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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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There is evidence that asthma and other allergic diseases are increasing and air pollution has been considered an important contributing factor to this observation. Using a specially designed organ culture system,
we examined the influence of ozone (0.06 to 0.2 ppm) and nitrogen dioxide (NO2, 200 and 800 µg/m3) on
nasal mucosa exposed for 24 h. Tissue was obtained from 105 patients undergoing surgical therapy (septoplasty and reduction of the inferior turbinates) for chronic nasal obstruction. The histamine content in the
culture medium of ozone- and NO2-exposed samples was significantly elevated compared with the control
cultures. This elevation was correlated with the number of degranulated mast cells in the tissue determined
by histomorphometry (P < 0.001). Moreover, the cytokines interleukin (IL)-1
(P < 0.05), IL-6 (P < 0.01), IL-8 (P < 0.001), and tumor necrosis factor-
(TNF-, P < 0.001) were significantly increased
(ozone 0.1 ppm). Furthermore, we found significant increases in the release of IL-4, IL-6, IL-8, and TNF-
of ozone-exposed (0.1 ppm) samples of atopic versus nonatopic patients and to a lesser extent for histamine following exposure to 0.15 ppm ozone. These results indicate that low ozone concentrations and NO2
lead to an inflammation of human nasal mucosa in vitro and that priming factors such as atopy or preexisting inflammation do increase the sensitivity to ozone and NO2. This organ culture system proved to be a
good experimental design for studying pathophysiologic alterations of human nasal mucosa under different experimental conditions (e.g., air pollutants).
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Epidemiologic studies have suggested that the incidence of allergic diseases such as asthma, rhinitis, and eczema has increased over the last 50 yr (1). Several investigations have indicated that there may be a link between hospital emergency room visits with respiratory symptoms (2), impaired lung function (5), rhinorrhea, cough, and infections of the lower respiratory tract (6), and episodes of air pollution characterized by high concentrations of ozone (O3) or nitrogen dioxide (NO2). However, the main problem encountered in epidemiologic studies has been the difficulty in controlling confounding variables such as weather, other pollutants, aeroallergens, smoking habits, and individual factors relating to socioeconomic status, housing, occupation, and medical conditions.
Both in vivo and in vitro studies carried out in healthy normal subjects and animals have provided substantial evidence
that short periods of exposure to O3 at ambient concentrations results in airway inflammation. Controlled exposure
studies on patients have shown that at ambient levels O3 has
adverse effects on lung function and induces airway inflammation (7) that persists for up to 24 h. Other studies have
shown significant increases in polymorphonuclear leukocytes
(PMNs), albumin, prostaglandin (PG) E2, fibronectin, interleukin (IL)-6, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), lactate dehydrogenase, tryptase, C3a,
tissue factor, urokinase-type plasminogen activator, factor VII, and 1-antitrypsin (1AT) in bronchoalveolar lavage
fluid (BALF) and nasal lavage fluid (NALF) (8).
Certain kinds of experimental work in humans are not possible because of ethical limitations. Cultures of isolated epithelial or bronchial cells provide only a partial and potentially misleading picture of the inflammatory responses in the mucosa.
Therefore, appropriate in vitro models are still required to understand better the interactions of airways and air pollution. In this situation, organ culture models offer physiologically relevant assay systems. The upper airway, especially the nose, is a major target of toxic damage. Unfortunately, very little work on specifically human nasal mucosa has been reported. Previously, we described a culture system of human nasal mucosa (12, 13) on gelatin sponges at the air-liquid interface.
The aims of this study were (1) to examine the influence of O3 and NO2 on human nasal mucosa in vitro with respect to inflammatory mediators and cytokines; (2) to determine whether this histoculture technique, slightly modified for O3 exposure conditions, demonstrates the same findings as found in vivo mainly in BALF and NALF or in animal experiments; and (3) to determine whether this organ culture system can serve as a screening tool for biologically relevant substances (i.e., air pollutants).
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Patients and Preparation of Human Nasal Mucosa
Human nasal mucosa was obtained from 105 patients undergoing surgical therapy for chronic nasal obstruction; patients suffered from nasal obstruction caused by septum deviation and hyperplasia of the inferior turbinates. Surgical procedures performed were septoplasty and reduction of the inferior turbinates. Patients with chronic paranasal sinus infection were excluded.
With respect to atopy, all subjects underwent routine screening procedures including complete medical history, physical examination, skin-prick test, and serum chemistries with screening for immunoglobulin E (CAP and Pharmacia CAP System SX1-FEIA, Phadiatop; Pharmacia, Uppsala, Sweden). Atopy was defined as the presence of one or more positive skin-prick test reactions to the most common antigens.
Tissue samples used for culture were taken from the inferior nasal turbinate and immediately transferred into N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (Hepes) buffer containing 137 mM NaCl, 4 mM KCl (Merck, Darmstadt, Germany), 11 mM glucose (Braun, Melsungen, Germany), 10 mM Hepes, and 100 µg/ml gentamicin (Sigma, St. Louis, MO) after surgical excision. The obtained tissue was stored maximally 30 min at 4°C for transport to the laboratory. Small pieces of complete nasal mucosa were cut with a 4-mm biopsy punch (Stiefel, Offenbach, Germany). Six biopsies were taken from each surgical specimen. Thereafter, the punches were washed three times in Hepes buffer to remove blood cells and mucus.
Histoculture of Human Nasal Mucosa and Exposure to O3 or NO2
The 4-mm punches of intact, full-thickness human nasal
mucosa and submucosa were implanted at the air-liquid
interface with the epithelium up and submucosa down in
1 × 1 × 0.7-cm pieces of collagen-containing gelatin
sponges (Gelfoam; Upjohn, Kalamazoo, MI) that had
been prehydrated for at least 24 h with culture medium. The mucosa fragments (one per sponge gel) were placed
individually in six-well plates (Nunclon; Nunc, Roskilde,
Denmark) filled with 4 ml culture medium per well, so that
the epithelium was above the liquid phase. For assessment
of mast cell degranulation (density) and histology, the culture medium consisted of Dulbecco's modified Eagle's
medium with NaHCO3 (3.7 g/liter), Na pyruvate (110 mg/ liter), and D-glucose (1 g/liter) (GIBCO, Uxbridge, UK)
supplemented with L-glutamine (2 mM) (Seromed, Berlin,
Germany), gentamicin (50 µg/ml), penicillin G (100 U/ml),
streptomycin (100 µg/ml), amphotericin B (0.25 µg/ml)
(Sigma), and 10% heat-inactivated fetal bovine serum
(Seromed). To determine the concentrations of inflammatory mediators, we used Hepes buffer containing 10 mM
Hepes, 4 mM KCl, 1.1 mM MgCl2 × 6 H2O (Sigma), 1 mM
CaCl2 (Merck), 11 mM glucose, and 50 µg/ml gentamicin.
The mucosa surface profile was assessed under a stereomicroscope (Zeiss, Oberkochen, Germany). Three punches
per surgical specimen were maintained as control cultures
for 24 h in a humidified incubator under 37°C, 95% air, 5% CO2; the O3/NO2 samples (three punches per patient)
were incubated in parallel under the same culture conditions as described previously in another humidified incubator connected with Ozocontrol 100/1,000 UV (Sorbios,
Berlin, Germany) and exposed to O3 (0.06, 0.08, 0.1, 0.15, and 0.2 ppm) or NO2 (800 and 200 µg/m3) for 24 h. We removed the covers of the culture plates so that the samples
were in direct contact with O3 or NO2. Aliquots of the supernatant were taken from the samples at 1, 2, and 24 h and stored immediately at 
70°C until analysis for inflammatory mediators. The tissue samples were weighed at the
beginning and the end of the incubation period; a representative tissue fragment was frozen for immunohistology
and the rest were processed for routine histology.
Histologic Studies and Mast Cell Histomorphometry
The nasal mucosa tissue was fixed with buffered formalin (4% formalin, 43.3 mM NaH2PO4/176 mM Na2HPO4, pH 7.0) and processed for routine histology. Four-micrometer sections were prepared from paraffin-embedded tissue blocks and stained with hematoxylin and eosin, Giemsa reagent, and periodic acid Schiff reagent following standard procedures. The percentage of histochemically visualized "degranulated" versus "granulated" mast cells was quantitated by morphometry. Mast cells were defined as degranulated when more than eight metachromatic extracellular granules were detected by light microscopy in close proximity to a cell with mast cell morphology. At least 10 sections per sample, with 10 microscopic views (×400 magnification) per section, were examined.
Determination of Inflammatory Mediators in the Culture Medium
For histamine analysis, the supernatants were assayed after protein precipitation with one volume of 2% HClO4 by
an automated fluorimetric technique capable of detecting
1 ng/ml histamine (14). The cytokines IL-1 (detection
limit: 0.083 pg/ml), IL-4 (0.065 pg/ml), IL-6 (0.104 pg/ml),
IL-8 (10 pg/ml), tumor necrosis factor (TNF)- (0.112 pg/
ml) (BioSource, Camarillo, CA), and interferon (IFN)-
(0.1 pg/ml; Amersham, Little Chalfont, UK) were assayed
with commercial kits (ultrasensitive enzyme-linked immunosorbent assays with the exception of IL-8). The measured mediator concentrations were calculated in nanograms or picograms per milligram wet weight.
Statistical Analysis
Data are expressed as medians with the 25 to 75 interquartile range (lower and upper quartile). The mediator concentrations of controls versus O3- or NO2-exposed samples were compared at each time point using the distribution-independent Wilcoxon signed-rank test for paired samples. The Mann-Whitney U test was used to compare data between groups (nonatopics versus atopics). P < 0.05 was considered significant. Spearman's correlation coefficient (r) was calculated to analyze possible correlations between two different parameters, and significant correlation was accepted at P < 0.05 according to the Spearman rank correlation analysis. Statgraphics (Statistical Graphics Corp., Rockville, MD) was used for statistical analysis.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A significantly higher histamine content was found in the supernatant of tissue samples exposed to 0.2 ppm (P < 0.001), 0.15 ppm (P < 0.05), 0.1 ppm (P < 0.001), or 0.08 ppm (P < 0.01) O3 for 24 h in comparison with controls (Table 1). Moreover, we found a significantly different release of histamine of cultures of atopic versus nonatopic patients following O3 exposure for 24 h with 0.15 ppm (P = 0.0209), but not following ozone exposure for 24 h with 0.1, 0.08, or 0.06 ppm (Table 2). Comparing the ozone-induced increases as well as the mean percent increases for atopics and nonatopics in response to O3, no statistical differences were observed.
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For assessment of mast cell density, human nasal mucosa was stained with Giemsa reagent and examined by histomorphometry. With this staining, mast cells were readily detected. In specimens exposed to O3 (0.1 ppm) for 24 h, 57.7% of all mast cells were found to be degranulated versus 40.1% in control sections. In the presence of O3, a significantly increased number of degranulated mast cells (P < 0.001) and a significantly decreased density of granulated mast cells (P < 0.01) were detected (Figure 1).
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The time course of the histamine concentration in the culture medium of nasal mucosa following O3 exposure (0.1 ppm) compared with control cultures at the time points 1 h (P < 0.01), 2 h (P < 0.01), and 24 h (P < 0.001) is illustrated in Figure 2.
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The cytokines IL-1 (P < 0.05), IL-6 (P < 0.01), IL-8
(P < 0.001), and TNF- (P < 0.05) were significantly increased in the culture supernatant of nasal mucosa exposed to 0.1 ppm ozone for 24 h (Table 3). A positive correlation was found between IL-6 and IL-8 (r = 0.75; P < 0.00001). Furthermore, a significantly different release of
IL-4 (P < 0.05), IL-6 (P < 0.05), IL-8 (P < 0.05), and
TNF- (P < 0.05) of O3-exposed (0.1 ppm, exposed for
24 h) cultures of atopic versus nonatopic patients were observed (Table 4). Comparing the O3-induced increases for
atopics and nonatopics in response to O3, a statistically
higher release of IL-4 and IL-6 was observed. Despite the
use of an ultrasensitive assay, the concentrations of IFN-
were close or below the detection limits and therefore not
reliable.
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In addition we observed a significant increase of histamine in the supernatant of cultures exposed to NO2 (200 µg/m3, P < 0.001; 800 µg/m3, P < 0.01) for 24 h versus control cultures (Table 5).
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Discussion |
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Materials and Methods
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There is increasing evidence from epidemiologic and laboratory-based studies that exposure to gas-derived air pollutants such as O3 and NO2 may play a role in the clinical manifestation of allergic and nonallergic airway disease. Both in vivo and in vitro studies carried out in healthy, normal subjects, patients, and animals have provided substantial evidence that short periods of exposure to O3 at ambient concentrations results in airway inflammation (7).
The ozone concentration required for this effect is still unknown and consequently is of general interest. In the organ culture model presented here, we used O3 concentrations of 0.06 ppm (120 µg/m3) to 0.2 ppm (400 µg/m3), which reflect environmental conditions.
Considering the fact that the nose is the first region of the respiratory tract in contact with airborne pollutants, relatively little research has been done on human nasal mucosa.
The culture supernatant of O3-exposed nasal mucosa revealed a significant increase of various inflammatory mediators. Histamine, a marker of mast cell degranulation, was significantly elevated after exposure to O3 (0.08 to 0.2 ppm) and after exposure to NO2 (200 and 800 µg/ m3) compared with control cultures. In parallel, histomorphometry showed a significantly decreased density of granulated mast cells and a significantly increased number of degranulated mast cells in response to 0.1 ppm O3. Koren and colleagues (11) found increased NALF concentrations of tryptase after O3 exposure (2 h, 0.4 ppm). Because tryptase is also an indicator for mast cell degranulation, it is conceivable that mast cell function is influenced by ozone.
Devlin and coworkers (8) have reported that concentrations of ozone as low as 0.08 ppm are sufficient to induce airway inflammation. This and other studies have revealed that O3 induces airway inflammation especially in
the proximal airways (15). These studies have also shown
a significant increase in PMNs, albumin, PGE2, PGF2
,
8-epi-PGF2, thromboxane B2 (TXB2), fibronectin, IL-6,
IL-8, GM-CSF, lactate dehydrogenase, C3a, tissue factor,
factor VII, and 1AT in BALF (8, 15).
Products of the arachidonic acid pathway have been
shown to be potent mediators of inflammation (16). Recently, we have reported significant increases in PGF2,
TXB2, leukotriene B4 (LTB4), and elevated concentrations
of LTC4/D4/E4 in the supernatant of O3-exposed (0.1 ppm,
24 h exposure) nasal mucosa using the organ culture described here (20). These studies are consistent with the hypothesis that O3 can augment eicosanoid metabolism in
the human nasal airways.
With regard to the influence of proinflammatory cytokines in the development of airway diseases, we have
studied a range of different inflammatory cytokines. So
far, increased concentrations of IL-6, IL-8, GM-CSF, and
TNF- were found in cultures of bronchial epithelial cells
(18), nasal epithelial cells (19), and BALF or NALF (8,
9, 21) after O3 exposure. In this study, significant increases
of IL-1, IL-6, IL-8, and TNF- after O3 exposure (0.1 ppm) were found.
Exposure to O3 induces neutrophilic inflammation in
both the nasal and bronchial airways of nonasthmatic/
nonatopic individuals as well as in asthmatic/atopic individuals. Subsequent investigations have employed bronchoscopy to examine the effect of O3 on bronchial inflammation in asthmatics. Basha and colleagues (22) reported that a cohort of five atopic asthmatics had a greater PMN
response to 0.2 ppm O3 (6 h) than a cohort of five nonasthmatics, as reflected in BALF. Furthermore, there was also
an enhanced IL-8 response in the asthmatics. The BALF
albumin, TNF-, and IL-1 levels were not significantly different among the two groups. In a larger study, Scannell
and coworkers (23) also compared the effect of exposure
to 0.2 ppm ozone for 4 h on lower airway inflammation in
18 asthmatic subjects and 20 nonasthmatic volunteers. Asthmatic subjects also had a greater PMN response in the BALF
than nonasthmatic subjects, and also had a trend for increased IL-8 levels. These two studies suggest that asthmatic individuals may be more sensitive to the inflammatory effects of O3 than nonasthmatic individuals.
In view of a possible link between O3 and the development of allergic diseases, we compared the results of
atopic and nonatopic patients. There was a significantly
higher release of IL-4, IL-6, IL-8, TNF- (0.1 ppm O3)
and, to a lesser extent, of histamine (0.15 ppm O3) of exposed cultures of atopic versus nonatopic patients.
The mechanisms and cell types involved in pollutant-mediated effects in the airways, however, are so far not clear. In vitro studies have suggested that human fibroblasts (24), alveolar macrophages (25), and epithelial cells/ cell lines may be involved (26). Based on our results, the interleukin-producing cells (e.g., T cells, neutrophils, and epithelial cells) are responsible for the interleukin- dependent inflammatory response mechanisms.
The possible mechanisms involved in pollution-induced effects may be due to decreasing ciliary activity (30, 31), increasing epithelial damage, and increasing epithelial permeability (34). This could be exaggerated in atopic/asthmatic patients with underlying mucosal inflammation, decreased clearance, and epithelial shedding resulting in greater absorption of inhaled allergen and hence more intensive contact between antigens and antigen-presenting cells. Increasing epithelial permeability also leads to increased accessibility of irritants to the subepithelial tissue, where they may react with and activate inflammatory cells, such as mast cells. Pollutants may also act by depleting naturally occurring antioxidants, which are important in the maintenance of epithelial cell membrane integrity. Moreover, pollution induces the increase of proinflammatory cytokines and cell adhesion molecules, which coordinate the detrimental functions of inflammatory cells such as eosinophils, mast cells, and lymphocytes (33).
In conclusion, our results show that low O3 concentrations and NO2 trigger an inflammatory reaction of human nasal mucosa in organ culture. The presented data suggest that the response to O3 seems to be increased in atopic mucosa. The results presented here using low O3 concentrations confirm the previous results from in vivo and in vitro studies, in many of which higher O3 concentrations were used.
In controlled exposure studies, BAL and NAL may serve as sensitive and reliable techniques to detect inflammation in the airways of subjects exposed to O3 under standardized conditions. Experimental work in humans, however, is limited by ethical considerations. Cultures of isolated epithelial cells probably reflect only a partial picture of the inflammatory response in the mucosa, because inflammatory cells such as mast cells, eosinophils, neutrophils, macrophages, lymphocytes, endothelial cells, and glandular elements are also involved in this process. There is still a need to establish human in vitro models that are safe, reliable, and physiologically relevant. The described histoculture technique allows the parallel assessment of biochemical mediator measurements, histomorphology, electron microscopy, and immunohistologic studies, and has proved its suitability as screening assay for biologically relevant substances such as O3 to study complex tissue and cell-cell interactions in the organ microenvironment of human nasal mucosa under physiologically relevant conditions.
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Footnotes |
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Address correspondence to: Prof. Dr. G. Kunkel, Department of Clinical Immunology and Asthma-OPD, Humboldt-University Berlin, Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany. E-mail: katrin.schierhorn{at}charite.de
(Received in original form December 10, 1997 and in revised form October 19, 1998).
Abbreviations: bronchoalveolar lavage fluid, BALF; granulocyte macrophage colony-stimulating factor, GM-CSF; N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid, Hepes; interleukin, IL; nasal alveolar lavage fluid, NALF; nitrogen oxide, NO2; ozone, O3; prostaglandin, PG; polymorphonuclear leukocyte, PMN; tumor necrosis factor, TNF.Acknowledgments: This work was supported by grant no. PUG L 94005, Program "Environment and Health" (PUG) at the Research Center Karlsruhe with funds of the State of Baden-Württemberg, Germany.
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References |
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Introduction
Materials and Methods
Results
Discussion
References
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  Y. Zhao, D. He, B. Saatian, T. Watkins, E. Wm. Spannhake, N. J. Pyne, and V. Natarajan Regulation of Lysophosphatidic Acid-induced Epidermal Growth Factor Receptor Transactivation and Interleukin-8 Secretion in Human Bronchial Epithelial Cells by Protein Kinase C{delta}, Lyn Kinase, and Matrix Metalloproteinases J. Biol. Chem., July 14, 2006; 281(28): 19501 - 19511. [Abstract] [Full Text] [PDF]
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 D. K. Bhalla, P. G. Reinhart, C. Bai, and S. K. Gupta Amelioration of Ozone-Induced Lung Injury by Anti-Tumor Necrosis Factor-{alpha} Toxicol. Sci., October 1, 2002; 69(2): 400 - 408. [Abstract] [Full Text] [PDF]
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J. G. Wagner, J. A. Hotchkiss, and J. R. Harkema Enhancement of Nasal Inflammatory and Epithelial Responses after Ozone and Allergen Coexposure in Brown Norway Rats Toxicol. Sci., June 1, 2002; 67(2): 284 - 294. [Abstract] [Full Text] [PDF]
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 L. Calderón-Garcidueñas, G. Valencia-Salazar, A. Rodríguez-Alcaraz, T. M. Gambling, R. García, N. Osnaya, A. Villarreal-Calderón, R. B. Devlin, and J. L. Carson Ultrastructural Nasal Pathology in Children Chronically and Sequentially Exposed to Air Pollutants Am. J. Respir. Cell Mol. Biol., February 1, 2001; 24(2): 132 - 138. [Abstract] [Full Text]
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