Interaction of rat alveolar macrophages with pulmonary epithelial cells following exposure to lipopolysaccharide

 Because both alveolar macrophages and pulmonary epithelial cells are primary target cells of inhaled endotoxin, it is of interest to study the interaction of alveolar macrophages with epithelial cells following exposure to lipopolysaccharide (LPS). Repeated bronchoalveolar lavage suggested that rat alveolar macrophages became adhesive to epithelial cells in response to intratracheally instilled LPS at 0.5 h post-exposure in vivo. Adherence of alveolar macrophages to SV40T2 (rat type II pulmonary epithelial cells transformed by SV40) was also increased following 0.5-h stimulation with LPS in vitro. The adherence was increased with dose and reached a plateau at 2 μg/ml LPS. The transepithelial resistance of the SV40T2 monolayer was decreased by coculture with macrophages in the presence of LPS for 4 h. The transepithelial resistance was not changed by exposure of SV40T2 alone to LPS or conditioned medium obtained from LPS-stimulated macrophages, suggesting that the intercellular interaction between alveolar macrophages and epithelial cells or unstable products of LPS-exposed alveolar macrophages was associated with the decrease in transepithelial resistance. Following the decrease in transepithelial resistance, lysis of SV40T2 was observed in the LPS-exposed coculture system. However, the lysis of SV40T2 did not occur until 12 h after the addition of LPS, indicating that the junctional change of the monolayer preceded cell death in SV40T2. Cytotoxicity of LPS-stimulated macrophages was significantly reduced by N ^G -monomethyl-L-arginine (NMMA), an inhibitor of nitric oxide synthase, but NMMA did not reduce the decrease in transepithelial resistance. These results suggest that the greater adherence of alveolar macrophages to epithelial cells, the junctional change of the epithelial monolayer and the lysis of epithelial cells occur in this order in the LPS-exposed alveolar macrophage-SV40T2 coculture system, and the greater adherence of alveolar macrophages may play a role in LPS-induced inflammation in the rat lung. Received: 17 May 1995 / Accepted: 21 August 1995     

Interaction between primary alveolar macrophages and primary alveolar type II cells under basal conditions and after lipopolysaccharide or quartz exposure

Intercellular communications between alveolar macrophages (AM) and alveolar epithelial type II (TII) cells have been suggested to be important in cellular responses. The main objective of this study was to improve our understanding of the interactions between AM and TII cells that might occur in the lung. In the present investigation, this interaction was studied under different interaction conditions (transwell or mixed coculture) and different exposure conditions (basal, lipopolysaccharide [LPS] exposure, or silica exposure). Studies also attempted different approaches to identify specific mediator(s) involved in this interaction. The following findings were made: (1) Surfactant released from TII cells appears to exert an inhibitory effect on AM activity. (2) Basal transwell coculture conditions are better than mixed coculture conditions to study AM/TII cell interactions, since the inhibitory effect of the surfactant in the transwell coculture is minimized. (3) AM/TII cell interaction is dependent on cell culture (transwell vs mixed) and exposure conditions. (4) Oxidants, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, prostaglandins, and leukotrienes probably do not independently affect the AM/TII intercellular interaction; instead, they appear to indirectly modulate the complex pathways of AM/TII communication.     

In vitro cytokine release from rat type II pneumocytes and alveolar macrophages following exposure to JP-8 jet fuel in co-culture

Exposure of Chinese hamster V79 cells to extracts of airborne pollutants induced formation of multipolar or incomplete mitotic spindles. To find out whether overexpression of the HSP70 chaperone protein could protect spindles against airborne toxins we constructed V79 cells stably transfected with an expression vector containing rat heat-inducible hsp70.1 gene under the control of a constitutive CMV promoter. When cells were incubated with extracts of airborne pollutants (5–20 μg/ml) no protective effect of the HSP70 protein against mitotic spindle damage was observed. Moreover, at 20 μg/ml of extracts of airborne toxins the frequency of mitotic malformations was even higher in HSP70-overexpressing cells than in control ones. Extracts of airborne pollutants of 50 μg/ml blocked the formation of mitotic figures both in control and HSP70-overexpressing cells and led to destruction of cell nuclei. However, the HSP70-overproducing cells exhibited higher survival rates when exposed to heat shock and airborne toxins than the control ones, as determined by MTT assay. This suggests that HSP70 overexpression—a frequent feature of cancer cells—should be considered as a factor facilitating survival of cells with damaged mitotic spindles and aberrantly segregated chromosomes.     

Response of alveolar macrophages from inducible nitric oxide synthase knockout or wild-type mice to an in vitro lipopolysaccharide or silica exposure

The role of nitric oxide (NO) in pulmonary disease has been controversial with both antiinflammatory (scavenging radicals and inhibiting NF-êB activation) and proinflammatory (forming highly reactive peroxynitrite and augmenting NF-êB activation by inflammatory agents) actions reported. Therefore, a study has been initiated to determine whether deletion of the inducible nitric oxide synthase (iNOS) gene in the C57BL/6J mouse alters the pulmonary macrophage response to lipopolysaccharide (LPS) or silica. The objective of the initial phase of this study was to determine the difference in responsiveness of alveolar macrophages (AMs), harvested from naive wild-type (WT) or iNOS knockout (iNOS KO) mice, to an in vitro LPS or silica exposure. Primary AMs were obtained by bronchoalveolar lavage (BAL) from age- and weight-matched iNOS KO and WT mice. The cells were treated with interferon-gamma (IFN-?) (50 U/ml), IFN-? (50 U/ml) + LPS (1 microg/ml), LPS (0.01-100 microg/ml), or silica (25-250 microg/ml). The following parameters were measured: nitrate and nitrite (NOx), tumor necrosis factor-á (TNF-á), macrophage inflammatory protein-2 (MIP-2), intracellular generation of the reactive oxygen species (ROS) hydrogen peroxide (H(2)O(2) and superoxide (O(*-2)), and basal (unstimulated) total antioxidant capacity. Data show a significant increase in NOx production upon exposure to IFN-? +/- LPS in the WT but not iNOS KO AMs. NOx production by iNOS KO or WT AMs was not altered by in vitro exposure to LPS or silica alone. LPS, but not silica, induced TNF-á and MIP-2 production in both iNOS KO and WT AMs. Statistical analysis of concentration response curves found a significant tendency for greater mediator production in the iNOS KO versus WT AMs. Basal intracellular production of H(2)O(2) and O(*- 2) was significantly greater in the iNOS KO compared to WT AMs. In contrast, LPS- (10 microg/ml) or silica- (100 microg/ml) stimulated intracellular oxidant production was lower in iNOS KO AMs, but overall (basal + stimulated) inflammatory capacity was similar between the cell types. The basal total antioxidant production of the iNOS KO AMs was approximately twofold higher than the WT AMs. In conclusion, certain compensatory changes appear to occur in AMs from iNOS KO mice. In response to the inability to induce NO production, iNOS KO AMs exhibit significantly higher basal generation of H(2)O(2) and (O(*- 2)) as well as higher total antioxidant levels. In addition, LPS induced TNF-á and MIP-2 production tend to be higher in AMs from iNOS KO mice. Such compensatory changes in the AM response may affect the response of iNOS KO mice to inflammatory exposures.     

Chemiluminescence from rat alveolar macrophages following induction of phospholipidosis with chlorphentermine

Rats were treated with chlorphentermine (30 mg/kg) 5 days a week for 4 weeks during which time a phospholipidosis developed in the alveolar macrophages of the lung. Compared to control macrophages, an enhanced chemiluminescence (CL), with altered kinetics, was generated when cells from treated rats were challenged with zymosan. The role of reactive oxygen species, particularly superoxide anion, in the increased CL was studied. Superoxide dismutase inhibited the CL from control cells correspondingly more than with lipidotic cells indicating processes other than the release of superoxide anion from the cells were involved in the enhanced CL response. While total oxygen consumption was similar for both cell populations, lipidotic cells released less superoxide anion during challenge than controls. Concomitantly, lipidotic cells showed an increase in lipid peroxidation. These results are consistent with an explanation that rather than being released by the cell, superoxide anion may become involved in a secondary reaction within the cell such as lipid peroxidation which is then reflected in the altered CL response.     

PGJ2 inhibition of LPS-induced inflammatory mediator expression from rat alveolar macrophages

Studies suggested that 15-deoxy-delta-(12,14)-prostaglandin J2 (PGJ2) may exert anti-inflammatory effects, including in the lung. Thus, in vitro studies were conducted to (1) investigate whether PGJ2 inhibited the production of inflammatory mediators from lipopolysaccharide (LPS)-exposed primary rat alveolar macrophages (AM), and (2) investigate possible mechanisms underlying PGJ2-mediated inhibition of inflammatory mediator production. These studies determined that PGJ2 inhibited LPS-induced nitric oxide (NO) production in a concentration- and time-dependent manner. PGJ2-mediated inhibition of NO, as well as of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2), was also determined to be dependent on the time of addition of PGJ2 relative to LPS, and suggested the PGJ2 inhibitory mechanism is an early event. PGJ2 was shown not to interfere with binding or internalization of LPS by AM, indicating this was not responsible for PGJ2 inhibitory effects. Another possible mechanism underlying PGJ2-mediated inhibition was via peroxisome proliferator-activated receptor-gamma (PPAR-gamma). However, biochemical studies suggested that PGJ2-mediated inhibition was not occurring through PPAR-gamma dependent mechanism, and molecular studies further established that both LPS and PGJ2 decrease PPAR-gamma mRNA expression. A third possible mechanism underlying PGJ2-mediated inhibition was by alteration of nuclear factor (NF)-kappaB. Molecular studies confirmed that LPS stimulated NF-kappaB mRNA expression, and PGJ2 reduced this stimulation, which is consistent with PGJ2 effect on LPS-induced production of NO, TNF-alpha and MIP-2. Thus, data in this study established that PGJ2 inhibited LPS-induced inflammatory mediator production in rat AM, and this inhibition is mediated, at least in part, by reducing the expression of NF-kappaB mRNA.     

PM2.5‐induced lung inflammation in mice: Differences of inflammatory response in macrophages and type II alveolar cells

Particulate matter 2.5 (<PM2.5 μm) leads to chronic obstructive pulmonary disease. In this study, biomarkers related to inflammation and oxidative stress in vitro and in vivo experiments were investigated to clarify the PM2.5‐induced lung inflammation mechanisms. In an in vitro study using RAW264.7 cells, PM2.5 caused phosphorylation of nuclear factor‐κB, p38 mitogen‐activated protein kinase and extracellular response kinases, an increase of proinflammatory gene and protein expressions (e.g. monocyte chemotactic protein‐1, tumor necrosis factor‐α). These biomarkers were substantially attenuated by polymyxin B (PMB). PM2.5 induced heme oxygenase‐1 (HO‐1) gene, which was attenuated by N‐ acetylcysteine (NAC). However, the suppressive effects of NAC on inflammatory biomarkers were very weak. In bone marrow‐derived macrophages (BMDMs) of wild‐type BALB/c mice, the effects of PMB and NAC on PM2.5‐induced inflammatory responses were similar to RAW264.7 cells. In BMDMs of MyD88^−/− mice, PM2.5‐induced proinflammatory mediators were substantially more attenuated. PM2.5 caused an increase of proinflammatory gene expressions (interleukin‐6, cyclooxygenase 2) and HO‐1 gene in MLE‐12 cells (mouse alveolar cell line). These biomarkers were substantially attenuated by NAC, but not by PMB. When BALB/c mice were exposed intratracheally to 0.2 mg PM2.5, PM2.5 caused severe lung inflammation, an increase of neutrophils along with proinflammatory mediators in bronchoalveolar lavage fluid. The inflammation was attenuated by NAC, particularly by NAC + PMB, but not by PMB alone. These results indicate that macrophages may act sensitively to lipopolysaccharide (LPS) present in PM2.5 and release proinflammatory mediators via the LPS/MyD88 pathway. However, type II alveolar cells may react sensitively to oxidative stress induced by PM2.5 and cause inflammatory response. Therefore, overall, PM2.5 may cause predominantly oxidative stress‐dependent inflammation rather than LPS/MyD88‐dependent inflammation in type II alveolar cell‐rich lungs. Copyright © 2017 John Wiley & Sons, Ltd.     

Role of mitogen-activated protein kinase activation in the production of inflammatory mediators: differences between primary rat alveolar macrophages and macrophage cell lines

Stimulation of macrophages has been shown to activate all three families of mitogen activated protein kinases (MAPKs). However, variable results are reported in the literature with respect to the particular kinases activated with any given stimulus. In this study, the role of activation of MAPKs was examined in the production of inflammatory mediators by measuring the phosphorylation of the kinases and their ability to phosphorylate specific substrates in rat primary alveolar macrophages, a rat alveolar macrophage cell line (NR8383), and two mouse monocytic cell lines (RAW 264.7 and J774A.1). In the three cell lines examined, all three families of MAPKs were activated upon stimulation with either lipopolysaccharide (LPS) or LPS plus interferon-gamma; in contrast, only ERK1/2 was activated in primary rat alveolar macrophages upon stimulation with LPS. Inhibition of ERK1/2 activation by the MEK inhibitor PD98059 abrogated nitric oxide and tumor necrosis factor-alpha (TNF-alpha) production in primary rat alveolar macrophages, but the p38 inhibitor SB203580 had no effect on the production of these two inflammatory mediators. These observations indicate that MAPK activation is cell specific and explain some of the conflicting results reported in the literature. These studies emphasize the need to exercise caution in extrapolating data from cell lines to primary cells.     

Antigen challenge modifies the cyclic AMP response of inflammatory mediators and beta-adrenergic drugs in alveolar macrophages

Adenylate cyclase activity was determined in alveolar macrophages (AMs) obtained from bronchoalveolar lavage (BAL) fluids of naive and antigen-challenged guinea pigs. After the anaphylactic reaction in ovalbumin-sensitized guinea pigs, the basal levels of cyclic AMP in AMs were significantly increased compared to the levels in naive AMS (1.87 +/- 0.22 versus 5.26 +/- 0.45 pmol cyclic AMP/5.10(6) cells). Prostaglandin E2 (PGE2), prostacyclin (DC-PGI2), histamine, isoprenaline and salbutamol stimulated adenylate cyclase activity more effectively in AMs obtained from sensitized guinea pigs after the booster injection compared to AMs obtained from non-treated animals. Moreover, DC-PGI2 and histamine, which were hardly able to induce a rise in cyclic AMP levels in naive AMs, become effective activators in AMs obtained after antigen challenge (100 and 60% increase in the response, respectively). Using selective receptor ligands, we have shown that beta 2-adrenoceptors and H2-subtype histamine receptors are functionally coupled to macrophage adenylate cyclase activity. The present data indicate that sensitization does not affect the configuration of the receptor on the outer membrane (no change in affinity constants), but affects other parts of the transmembrane signal system leading to the intracellular production of cyclic AMP (e.g. regulatory binding proteins or increases in the number of receptors).     

Rat alveolar and interstitial macrophages in the fibrosing stage following quartz exposure

Exposure to quartz induces pulmonary inflammation and development of fibrosis. In order to study the fibrosing process, we investigated morphology, function and phenotype of alveolar (AMs) and interstitial (IMs) macrophages at an early stage of fibrosis in rats. Rats were exposed by intratracheal instillations of 10 mg quartz (n=8) or saline (n=8) and studied 3 months later. AMs were obtained by bronchoalveolar lavage and IMs by mechanical fragmentation, followed by enzymatic digestion of lung tissue. Histology revealed subacute silicosis, with early focal fibrosis and alveolar lipoproteinosis. AM quartz exposure increased phagocytic activity and expression of major histocompatibility complex (MHC) Ia antigens, the latter being associated with cellular antigen presenting capacity. IM had an even more pronounced expression of MHC than AM after quartz exposure. Both macrophage fractions had a higher expression of OX-42 (complement receptor 3, CR3) than controls, but the increase in the IM fraction might be explained by the remaining AM in the IM fraction. Exposed AM adhered less to extracellular matrix components (vitronectin and fibronectin) than controls. In contrast, the adhesion of IM to vitronectin increased after exposure. Besides increased adhesion, the effects on IM were scarce. Our results therefore do not support the hypothesis that IM has a key role in the process of inflammation, including fibrosis.     

Changes in phospholipid biosynthetic enzymes in Type II cells and alveolar macrophages isolated from rat lungs after NO2 exposure

The activities of phospholipid biosynthesizing enzymes and subcellular marker enzymes were measured in type II cells and alveolar macrophages isolated from rat lungs 48 hr after exposure to air or 40 ppm NO₂. DNA and protein increased in cell fractions from NO₂-exposed lungs, but NO₂ exposure had no effect on the percentage of type II cells in isolated cell fractions. A general increase in cell content of biosynthetic enzyme activities (units/mg DNA) was observed in type II cells from NO₂-exposed rats, but no change was detected in the activity of the microsomal marker enzyme, NADPH cytochrome c reductase. Glycerolphosphate acyltransferase and choline phosphotransferase increased 171 and 168%, respectively, and phosphatidate phosphohydrolase increased 69%. Glycerolphosphate phosphatidyltranferase increased 143% and succinate cytochrome c reductase, the mitochondrial marker enzyme, increased 111%. Type II cells from control lungs contained a greater than threefold higher activity of the phosphatidylglycerol synthesizing enzyme, glycerolphosphate phosphatidyltransferase, compared to alveolar macrophages, and comparable activities of the enzymes of phosphatidylcholine synthesis and of the microsomal and mitochondrial reductases. Exposure to NO₂ resulted in a significant increase in NADPH cytochrome c reductase activity (46%) in macrophages, but no change in any biosynthetic enzymes. The increases in protein content and activity of phospholipid biosynthetic enzymes in type II cells are consistent with a general hypertrophy of type II cells which includes stimulation of surfactant phospholipid biosynthesis 2 days after exposure to NO₂, when type II cell proliferation is occurring.     

Xenobiotic-metabolizing enzyme activities in primary cultures of rat type II pneumocytes and alveolar macrophages.

Because of the evidence for the involvement of xenobiotic bioactivation in pulmonary toxicity and carcinogenesis, it is important to improve our understanding of the xenobiotic-metabolizing enzymes in isolated and cultured specific pulmonary cell populations. Some phase I and phase II xenobiotic-metabolizing enzyme activities, reduced glutathione (GSH), and gamma-glutamyl transferase (gamma-GT) were studied in rat type II pneumocytes and alveolar macrophages cultured for up to 48 h and 3 h, respectively. In type II pneumocytes, 7-ethoxyresorufin activity was not detected. 7-Benzyloxyresorufin (BROD) and 7-pentoxyresorufin (PROD) O-dealkylation decreased at 24 h by 84 and 82%, respectively, and continued to decline over the next 24 h with no measurable PROD at 48 h. The activity of NADPH- and NADH-cytochrome c reductase at 48 h decreased by 31 and 67%, respectively. GST activity decreased by 25 and 42% at 24 and 48 h, respectively. A transient increase in DT-diaphorase activity was observed at 24 h (by 55%). GSH content and gamma-GT activity increased significantly with time in culture. In freshly isolated alveolar macrophages, BROD activity was the only cytochrome P450-dependent alkoxyresorufin-O-dealkylase activity measured. BROD activity decreased by 38% in 3-h-attached macrophages. There were no changes in NADPH- and NADH-cytochrome c reductase, GST, and DT-diaphorase. An increase of GSH (by 24%) was observed in attached macrophages. In conclusion, type II pneumocytes and to a lesser extent alveolar macrophages in primary cultures undergo changes in biotransformation-related enzyme activities and intracellular GSH level that may affect xenobiotic toxicity at different times in culture.     

Surface morphology and morphometry of rat alveolar macrophages after ozone exposure

As the ultrastructural data on the effects of ozone on pulmonary alveolar macrophages (PAM) are lacking, transmission (TEM) and scanning (SEM) electron microscopy were performed on rat PAM present in alveolar lavages following exposure to ozone. Rats were continuously exposed for 7 d to ozone concentrations ranging from 0.25 to 1.50 mg/m3 for 7 d followed by a 5-d recovery period. Additionally, morphometry on lung sections was performed to quantitate PAM. In a second experiment rats were continuously exposed to 1.50 mg O3/m3 for 1, 3, 5, or 7 d. To study the influence of concurrent ozone exposure and lung infection, due to Listeria monocytogenes, rats were exposed for 7 d to 1.50 mg O3/m3 after a Listeria infection. The surface area of lavaged control PAM was uniformly covered with ruffles as shown by SEM and TEM. Exposure to 0.5 mg ozone/m3 for 7 d resulted in cells partly covered with microvilli and blebs in addition to normal ruffles. The number of large size PAM increased with an increase in ozone concentration. After 1 d of exposure, normal-appearing as well as many small macrophages with ruffles and scattered lymphocytes were seen. Lavage samples taken after 5 or 7 d of exposure showed an identical cell composition to that taken after 3 d of exposure. After Listeria infection alone, lavage samples consisted of mainly lymphocytes and some macrophages. Small quantitative changes, such as an increase in the number of polymorphonuclear neutrophils and large-size PAM, occurred in lavages after ozone exposure and infection with L. monocytogenes. Morphometric examination of lung sections revealed a concentration-related increase in the number of PAM, even in animals exposed to 0.25 mg ozone/m3 for 7 d. Centriacinar regions were more severely affected than other regions of lung tissue. By 5 d after termination of exposure to ozone, the number of lysozyme-positive alveolar cells was still significantly increased in centriacinar areas of the lung. The results indicate that ozone exposure causes major changes in the number, size, and surface morphology of PAM in rat lung. Furthermore, the results presented here suggest that changes in alveolar macrophage function are reflected by morphological changes.     

Calcium mobilization and response recovery following P2-purinoceptor stimulation of rat isolated alveolar type II cells.

1. The effect of adenosine 5'-triphosphate (ATP) on surfactant phospholipid secretion, calcium mobilization, and the time course for recovery of the response system was studied in isolated alveolar Type II cells of the rat. 2. ATP (10 microM) stimulated a biphasic intracellular Ca2+ transient monitored by changes in Fura-2 fluorescence, from a basal level of 126 +/- 9 nM, to a rapid peak of 391 +/- 1 nM, followed by a prolonged plateau 26 +/- 4 nM above baseline (mean +/- s.e.mean, n = 26). 3. ATP-stimulated surfactant phospholipid secretion and peak Ca2+ levels had similar EC50s (1 x 10(-6) M), and were unaffected by chelation of extracellular Ca2+. However, the prolonged plateau phase was abolished by chelation of extracellular Ca2+. 4. There was a 15 min refractory period before full recovery of the Ca2+-response to ATP. Recovery was dependent on extracellular Ca2+, was accelerated by removing extracellular agonist and was prolonged following stimulation with the poorly hydrolyzed ATP analogue, ATP-gamma-S. 5. While the Type II cell was capable of multiple ATP-induced Ca2+ transients following recovery, no additional surfactant phospholipid was released with sequential stimulation. 6. These findings suggest initial exposure of Type II cells to ATP mobilizes intracellular Ca2+, stimulates phospholipid secretion and rapidly desensitizes the cell to further stimulation by ATP. Recovery of the ATP-induced Ca2+-response depends on presence of extracellular Ca2+ and removal of agonist.     

Paracetamol (acetaminophen) cytotoxicity in rat type II pneumocytes and alveolar macrophages in vitro

Paracetamol (acetaminophen, APAP) liver and kidney cytotoxicity is associated with bioactivation by P450 and/or prostaglandin H synthetase (PGHS) to a reactive metabolite, which depletes GSH, covalently binds to proteins, and leads to oxidative stress. Although APAP may also damage the lung, little is known about the mechanism by which this occurs. We studied the in vitro 24-hr-old type II pneumocytes. A time- and concentration-dependent decrease in intracellular GSH occurred in freshly isolated type II pneumocytes and alveolar macrophages exposed to subtoxic (相似文献   

P2-purinoceptors regulate surfactant secretion from rat isolated alveolar type II cells.

Rat isolated alveolar Type II cells were utilized to examine the effect of purine and pyrimidine analogues on secretion of pulmonary surfactant. ATP potently stimulated [3H]-phosphatidylcholine ([3H]-PC) secretion in a time- and dose-dependent manner. The effect of ATP was noted by one hour of exposure and persisted for three hours. The EC50 (concentration producing 1/2 the maximal response) for ATP-induced [3H]-PC secretion was 100 nM. ADP was also a potent secretagogue for surfactant secretion, but AMP and adenosine had no significant effect on surfactant secretion at concentrations less than or equal to 250 microM. The EC50 for ADP-induced [3H]-PC secretion was 250 nM. Other purine and pyrimidine nucleotides (ITP, GTP, CTP, TTP) were examined for their effect on [3H]-PC secretion. All purine and pyrimidine triphosphates examined significantly augmented [3H]-PC secretion, but were much less potent than ATP. The EC50s were ITP = 10 microM; GTP = 100 microM; CTP = 250 microM; TTP = 100 microM. Neither 8-phenyltheophylline (10 microM, a P1-purinoceptor antagonist), propranolol (100 microM, a beta-adrenoceptor antagonist), nor indomethacin (10 microM, a prostaglandin synthetase inhibitor) inhibited ATP-induced [3H]-PC secretion from isolated Type II cells. These data provide evidence for regulation of surfactant secretion from alveolar Type II cells by a P2-purinoceptor.     

The effect of stone-wool on rat lungs and on the primary culture of rat alveolar macrophages and type II pneumocytes

The effect of stone-wool has been studied in both in vivo long term sequential and in vitro methods in male Sprague-Dawley rats. Stone-wool was administered by single intratracheal instillation and the lungs were examined after 1, 3 and 6 months of exposure by morphological methods. UICC crocidolite was applied as a positive control. In addition, the effects of both fibres were examined in primary cultures of alveolar macrophages (AM) and type II pneumocytes (T2) by morphological, biochemical and immunological methods. By the end of 6 months stone-wool induced moderate pulmonary interstitial inflammation and fibrosis without progression, whereas crocidolite induced progressive interstitial inflammation and fibrosis as a function of time. Although stone-wool inhibited phagocytosis, it did not induce serious membrane damage to the cells examined and did not destroy their ultrastructure. It significantly reduced the activity of Cu,Zn/superoxide dismutase (SOD) and alkaline phosphatase (AP) in alveolar macrophages and significantly decreased the activity of AP and gamma-glutamyl transpeptidase (GGT) in type II pneumocytes. Crocidolite, on the other hand, decreased the activity of all enzymes (glutathione peroxidase, GSH-Px; glutathione reductase, GSH-Rd) of glutathione metabolism as well as alkaline phosphatase in alveolar macrophages. It decreased the activity of all enzymes in type II pneumocytes, except for Cu,Zn/SOD. On exposure to stone-wool, the production of inflammatory proteins, macrophage chemoattractant protein-1 (MCP-1) and macrophage inhibitory protein-1alpha (MIP-1alpha) increased in both cultured cells but did not reach the level induced by crocidolite. Our results suggested that stone-wool is less toxic than crocidolite. Whether it is carcinogenic or not, is still an open question.     

In vitro cytotoxicity of various forms of cobalt for rat alveolar macrophages and type II pneumocytes

It has been shown that cobalt (Co) and the mixture cobalt-tungsten carbide (CoWC) are cytotoxic for alveolar macrophages (AM) and alveolar type II cells (AT-II), but the exact mechanisms of toxicity are not entirely elucidated. In this study, we exposed primary cultures of AT-II and AM, in vitro, to different forms of Co (Co particles, CoWC particles, CoCl(2)) and assessed cell damage using the dimethylthiazol diphenyl tetrazolium bromide test. In some experiments, inserts were used to separate the particles from the cells. The results show that AT-II are twice as sensitive to the effects of 100 microg Co particles/well (1.88 cm(2)) than AM. For this latter cell type, the presence of WC almost doubled (at 25 microg Co/well) the toxic effects compared to pure Co, but this synergy between Co and WC only occurred if the particles were in close contact with the cells. Lactalbumin and, to a lesser degree, EDTA were able to reduce the toxicity of Co, CoWC, and CoCl(2) for AT-II and AM. CoCl(2) showed a similar toxicity for AT-II and AM. The use of Co-conditioned medium revealed that Co particles are "aged" after having been incubated for 24 h in an aqueous medium and are then no longer able to cause the same degree of cell damage as fresh Co particles (71 versus 15% viability for 100 microg Co/well). The time course of the toxicity of the different forms of Co for AT-II and AM showed different patterns in causing cell damage, suggesting different action mechanisms. Evaluation of cell damage by electron microscopy was consistent with biochemical indices. Overall, our results indicate that the Co ion does play a role in the toxicity of both Co particles and CoWC particles.     

Characterization of the oxidant generation by inflammatory cells lavaged from rat lungs following acute exposure to ozone

Following exposure to 2 ppm ozone for 4 hr, two distinct effects on rat lung inflammatory cell oxidant generation were observed. TPA- and opsonized zymosan-stimulated superoxide production by the inflammatory cell population was found to be maximally inhibited 24 hr following ozone exposure. In contrast, luminol-amplified chemiluminescence increased 24 hr following ozone exposure, coinciding with an increase in the percentage of neutrophils and myeloperoxidase in the inflammatory cell population. Supporting the involvement of myeloperoxidase in the enhanced oxidant-generating status of these cells, the luminol-amplified chemiluminescence was found to be azide-, but not superoxide dismutase-inhibitable. Additionally, this cell population was found to generate taurine chloramines, a myeloperoxidase-dependent function which was absent prior to the ozone exposure and also demonstrated enhanced activation of benzo[a]pyrene-7,8-dihydrodiol to its light-emitting dioxetane intermediate. Addition of myeloperoxidase to control alveolar macrophages resulted in enhanced luminol-amplified chemiluminescence, taurine chloramine generation, and enhanced chemiluminescence from benzo[a]pyrene-7,8-dihydrodiol demonstrating that, in the presence of myeloperoxidase, alveolar macrophages are capable of supporting myeloperoxidase-dependent reactions. The possibility of such an interaction occurring in vivo is suggested by the detection of myeloperoxidase activity in the cell-free lavagates of ozone-exposed rats. These studies suggest that neutrophils recruited to ozone-exposed lungs alter the oxidant-generating capabilities in the lung which could further contribute to lung injury or to the metabolism of inhaled xenobiotics.