Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Cytokine Profiles in Asthma Families Depend on Age and Phenotype

  • Katrin Pukelsheim,

    Affiliation Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany

  • Tobias Stoeger,

    Affiliation Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany

  • David Kutschke,

    Affiliation Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany

  • Koustav Ganguly,

    Affiliations Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America

  • Matthias Wjst

    m@wjst.de

    Affiliation Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany

Cytokine Profiles in Asthma Families Depend on Age and Phenotype

  • Katrin Pukelsheim, 
  • Tobias Stoeger, 
  • David Kutschke, 
  • Koustav Ganguly, 
  • Matthias Wjst
PLOS
x

Abstract

Background

Circulating cytokine patterns may be relevant for the diagnosis of asthma, for the discrimination of certain phenotypes, and prognostic factors for exacerbation of disease.

Methodology/Principal Findings

In this study we investigated serum samples from 944 individuals of 218 asthma-affected families by a multiplex, microsphere based system detecting at high sensitivity eleven asthma associated mediators: eotaxin (CCL11), granulocyte macrophage stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin-4 (IL-4), IL-5, IL-8, IL-10, IL-12 (p40), IL-13, IL-17 and tumor necrosis factor alpha (TNFα). Single cytokine levels were largely similar between asthmatic and healthy individuals when analysing asthma as single disease entity. Regulatory differences between parental and pediatric asthma were reflected by six of the eleven mediators analyzed (eotaxin, IL-4, IL-5, IL-10, IL-12, TNFα). IL-12 (p40) and IL-5 were the best predictor for extrinsic asthma in children with an increased odds ratio of 2.85 and 1.96 per log pg/ml increase (IL-12 (p40): 1.2–6.8, p = 0.019, and IL-5: 1.2–2.5, p = 0.025). Frequent asthma attacks in children are associated with elevated IL-5 serum levels (p = 0.013). Cytokine patterns seem to be individually balanced in both, healthy and diseased adults and children, with various cytokines correlating among each other (IL-17 and IFNγ (rs = 0.67), IL-4 and IL-5 (rs = 0.55), IFNγ and GM-CSF (rs = 0.54)).

Conclusion/Significance

Our data support mainly an age- but also an asthma phenotype-dependent systemic immune regulation.

Introduction

Asthma is the most common serious chronic lung disease and shows increasing incidence in industrialized as well as in developing countries, afflicting around 150 million people worldwide [1]. With airway hyper-responsiveness being the physiological hallmark of asthma, it is also characterized by chronic inflammation of the respiratory tract, allergen-specific IgE production, infiltration of eosinophils, the recruitment of T cells into the airways, and alterations in the fine balance between type 1 helper T lymphocytes (Th1) and type 2 helper T lymphocytes (Th2) responses towards Th2 bias [2].

Th2 cells secret a panel of cytokines with several overlapping functions including Interleukin-4 (IL-4), IL-5, IL-13, and granulocyte-macrophage colony stimulating factor (GM-CSF). By mediating differentiation of the Th2 subpopulation and eosinophils, as well as modulating B-cell proliferation and IgE switching, the Th2 cytokines are thought to play a prominent role in asthma [3], [4]. The sentinel Th1 cytokine, interferon gamma (IFNγ) and IL-12 reciprocally stimulate their production and function during cell-mediated immunity and development of naïve T lymphocytes into Th1 cells. Evidence suggests a contributory role of Th1 cells and their cytokines in asthmatic inflammation and airway hyperresponsiveness [5], [6]. The T cell subset of regulatory T cells (Treg) acts by expressing immunosuppressive cytokines, such as IL-10, of which impaired production has been reported in asthmatic patients [7]. Moreover, the lymphocyte lineage Th17 is increased in inflamed airways [8] and characterised by the production of IL-17. This pro-inflammatory cytokine is capable of causing the release of other pro-inflammatory cytokines, such as IL-8, tumor necrosis factor alpha (TNFα), and GM-CSF, which have been associated with asthma in murine models [9], in humans [10], [11], or with disease severity [12], [13], [14], [15].

Asthma – probably the most heterogeneous lung disease – may also demonstrate systemic patterns outside the respiratory system. Determination of serum cytokine levels in asthmatic patients could have potential utility in diagnosis of asthma and certain phenotypes, in prediction of attacks, and treatment choice. Although there are numerous in vitro, as well as in vivo data from animal studies, to date no information of large scale clinical studies exist that examines a correlation between circulating cytokine levels and asthma phenotypes or exacerbations of disease in different age groups.

Thus, the present study aimed at investigating circulating cytokine and chemokine levels of healthy and asthmatic children as well as their parents. We hypothesized that one of the analyzed cytokines and chemokines, or a combination is suited as a serological marker for asthma or certain phenotypes. To test our hypothesis we measured serum concentrations of eleven cytokines and chemokines that were recently described to play an important role in asthma pathogenesis: IL-4, IL-5, IL-8, IL-10, IL-12 (p40), IL-13, IL-17, TNFα, GM-CSF, eotaxin, and IFNγ.

Materials and Methods

Study Population

218 core families were selected during two study stages. The sample is identical to previous reports [16], [17], [18] describing explicitly the selection process, study participants and their clinical characteristics. In the first stage, 97 families consisting of at least two children with confirmed clinical asthma were selected for an initial genome-wide linkage scan [18]. The families were collected mainly in pediatric university clinical centers in Germany and Sweden. At least two children with confirmed clinical asthma were required, and prematurity or low birth weight of the children was excluded, along with any severe pulmonary disease other than asthma. In the second study stage, another set of asthma sib pair families was selected. Three university hospitals and six pediatric pulmonary practices carried out an identical phenotyping procedure as during the first stage.

The 218 families comprised 944 individuals, of whom 424 were parents with a mean age of 40 years and 499 were children with a mean age of 11 years; 21 individuals had insufficient clinical documentation and were excluded from further analysis. 275 of the children were male, 224 were female and 443 had physician diagnosed asthma. The youngest child diagnosed with asthma was 4 years old and the oldest child was 34 years old. Fifty-eight percent of the children with asthma were boys. Asthma was initially defined by clinical history and validated later by interview questions. In addition to a clinical asthma diagnosis, all affected children over age 3 had a history of at least 3 years of recurrent wheezing and with no other airway diseases diagnosed.

The phenotyping procedure contained detailed interviews of every family member, skin prick tests (SPT) of frequent allergens, blood samples (for IgE and allergen-specific IgE (RAST) measurements, eosinophil count), peak flow tests for a period of 10 days and dust collection at patients home. Total IgE was determined with an ELISA (Pharmacia Diagnostics, Uppsala, Sweden). SPT and RAST assays included several pollens, animal furs, mould, and house dust mite allergens (ALK-SCHERAX, Hamburg, Germany). Atopic (extrinsic) asthma was defined if patients demonstrated at least one positive skin prick test reaction or one increased specific IgE. Pulmonary function tests were performed by forced flow volume tests and bronchial challenge was done by a defined increasing methacholine exposure. Written consent was given by all study participants. The parent/guardians of all participants under 18 gave informed consent too. All study methods were approved in 1995 by the Ethics Commission of “Nordrhein-Westfalen” and again in 2001 by “Bayerische Landesärztekammer München”.

Cytokine Assays

Concentration of eotaxin, GM-CSF, IFN-γ, IL-4, IL-5, IL-8, IL-10, IL-12 (p40), IL-13, IL-17, TNF-α in the serum samples was quantified using a custom-made MILLIPLEX™ MAP (multiple analyte profile) Human Cytokine/Chemokine Panel (Millipore, Schwalbach/Ts, Germany). The assay was performed according to the recommendations of the manufacturer using a 8-point standard curve for every cytokine. Samples were analyzed on a Luminex 100 device (BioRad, München, Germany) and the data were evaluated using the Bio-Plex Manager software (BioRad). Standards and internal controls were reported as means of duplicate measurements. Samples were analyzed in single measurements, since the MILLIPLEX™ MAP assay shows good precision in pre-tests, with a stated intra-assay mean coefficient of variance (CV) of 4.5% to 10.4%. The lower detection limits for every cytokine and numbers of samples that were below the lower detection limit and above the upper detection limit are listed in Table S1 (online data supplement). Values below the detection limit were set to the half of the detection limit and to 3000 pg/ml if exceeding the maximum concentration of 3000 pg/ml.

Analysis

Statistical analyses were performed using R software. Following a descriptive analysis using cross-tabulation and distribution of single variables, linear and logistic regression analysis was used for determination of effect estimates.

Results

The serum concentrations of the 11 cytokines and chemokines IL-4, IL-5, IL-8, IL-10, IL-12 (p40), IL-13, IL-17, TNFα, GM-CSF, Eotaxin, and IFNγ could be obtained from 944 individuals (Figure S1) from 218 families, of whom 424 were parents and 499 were children. Main demographic and clinical characteristics of study participants were summarized in previous reports [16], [17], [18]. In table 1 healthy and asthmatic parents are compared as well as healthy and asthmatic children. Except of IL-8 (p = 0.012) and IL-10 (p = 0.045) in the adult group, none of the cytokine concentrations in serum differ significantly between healthy and diseased individuals. However, mean cytokine levels for six of the eleven cytokines are significantly different between asthmatic children and asthmatic adults. Values for Eotaxin (p = 0.006), IL-10 (p = 0.002), and TNFα (p = 0.038) are higher in parents than in children. In contrast IL-4 (p<0.001), IL-5 (p<0.001), and IL-12 (p40) (p = 0.001) serum levels are lower in diseased adults compared to diseased children.

thumbnail
Table 1. Case control analysis of parents and children with and without asthma.

https://doi.org/10.1371/journal.pone.0014299.t001

Results of serum cytokine measurements of the asthmatic children and distinct clinical parameters were then further analyzed in a regression model. In table 2 regression coefficients and p-values are calculated using logarithmic values. Of the blood cell counts, lymphocyte and eosinophil counts are inversely associated to their cooperating chemokines (lymphocyte numbers and IL-8: r = −0.019, p = 0.011; eosinophil numbers and eotaxin: r = −0.432, p = 0.033). Furthermore IL-5 is positively associated with eosinophilia (r = 0.119, p = 0.050), while IFNγ/IL-5 demonstrates an inverse association to eosinophil counts (r = −0.119, p = 0.030). Regression analysis of serum cytokine levels to established clinical parameters of asthma, such as current lung function (FEV1) and bronchial hyperreactivity (BHR), showed weak results. Of all cytokines measured only the serum concentrations of IL-4 (r = 0.016, p = 0.013), is associated with the current lung function FEV1. However, in spite of a remarkable result for IL-12 (p40) (p = 0.056), none of the eleven circulating cytokines is related to BHR. But, classifying the number of asthma attacks occurring in the last year to categories from one to four, revealed that frequent asthma attacks in children are associated with higher IL-5 serum levels (p = 0.013, Figure 1).

thumbnail
Figure 1. Box plot of serum IL-5 concentrations and asthma attack rates in one year.

Only asthmatic children (n = 443) were included in this analysis. The numbers of asthma attacks occurring in the last year were classified into continuous categories from one to four. Numbers of samples of the respective groups are 213 (group 1), 175 (group 2), 26 (group 3), and 29 (group 4). Children with the most frequent asthma attacks had the highest serum levels of IL-5 (p = 0.013).

https://doi.org/10.1371/journal.pone.0014299.g001

thumbnail
Table 2. Regression analysis of serum cytokines and clinical characteristics.

https://doi.org/10.1371/journal.pone.0014299.t002

In table 3 the odds ratios (OR) are used to describe the predictive value of serum cytokine levels to asthma subtypes, allergic rhinitis, eczema, actual medication and hospitalization frequency, while only asthmatic children were included in this analysis. Atopic (extrinsic) asthma was defined if patients demonstrated at least one positive skin prick test reaction or one increased specific IgE. IL-12 (p40) is the best predictor for extrinsic asthma in children with an increased OR of 2.85 per log pg/ml increase (1.19–6.84, p = 0.019) or vice versa with a decreased OR of 0.35 (0.15–0.84, p = 0.019) for intrinsic asthma. Moreover atopic and non-atopic asthma phenotypes were significantly influenced by IL-5 levels (OR for atopic asthma 1.96 (1.09–3.52, p = 0.025). A receiver operating characteristic (ROC) revealed a sensitivity of 67.1% for IL-12 (p40) and 66.5% for IL-5 and specificity of 66.7% for IL-12 (p40) and 58.3% for IL-5 serum levels to distinguish between the extrinsic and intrinsic asthma phenotype in children (Figure 2 A and B). The association of serum cytokine levels to other atopic diseases, such as allergic rhinitis and eczema, showed mixed results. For the latter no circulating cytokine was predictive and for allergic rhinitis a strong impact of IL-5 serum levels (OR: 1.42 (1.11–1.82), p = 0.005) could be detected.

thumbnail
Figure 2. Receiver operating characteristic for the diagnostic value of IL-12 (p40) (A) and IL-5 (B).

Only asthmatic children (n = 443) were included in this analysis. Area under the curve of the ROC plot produced a sensitivity of 67.1% and specificity of 66.7% for IL-12(p40), and a sensitivity of 66.5% and a specificity of 58.3% for IL-5 to distinguish between the extrinsic and intrinsic asthma phenotype.

https://doi.org/10.1371/journal.pone.0014299.g002

thumbnail
Table 3. Predictive value of serum cytokine levels for extrinsic asthma, allergic rhinitis, eczema, actual medication and hospitalization frequency.

https://doi.org/10.1371/journal.pone.0014299.t003

Generalized estimating equations of serum cytokine measurements to clinical parameters as well as to atopic diseases, medication, and hospitalization were carried out to adjust for intra-cluster (intra-household) correlation (Table 4 and 5). In Figure S2 (supplementary data) a regression analysis was carried out to identify relations between cytokines. Positive correlations could be found between IFNγ and IL-17 levels (r = 0.67), within the Th2 cytokines IL-4 and IL-5 (r = 0.55), and between IFNγ and GM-CSF (r = 0.54). The division into healthy and asthmatic individuals resulted in similar correlation patterns (data not shown). Figure S3 (supplementary data) shows a principal component analysis of cytokine concentrations.

thumbnail
Table 4. Generalized estimating equations of serum cytokines and clinical characteristics.

https://doi.org/10.1371/journal.pone.0014299.t004

thumbnail
Table 5. Generalized estimating equations of serum cytokine levels and extrinsic asthma, allergic rhinitis, eczema, actual medication and hospitalization frequency.

https://doi.org/10.1371/journal.pone.0014299.t005

Discussion

In adults and children affected with asthma, several pathophysiological effects have been associated with imbalanced T cell activation and the presence or absence of distinct immune mediators. Thus cytokines and chemokines evoke increasing interest for diagnosis and therapy. The present study aimed at understanding the pattern of circulating cytokines in asthmatic adults and children. To assess the potential value of serum cytokines as biomarker for asthma, or certain asthma phenotypes, or prediction of exacerbation of disease, we compared serum levels of eleven distinct asthma associated mediators of 218 families where at least two children have asthma. A number of authors have studied serum cytokines in asthmatic patients [15], [19], [20], [21], [22], [23]. However, no study covered such a large set of individuals with inclusion of a large number of cytokines and chemokines.

The results presented here concern asthmatic and healthy siblings and their parents and provide reference values for serum cytokine levels of asthma families and the respective age group. Like in measurements of a similar design, the results demonstrate a high variability of serum cytokine amounts between individuals [24] and at the same time, reveal distinct characteristics in the distributions of the single cytokines and chemokines analyzed (Figure S1). No differences were observed between healthy and diseased individuals in the cytokines analyzed, except IL-8 in adults (table 1). In this analysis asthma was referred to as one disease and was not divided into distinct subgroups. Our data differ from those of the exploratory study of ten Hacken et al. who found that the median levels of serum IL-4, IL-5, and IFNγ were significantly higher in asthmatic patients than in healthy controls [25]. Instead, our results support the hypothesis of asthma not being associated with changes of serum cytokine amounts, when regarded as single disease entity. A most likely explanation could be that asthma is a complex and heterogeneous disease consisting of many phenotypes [1]. On the contrary, it might be that systemic patterns of Th1-, Th2-, Th17-, and Treg -cytokines in serum do not reflect the same immune imbalances proposed for the diseased organs of immune mediated disorders. In this respect, it has been suggested, that findings concerning inflammatory mediators of asthmatic patients differ in serum and bronchial lavage fluid [19]. Additionally, frequencies of cytokine producing T cells in blood do not reflect those in sputum [26]. It should be mentioned that the study design of the German Asthma Family Study may not be advantageous for comparing healthy and diseased children, due to a small proportion of healthy children (n = 56), resulting in a loss of power to discriminate between health and disease. However, since the healthy children are sharing genes and environment with the asthmatic children, the discordance for disease may be of particular interest, even with low nominal power.

Serum cytokine patterns showed marked differences between parental and pediatric asthma. In asthmatic children cytokine levels demonstrated significant elevation in IL-4, IL-5, and IL-12(p40), and a decrease in eotaxin, IL-10, and TNFα, compared to asthmatic adults (table 1). This points at age being important factors in systemic immune regulation of asthma. The impact of age was also recently shown for mitogen-stimulated cytokine production in a general population [27]. Further, our data and literature data point at the importance of evaluating circulating cytokine levels in specific asthma phenotypes. Wong et al. found higher plasma concentrations of IL-18, IL-12, IL-10, and IL-13 in allergic asthmatic patients than in healthy controls [28]. Silvestri et al. stated that, IL-8 and TNFα, but not IL-16 and IL-13, serum levels were higher in severe asthmatic compared to mild-moderate asthmatics or controls [15]. Our study includes mainly individuals from university hospitals and families with two affected children. To control for correlation across family members generalized estimating equations of serum cytokine measurements to clinical parameters as well as to atopic diseases, medication, and hospitalization were carried (Table 4 and 5). Nevertheless, the study design certainly leads to a biased sample towards children with a strong familiar/genetic component and towards children with more severe disease. But, more extreme phenotypes may be more informative for detecting any biochemical or genetic abnormalities.

The most common classification of asthma is the division into extrinsic (atopic, allergic) and intrinsic (non-allergic) phenotype. The finding that systemic levels of IL-12 (p40), IL-5, and IFNγ/IL-5 reflect the regulatory differences between the allergic and non-allergic pediatric asthma subgroups (table 3), led us to address this issue more closely. The expression of IL-12, a heteromeric protein consisting of the two subunits p35 and p40, and the monomeric and homodimeric form of p40 respectively, has proven to be associated with asthma [5]. Furthermore, anti-IL-12-p35 or anti-IL-12-p40 monoclonal antibodies were shown to abolish asthma symptoms during the challenge phase in a mouse model of experimental asthma [29]. In contrast to IL-12, a substantial body of data in mice and humans support the importance of IL-5 in asthma [3], [30]. Frequent asthma attacks were significantly associated with higher circulating IL-5 levels (Figure 2). To determine, if any of the analyzed serum cytokine levels could be used as a potential disease indicator, the diagnostic value of IL-12 (p40) and IL-5 were directly tested in a receiver operating characteristic (Figure 2A, B). IL-12 and IL-5 demonstrate only moderate marker characteristics to distinguish between the extrinsic and intrinsic asthma phenotype. The clinical use of these cytokines is still hampered by the fact that there would be too high false negatives and positives.

In the present study serum cytokine levels and its correlations to clinical symptoms, such as blood cell count, total and specific IgE, asthma onset, and pulmonary function was evaluated to assess practical applicability (table 2). Overall correlating circulating cytokine concentrations to established clinical parameters of asthma did not reveal any clear pattern. Our results may indicate that serum cytokine levels and functional tests reflect distinct aspects of the pathophysiology of asthma, and clinical symptoms occur independently of systemic cytokine levels. However, this contrasts with results of Nakamura et al. who found that plasma eotaxin levels were inversely related to percent predicted FEV1 and directly related to asthma diagnosis in a large population [22]. Unlike results of some exploratory studies [21], [25], [31], [32], in our study asthmatic children did not demonstrate an association of serum IFNγ to clinical manifestations of asthma, possibly due to distinct study designs and inclusion criteria for the selection of cases, again outlining the crucial role of subdividing asthma into defined phenotypes.

IL-8 is a pro-neutrophilic chemokine that is secreted by various cell types. For more than 10 years, IL-8 has been suggested to play an important role in asthma, and especially in severity of disease [11], [13], [15]. In our study, analysing IL-8 serum levels in regard to clinical parameters of asthma, demonstrated several significant results. It correlated negatively with lymphocyte counts, asthma attack frequency and positively with specific IgE for GX1 (table 2) and, additionally, IL-8 serum concentrations differed between healthy and asthmatic adults (table 1). This points towards an important systemic role for IL-8. However, our analysis was hampered by the observation that IL-8 levels of the first funding period, which took place from 1995 to 1998, were markedly lower than IL-8 concentrations of the second part of the study, which took place from 2000 to 2001. This contrasts with the homogenous measurements for the other analyzed cytokines (Figure S1). Due to the fact that all cytokines were quantified in one assay and originate of the same serum sample, which was thawed only once, we can largely exclude laboratory procedures to be responsible for this phenomenon. The only difference between samples of the first study and the second study half is the time of storage at −80°C (14-11 years versus 9-8 years). IL-8 may be the most unstable of the analyzed cytokines, which is supported by the conclusion of Friebe et al., who recently proposed that there are major differences in the stability of cytokines in blood samples [33].

For a better understanding of the regulation of cytokine networks in asthmatic patients, we considered further the possibility of a direct or inverse relationship between Th2, Th1, Treg and Th17 cytokines. Pair wise assessment of correlations among the analyzed cytokines, revealed a strong positive correlation between IFNγ and IL-17 within healthy and asthmatic individuals (Figure S2). This is in accordance with previous data where elevation of the dominant Th1 cytokine IFNγ and the strong pro-inflammatory cytokine IL-17, have been reported in sputum of asthmatic patients [26], [34]. It is obvious in our analysis that the cytokines are correlated in healthy controls and asthmatic patients. Based on the knowledge that IFNγ, has generally been considered having a negative role on Th17 cell differentiation and expansion [35], this direct relation could be due to an individually balanced cytokine pattern. This is favored also by a direct relation for the Th2 cytokines IL-4 and IL-5, and IFNγ and GM-CSF. The hypothesis of a relative balance appearing more important than actual concentrations was suggested also recently by Halonen et al. for cytokine production in mothers, fathers, and infants of a large birth cohort [27].

Our data do not provide information about the origin of the analyzed cytokines. It is known that during allergic reactions, no activated lymphocytes are found in the circulation, although there is an increase in local lymphocyte activity [36]. Such factors would favor the importance of the local environment, such as lung tissue, to search for a marker of practical applicability, but less invasive measures, such as serum, represent the great advantage of being much easier to obtain, especially in children. Considering the inconclusive overall pattern in obtaining biomarker characteristics for any of the analyzed cytokines, our data do not support the idea of a routine evaluation of serum cytokines in asthmatic patients and point at the importance of precisely defining asthma subtypes. Little is known about the dynamics of circulating cytokines, including degradation, regulation and half life, which was not addressed in this study and remain to be elucidated in further studies. This knowledge would pave the way for a conclusive interpretation and validation of systemic immune mediators and the discovery of a serological marker for asthma or asthma phenotypes or severity respectively.

In conclusion, the present study demonstrates main characteristics of serum cytokine patterns in healthy and asthmatic children and adults, and provides for the first time reference values for these groups in a large population. Serum patterns were not different between healthy and asthmatic individuals, with regard to absolute amounts. By looking at distinct phenotypes of asthma, patterns of circulating immune mediators demonstrated major differences. Serum levels of six out of eleven mediators analyzed differed in parental and pediatric asthma. IL-12 (p40), IL-5, and IFNγ/IL-5 showed differences between the extrinsic and intrinsic asthma phenotype. High IL-5 serum levels are associated with frequent asthma attacks in children. Furthermore, cytokine patterns seem to be individually balanced both in healthy and diseased adults and children. The precise mechanism of how systemic cytokines and their coactive interactions might be involved in driving asthma processes, however, remains elusive. A further expansion of the cytokine panel tested, possibly coupled even with direct local measurements and clearly defined asthma phenotypes is warranted.

Supporting Information

Table S1.

Assay Sensitivities of MILLIPLEX MAP for the 11 analyzed Cytokines and Chemokines.

https://doi.org/10.1371/journal.pone.0014299.s001

(0.05 MB DOC)

Figure S1.

Histogram of serum cytokine levels of all individuals. Each of the cytokines has its own scale and concentrations are in the range of pg/ml. The 944 study participants are ordered chronologically by the time of examination (on the x axis). There is a high variability of serum cytokine amounts between individuals. One individual showed extremely high levels at nearly all cytokines. Single cytokines demonstrate distinct characteristics in their distributions.

https://doi.org/10.1371/journal.pone.0014299.s002

(0.38 MB TIF)

Figure S2.

Correlation matrix of the analyzed cytokine concentrations. Pair wise assessment of correlations, revealed a strong positive relation for IL-17 and IFNγ levels (rs = 0.67), GM-CSF and IFNγ levels (rs = 0.54), as well as IL-4 and IL-5 (rs = 0.55).

https://doi.org/10.1371/journal.pone.0014299.s003

(2.55 MB TIF)

Figure S3.

Principal component analysis of serum cytokine concentrations.

https://doi.org/10.1371/journal.pone.0014299.s004

(0.13 MB TIF)

Acknowledgments

We thank all participating families for their help and the members of the clinical centers for their work: R. Nickel, K. Beyer, R. Kehrt, U.Wahn (Berlin), K. Richter, H. Janiki, R. Joerres, H. Magnussen (Grosshansdorf), I. M. Sandberg, L. Lindell, N.I.M. Kjellman (Linkoeping), C. Frye, G. Woehlke, I. Meyer, O. Manuwald (Erfurt), A. Demirsoy, M. Griese, D. Reinhardt (München), G. Oepen, A. Martin, A. von Berg, D. Berdel (Wesel), Y. Guesewell, M. Gappa, H. von der Hardt (Hannover), J. Tuecke, F. Riedel (Bochum), M. Boehle, G. Kusenbach, H. Jellouschek, M. Barker, G. Heimann (Aachen), S. van Koningsbruggen, E. Rietschel (Köln), P. Schoberth (Köln), G. Damm, R. Szczepanski, T. Lob-Corzilius (Osnabrück), L. Schmid, W. Dorsch (München), M. Skiba, C.Seidel, M. Silbermann (Berlin), A. Schuster (Düsseldorf), J. Seidenberg (Oldenburg), W. Leupold, J. Kelber (Dresden), W. Wahlen (Homburg), F. Friedrichs, K. Zima (Aachen), P. Wolff (Pfullendorf), D. Bulle (Ravensburg), W. Rebien, A.Keller (Hamburg) and M. Tiedgen (Hamburg). We wish to thank M. Hoeltzenbein who helped start the study and J. Altmüller who managed the second part of the study, G. Schlenvoigt and L.Jaeger for IgE determination; L. Thaller, G. Fischer, T. Illig, N. Klopp, C. Vollmert and M. Werner and doctoral students H. Gohlke, N. Herbon and G. Dütsch for their contribution in previous genotyping projects; E. André, M. Bahnweg, A. Luze, C. Braig and B. Wunderlich for excellent laboratory work.

Author Contributions

Conceived and designed the experiments: KP MW. Performed the experiments: KP DK. Analyzed the data: KP TS KG MW. Contributed reagents/materials/analysis tools: MW. Wrote the paper: KP.

References

  1. 1. Anderson GP (2008) Endotyping asthma: new insights into key pathogenic mechanisms in a complex, heterogeneous disease. Lancet 372: 1107–1119.GP Anderson2008Endotyping asthma: new insights into key pathogenic mechanisms in a complex, heterogeneous disease.Lancet37211071119
  2. 2. Larche M, Robinson DS, Kay AB (2003) The role of T lymphocytes in the pathogenesis of asthma. J Allergy Clin Immunol 111: 450–463; quiz 464.M. LarcheDS RobinsonAB Kay2003The role of T lymphocytes in the pathogenesis of asthma.J Allergy Clin Immunol111450463; quiz 464
  3. 3. Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, et al. (1992) Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 326: 298–304.DS RobinsonQ. HamidS. YingA. TsicopoulosJ. Barkans1992Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma.N Engl J Med326298304
  4. 4. Wills-Karp M, Finkelman FD (2008) Untangling the complex web of IL-4- and IL-13-mediated signaling pathways. Sci Signal 1: pe55.M. Wills-KarpFD Finkelman2008Untangling the complex web of IL-4- and IL-13-mediated signaling pathways.Sci Signal1pe55
  5. 5. Cooper AM, Khader SA (2007) IL-12p40: an inherently agonistic cytokine. Trends Immunol 28: 33–38.AM CooperSA Khader2007IL-12p40: an inherently agonistic cytokine.Trends Immunol283338
  6. 6. Kumar RK, Webb DC, Herbert C, Foster PS (2006) Interferon-gamma as a possible target in chronic asthma. Inflamm Allergy Drug Targets 5: 253–256.RK KumarDC WebbC. HerbertPS Foster2006Interferon-gamma as a possible target in chronic asthma.Inflamm Allergy Drug Targets5253256
  7. 7. John M, Lim S, Seybold J, Jose P, Robichaud A, et al. (1998) Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colony-stimulating factor, and interferon-gamma release from alveolar macrophages in asthma. Am J Respir Crit Care Med 157: 256–262.M. JohnS. LimJ. SeyboldP. JoseA. Robichaud1998Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colony-stimulating factor, and interferon-gamma release from alveolar macrophages in asthma.Am J Respir Crit Care Med157256262
  8. 8. Pene J, Chevalier S, Preisser L, Venereau E, Guilleux MH, et al. (2008) Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes. J Immunol 180: 7423–7430.J. PeneS. ChevalierL. PreisserE. VenereauMH Guilleux2008Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes.J Immunol18074237430
  9. 9. Yamashita N, Tashimo H, Ishida H, Kaneko F, Nakano J, et al. (2002) Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF). Cell Immunol 219: 92–97.N. YamashitaH. TashimoH. IshidaF. KanekoJ. Nakano2002Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF).Cell Immunol2199297
  10. 10. Dente FL, Carnevali S, Bartoli ML, Cianchetti S, Bacci E, et al. (2006) Profiles of proinflammatory cytokines in sputum from different groups of severe asthmatic patients. Ann Allergy Asthma Immunol 97: 312–320.FL DenteS. CarnevaliML BartoliS. CianchettiE. Bacci2006Profiles of proinflammatory cytokines in sputum from different groups of severe asthmatic patients.Ann Allergy Asthma Immunol97312320
  11. 11. Shute JK, Vrugt B, Lindley IJ, Holgate ST, Bron A, et al. (1997) Free and complexed interleukin-8 in blood and bronchial mucosa in asthma. Am J Respir Crit Care Med 155: 1877–1883.JK ShuteB. VrugtIJ LindleyST HolgateA. Bron1997Free and complexed interleukin-8 in blood and bronchial mucosa in asthma.Am J Respir Crit Care Med15518771883
  12. 12. Berry MA, Hargadon B, Shelley M, Parker D, Shaw DE, et al. (2006) Evidence of a role of tumor necrosis factor alpha in refractory asthma. N Engl J Med 354: 697–708.MA BerryB. HargadonM. ShelleyD. ParkerDE Shaw2006Evidence of a role of tumor necrosis factor alpha in refractory asthma.N Engl J Med354697708
  13. 13. Jatakanon A, Uasuf C, Maziak W, Lim S, Chung KF, et al. (1999) Neutrophilic inflammation in severe persistent asthma. Am J Respir Crit Care Med 160: 1532–1539.A. JatakanonC. UasufW. MaziakS. LimKF Chung1999Neutrophilic inflammation in severe persistent asthma.Am J Respir Crit Care Med16015321539
  14. 14. Saha SK, Doe C, Mistry V, Siddiqui S, Parker D, et al. (2009) Granulocyte macrophage colony stimulating factor expression in induced sputum and bronchial mucosa in asthma and COPD. Thorax. SK SahaC. DoeV. MistryS. SiddiquiD. Parker2009Granulocyte macrophage colony stimulating factor expression in induced sputum and bronchial mucosa in asthma and COPDThorax
  15. 15. Silvestri M, Bontempelli M, Giacomelli M, Malerba M, Rossi GA, et al. (2006) High serum levels of tumour necrosis factor-alpha and interleukin-8 in severe asthma: markers of systemic inflammation? Clin Exp Allergy 36: 1373–1381.M. SilvestriM. BontempelliM. GiacomelliM. MalerbaGA Rossi2006High serum levels of tumour necrosis factor-alpha and interleukin-8 in severe asthma: markers of systemic inflammation?Clin Exp Allergy3613731381
  16. 16. Altmuller J, Seidel C, Lee YA, Loesgen S, Bulle D, et al. (2005) Phenotypic and genetic heterogeneity in a genome-wide linkage study of asthma families. BMC Pulm Med 5: 1.J. AltmullerC. SeidelYA LeeS. LoesgenD. Bulle2005Phenotypic and genetic heterogeneity in a genome-wide linkage study of asthma families.BMC Pulm Med51
  17. 17. Wjst M, Altmuller J, Faus-Kessler T, Braig C, Bahnweg M, et al. (2006) Asthma families show transmission disequilibrium of gene variants in the vitamin D metabolism and signalling pathway. Respir Res 7: 60.M. WjstJ. AltmullerT. Faus-KesslerC. BraigM. Bahnweg2006Asthma families show transmission disequilibrium of gene variants in the vitamin D metabolism and signalling pathway.Respir Res760
  18. 18. Wjst M, Fischer G, Immervoll T, Jung M, Saar K, et al. (1999) A genome-wide search for linkage to asthma. German Asthma Genetics Group. Genomics 58: 1–8.M. WjstG. FischerT. ImmervollM. JungK. Saar1999A genome-wide search for linkage to asthma. German Asthma Genetics Group.Genomics5818
  19. 19. Hollander C, Sitkauskiene B, Sakalauskas R, Westin U, Janciauskiene SM (2007) Serum and bronchial lavage fluid concentrations of IL-8, SLPI, sCD14 and sICAM-1 in patients with COPD and asthma. Respir Med 101: 1947–1953.C. HollanderB. SitkauskieneR. SakalauskasU. WestinSM Janciauskiene2007Serum and bronchial lavage fluid concentrations of IL-8, SLPI, sCD14 and sICAM-1 in patients with COPD and asthma.Respir Med10119471953
  20. 20. Krogulska A, Wasowska-Krolikowska K, Polakowska E, Chrul S (2009) Cytokine profile in children with asthma undergoing food challenges. J Investig Allergol Clin Immunol 19: 43–48.A. KrogulskaK. Wasowska-KrolikowskaE. PolakowskaS. Chrul2009Cytokine profile in children with asthma undergoing food challenges.J Investig Allergol Clin Immunol194348
  21. 21. Litonjua AA, Sparrow D, Guevarra L, O'Connor GT, Weiss ST, et al. (2003) Serum interferon-gamma is associated with longitudinal decline in lung function among asthmatic patients: the Normative Aging Study. Ann Allergy Asthma Immunol 90: 422–428.AA LitonjuaD. SparrowL. GuevarraGT O'ConnorST Weiss2003Serum interferon-gamma is associated with longitudinal decline in lung function among asthmatic patients: the Normative Aging Study.Ann Allergy Asthma Immunol90422428
  22. 22. Nakamura H, Weiss ST, Israel E, Luster AD, Drazen JM, et al. (1999) Eotaxin and impaired lung function in asthma. Am J Respir Crit Care Med 160: 1952–1956.H. NakamuraST WeissE. IsraelAD LusterJM Drazen1999Eotaxin and impaired lung function in asthma.Am J Respir Crit Care Med16019521956
  23. 23. Tateno H, Nakamura H, Minematsu N, Nakajima T, Takahashi S, et al. (2004) Plasma eotaxin level and severity of asthma treated with corticosteroid. Respir Med 98: 782–790.H. TatenoH. NakamuraN. MinematsuT. NakajimaS. Takahashi2004Plasma eotaxin level and severity of asthma treated with corticosteroid.Respir Med98782790
  24. 24. Prokesova L, Lodinova-Zadnikova R, Zizka J, Kocourkova I, Novotna O, et al. (2006) Cytokine levels in healthy and allergic mothers and their children during the first year of life. Pediatr Allergy Immunol 17: 175–183.L. ProkesovaR. Lodinova-ZadnikovaJ. ZizkaI. KocourkovaO. Novotna2006Cytokine levels in healthy and allergic mothers and their children during the first year of life.Pediatr Allergy Immunol17175183
  25. 25. ten Hacken NH, Oosterhoff Y, Kauffman HF, Guevarra L, Satoh T, et al. (1998) Elevated serum interferon-gamma in atopic asthma correlates with increased airways responsiveness and circadian peak expiratory flow variation. Eur Respir J 11: 312–316.NH ten HackenY. OosterhoffHF KauffmanL. GuevarraT. Satoh1998Elevated serum interferon-gamma in atopic asthma correlates with increased airways responsiveness and circadian peak expiratory flow variation.Eur Respir J11312316
  26. 26. Cho SH, Stanciu LA, Holgate ST, Johnston SL (2005) Increased interleukin-4, interleukin-5, and interferon-gamma in airway CD4+ and CD8+ T cells in atopic asthma. Am J Respir Crit Care Med 171: 224–230.SH ChoLA StanciuST HolgateSL Johnston2005Increased interleukin-4, interleukin-5, and interferon-gamma in airway CD4+ and CD8+ T cells in atopic asthma.Am J Respir Crit Care Med171224230
  27. 27. Halonen M, Lohman IC, Stern DA, Spangenberg A, Anderson D, et al. (2009) Th1/Th2 patterns and balance in cytokine production in the parents and infants of a large birth cohort. J Immunol 182: 3285–3293.M. HalonenIC LohmanDA SternA. SpangenbergD. Anderson2009Th1/Th2 patterns and balance in cytokine production in the parents and infants of a large birth cohort.J Immunol18232853293
  28. 28. Wong CK, Ho CY, Ko FW, Chan CH, Ho AS, et al. (2001) Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin Exp Immunol 125: 177–183.CK WongCY HoFW KoCH ChanAS Ho2001Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma.Clin Exp Immunol125177183
  29. 29. Meyts I, Hellings PW, Hens G, Vanaudenaerde BM, Verbinnen B, et al. (2006) IL-12 contributes to allergen-induced airway inflammation in experimental asthma. J Immunol 177: 6460–6470.I. MeytsPW HellingsG. HensBM VanaudenaerdeB. Verbinnen2006IL-12 contributes to allergen-induced airway inflammation in experimental asthma.J Immunol17764606470
  30. 30. Nakajima H, Takatsu K (2007) Role of cytokines in allergic airway inflammation. Int Arch Allergy Immunol 142: 265–273.H. NakajimaK. Takatsu2007Role of cytokines in allergic airway inflammation.Int Arch Allergy Immunol142265273
  31. 31. Corrigan CJ, Kay AB (1990) CD4 T-lymphocyte activation in acute severe asthma. Relationship to disease severity and atopic status. Am Rev Respir Dis 141: 970–977.CJ CorriganAB Kay1990CD4 T-lymphocyte activation in acute severe asthma. Relationship to disease severity and atopic status.Am Rev Respir Dis141970977
  32. 32. Saito H, Hayakawa T, Mita H, Yui YS, Shida T (1988) Augmentation of leukotriene C4 production by gamma interferon in leukocytes challenged with an allergen. Int Arch Allergy Appl Immunol 87: 286–293.H. SaitoT. HayakawaH. MitaYS YuiT. Shida1988Augmentation of leukotriene C4 production by gamma interferon in leukocytes challenged with an allergen.Int Arch Allergy Appl Immunol87286293
  33. 33. Friebe A, Volk HD (2008) Stability of tumor necrosis factor alpha, interleukin 6, and interleukin 8 in blood samples of patients with systemic immune activation. Arch Pathol Lab Med 132: 1802–1806.A. FriebeHD Volk2008Stability of tumor necrosis factor alpha, interleukin 6, and interleukin 8 in blood samples of patients with systemic immune activation.Arch Pathol Lab Med13218021806
  34. 34. Bullens DM, Truyen E, Coteur L, Dilissen E, Hellings PW, et al. (2006) IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx? Respir Res 7: 135.DM BullensE. TruyenL. CoteurE. DilissenPW Hellings2006IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?Respir Res7135
  35. 35. Park H, Li Z, Yang XO, Chang SH, Nurieva R, et al. (2005) A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6: 1133–1141.H. ParkZ. LiXO YangSH ChangR. Nurieva2005A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17.Nat Immunol611331141
  36. 36. Perez-Machado MA, Ashwood P, Torrente F, Salvestrini C, Sim R, et al. (2004) Spontaneous T(H)1 cytokine production by intraepithelial but not circulating T cells in infants with or without food allergies. Allergy 59: 346–353.MA Perez-MachadoP. AshwoodF. TorrenteC. SalvestriniR. Sim2004Spontaneous T(H)1 cytokine production by intraepithelial but not circulating T cells in infants with or without food allergies.Allergy59346353