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It has been proposed that oxidant stress of cells in the lung is one of the underlying mechanisms of particulate pollution-induced exacerbation of lung disease. Individuals who are considered most sensitive to particulate pollution are those with pre-existing airways inflammation, such as chronic obstructive pulmonary disease (COPD), lung infection or asthma. These diseases are characterized by a presence of inflammatory cells in the airways including neutrophils (PMN), eosinophils and monocytes (Mo), and increased numbers of alveolar macrophages (AM). These cells have a high capacity for production of oxygen radicals, as compared to other cell types of the lung. To assess the oxidative response of these various cell types to pollution particles of various sources, luminol-dependent chemiluminescence was employed. Particles including transition metal-rich residual oil fly ashes (ROFAs), coal fly ashes, diesel, SiO2, TiO2 and fugitive dusts were co-cultured with AM, Mo and PMN in a dose range of 10-100 microg/2 x 10(5) cells and chemiluminescence determined following a 20-min interaction. A strong oxidant response of AM was restricted to oil fly ashes, while the PMN were most reactive to the dusts containing aluminium silicate. In general, the Mo response was less vigorous, but overlapped both AM- and PMN-stimulating dusts. However, in response to SiO2 and volcanic ash the Mo chemiluminescence exceeded that of the other cell types. Oxygen radicals generated in response to ROFA by the AM were likely to be dependent on mitochondrial processes, while the response in PMN involved the membrane NADPH oxidase complex, as determined by targeting inhibitors. The response of AM to SiO2 of various sizes and TiO2 in the fine size range obtained from different commercial sources, was highly variable, implying that composition rather than size was responsible for the oxidant response. A strong chemiluminescence response was not consistently associated with cytotoxicity in the responsive cell. Taken together, these results suggest that oxidant activation by various sources of particulate matter is cell specific. Therefore, the inflamed lung is likely to be more susceptible to harm of ambient air particulates because of the oxidant stress posed by a broader range of particles.

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We tested the hypothesis that exposure of healthy volunteers to concentrated ambient air particles (CAPS) between 0.1 and 2.5 microm in diameter is associated with modulation of human alveolar macrophage (AM) function, cytokine production, and immune phenotype in both blood and lung. Thirty-eight volunteers were exposed to either filtered air or CAPS from the immediate environment of the U.S. Environmental Protection Agency human studies facility in Chapel Hill, North Carolina, USA. Particle concentrations in the chamber during the exposures ranged from 23.1 to 311.1 microg/m3. No symptoms were noted by volunteers after the exposure. Eighteen hours after exposure, analysis of cells obtained by bronchoalveolar lavage (BAL) showed a mild increase in neutrophils in both the bronchial (8.4 +/- 2%) and alveolar fractions (4.2 +/- 1.7%) in subjects exposed to the highest concentration of CAPS compared to neutrophils in the fluids of those exposed to filtered air (bronchial fraction 2.7 +/- 0.6%; alveolar fraction 0.8 +/- 0.3%). There was no change in the percentage of lymphocytes or AMs recovered in the lavage after inhalation of the highest particle levels (mean 207 microg/m3). There was also no change in the proportion of lymphocytes in the BAL expressing CD3, CD4, CD8, CD19, nor activation markers CD25 or CD69. Particle inhalation did not affect the expression of CD11b, CD64, CD16, CD14, CD71 on AM, nor was there an effect on phagocytosis or oxidant generation following stimulation with zymosan A. IL-6 and IL-8 levels detected by enzyme-linked immunoabsorbent assay in the BAL were unrelated to inhaled particle levels. The distribution of lymphocyte subsets in blood obtained 18 hr after exposure to CAPS did not differ from that found before exposure. We conclude that ambient air particles are capable of inducing a mild inflammation in the lower respiratory tract but have no effect on immune phenotype or macrophage function under the conditions tested.

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Inhalation of particulate matter in the ambient air has been shown to cause pulmonary morbidity and exacerbate asthma. Alveolar macrophage (AM) are essential for effective removal of inhaled particles and microbes in the lower airways. While some particles minimally effect AM function others inhibit antimicrobial activity or cause cytokine and growth factor production leading to inflammation and tissue remodeling. This study has investigated the effects of water soluble (s) and insoluble (is) components of Chapel Hill, North Carolina ambient particulate matter in the size ranges 0.1-2.5 microm (PM2.5) and 2.5-10 microm (PM10) diameter, on human AM IL-6, TNFalpha, and MCP-1 cytokine production and host defense mechanisms including phagocytosis and oxidant production. Cytokines were found to be induced by isPM10 to a much higher extent (>50-fold) than sPM10, which in turn stimulated production better than isPM2.5, while sPM2.5 was inactive. Previous studies have indicated that endotoxin (ETOX) is a component of sPM10 responsible for cytokine production. Here, it is shown that inhibition of isPM10-induced cytokine production was partially achieved with polymyxin B and LPS-binding protein (LBP), but not with a metal chelator, implicating ETOX as a cytokine-inducing moiety also in isPM10. In addition to inducing cytokines, exposure to isPM10, but not the other PM fractions, also inhibited phagocytosis and oxidant generation in response to yeast. This inhibition was ETOX independent. The decrease in host defenses may be the result of apoptosis in the AM population, which was also found to be specifically caused by isPM10. These results show that the functional capacity of AM is selectively modulated by insoluble components of coarse PM, including the biocontaminant ETOX.

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Water-soluble extracts of Utah Valley dust (UVD) have been found to cause inflammatory injury of the lung in both humans and rodents. The degree of lung damage found correlated with the metal content in the extracts. In the present study, extracts of a set of UVD PM(10) filters collected over a 3-yr span, varying in total metal content with yr 1 = yr 3 > yr 2, were used to assess effects on human alveolar macrophage (AM) function. The phagocytic activity and oxidative response of AM was investigated 24 h after segmental instillation of UVD, or after overnight in vitro culture of the extracts with AM. Using flow cytometry analysis, AM phagocytosis of fluorescently (FITC)-labeled Saccharomyces cerevisiae was inhibited following instillation of UVD1 (61%) but not by yr 2 and 3. Neither baseline oxidant activity nor phorbol ester-induced oxidant generation was affected by the dust extracts in vivo. Overnight culture of AM with UVD1 resulted in a significant decrease in the percentage of AM phagocytizing particles (30%), while no significant effect on this function was found with the other two extracts. Furthermore, only UVD1 caused an immediate oxidative response in AM, although both UVD1 and UVD3 inhibited oxidant activity in AM when the cells were incubated with the extracts overnight. The detrimental effects on AM host defenses could be due to apoptosis, which was evident in cells exposed to the UVD1 and to a much lesser extent with AM exposed to yr 2 and 3. The component(s) responsible for the toxic effects on AM in vitro were removed by pretreatment of the UVD extracts with a polycation chelating resin, chelex-100. However, since yr 1 and 3 are similar in their soluble metal content, but differ in their effects on AM phagocytosis, it is possible that the metals may not be the culprit in effects of particulate matter on AM host defense.

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The early inflammatory events in respiratory syncytial viral (RSV) infection are likely to be crucial in the development of clinical disease, which is characterized by bronchiolitis with mononuclear cell inflammation, some eosinophil involvement and airway hyperreactivity. Since RSV replication is restricted to airway epithelial cells, our working hypothesis is that inflammatory cell recruitment by the infected cells will set the stage for late immunopathology. We have identified the selective induction and release of mononuclear cell and eosinophil-attracting beta-chemokines MIP-1alpha and RANTES, but not eotaxin, by RSV-infected airway epithelial cells and herein demonstrated the recruitment of eosinophils and monocytes, but not neutrophils, in response to chemokines produced by infected epithelial cells during viral replication and dissemination. The chemotactic response of both eosinophils and monocytes was inhibited by antibodies to RANTES but not to MIP-1alpha. Interaction of eosinophils or monocytes with RSV-infected epithelial cells resulted in the production of additional beta-chemokines MCP-1 and MIP-1beta, and increased levels of MIP-1alpha. The monocyte containing cultures produced >10 fold the amount of these chemokines compared to eosinophil containing cultures. On the other hand, the levels of RANTES and the lack of eotaxin were not altered in the cocultures, RSV-infected monocytes appeared to be the main source of MIP-1alpha and MIP-1beta, while MCP-1 was derived from monocytes as well as epithelial cells following coculture. These data implicate RANTES as the primary chemokine responsible for selectively recruiting eosinophils and monocytes to the site of RSV infection. This inflammatory response results in the production of high levels of additional chemokines capable of setting up a full-fledged inflammatory response including lymphocytes.

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Epidemiology studies associate increased pulmonary morbidity with episodes of high particulate air pollution (size range 0.1-10 microm diameter, PM10). Pneumonia, often viral in origin, is increased following episodes of high PM10 pollution. Therefore, this study was undertaken to investigate how PM10 alters airway inflammatory responses to respiratory syncytial virus (RSV), a frequent cause of viral pneumonia in infants and the elderly. Supernatants of unexposed and PM10-exposed alveolar macrophage (AM) cultured with uninfected or RSV-infected airway epithelial cells were assessed for a number of chemokines responsible for inflammatory responses in the lung. AM exposure to PM10 in the absence of infection resulted in a significant increase in interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1alpha production but not in MIP-1beta or monocyte chemotactic protein (MCP)-1. AM responded to RSV infection by the production of IL-8, MIP-1alpha, MIP-1beta, and MCP-1, while RANTES was derived solely from the RSV-infected bronchial epithelial cell line BEAS-2B. In the presence of PM10, the AM response to RSV was blunted. RSV-induced MCP-1 was significantly decreased, and the levels of MIP-1 and IL-8 were lower than expected from a combined response to PM10 and RSV. Furthermore, AM analyzed for uptake of virus showed a 50% decrease in viral antigen when exposed to PM10 RSV-induced production of RANTES by epithelial cells was decreased in the presence of AM but not affected by PM10 exposure. Taken together, these results suggest that AM-regulated inflammatory responses to viral infection are altered by exposure to PM10 in a manner that may result in increased spread of infection and thus may increase viral pneumonia-related hospital admissions.

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Nitrogen dioxide (NO(2)) is a common air pollutant outdoors and indoors in homes with unvented combustion sources. It is also a constituent of tobacco smoke. Epidemiological studies suggest that children exposed to NO(2), or living with smoking parents, have an increased incidence of respiratory viral infections. The most common virus causing severe respiratory symptoms in infants and young children is respiratory syncytial virus (RSV). In the present study we investigated whether NO(2) exposure affects RSV infection in airway epithelial cells, the host cells for viral replication and virus-induced cytokine production. Cultures of the bronchial epithelial cell line BEAS-2B exposed to 0.5, 1.0, and 1.5 ppm NO(2) for 60 min were infected with RSV. Viral replication, as well as RSV-induced interleukin (IL)-6 and IL-8, was assessed at various times postinfection. The NO(2) doses used were not toxic to the BEAS-2B cells as measured by release of lactic dehydrogenase (LDH). The internalization of RSV was increased by exposure to 0.5 ppm NO(2) and decreased by exposure to 1.5 ppm NO(2). On the other hand, the release of infectious virus 48 h postexposure was not affected by the two lower doses of NO(2), but was significantly reduced in cells exposed to 1.5 ppm NO(2). Virus-induced cytokine production was also significantly reduced in cells exposed to 1.5 ppm NO(2), and not affected by 0.5 and 1.0 ppm. It is likely that the decrease in cytokine production is related to the decrease in viral burden. These data suggest that possible increases in viral clinical symptoms associated with NO(2) may not be caused by increased susceptibility of the epithelial cells to infection but may result from effects of NO(2) on host defenses that prevent the spread of virus.

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Epidemiologic studies have reported causal relationships between exposures to high concentrations of ambient air particles (AAP) and increased morbidity in individuals with underlying respiratory problems. Polymorphonuclear leukocytes (PMN) are frequently present in the airways of individuals exposed to particles. Upon particulate stimulation the PMN may release reactive oxygen species (ROS), which can result in tissue damage and injury. In this study a wide range of AAP samples from divergent sources (1, natural dust; 2, oil fly ash; 2, coal fly ash; 5, ambient air; and 1, carbon black) were analyzed for elemental content and solubility in relation to their ability to generate ROS. Elemental analyses were carried out in AAP and dH(2)O-washed AAP using energy dispersive x-ray fluorescence (XRF). Percent of sample mass accounted for by XRF-detectable elements was 1.2% (carbon black); 22-29% (natural dust and ambient air particles); 13-22% (oil fly ash particles); 28-49% (coal fly ash particles). The major proportion of elements in most of these particles were aluminosilicates and insoluble iron, except oil-derived fly ash particles in which soluble vanadium and nickel were in highest concentrations, consistent with particle acidity as measured in the supernatants. Human blood-derived monocytes and PMN were exposed to AAP and dH(2)O-washed particles, and generation of ROS was determined using luminol-enhanced chemiluminescence (LCL) assay. All the particles induced chemiluminescence response in the cells, except carbon black. The oxidant response of monocytes induced by AAP (with the exception of oil fly ash particles) was less than the response elicited by PMN. The LCL response of PMN in general increased with all washed particles, with oil fly ash (OFA) and one urban air particle showing statistically significant (p < 0. 05) differences between dH(2)O-washed and unwashed particles. The LCL activity in PMN induced by both particles and dH(2)O-washed particles was significantly correlated with the insoluble Si, Fe, Mn, Ti, and Co content of particles (p < 0.05). No relationship between LCL activity in PMN and soluble transition metals such as V, Cr, Ni, and Cu was noted. Pretreatment of the particles with a metal ion-chelator, deferoxamine, did not affect LCL in PMN, suggesting that metal ions are not related to the induction of LCL in PMN. Particulate S content and acidity of the particles as measured in the supernatants did not relate to LCL activity in PMN. These results point to the possibility that the insoluble constituents of the particles are related to LCL in PMN. Since some of these dusts are capable of depositing in the lungs and can cause infiltration of PMN, the ability to activate those cells may contribute to particulate toxicity.

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Elevated levels of air pollution particulates < or = 10 microm in diameter (PM10) have been associated with an increase in mortality and morbidity due to pulmonary complications, including pneumonia. Impairment of inflammatory and host defense functions of the alveolar macrophage (AM) may be a precipitating factor. The present study was undertaken to determine whether human AM and blood derived monocytes (MO) modulate the expression of receptors important for phagocytosis of opsonized microbes (CD11b, CD11c), gram-negative bacteria (CD14), extracellular matrix interaction (CD29), and immune responses (CD11a, CD54, HLA-DR) when exposed to particulates obtained from urban air (UAP). Furthermore, phagocytosis of and oxidant generation by opsonized yeast were investigated in particle-exposed cells. AM and MO exposed to UAP for 18 h expressed significantly lower levels of CD11b and CD29. CD14 expression was markedly decreased in MO but not in AM, and CD11c was reduced in AM but not in MO. CD11a, CD54, and HLA-DR were unaltered in both phagocyte populations. Decreased receptor expression was not dependent on particle load in the cells. Phagocytosis of Saccharomyces cerevisiae and the chemiluminescence response were also significantly inhibited by UAP. Time-course studies revealed that decreased oxidant generation was evident already at 3 h postexposure, while significant effects on phagocytosis and CD11b expression were found at 18 h. These data indicate that exposure to particulate pollution is likely to impair host defense functions of AM and MO, which are important in elimination of a variety of pathogens in the lung.

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Airway epithelium is the primary target tissue for respiratory viruses as well as an important target of ozone (O(3)) toxicity. A change in the severity of viral airway infection may result from changes in epithelial cell susceptibility to infection, metabolic interference with viral replication, or altered production of immune regulatory molecules by the infected cells as a result of exposure to O(3). In this study we have investigated whether O(3) exposure alters the susceptibility of human airway epithelial cells to respiratory syncytial virus (RSV) infection, the production of infectious virus, and/or release of virus-induced cytokines IL-6 and IL-8. The epithelial cell line BEAS-2B grown on collagen-impregnated filters was exposed to O(3) (0.5 ppm for 60 min) or filtered air immediately before or 24 h after infection with RSV. Cells exposed to O(3) before RSV infection released 44% less virus over 4 days of infection while O(3) exposure post RSV infection had no effect on virus production. O(3) exposure preceding RSV infection showed short term additive effects of these treatments on epithelial cell IL-6 and IL-8 production, a decrease in cytokines at 48 h, but did not affect long term cytokine production by RSV-infected cells. Furthermore, O(3) exposure did not affect long term cytokine production by cells with an established RSV infection at the time of exposure. These data suggest that O(3) does not adversely affect viral airway infection, at least not on the level of the host cell for viral replication.

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A number of epidemiological studies have associated increased cardiopulmonary mortality and hospital admissions with episodes of high particulate air pollution. Inhaled particles, with a mass median aerodynamic diameter <10 microm (PM10) reach the lower respiratory tract where they are phagocytized by alveolar macrophages (AM). Depending on particle composition, exposed AM may produce reactive oxygen species and inflammatory mediators resulting in vascular permeability changes, airway constriction, tissue injury, and inflammation. In the present study human and rat AM were reacted with a range of environmental particles, including oil fly ash (OFA), diesel dust (DD), and ambient air particles (UAP) collected in four urban centers. AM were tested for a chemiluminescence response induced by the particles as well as IL-6 and TNF production. While OFA in a dose range of 1000-10 microg/2-3 x 10(5) AM caused acute cytotoxicity above 100 microg in both human and rat AM (LDH release at 2 hr), DD and UAP were found to be nontoxic in the same dose range. However, after 20 hr of coincubation, UAP concentrations >167 microg/ml were also cytotoxic. Subcytotoxic concentrations of OFA induced a strong immediate chemiluminescence response by AM. A small but significant chemiluminescence response was induced by two out of three UAP tested, while no chemiluminescence was generated in response to DD. The magnitude of particle-induced chemiluminescence was not predictive of a cytokine response by either human or rat AM. TNF and IL-6 production was strongly induced by UAP over a range of noncytotoxic concentrations of particles. OFA induced only small amounts of TNF in a subset of human AM preparations, but not in rat AM. The AM cytokine response to UAP was partly inhibitable by polymyxin B, but not by the iron chelator deferoxamine, indicating that endotoxins but not transitional iron were cytokine-inducing moieties in the tested UAP preparations.

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In our private clinic-hospital setting, respiratory syncytial virus (RSV) was isolated from infants more frequently and sooner from nasal washes (84%; 4.2 days) than from throat swabs (45%; 5.5 days) or nasopharyngeal swabs (39%; 5.7 days). Immunofluorescence of nasal wash cells identified 72% of the infants with virus isolations from nasal washes in less than one day. We therefore recommend the combination of isolation and immunofluorescence on nasal wash specimens for optimal detection of RSV-infected infants. Immunofluorescence of respiratory tract cells was also useful for monitoring the presence of RSV antigen in intubation secretions during ribavirin antiviral therapy. RSV infectivity was maintained in phosphate-buffered saline at room temperature for 6 h. Transport and inoculation of specimens in less than 6 h yielded RSV isolates from 50% of sampled infants during the two RSV seasons examined. For optimal RSV isolation, we recommend inoculation of HEp-2 tubes less than or equal to 4 days old. Replacing medium after 3 days as compared with 7 days did not increase recovery of RSV and provided little practical reduction in time to detection of cytopathology.

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