Interactive effects of cerium oxide and diesel exhaust nanoparticles on inducing pulmonary fibrosis.

Cerium compounds have been used as a fuel-borne catalyst to lower the generation of diesel exhaust particles (DEPs), but are emitted as cerium oxide nanoparticles (CeO2) along with DEP in the diesel exhaust. The present study investigates the effects of the combined exposure to DEP and CeO2 on the pulmonary system in a rat model. Specific pathogen-free male Sprague-Dawley rats were exposed to CeO2 and/or DEP via a single intratracheal instillation and were sacrificed at various time points post-exposure. This investigation demonstrated that CeO2 induces a sustained inflammatory response, whereas DEP elicits a switch of the pulmonary immune response from Th1 to Th2. Both CeO2 and DEP activated AM and lymphocyte secretion of the proinflammatory cytokines IL-12 and IFN-γ, respectively. However, only DEP enhanced the anti-inflammatory cytokine IL-10 production in response to ex vivo LPS or Concanavalin A challenge that was not affected by the presence of CeO2, suggesting that DEP suppresses host defense capability by inducing the Th2 immunity. The micrographs of lymph nodes show that the particle clumps in DEP+CeO2 were significantly larger than CeO2 or DEP, exhibiting dense clumps continuous throughout the lymph nodes. Morphometric analysis demonstrates that the localization of collagen in the lung tissue after DEP+CeO2 reflects the combination of DEP-exposure plus CeO2-exposure. At 4 weeks post-exposure, the histological features demonstrated that CeO2 induced lung phospholipidosis and fibrosis. DEP induced lung granulomas that were not significantly affected by the presence of CeO2 in the combined exposure. Using CeO2 as diesel fuel catalyst may cause health concerns.

Crystalline silica is a negative modifier of pulmonary cytochrome P-4501A1 induction.

Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion that are commonly inhaled by workers in the dusty trades. Many PAHs are metabolized by cytochrome P-4501A1 (CYP1A1), which may facilitate excretion but may activate pulmonary carcinogens. PAHs also stimulate their own metabolism by inducing CYP1A1. Recent studies suggest that respirable coal dust exposure inhibits induction of pulmonary CYP1A1 using the model PAH beta-naphthoflavone. The effect of the occupational particulate respirable crystalline silica was investigated on PAH-dependent pulmonary CYP1A1 induction. Male Sprague-Dawley rats were exposed to intratracheal silica or vehicle and then intraperitoneal beta-naphthoflavone, a CYP1A1 inducer, and/or phenobarbital, an inducer of hepatic CYP2B1, or vehicle. Beta-naphthoflavone induced pulmonary CYP1A1, but silica attenuated this beta-naphthoflavone-induced CYP1A1 activity and also suppressed the activity of CYP2B1, the major constitutive CYP in rat lung. The magnitude of CYP activity suppression was similar regardless of silica exposure dose within a range of 5 to 20 mg/rat. Phenobarbital and beta-naphthoflavone had no effect on pulmonary CYP2B1 activity. Both enzymatic immunohistochemistry and immunofluorescent staining for CYP1A1 indicated that sites of CYP1A1 induction were nonciliated airway epithelial cells, endothelial cells, and the alveolar septum. Using immunofluorescent colocalization of CYP1A1 with cytokeratin 8, a marker of alveolar type II cells, the proximal alveolar region was the site of both increased alveolar type II cells and decreased proportional CYP1A1 expression in alveolar type II cells. Our findings suggest that in PAH-exposed rat lung, silica is a negative modifier of CYP1A1 induction and CYP2B1 activity.

Suppression of phagocytic and bactericidal functions of rat alveolar macrophages by the organic component of diesel exhaust particles.

Exposure to diesel exhaust particles (DEP) was shown to increase the susceptibility of the lung to bacterial infection in rats. In this study, the effects of DEP on alveolar macrophage (AM) phagocytic and bactericidal functions and cytokine secretion by AM and lymphocytes in response to Listeria monocytogenes infection were investigated in vitro and the roles of different DEP components in these processes were compared. Exposure to DEP or the organic extracts of DEP (eDEP) significantly decreased the phagocytosis and killing of L. monocytogenes by AM obtained from normal rats. Washed DEP (wDEP) also decreased AM phagocytosis and bacterial killing to a lesser extent, whereas carbon black (CB) reduced AM phagocytosis but had no significant effect on AM bactericidal activity. DEP or eDEP concentration-dependently suppressed L. monocytogenes-induced secretion of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-12 by AM and of IL-2 and interferon-gamma by lymphocytes obtained from L. monocytogenes-infected rats, but augmented the AM secretion of IL-10. wDEP or CB, however, exerted little or no effect on these L. monocytogenes-induced cytokines. These results provide direct evidence that DEP, through the actions of organic components, suppresses AM phagocytic and bactericidal functions in vitro. Inhibition of AM phagocytic function and alterations of AM and lymphocyte cytokine secretion by DEP and DEP organic compounds may be implicated in the diminished AM bactericidal activity and the lymphatic arm of the host immune system, thus resulting in an suppressed pulmonary clearance of L. monocytogenes and an increased susceptibility of the lung to bacterial infection.

Apoptosis and Bax expression are increased by coal dust in the polycyclic aromatic hydrocarbon-exposed lung.

BACKGROUND: Miners inhaling respirable coal dust (CD) frequently develop coal workers' pneumoconiosis, a dust-associated pneumoconiosis characterized by lung inflammation and variable fibrosis. Many coal miners are also exposed to polycyclic aromatic hydrocarbon (PAH) components of diesel engine exhaust and cigarette smoke, which may contribute to lung disease in these workers. Recently, apoptosis was reported to play a critical role in the development of another pneumoconiosis of miners, silicosis. In addition, CD was reported to suppress cytochrome P450 1A1 (CYP1A1) induction by PAHs. METHODS: We investigated the hypothesis that apoptosis plays a critical role in lung injury and down-regulation of CYP1A1 induction in mixed exposures to CD and PAHs. We exposed rats intratracheally to 0.0, 2.5, 10.0, 20.0, or 40.0 mg/rat CD and, 11 days later, to intraperitoneal beta-naphthoflavone (BNF) , a PAH. In another group of rats exposed to CD and BNF, caspase activity was inhibited by injection of the pan-caspase inhibitor Q-VD-OPH [quinoline-Val-Asp (OMe) -CH2-OPH]. RESULTS: In rats exposed to BNF, CD exposure increased alveolar expression of the proapoptotic mediator Bax but decreased CYP1A1 induction relative to BNF exposure alone. Pan-caspase inhibition decreased CD-associated Bax expression and apoptosis but did not restore CYP1A1 activity. Further, CD-induced lung inflammation and alveolar epithelial cell hypertrophy and hyperplasia were not suppressed by caspase inhibition. CONCLUSIONS: Combined BNF and CD exposure increased Bax expression and apoptosis in the lung, but Bax and apoptosis were not the major determinants of early lung injury in this model.

Cooperation of the inducible nitric oxide synthase and cytochrome P450 1A1 in mediating lung inflammation and mutagenicity induced by diesel exhaust particles.

Diesel exhaust particles (DEPs) have been shown to activate oxidant generation by alveolar macrophages (AMs), alter xenobiotic metabolic pathways, and modify the balance of pro-antiinflammatory cytokines. In this study we investigated the role of nitric oxide (NO) in DEP-mediated and DEP organic extract (DEPE) -mediated inflammatory responses and evaluated the interaction of inducible NO synthase (iNOS) and cytochrome P450 1A1 (CYP1A1). Male Sprague-Dawley rats were intratracheally (IT) instilled with saline, DEPs (35 mg/kg), or DEPEs (equivalent to 35 mg DEP/kg), with or without further treatment with an iNOS inhibitor, aminoguanidine (AG; 100 mg/kg), by intraperitoneal injection 30 min before and 3, 6, and 9 hr after IT exposure. At 1 day postexposure, both DEPs and DEPEs induced iNOS expression and NO production by AMs. AG significantly lowered DEP- and DEPE-induced iNOS activity but not the protein level while attenuating DEPE- but not DEP-mediated pulmonary inflammation, airway damage, and oxidant generation by AMs. DEP or DEPE exposure resulted in elevated secretion of both interleukin (IL) -12 and IL-10 by AMs. AG significantly reduced DEP- and DEPE-activated AMs in IL-12 production. In comparison, AG inhibited IL-10 production by DEPE-exposed AMs but markedly increased its production by DEP-exposed AMs, suggesting that NO differentially regulates the pro- and antiinflammatory cytokine balance in the lung. Both DEPs and DEPEs induced CYP1A1 expression. AG strongly inhibited CYP1A1 activity and lung S9 activity-dependent 2-aminoanthracene mutagenicity. These studies show that NO plays a major role in DEPE-induced lung inflammation and CYP-dependent mutagen activation but a lesser role in particulate-induced inflammatory damage.

Exposure of brown Norway rats to diesel exhaust particles prior to ovalbumin (OVA) sensitization elicits IgE adjuvant activity but attenuates OVA-induced airway inflammation.

Exposure to diesel exhaust particles (DEP) during the sensitization process has been shown to increase antigen-specific IgE production and aggravate allergic airway inflammation in human and animal models. In this study, we evaluated the effect of short-term DEP exposure on ovalbumin (OVA)-mediated responses using a post-sensitization model. Brown Norway rats were first exposed to filtered air or DEP (20.6 +/- 2.7 mg/m3) for 4 h/day for five consecutive days. One day after the final air or DEP exposure (day 1), rats were sensitized with aerosolized OVA (40.5 +/- 6.3 mg/m3), and then again on days 8 and 15, challenged with OVA on day 29, and sacrificed on days 9 or 30, 24 h after the second OVA exposure or the final OVA challenge, respectively. Control animals received aerosolized saline instead of OVA. DEP were shown to elicit an adjuvant effect on the production of antigen-specific IgE and IgG on day 30. At both time points, no significant airway inflammatory responses and lung injury were found for DEP exposure alone. However, the OVA-induced inflammatory cell infiltration, acellular lactate dehydrogenase activity and albumin content in bronchoalveolar lavage (BAL) fluid, and numbers of T cells and their CD4+ and CD8+ subsets in lung-draining lymph nodes were markedly reduced by DEP on day 30 compared with the air-plus-OVA exposure group. The OVA-induced nitric oxide (NO) in the BAL fluid and production of NO, interleukin (IL)-10, and IL-12 by alveolar macrophages (AM) were also significantly lowered by DEP on day 30 as well as day 9. DEP or OVA alone decreased intracellular glutathione (GSH) in AM and lymphocytes on days 9 and 30. The combined DEP and OVA exposure resulted in further depletion of GSH in both cell types. These results show that short-term DEP exposure prior to sensitization had a delayed effect on enhancement of the sensitization in terms of allergen-specific IgE and IgG production, but caused an attenuation of the allergen-induced airway inflammatory responses.

Effect of diesel exhaust particles on allergic reactions and airway responsiveness in ovalbumin-sensitized brown Norway rats.

We have previously demonstrated that exposure to diesel exhaust particles (DEP) prior to ovalbumin (OVA) sensitization in rats reduced OVA-induced airway inflammation. In the present study, Brown Norway rats were first sensitized to OVA (42.3 +/- 5.7 mg/m3) for 30 min on days 1, 8, and 15, then exposed to filtered air or DEP (22.7 +/- 2.5 mg/m3) for 4 h/day on days 24-28, and challenged with OVA on day 29. Airway responsiveness was examined on day 30, and animals were sacrificed on day 31. Ovalbumin sensitization and challenge resulted in a significant infiltration of neutrophils, lymphocytes, and eosinophils into the lung, elevated presence of CD4+ and CD8+ T lymphocytes in lung draining lymph nodes, and increased production of serum OVA-specific immunoglobulin (Ig)E and IgG. Diesel exhaust particles pre-exposure augmented OVA-induced production of allergen-specific IgE and IgG and pulmonary inflammation characterized by marked increases in T lymphocytes and infiltration of eosinophils after OVA challenge, whereas DEP alone did not have these effects. Although OVA-sensitized rats showed modest response to methacholine challenge, it was the combined DEP and OVA exposure that produced significant airway hyperresponsiveness in this animal model. The effect of DEP pre-exposure on OVA-induced immune responses correlated with an interactive effect of DEP with OVA on increased production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM) and alveolar type II (ATII) cells, NO levels in bronchoalveolar lavage fluid, the induction of inducible NO synthase expression in AM and ATII cells, and a depletion of total intracellular glutathione (GSH) in AM and lymphocytes. These results show that DEP pre-exposure exacerbates the allergic responses to the subsequent challenge with OVA in OVA-sensitized rats. This DEP effect may be, at least partially, attributed to the elevated generation of ROS in AM and ATII cells, a depletion of GSH in AM and lymphocytes, and an increase in AM and ATII cell production of NO.

Sustained effect of inhaled diesel exhaust particles on T-lymphocyte-mediated immune responses against Listeria monocytogenes.

Studies have shown that exposure to diesel exhaust particles (DEP) suppresses pulmonary host defense against bacterial infection. The present study was carried out to characterize whether DEP exposure exerts a sustained effect in which inhaled DEP increase the susceptibility of the lung to bacterial infection occurring at a later time. Brown Norway rats were exposed to filtered air or DEP by inhalation at a dose of 21.2 +/- 2.3 mg/m3, 4 h/day for 5 days, and intratracheally instilled with saline or 100,000 Listeria monocytogenes (Listeria) 7 days after the final DEP exposure. Bacterial growth and cellular responses to DEP and Listeria exposures were examined at 3 and 7 days post-infection. The results showed that inhaled DEP prolonged the growth of bacteria, administered 7 days post DEP exposure, in the lung as compared to the air-exposed controls. Pulmonary responses to Listeria infection were characterized by increased production of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12, and IL-10 by alveolar macrophages (AM) and increased presence of T lymphocytes and their CD4+ and CD8+ subsets in lung draining lymph nodes that secreted elevated levels of IL-2, IL-6, IL-10, and interferon (IFN)-gamma. Diesel exhaust particles were found to inhibit Listeria-induced production of IL-1beta and TNF-alpha, which are responsible for the innate immunity, and IL-12, which initiates the development of T helper (Th)1 responses, but enhance Listeria-induced AM production of IL-10, which prolongs Listeria survival in these phagocytes. The dual action of DEP on AM production of IL-12 and IL-10 correlated with an inhibition of the development of bacteria-specific T lymphocytes by DEP. Cytokine production by lymphocytes from DEP- and Listeria-exposed rats showed a marked decrease in the production of IL-2, IL-10, and IFN-gamma compared to Listeria infection alone, suggesting either that DEP inhibit the production of cytokines by lymphocytes or that these lymphocytes contained T-cell subsets that are different from those of Listeria infection alone and less effective in mediating Th1 immune responses. This study demonstrates that inhaled DEP, after a 7-day resting period, increase the susceptibility of the lung to bacterial infection occurring at a later time by inhibiting macrophage immune function and suppressing the development of T-cell-mediated immune responses. The results support the epidemiological observations that exposure to DEP may be responsible for the pulmonary health effects on humans.

Time course of gene expression of inflammatory mediators in rat lung after diesel exhaust particle exposure.

Diesel exhaust particles (DEPs) at three concentrations (5, 35, and 50 mg/kg body weight) were instilled into rats intratracheally. We studied gene expression at 1, 7, and 30 days postexposure in cells obtained by bronchoalveolar lavage (BAL) and in lung tissue. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we measured the mRNA levels of eight genes [interleukin (IL)-1beta, IL-6, IL-10, iNOS (inducible nitric oxide synthase), MCP-1 (monocyte chemoattractant protein-1), MIP-2 (macrophage inflammatory protein-2), TGF-beta1 (transforming growth factor-beta1), and TNF-alpha (tumor necrosis factor-alpha )] in BAL cells and four genes [IL-6, ICAM-1 (intercellular adhesion molecule-1), GM-CSF (granulocyte/macrophage-colony stimulating factor), and RANTES (regulated upon activation normal T cell expressed and secreted)] in lung tissue. In BAL cells on day 1, high-dose exposure induced a significant up-regulation of IL-1beta, iNOS, MCP-1, and MIP-2 but no change in IL-6, IL-10, TGF-beta1, and TNF-alpha mRNA levels. There was no change in the mRNA levels of IL-6, RANTES, ICAM-1, and GM-CSF in lung tissue. Nitric oxide production and levels of MCP-1 and MIP-2 were increased in the 24-hr culture media of alveolar macrophages (AMs) obtained on day 1. IL-6, MCP-1, and MIP-2 levels were also elevated in the BAL fluid. BAL fluid also showed increases in albumin and lactate dehydrogenase. The cellular content in BAL fluid increased at all doses and at all time periods, mainly due to an increase in polymorphonuclear leukocytes. In vitro studies in AMs and cultured lung fibroblasts showed that lung fibroblasts are a significant source of IL-6 and MCP-1 in the lung.

Roles of reactive oxygen species and heme oxygenase-1 in modulation of alveolar macrophage-mediated pulmonary immune responses to Listeria monocytogenes by diesel exhaust particles.

Diesel exhaust particles (DEP) have been shown to suppress alveolar macrophage (AM)-mediated pulmonary immune responses to Listeria monocytogenes in vivo. In this study, effects of DEP-derived reactive oxygen species (ROS) and heme oxygenase (HO)-1 on AM-mediated immune responses to L. monocytogenes were investigated. Brown Norway rats were intratracheally inoculated with 100,000 L. monocytogenes, and AM were isolated at 7 days post-infection. Exposure to DEP or their organic extract (eDEP), but not the washed DEP (wDEP) or carbon black, increased intracellular ROS and HO-1 expression in AM. Induction of ROS and HO-1 by eDEP was partially reversed by alpha-naphthoflavone, a cytochrome P450 1A1 inhibitor, and totally blocked by N-acetylcysteine. In addition, exposure to eDEP, but not wDEP, inhibited lipopolysacchride-stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-12 (IL-12), but augmented production of IL-10 by AM. Kinetic studies showed that modulation of cytokines by eDEP was preceded by ROS and HO-1 induction. Furthermore, pretreatment of AM with superoxide dismutase (SOD) or zinc protoporphrin IX (Znpp), which attenuated eDEP-induced HO-1 expression/activity, substantially inhibited eDEP effect on IL-10. Finally, direct stimulation with pyrogallol (PYR), a superoxide donor, upregulated HO-1 and IL-10 but decreased secretion of IL-12 in L. monocytogenes-infected AM. These results show that DEP, through eDEP-mediated ROS, induce HO-1 expression and IL-10 production and at the same time inhibit AM production of TNF-alpha and IL-12 to dampen the host immune responses. The results also suggest that HO-1 may play an important role in regulating production of IL-10 by DEP-exposed and L. monocytogenes-infected AM.

Effect of diesel exhaust particulate (DEP) on immune responses: contributions of particulate versus organic soluble components.

The effect of diesel exhaust particulate (DEP) exposure on innate, cellular and humoral pulmonary immunity was studied using high-dose, acute-exposure rat, mouse, and cell culture models. DEP consists of a complex mixture of petrochemical-derived organics adsorbed onto elemental carbon particles. DEP is a major component of particulate urban air pollution and a health concern in both urban and occupational environments. The alveolar macrophage is considered a key cellular component in pulmonary innate immunity. DEP and DEP organic extracts have been found to suppress alveolar macrophage function as demonstrated by reduced production of cytokines (interleukin-1 [IL-1], tumor necrosis factor- alpha [TNF- alpha]) and reactive oxygen species (ROS) in response to a variety of agents, including lipopolysaccharide (LPS), interferon- gamma (IFN- gamma), and bacteria. Fractionation of DEP organic extract suggests that this activity was predominately in polyaromatic-containing and more polar (resin) fractions. Organic-stripped DEP did not alter these innate pulmonary immune responses. DEP also depressed pulmonary clearance of Listeria monocytogenes and Bacillus Calmette-Guerin (BCG). The contribution of the organic component of DEP is less well defined with respect to acquired and humoral immunity. Indeed, both DEP and carbon black enhanced humoral immune responses (specific immunoglobulin [Ig] E and IgG) in an ovalbumin-sensitized rat model. It is concluded that both the particulate and adsorbed organics may contribute to DEP-mediated immune alterations.

Suppression of cell-mediated immune responses to listeria infection by repeated exposure to diesel exhaust particles in brown Norway rats.

Diesel exhaust particles (DEP) have been shown to alter pulmonary immune responses to bacterial infection. Exposure of rats to 100 mg/m(3) DEP for 4 h was found to aggravate Listeria monocytogenes(Listeria) infection at 3 days postinfection, but the bacteria were largely cleared at 7 days postinfection due to the development of a strong T cell-mediated immunity. In the present study, we examined the effects of repeated DEP exposure at lower doses on pulmonary responses to bacterial infection. Brown Norway rats were exposed to DEP by inhalation at 20.62 +/- 1.31 mg/m 3 for 4 h/day for 5 days, followed by intratracheal inoculation with 100,000 Listeria at 2 h after the last DEP exposure. DEP-exposed rats showed a significant increase in lung bacterial load at both 3 and 7 days postinfection. The repeated DEP exposure was shown to suppress both the innate, orchestrated by alveolar macrophages (AM), and T cell-mediated responses to Listeria. DEP inhibited AM production of interleukin- (IL-) 1beta, tumor necrosis factor- (TNF-) alpha, and IL-12 but enhanced Listeria-induced AM production of IL-10, which has been shown to prolong the survival of intracellular pathogens such as Listeria. DEP exposure also suppressed the development of bacteria-specific lymphocytes from lung-draining lymph nodes, as indicated by the decreased numbers of T lymphocytes and their CD4(+) and CD8(+) subsets. Furthermore, the DEP exposure markedly inhibited the Listeria-induced lymphocyte secretion of IL-2 at day 7, IL-10 at days 3 and 7, and interferon- (IFN-) gamma at days 3 to 10 postinfection when compared to air-exposed controls. These results show a sustained pattern of downregulation of T cell-mediated immune responses by repeated low-dose DEP exposure, which is different from the results of a single high-dose exposure where the acute effect of DEP aggravated bacteria infection but triggered a strong T cell-mediated immunity.

Effect of asphalt fume inhalation exposure at simulated road paving conditions prior to bacterial infection on lung defense responses in rats.

Asphalt fume inhalation has been suspected of affecting immune function in exposed workers. The objective of this study was to evaluate the effect of asphalt exposure on lung immune responses in rats using a bacterial infectivity model. Pathogen-free male Sprague-Dawley rats were exposed by inhalation to asphalt fumes (72.6 +/- 4.95 mg/m3) or filtered air for 6 h/day for 5 days. One day after the final asphalt exposure, rats were intratracheally inoculated with 5 x 10(5) Listeria monocytogenes. At 0 (prior to bacterial inoculation), 3, and 7 days after L. monocytogenes instillation, the lungs of each animal were divided. Bronchoalveolar lavage (BAL) was performed on right lungs. The recovered BAL cells were then differentiated and counted, and alveolar macrophage (AM) function was determined. Albumin and lactate dehydrogenase (LDH), two indices of lung injury, were measured in the acellular BAL fluid. To assess bacterial clearance, the left lungs were removed, homogenized, and bacterial colony-forming units (CFUs) were counted. In addition, lung-draining lymph nodes were removed, and lymphocyte phenotype and lymphocyte-induced cytokine production were examined. Asphalt fume exposure did not cause lung injury or inflammation in rats in the absence of infection. Infection induced elevations in AMs, neutrophils (PMNs), albumin, and LDH. Importantly, no significant differences were seen when comparing the asphalt group with the air and nonexposed naive groups at any time before or after infection. Also, asphalt fume inhalation exposure did not affect the rate of pulmonary clearance of L. monocytogenes or AM production of reactive oxygen and nitrogen species. However, asphalt-related increases in lymphocyte secretion of interferon (IFN)-gamma, interleukin (IL)-6, and IL-10 were observed at different times after bacterial infection, whereas the total number of lymph-node cells and the percentage of CD4+ and CD8+ cells were not significantly different among the treatment groups. Despite the asphalt-induced changes observed in lymphokine secretion, adaptive immune function seemed to function properly in lung defense against bacterial infection. Because innate nonspecific lung responses and pulmonary clearance of L. monocytogenes were unaffected by asphalt fume exposure, lung defenses were sufficient to control the infection. It was concluded that acute inhalation of asphalt fumes at a high concentration had a minimal effect on lung immune responses to infection in rats.

Inhalation exposure of rats to asphalt fumes generated at paving temperatures alters pulmonary xenobiotic metabolism pathways without lung injury.

Asphalt fumes are complex mixtures of various organic compounds, including polycyclic aromatic hydrocarbons (PAHs). PAHs require bioactivation by the cytochrome P-450 monooxygenase system to exert toxic/carcinogenic effects. The present study was carried out to characterize the acute pulmonary inflammatory responses and the alterations of pulmonary xenobiotic pathways in rats exposed to asphalt fumes by inhalation. Rats were exposed at various doses and time periods to air or to asphalt fumes generated at paving temperatures. To assess the acute damage and inflammatory responses, differential cell counts, acellular lactate dehydrogenase (LDH) activity, and protein content of bronchoalveolar lavage fluid were determined. Alveolar macrophage (AM) function was assessed by monitoring generation of chemiluminescence and production of tumor necrosis factor-alpha and interleukin-1. Alteration of pulmonary xenobiotic pathways was determined by monitoring the protein levels and activities of P-450 isozymes (CYP1A1 and CYP2B1), glutathioneS-transferase (GST), and NADPH:quinone oxidoreductase (QR). The results show that acute asphalt fume exposure did not cause neutrophil infiltration, alter LDH activity or protein content, or affect AM function, suggesting that short-term asphalt fume exposure did not induce acute lung damage or inflammation. However, acute asphalt fume exposure significantly increased the activity and protein level of CYP1A1 whereas it markedly reduced the activity and protein level of CYP2B1 in the lung. The induction of CYP1A1 was localized in nonciliated bronchiolar epithelial (Clara) cells, alveolar septa, and endothelial cells by immunofluorescence microscopy. Cytosolic QR activity was significantly elevated after asphalt fume exposure, whereas GST activity was not affected by the exposure. This induction of CYP1A1 and QR with the concomitant down-regulation of CYP2B1 after asphalt fume exposure could alter PAH metabolism and may lead to potential toxic effects in the lung.

Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs). II. Effects of DEPs on T-cell-mediated immune responses in rats.

Previously, we showed that diesel exhaust particles (DEPs) suppressed pulmonary clearance of Listeria monocytogenes (Listeria) and inhibited the phagocytosis of alveolar macrophages and their response to Listeria in the secretion of interleukin (IL)-1 beta, tumor necrosis factor alpha, and IL-12. In this report we examined the effects of DEPs and/or Listeria on T-cell development and secretion of IL-2, IL-6, and interferon (IFN)-gamma. We exposed Brown Norway rats to clean air or DEPs at 50 or 100 mg/m3 for 4 hr by nose-only inhalation and inoculated with 100,000 Listeria. Lymphocytes in the lung-draining lymph nodes were isolated at 3 and 7 days postexposure, analyzed for CD4+ and CD8+ cells, and measured for cytokine production in response to concanavalin A or heat-killed L. monocytogenes. Listeria infection induced lymphocyte production of IL-6. At 7 days postinfection, lymphocytes from Listeria-infected rats showed significant increases in CD4+ and CD8+ cell counts and the CD8+/CD4+ ratio and exhibited increased production of IFN-gamma and IL-2 receptor expression compared with the noninfected control. These results suggest an immune response that involves the action of IL-6 on T-cell activation, yielding Listeria-specific CD8+ cells. DEP exposure alone enhanced lymphocyte production of both IL-2 and IL-6 but inhibited lymphocyte secretion of IFN-gamma. In rats exposed to 100 mg/m3 DEPs and Listeria, a 10-fold increase occurred in pulmonary bacterial count at 3 days postinfection when compared with the Listeria-only exposure group. The isolated lymphocytes showed a significant increase in the CD4+ and CD8+ cell counts and the CD8+/CD4+ ratio and exhibited increased IL-2 responsiveness and increased capacity in the secretion of IL-2, IL-6, and IFN-gamma. This T-cell immune response was sufficient to allow the Brown Norway rats to clear the bacteria at 7 days postinfection and overcome the down-regulation of the innate immunity by the acute DEP exposure.

Alteration of pulmonary immunity to Listeria monocytogenes by diesel exhaust particles (DEPs). I. Effects of DEPs on early pulmonary responses.

It has been hypothesized that diesel exhaust particles (DEPs) aggravate pulmonary bacterial infection by both innate and cell-mediated immune mechanisms. To test this hypothesis, we investigated the effects of DEP exposure on the functions of alveolar macrophages (AMs) and lymphocytes from lung-draining lymph nodes using a rat Listeria monocytogenes infection model. In the present study, we focused on the effects of DEP exposure on AM functions, including phagocytic activity and secretion of proinflammatory cytokines. The Listeria infection model was characterized by an increase in neutrophil count, albumin content, and acellular lactate dehydrogenase activity in the bronchoalveolar lavage (BAL) fluid at 3 and 7 days postinfection. Short-term DEP inhalation (50 and 100 mg/m(3), 4 hr) resulted in a dose-dependent suppression of lung clearance of Listeria, with the highest bacteria count occurring at day 3. This aggravated bacterial infection was consistent with the inhibitory effect of DEPs on macrophage functions. DEPs suppressed phagocytosis and Listeria-induced basal secretion of interleukin-1ss (IL-1ss) and IL-12 by AMs in a dose-dependent manner. The amount of IL-1ss and IL-12 in the BAL fluid was also reduced by DEP exposure. In addition, DEPs decreased Listeria-induced lipopolysaccharide-stimulated secretion of tumor necrosis factor-alpha (TNF-alpha), IL-1ss, and IL-12 from AMs. These results suggest that DEPs retard bacterial clearance by inhibiting AM phagocytosis and weaken the innate immunity by inhibiting AM secretion of IL-1ss and TNF-alpha. DEPs may also suppress cell-mediated immunity by inhibiting AM secretion of IL-12, a key cytokine for the initiation of T helper type 1 cell development in Listeria infection.

Residual oil fly ash increases the susceptibility to infection and severely damages the lungs after pulmonary challenge with a bacterial pathogen.

Inhalation of residual oil fly ash (ROFA), a component of ambient particulate matter, has been shown to increase pulmonary morbidity and impair lung defense mechanisms in exposed workers. Our objective was to evaluate the effect of ROFA preexposure on lung defense and injury after pulmonary challenge with a bacterial pathogen. Male Sprague-Dawley rats were dosed intratracheally at day 0 with saline (control) or ROFA (0.2 or 1 mg/100 g body weight). Three days later, a low (5 x 10(3)) or high (5 x 10(5)) dose of Listeria monocytogenes was instilled intratracheally into the ROFA- and saline-treated rats. Bronchoalveolar lavage was performed on the right lungs at days 6, 8, and 10. The recovered cells were differentiated, and chemiluminescence (CL) and nitric oxide (NO) production, two indices of alveolar macrophage (AM) function, were measured. At the same time points, the left lung and spleen were removed, homogenized, and cultured, and colony-forming units were counted after an overnight incubation. Exposure to ROFA and the high dose of L. monocytogenes led to marked lung injury and inflammation as well as to an increase in mortality, compared with rats treated with saline and the high dose of L. monocytogenes. Preexposure to ROFA significantly enhanced injury and delayed the pulmonary clearance of L. monocytogenes at both bacterial doses when compared to the saline-treated control rats. ROFA had no effect on AM CL but caused a significant suppression of AM NO production, as compared to the saline control rats. We have demonstrated that acute exposure to ROFA slowed the pulmonary clearance of L. monocytogenes. The suppression in AM NO production by ROFA pretreatment likely plays an important role. These results suggest that pulmonary exposure to ROFA may alter AM function and lead to increased susceptibility to lung infection in exposed populations.

Effect of inhaled crystalline silica in a rat model: time course of pulmonary reactions.

Numerous investigations have been conducted to elucidate mechanisms involved in the initiation and progression of silicosis. However, most of these studies involved bolus exposure of rats to silica, i.e. intratracheal instillation or a short duration inhalation exposure to a high dose of silica. Therefore, the question of pulmonary overload has been an issue in these studies. The objective of the current investigation was to monitor the time course of pulmonary reactions of rats exposed by inhalation to a non-overload level of crystalline silica. To accomplish this, rats were exposed to 15 mg/m3 silica, 6 h/day, 5 days/week for up to 116 days of exposure. At various times (5-116 days exposure), animals were sacrificed and silica lung burden, lung damage, inflammation, NF-KB activation, reactive oxygen species and nitric oxide production, cytokine production, alveolar type II epithelial cell activity, and fibrosis were monitored. Activation of NF-KB/DNA binding in BAL cells was evident after 5 days of silica inhalation and increased linearly with continued exposure. Parameters of pulmonary damage, inflammation and alveolar type II epithelial cell activity rapidly increased to a significantly elevated but stable new level through the first 41 days of exposure and increased at a steep rate thereafter. Pulmonary fibrosis was measurable only after this explosive rise in lung damage and inflammation, as was the steep increase in TNF-alpha and IL-1 production from BAL cells and the dramatic rise in lavageable alveolar macrophages. Indicators of oxidant stress and pulmonary production of nitric oxide exhibited a time course which was similar to that for lung damage and inflammation with the steep rise correlating with initiation of pulmonary fibrosis. Staining for iNOS and nitrotyrosine was localized in granulomatous regions of the lung and bronchial associated lymphoid tissue. Therefore, these data demonstrate that the generation of oxidants and nitric oxide, in particular, is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and fibrosis. This suggests an important role for nitric oxide in the initiation of silicosis.

Transport and utilization of arginine and arginine-containing peptides by rat alveolar macrophages.

PURPOSE: To demonstrate that rat alveolar macrophages (AM) exhibited the PepT1-like transporter for the uptake of arginine (Arg)-containing small peptides and utilized these peptides as direct substrates for nitric oxide (NO) production. NO is an important mediator that, on one hand, protects the lung from bacteria infection and, on the other hand, augments inflammatory lung injury. METHOD: The uptake of small peptides by rat AM was evaluated using fluorescein isothiocyanate (FITC)-labeled (*) peptides (Arg-Lys*, Gly-Sar-Lys*, and beta-Ala-Lys*), high-performance liquid chromatography (HPLC) analysis of potential peptide degradation, and known inhibitors of Arg and PepT1 transport. NO production by AM through Arg and Arg-containing peptides was studied with and without inhibition by transport inhibitors. The presence of PepT1-like transporter on AM was evaluated using anti-PepT1 antisera and Western blot analysis. The substrate specificity of Arg-Gly and Arg-Gly-Asp was determined using purified inducible NO synthase (iNOS). The availability of Arg-containing peptides in the lung was determined by HPLC analysis of bronchoalveolar lavage (BAL) fluid. RESULTS: The FITC-labeled peptides were internalized by AM without degradation. The uptake of Arg-Lys*, beta-Ala-Lys*, and Gly-Sar-Lys* was blocked (approximately 50%) by cephradine (an inhibitor of PepT1 for peptide transport) but not by Lys (an inhibitor on cationic amino acid transporter 2B for Arg transport). The NO production by AM through Arg-containing peptides was significantly blocked only by PepT1 inhibitors and by an anti-PepT1 antibody in a dose-dependent manner. These inhibitors had no effect on the AM production of NO using Arg as a substrate. Arg-Gly and Arg-Gly-Asp were found to be direct substrates for iNOS with similar Km and Vmax values to those of Arg. But, the production of NO by AM using these peptides as substrates was 2-fold higher than using Arg as a substrate. Both Arg-Gly and Arg-Gly-Asp were found in the BAL fluid. The presence of a PepT1-like transporter on AM was confirmed by Western blotting. CONCLUSION: This study shows that AM exhibit PepT1-like transporter for small peptide uptake. Arginine-containing peptides, through the PepT1 transporter system, can serve as direct substrates of iNOS for the production of NO by AM.

The effect of diesel exhaust particles (DEP) and carbon black (CB) on thiol changes in pulmonary ovalbumin allergic sensitized Brown Norway rats.

Brown Norway rats were exposed by intratracheal instillation of saline, carbon black (CB), or diesel exhaust particles (DEP) (5 mg/kg) on day 1, followed by exposure to ovalbumin (OVA, 90 mg/m(3)) or saline for 30 minutes on days 1, 8, 15, and 29. Animals were sacrificed on day 30. The DEP, CB, or OVA exposure alone did not result in abnormal levels of inflammatory cells, lactate dehydrogenase (LDH), or total protein in the lavage fluid. In combined OVA-DEP or OVA-CB exposure, however, these markers were significantly increased. The adjuvant effect of CB and DEP on OVA sensitization was evidenced by the marked increases in serum OVA-specific IgG (5.6-fold) and IgE (3.5-4 fold) levels, and the increase in interleukin-4 (IL-4) mRNA levels in lung tissue. The OVA exposure markedly reduced glutathione (GSH) levels in both cell types. In combined DEP-OVA exposure, the level of GSH in lymphocytes was further decreased, indicating a possible interactive effect between DEP and OVA exposures. These results show that both DEP and CB augmented OVA-induced allergic sensitization, and that particle composition of DEP may not be a critical factor for the adjuvant effect. OVA exposure causes significant depletion of intracellular GSH in lymphocytes, which may play a key role in OVA-mediated immune responses.