abnormalities in t - helper ( th)2/th1 balance are considered to be involved in the pathogenesis of several chronic diseases including atopy and asthma .
there are at least two types of t - helper cells that regulate the th2/th1 balance through their corresponding cytokines , which have reciprocal functions to balance the immune system and protect organisms from infection
. the th1 cytokines interferon - gamma ( ifn- ) and interleukin ( il)-2 activate macrophages and cytotoxic tcells to kill intracellular organisms , while il-5 , il-4 , and other th2 cytokines help b cells to secrete protective antibodies [ 13 ] .
il-10 , which is produced in recently differentiated primary th1 and th2 cells , regulatory t cells , b cells , monocytes , macrophages , and dendritic cells , is a pleiotropic cytokine and is considered a major regulator of the homeostasis of the immune system [ 4 , 5 ] .
multifaceted inflammatory processes , orchestrated and regulated by a complex network of mutually interacting cytokines , determine the prognosis of chronic disorders , such as atopy and asthma [ 6 , 7 ] .
experiments on animals clearly established the concept that th1/th2 balance is involved in the etiology of atopy and asthma [ 2 , 7 , 8 ] .
th2 and th1 cytokines , representative of the two polarized immune processes , have been extensively investigated with regard to the pathogenesis of asthma and allergy . however ,
the mechanisms underlying the establishment and maintenance of this th1/th2 balance are still poorly understood in humans .
consistent with the th1/th2 balance concept , allergic individuals would be expected to have enhanced th2 immunity with a depressed th1 response   and many studies have reported that atopy is associated with a th2-dominated response , with increased il-5 , il-13 , il-9 , and il-4 cytokine responses [ 10 , 11 ] .
however , the levels of the th1 cytokine ifn- and regulator il-10 were not found to be significantly different between atopics and non - atopics . as the immune response involves complex direct / indirect interrelationships among cytokines
, these interactions may distort the true associations of cytokine responses with allergic phenotypes . the present study aimed to clarify the effects of the regulatory cytokine il-10 and th1 cytokine ifn- on allergic phenotypes after adjusting for covariance with th2 cytokines .
we expected that atopic phenotypes would be associated with a decreased level of a th1 cytokine such as ifn- , based on the established knowledge on th1/th2 balance with atopic phenotypes , however , this was not found in our previous analyses .
in addition , we recently found that the ratios of il-10 and il-5 capacities in cord blood were protective against acute respiratory infections during infancy and early childhood in a high - risk cohort .
as il-10 is a multifactorial cytokine regulating the th1/th2 balance , we also expected that il-10 would be significantly associated with atopic phenotypes , possibly with a contrasting effect on expression of atopic phenotypes with th2 cytokines . with a simple correlation for those cytokine data
correlations between il-10 and ifn- and th2 cytokines , ( tables 1 and 2 ) .
we assume that these correlations may confound finding any associations of il-10 and ifn- with atopic phenotypes .
therefore , our present study aimed to examine our hypotheses based on the previously ascertained cytokine data by accounting for the complex inter - relationships of il-10 and ifn- with several important th2 cytokines .
the perth infant asthma followup ( piaf ) longitudinal birth cohort has been previously described in several publications [ 1214 ] .
this study included 130 unselected probands for whom cytokine measurements were available from the 11-year review of the cohort .
phenotypes of atopy ( defined by skin prick test ( spt ) ) , total ige , asthma , wheezing , and bronchial hyperresponsiveness ( bhr ) in the population have been described in previous publications [ 1215 ] .
the present study was approved by the institutional ethics committee of princess margaret hospital , perth . as described previously
[ 11 , 16 ] , peripheral blood mononuclear cells ( pbmcs ) responses were determined on cryobanked samples .
the pbmc were cultured for 48  h in aim - v medium with 410  m 2-me alone or with 10  g / ml house - dust mite extract or purified der p1 dust mite allergen at 30  g / ml , or 1  g / ml phytohaemagglutinin a ( pha ) , or staphylococcal enterotoxin b ( seb ) .
cell pellets were resuspended in rnalater ( ambion , austin ) and stored at 20c .
levels of cytokines il-5 , il-13 , il-10 , and ifn- were determined by means of elisa or time - resolved fluorescence in cell culture supernatants . for hdm culture ,
levels of cytokine and total serum ige were natural log - transformed prior to further parametric analyses .
pearsons'correlations were employed to investigate the relationships between cytokines . as shown in ( tables 1 and 2 ) ,
there were significant correlations between il-10 and ifn- with th2 cytokines indicating that there was a direct / indirect correlation between them .
thus , a proportion of il-10 and ifn- levels may be explained by the direct / indirect relationship with th2 cytokines .
the positive co - relationships ( covariance ) of il-10 , and ifn- with th2 cytokines : il-5 , il-13 , il-9 , and il-4 may tend to result in atopic subjects with relatively higher levels of il-10 and ifn-. this may distort the true picture of association of the two cytokines with atopic phenotypes . to adjust for the covariance , we employed two statistical methods . in method one ,
adjusted levels of il-10 and ifn- were computed by adding residuals to the mean of raw log values of the cytokines for individual subjects .
the residual was estimated in a linear regression model with either log value of il-10 or log value of ifn- as dependent variable and one th2 cytokine as independent variable ( adjusted for ) .
the adjusted il-10 and ifn- values were further used for analyses to investigate the association with allergic phenotypes . in method two , we fitted either il-10 ( log value ) or ifn- ( log value ) and one th2 cytokine ( adjusted for ) in a logistic regression model to estimate the adjusted odds ratios ( or ) and their 95% confidence intervals ( 95% ci ) for binary atopic phenotypes .
independent sample t - test was utilized to explore the difference in the adjusted il-10 and ifn- levels for the binary phenotypic variables .
the mean age of the children was 11.2 yrs ( 95% ci : 11.011.3 yrs ) .
the correlation coefficients between natural log values of cytokines il-5 , il-13 , il-9 , il-4 , il-10 , and ifn- are shown in ( tables 1 and 2 ) .
there were significant positive correlations between cytokines ; unexpectedly , such correlations were also significant between th2 cytokines , il-10 , and ifn-. regarding the raw means of il-10 and ifn- stimulated with hdm , no significant difference was found between individuals with and without atopy .
however , after adjusting for individual th2 cytokines , the differences in levels of il-10 and ifn- between atopic and non - atopic children were significant for il-10 adjusting for il-5 ( p = 0.002 ) , il-13 ( p = 0.012 ) , il-9 ( p = 0.016 ) , and il-4 ( p = 0.043 ) , respectively , and for ifn- adjusting for il-5 ( p = 0.005 ) , il-13 ( p = 0.005 ) , and il-9 ( p = 0.037 ) , respectively ( table 3 ) .
atopic subjects appeared to have low levels of il-10 and ifn- compared with non - atopics .
consistent with the findings on il-10 and ifn- stimulated with hdm , il-10 and ifn- stimulated with pha and seb exhibited a similar pattern ( table 4 ) . except for ifn- ( seb ) ,
the adjusted il-10 and ifn- were significantly negatively correlated with total ige , but not significantly raw levels of the two cytokines .
the pattern was similar for allergen - specific cytokine response as well as polyclonal stimulator response ( table 5 ) .
the association of adjusted il-10 and ifn- with asthma , wheezing , and bronchial hyperresponsiveness was also investigated in this population . il-10 and
ifn- were not significantly associated with these phenotypes in the whole population either before or after the adjustment for th2 cytokines .  
as described in the statistical analysis section , we further investigated the association of il-10 and ifn- with atopy in the logistic regression analysis using the two adjusted methods . before the adjustment , il-10 and ifn-
were not significantly associated with atopy except for raw levels of pha il-10 and ifn-. however , after the adjustment , both il-10 and ifn- were clearly protective against atopy .
the present study is the first to investigate the relationships of th1 and regulatory cytokines with atopic phenotypes after adjusting for the correlative effects of th2 cytokines in a community population . in this study we found that il-10 , major findings shown in ( figure 1 ) , and ifn- were protective against atopy after accounting for their correlations with th2 cytokines . after taking out the shaded area of il-10 ( adjusting for th2 cytokines ) ,
these findings provide important insights into better understanding the function of the th1 cytokine ifn- and regulatory cytokine il-10 .  
th2/th1 balance was postulated after the discovery of the mutual inhibitory effects of th1 and th2 cells in mice , thus , a negative correlation would be expected between th1 and th2 cytokines [ 2 , 8 ] . however , as we have shown , there was a strong positive correlation between il-10 and ifn- and th2 cytokines .
consistent with our findings , a recent publication reported all cytokines including th1/th2 groups showed marked positive intraindividual correlations .
there are several possible explanations for these positive correlations : childhood immunity is primed by environmental factors in specific domiciliary environments ( nutrients , environmental allergen exposure , and infections ) .
furthermore , in the process of pathogenesis of atopic conditions , polarized th2 cytokine increases may result in increased th1 cytokines and regulatory cytokines through a self - balance / correction mechanism .
whatever the reasons for these correlations , they may distort the associations between th1 , regulatory cytokines , and allergic phenotypes . indeed , in the present study , after adjusting for the th2 cytokines il-5 , il-13 , or il-9 we found that both il-10 and ifn- levels were significantly and consistently lower in atopic subjects .
it is not unexpected that th1 cytokines and il-10 have a protective effect on allergy - related phenotypes .
however , if cytokine data in a population report that il-10 and ifn- are at risk for allergy and asthma , the confounding effects of th2 cytokines should be accounted and the corresponding findings should be interpreted with caution .   as we found that adjusting for th2 cytokines clarified the effects of il-10 and ifn- on atopy , we further examined the consequence of mutually adjusting th2 cytokines . for example
, we calculated the mean of il-5 adjusting for il-13 and found that the adjusted il-5 ( by il-13 ) was not significantly associated with atopy .
similarly , when we fitted il-5 and il-13 together in a logistic regression model using atopy as the dependent variable , we did not find a significant association of these cytokines with atopy , possibly due to collinearity .
these results ( data not shown ) were expected considering the pathogenic relationship of th2 cytokines and further indicated that the adjustment of il-10 and ifn- for th2 cytokines is reliable and meaningful .  
our community population was recruited in perth , australia , a developed country with a high prevalence of allergic diseases . in our population
, we found that th2 cytokines distorted the association of il-10 and ifn-with allergic phenotypes .
however , in a th1-dominant milieu , th1 cytokines may also distort the association of th2 cytokines with complex diseases of interest .
il-10 is a pleiotropic cytokine with regulatory effects on th1/th2 balance [ 4 , 5 , 19 ] .
the precise functional role of il-10 , however , has not been clarified . whether il-10 expression is altered in asthmatics
is uncertain , as it has been reported to be reduced in some studies but increased in others [ 11 , 20 , 21 ] . in a high - risk cohort
, we have previously reported that the ratio of il-10 and il-5 was protective against the susceptibility to respiratory infections during infancy and early childhood . in the current study
, we found that adjusted il-10 expression was clearly decreased in children with atopy or high total serum ige levels . however , in individuals with asthma and wheezing , the effects of il-10 still remain unclear , indicating the complex effects of regulatory cytokines in relation to the pathogenesis of asthma .   in conclusion , in a western - community - based cohort ,
levels of th2 cytokines distorted the association of il-10 and ifn- with allergic phenotypes . after adjustment for th2 cytokines ,
 the expression of allergic phenotypes involves complex inter - relationships among several th2 and th1 cytokines as well as the regulator cytokine interleukin ( il)-10 . 
 these direct or indirect interrelationships may distort the true associations of cytokine responses with these phenotypes . in this study 
 , we aimed to clarify the effects of the regulatory cytokine il-10 and th1 cytokine interferon - gamma ( ifn- ) on allergic phenotypes after adjusting for the correlations with th2 cytokines . after adjusting for th2 cytokines , il-10 and ifn- were protective against atopy . 
 adjusted levels of il-10 and ifn- stimulated with house - dust mite ( hdm ) were significantly lower in atopics than non - atopics , for il-10 adjusting for il-5 ( p = 0.002 ) , il-13 ( p = 0.012 ) , il-9 ( p = 0.016 ) , and il-4 ( p = 0.043 ) , and for ifn- adjusting for il-5 ( p = 0.005 ) , il-13 ( p = 0.005 ) , and il-9 ( p = 0.037 ) . il-10 and ifn- levels stimulated with phytohaemagglutinin ( pha ) and staphylococcal enterotoxin b ( seb ) exhibited a similar pattern . 
 the adjusted levels of il-10 and ifn- stimulated with hdm , pha or seb were all significantly negatively correlated with total serum ige , except for ifn- stimulated with seb . 
 levels of th2 cytokines distort the associations of il-10 and ifn- with allergic phenotypes . removing the covariance with th2 cytokines , 
 both il-10 and ifn- were protective against atopy . 
1. Introduction 
2. Method
3. Results
4. Discussion