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Thymus and activation regulated chemokines in children with atopic dermatitis: Kyushu University Ishigaki Atopic Dermatitis Study (KIDS)

European Journal of Dermatology. Volume 17, Number 5, 397-404, September-October 2007, Investigative report

DOI : 10.1684/ejd.2007.0237

Summary

Author(s) : Norihiro Furusyo, Hiroaki Takeoka, Kazuhiro Toyoda, Masayuki Murata, Shinji Maeda, Hachiro Ohnishi, Noriko Fukiwake, Hiroshi Uchi, Masutaka Furue, Jun Hayashi , Department of General Medicine, Kyushu University Hospital, Higashi-Ku, Fukuoka, 812-8582 Japan, Department of Environmental Medicine and Infectious Diseases, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan, Department of Dermatology, Kyushu University Hospital, Fukuoka, Japan.

Summary : The purpose of this population-based study was to investigate the clinical significance of serum thymus and activation-regulated chemokine (TARC) in children with atopic dermatitis (AD). Between 2003 and 2004, 1359 Japanese children aged 5 years and under were prospectively followed. Serum levels of TARC by using an ELISA in each child were monitored throughout the study period. The first tested year, the mean serum level of TARC in children with sustained AD (mean\; 691.7 pg/mL) was significantly higher than that of regressed AD children (569.9 pg/mL), newly developed AD children (380.1 pg/ mL), and healthy children (506.3 pg/mL). The changes of TARC levels in sustained AD children found no significance between 2003 (691.7 pg/mL) and 2004 (682.0 pg/mL). The mean levels of TARC of both regressed AD and healthy children significantly decreased from 2003 to 2004 (644.2 pg/mL to 448.7 pg/mL and 506.3 pg/mL to 442.1 pg/mL, respectively). The mean TARC level of newly developed AD children significantly increased from 2003 to 2004 (380.1 pg/mL to 491.8 pg/mL). We demonstrated strong associations between TARC levels and the natural course of childhood AD. Monitoring serum TARC levels of AD children may be useful for the biological evaluation of AD.

Keywords : atopic dermatitis, thymus and activation regulated chemokine, epidemiology

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ARTICLE

Auteur(s) : Norihiro Furusyo^1,2, Hiroaki Takeoka², Kazuhiro Toyoda², Masayuki Murata¹, Shinji Maeda², Hachiro Ohnishi², Noriko Fukiwake³, Hiroshi Uchi³, Masutaka Furue³, Jun Hayashi^1,2

¹Department of General Medicine, Kyushu University Hospital, Higashi-Ku, Fukuoka, 812-8582 Japan
²Department of Environmental Medicine and Infectious Diseases, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan
³Department of Dermatology, Kyushu University Hospital, Fukuoka, Japan

accepté le 3 Mai 2007

Atopic disorders affect up to 20% of the general populations worldwide and the incidence of allergic diseases and atopic dermatitis (AD) appears to be increasing [1]. AD is a chronic inflammatory and recurrent pruritic eczematous skin disease that involves peripheral blood eosinophilia and high level of serum immunoglobulin E (IgE) [2, 3]. Acute lesional skin of patients with AD is characterized by eosinophil-derived granule deposits and T cell infiltration, which are predominantly T-helper (Th) 2-type cells expressing such cytokines as interleukin (IL)-4, IL-5 and IL-13 [2, 4, 5].Chemokines have been identified as attractants of different types of leukocytes to the sites of infection and inflammation. They are divided into four subfamilies, CXC, CC, CX3C and C, depending on the position of the first two N-terminal cysteine residues [6]. They are produced locally in the tissues and act on leukocytes through specific receptors. They also function as regulatory molecules in leukocyte maturation, trafficking and homing in the development of lymphoid tissues [7]. Thymus and activation-regulated chemokine (TARC/CCL17) is a member of CC chemokines and is produced by monocyte-derived dendritic cells, endothelial cells, bronchial epithelial cells, and epidermal keratinocytes [8, 9]. In humans with AD, TARC, designated as a Th2 type chemokine, is produced mainly by keratinocytes distributed in lesional skin [10]. The serum TARC levels are significantly elevated and reflect the disease activity of patients with AD [10, 11].Numerous studies have been done on the prevalence and incidence of AD [12-16]. To investigate the natural history of AD in children of this age group, a population-based cohort study, the Kyushu University Ishigaki Atopic Dermatitis Study (KIDS), was launched in 2001 [17, 18]. Prior research indicated that more than 70% of children with AD experienced spontaneous regression within 3-year follow-up period, and that de novo occurrence of AD in these children was estimated as 3.67%/person-year [17, 18]. In the present study, we investigated the association of the serum TARC levels with the natural course of children with AD by analysis of the stored serum samples of children enrolled in the KIDS trial.

Materials and methods

Study populations

The population-based KIDS trial began in 2001 and has been previously described [17, 18]. The candidates of the present study were 1359 children who had received our free physical examinations in 2003 and 2004, when they were aged 0 to 5 years. In the present study, a blood sample was obtained from each child for analysis of the serum TARC level and other values.

Free physical examinations were given to children of 16 nursery schools in Ishigaki City, which has a population of 45,000, in Okinawa Prefecture, Japan. The yearly average temperature and humidity are 25.4 °C and 76%, respectively. The physical and laboratory examinations were done annually in July and August. For the present study, 749 children in 2003 and 610 children in 2004, a total of 1359 children, which represented 33.0% of nursery school children in Ishigaki City, were analyzed. One thousand and nine children were examined only once and 350 were followed for one year.

The study was done in accordance with the principles of the Declaration of Helsinki and its appendices and was approved by the ethics committee of Kyushu University Hospital as well as by the directors and class teachers of the schools. Written informed consent to allow participation of the children was obtained from the parents or guardians.

Physical and laboratory examination

The medical examinations for all children were done by two experienced dermatologists from the Department of Dermatology at Kyushu University Hospital. AD was diagnosed according to the Japanese Dermatological Association criteria (table 1) [16, 19]. These criteria are very similar to those of Hanifin and Rajka [20]. The difference is that most of the minor features in the Hanifin and Rajka criteria are referred to as diagnostic aids, clinical types or significant complications in Japanese criteria.

The severity of AD was graded as mild, moderate, severe or very severe according to the following criteria [16]: (1) mild, skin involvement of mild eruption only; (2) moderate, under 10% surface area involvement of eruption with severe inflammation (severe eruption); (3) severe, over 10% but under 30% skin involvement of severe eruption; and (4) very severe, over 30% of body involvement of severe eruption.

Serum TARC and total IgE levels were measured and monitored in sera of all the studied children.
Table 1 Definition and diagnostic criteria for atopic dermatitis (AD) by the Japanese Dermatological Association

Definition

AD is a pruritic, eczematous dermatitis, the symptoms of which fluctuate chronically with remissions and relapses. Most individuals with AD have atopic diathesis

Atopic diathesis: 1) personal or family history (asthma, allergic rhinitis, and/or conjunctivitis and AD), and/or 2) predisposition to overproduction of immunoglobulin E (IgE) antibodies

Diagnostic criteria for atopic dermatitis

1. Pruritus

2. Typical morphology and distribution

(1) Eczematous dermatitis

(a) acute lesions: erythema, exudation, papules, vesiculopapules, scales, crusts

(b) chronic lesions: infiltrated erythema, lichenification, prurigo, scales, crusts

(2) Distribution

(a) symmetrical: predilection sites: forehead, periorbital area, perioral area, lips, periauricular area, neck, joint areas of limbs, trunk

(b) age-related characteristics

Infantile phase: starts on the scalp and face, often spreads to the trunk and extremities

Childhood phase: neck, the flexural surfaces of the arms and legs

Adolescent and adult phase: tendency to be severe on the upper half of body (face, neck, anterior, chest and back)

3. Chronic or chronically relapsing course (usually coexistence of old and new lesions)

(1) More than 2 months in infancy

(2) More than 6 months in childhood, adolescence and adulthood

Define diagnosis of AD requires the presence of all three features

Measurement of serum TARC level

Serum TARC levels were measured by ELISA using a flexible, 96-well polyvinyl chloride plate coated with a murine monoclonal antibody against human TARC (SD-8864, Shionogi & Co., Ltd, Osaka, Japan). The ELISA was performed according to the manufacturers’ directions. After addition of 100 μL of assay buffer (50 mmol/L phosphate buffer containing 0.3 mol/L NaCl, 0.5 mL/l Tween 20, 1 g/L BSA, and 0.2 g/L NaN₃) to each well of the immunoplates, aliquots of human TARC standards (0-8000 pg/mL in assay buffer) or samples (25 μL each) were added to the wells and incubated for 2 hours at 25 °C. Each well was washed three times with PBST, 100 μL of Fab’-HRP (75 ng) in PBS containing 4 g/L BlockAce (Snow Brand, Tokyo, Japan) and 1 g/L Kathon CG (Rohm and Haas, Philadelphia, PA) was added to each well and incubated for 1 hour at 25 °C. Following five-time washing of the assay plates with PBST, the immunoreactivity was visualized by addition of 100 μL/well of substrate solution (ColorBurstBlue; AlerCHEK, Portland, ME) for 15 minutes at 25 °C. The reaction was stopped by the addition of 50 μL of 0.18 mol/L sulfuric acid to each well, and absorbance was measured at 405 nm using an immunoreader MTP-32 (Corona Electric, Ibaragi, Japan). The TARC level was calculated based on the standard curve generated by a curve-fitting program on each assay plate. The minimum detectable dose of TARC was 20 pg/mL. The above method was similar to that previously reported [21].

Measurement of serum IgE level

Total IgE levels were determined by a radioimmunoassay with a detection limit of 20 IU/mL (Shionoria IgE, Shionogi & Co., Ltd. Japan).

Statistical analysis

Continuous data were expressed as mean values, mean ± standard deviation (SD), or values ± standard error (SE) of the mean. Statistical differences in the continuous data were determined by paired t-test, unpaired t-test, Mann-Whitney U test, Kruskal-Wallis test, or Wilcoxon test. The differences in the categorical data were compared by chi-square test and Fisher’s exact test. A P value less than 0.05 was regarded as statistically significant.

Results

Correlation between serum levels of TARC and total IgE in children with or without AD

In 2003 and 2004, 883 healthy children and 115 AD children were identified. Because all the children diagnosed as AD had mild activity of the disease, they were not treated with topical or systematic drugs. The mean ± SD serum TARC level of the healthy children was 485.3 ± 216.3 pg/ mL, with a minimum of 169 pg/mL and a maximum of 2101 pg/mL. No significant difference in the mean ± SE serum TARC levels were found between the healthy and AD children (485.3 ± 7.3 pg/mL vs. 535.9 ± 24.9 pg/mL, P = 0.0614). The mean ± SE serum total IgE level of the AD children (565.4 ± 131.4 IU/mL) was significantly higher than that of the healthy children (130.9 ± 12.5 IU/ mL) (P < 0.0001). The serum TARC levels of both children with and without AD showed significant correlations with the total IgE levels (r = 0.430, P < 0.0001 in children with AD, and r = 0.147, P < 0.0001 in the healthy children, data not shown).

Figure 1 shows age-related differences in serum TARC levels. The mean ± SD TARC (pg/ mL) levels of the healthy children significantly decreased with age: 630.8 ± 329.6 in 24 children aged under one year, 524.9 ± 250.7 in 129 children one year of age, 494.3 ± 190.9 in 229 children two years of age, 479.0 ± 224.6 in 204 children three years of age, 465.3 ± 203.7 in 209 children four years of age, and 426.0 ± 165.7 in 88 children five years of age (P < 0.0001 by Kruskal-Wallis test) (figure 1A). However, the mean ± SD TARC levels (pg/mL) of the children with AD did not significantly change in accordance with age (P = 0.2465 by Kruskal-Wallis test): 515.5 ± 57.3 in 2 children under one year, 604.6 ± 412.9 in 8 children one year of age, 539.2 ± 270.3 in 33 children two years of age, 604.3 ± 307.3 in 35 children three years of age, 467.8 ± 169.1 in 28 children four years of age, and 412.8 ± 122.3 in 9 children five years of age (figure 1B). Except for in the three year old age group (P = 0.0085), no significant age related differences in the serum TARC level were found between healthy and AD children (P = 0.9616 in children under one year, P = 0.8149 in children one year of age, P = 0.7015 in children two years of age, P = 0.6736 in children four years of age, and P = 0.9306 in children five years of age) by using a Mann-Whitney U test. Based on the definition of a level over a mean value + 2 SD being an elevated TARC abnormality form data of healthy children, the normal and abnormal cut off values were 1290, 1027, 877, 929, 873, and 758 pg/mL in 0, 1, 2, 3, 4, and 5 year-aged children, respectively.

Figure 2 shows age-related differences in serum total IgE levels. The mean ± SD levels of the total IgE (IU/mL) of the healthy children significantly increased with age (P = 0.0109 by Kruskal-Wallis test) (figure 2A). However, the mean ± SD levels of total IgE (IU/mL) of the AD children did not significantly change in accordance with age (P = 0.6641 by Kruskal-Wallis test) (figure 2B).

Follow-up study of the natural course of AD

Of the 1359 children examined, 350 were followed in both 2003 and 2004, 44 of them were diagnosed as having AD at the initial physical examination in 2003. Of these 44, 30 were confirmed to have regressed AD in 2004. Of the 306 children without AD, 17 newly developed AD. In 2004, 14 children had sustained AD (Group A), 30 had regressed AD (Group B), 17 had newly developed AD children (Group C), and 289 children were healthy without AD (Group D).

Figure 3 shows the mean serum TARC levels of the sustained, regressed, newly-developed AD and non-AD children in 2003. The mean ± SE serum TARC level in Group A (691.7 ± 84.4 pg/mL) was significantly higher than that of the other groups (569.9 ± 57.6 pg/mL in Group B, 380.1 ± 27.7 pg/mL in Group C, and 506.3 ± 18.1 pg/mL in Group D) (all P < 0.05, Group A vs. other groups). These findings suggest that a higher TARC level correlates with the sustained AD.

Figure 4 shows the mean serum IgE levels of the sustained, regressed, newly-developed AD and non-AD children in 2003. The mean ± SE serum IgE levels in Groups A, B, and C (800.8 ± 494.1 IU/mL, 461.9 ± 260.1 IU/mL, 369.4 ± 217.2 IU/mL, respectively) were significantly higher than that of Group D (108.3 ± 14.4 IU/mL) (all P < 0.05, Group D vs. other groups).

Figure 5 shows changes in the mean serum TARC levels of the sustained, regressed, newly-developed AD and non-AD children. In Group A children, the mean ± SE levels of serum TARC in 2003 and 2004 were stably high (691.7 ± 84.4 pg/mL in 2003, 682.0 ± 100.9 pg/mL in 2004) without any significant difference (P = 0.0747). In Group B and D children, the mean ± SE serum TARC levels significantly decreased from 2003 to 2004 (644.2 ± 57.6 pg/mL to 448.7 ± 65.7 pg/mL in Group B, P = 0.0145 and 506.3 ± 18.1 pg/mL to 442.1 ± 10.9 pg/mL in Group D, P < 0.0001). In Group C children, the mean ± SE serum TARC levels significantly increased from 2003 to 2004 (380.1 ± 27.7 pg/mL to 491.8 ± 30.4 pg/mL) (P = 0.0203). These findings suggest that an increase in the TARC level correlates with the sustained and developed AD.

Figure 6 shows changes of mean serum IgE levels in the sustained, regressed, newly-developed AD and non-AD children. In Groups A, B, and D children, the mean ± SE serum IgE levels significantly increased from 2003 to 2004 (800.8 ± 494.1 IU/mL to 2375.3 ± 1613.6 IU/mL in Group A, P = 0.0277, 461.9 ± 260.1 IU/mL to 541.4 ± 212.5 IU/mL in Group B, P = 0.0068, and 108.3 ± 14.4 IU/mL to 196.6 ± 27.1 IU/mL in Group D, P < 0.0001). In Group C children, even though their AD lesions newly developed, the change of mean ± SE serum IgE levels did not significantly increase from 2003 (369.4 ± 217.2 IU/mL) to 2004 (356.4 ± 90.5 IU/mL) (P = 0.9434). These findings suggest that a change in the IgE level dose not apparently correlate with the regressed AD.

Discussion

AD represents a major public health problem worldwide and usually develops in early childhood [22]. To date, there has been no gold standard for assessing the clinical course of AD. The previous studies on adult AD patients showed that an evaluation has been carried out of the association between the disease activity of AD and serum TARC levels as an objective chemokine marker [10, 11]. In the present study, we demonstrated strong associations between the serum TARC levels and the natural course (newly developed, regressed, and maintained AD) of childhood AD through an analysis of data from our KIDS trial, a population-based cohort study with a large number of children. These findings indicated that serum TARC played an important role in the natural course of AD in children.

Th2 cells and eosinophils are the most prominent cells of allergic inflammation that selectively express CC chemokine receptor (CCR) 4 and CCR3, respectively. TARC, the chemokine ligand of CCR4, implicates the mechanism of inflammation in allergic diseases [23, 24]. Keratinocytes, T cells and dendritic cells are major sources of TARC in AD patients [10, 22]. The serum TARC levels of AD children were significantly higher than those of healthy children in the present study. In a study of adults [10, 25], the increases in serum TARC levels of AD patients were greater in a severely affected patient group than in moderate or mild groups. The TARC levels of adult AD patients decreased after the treatment in accordance with an improvement in clinical symptoms. In addition, the serum TARC levels were significantly correlated with number of eosinophils and other inflammation markers. These results strongly suggest that serum TARC levels are closely related to the disease activity of AD.

In the present study, serum TARC levels of both children with and without AD showed significant correlations with total IgE levels. AD is characterized by the predominant inflammation of mononuclear cells, especially T cells, eosinophils and macrophages in lesional skin and is associated with a high serum level of IgE [11]. Our study showed that serum total IgE level of AD children was significantly higher than that of healthy children. However, we also demonstrated that the changes in the IgE level did not apparently correlate with the regressed AD and healthy children. Moreover, total IgE level of healthy children was confirmed to significantly increase with age, as was found in previous studies [17, 18]. Therefore, serum total IgE would not be an appropriate marker for assessing the natural course of AD in children, although a high IgE level may indicate the possible persistence of AD.

The TARC gene is located at chromosome 16q13 [26], where total serum IgE level has been reportedly linked [27]. Although a single nucleotide polymorphism (SNP) of TARC gene is a candidate of the genetic factors in allergic diseases, no significant association of the SNP with susceptibility to AD and bronchial asthma was reported [23, 28]. The elevation of serum TARC levels could be induced by other cytokines that enhance the TARC production, but not by this TARC SNP as the promoter.

In the present study, we found a significant positive correlation between TARC, total IgE and the natural course of AD. In a study of a murine model of bacteria-induced hepatic failure, TARC mediated infiltration of CCR4 cells in hepatic lesions was inhibited by administration of a monoclonal antibody to TARC [29]. The use of a neutralizing monoclonal antibody to TARC suppresses allergic inflammation and attenuates the accumulation of eosinophils in a murine model of allergen-induced asthma [30]. In addition, basic studies demonstrated the intracellular signaling pathways linked to TARC and CCR4-mediated chemotaxis [31, 32]. In a clinical study, the production of TARC by peripheral blood mononuclear cells in adult AD patients was dramatically inhibited by the administration of an antagonistic drug against the histamine H₁ receptor, Olopatadine [33]. Taken together, chemokine-target treatment may be an effective strategy for the treatment of allergic diseases including AD.

Serum chemokine markers for AD have been extensively studied in both adults and children. Specially, the levels of AD-associated chemokines such as cutaneous T-cell attracting cytokine (CTACK), macrophage-derived chemokine (MDC), and interleukin (IL)-18 were correlated with various clinical signs and severity of eczema in children [5, 34-36]. However, it was not reported which was the most useful for evaluating the disease state. The solution awaits further study.

Our patients diagnosed as AD had not been treated with topical or systematic drugs due to their mild AD lesions. During the present study, we have held the annual educational meetings for their parents, guardians, and school teachers only about the basic management for AD, addressing the skin barrier defect with skin hydration along with identification and avoidance of specific and nonspecific irritating trigger factors [37, 38]. Therefore, this study did not include the children with AD who have received medical interventions.

Japanese investigators reported that the prevalence (17.3%) of AD was significantly higher in the cooler climate of Gifu Prefecture, the middle area of Honshu (main island of Japan), than in the warmer climate of Itoman, Okinawa (3.4%), even after controlling for genetic and environmental factors [39, 40]. In our previous studies [17, 18], the prevalence of AD (6.9%) in children aged 5 years and younger in Ishigaki Island, which is located in the subtropical zone of Japan, was lower than the average rate on the mainland of Japan, possibly suggesting that the AD prevalence can depend on climate state. A worldwide survey has reported that AD is increasing in the developed countries in cooler climates [41], although the reason for the lower prevalence in the warmer climate areas remains to be elucidated.

The limitation of our study is that we did not perform any scoring of AD so that a correlation with the activity or severity has not been carried out, which would increase and strengthen the relevance of the data. In addition, concomitant atopic diseases such as allergic rhinitis or asthma have not been considered in the present analysis. However, we believe that the topic of our study, the relevance of chemotactic signals in the natural course of AD, will be interesting and important for physicians and researchers, because the study power of this longitudinal and population-based approach is very high.

In conclusion, we demonstrated strong associations between serum TARC levels and the natural course of childhood AD in this population-based cohort study with a large number of children. Monitoring serum TARC levels of AD children may be useful for the biological evaluation of AD.

Acknowledgements

We greatly thank Maki Hamada, Makiko Shimizu, Masataka Etoh, Mai Yanagi, and the members of our laboratory and our dermatology colleagues for their assistance with this study. This study was supported by a grant from the 21st century COE program of the Japanese Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.

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