American Journal of Respiratory and Critical Care Medicine

Rationale: High endotoxin exposure may reduce the risk of allergic sensitization.

Objective: To determine the relationship between a promoter polymorphism in the CD14 gene (CD14/−159 C to T) and endotoxin exposure in relation to the development of allergic sensitization, eczema, and wheeze within the setting of a birth cohort.

Methods: We genotyped 442 children (CD14/−159 C to T; rs2569190). We assessed children for allergic sensitization (IgE > 0.2 kU/L to at least one of seven allergens), eczema (physical examination), and parentally reported wheeze. Endotoxin was measured in house dust.

Main Results: Genotype frequencies were consistent with other populations (TT, 25%; CT, 47%; CC, 28%). Sensitization (present in 33% of children) was not associated with genotype. For children with TT and CT genotypes, there was no association between endotoxin and sensitization (odds ratio [OR], 0.95; 95% confidence interval [CI], 0.71–1.23; p = 0.7; and OR, 0.90; 95% CI, 0.77–1.04; p = 0.16, respectively) or endotoxin and eczema (OR, 0.99; 95% CI, 0.81–1.20; p = 0.89; and OR, 1.38; 95% CI, 0.83–2.30; p = 0.22, respectively). In children with the genotype CC, increasing endotoxin load was associated with a marked and significant reduction in the risk of sensitization (OR, 0.70; 95% CI, 0.55–0.89; p = 0.004) and eczema (OR, 0.73; 95% CI, 0.56–0.95; p = 0.02). However, we observed an increased risk of nonatopic wheeze with increasing endotoxin exposure in children with the CC genotype (OR, 1.42; 95% CI, 1.01–1.99; p = 0.04) but not other genotypes. No effect was seen for atopic wheeze.

Conclusions: Increasing endotoxin exposure is associated with reduced risk of allergic sensitization and eczema but with increased risk of nonatopic wheeze in children with the CC genotype at −159 of the CD14 gene. The impact of environmental endotoxin may be enhanced in individuals with this genotype.

Allergic sensitization is neither necessary nor sufficient for asthma development, but the association between the two is strong (1). Twin and family studies have confirmed a strong genetic component of both conditions (2). The increase in prevalence of asthma and sensitization suggests an important environmental component. Until recently, genetic and environmental factors have generally been studied independently. To increase the probability of identifying causative factors for complex diseases, it is necessary to study the genes and the environment to determine their interactions (3, 4).

A locus on chromosome 5q31.1 is linked to atopy and asthma (5). One gene mapping to this region is CD14, part of the receptor complex for lipopolysaccharide, the major component of endotoxin (6, 7). Activation of this pathway results in strong interleukin (IL)-12 expression in early life, which favors Th1 differentiation of naive T cells, reducing the probability of an IgE response to allergens (8, 9). Although a promoter single nucleotide polymorphism (SNP) in this gene (CD14/−159 C to T, rs2569190) was not associated with the frequency of sensitization, among sensitized children, C-allele homozygotes had higher total IgE levels and more positive skin tests (10). Similar results were reported in studies of Dutch adults with asthma (11), in Australian (12) and Chinese children (13), and in northern Indian populations (14). No association was found between CD14/−159 polymorphism and atopy or total IgE in two German populations (15, 16) or among Japanese subjects (17). In contrast, in a founder population in the United States (the Hutterites), the T allele was associated with sensitization (18). This allele has recently been identified as a risk for eczema at 2 yr of age in high-risk children (19). However, a study of a high–risk, 1-yr-old children (including 14% ethnic minorities) found that T-allele homozygotes had a reduced risk of eczema but only if they were dog owners (20). The results of previous studies that investigated the association between CD14 polymorphisms and clinical outcomes are presented in Table E1 of the online supplement.

The T allele was also associated with higher soluble CD14 levels. In vivo (10) and in vitro studies showed that the T allele was transcribed more efficiently in monocytes (21), suggesting that this polymorphism is functional.

Conflicting results of the association between CD14 polymorphisms and sensitization may be due to differing environmental exposures in the populations studied. Endotoxin exposure may be a plausible candidate and has been associated with allergic disease. Lower early-life endotoxin load was reported in sensitized compared with nonsensitized, high-risk children (22). Higher exposure reduced the risk of eczema at 6 mo (23) and 12 mo of age (24) and of sensitization and atopic asthma among schoolchildren (25). However, this association was not reproduced in all populations (26, 27).

Endotoxin exposure and the CD14 polymorphism have been associated with allergy in some populations but not in others. Because the effect of endotoxin is mediated through CD14, we aimed to investigate the relationship between a promoter polymorphism in the CD14 gene (CD14/−159 C to T) and endotoxin exposure with allergic sensitization, eczema, and wheeze in a population-based birth cohort study. Some of the results of this study were reported in the form of an abstract (28, 29).

Additional details on study population, environmental exposures, and genotyping are available in the online supplement.

Study Population

The Manchester Asthma and Allergy Study is a population-based birth cohort study (3032) set in Greater Manchester, United Kingdom (population, 6 million). Participants were recruited prenatally (30) and reviewed at 5 yr (± 4 wk) of age. We collected domestic dust samples for measurement of environmental exposures at this time. The study was approved by the local research ethics committee. We obtained informed consent from all parents.

Outcomes
Allergic sensitization.

We measured specific serum IgE (mite, cat, dog, grass, milk, egg, and peanut; ImmunoCAP; Pharmacia, Uppsala, Sweden) and defined sensitization as specific IgE greater than 0.2 kU/L to any allergen. We used a skin-prick test for Dermatophagoides pteronyssinus, cat, dog, grasses, molds, milk, and egg (Bayer, Elkahrt, IN) and defined sensitization as a weal diameter 3 mm greater than negative control.

Clinical assessment.

A validated questionnaire (33) was interviewer administered to collect information on parentally reported symptoms, physician-diagnosed illnesses, and treatments received. Children were defined as having recent wheeze if they had wheezed within the previous 12 mo. Wheeze was defined as atopic if it occurred within the context of allergic sensitization and nonatopic if it was reported in children who were not sensitized.

Physical examination was conducted in a standardized manner. Eczema was defined using the U.K. diagnostic criteria (34).

Environmental Exposures

We collected two living-room floor dust samples by vacuuming 1-m2 areas for 2 min (32). Endotoxin content was measured by a kinetic limulus assay (35). Results were expressed as endotoxin load (EU/m2) and level (EU/mg). Dust mite allergen Der p 1 was measured using ELISA (32). Results were expressed as allergen concentration (μg/g).

Genotyping

We genotyped an SNP in the promoter region of CD14 (−159 T > C rs2569190) using a fluorescence-based primer extension method (SNaPshot; ABI, Warrington, UK). Polymerase chain reactions were performed in 10-μl reaction volumes containing 10 ng genomic DNA (isolated from whole blood) per reaction. Three positive controls of each genotype and negative water controls were included on each plate.

Soluble CD14

Soluble CD14 (sCD14) was measured in plasma collected at 5 yr of age (R&D Systems, Abingdon, UK). Results were expressed as μg/ml.

Statistical Analysis

Statistical analysis was performed using SPSS for Windows (version 11.0). Endotoxin and mite allergen data followed a log-normal distribution; results are presented as geometric means (GM) and 95% confidence intervals (CI). Deviation from Hardy-Weinberg equilibrium was tested for using a χ2 test. Logistic regression was used to ascertain the effect of endotoxin load on sensitization, eczema, wheeze, and sCD14 in the whole population and within different genotype groups. The results are presented as odds ratios (OR) and 95% CI.

The study profile is shown in Figure 1. We reviewed 1,025 children; 122 children randomized to an environmental control regime (36) were excluded from this analysis. A full dataset was available in 442 children of mixed European ancestry. Where genotyping was not performed, this was generally because the child refused venipuncture. Children excluded from the analysis did not differ from those included in terms of family history, parental smoking, maternal age, socioeconomic status, gestational age, history of wheeze, allergic sensitization, and endotoxin exposure (data available on request).

Demographic data of the subjects are presented in Table 1; 33% were sensitized by IgE (17% to mite) and 25% by skin tests (14% to mite), 38% reported wheeze ever, 21% reported current wheeze, and 14% had eczema on clinical examination. Endotoxin exposure covered a large range (∼ 200,000-fold; load GM, 2,856 EU/m2; 95% CI, 2,420–3,370, and level GM, 16.1 EU/mg; 95% CI, 14.3–18.1) and was not associated with family history of allergic disease, socioeconomic status, position in the sibship, or parental smoking (p > 0.2). sCD14 was detectable in all children (mean, 1.77 μg/ml; 95% CI, 1.73–1.80 μg/ml). Genotype frequencies were in Hardy-Weinberg equilibrium and consistent with other populations (TT, 25%; CT, 47%; CC, 28%) (10).

TABLE 1. DEMOGRAPHIC DATA OF THE STUDY POPULATION




Genotype (%)

Variable
Whole Group*
TT (n = 109)
CT (n = 207)
CC (n = 126)
p Value
Male566252560.2
Maternal asthma18.318.318.418.31
Paternal asthma11.515.69.711.10.3
Maternal smoking14.311.91515.10.7
Paternal smoking25.12127250.5
Maternal atopy52.846.854.954.80.3
Paternal atopy56.863.953.456.30.2
Cat owner25.631.223.823.80.3
Dog owner19.518.319.420.60.9
Older siblings56.3565755.60.9
Attended nursery6868.966.170.10.7
Breast fed > 16 wk39.242.939.335.70.1
Wheeze ever38.234.939.139.70.7
Current wheeze20.615.624.219.00.2
Eczema14.18.316.415.20.14
Sensitization (IgE)32.828.435.731.70.4
Sensitization (SPT)25.322.228.622.70.3
Mite sensitization (IgE)1716.517.416.71
Mite sensitization (SPT)141314141
sCD14, mg/ml1.771.831.781.700.02
Total IgE, kU/L26.9924.7924.6733.670.18
Endotoxin load, EU/m2
2856
2885
2974
2647
0.84

Definition of abbreviation: SPT = skin prick test.

*Values are percentages unless otherwise noted.

p Value for χ2 by genotype.

p value by analysis of variance.

Allergic Sensitization

There was no association between CD14/−159 genotype and sensitization defined by IgE (p = 0.4). For the whole population, increasing endotoxin load was significantly associated with a decreased risk of sensitization (OR, 0.85; 95% CI, 0.76–0.95; p = 0.005). However, when this analysis was repeated by genotype, there was no association between endotoxin load and sensitization for the TT and CT genotypes (OR, 0.95; 95% CI, 0.71–1.3; p = 0.7; and OR, 0.90; 95% CI, 0.77–1.04; p = 0.16, respectively). In contrast, in children with the CC genotype, increasing endotoxin load was associated with a marked and significant decrease in the risk of sensitization (OR, 0.70; 95% CI, 0.55–0.89; p = 0.004; Figure 2). For the CC genotype group only, for each log unit increase in endotoxin exposure in the home, there was a 30% decrease (95% CI, 11–45) in the odds of being sensitized to one or more allergen. We found some limited evidence of a statistical interaction between genotype and endotoxin load in their relationship with allergic sensitization when an interaction term was included in the logistic regression model (overall interaction, p = 0.09).

None of these relationships was significantly altered when sensitization was defined as one or more specific IgE greater than 0.35 kU/L. Similarly, defining allergic sensitization by skin tests or expressing endotoxin exposure as level or quartiles of load (Table 2) or inclusion of the environmental tobacco smoke exposure at 5 yr of age or maternal smoking during pregnancy did not materially alter the results.

TABLE 2. EVIDENCE FOR ASSOCIATION AMONG THREE DIFFERENT DEFINITIONS OF ALLERGIC SENSITIZATION AND THREE DIFFERENT MEASURES OF ENDOTOXIN EXPOSURE IN CHILDREN WITH THE CC GENOTYPE (n = 126)



IgE > 0.2 kU/L (n = 31 with allergic sensitization)

IgE > 0.35 kU/L (n = 27 with allergic sensitization)

Skin Prick Test (n = 24 with allergic sensitization)

OR (95% CI)
p Value
OR (95% CI)
p Value
OR (95% CI)
p Value
Endotoxin load, EU/m20.70 (0.55–0.89)0.0040.77 (0.61–0.96)0.0230.81 (0.64–1.02)0.067
Endotoxin level, EU/mg0.61 (0.43–0.87)0.0060.70 (0.51–0.97)0.0320.76 (0.55–1.05)0.093
Endotoxin load, quartiles
0.60 (0.42–0.87)
0.007
0.62 (0.43–0.91)
0.015
0.63 (0.42–0.94)
0.023

Definition of abbreviations: CI = confidence interval; OR = odds ratio.

To further explore this association with sensitization to individual allergens, we investigated the relationship between the CD14 polymorphism, exposure to mite allergen, and endotoxin- and mite-specific IgE. There was no association between CD14 genotype and mite sensitization (p = 1; see Table 1). In a multivariate logistic regression analysis, for the whole population there was a trend toward an increase in the risk of sensitization to mite with increasing mite allergen exposure (OR, 1.14; 95% CI, 1.00–1.30; p = 0.06) and a significant decrease in the risk with increasing endotoxin exposure (OR, 0.84; 95% CI, 0.73–0.98; p = 0.02). In children with the CC genotype, an increase in the risk of mite sensitization was significantly associated with increasing mite allergen exposure and decreasing endotoxin exposure (OR, 1.34; 95% CI, 1.03–1.75; p = 0.03; and OR, 0.67; 95% CI, 0.50–0.90; p = 0.007, respectively; Figure 3). No such effect was seen in children with the CT and TT genotypes (p > 0.4). Defining mite sensitization by skin tests or using early-life or cumulative exposure did not alter the results (data not shown).

Eczema

For the whole population, there was no association between eczema and CD14 genotype (p = 0.14) or endotoxin exposure (p = 0.28). However, in children with the CC genotype, increasing endotoxin load was associated with a decreased risk of eczema (OR, 0.73; 95% CI, 0.56–0.95; p = 0.02), which was not seen for the CT or TT genotypes (OR, 0.99; 95% CI, 0.81–1.20; p = 0.89; and OR, 1.38; 95% CI, 0.83–2.30; p = 0.22, respectively; Figure 4a). For the CC genotype group only, for each log unit increase in endotoxin exposure in the home there was a 27% decrease (95% CI, 5–44) in the odds of having eczema. We found a statistically significant interaction between genotype and endotoxin load, with evidence of a difference between the genotypes with respect to the relationship between eczema and endotoxin load (overall interaction, p = 0.05).

Wheeze

For the whole population, we found no association between wheeze and CD14 genotype (wheeze ever, p = 0.7; recent wheeze, p = 0.2) or between wheeze and endotoxin exposure (wheeze ever, p = 0.1; recent wheeze, p = 0.3). However, in the CC genotype subgroup, increasing endotoxin load was associated with a significant increase in the risk of recent wheeze (OR, 1.31; 95% CI, 1.00–1.73; p = 0.05), which was not seen in the CT or TT genotypes (OR, 1.00; 95% CI, 0.84–1.18; p = 1.0; and OR, 1.04; 95% CI, 0.72–1.50; p = 0.8, respectively). Children with recent wheeze were divided into atopic (n = 42) and nonatopic (n = 49) groups. Using nonwheezers as the reference group, there was no association between atopic wheeze and endotoxin exposure for the whole population (p = 0.8) or for individual genotypes (p > 0.4). For nonatopic wheeze, there was no association between endotoxin exposure and wheeze for the whole population (p = 0.2), but in children with CC genotype, increasing endotoxin was associated with an increase in wheeze (OR, 1.42; 95% CI, 1.01–1.99; p = 0.04; Figure 4b). This was not seen for the CT (OR, 1.05; 95% CI, 0.81–1.34; p = 0.7) or TT genotypes (OR, 0.97; 95% CI, 0.62–1.52; p = 0.9). We found no association between endotoxin exposure and wheeze ever for individual genotypes.

Potential Confounders

We found no association between CD14 genotypes and factors previously shown to be associated with allergic sensitization and wheeze (see Table 1).

sCD14

sCD14 was lower among children with the CC genotype (p = 0.02; see Table 1). There was no linear association between endotoxin exposure and sCD14 level for the whole group (p = 0.6) or within genotype groups (p > 0.2). There was no association between sCD14 level and allergic sensitization or current wheeze (p > 0.14). Children with eczema had lower sCD14 (mean, 1.67 μg/ml; 95% CI, 1.59–1.74, vs. mean, 1.78; 95% CI, 1.74–1.82; p = 0.02).

This study provides clear evidence of the differing effect of domestic endotoxin exposure on allergic sensitization, eczema, and wheeze in children with different CD14 genotypes. Children homozygous for the C allele at position −159 in the promoter region of the CD14 gene showed a dose-dependent response to endotoxin: high exposures were associated with a decreased risk of allergic sensitization and eczema, and low exposures were associated with an increased risk relative to the rest of the population. However, in the same children, high endotoxin was associated with an increase in nonatopic wheeze. Among the remaining children, there was no effect of endotoxin on sensitization, wheeze, or eczema.

Limitations

One potential limitation is that endotoxin was measured once at age 5 yr, raising a question whether this single level is an appropriate index of early-life exposure (arguably the most critical time). In 70 homes, we also measured early-life endotoxin load and demonstrated a good correlation between the two measures (r = 0.54), similar to that reported by others over a 6-yr period (r = 0.51) (37). We also measured mite allergen at birth and at 3 and 5 yr of age and demonstrated the same relationship between exposure and mite sensitization when we used allergen levels at the individual time points or cumulative level. Thus, when assigning “index of exposure” to an environmental factor with huge between-individual variability within a population (∼ 200,000-fold for endotoxin in our study), comparatively small within-individual variability due to timing of the measurement is outweighed by large between-individual differences. We therefore argue that endotoxin exposure measured at age 5 yr is a good estimate of early-life exposure. The levels of endotoxin in our study were similar to those in the nonfarming households but lower compared with the farming households in the ALEX (ALlergy and EndotoXin) study (25). The reported relationships seemed to be specific for endotoxin and were not observed when we repeated the analysis using total weight of dust.

CD14 contributes not only to TLR4-mediated recognition of endotoxin (more generally, Toll-like receptor-4 [TLR4] ligands) but is also involved in the detection of gram-positive and other products through TLR2 (38). Thus, endotoxin may act as a surrogate marker for a loosely related ligand rather than the CD14 ligand itself, which may be an alternative explanation for the lack of consistency between studies.

We have investigated the effect of a single SNP in the promoter region of the CD14 gene (CD14/−159 C to T; rs2569190). It is possible that the effects seen are a result of linkage disequilibrium between this and other SNPs in this gene or in the surrounding region. A further line of investigation might be to study the effects of maternal CD14 genotype, endotoxin exposure during pregnancy, and allergic outcomes in offspring.

Although retention in this population-based observational birth cohort is excellent, we analyzed data from approximately half of the subjects (mostly because some children refused venipuncture). There was no difference between children excluded or included in the analysis in any relevant parameter. Furthermore, the prevalence of allergic sensitization among the parents of the children is similar to that of young adults in the United Kingdom (39), suggesting the subjects are representative of the general population.

“Endotoxin Switch”

In 2003, Vercelli proposed the “endotoxin switch,” suggesting a bimodal relationship between environmental exposures to bacterial products and immune responses (being Th2-like at low and Th1-like at high exposures) (4). Our data support this hypothesis. We identified an endotoxin load above which children with CC genotype were at a reduced risk of sensitization relative to the remainder of the population. Immune responses to endotoxin in different CD14/−159 genotypes may be represented as a series of parallel curves positioned at different points along the bacterial load axis, implying that the dose of endotoxin at which they switch from a Th2 to a Th1 response is shifted in different genotypes (4). Rather than identifying the parallel response curves at differing endotoxin loads for the three CD14/−159 genotypes, our data suggest that the difference between genotypes in the relationship between endotoxin load and immune response is the gradient (not the position) of the dose–response curve. This may suggest that individuals with TT and CT genotypes are much less susceptible to the effect of endotoxin compared with C-allele homozygotes.

In most studies that reported an association between CD14/−159 genotype and severity of sensitization, the increase in severity was associated with the C allele (10, 11). In contrast, a study in the Hutterites of a farming community likely to be exposed to high endotoxin levels (40) suggested that the T allele was associated with sensitization (18). Extrapolating from our results, in populations with high endotoxin exposure, CC homozygotes would be at reduced risk of sensitization relative to the T-allele carriers, making this the risk genotype. In studies where the C allele conferred increased risk, it is likely that the population exposures to endotoxin were generally lower. Our data can explain how, in cross-sectional studies, the same genotype may confer an increase, a decrease, or no effect on sensitization, dependent on the population endotoxin exposure. A recent family-based study of adults with asthma from Barbados suggested that T-allele homozygotes were less likely to have asthma as a group. However, when stratified by endotoxin exposure (highest quartile vs. rest), T-allele homozygotes with high exposure were at increased risk compared with the CC genotype group (albeit in a small number of subjects) (41). This finding is in agreement with our data. Further confirmation of this important effect was reported in children of the ALEX study, where carriers of the C allele exposed to the highest tertile of endotoxin had less allergic sensitization (42). Although the studies are of different design, there are important similarities replicating the findings.

Although we confirmed previous reports of lower sCD14 in the CC genotype subgroup (10), we found no effect of endotoxin on sCD14, suggesting that the mechanism by which endotoxin exposure affects sensitization is not by altering sCD14 levels. As well as sCD14, in humans CD14 is expressed on the membrane of monocytes and macrophages (mCD14). Studies of the effect of mutations on the function of CD14 protein as an LPS receptor suggest that the critical regions may be different for the membrane and the soluble form of CD14 (43). The relationship between the density of mCD14 on monocytes and CD14 genotype is also variable (4446). Therefore, our findings of an effect of endotoxin on allergic sensitization in the CC genotype group may be mediated by complex effects on the function of membrane-bound CD14.

Environmental Exposures and Mite-specific Sensitization

A dose–response relationship between mite allergen exposure and specific sensitization has been shown for some populations (47) but not others (48). In our study, increasing allergen exposure was associated with increased risk of sensitization, but the size of this effect was dampened at higher endotoxin exposure. When the analysis was repeated by genotype, this complex relationship was seen only among children with CC genotype at CD14/−159. Thus, the development of allergen-specific sensitization is influenced not only by allergen exposure, but also by other environmental exposures (e.g., endotoxin) and genetic predisposition. We found no such effect for cat and dog sensitization and exposure to the respective allergens (data not shown). This may be a consequence of lower rates of sensitization to pet allergens (cat, 9%; dog, 5.9%).

Symptomatic Disease

Similar to the relationship between endotoxin and sensitization, eczema was decreased in children with the CC genotype exposed to high endotoxin levels. We confirmed eczema by clinical examination rather than by relying only on parental reports of itchy rash.

In marked contrast to the findings for eczema, nonatopic wheeze was increased in children with the CC genotype with increasing endotoxin exposure. No such association was seen for atopic wheeze. Others have also reported an increase in nonatopic wheeze with increasing endotoxin exposure (25). The hygiene hypothesis implicates reduced exposure to microbials as a cause for the increase in prevalence of allergy (49). The results of the current study may help explain why this hypothesis is more consistent in its association with allergic sensitization than with wheeze and asthma (50).

One aim of epidemiologic studies is to identify potentially modifiable risk factors and to design primary prevention strategies for asthma and allergic diseases. Success in the identification of genetic polymorphisms that confer an increase in risk for sensitization and asthma has been limited. This reflects the difficulties in phenotype definition and the fact that the relevant environmental exposures have rarely been taken into account. We have shown that exposure to a specific environmental factor (endotoxin) may be relevant only in individuals with a particular genetic polymorphism (CC genotype for CD14/−159). Therefore, within the context of primary prevention strategies, only individuals with particular genotypes may benefit from an intervention directed at an increase in endotoxin (or microbial) exposure. Primary prevention requires the development of tailor-made measures targeting subjects with specific susceptibilities who will benefit from a particular intervention rather than blanket advice aimed at the whole population. Thus, in assessing the impact of genetic factors on complex disease, it is imperative not to investigate genetic factors in isolation but to consider potential gene–environment interactions.

The authors thank the children and parents of the Manchester Asthma and Allergy Study for their participation; Bridget Simpson and Patricia Kissen for the clinical follow-up of the cohort; Dr. Gael Tavernier for the endotoxin measurements; Mark Craven, M.Sc., for Der p 1 measurement; and Julie Morris, M.Sc., and Lars Soderstrom for help in the statistical analysis.

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Correspondence and requests for reprints should be addressed to Adnan Custovic, M.D., Ph.D., North West Lung Centre, Wythenshawe Hospital, Manchester M23 9LT, UK. E-mail:

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