Eosinophils and basophils are typical effectors of allergic inflammation [
The mother-child study LINA was designed to investigate how environmental factors in the pre- and postnatal period influence the immune system and whether they are determinants of increased allergy risk later in children’s life. For this study, 629 mother-child pairs (including 7 twins) were recruited between May 2006 and December 2008 in Leipzig, Germany. Mothers suffering from immune or infectious diseases during pregnancy were excluded from the study. Six hundred six mother-child pairs participated in the one-year and 546 pairs in the two-year follow-up (one scheduled visit/year around the child’s birthday). Blood samples from 397 mothers and 340 children were obtained at children’s age of two (mean age: 2 years and 26 days, min–max: 1 year and 343 days–2 years and 161 days) as part of the scheduled visit. Sufficient peripheral blood mononuclear cells (PBMCs) for methylcellulose cultures were available for a subset of 68 corresponding mother-child pairs (66 mothers, 68 children; 2 sets of twins). All relevant
Information concerning general aspects of life and environmental conditions during pregnancy was collected by detailed questionnaires during the 36th week of pregnancy. Further, information about respiratory outcomes of the child in the last 12 months as well as information about housing and environmental conditions (e.g., environmental tobacco smoke (ETS) exposure, and usage of cleaning agents) was obtained annually. All questionnaires were self-administered by the parents. For more detailed information, please see the methods section of Supplementary Data.
PBMCs were isolated within six hours after collection of about 3 mL (child) to 5 mL (mother) fresh heparinised peripheral blood via Ficoll Paque density centrifugation. PBMCs were frozen in 1 mL aliquots of 90% fetal bovine serum and 10% dimethylsulfoxide with 10–30 × 106 cells. The cell thawing protocol and isolation of nonadherent mononuclear cells (NAMNCs) were performed according to Reece et al. [
In the present paper a well-established and prevalidated method of functional methylcellulose assay was used to assess Eo/B CFUs [
To measure the individual exposure to volatile organic compounds (VOCs) in the homes, passive samplers (3M monitors, type OVM 3500; 3M GmbH, Neuss, Germany) were placed in the child’s bedroom (or alternatively in the room where the child preferentially spent most of their time) around the first birthday of the child. Concentrations of VOCs were analysed as described earlier [
Statistical tests were performed using STATISTICA for Windows Version 10.0 (StatSoft Inc. Europe, Hamburg, Germany). The chi squared test was performed to compare parameters of the analysed subcohort with the remaining LINA cohort (
Analyses related to Eo/B CFUs are calculated within the described subcohort (
It was tested whether the available sample size for Eo/B analyses has the power to detect expected effect of indoor pollutants (in particular VOCs). With a type one error rate (alpha) of 0.05, a power goal of 0.9, and an expected population correlation of 0.460 (according to our earlier paper [
Since the majority of parameters were not normally distributed, analyses were performed using nonparametric tests in general. To address the relationship between the numbers of Eo/B CFUs and atopic outcomes or exposure to indoor ETS or disinfectants, medians were compared using the Mann-Whitney
Characteristics of the study population are shown in Table
Characteristics of the analysed subcohort and the remaining LINA cohort. (A) General characteristics assessed during the 36th week of pregnancy. (B) Indoor chemical exposures during the second year of life. (C) Infant’s respiratory outcomes during the second year of life.
Parameters | Analysed subcohort |
Remaining cohort |
|
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(A) General characteristics | |||
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|||
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16 (23.5) | 140 (29.3) | 0.502 |
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28 (41.2) | 162 (33.9) | |
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24 (35.3) | 176 (36.8) | |
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29 (42.6) | 235 (49.2) | 0.349 |
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39 (57.4) | 243 (50.8) | |
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|||
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0 (0) | 6 (1.3) | 0.294 |
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19 (27.9) | 101 (21.1) | |
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49 (72.1) | 371 (77.6) | |
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18 (26.5) | 80 (16.7) | 0.167 |
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26 (38.2) | 234 (49.0) | |
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24 (35.3) | 164 (34.3) | |
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11 (16.2) | 59 (12.3) | 0.430 |
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57 (83.8) | 419 (87.7) | |
Dampness in dwelling during the second year of life | |||
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6 (9.2) | 46 (10.9) | 0.700 |
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59 (90.2) | 377 (89.1) | |
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(B) Indoor chemical exposure during the second year of life | |||
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7 (10.4) | 27 (5.7) | 0.222 |
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60 (89.6) | 445 (94.3) | |
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3 (4.5) | 7 (1.5) | 0.173 |
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4 (6.0) | 10 (2.2) | |
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60 (89.5) | 447 (96.3) | |
Usage of disinfectants | |||
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40 (61.5) | 294 (65.0) | 0.608 |
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25 (38.5) | 158 (35.0) | |
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(C) Respiratory outcomes during second year of life | |||
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15 (22.7) | 101 (21.8) | 0.878 |
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51 (77.3) | 362 (78.2) | |
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12 (19.0) | 54 (12.5) | 0.207 |
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51 (81.0) | 377 (87.5) | |
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7 (10.3) | 26 (5.7) | 0.231 |
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61 (89.7) | 432 (94.3) | |
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18 (28.6) | 125 (27.7) | 0.887 |
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45 (71.4) | 326 (72.3) | |
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6 (9.4) | 42 (9.6) | 0.962 |
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58 (90.6) | 396 (90.4) |
In general, a positive correlation was found between corresponding maternal and infant IL-3- (
Numbers of maternal and infant IL-3-, IL-5-, or GM-CSF-stimulated eosinophil/basophil (Eo/B) colony forming units (CFUs), presented as median and interquartile range (IQR). Correlation between maternal and infant Eo/B CFUs was calculated using Spearman’s rank correlation test;
Eo/B CFUs | Mother | Child |
|
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Median (IQR) | ||||
IL-3 | 8.5 (3.5–17.5) | 9.0 (4.0–15.0) |
|
0.447 |
IL-5 | 0.5 (0.3–1.5) | 0.5 (0–1.5) | 0.565 | 0.101 |
GM-CSF | 5.5 (1.0–7.0) | 2.5 (1.0–8.3) |
|
0.686 |
Associations among indoor chemical exposures, Eo/B CFUs of mother-child pairs, and clinical outcomes of 2-year-old children.
During the second year of life 61.5% of the families in the analysed subcohort declared their usage of disinfectants in the household as “once a week or more,” “once a month or more,” or “occasionally,” with no differences when compared to the remaining LINA cohort (
Within the analysed subcohort, 10.4% of the participants were exposed to indoor ETS “(almost) daily,” “once a week or more,” or “occasionally,” with no differences when compared to the remaining LINA cohort (
Two-year-old children exposed to ETS at home had significant higher numbers of IL-3- (
Association between indoor chemical exposures and the number of IL-3-, IL-5-, or GM-CSF-stimulated eosinophil/basophil (Eo/B) colony forming units (CFUs) of two-year-old children and their mothers. Data are shown as median and interquartile range (IQR); analyses were performed using Mann-Whitney
Indoor exposures | Exposure to ETS in dwelling | Usage of disinfectants in the household | ||||
---|---|---|---|---|---|---|
Yes | No |
|
Yes | No |
| |
Eo/B CFUs | Median (IQR) | Median (IQR) | ||||
Mother | ||||||
IL-3 | 18.8 (5.0–55.5) | 8.5 (3.8–17.0) | 0.116 | 10.5 (4.0–16.5) | 7.8 (4.0–17.5) | 0.790 |
IL-5 |
|
0.5 (0.3–1.3) | 1.0 (0.5–1.5) | 0.597 | ||
GM-CSF |
|
7.0 (5.5–7.0) | 4.8 (1.0–5.5) | 0.312 | ||
Child | ||||||
IL-3 | 23.0 (9.5–32.0) | 8.0 (3.5–14.0) |
|
9.8 (4.8–19.8) | 6.0 (3.0–12.5) | 0.067 |
IL-5 | 1.0 (0.5–4.5) | 0.5 (0–1.5) | 0.100 | 0.5 (0–1.8) | 0.5 (0–1.5) | 0.930 |
GM-CSF | 12.0 (3.0–16.0) | 2.5 (1.0–6.5) |
|
3.8 (2.0–12.3) | 1.8 (1.0–5.5) |
|
ETS: environmental tobacco smoke.
Correlation of indoor smoked cigarettes per day and IL-3-, IL-5-, or GM-CSF-stimulated eosinophil/basophil (Eo/B) colony forming units (CFUs) in peripheral blood of two-year-old children and their mothers. Data are shown as Spearman’s rank correlations;
Eo/B CFUs mother | Eo/B CFUs child | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
IL-3 | IL-5 | GM-CSF | IL-3 | IL-5 | GM-CSF | |||||||
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| |
Number of smoked cigarettes/day in dwelling | 0.131 | 0.194 |
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0.096 | 0.208 |
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|
With respect to VOCs, positive correlations were found between children’s IL-3-, IL-5-, or GM-CSF-stimulated Eo/B CFUs and the sum of all measured VOCs as well as for the single smoking related VOCs benzene, m + p-xylene, and o-xylene (
Correlation of indoor volatile organic compound (VOC) concentrations and IL-3-, IL-5-, or GM-CSF-stimulated eosinophil/basophil (Eo/B) colony forming units (CFUs) in peripheral blood of two-year-old children and their mothers. Shown are aromatic VOCs as well as the sum of all analysed VOCs. Data are presented as Spearman’s rank correlations;
VOCs | Eo/B CFUs mother | Eo/B CFUs child | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
IL-3 | IL-5 | GM-CSF | IL-3 | IL-5 | GM-CSF | |||||||
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| |
Benzene |
|
|
0.076 | 0.299 | 0.544 | 0.206 | 0.101 | 0.202 |
|
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|
Toluene | 0.899 | 0.016 | 0.834 | 0.036 | 0.779 | 0.096 | 0.750 | 0.040 | 0.314 | 0.126 | 0.433 | 0.107 |
m + p-Xylene | 0.872 | 0.021 | 0.957 | 0.009 | 0.447 | 0.256 | 0.418 | 0.101 |
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|
o-Xylene | 0.483 | 0.090 | 0.882 | −0.026 | 0.472 | 0.243 | 0.174 | 0.168 |
|
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|
Styrene | 0.777 | 0.037 | 0.282 | −0.184 | 0.728 | 0.119 | 0.830 | 0.027 | 0.507 | 0.083 | 0.807 | 0.033 |
|
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Sum of all VOCs | 0.093 | 0.213 | 0.848 | 0.033 | 0.148 | 0.467 |
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The usage of disinfectants was found to be associated with increased numbers of GM-CSF-stimulated Eo/B CFUs among infants (
Within the analysed subcohort, 22.7% of the children were positive for wheezing ever, 19.0% for recurrent wheezing, and 10.3% for wheezing requiring medical treatment during the second year of life. Furthermore a physician-diagnosed bronchitis was seen in 28.6% of the children and obstructive bronchitis in 9.4%. There were no differences in the distribution of considered respiratory outcomes in the analysed subcohort (
Children who suffered from wheezing requiring medical treatment during the second year of life had significantly more IL-3- (
IL-3-, IL-5-, or GM-CSF-stimulated Eo/B CFUs in peripheral blood of two-year-old children with (pos) or without (neg) wheezing requiring medical treatment during the second year of life. Data are shown as box plots with median and 25th to 75th percentile.
Within earlier studies, we showed that there are increases in blood eosinophil/basophil progenitor cells in one-year-old children in association with exposure to environmental chemicals [
To our knowledge absolute Eo/B colony numbers of mothers and their infants have not been compared before. It is well known that numbers of progenitor cells, except in bone marrow, are highest in cord blood and decrease in peripheral blood later in life [
According to our hypothesis we could demonstrate with the present data that infant’s progenitor cells seem to respond with more sensitivity to environmental pollutants (ETS, VOCs, and disinfectants) compared to maternal progenitor cells. This is in agreement with results shown earlier within the LINA study: VOCs emitted due to renovation activities were observed to influence the child’s but not the mother’s immune response. In cord blood but not in peripheral blood of the mothers increased IL-4 and IL-5 serum levels [
In addition, our data support results showing that enhanced numbers of Eo/B progenitor cells as a consequence of environmental pollutants may increase the risk for wheeze and skin manifestations in early infancy [
The fact that exposure to tobacco smoke/VOCs seems to influence the development of respiratory diseases has been shown before. There are several epidemiological studies demonstrating that exposure to prenatal smoke or passive tobacco smoke early in life is associated with an increased risk of wheezing in early infancy [
There is furthermore evidence that exposure to tobacco smoke/VOCs provokes an immunological imbalance. Newborn children from smoking mothers were reported to have fewer cord blood regulatory T cell numbers [
Finally, the present data demonstrate that Eo/B progenitors of two-year-old children which correlated positively with wheezing in early childhood were progenitors stimulated by IL-3 and GM-CSF: cytokines which are known to influence early eosinophil/basophil lineage differentiation [
The strength of our study derives from the fact that the analyses of Eo/B progenitor cells were performed in mother-child pairs which are well characterised regarding their immune parameters, atopic outcomes, and indoor air exposure to environmental chemicals. For example, individual ETS exposure was assessed not only by questionnaire data, which are always dependent on honesty and compliance of the participants [
A weakness of the LINA study in general is the potential bias by high rates of participating atopic parents (about 65%). We addressed this point by including family history of atopy as a confounding variable in the regression models. The high prevalence of atopic parents (who are already aware of their atopic disease and probably avoid potential hazards more than others) might also be one reason for the quite low prevalence of children exposed to ETS during the second year of life (10% versus 18.7% shown earlier for Germany [
In the present study we could confirm our earlier published data [
Confidence interval
Colony forming unit
Eosinophil/basophil
Environmental tobacco smoke
Granulocyte macrophage colony-stimulating factor
Interleukin
Interquartile range
Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk
Nonadherent mononuclear cell
Odds ratio
Peripheral blood mononuclear cell
Spearman’s rank correlation coefficient
Volatile organic compound.
The authors declare that there are no competing interests regarding the publication of this paper.
Irina Lehmann and Kristin M. Junge contributed equally to this work.
The authors thank all the participants of the LINA study, Anne Hain, Anika Lüdemann for her excellent technical support, and Melanie Nowak for her motivated study organisation assistance. The LINA study is supported by Helmholtz institutional funding (Helmholtz Centre for Environmental Research (UFZ)). This paper is further supported by Leipzig Research Center for Civilization Diseases (LIFE), University of Leipzig. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERDF), and by means of the Free State of Saxony within the framework of the excellence initiative (SAB 713-257022).