Ozone Effects on the Immediate-Phase Response to Allergen in the Nasal Airways of Allergic Asthmatic Subjects

Abstract

Epidemiologic and clinical trials have suggested that exposure to ozone increases airway hyperre-sponsiveness and inflammatory response to inhaled nasal allergen challenge in allergic asthmatic subjects. Previous studies have demonstrated an increased late-phase response to nasal allergen challenge; however, the early-phase response is unknown. We sought to characterize the early-phase response by measuring mast-cell inflammatory mediators and cellular influx at time points immediately following ozone exposure and subsequent allergen challenge. A cohort of mild, asymptomatic dust mite–sensitive asthmatic subjects was identified. Each subject underwent two separate exposures to both 0.4 ppm ozone and clean air in a randomized manner. Nasal lavage was performed before and after each exposure. Nasal allergen was then administered to a defined clinical end point, followed by nasal lavage. Differential cell counts and mast-cell products were identified in each lavage specimen. The mast-cell mediators tryptase and prostaglandin D₂ were analyzed, as was a marker of epithelial cell permeability, albumin. Although allergen produced an increase in early-onset mediator release (mast cell–derived), no enhancement was noted after exposure to ozone. Neutrophil and eosinophil inflammatory mediators were not increased after ozone exposure or enhanced after allergen exposure, although ozone did enhance eosinophilic influx after exposure to allergen. Ozone exposure does not promote early-phase–response mediator release or enhance the response to allergen challenge in the nasal airways of extrinsic asthmatic subjects. Ozone, however, may promote an inflammatory cell influx, which helps induce a more significant late-phase response in this population.

Epidemiologic investigations suggest that asthmatic individuals have increased morbidity after exposure to ozone (O₃). 1 , 2 It has been suggested that the link between asthma morbidity and increased ambient ozone levels is due to an enhancement of the effects of allergen in this population. 3 Molfino et al 4 reported that a resting exposure to 0.12 ppm ozone for one hour enhanced immediate bronchial sensitivity to inhaled allergen in six of seven allergic asthmatic subjects, as determined by decrements in 1-second forced expiratory volume (FEV₁). Likewise, Jorres and colleagues 5 observed that exposure to 0.25 ppm ozone for 3 hours with moderate exercise increased immediate bronchial sensitivity to allergen in allergic asthmatic subjects. Therefore ozone exposure appears to enhance the immediate-phase responses of the airway to inhaled allergen.

Previously we reported that exposure of mite-sensitive allergic asthmatics to 0.4 ppm ozone enhances the late-phase responses to allergen. 6 In that study, after separate exposures to clean air and ozone, subjects underwent graded allergen challenge until a predetermined level of nasal symptoms was observed. Nasal allergen challenge can induce both immediate-phase responses—mediated largely by mast cells—and late-phase responses, which involve influx of neutrophils and eosinophils. 7 After exposure to ozone, many subjects required less allergen to induce immediate symptoms, suggesting that both the early- and late-phase responses to allergen are enhanced after exposure to ozone.

The mechanism by which ozone enhances the response to allergen challenge is not known. In this study, we examined the effect of ozone on the immediate-phase response to allergen in the nasal airways of mite-sensitive asthmatic subjects. The ozone exposure/nasal allergen challenge protocol we used was a modification of one previously reported. 6 Nasal lavage was performed immediately after allergen challenge, with the principal end points being the symptomatic response to allergen; mast-cell responses, as indicated by tryptase and prostaglandin D₂ (PGD₂); and, as a measure of mucosal permeability, albumin. The results reported herein demonstrate that ozone does not enhance mediator release during the immediate-phase response to allergen in the nasal airway of extrinsic asthmatic subjects.

Table 1

Allergen doses given to induce nasal symptoms after air and ozone exposures

Subject no.	Clean air (AU)	Ozone (AU)
1	10	10
2	10	10
3	100	10
4	10	100
5	100	100
6	10	10
7	10	10
8	10	100
9	100	100
10	100	100
11	10	10
12	10	10

METHODS AND MATERIAL

The protocol used in this study is detailed in our previous report. 6 It was reviewed and approved by the Committee for the Protection of the Rights of Human Subjects of the University of North Carolina. Fifteen mildly asthmatic subjects between the ages of 18 and 35, with skin-test reactivity to Dermatophagoides farinae extract (Greer Laboratories, Lenoir, N.C.), were recruited and their informed consent obtained. All subjects were otherwise healthy and had sustained no illnesses within 6 weeks of enrolling in the study.

A diagnosis of asthma was based on one of the following criteria:

(1) symptoms of variable wheeze, shortness of breath, chest tightness, or cough; and (2) documentation of spontaneous or β-agonist–induced reversibility of airflow obstruction as determined by a 15% or greater change of either 1-second forced expiratory volume (FEV₁) or peak expiratory flow rate (PEFR).

None of the subjects had smoked in the 5 years preceding the study, and none had a history of chronic pulmonary disease (other than asthma) or acute respiratory illness within 8 weeks of the study. Limited use of anti-inflammatories and antioxidant medications was allowed before participation in the study. No use of any aspirin, vitamin C or E, or antihistamine was allowed for at least 1 week before the study. Systemic or topical steroids were acceptable for up to 6 weeks before the study. Astemizole was not permitted in the 3 months preceding the study. Subjects were allowed to use β-agonists, theophylline, and inhaled cromolyn or steroids (although nasal steroids and nasal cromolyn were not acceptable).

Recent or recurring exposure to dusty, irritating, or allergic environments was not permitted in the study subjects. Their forced vital capacity (FVC) and FEV1 had to be within 65% of predicted normal limits for age and height.

Enrollment was denied subjects who had been exposed to either a pollutant or an allergen as part of another Environmental Protection Agency study within 5 weeks of entry into this study.

Each subject underwent two separate 2-hour chamber exposures to clean filtered air or 0.4 ppm ozone in a randomized, double-blind manner. 8 A baseline spirometry reading was taken and nasal lavage performed before each exposure. The subjects were at rest throughout the exposures. Exposures were conducted in a 13 × 20-foot chamber at the U.S. EPA Human Studies Facility in Chapel Hill, N.C. They were carried out with computer-controlled temperature (21° C), humidity (40%), light, and pollutant concentrations. Technicians monitored the chambers at all times during the studies to ensure that actual pollutant gas did not exceed ±5% of the desired concentrations. Each subject was directly observed and monitored by way of electrocardiographic telemetry throughout the study.

Nasal lavage was performed with a metered-dose atomizer, delivering 0.1 mL with each spray. Each subject self-administered 40 sprays, in eight five-spray sequences, to deliver a total of 4 mL. After each five-spray sequence, the fluid (nasal-lavage fluid) was collected in a sterile specimen container, held below the naris, until the entire sequence was completed. This protocol was identical to techniques reported by Peden et al 6 and was done both before and immediately after each chamber exposure. Each subject was then administered, by way of nasal atomizer, either allergen (D. farinae extract, Greer Laboratories), in increasing doses (10, 100, 1000, 10,000 allergen units [AU]) or sham normal saline solution control using the “split-nose” technique of our previous study. This technique, of concomitantly delivering allergen and sham normal saline solution to each subject, in alternate sides of the nose, permits each individual to serve as his or her own control. The dose (concentration) administered increased in 15-minute intervals, on the “allergen” side only, until nasal-symptom scores exceeded 5 (based on subjective scoring of rhinorrhea, burning or itching, congestion and sneezing on a scoring scale of 0 [no symptoms], 0.5 [trace symptoms], 1 [mild symptoms], 2 [moderate symptoms], and 3 [significant symptoms]), or until 10,000 AU (61 mg of group I allergen) had been administered.

Fig 1

Soluble mast-cell mediators in nasal lavage fluid from the allergen nostril after air and ozone exposure. A, Tryptase; B, PGD₂.O₃ , Ozone.

In an attempt to keep the allergen doses uniform for both exposures, we modified the allergen challenge from the technique used in our earlier study. 6 For a given subject, the allergen given during the second challenge was advanced until the dose given equaled that administered for the first challenge (Table 1). The only exceptions were made when subjects became symptomatic with lesser amounts of allergen on the second challenge.

Fifteen minutes after completion of allergen administration, a third nasal lavage, of both nares, was performed. Fluid from the final bilateral nasal lavage was collected 4 hours after the allergen challenge, and the subjects were then dismissed. In this manner, we were able to analyze immediate-phase responses by focusing on bilateral nasal lavage fluid collected at time 0 (before exposure), 2 hours after exposure, and 2.25 hours after allergen challenge.

We assessed cell viability with the use of trypan blue exclusion, and the numbers of live cells, dead cells, and total cells were determined from unprocessed nasal-lavage fluid with the use of a hemocytometer. A 0.4-mL aliquot of lavage fluid was cytocentrifuged onto a glass slide and stained with a modified Wright stain (Fisher Leukostat Stain Kit; Fisher Scientific, Pittsburgh, PA). Differential counts were then made on a minimum of 200 cells and the percentages of eosinophils, neutrophils, and epithelial cells determined. The percentage of the total cell count was expressed as number of cells per milliliter of lavage fluid.

Mast-cell activity was assayed on the basis of tryptase and histamine levels. We determined tryptase concentration using an ¹²⁵I-tryptase radioimmunoassay kit (Kabi Pharmacia, Inc, Piscataway, NJ). PGD₂ was measured with the use of a ³H-radioimmunoassay system (Amersham Life Sciences, Arlington Heights, IL). Tumor necrosis factor (TNF)-α was assayed with an ELISA kit purchased from R&D Systems (Minneapolis, MN). Albumin was assayed by means of competitive-ELISA techniques described previously with the use of reagents purchased from Calbiochem (San Diego, CA).

Statistical analysis was conducted as in our previous study. 6 We analyzed the effect of ozone by comparing the difference between postexposure or postchallenge time points and the baseline value for a given component of nasal lavage fluid. Comparison between ozone exposure and clean-air exposures were assessed with the same formulas previously reported. The null hypothesis was again that ozone has no effect on the given component of nasal lavage fluid. The Wilcoxon signed-rank statistic was again used in a one-sided test of the null hypothesis, and approximate P values were calculated.

Fig 2

. Soluble cytokines and albumin in nasal lavage fluid from the allergen nostril after air and ozone exposure. A, TNF-α; B, albumin.O₃ , Ozone.

RESULTS

Fifteen allergic asthmatic subjects were identified and enrolled in the study. Twelve completed both clean-air and ozone exposure protocols. Each subject underwent 12 separate lavages of 4 mL each. Each subject underwent lavages on both allergen-exposed and sham-exposed nostrils, at each of three time points, on both ozone- and air-exposure days, resulting in 12 total specimens. Of the total 144 samples, the average volume was 2.8 ± 0.1 mL, a recovery comparable to that in our previous study.

Dosing of nasal allergen was achieved by incremental increases of D. farinae extract, up to a clinical score of ≥5, as described in the Method section. Because we were more interested in comparing end points—both soluble mediators and cell counts, at equivalent allergen dosages—upon second exposure, the subjects received the same dosage of allergen as they did previously. As noted, the exception to this, was in those subjects who became clinically symptomatic at a dosage less than that which they had received during their first challenges. The amount of allergen required after clean-air exposure was 40.9 ± 12.8 AU, with the amount increased to 47.5 ± 13.4 AU after ozone exposure. Allergen doses given to each individual subject are depicted in Table 1.

Table 2

Cell recovery in nasal lavage fluid from allergen and nonallergen (control) nostrils after air and ozone exposure

	Live cells	Dead cells	Neutrophils	Eosinophils
Lavage	Clean air	Ozone	Clean air	Ozone	Clean air	Ozone	Clean air	Ozone
Allergen
Baseline	438 ±119	2228 ± 1822	395 ± 67	2273 ± 2000	176 ± 64	685 ± 496	26 ± 11	96 ± 84
2	449 ± 210	475 ±152	390 ± 172	273 ±114	207 ± 85	188 ± 63	77 ± 63	65 ± 36
3	118 ± 51	134 ± 66	95 ± 21	170 ± 73	38 ± 28	47 ± 24	4.0 ± 1.4	13.2 ± 4.7
Nonallergen
Baseline	455 ± 193	716 ± 376	365 ± 78	500 ± 270	321 ± 164	369 ± 248	46 ± 31	255 ± 240
2	236 ± 103	1424 ± 1068	158 ± 49	512 ± 265	181 ±66	523 ± 381	22 ± 11	114 ± 79
3	67 ± 32	1182 ± 1069	88 ± 21	1689 ± 1612	16 ± 5	355 ± 301	3.9 ± 2.3	147 ± 137
Data expressed as mean ± SD cellular constituents (cells/milliliter nasal lavage fluid × 1000) after allergen challenge.

Table 3

Soluble mediators in nasal lavage fluid from allergen and nonallergen (control) nostrils after air and ozone exposure

		Tryptase (U/mL)	PGD₂ (pg/mL)	TNF-α (pg/mL)	Albumin (mg/mL)
Lavage	Clean air	Ozone	Clean air	Ozone	Clean air	Ozone	Clean air	Ozone
Nonallergen
Baseline	10.1 ± 5.4	5.3 ± 1.5	36.5 ± 3.9	27.6 ± 2.8	110 ± 30	87 ± 29	752 ± 340	618 ± 245
2	4.8 ± 2.7	4.6 ± 1.2	30.5 ± 2.8	31.9 ± 4.1	84 ± 28	65 ± 28	2869 ± 2036	1412 ± 987
3	14.1 ± 5.1	5.4 ± 1.1	47.7 ± 14.1	37.9 ± 6.2	148 ± 60	92 ± 42	2903 ± 2032	826 ± 258
Allergen
Baseline	9.8 ± 4.8	5.6 ± 1.7	30.2 ± 4.0	36.0 ± 8.1	112 ± 40	124 ± 36	611 ± 242	666 ± 275
2	4.7 ± 1.8	3.9 ± 1.0	39.4 ±7.1	29.8 ± 2.7	97 ± 43	62 ± 17	1694 ± 577	994 ± 485
3	25.5 ± 7.2	27.5 ± 6.6	61.0 ± 10.9	72 ± 11.1	114 ± 47	98 ± 43	1953 ± 613	3376 ± 1986
Data expressed as mean ± SD.

As shown in Fig 1 A, there was a significant increase in tryptase levels recovered in nasal lavage fluid allergen challenge, demonstrating that a mast-cell response to allergen did occur. However, we noted no difference in allergen-induced tryptase release after the air- and ozone-exposure sessions, indicating that ozone exposure had no effect on mast-cell responses to ozone. Likewise, concentrations of PGD₂, compared with baseline values, were equally increased immediately after allergen challenge following either air or ozone exposure, again showing a failure of ozone to exert an effect on mast-cell response to allergen (Fig 1 B). Histamine values were unchanged in samples throughout the experiment, regardless of whether the samples were collected after allergen challenge or ozone exposure (data not shown).

Influxes of neutrophils and eosinophils were also examined in nasal lavage fluid at three time points. They are depicted in Table 2. Remarkable variation in baseline values persisted through the second time point (after exposure but before allergen challenge), making comparison with baseline values difficult. However, after the first two lavages, variability was decreased, permitting more direct comparison of the lavage fluid after air and ozone exposure. When we directly compared eosinophil counts obtained after allergen challenge, we noted a clear ozone enhancement of eosinophil influx after allergen challenge (4.0 ± 1.4 vs. 13.2 ± 4.7 × 1000 cells/mL lavage fluid, air vs. ozone, P < 0.05).

We also examined the combined effect of ozone exposure and allergen challenge on TNF-α. As shown in Fig 2 A, neither an allergen- nor an ozone-related increase in TNF-α was found. We did note a nonsignificant trend for albumin levels to be increased after allergen challenge in the wake of either air or ozone exposure (Fig 2 B). There was no pre–allergen challenge increase in albumin after ozone exposure, indicating no ozone-induced increase in mucosal permeability before allergen challenge. There was also no suggestion of enhancement of allergen-induced increases in permeability after ozone exposure.

Nasal lavage fluid was collected from the contralateral, non–allergen-challenged nostril at the same time points as described for the allergen-challenged nostril. However, at these time points, the nonallergen nostril was sham-challenged with saline rather than allergen, as previously described. 6 As described earlier, nasal lavage fluid was assayed for mast-cell products (tryptase, PGD₂, histamine), inflammatory cells (neutrophils, eosinophils), albumin, and TNF-α (Table 3).

Fig 3

. Soluble mast-cell mediators in nasal lavage fluid from the nonallergen nostril after air and ozone exposure. A, Tryptase; B, PGD₂.O₃ , Ozone.

As depicted in Fig 3, ozone exposure did not induce release of tryptase or PGD₂. We found substantial variability in both PMN and eosinophil counts in nasal lavage fluid recovered at each time point; therefore there was no significant effect of ozone alone on inflammatory cell influx (Table 2). Finally, we detected no ozone-induced increases in TNF-α, or albumin (Fig 4 A and 4B).

DISCUSSION

Previous investigators have observed that in asthmatic subjects, lower-airway reactivity to allergen is increased after exposure to ozone. 9 Potential mechanisms for this phenomenon include increased sensitivity of mast cells to IgE-mediated stimulation or increased mucosal permeability, which would permit increased delivery of allergen to resident mast cells for a given dose of allergen. Alternatively, decreased metabolism of mast cell–derived mediators, as a result of ozone exposure or increased end-organ sensitivity to these mediators, could account for increased bronchial sensitivity to allergen.

In this study, we found no difference in tryptase (a mast-cell granule constituent) or PGD₂ (a mediator generated de novo after IgE stimulation of mast cells) recovered in nasal lavage fluid after allergen challenge. This is consistent with findings made by Wang et al, 10 who reported that NO₂, another oxidizing pollutant, did not enhance allergen-induced mast-cell degranulation in atopic subjects. These findings are also consistent with in vitro studies in which ozone inhibited, rather than augmented, both IgE-mediated degranulation and A23187-stimulated eicosanoid release of the RBL-2H3 mast-cell line. 11 Thus a direct priming of mast cells to IgE-mediated stimulation probably does not account for the effect of this pollutant on bronchial reactivity to inhaled allergen.

Fig 4

. Soluble cytokines and albumin in nasal lavage fluid from the nonallergen nostril after air and ozone exposure. A, TNF-α; B, albumin.O₃ , Ozone.

Using albumin as a marker for mucosal permeability, 12 we could find no enhancement of nasal mucosal permeability after ozone exposure. Therefore, in the nasal airway, increased allergen interaction with mast cells as a result of increased permeability seems unlikely after ozone exposure. This finding is also consistent with the failure of ozone to augment allergen-induced mast-cell activation. However, high-level ozone exposure of normal subjects has been found to increase pulmonary permeability. 13 Likewise, albumin has been reported to be increased in bronchoalveolar lavage fluid of normal subjects following exposure to ozone. 14 It is possible that the nasal epithelium is more resistant to the effect of ozone on mucosal permeability than the bronchial mucosa. Such differential susceptibility could account for differences in the effect of ozone on immediate-phase responses to allergen in these two regions of the airway.

Ozone may also act by blunting degradation of mediators released by the nasal epithelium and submucosal cells. Several mediators are released during the immediate phase response to allergen, including mast cell–granule constituents, such as histamine and tryptase, as well as other mediators such as neuropeptides. 15 Additionally, there is preliminary evidence that substance P is released from airway neurons after ozone exposure. 16 If enzymes that degrade mediators released after allergen or ozone exposure are diminished by such ozone exposure, then the effect of these mediators would be magnified. Epithelial cells are a significant source of N-methyl histamidase, neutral endopeptidase and secretory leukocyte protease inhibitor. 17 Ozone can result in epithelial injury and loss of these epithelial cell-derived enzymes could be a mechanism by which immediate-phase reactivity to allergen could be augmented. Further studies specifically examining the effect of ozone on anti-inflammatory properties of the epithelium are indicated.

The effect of ozone alone on the nasal mucosa was also examined with the use of the same endpoints outlined. Ozone exposure, without allergen challenge, result in neither mast-cell degranulation nor PGD₂ release in allergic asthmatic subjects. These findings contrast with those of Graham and Koren, 14 who found ozone to increase mast cell tryptase and PMN number in nasal lavage fluid of normal subjects shortly after ozone exposure. It is unclear why such differences are found when contrasting normal and asthmatic subjects.

CONCLUSION

In summary, we report that ozone does not enhance immediate-phase mediator release from mast cells after allergen challenge, in the nasal airway of allergic asthmatic subjects. However, we did observe an enhancement of allergen-induced eosinophil influx to the nasal mucosa, consistent with results of others and reported in our previous study.

Taken together, these results indicate that ozone enhances late-phase inflammation due to allergen without altering mast-cell responsiveness. This, in turn, suggests that airway epithelial cells and neurons, which interact with mast-cell mediators and participate in the late-phase inflammation, are likely targets of ozone.

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Subject no.	Clean air (AU)	Ozone (AU)
1	10	10
2	10	10
3	100	10
4	10	100
5	100	100
6	10	10
7	10	10
8	10	100
9	100	100
10	100	100
11	10	10
12	10	10

Subject no.	Clean air (AU)	Ozone (AU)
1	10	10
2	10	10
3	100	10
4	10	100
5	100	100
6	10	10
7	10	10
8	10	100
9	100	100
10	100	100
11	10	10
12	10	10

Subject no.	Clean air (AU)	Ozone (AU)
1	10	10
2	10	10
3	100	10
4	10	100
5	100	100
6	10	10
7	10	10
8	10	100
9	100	100
10	100	100
11	10	10
12	10	10