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Original Paper Int Arch Allergy Immunol 2010;152:151–158 DOI: 10.1159/000265536

Received: June 2, 2009 Accepted after revision: August 11, 2009 Published online: December 16, 2009

Occupational Endotoxin Exposure Reduces the Risk of Atopic Sensitization but Increases the Risk of Bronchial Hyperresponsiveness Lidwien A.M. Smit a Dick Heederik a Gert Doekes a Jan-Willem J. Lammers b Inge M. Wouters a a b

Institute for Risk Assessment Sciences, Division of Environmental Epidemiology, Utrecht University, and Department of Pulmonary Diseases, University Medical Centre Utrecht, Utrecht, The Netherlands

Key Words Asthma ⴢ Atopy ⴢ Bronchial responsiveness ⴢ Epidemiology ⴢ Farming ⴢ Hygiene hypothesis ⴢ Occupational exposure

Abstract Background: Microbial exposures in both childhood and adult life are protective against atopy, allergic rhinitis and atopic asthma. In adults, this protective effect is paralleled by an increased prevalence of non-atopic asthma. This study was undertaken to investigate associations between occupational endotoxin exposure and atopic sensitization and bronchial hyperresponsiveness to methacholine (BHR) in agricultural workers. In addition, the role of atopy in endotoxinrelated respiratory effects was studied. Methods: Data were available for 427 farmers and agricultural industry workers, for whom airborne endotoxin exposure levels were estimated by 249 personal exposure measurements. Atopy was assessed as specific serum IgE to common inhalant allergens, and respiratory symptoms and personal characteristics by standardized questionnaires. BHR was determined in a subset of 113 subjects. Associations were adjusted for age, sex, smoking and living on a farm during childhood. Results: Endotoxin exposure was positively associated with BHR and wheeze (p ! 0.05). In contrast, endotoxin exposure was inversely associated with atopy and IgE to grass pollen (p !

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0.001). The proportions of wheeze and BHR that were attributable to atopy were only 16.6 and 32.8%, respectively. Conclusions: High endotoxin exposure is a risk factor for BHR and wheeze, which were characterized by a predominantly non-atopic phenotype. At the same time, endotoxin exposure is related to a reduced risk of atopy and IgE to grass pollen in adults. It is unlikely that this is entirely a result of healthy worker selection, as significant inverse associations between endotoxin and IgE to grass pollen were found regardless of reported allergic symptoms. Copyright © 2009 S. Karger AG, Basel

Introduction

There is increasing evidence that farm exposures throughout life are protective against atopy, allergic rhinitis and atopic asthma [1]. Several studies have observed a strongly decreased prevalence of allergic sensitization [2–4], hay fever [5] and asthma [6] among adults with childhood and current farm exposures. Farmers, and children growing up on farms, are exposed to high levels of microbial agents, such as bacterial endotoxin and fungal ␤(1]3)-glucans [7, 8]. It has been hypothesized that exposure to such agents may induce a shift from atopic Th2 responses to Th1 responses through stimulation of Correspondence to: Dr. Lidwien A.M. Smit Institute for Risk Assessment Sciences Division of Environmental Epidemiology, Utrecht University PO Box 80178, NL–3508 TD Utrecht (The Netherlands) Tel. +31 30 253 9494, Fax +31 30 253 9499, E-Mail L.A.Smit @ uu.nl

the innate immune system and regulatory T cells [9]. Protective effects of house dust endotoxin on the development of atopy and asthma have been shown in children [10–12], and more recently, studies among adults have shown similar inverse relationships between endotoxin exposure and atopic asthma [7], allergic sensitization [13, 14] and hay fever [15]. Since endotoxin is also a potent pro-inflammatory agent, the downside of increased exposure can be an elevated risk of non-allergic respiratory health effects [16– 18]. Only a few studies have explicitly reported the Janusfaced nature of endotoxin – a protective effect on atopic disease, paralleled by an increased risk of non-atopic asthma and nonspecific airway hyperresponsiveness – in the same population [7, 11, 13]. We recently observed this dual nature, specifically associated with adult exposure in farmers and agricultural processing workers. We found a significantly increasing prevalence of wheeze at high endotoxin exposure, but an inverse association between endotoxin exposure and selfreported hay fever [15]. The latter finding is compatible with a presumed down-regulatory effect of endotoxin exposure on atopic immune responses, since hay fever symptoms are usually strongly associated with atopic sensitization [19]. The dose-response relationship between endotoxin and wheeze was independent of self-reported allergy, a surrogate for atopy [15], suggesting that wheezing reported in this population may not be due to atopic airway inflammation. This might explain the paradoxical association with wheezing at high endotoxin exposure, since wheeze is usually associated with atopy [20]. The aim of the present study was to further elucidate this apparent paradox by using objectively measured health data: serum IgE to common allergens and nonspecific bronchial hyperresponsiveness (BHR). Specific objectives were to test the hypotheses that (1) occupational endotoxin exposure in agricultural workers is positively associated with airway hyperresponsiveness, but inversely with atopic sensitization, and (2) atopy plays a minor role in endotoxin-related respiratory effects.

Subjects and Methods Study Population Figure 1 depicts a flow diagram illustrating study population recruitment. As described earlier [15], questionnaires were sent to employees of 23 companies in 4 agricultural processing sectors (onion trade, flower bulb trade, animal feed industry and vegetable seed industry). We received 376 completed questionnaires (re-

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Int Arch Allergy Immunol 2010;152:151–158

sponse rate 90%). Farmers were recruited by sending questionnaires to 748 farm owners, and we received 525 completed questionnaires from 418 farms (farm owners’ response rate 61%). Selection of the present study population has been described in detail elsewhere [21, 22]. Briefly, a total of 342 agricultural processing workers (91% of 376 eligible) consented to blood sampling. A total of 340 farmers (65% of 525 eligible) indicated that they would not object being contacted for a medical examination. However, for logistical reasons, only approximately 100 farmers could be included. A random sample of 122 farmers was approached by telephone, and 92 farmers participated. Data from 427 of 434 participating subjects were used (338 agricultural processing workers, 89 farmers), as we excluded 4 subjects aged !18 or 1 65 years, and 3 subjects with incomplete data. Farmers who participated in the medical examination did not differ significantly from farmers in the original sample with respect to endotoxin exposure, potential confounders and wheeze, but hay fever was more prevalent in the farmers who participated (17 vs. 9%; p = 0.02, ␹2 test). Selection bias was a potential concern, and therefore, sensitivity analyses were performed to see whether results changed after excluding farmers. The study protocol was approved by the institutional ethics committee and all participants gave their written informed consent. Endotoxin Exposure Endotoxin sampling, quantification and modelling has been described before [15]. Briefly, 249 personal full-shift inhalable dust samples were collected in a sample of participants. Endotoxin was analysed by the quantitative kinetic chromogenic Limulus amebocyte lysate assay. Endotoxin exposure levels for each participant were estimated by work site and job title. Endotoxin exposure levels were expressed as endotoxin units (EU) per m3. IgE Serology Specific IgE to common allergens house dust mite, grass pollen (mix of timothy and perennial ryegrass), cat and dog, as well as total serum IgE were determined by enzyme immunoassays as described previously [13, 23]. Total IgE levels were dichotomized by using 100 IU/ml as cut-off level. Atopy was defined as the presence of specific serum IgE antibodies to 1 or more of the common allergens. Bronchial Hyperresponsiveness BHR to methacholine was assessed in subjects from 7 randomly selected agricultural processing companies that were visited on a second occasion, as shown in figure 1. In total, 113 of 119 subjects who were present again on the second occasion consented to BHR (response 95%). Forced expiratory lung function and BHR were assessed according to the European Respiratory Society guidelines [24, 25]. Methacholine was inhaled from 0.019 mg up to a cumulative dose of 2.5 mg, as described before [26]. The test was stopped after the maximum cumulative dose was reached, or when the forced expiratory volume in 1 s (FEV1) fell by 20% or more. Data Analysis BHR was defined as a fall in FEV1 of at least 20% at a methacholine dose of 2.5 mg or less. Bronchial challenge test results were also expressed by the dose-response slope (DRS), calculated

Smit /Heederik /Doekes /Lammers / Wouters

Farmers

Questionnaire

Agricultural processing workers

Questionnaire n = 525 (response rate 61%)

Complete data: n = 877 Farmers: n = 504 Flower bulb workers: n = 126 Animal feed workers: n = 114 Onion workers: n = 97 Vegetable seed workers: n = 36

Questionnaire n = 376 (response rate 90%)

Consent to medical examination n = 340

Consent to medical examination n = 358

IgE Included in medical examination: n = 96 Consent to blood collection: n = 92 Complete data: n = 89 Endotoxin exposure measurements n = 82

Complete data: n = 427 Farmers: n = 89 Flower bulb workers: n = 120 Animal feed workers: n = 102 Onion workers: n = 80 Vegetable seed workers: n = 36

Included in medical examination: n = 358 Consent to blood collection: n = 342 Complete data: n = 338 Endotoxin exposure measurements n = 167

BHR Workers from 7 randomly selected companies were invited: n = 119 Consent: n = 113 Flower bulb workers: n = 34 Animal feed workers: n = 23 Onion workers: n = 25 Vegetable seed workers: n = 31

Fig. 1. Flow diagram representing study population recruitment. The questionnaire and endotoxin exposure

measurements are described in detail by Smit et al. [15].

as the maximum percentage fall in FEV1 per mg inhaled methacholine [27]. Natural log-transformed DRS values were used in calculations. Associations between natural log-transformed endotoxin levels and dichotomous and continuous health outcomes were assessed by multiple logistic and linear regression analysis. Odds ratios (ORs) for the interquartile range increase in endotoxin exposure were calculated and were adjusted for age, sex, smoking habits and farm childhood, unless stated otherwise. Exposureresponse relationships between endotoxin exposure and health outcomes were further studied by assessing the shape of the exposure-response relationship by means of generalized additive modelling (smoothing; SAS Proc GAM), using Akaike’s information criterion to select the degree of smoothing [13]. The proportion of health outcomes attributable to atopy (i.e. population attributable risk) was calculated as described by Pearce et al. [28].

Endotoxin Exposure and Atopy in Adults

Results

Characteristics of the study population are presented in table 1. Endotoxin exposure levels ranged over 3 orders of magnitude (approximately 10–10,000 EU/m3) with a geometric mean endotoxin exposure level of 319 EU/m3, which was similar in the original sample (265 EU/m3). First, the association between endotoxin and wheeze and BHR was explored. Positive and statistically significant relationships between endotoxin exposure and wheezing and BHR (as a dichotomous variable) were found (table 2). The association between endotoxin exposure and wheeze hardly changed when we only considered the processing workers and removed farmers from Int Arch Allergy Immunol 2010;152:151–158

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Table 1. Characteristics of the study population

Total Male Age, years Smoking habits Never Former Current Farm childhood Endotoxin exposure, EU/m3 Geometric mean Geometric SD Interquartile range Range

Table 3. Association between occupational endotoxin exposure and atopy or grass pollen sensitization, stratified by self-reported allergy

427 371 (86.9) 41.8810.5

n/N

173 (40.5) 134 (31.4) 120 (28.1) 193 (45.2)

Figures in parentheses are percentages.

Table 2. Associations between occupational endotoxin exposure and wheezing, BHR, atopic sensitization, elevated total IgE and hay fever in 427 agricultural workers

n

55 Wheezing2 30 BHR3 Atopy 81 Grass pollen IgE 49 House dust mite IgE 46 Total IgE (≥100 IU/ml) 104 51 Hay fever2

%

Endotoxin exposure1

p value

OR

95% CI

12.9 26.6 19.0 11.5 10.8

1.30 1.55 0.67 0.59 0.84

1.01–1.67 1.03–2.35 0.54–0.84 0.45–0.79 0.65–1.09

0.04 0.04 0.0005 0.0003 0.18

24.4 11.9

0.87 0.57

0.72–1.05 0.43–0.75

0.15