Published ahead of print on April 7, 2006, doi:10.1164/rccm.200506-964OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200506-964OC
Modified Th2 Responses at High-Dose Exposures to AllergenUsing an Occupational ModelDepartment of Occupational and Environmental Medicine, Imperial College (NHLI); and St. George's Hospital, London, United Kingdom Correspondence and requests for reprints should be addressed to Meinir Jones, Ph.D., Department of Occupational and Environmental Medicine, Faculty of Medicine, Imperial College (NHLI), 1B Manresa Road, London SW3 6LR, United Kingdom. E-mail: meinir.jones{at}imperial.ac.uk
Rationale: The relationships between allergen exposures and allergy and asthma are complex. High exposure levels to cat allergen are associated with IgG- and IgG4-specific antibody responses without sensitization or risk of asthma, a process described as a "modified Th2 response." Attenuation of risk of allergy and asthma at high exposure levels has been reported in longitudinal studies of both childhood and occupational asthma. Objectives: To investigate, using an occupational model, the relationships among estimated exposure to aeroallergens, the production of specific IgE, IgG and IgG4 antibodies, and the prevalence of associated symptoms. Methods: Cross-sectional survey of employees exposed to rats at work on six pharmaceutical sites across the United Kingdom. A total of 689 (89%) provided a blood sample and completed a questionnaire. Measurements and Main Results: At highest exposure to rats, there was an attenuation of the exposure response for sensitization and symptoms. In contrast, the frequency of individuals producing high quantities of specific IgG and IgG4 increased with exposure intensity. Ratios of IgG4/IgE were highest in those handling the greatest number of rats. Risk of developing work-related chest symptoms was lower for those who produced both specific IgE and IgG4 compared to those with specific IgE only.. Conclusions: High exposure to rats is associated with lower rates of specific IgE and symptoms but an increased frequency of high specific IgG and IgG4 production. Specific IgG4 produced together with specific IgE may reduce the risk of developing work-related chest symptoms compared with when specific IgE is produced alone.
Key Words: IgG • IgG4 • IgE • laboratory animal allergy • occupational allergy The relationship between allergen exposures and the induction of allergy and asthma is complex. On cross-sectional analysis, high current exposures to cat allergens are associated with specific IgG and IgG4 antibody responses in the absence of specific sensitization or risk of asthma, a state that has been described as a "modified Th2 response" (1). We have reported high-dose attenuation in a prospectively studied cohort investigating the relationship between early allergen exposure and childhood allergy (2). The risks of IgE sensitization and atopic wheeze at age 5.5 yr increased at very low levels of exposure to cat (and house dust mite) allergen but were attenuated thereafter. Occupational asthma, such as caused by laboratory animal allergy (LAA), provides a useful model of allergic asthma because its phenotypes are well defined and exposure can be readily characterized and measured. Again, we have observed in a prospective study of a cohort of laboratory animal workers an attenuation at high dose of the relationships between exposure to rat urinary proteins and specific IgE antibodies (3). The findings of previous studies of specific IgG antibodies in LAA have been difficult to reconcile. In some, the presence of specific IgG antibodies was significantly associated with intensity, but not duration, of exposure to rats (4, 5); in others, cumulative exposure (to mice) was associated with high specific IgG production (6). A protective role for specific IgG4 was suggested by one study in which symptomatic individuals, with rat-specific IgE, had lower titers of IgG4 than corresponding asymptomatic, IgE-positive subjects (7). More recently, the longitudinal relationship between specific IgG4 and rat allergy was examined using information obtained over 2 yr of follow-up. High levels of specific IgG4 antibodies to rat urine were a strong predictor of prevalent and incident sensitization and symptomatic rat allergy in atopic and rat-sensitized subjects (8). We carried out this study to help us understand the basis for our previous longitudinal findings (3). Using a cross-sectional design, we have investigated the relationships among workplace exposure to rat aeroallergens, the production of specific antibodies (IgE, IgG, and IgG4), and the prevalence of associated symptoms.
Subjects We surveyed 776 employees of six U.K. pharmaceutical companies who were undergoing routine health surveillance for LAA. Employees were eligible for this analysis if they had been exposed to rat proteins at work for at least 1 mo (n = 718). All were invited to complete a questionnaire and undergo skin-prick tests and venesection. Nineteen employees of Imperial College who had never had exposure to rats were also studied. Serum antibodies were measured in 689 (96%) of the 718 eligible employees and all 19 unexposed individuals. The Royal Brompton Hospital/National Heart and Lung Institute (NHLI) ethics committee approved the study; written, informed consent was obtained from all participants.
Exposure and Symptom Assessment
Atopy
Measurement of Specific IgE
Measurement of Rat-specific IgG and IgG4
Statistical Analyses
Subjects The employees included in this analysis were, on average, 35 yr old (range, 17–65 yr); 363 (53%) were male. There was a close agreement between the exposure categorization based on job title and that based on the maximum number of rats handled in 1 d (Table 1). Thirty-eight percent had handled more than 50 rats a day, whereas 17% had never handled any; equal proportions (23 and 22%) had handled up to 1 to 10 or 11 to 50 rats. For most (464 = 68%), their highest exposure to rat proteins had been during work as a scientist. Twenty percent (n = 140) had worked in animal husbandry, whereas 12% (n = 81) had only ever worked in low-intensity jobs. These proportions were not greatly different (63, 16, and 22%, respectively) among those employed for 3 yr or less, although the proportion of low-intensity employees was a little higher.
Forty-three percent of all eligible workers were atopic; this proportion varied by exposure category (p = 0.01), being lowest among those in the highest intensity job category. We found the same pattern when we restricted the population to those with no more then 3 yr of exposure (Table 1), and when we restricted it further to those with less than 2 yr, 1 yr, and 6 mo of exposure (data not shown).
Antibody Measurements When measured on a continuous scale, rat-specific IgG and IgG4 antibody levels were significantly higher in employees with IgE sensitization to rats and in those with both rat sensitization and work-related chest or eye/nose symptoms (Table 2). Predictably, ratios of rat-specific IgG/IgE and specific IgG4/IgE were significantly higher in those who were not sensitized to rats and in those without symptoms.
Ninety employees reported work-related nasal or chest symptoms, in the absence of rat-specific IgE sensitization. Their median levels of rat-specific IgG (0.21) and IgG4 (0.09) and ratios of IgG/IgE (0.33) and IgG4/IgE (0.12) were not different from those without IgE sensitization to rats and without any work-related symptoms.
Short-duration employees.
Unexposed control subjects.
Antibody Production and Allergen Exposure
Restriction of our analyses to employees of short duration (  3yr) showed a similar bell-shaped pattern of exposure–response for IgE sensitization to rats (Figure 2B). In contrast, as with the full population, the proportion of individuals producing high levels of rat-specific IgG and IgG4 was greatest in the highest exposure category. Again, ratios of specific IgG4/IgE were highest in short-duration employees who had worked with the highest numbers of rats, a difference that was statistically significant (p = 0.05).
Work-related Symptoms and Allergen Exposure Through multivariate logistic regression analysis, we confirmed the strong relationship between rat-specific IgE sensitization and work-related chest symptoms, and a weaker relationship with atopy. There was a reduction in the risk of work-related chest symptoms among those employees whose rat-specific IgE sensitization was accompanied by high levels of rat-specific IgG4, compared with those producing specific IgE alone (Table 3). We did not find these relationships when we examined the risk of eye/nose symptoms.
Within this cross-sectional examination of laboratory animal workers, we observed complex patterns between exposure to rats and both sensitization and work-related symptoms. The findings are consistent with those from our previous cohort study (3), whereby there appear to be increasing risks of sensitization and work-related symptoms with increasing exposures to rats, except at the highest exposure levels where the risks of both outcomes may be lower. In parallel, in this study, we found an increase in the prevalence of individuals producing high levels of rat-specific IgG and IgG4 antibodies with increasing exposure intensity. We were specifically interested to explore whether rat-specific IgG4 was associated with a reduction in the risk of symptoms of LAA. We found a twofold reduction in the risk of developing work-related chest symptoms in those who produced both rat-specific IgG4 and IgE as compared with those producing only specific IgE (Table 3). This is, to the best of our knowledge, the first time that this interaction has been demonstrated. Atopic individuals are at higher risk of LAA. In our population, there were fewer atopic employees among those in jobs with the highest exposure, a pattern that could have explained the reduced prevalence of rat-specific sensitization in this group. The pattern might, in theory, be the result of a higher proportion of nonatopic individuals entering high-exposure jobs (selection bias) or of a higher proportion of atopic individuals leaving them (survival bias). However, our extended analyses indicate that neither of these is a sufficient explanation for our findings. To investigate the potential role of a survivor effect in the current study, we restricted analysis to those employees with no more than 3 yr of exposure to rats, and observed the same pattern of high-dose attenuation. This was true also for individuals who had less than a single year of exposure to rats (data available on request). In addition, we found a reduction in the prevalence of IgE positive/asymptomatic individuals at high exposure (data available on request) where the observations cannot be explained by individuals leaving employment because of their work-related symptoms. Finally, we observed a similar exposure–response pattern in a 7-yr longitudinal study of laboratory animal workers with high rates of follow-up (3). It is not likely that a selection bias of nonatopic individuals into heavily exposed jobs can explain the exposure–response pattern seen in our study. We demonstrated the same bell-shaped curve for sensitization in both atopic and nonatopic individuals. Although there were fewer susceptible (atopic) individuals in higher exposure jobs, the pattern of risk in the susceptible population is the same as that in the nonsusceptible group. Thus, we believe it is unlikely that either survival pressures or selection bias explains the patterns of exposure–response we have observed. Inverse relationships between IgE sensitization and increased specific IgG and IgG4 have been described as representing a "modified Th2 response," because both antibody classes require the Th2 cytokine interleukin 4 for their production. The term was first used to describe the apparent tolerance observed in children exposed to high levels of cat allergen who produced specific IgG and IgG4 antibodies without IgE sensitization or asthma (1). A further study has confirmed cat-keeping to be associated with a modified Th2 response, which was not associated with an increased risk of either asthma or allergy. It was suggested in that study that the shift, from IgE to IgG4, resulted in less asthma and allergy due to lower IgE levels rather than because of a protective effect directly mediated by IgG4 antibodies (11). Ratios of specific IgE to IgG4 change during specific immunotherapy. Specific serum levels of both IgE and IgG isotypes increase during the early phase of therapy, but the increase in specific IgG4 is more pronounced and the ratio of specific IgG4 to IgE increased by 10- to 100-fold, suggesting a protective role for IgG4 (12). In our study, the ratios of specific IgG4 to IgE were increased in those with the highest exposure. Laboratory animal workers may, at very high exposures, be experiencing a natural form of immunotherapy; interestingly, this does not seem to be the case for other groups at risk of occupational asthma—for example, bakers (13) and detergent manufacturers (14). We propose that the differences arise because laboratory animal workers experience exposures not only through inhalation but also through an intradermal route after bites and scratches. The same mechanism may be relevant, as above, in the responses to domestic cat exposures—which likely include scratches—described by Platts Mills and colleagues (1), where high-dose tolerance was observed to cat allergen but not to house dust mite, for which instead there was a linear dose–response association. Consistent with our hypothesis is the state of naturally induced tolerance observed among healthy, hyperimmune subjects who have been frequently stung by bees. Beekeepers showed similar changes in their immune responses to those receiving therapeutic immunotherapy (15). Furthermore, repeated inhalation of cat peptides, as an experimental method of immunotherapy, was not—in contrast to an intradermal method—associated with the induction of hyporesponsiveness ("tolerance") in the skin or the lung (16).
A recent study in which the role of IgG4 in LAA was investigated also reported that high exposure to rats was associated with a strong allergen-specific IgG4 antibody response (8). These authors concluded that IgG4 could not explain the absence of a dose response between allergen exposure and allergy in long-term exposed workers but was more likely to be a combined marker of exposure and susceptibility. Our findings, however, suggest that IgG antibodies themselves are protective. If this is so, then the question of a mechanism arises. Some studies have suggested that IgG antibodies could play a protective role by blocking leukocyte histamine release, inhibiting signal transduction and mediator release through the high-affinity IgE receptor (Fc Our findings indicate that the relationship between allergen exposure and induction of allergy and asthma is complex. In particular, the attenuation of risk of IgE sensitization and asthma among those most heavily exposed is likely to be explained, at least in part, by a modified Th2 response. We have shown that IgG4 production is driven by increasing allergen exposure and have demonstrated, for the first time, an interaction between IgE and IgG4 whereby their coproduction is associated with a reduction in risk of IgE-associated, work-related respiratory disease. These findings require detailed, prospective study before their implications for the prevention of LAA can be interpreted.
The authors thank the employees who took part in this study, and the management of the survey sites for allowing us to undertake research on their premises. They are grateful to the individuals who carried out the field work, and to Asthma UK for funding.
Supported by Asthma UK. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200506-964OC on April 7, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form June 22, 2005; accepted in final form April 6, 2006
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ABSTRACT
METHODS
3 mm wheal) to tests with one or more of the above extracts. We carried out further prick testing with an extract of rat urine and categorized as a positive response in the same way. 
= 0.78). RAST results are expressed as the percentage of binding of the total 125I anti-IgE added, with a figure of 2% or greater considered positive. 
2 tests, and used 
RI) and IgG (Fc
RIIB) receptors (
