Nitric Oxide and Proinflammatory Cytokines in Nasal Lavage Fluid Associated with Symptoms and Exposure to Moldy Building Microbes MAIJA-RIITTA HIRVONEN, MARJO RUOTSALAINEN, MARJUT ROPONEN, ANNE HYVÄRINEN, TUULA HUSMAN, VELI-MATTI KOSMA, HANNU KOMULAINEN, KAI SAVOLAINEN, and AINO NEVALAINEN Division of Environmental Health, National Public Health Institute, Kuopio, Finland; Department of Pathology and Forensic Medicine, and Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland; and Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland
Epidemiological data indicate that living or working in a moldy building is associated with increased risk of respiratory symptoms and disease related to inflammatory reactions, but biochemical evidence linking cause and effect is still scarce. The staff working in a mold-contaminated school, and a reference group without such exposure, were studied. Nasal lavage was performed and health data were collected with a questionnaire at the end of the spring term, after a 2.5-mo summer vacation, and at the end of the fall term. Here we show that concentrations of tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), and nitric oxide (NO) in nasal lavage fluid were significantly higher in the exposed than in the control subjects at the end of the first exposure period. These inflammatory mediators decreased to reference group concentrations during the period when there was no exposure and the production of NO and IL-6 increased again during the reexposure in the fall term. Reports of cough, phlegm, rhinitis, eye irritation, and fatigue paralleled the changes in the measured inflammatory markers. These results point to an association between inflammatory markers in the nasal lavage fluid, the high prevalence of respiratory symptoms among the occupants, and chronic exposure to molds in the indoor environment. Hirvonen M-R, Ruotsalainen M, Roponen M, Hyvärinen A, Husman T, Kosma V-M, Komulainen H, Savolainen K, Nevalainen A. Nitric oxide and proinflammatory cytokines in nasal lavage fluid associated with symptoms and exposure to moldy building microbes. AM J RESPIR CRIT CARE MED 1999;160:1943–1946.
There is increasing concern about the adverse health effects of bioaerosols on people who live or work in buildings with moisture and mold problems. Recent epidemiological data indicate that those individuals complain of a variety of symptoms related to inflammatory reactions, i.e., recurrent respiratory tract infections, rhinitis, bronchitis, and asthma (1, 2). However, the biochemical evidence linking exposure to microbes present in moldy buildings to inflammatory reaction has not been previously presented. Increased production of nitric oxide (NO) and proinflammatory cytokines including tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6), are important markers of immunological activation of the cells in the respiratory tract. TNF-a and IL6 are soluble proteins that are involved in communication between the cells of the immune system. Many cytokines can activate the production of NO, a highly reactive radical, which plays a critical role in cell signaling in both physiological and patho-
(Received in original form March 1, 1999 and in revised form June 14, 1999) Supported by The Finnish Work Environment Fund and The Academy of Finland. Correspondence and requests for reprints should be addressed to Dr. Maija-Riitta Hirvonen, Division of Environmental Health, National Public Health Institute, P.O. Box 95, FIN-70701 Kuopio, Finland. E-mail:
[email protected] Am J Respir Crit Care Med Vol 160. pp 1943–1946, 1999 Internet address: www.atsjournals.org
physiological processes (3). Constitutive NO synthase (cNOS) produces picomolar concentrations of NO from L-arginine for intracellular signaling, whereas the high concentration of NO generated over long periods by inducible NO synthase (iNOS) is potentially proinflammatory and can damage the surrounding cells and tissues. NO-induced proinflammatory effects include vasodilatation, edema, cytotoxicity, and mediation of cytokinedependent processes (4, 5). We have previously demonstrated the dose- and time-dependent potential of certain moldy house microbes to induce NO and cytokine production and to cause cytotoxicity in immunological cells in vitro (6–8). It has been shown that NO is continuously produced in the nasal cavity (9, 10). Human upper airway cells express cNOS, both in inflamed and noninflamed tissues, but iNOS is expressed only in inflamed tissues (11). Because many cell types found in the epithelial lining of the nasopharyngeal region are similar to the cells of the tracheal and bronchial lining, it has been suggested that cellular responses in the nose to harmful agents are likely to be similar to those in the lower airways (12). The technique of nasal lavage is relatively noninvasive, easy to carry out, well tolerated and without adverse side effects (13, 14). The use of cytokines in nasal lavage fluid as biomarkers for health effects has been explored in previous epidemiological studies on gaseous air pollution (15). In the present study, we investigated the production of inflammatory mediators, i.e., NO, TNF-a, and IL-6 in nasal la-
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vage fluid and their association with respiratory symptoms as well as with a prolonged working period in a mold-damaged building. Moreover, we evaluated the value of nasal lavage in the assessment of health effects induced by exposure to a moldy indoor environment.
METHODS Subjects and Questionnaire on Health Status Thirty-two volunteers, 26 to 58 yr of age, working full-time in a school building with visible mold growth in the structures were studied. The subjects were teachers (22), kitchen workers (4), cleaners (3), a nurse, a janitor, and a secretary. The subjects were contacted three times during the study. The first contact was at the end of the spring term in May to evaluate the effects of a prolonged exposure period during the spring term (5 mo). The second time, at the end of the summer vacation (2.5 mo) in August, was selected to explore the effects of the absence from the moldy school. The third sampling was at the end of the fall term in December to characterize the effects of the reexposure (4 mo) after the vacation. To study the basic level and seasonal variation of the biochemical responses, eight healthy persons age 24 to 48 yr, from our institute, who had no known exposure to moldy indoor environments served concurrently as controls. Before nasal lavage, the subjects were interviewed using a onepage questionnaire concerning their current health with the emphasis on respiratory symptoms, i.e., cough, wheezing, and dyspnea and nonrespiratory symptoms such as fever, headache, muscle and joint pain, and eye symptoms during the preceding week. Moreover, use of medicines during the preceding week, and the working hours spent in the school building that day and the previous day were noted as well. The research plan was approved by the ethical committee of the National Public Health Institute.
Characterization of the Microbial Exposure in the School Building Both the school and the control building were inspected for visible signs of moisture and mold growth by a civil engineer using a checklist and a surface moisture recorder. No repairs or changes in the indoor conditions were made during the study period. To determine the airborne concentrations of viable fungi and flora in the school building, microbial samples were collected at the end of the fall term in December, when outdoor fungi do not contribute to the indoor mycoflora (16). Samples of airborne microbes were collected (10 min) with a six-stage impactor (Andersen 10-800) in 17 different rooms. The indoor surfaces (100 cm2) were sampled with a sterile swab and building material samples were taken from visibly contaminated structures. Suspensions were prepared from surface and material samples in sterile buffer with Tween 80. The media used in all environmental sampling were 2% malt extract agar with streptomycin and dichloran glycerol agar with chloramphenicol for mesophilic fungi, and tryptone–yeast extract–glucose agar with cycloheximide for mesophilic bacteria. After a 7-d incubation at 258 C, fungal colonies were counted and the genera were identified using optical microscope. Bacterial colonies were counted after 5-d and 14-d incubation at room temperature (20 to 228 C).
Nasal Lavage Nasal lavage was performed as described earlier with some modifications (12). Briefly, 4.5 ml of prewarmed Hanks’ balanced salt solution (HBSS) (378 C) was instilled through a heat-softened catheter into the nare. The subject held his or her chin down toward the chest and held the catheter in place by pinching the nares closed. The cartilaginous bridge of the nose was vibrated by a pediatric precursor while the fluid was refluxed three times. The lavage fluid was recovered and the procedure repeated in the opposite nare. The lavage fluid was placed immediately on ice until processing. Thereafter the lavage fluid sample was centrifuged, the cells were resuspended in 2 ml of supernatant of the lavage fluid, and the rest of the cell-free supernatant was aliquoted and frozen. A volume of 100 ml of resuspended cell suspension was used for cytospin; the remaining cell suspension was incubated for 20 h at 378 C, and centrifuged. The cells were frozen for Western blot analysis of iNOS. Nitrite production was measured in 100 ml of the supernatant, with the remainder being collected and frozen for later cytokine analysis.
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Cytospin Cytocentrifuge preparations were made by using 100 ml of resuspended cell suspension, in which the mucus was broken by 0.5% dithiothreitol/0.1% bovine serum albumin. The solution was centrifuged and the slides were stained with May-Grunwald-Giemsa staining (17) for the cell differential counts.
Analysis of Nitric Oxide Nitric oxide in the supernatant was assayed by the Griess reaction as the stable NO oxidation product nitrite, which produces a chromophore with the Griess reagent (18). Expression of iNOS in the cells in nasal lavage fluid was measured by Western blot analysis as described earlier (18).
TNF-a and IL-6 Analysis TNF-a and IL-6 were analyzed by using Pelikine Compact human TNF-a and IL-6 ELISA kits obtained from CLB (Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam). The assay was performed as described by the manufacturer. Reported detection limits were 3 pg/ml for TNF-a and 0.4 pg/ml for IL-6.
Statistical Analysis The data were analyzed statistically using Kruskal-Wallis one-way analysis of variance and logistic regression analysis.
RESULTS The Microbial Exposure
Visible signs of moisture damages and mold growth both on the ceilings and the base floor structures were recorded by the civil engineer in the school but not in the control building. Microbiological analysis confirmed the presence of several microbial types indicative of moisture and mold problems (19). Aspergillus fumigatus, Aspergillus versicolor, Eurotium, Exophiala, Fusarium, Phialophora, Rhodotorula, Stachybotrys, Trichoderma, Ulocladium, Wallemia, and actinomycetes were detected in the indoor air, surfaces, and/or in the building materials obtained from the school. The airborne concentrations of viable fungi varied between 7 and 100 colony-forming units (cfu)/ m3. The concentrations of airborne microbes in the control building have been low and no such indicators have been present. Symptoms
The subjects reported both respiratory and nonspecific symptoms, such as cough and phlegm production, rhinitis, eye irritation, and fatigue (Table 1). These symptoms were significantly more frequent at the end of both exposure periods, i.e., the spring and fall terms, than at the end of nonexposure summer vacation period. The symptoms after the nonexposure period did not differ from those of control subjects (Table 1). There were no statistically significant seasonal differences in the reporting of symptoms among the control subjects. NO Production and Expression of iNOS
Production of NO, assessed as nitrite levels in nasal lavage fluid, was significantly increased both at the end of the spring term (mean 2.1 mM) and at the end of the fall term (mean 3.0 mM), as compared with value at the end of the summer vacation (mean 0.8 mM) (Table 2). Moreover, at the end of the summer vacation, the mean nitrite levels in nasal lavage fluid had regressed down to the level of the control subjects (0.7 mM) (Table 2, Figure 1). Because there were no statistically significant seasonal differences in the concentrations of any of the analyzed biomarkers in the nasal lavage fluid among the control subjects, the mean values of the pooled data are presented (Table 2). Consistent with the elevated nitrite levels in the nasal lavage fluid, Western blot analysis revealed increased expression
Hirvonen, Ruotsalainen, Roponen, et al.: Inflammatory Markers and Exposure to Molds
DISCUSSION
TABLE 1 SYMPTOMS REPORTED BY THE SUBJECTS FROM THE SCHOOL AND THE CONTROL SUBJECTS* Subjects from the School End of Spring Term (n 5 32) n
Symptom Cough Phlegm Dyspnea Rhinitis Eye irritation Skin symptoms Fever Headache Fatigue Nausea
n
§
14 22 5 20 14 6 1 12 23 2
End of Fall Term (n 5 27)
End of Vacation (n 5 28)
% 44 69† 16§ 63† 44‡ 19 3 38 72† 6
%
n
¶
3 6 3 7 7 0 0 5 2 0
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11 21i 11§ 25¶ 25 0¶ 0 18 7i 0
10 16 3 13 9 5 1 11 18 1
% §§
37 59‡ ‡‡ 11§ 48§ 33§ 19‡‡ 4 41§ 67† †† 4
Control Subjects (n 5 25) n
%
4 5 0 5 2 3 0 4 1 1
16 20 0 20 8 12 0 16 4 4
* n represents individuals complaining of the symptoms. Logistic regression: † p , 0.001, ‡ p , 0.01, § p , 0.05 compared with control subjects; i p , 0.001, ¶ p , 0.01, **p , 0.05 end of vacation compared with the end of spring term; †† p , 0.001, ‡‡ p , 0.01, §§ p , 0.05 end of fall term compared with the end of vacation.
of iNOS in cells present in the nasal lavage, whereas iNOS could not be detected in the samples from control subjects (data not shown). Production of TNF-a and IL-6
Several epidemiological cross-sectional studies (20–22) and case-control studies (23) based on questionnaire data have demonstrated an association between moisture and mold growth in structures of buildings and increased frequency of respiratory symptoms among the inhabitants or occupants. Currently there is a serious lack of data based on biochemical evidence of a link between objective biomarkers, mold exposure, and subjective symptoms. The present data clearly indicate that occupational exposure in a school building with mold growth leads to an increased production of proinflammatory mediators in the nasal lavage fluid of the exposed individuals. Both technical building inspection and microbial assessment confirmed the presence of moisture problems, and highlighted the unusual microbial status of the school building. Our data indicate that there was an association between exposure, symptoms, and the proinflammatory mediators detected, especially IL-6- and iNOS-induced NO production, and therefore we propose that moldy house microbes may cause adverse respiratory health effects. Expression of iNOS in the cells obtained from nasal lavage and elevated concentrations of NO, TNF-a, and IL-6 in nasal lavage fluid suggest that there was an ongoing inflammatory process in the respiratory tract of those subjects who were working in the school building. This interpretation is supported by previous findings showing that there is an enhanced expression of iNOS in inflamed upper airways (11). We have earlier reported that the spores of actinomycetes and mycobacteria (6–8), isolated from mold problem houses, induce iNOS expression and subsequent NO produc-
The concentration of IL-6 in nasal lavage fluid was statistically significantly higher at the end of both the spring term and the fall term than that present at the end of the vacation or the corresponding values in the control subjects (Table 2). Production of the proinflammatory cytokine TNF-a was significantly increased in nasal lavage fluid at the end of the spring term (Table 2). At the end of the summer vacation, TNF-a concentrations had decreased to control levels. However, at the end of the fall term, TNF-a was not significantly different from the values at the end of the vacation and those of control subjects (Table 2). Cell Differential Count
The relative share of lymphocytes, macrophages, neutrophils, and eosinophils in nasal lavage did not differ between the periods nor did it differ from the control subjects (data not shown). No differences in cell morphology were seen between the exposed workers and the control subjects. TABLE 2 NO, TNF-a, AND IL-6 CONCENTRATIONS IN NASAL LAVAGE FLUID* Subjects from the School End of Spring Term (n 5 30) NO TNF-a IL-6
2.1 mM (0.2–8.8) 6.8 pg/ml† (0–77.5) 9.9 pg/ml† (0.4–89.0) †
End of Vacation (n 5 28) i
0.8 mM (0.2–10.1) 1.9 pg/mli (0–1.1) 2.5 pg/mli (0.2–19.5)
Control Subjects
End of Fall Term (n 5 27)
(n 5 25)
3.0 mM ** (0.5–13.9) 0.5 pg/ml (0–7.7) 17.5 pg/ml†¶ (0.3–456.1)
0.7 mM (0–3.0) 0.6 pg/ml (0–3.9) 2.0 pg/ml (0.1–6.0)
†
* The values are group means, the range is given in parentheses. Kruskal-Wallis one-way ANOVA: † p , 0.001, ‡ p , 0.01 compared with control subjects; § p , 0.001, i p , 0.01 end of vacation compared with the end of spring term; ¶ p , 0.001, **p , 0.01 end of fall term compared with the end of vacation.
Figure 1. NO production, assessed as nitrite (mM) in nasal lavage fluids of the occupants of the school and the control subjects. The occupants are identified by same symbol at different time points. The mean value is noted with a bar.
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tion and can also induce dose-dependent cytokine production and cytotoxicity in murine macrophages in vitro. The role of TNF-a and IL-6 in inflammation is widely acknowledged. In the present study, TNF-a was elevated at the end of the spring term, dropped during the summer vacation but was not significantly elevated at the end of the fall term in the same way as NO and IL-6. Differences in the induced cytokine profiles in the nasal lavage fluid of the school personnel between the two exposure periods may have been caused by different total exposure leading to activation of different cell types in the airways. Intense exposure to moldy house microbes and also outdoor microbial pollutants in the spring may have caused TNF-a production in addition to IL-6. This effect was not detected when the subjects did not have the joint exposure, i.e., in winter when they were exposed only to indoor mycoflora or after the summer vacation when they had not been exposed to the microbes present in the school building. Cytokine-producing cells and target cells form complex cellular networks within the immune system. Thus, several distinct cytokines exhibit similar biological activities and have overlapping functions; therefore, they can cause similar symptoms. The present result is also in line with our recent preliminary in vitro data showing that actinomycetes isolated from moldy houses stimulate NO and IL-6 production and decrease cell viability, but do not cause TNF-a secretion in human lung epithelial type II cells (24). This is of interest because actinomycetes were detected also from this school building. Hence, the microbes or their spores present in the school building could well have activated cells in the upper airways of the school staff. However, the specific cellular mechanisms and causality of the adverse health effects induced by each indicator microbe present in the moldy building remains to be studied. The symptoms most frequently reported by the subjects, i.e., cough, phlegm, and rhinitis, have been observed in epidemiological studies in people living in moldy houses (1, 2). Respiratory symptoms in the present subjects were more common than in staff working in a Finnish day care center with a moisture problem (25–27). In particular, rhinitis and phlegm seem to be associated with inflammatory processes in the respiratory mucosa. The symptoms disappeared and the concentrations of the inflammatory mediators in nasal lavage fluid dropped to the background level when the subjects were away from the building for 2.5 mo. It remains to be studied how quickly these changes reappear after the holidays when the subjects are reexposed to microbes. Moreover, the specific species among the moldy house microbes responsible for these inflammatory changes are not yet known, but experimental evidence suggests that these effects may be specific for certain groups of microbes (6–8). Acknowledgment : The authors thank the personnel of the school building and the volunteers from our institute for participation in the study. Virpi Koponen, Tuula Wallenius, and Heli Martikainen are acknowledged for their excellent technical assistance and Mikko Vahteristo and Asko Vepsäläinen for their statistical analyses. They also thank Dr. Ewen MacDonald and Dr. Juha Pekkanen for reading and commenting on the manuscript.
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