Effects of Ozone on the Respiratory Health, Allergic Sensitization, and Cellular Immune System in Children 1- 3

HARTMUT ZWICK, WOLFGANG POP~ CHRISTIAN WAGNER, KLAUS REISER, JOSEF SCHMOGER, ANDREAS BOCK, KURT HERKNER, and KLAUS RADUNSKY

Introduction Increased levels of ozone (0 3 ) are found in many places with highly polluted air, especially during the summer season (1). The acute physiologic effects of exposure to ozone have been examined by numerous investigators in both human and animal experiments. It is evident that exposure to ozone has toxic effects and leads to immediate and sometimes lasting dose-dependent disturbances of biochemical functions (2-8) and to morphologic changes in the respiratory tract (9-15). Several studies have demonstrated not only pulmonary function impairment caused by exposure to ozone at rest, showing that pulmonary function is impaired even more severely during exercise, but also some degree of adaptation. Ozone may also intensify bronchial hyperresponsiveness to histamine and methacholine in healthy and atopic humans and in animals (16-21); repeated exposures to ozone, however, may give rise to adaptation (22, 23). These studies were carried out in subjects who were exposed to ozone in several repeated challenges; in epidemiologic studies, however, the lasting effects of ozone on bronchial hyperresponsiveness, allergic sensitization, and the cellular immune system have remained unexplored. The object of this study was to investigate persistent bronchial hyperresponsiveness, allergic sensitization, and changes in lymphocyte subpopulations in children who, under environmental conditions, were exposed to high and others who were exposed to low concentrations of ozone in the preceding summer. Methods 'lest Subjects and Questionnaire The target group was the school population of four Austrian schools (n = 664), two of which were located in an area high in ozone concentration (Seewinkel/Burgenland; Group A); the other two schools were located in an area low in ozone concentration, which was

SUMMARY To Investigate the lasting effects of high ozone concentrations under environmental conditions, we examined the respiratory health, pulmonary function, bronchial hyperresponsiveness to methacholine, allergic sensitization, and lymphocyte sUbpopulatlons of 10-to 14-yr-old children. A total of 218 children recruited from an area with high ozone concentrations (Group A) were tested against 281 children coming from an area with low ozone concentrations (Group B). As to SUbjective complaints, categorized as "usually cough with or without phlegm," "breathlessness; and "susceptibility to chest colds," there was no difference between the two groups. The lung function parameters were similar, but In Group A subjects' bronchial hyperresponslveness occurred more frequently and was found to be more severe than In Group B (29.4 versus 19.9%, p < 0.02; P0 20 2,100 ± 87 versus 2,350 ± 58 I1g, P < 0.05). In both groups the number of children who had been suffering from allergic diseases and sensitization to aeroallergens, found by means of the skin test, was the same. Comparison of the totallgE levels showed no difference at all between the two groups. As tar as the white blood cells are concerned, the total and differential cell count was the same, whereas lymphocyte subpopulatlons showed readily recognizable changes. In Group A subjects the absolute and relative numbers of T-helper cells (OKT4+) had decreased and those of T-suppressor cells (OKT8+) had increased, which resulted In a significantly low helper/suppressor cell index (OKT4+/0KT8+: 1.44 ± 0.45 versus 1.75 ± 0.57, P < 0.0001); moreover, the absolute and relative numbers of natural killer cells (OKNK+)dropped significantly (p < 0.001and p < 0.0001).These findings suggest that long-term exposure to high ozone concentrations may lead to persistent bronchial hyperresponslveness and subclinical effects on lymphocyte subpopulations in children. AM REV RESPIR DIS 1991; 144:1075-1079

about 100 km away (Laaberg/Vienna; Group B). Both areas were characterized by the absence of large-scale industry and heavy traffic, high-quality air in view of other air pollutants, such as S02 or N0 2, and equivalent meteorologic conditions. The number of children finally participating in our investigation upon informed consent was 218 (Group A) and 281 (Group B), which amounted to almost equal participation rates (77.0% as against 73.8010). This study was performed in a 5-wk period under identical meteorologic conditions in the fall of 1989, when the pollen season was over. At the time of investigation, there was no sign of epidemic disease caused by viral infection or other pathogens in the areas studied. At least for the 2 wk before investigation, the ozone concentrations were extremely low « 60 ppb). The team of investigators remained unchanged throughout the study. A standardized pediatric questionnaire on personal and environmental conditions, which was simplified in view of local necessities, was handed to the parents for joint evaluation (24). We particularly focused on "usually cough with colds or apart from that" (ATSDLD-78-C, Questions 14A or 14B), "phlegm production with or without colds" (Questions 15A or 15B),"breathlessness" (Question 18A),

and "susceptibility to chest colds," which by definition was considered present in one or more bouts of illness a year during the preceding 3 yr (Question 16A). Before investigation the respective case history was discussed with each test person.

Data on Meteorologic Conditions and A ir Pollution As to the meteorologic conditions in the two areas, we studied the differences in temperature (mean, minimum, and maximum values and degree of change), atmospheric pressure, atmospheric humidity, steam pressure, degree

(Received in originalform December 18, 1990and in revised form June ll, 1991) 1 From the Pulmonary Department, Krankenhaus Lainz, the Pediatrics Clinic, Ludwig Boltzmann Institute for Pediatric Endocrinology and Immunology, Universityof Vienna, and the Department of Air Hygiene, Federal Environmental Office, Vienna, Austria. 2 Supported by the Lunge und Umwelt Research Project. 3 Correspondence and requests for reprints should be addressed to Dr. Hartmut Zwick, Krankenhaus Lainz, Pulmonary Department, Wolkersbergenstrasse 1, A-I130 Vienna, Austria.

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of cloudiness, wind speed, and precipitation in the past 2 yr. For the same period, air pollution data with respect to the content of S02 and N0 2, which were recorded continuously, were found to be equal in both areas. Ozone was measured continuously in the preceding 2 yr by means of a Labs Monitor 8810installed 4 m above the ground. Local emissions and artificial origins of high ozone levels were excluded by installing the monitor in a flat area at a distance of 1 km from the nearest road and 2 km from the nearest village. In both areas, the monitors were positioned downwind; in the area with high ozone concentrations, the distance between each school and the respective monitoring system was 2 km and about 13 km and in that with low ozone concentrations it was about 2 km. Repeated control measurements within the areas revealed results slightly different from those yielded by the stationary monitors « 5 ppb, or 100,70). In both areas, the periods in which the ozone concentration was above 60, 100, and 120 ppb were recorded and then compared.

Lung Function and Methacholine Provocation Spirometry was performed by means of pneumotachograph (Spirflows; Jaeger, Wurzburg, Germany) in the sitting position with the use of noseclips. FVC, FEVh and maximal expiratory flow at 500,70 expiration (MEF50) were measured according to standardized methods and related to the normal values for Austria (24, 25). In all subjects, the methacholine challenge test was performed by means of an inspiration-triggered dosimeter (Bronchoscreen's; Jaeger, Wurzburg, Germany) releasing 5 ul of methacholine inhaled during each inspiration while the subjects were quietly breathing without breath holding. The methacholine concentrations used were 0.10,70 for 28 inhalations up to a total dose of 140 ug, 1070 for 28 inhalations up to a total dose of 1,540 ug, and 50,70 for six inhalations up to a total dose of 3,040 ug, Spirometry was performed 3 min after each concentration of methacholine was administered. The provocation test was considered positive if the FEV 1 declined by 200,70. The dose required to provoke a 200,70 decline in FEV 1 (PD 20)was calculated according to the logarithmic linear model.

ZWICK, POPP, WAGNER, ET AL.

of the Phadezym'" PRIST method (Pharmacia, Uppsala, Sweden) (27, 28). IgG was measured by using a BNA nephelometer (Behring Institute, Marburg, Germany). The white blood cell count and the differential cell count were determined for each test subject in the same way: the whole blood was collected into a Vacutainers (Becton Dickinson, Meylan Cedex, France) containing 15% EDTA, stored at 4 to 8° C, and analyzed between 4 and 6 h after blood drawing. Lymphocyte subpopulations were examined by employing immunofluorescent staining with fluorescein-isothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies. Aliquots of 100 ul whole blood and 100 ul phosphate-buffered saline were incubated with 10 ul of each of the following combinations of antibodies: OKT3(CD3)FITC and OKB7(CD21)-PE, OKT4(CD4)FITC and OKT8(CD8)-PE, OKT4-FITC and OKDR-PE, and OKNK (CD16)-FITC (Ortho Diagnostic). The lymphocytes were identified by their light-scattering properties. Simultaneous immunofluorescence analysis was carried out in a cytofluorometer (Cytorons; Ortho Diagnostic, Raritan, NJ) after lysis of the red blood cells (29). The gates for the doublefluorescent signals were set in accordance with the results of autofluorescence, nonspecific immunofluorescence of FITC- or PE-conjugated nonspecific mouse antibodies, and single green or single red immunofluorescent staining.

Statistical Methods Normal distributions (Gaussian distribution) are given as mean values ± standard deviation and, in the absence of normal distribution, as the median ± standard error of the median (age, PD 20, total IgE, and eosinophil granulocytes). For qualitative statistical analysis we used the chi-square test and for quantitative group comparisons the KolmogoroffSmirnoff test (30). The influence of active and/or passive smoking on the investigated parameters was studied using multivariant analysis. A p value < 0.05 was considered statistically significant.

In the 2-yr period preceding this study, the meteorologic conditions were the same in both areas with respect to all parameters investigated: temperature (mean, minimum, and maximum degree of change), atmospheric pressure, atmospheric humidity, steam pressure, degree of cloudiness, wind speed, and precipitation. In the same period, the content of S02 and N0 2was equally low in both areas: the maximum concentrations of S02 and N0 2were 0.21 and 0.22 mg/rrr', respectively, both of which were recorded in the area with low ozone concentrations. The small amounts of these air pollutants seem to have been a result of local environmental factors, such as domestic heating and traffic. The differences in the periods in which the ozone concentrations determined by instantaneous measurements were above 60, 100, and 120 ppb in the past 2 yr proved to be highly significant (p < 0.0001 for all levels)in both areas investigated (table 1). The characteristics of the test subj ects are given in table 1. All children included in this study weremembers of the same ethnic group. The area with low ozone concentrations was a green belt in the outskirts of a city; that with high ozone concentrations was of a more rural type, with larger villages. With regard to socioeconomic groups and environmental conditions both areas were similar. They showed no difference with respect to sex and age ratios or anthropometric data; active smoking of one or more cigarettes a day was reported in similarly low percentages. The frequency of subjective complaints reported - cough, cough with phlegm, breathlessness, and susceptibility to chest colds - was the same for both

TABLE 1 STUDY SUBJECT CHARACTERISTICS AND ENVIRONMENTAL CONDITIONS· Group A, High Ozone Concentration

A//ergologic and Immunologic Tests Skin prick testing was carried out by one experienced investigator according to standardized skin prick test methods with the use of the following solutions (26): Dermatophagoidespteronyssinus, Dermatophagoides farinae, grass pollen, rye pollen, birch pollen, mugwort pollen, cat dander, dog dander, horse dander, Alternaria, Aspergillus, Cladosporium, Penicillium, negative control, and 1% histamine control (Pangramins; Bender). The skin prick test was considered positive if the diameter of the wheal was 2 mm larger than that of the negative control. Total serum IgE was determined by means

Results

(n = 218)

Subjects, M/F (%) Age, yr Height, cm Weight, kg Active smokers (%) Mean S02' mg/m3 Mean N02, mg/m 3 Maximum ozone concentration, ppb Percentage of time with ozone> 60 ppb Percentage of time with ozone > 100 ppb Percentage of time with ozone> 120 ppb

122 (56.0)/96 (44.0) 11.6 ± 0.3 152.5 ± 9.3 46.3 ± 11.7 11 (5.1)

0.010 ± 0.007 0.011 ± 0.004 188 45.39 9.68 2.5

Group B, Low Ozone Concentration (n

= 281)

173 (61.6)/108 (38.4) 11.7±0.3 153.9 ::':: 13.1 45.9 ± 11.0 22 (7.8) 0.016 ± 0.008 0.042 ± 0.012 95 0.33

o o

• Age is given as median ± standard error of median, height and weight as mean ± standard deviation. The values of air pollutants are given as mean values calculated from the 2-yr period before investigation.

EFFECTS OF OZONE ON RESPIRATORY HEALTH

1077 TABLE 2

SUBJECTIVE COMPLAINTS IN STUDY SUBJECTS· Group A, High Ozone Concentration (n = 218) Cough Cough with phlegm Breath lessness Susceptibility to chest colds

23 3 14 54

Group B, Low Ozone Concentration (n = 281)

(10.6%) (1.4%) (6.4%) (24.8%)

38 4 24 60

(13.5%) (1.4%) (8.5%) (21.4%)

p Value NS NS NS NS

• The prevalence of symptoms in the two groups was compared using the chi-square test.

TABLE 3 LUNG FUNCTION DATA AND BRONCHIAL HYPER RESPONSIVENESS·

FVC, % of predicted FEV" % of predicted FEV,/FVC, % MEFso/FVC, % Bronchial hyperresponsiveness (%) PD 2o, Ilg

Group A, High Ozone Concentration (n = 218)

Group B, Low Ozone Con centration (n = 281)

p Value

98.7 ± 10.3 98.0 ± 10.7 84.8 ± 5.0 105.5 ± 19.8 64 (29.4) 2,100 ± 87

98.5 ± 11.2 98.1 ± 11.6 84.9 ± 5.5 104.7 ± 20.6 56 (19.9) 2,350 ± 58

NS NS NS NS < 0.02 < 0.05

• Spirometric data are given as mean values ± standard deviations. FEV,/FVC (%) and MEFsolFVC(%) are given as percentage of FVC. Bronchial hyperresponsiveness as prevalence and PD20 as median ± standard error of the median.

groups (table 2). All these complaints were found to be independent of sex, age, and environmental influences, such as active or passive smoking and cooking and heating modes. In both groups, the lung function parameters were similar (table 3). No difference was found with respect to FVC, FEV i t and the FEV l/FVC. Even the MEF so/FVC, which was equally high in all children, proved to be independent of the differences in ozone conentration. Bronchial hyperresponsiveness to methacholine was found in 78 male and in 42 female children (26.4 versus 20.6010; NS). It was detected in 64 of 218Group A subjects and in 56 of 281 Group B subjects (29.4 versus 19.9%; p < 0.02). What is

more, bronchial hyperresponsiveness was more severe in the Group A subjects. The results of bronchial hyperresponsiveness were influenced neither by other factors, such as passive smoking or cooking and heating modes, nor by colds during the 6 wk before this investigation. In both groups, allergic diseases of the conjunctiva and respiratory tract were reported in equal numbers (table 4): the total numbers of manifest allergies were 31of 218Group A subjects and 41 of 281 Group B subjects (14.2 versus 14.6%; NS). In Group A, allergic rhinitis was reported by 13.8% (Group B, 13.9%), allergic conjunctivitis by 2.3% (Group B, 5.3010), and bronchial asthma by 2.8010 (Group B, 3.6%) of the test subjects. In

TABLE 4 ALLERGOLOGIC RESULTS· Group A, High Ozone Concentration (n = 218) Allergic disease Rhinitis Conjunctivitis Bronchial asthma Positive skin prick test Total IgE, kUlL Total IgE, > 100 kU/L Total IgG, mg/100 ml

31 (14.2%) 30 (13.8%) 5 (2.3%) 6 (2.8%) 45 (20.6%) 62 ± 7 82 (37.6%) 1,229 ± 423

Group B, Low Ozone Concentration (n = 281) 41 39 15 10 68

(14.6%) (13.9%) (5.3%) (3.6%) (24.2%) 59 ± 6 104 (37.0%) 1,178 ± 310

P Value NS NS NS NS NS NS NS NS

• Results are prevalences, excepttotallgE, which is also given as median ± standard error of the median and total IgG, which is given as mean ± SO.

Group A, 24.2 versus 20.6% of the test subjects in Group B were found positive to the skin prick test. In both groups, sensitization to grass pollen was found most frequently, followed by sensitization to house dust mite, tree pollen, mold spores, and animal dander. Furthermore, there were no differences between the two groups in the total serum IgE levels with respect to the median, distribution, or number of increased values (>100kU/L). IgG levels did not differ at all. As to the total leukocyte and differential count, there was no difference between Group A and Group B subjects (table 5). The cellular immune system revealed the following changes. Group A children showed significantly decreased absolute and relative numbers of OKT4+ cells and increased absolute and relative numbers of OKT8+ cells, which consequently resulted in a lower OKT4+/ OKT8+ cell index (p < 0.0001). In these children the absolute and relative numbers of OKNK+ cells also dropped significantly (p < 0.0001). All allergologic and immunologic results were analyzed for the influence of active and/or passivesmoking, which, however,had no impact on the results of our study, neither within a group nor when comparing the two groups. Discussion

Ozone is a common air pollutant (1),and it may account for many acute physiologic effects in humans exposed to it (31-34). Recent studies reported on subjects who were exposed to ozone in several repeated challenges (16, 17, 35-46). The object of our study was to examine, in children, the lasting effects of perennial exposure to high and low concentrations of ozone under environmental conditions on bronchial hyperresponsiveness, allergic sensitization, and lymphocyte subpopulations. Both areas studied were characterized by the absence of large-scale industry and heavy traffic and by high-quality air with respect to S02 and N0 2, which obviously were the result of local emissions. Ozone itself seems to be caused by local rather than anthropogenic factors of air pollution in the summer season. The area with high ozone concentrations lies in the vicinity of a large steppe lake (Neusiedler See), which is deemed the source of high ozone concentrations because of volatile organic compounds. As far as reported symptoms - cough, phlegm production, breathlessness, and the susceptibility to chest colds - are con-

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ZWICK, POPP, WAGNER, ET AL.

TABLE 5 CHANGES IN LYMPHOCYTE SUBPOPULATIONS·

Leukocytes per microliter Lymphocytes (absolute) Lymphocytes, % Eosinophils (absolute) Eosinophils, % OKT3' (absolute) OKT3., 0/0 OKB7' (absolute) OKB7', % OKT4' (absolute) OKT4', % OKT8' (absolute) OKT8', % OKT4'/OKT8' OKDR' (absolute) OKDW, % OKNK+ (absolute) OKNK., %

Group A, High Ozone Concentration (n = 218)

Group B, Low Ozone Concentration (n = 281)

7,418 2,938 40.6 186 3.3 2,010 68.3 261 8.9 1,114 38.1 831 28.0 1.44 375 12.9 591 20.0

7,152 2,940 42.1 201 3.4 2,053 69.8 257 8.6 1,197 40.7 728 24.8 1.75 392 13.1 676 23.1

± 1,880 ± 824 ± 9.6 ± 14 ± 0.3 ± 619 ± 6.8 ± 133 ± 3.9 ± 341 ± 6.3 ± 324 ± 5.5 ± 0.45 ± 197 ± 5.8 ± 266 ± 6.8

± ± ± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ±

1,861 821 10.0 13 0.3 623 6.8 147 3.7 386 6.6 260 5.4 0.57 198 4.9 255 6.0

NS NS NS NS NS NS NS NS NS P < 0.05 P < 0.01 P < 0.001 P < 0.0001 P < 0.0001 NS NS P < 0.001 P < 0.0001

• The percentages of lymphocyte subpopulations were derived from the total lymphocyte count. The values are given as mean

:r standard deviation, except for eosinophils, which are given as the median ± standard error of the median.

cerned, the high ozone concentrations did not influence these variables lastingly enough to make the resulting changes statistically significant. We do not doubt that acute symptoms may be caused by exposure to high ozone concentrations, but the resultsof this epidemiologic study suggest that lasting effects on the subjective feeling or the susceptibility to colds may be precluded. The lung function parameters were similar to the clinical data. Exposure to high concentrations of ozone for at least the 2 yr before investigation had no lasting influence on the spirometric results. This phenomenon may be due to the absence of acute exposure to ozone during the investigation rather than to adaptation (18, 22, 42-44). On the contrary, we found bronchial hyperresponsiveness to occur more frequently and more severely in Group A subjects, which indicates a subclinical, persistent, and noxious effect (47). These findings on bronchial hyperresponsiveness may be an indication of lasting airway inflammation, which was recently described for the upper respiratory tract (48). Previous colds had no influence whatsoever on the incidence of bronchial hyperresponsiveness. Allergic diathesis and sex (male) increased the number and the severity of bronchial hyperresponsiveness, but these two factors did not account for the differences in the findings between Group A and Group B subjects. Because both test groups were identical, we precluded other possible influences on bronchial hyperrresponsiveness.

With regard to allergologic diseases, sensitization to aeroallergens, and total serum IgE, we found no significant difference between the two groups. On the one hand, one might suspect an increase in the sensitization to aeroallergens because of the undermining effect of the mucosal defense barrier and the increased absorption of antigens via the irritated membrane (49, 50); on the other hand, an immunosuppressive or toxic effect might be expected. It has recently been shown that the responsiveness to allergen challenge in the upper respiratory tract is not increased by the inhalation of ozone (48). As far as the lymphocyte subpopulations are concerned, the decrease in helper cells (OKT4+) and natural killer cells (OKNK+), together with the increase in suppressor cells (OKT8+), may be interpreted as an alternative effect of ozone on lymphocyte subpopulations. Further functional studies could support these findings. In addition, no influence was detected in the two groups that may have been traceable to IgG levels. The results regarding lymphocyte subpopulations were not traceable to colds shortly before investigation or to other anamnestic or clinical findings. As to the changes in lymphocyte sub populations, no definite explanation can be given. It may be speculated that alterative effects of ozone are involved,however, since following the exposure to ozone such effects occur in other biochemical systems as well (2, 4-7, 47, 51, 52). From these findings we conclude that frequent and long-term exposure to in-

creased concentrations of ozone may lead to lasting effects on the respiratory tract system and the levels of lymphocyte subpopulations of children. Even though these changes remained subclinical, we believe that they are sensitive indicators for the assessment of the influence of air pollution. Acknowledgment The authors thank Mrs. E. Handler and Mrs. B. Schreiber for their excellent technical assistance.

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EFFECTS OF OZONE ON RESPIRATORY HEALTH

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Effects of ozone on the respiratory health, allergic sensitization, and cellular immune system in children.

To investigate the lasting effects of high ozone concentrations under environmental conditions, we examined the respiratory health, pulmonary function...
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