavage cells. Interestingly, inhalation of urban particulate matter increased the plasma levels of endothelin-1, but this response was not influenced by the synergistic effects of ozone and particles on centriacinar septal tissue changes. This suggests an impact of the distally distributed particulate dose on capillary endothelial production or filtration of the vasoconstrictor. Overall, equivalent patterns of effects were observed after a single exposure or three consecutive daily exposures to the pollutants. The experimental data are consistent with epidemiological evidence for acute pulmonary effects of ozone and respirable particulate matter and suggest a possible mechanism whereby cardiovascular effects may be induced by particle exposure. In a broad sense, acute biological effects of respirable particulate matter from ambient air appear related to paracrine/endocrine disruption mechanisms.

Dose response of the pulmonary macrophagic system to various particulates and its relationship to transepithelial passage of free particles.

Alveolar macrophages are thought to arise from both marrow-derived monocytes and pulmonary interstitial cells. Macrophage kinetics are now studied under various conditions of alveolar loading using several doses of carbon (0.03 micrometers diameter), polystyrene latex (0.1 and 1.0 micrometers), and heat-killed bacteria. In serial studies we examined the number of macrophages recovered by lavage, DNA synthesis by lung cells on autoradiographs, and the passage of free particles into lung tissues by electron microscopy. The dual origin of the alveolar macrophage was confirmed for each particulate. The observed peak in macrophagic output at 1 day may be explained by monocytic egress. With greater loads, the peak value did not increase, but the continuing macrophagic production correlated with a period of interstitial cell proliferation. For all particles used, the number of new macrophages was related more closely to number of particles instilled than to the total dose by weight delivered to the lungs. With increasing number, more free particles of carbon and latex crossed the Type 1 epithelium to be phagocytized by interstitial macrophages. The results suggest that the adaptive outpouring of alveolar macrophages occurs by an acceleration of the normal biphasic pathway; when the adaptive response is prolonged, the interstitial compartment appears to be the predominant source of new cells.

COMPARATIVE PULMONARY TOXICITY OF VARIOUS SOLUBLE METALS FOUND IN URBAN PARTICULATE DUSTS

The potential toxicity of an atmospheric dust sample EHC-93 has been attributed to the soluble fraction and, more specifically, to the zinc component. The concentration of Zn is the highest among the metals present in the soluble EHC-93 fraction. We now determine whether other metal components of this dust could cause similar lung injury if present at the same concentration as Zn (4.8 mg/g dust). Solutions of Zn, Cu, V, Ni, Fe, and Pb salts in 0.1 mL water were instilled to mouse lung and animals were killed at intervals up to 2 weeks later; each mouse received tritiated thymidine 1 hour before death. Solutions containing Zn and to a lesser degree Cu induced lung injury; in addition, increased numbers of alveolar macrophages and polymorphonuclear leukocytes were found in the lavage fluid, which also contained increased protein levels up to 1 week later. The magnitude of response was similar to that seen after administering EHC-93 dust at 1 mg in 0.1 mL water, whereas the response to other metal solutions containing Ni, Fe, Pb, and V was minimal. Morphologic evidence of lung injury and inflammation was also seen after EHC dust and the Zn or Cu solutions only. Reparative cell proliferation was measured after thymidine uptake and autoradiographs showed increased labeling of lung cells, particularly at 3 and 7 days. Labeling was confined to bronchiolar and type 2 alveolar epithelial cells, indicating previous epithelial cell necrosis in response to Zn or Cu. The results indicate that atmospheric contaminant metals Zn and Cu are most likely to cause lung injury and inflammation as compared to metals such as Ni, Fe, Pb, and V at the same concentrations. It appears that similar toxicity occurs when both redox (Cu) and nonredox (Zn) reactions are involved.

Relationship of Keratinocyte Growth Factor and Hepatocyte Growth Factor Levels in Rat Lung Lavage Fluid to Epithelial Cell Regeneration after Bleomycin

Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are known mitogens for normal alveolar Type 2 cells in vitro and in vivo. We wished to determine whether these two growth factors are involved in lung repair after epithelial cell necrosis by determining the levels of each factor in lung lavage fluid collected serially after bleomycin-induced injury, and how these values relate specifically to proliferation of bronchiolar and alveolar epithelial cells. Rats received an intratracheal injection of 1 unit bleomycin in 0.5 ml water and were killed at intervals up to 4 weeks with 1 muCi/g tritiated thymidine injected 1 hour before death. Early necrosis of bronchiolar epithelial (BR) cells and Type 1 alveolar epithelium was followed by an increase in inflammatory cell numbers and high protein levels in bronchoalveolar lavage (BAL) fluids. In addition, the levels of KGF and HGF, measured by enzyme-linked immunosorbent assay in BAL, increased as early as 3 days and peaked at 7-14 days, when KGF was measured at 160 pg/ml (n = 50) and HGF reached 460 pg/ml (n = 40). Both values dropped sharply after 2 weeks. Epithelial cell proliferation was quantitated as percentage of labeled cells in autoradiographs of methacrylate sections. Labeling of BR cells predominated in the first week and peaked at 7% at 3 days. Type 2 cell proliferation was delayed somewhat but occurred in 3 to 10 days with a peak of 7% labeled cells at 1 week. The results demonstrate that both HGF and KGF are present in the lung in greatly increased amounts soon after bleomycin-induced epithelial cell necrosis. These high levels are associated with both BR and alveolar epithelial cell proliferation.

Reciprocal Epithelial: Fibroblast Interactions in the Control of Fetal and Adult Rat Lung Cells in Culture

Intercellular communication between epithelial cells and fibroblasts of the alveolar wall has been postulated from studies of lung development and repair. We examined the epithelial cell-fibroblast interactions with respect to growth control and epithelial cell function using cultured fetal and adult lung cells. The role of diffusing factor(s) as compared to direct cell-to-cell contact was studied by culturing epithelial cells either on a permeable culture well insert over fibroblasts or in co-culture with fibroblasts. The results show that the normal low proliferative rate of epithelial cells in culture is increased when exposed to fibroblast supernatants. In contrast, epithelial cells (particularly from adult lung) secrete a factor that suppresses fibroblast growth when cultured with a filter between the cell types. However, when cell-cell contact occurs in co-culture, the growth rate of fibroblasts is greatly increased. Synthesis of disaturated phosphatidylcholine by epithelial cells is increased under serum-free conditions and further rises when fetal epithelial cells are exposed to steroid-treated fibroblasts, when the cell types are separated, and when cells are in contact. This indicates that a fibroblast-derived factor stimulates epithelial differentiation, and morphologic evidence relating the appearance of lamellar bodies to the areas of direct epithelial cell-fibroblast contact was found. The results indicate the complex interdependence of these two types of cell where a secretory product of one cell or direct cell-cell contact may alter when regulatory control of the other cell type. These interactions are likely to be important in orderly development and in the reparative response of the lung to injury.

Epithelial-Mesenchymal Interactions in Postnatal Rat Lung Growth

Epithelial-mesenchymal interactions have been implicated in epithelial cell differentiation in fetal lung. The role of such intercellular communication during postnatal lung growth is now evaluated to correlate intercellular contacts with cell division and with biochemical function of epithelial and interstitial cells. Sexed newborn rats, killed at frequent intervals to 8 weeks, received 3H-thymidine 1 h before death. A period of rapid cell proliferation between 3 and 10 days involved a five-fold increase in labeling index of both interstitial and type 2 epithelial cells. Hydroxyproline levels increased rapidly as interstitial cell division slowed, and epithelial growth was associated with elevated levels of disaturated phosphatidylcholine. Compared to the time of birth when epithelial cell division was slow, continuous basement membrane (BM) was more frequently found beneath type 2 cells during the postnatal proliferative phase, and fewer foot processes (FP) penetrated the BM; type 1 cells had few FP and uninterrupted BM. The number of intercellular contacts between type 2 cells and interstitial cells, one per two epithelial cell profiles at birth, decreased rapidly during the postnatal proliferative phase. There was a subsequent increase between 2 and 4 weeks that may reflect postmitotic epithelial differentiation. These observations support the hypothesis that transfer of mesenchymal factors may be important in the control of pulmonary epithelial growth and differentiation. The low incidence of cell-cell contacts seen after 4 weeks, one per five epithelial cell profiles, may reflect base levels of communication necessary to retain type 2 cell function in a resting cell population.

Collagenase and gelatinase activities in bronchoalveolar lavage fluids during bleomycin‐induced lung injury

Basement membrane degradation can be indicative of tissue injury, but the process may also release matrix‐bound cytokines to stimulate cell regeneration. To investigate this process, acute lung injury was induced in rats by intratracheal bleomycin and animals were killed from 3 days to 8 weeks later. The lungs were lavaged with saline to collect bronchoalveolar lavage (BAL) fluid and cell proliferation was assessed by pulse incorporation of tritiated thymidine. Bleomycin induced rapid inflammation with increased cell numbers and protein levels in BAL. Collagen degradation products were also increased in BAL fluid from 3 days to 4 weeks. Incubating samples of BAL fluid with radiolabelled collagens I and IV showed that high levels of activity, particularly for the degradation of type IV collagen, were present as early as 3 days post‐bleomycin and persisted over the 8‐week period. Zymograms demonstrated the highest level of gelatinase A (MMP‐2) activity in BAL fluid in the first 2 weeks after bleomycin. Coincident with peak basement membrane degradative activity was the onset of a phase of epithelial cell proliferation, as measured by labelled nuclei in autoradiographs. The results show that enzymes capable of degrading the alveolar basement membrane are secreted early in the lung injury phase and that their presence in BAL fluid can be used as a measure of alveolar wall damage. It is possible that this enzyme action may release bound cytokines from the basement membrane, since maximal gelatinase activity correlates with alveolar epithelial cell proliferation.

The intracellular site of surfactant synthesis: Autoradiographic studies on murine and avian lung explants

Abstract In murine and avian lungs maintained in organ culture, the Type II epithelial cells selectively incorporate labelled precursors of lecithin; the Clara cells, alveolar macrophages and other pulmonary cells show a low isotopic uptake. Within Type II cells, the silver grains, though associated with the lamellar bodies, are predominantly located at the periphery of these structures; saturated phospholipids are also concentrated at these sites. It is concluded that the perilamellar membrane of the Type II cell is the active site of surfactant synthesis.

KGF and HGF are growth factors for mesothelial cells in pleural lavage fluid after intratracheal asbestos.

Mesothelial cells proliferate soon after asbestos deposition in the lung. The present study investigates whether the known mesothelial cell mitogens keratinocyte growth factor (KGF)and hepatocyte growth factor (HGF)are present in the lung and specifically in the pleural cavity during the phase of mesothelial cell growth. Rats received 1 mg crocidolite asbestos in 0.5 mL water by intratracheal instillation and were killed up to 2 weeks later; tritiated thymidine was injected 1 hour before death. Bronchoalveolar lavage (BAL)and pleural lavage (PLL)were performed. Increased inflammatory cell numbers and protein levels were found in BAL but also in PLL at 1 day after asbestos deposition. In lung sections, labeling of mesothelial cells increased > 10-fold at day 1 and stayed above normal for 1 week. During this period, the levels of HGF and KGF were significantly raised in both BAL and PLL fluids. The PLL fluid had mitogenic activity for mesothelial cells in culture and this effect was significantly reduced by antibodies to HGF and KGF. The results indicate that fiber deposition in the airspaces rapidly induces lung injury and inflammation, in which growth factors for mesothelial cells KGF and HGF are secreted. These factors reach the pleural cavity at the time when mesothelial cell proliferation occurs. It is possible that the activated, dividing mesothelial population may then be more susceptible to DNA damage by any translocated fibers.Mesothelial cells proliferate soon after asbestos deposition in the lung. The present study investigates whether the known mesothelial cell mitogens keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are present in the lung and specifically in the pleural cavity during the phase of mesothelial cell growth. Rats received 1 mg crocidolite asbestos in 0.5 mL water by intratracheal instillation and were killed up to 2 weeks later; tritiated thymidine was injected 1 hour before death. Bronchoalveolar lavage (BAL) and pleural lavage (PLL) were performed. Increased inflammatory cell numbers and protein levels were found in BAL but also in PLL at 1 day after asbestos deposition. In lung sections, labeling of mesothelial cells increased > 10-fold at day 1 and stayed above normal for 1 week. During this period, the levels of HGF and KGF were significantly raised in both BAL and PLL fluids. The PLL fluid had mitogenic activity for mesothelial cells in culture and this effect was significantly reduced by antibodies to HGF and KGF. The results indicate that fiber deposition in the airspaces rapidly induces lung injury and inflammation, in which growth factors for mesothelial cells KGF and HGF are secreted. These factors reach the pleural cavity at the time when mesothelial cell proliferation occurs. It is possible that the activated, dividing mesothelial population may then be more susceptible to DNA damage by any translocated fibers.

SOLUBLE AND INSOLUBLE AIR PARTICLE FRACTIONS INDUCE DIFFERENTIAL PRODUCTION OF TUMOR NECROSIS FACTOR α IN RAT LUNG

Altered cytokine production in the lung follows the deposition of urban air particles. The present study was designed to measure changes in tumor necrosis factorα (TNFα) and endothelin-1 (ET-1) levels in rat lung after instilling various fractions of the dust EHC-93, while in vitro, alveolar macrophages (AMs) and type 2 epithelial cells were studied to determine relative production of these molecules in response to the same particles. Whole dust and its soluble and leached components were instilled into rat lung and the animals were killed at intervals to 2 weeks; they received tritiated thymidine by intraperitoneal injection 1 hour before death. All samples induced some inflammation, with the highest cellular efflux being found by bronchoalveolar lavage 1 day after leached particles. Lung injury, illustrated by protein levels in lavage fluid, was maximal after instilling the soluble fraction and subsequently epithelial regeneration was also maximal in this group. TNFα levels were highest after instilling whole dust or its leached fraction at 4 hours and 1 day, and cell culture studies indicated a predominant AM source for this cytokine. ET-1 levels were also increased in BAL from 4 hours to 3 days and were mostly associated with the instillation of leached particles. The results demonstrate that the rapid production/release of TNFα and ET-1 after particle deposition is largely due to the insoluble particulate fraction. There appears to be a differential response to whole dust where the soluble components cause some inflammation and epithelial cell necrosis, whereas the leached particles are more likely to react with macrophages to induce the production of proinflammatory cytokines such as TNFα.