K. Kienast

Production of Reactive Oxygen Intermediates by Human Macrophages Exposed to Soot Particles and Asbestos Fibers and Increase in NF-kappa B p50/p105 mRNA

Abstract. Alveolar macrophages (AM) play a decisive role in the immunologic defense system of the lung and in inflammatory pulmonary pathomechanisms. AM and blood monocytes (BM) were exposed to Chrysotile B, Soot FR 101, and Printex 90 (P 90). We evaluated the reactive oxygen intermediate (ROI) release of AM and BM after particle exposure. ROI release was measured by chemiluminescence. Thirty-minute exposure caused a significant (up to 2.5-fold) increase in ROI release of AM (100 μg/106 cells) compared with control experiments (p < 0.01). Identical exposure conditions for BM resulted in a similar reaction pattern (maximum 2.2-fold increase in ROI release; p < 0.05). After a 90-min particle exposure at concentrations of 10 and 100 μg/106 cells, we investigated the steady-state level of p50/p105 mRNA encoding for the precursor protein of the p50 subunit of nuclear factor kappa B (NF-κB) by semiquantitative reverse transcription–polymerase chain reaction. One hundred μg Chrysotile B, FR 101, or P 90 induced a significant maximum 4.0-fold up-regulation of NF-κB gene expression in AM and a 3.3-fold up-regulation in BM (p < 0.05). The addition of superoxide dismutase (200 U/ml) to particle- and fiber-exposed macrophages resulted in inhibition of ROI release and a decrease in NF-κB mRNA expression (70%). NF-κB is an important transcription factor involved in the regulation of numerous genes (e.g., for inflammatory cytokines, and cytokine receptors). These cytokines are supposed to be involved in inflammatory pathomechanisms in bronchial epithelial cells, which result, for example, in chronic obstructive pulmonary disease. Our results suggest that particle-induced ROI release is associated with an increase in NF-κB (p50/p105) mRNA steady-state level.

Indoor air pollutants stimulate interleukin-8-specific mRNA expression and protein secretion of alveolar macrophages.

Abstract. Indoor air pollutants may cause inflammatory changes of the airways and adjacent pulmonary tissue. After phagocytosis of inhaled particles alveolar macrophages (AM) release chemotactic mediators capable of attracting inflammatory cells into the lung tissue. To evaluate these mechanisms further peripheral blood mononuclear cells (PBMNC) and human AM (freshly recovered from the lower respiratory tract) were exposed to the indoor particles Soot FR 101 and Printex 90, the asbestos fiber Chrysotile B, and titanium dioxide (TiO2) at concentrations of 10 or 50 μg/106 cells for up to 8 h. The migration of granulocytes into the conditioned supernatants of AM and PBMNC was quantified by chemotaxis assay in a Boyden chamber. Granulocyte migration increased by 42.3 ± 25.8% (Chrysotile B), 64.6 ± 18.3% (FR 101), 74.2 ± 16.5% (P 90), and 86.7 ± 25.6% (TiO2) in AM-conditioned supernatants (p < 0.05). Qualitative, Interleukin (IL)-8 specific reverse transcriptase-polymerase chain reaction was performed after exposure of AM or PBMNC to Chrysotile B, FR 101, P 90, and TiO2 at concentrations of 10 and 50 μg/106 cells for 90 min. Each of the tested particles caused an increase in IL-8-specific mRNA expression of AM or PBMNC after particle exposure compared with the unexposed control. To find out if IL-8, the most powerful granulocyte chemokine, is involved, supernatants were preincubated with anti-IL-8. Granulocyte migration decreased by up to 35 ± 15% (50 ng/ml anti-IL-8) and 41.5 ± 16% (100 ng/ml anti-IL-8) (p < 0.0625) in AM-conditioned supernatants. Pretreatment of the granulocytes with human IL-8 decreased by up to 59 ± 18% (10 ng/ml) (p < 0.0625) in AM-conditioned supernatants. Similar reaction patterns were observed using anti-IL-8-pretreated supernatants of particle-exposed PBMNC. In conclusion, indoor air pollutants may promote inflammatory changes in the lung via IL-8 release by alveolar macrophages.

Effect of Sulfur Dioxide on Cytokine Production of Human Alveolar Macrophages in Vitro

Tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and transforming growth factor-beta are cytokines synthesized by alveolar macrophages. We investigated the effect of sulfur dioxide, a major air pollutant, on the production of these cytokines by alveolar macrophages. The cells were layered on a polycarbonate membrane and exposed for 30 min to 0.0, 1.0, 2.5, and 5.0 ppm sulfur dioxide at 37 degrees C and 100% air humidity. The cells were incubated for 24 h after exposure, thus allowing cytokine release. Cytotoxic effects of sulfur dioxide were evaluated by trypan blue exclusion. Cytokines were measured with enzyme-linked immunosorbent assays (i.e., tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) or by use of a specific bioassay (i.e., transforming growth factor-beta). The toxicity of sulfur dioxide for alveolar macrophages ranged from 3.1 % to 9.5 %. A 30-min exposure to sulfur dioxide induced a significant decrease in spontaneous and lipopolysaccharide-stimulated tumor necrosis factor-alpha (p < .001) and lipopolysaccharide-stimulated interleukin-1beta release (p < .05). The release of interleukin-6 and transforming growth factor-beta was not affected significantly by sulfur dioxide exposure. Our results demonstrated a functional impairment of alveolar macrophages after sulfur dioxide exposure (i.e., release of tumor necrosis factor-alpha and interleukin-1beta). Neither spontaneous nor stimulated release of interleukin-6 and transforming growth factors were influenced by exposure to sulfur dioxide.

Spontaneous interleukin 2 release of bronchoalveolar lavage cells in sarcoidosis is a codeterminator of prognosis

There is mounting evidence that activated interleukin 2 (IL-2)-releasing lymphocytes play a central role in the immunopathogenesis of sarcoidosis by directing inflammatory reactions and granuloma formation. In the context that a significant proportion of these cells accumulates in the lung and releases mediators, we hypothesized that different immunologically defined stages of sarcoidosis can be identified. A cohort of 89 sarcoidosis patients was allocated to four groups according to the following criteria: stage A, a low number of bronchoalveolar lavage (BAL) lymphocytes (<20%) without IL-2 release (<1 unit/ml in BAL cell culture supernatant); stage B, BAL lymphocytes <20%, with IL-2 release (≥1 unit/ml); stage C, BAL lymphocytes ≥20% with IL-2 release; and stage D, ≥20% BAL lymphocytes without IL-2 release. Although patients of stages C and D (n = 49) exhibited lymphocytic inflammation, only 20/49 of these patients had activated IL-2-releasing alveolar lymphocytes. BAL of groups A and B showed a low number of lymphocytes, but the lymphocytes were activated in 20/40 patients. Forty-four patients not receiving therapy were reevaluated by pulmonary function tests 8 ± 1 months after BAL. Progressive disease was found in 9/12 patients of group C and stable or regressing disease in 13/13 patients of group A. These results demonstrate that a combination of BAL parameters can yield prognostic information.

An experimental model for the exposure of human ciliated cells to sulfur dioxide at different concentrations

Mucociliary transport is an important nonimmunological defense mechanism of the respiratory tract. The aim of this study was to investigate the effect of sulfur dioxide (S02) at different concentrations on ciliary beat frequency (CBF). Ciliated cells were obtained from 12 volunteers by nose brush. CBF was quantified using video-interference microscopy. The cells were placed on a polycarbonate membrane in contact with the surface of a reservoir filled with RPMI 1640 (bicarbonate buffered) or Ringers (electrolyte) solution, allowing the cells to be supplied by capillarity. In an exposure chamber the cells were exposed for 30 min to SO2 2.5–12.5 ppm at 37°C and 100% air humidity. SO2 induced a dose-dependent decrease in CBF of the cells cultured in Ringers solution. SO2 at 2.5 ppm caused a 42.8 % decrease and at 12.5 ppm a 96.5% decrease (8.1 ± 0.24 versus 0.28 ± 0.20 Hz). CBF of cells cultured in RPMI 1640 was reduced only moderately after 12.5 ppm SO2 exposure (7.9 ± 0.26 versus 6.70 ± 0.30 Hz). In Ringers solution a decrease in pH was observed after 30 min of S02 exposure to 12.5 ppm to a minimum value of 3.6. By contrast, the pH of RPMI 1640 remained constant at 7.5 under identical conditions. After adding RPMI 1640 to Ringers solution, CBF increased in parallel to the pH to control values (5.0 ppm: 4.64 ± 0.45 to 8.51 ± 0.60 Hz). These data suggest that the highly water-soluble SO2 reversibly eliminates CBF in correlation with a decrease in pH.

Effect of sulfur dioxide on mucociliary activity and ciliary beat frequency in guinea pig trachea

SummaryThe effects of 30 min exposure to sulfur dioxide on mucociliary activity (MCA) and ciliary beat frequency (CBF) were studied in 31 guinea pig tracheas. MCA was measured by recording the light reflected from ciliated mucous membranes using an infrared bar code reader. CBF of single ciliated cells obtained by brushing was measured with phase-contrast microscopy. Each tracheal sample was exposed to SO2 at concentrations ranging from 2.5 to 12.5 ppm, or to air for control purposes. MCA and CBF were measured before and immediately after gas exposure. A reduction in mean MCA of 63% (P = 0.0007) and statistically insignificant changes in CBF (P > 0.05) were recorded at concentrations of 2.5 PPM SO2. Higher SO2 concentrations caused a further impairment of MCA as well as a dose-dependent decrease in CBF (P = 0.002). A concentration of 12.5 PPM SO2 induced a decrease from baseline values of approximately 80% in mean MCA and of roughly 70% in mean CBE This study demonstrates a dose-dependent SO2-induced decrease in MCA of guinea pig tracheas. The decrease in MCA was associated with an impairment of CBF only at SO2 concentrations higher than 5.0 ppm.

In vitro study of human alveolar macrophage and peripheral blood mononuclear cell reactive oxygen-intermediates release induced by sulfur dioxide at different concentrations

Sulfur dioxide (SO2) is a major air pollutant in urban areas. Alveolar macrophages (AM) located on the alveolar surface are in direct contact with this inhaled gas. We evaluated the dose-dependent effect of SO2 exposure on the oxidative metabolism of AM and peripheral blood mononuclear cells (PBMNC) by measuring the spontaneous and stimulated reactive oxygen intermediates (ROI) release. AM or PBMNC were placed on a polycarbonate membrane, which was in direct contact with the surface of a nutrient reservoir. For exposure of the cells to SO2 a special chamber was employed, in which humidified standard air with 5% CO2 at 37°C was mixed with SO2 at the desired concentration. Periods of time between 30 and 120 minutes and concentrations between 0.3 and 1.5 ppm SO2 were chosen for exposure. Thirty minutes exposure of AM to SO2 (0.3-1.5 ppm) yielded a dose-dependent stimulation of ROI release; 2.0- to 3.6-fold of control (r = 0.965, p < 0.005). An exposure of 120 minutes to SO2 resulted in a similar ROl production of about 2.5-fold at all tested concentrations. These experiments provide evidence that AM and PBMNC become activated by SO2 producing large amounts of ROl.

Modulation of IL-1β, IL-6, IL-8, TNF-α, and TGF-β secretions by alveolar macrophages under NO2 exposure

Activated alveolar macrophages (AMs) secrete interleukine (IL)1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β), whose inflammatory and fibroblast-activating characteristics may play a role in the maintenance of pulmonary inflammatory processes and subsequent fibrosis. Human AMs were transferred to a gas cylinder and exposed to NO2 in concentrations ranging from 0.1 to 0.5 ppm in synthetic air for 30 min at 37°C. AMs were fixed on a polycarbonate membrane and placed on culture medium. A culture was established, with the exposed AM (nonstimulated or stimulated with 1 μg/ml lipopolysaccharide [LPS]), and the remaining cells were used to determine the cytokines. IL-1β, IL-6, and IL-8 were quantified by commercial enzyme-linked immunosorbent assay kits (ELISA kits). TNF-α was determined with a “sandwich” ELISA, using the biotin-streptavidin system. NO2 exposure of nonstimulated AM did not result in changes in IL-1β, IL-6, TNF-α, and TGF-β release, compared to the situation with control experiments. Exposure for 30 min to NO2 induced a significant decrease of LPS-stimulated IL-1β, IL-6, IL-8, and TNF-α (p < .05). The release of TGF-β was not significantly affected by NO2 exposure. Cytotoxicity of AM was checked by trypan blue exclusion, with values ranging from 1.3 to 3.0%. NO2 exposure of LPS-stimulated AM resulted in a functional impairment of AM after NO2 exposure regarding IL-1β, IL-6, IL-8, and TNF-α. Neither the spontaneous nor the stimulated release of TGF-β were influenced by NO2.

Chemotactic response of human alveolar macrophages and blood monocytes elicited by exposure to sulfur dioxide

An experimental study was undertaken to investigate the in vitro effect of sulfur dioxide on the chemotactic activity of alveolar macrophages (AM) and blood monocytes (BM). The cells were placed on a polycarbonate membrane and exposed to SO2 0.5, 1.5 and 2.5 ppm for 15 min. Control experiments were performed with exposure of the cells to synthetic air with 5% CO2. After gas exposure the cells were incubated with the chemotactic active agent C5a in 5% carbon dioxide (CO2) at 37°C for 60 min. The numbers of AM and BM passing actively through the membrane were quantified using light microscopy. Our results show a dosedependent reduction in the migration rate of cells under SO2 exposure. SO2 0.5 ppm induced a 29% and SO2 2.5 ppm a 53% decrease in migration of AM compared with the control exposure to synthetic air (P<0.01). Identical experiments with BM resulted in a decrease in migration of up to 57% (P<0.01). At SO2 concentrations of up to 2.5 ppm no significant cytotoxic effects were observed for AM or BM. The data demonstrate that exposure to SO2 may reduce the chemotactic activity of AM and BM. Our results further suggest that the decrease in cell migration induced by SO2 is due to changes in chemotactic mechanisms and not to cell death.