IFN-
-inducible expression of thymus and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in epidermal keratinocytes and their roles in atopic dermatitis
Tatsuya Horikawa1,
Takashi Nakayama2,
Ichiro Hikita3,
Hidekazu Yamada4,
Ryuichi Fujisawa2,
Toshinori Bito1,
Susumu Harada1,
Atsushi Fukunaga1,
David Chantry5,
Patrick W. Gray5,
Atsushi Morita3,
Ryuji Suzuki3,
Tadashi Tezuka4,
Masamitsu Ichihashi1 and
Osamu Yoshie2
1 Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan 2 Departments of Microbiology and 4 Dermatology, Kinki University School of Medicine, Osaka 589-8511, Japan 3 Shionogi Institute for Medical Science, Osaka 566-0022, Japan 5 ICOS Corp., Bothell, WA 98021, USA
Correspondence to: O. Yoshie; E-mail:o.yoshie{at}med.kindai.ac.jp.
Transmitting editor: M. Miyasaka
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Abstract
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Thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 are a pair of CC chemokines known to selectively attract Th2 type memory T cells via CCR4. Here we examined circulating levels of TARC and MDC in patients with atopic dermatitis (AD) and control subjects by using plasma samples, which reflect blood contents of chemokines more accurately than serum samples. The plasma levels of TARC and MDC were significantly elevated in AD patients. These values also strongly correlated with disease severity and serum lactate dehydrogenase levels, and weakly correlated with serum total IgE levels and blood eosinophilia. Previous studies demonstrated TARC immunoreactivity in the epidermal layer of AD lesional skin and production of TARC by a human keratinocytic cell line HaCaT upon stimulation with IFN-
. Here we demonstrated MDC immunoreactivity in the epidermal layer of AD skin at levels stronger than that of TARC. Furthermore, primary epidermal keratinocytes expressed both TARC and MDC mRNA upon stimulation with IFN-
, but efficiently secreted only MDC. These results suggest a post-transcriptional regulation in TARC production. IFN-
also induced TARC and MDC mRNA in mouse skin. Collectively, both TARC and MDC play important roles in the local accumulation of Th2 cells in AD lesional skin. Production of Th2-attracting chemokines by epidermal keratinocytes upon treatment with IFN-
, which is also the potent inducer of Th1-attracting chemokines, may underline the pivotal role of IFN-
in the chronic phase of AD where both Th1 and Th2 responses are mixed.
Keywords: atopy, chemokine, chemokine receptor, IFN-
, keratinocyte
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Introduction
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Atopic dermatitis (AD) is a chronic inflammatory disease of the skin based on still unknown genetic predispositions, and is commonly characterized with dry skin, severe pruritis, high serum IgE levels and eosinophilia (1). The histological features of AD include epidermal hyperplasia, thickening of the papillary dermis and prominent perivascular infiltrates consisting predominantly of T cells (2). It is now considered that disregulated Th2-dominant immune responses to environmental allergens in the skin are the central features of AD (1,2). In this context, it is likely that a group of chemotactic cytokines collectively called chemokines play important roles in AD pathogenesis by attracting various types of leukocytes into the lesional skin (3).
Previously, we have shown that LARC/CCL20, which is known to attract immature dendritic cells and effector/memory T cells via CCR6, is immunologically stained in epidermal layers of AD skin lesion, and can be induced in primary human epidermal keratinocytes upon stimulation with proinflammatory cytokines such as IL-1
and tumor necrosis factor (TNF)-
(4). Thus, LARC is likely to play an important role in AD pathogenesis through coupling innate and acquired immune responses in the skin (3). We have also shown that thymus and activation-regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22, a pair of chemokines commonly acting on CCR4, selectively attract a subset of CD4+ memory T cells, which are mostly polarized to Th2 (5). In vitro polarization of naive CD4+ T cells into Th2 cells also selectively induced CCR4 expression (5). Thus, TARC and MDC are likely to play important roles in the Th2-type immune responses by selectively recruiting Th2-polarized memory/effector T cells into inflamed tissues (3). This notion has been amply supported by recent studies on murine models of AD and asthma (68). Furthermore, elevated serum levels of MDC and TARC in AD patients as well as selective infiltration of CCR4-expressing T cells in AD skin lesions have been reported (912). TARC was also shown to be induced in a human keratinocytic cell line HaCaT upon stimulation with IFN-
and TNF-
, and was immunologically stained in epidermal keratinocytes of AD lesional skin (13). Collectively, it is likely that TARC and MDC are produced in large quantities in lesional skin of AD patients.
The use of serum samples in previous studies for evaluation of blood levels of TARC and MDC (9,10), however, had potential problems due to possibilities such as release of stored chemokines from platelets (14,15), release of chemokines from DARC, the chemokine scavenger receptor on erythrocytes (16,17) and/or adsorption of chemokines to newly formed blood clots (18). In fact, we have recently observed a substantial release of TARC from platelets during clotting (Fujisawa et al., submitted). In the present study, therefore, we re-evaluated circulating levels of TARC and MDC in AD patients and control subjects by using plasma samples. We have found that plasma levels of TARC and MDC are significantly elevated in AD patients, and correlate well with disease severity and serum LDH levels. We have also shown that both TARC and MDC are immunologically stained in the epidermal keratinocytes of AD skin lesions. Furthermore, we have demonstrated for the first time that primary human epidermal keratinocytes are induced to express not only TARC, but also MDC upon stimulation with IFN-
. Unexpectedly, MDC, but not TARC, was efficiently secreted by IFN-
-stimulated epidermal keratinocytes in vitro, suggesting a post-transcriptional regulation in the production/secretion of the latter.
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Methods
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Subjects
AD was diagnosed according to the criteria of Haniffin and Rajka (19). Fifty-two patients with AD aged from 14 to 56 years old (25 males and 27 females), all seen in Kobe University Hospital, were enrolled in this study. Levels of total IgE and lactate dehydrogenase (LDH) in patients sera, and the numbers of eosinophils in peripheral blood were determined by routine laboratory tests. Skin symptoms of the patients were recorded and assessed using the eczema area and severity index (EASI) score (20). This scoring system evaluates area of involvement and erythema, infiltration/papulation, excoriation and lichenification in each area of head/neck, trunk, upper limbs and lower limbs. Based on the EASI score, AD patients were divided into three groups: mild (score <10), moderate (score 1020) and severe (score >20). Healthy control subjects (N = 8) had no history of allergic diseases. Serum levels of IgE in control subjects were <160 IU/ml and no specific IgE antibodies to common inhaled allergens were detected using Phadiatope (Pharmacia Upjohn, Uppsala, Sweden). Peripheral blood samples were collected from AD patients and control subjects using sodium EDTA-containing tubes. After centrifugation, the plasma samples were store at 80°C until assay. Skin biopsies were taken from several donors as described previously (4). Informed consents were obtained from all subjects. This study was approved by the Ethical Committee of Kobe University Graduate School of Medicine.
Cells and reagents
Neonatal human epidermal keratinocytes were purchased from Clonetics (Walkersville, MD). The immortalized human keratinocytic cell line HaCaT was generously provided by Professor N. Fusenig (German Cancer Research Center, Heidelberg, Germany). Recombinant human and murine IL-1
, TNF-
, IFN-
and IL-4 were all purchased from PeproTech (Rocky Hill, NJ). Lipopolysaccharide from Escherichia coli was purchased from Sigma (St Louis, MO). Recombinant human TARC/CCL17 and MDC/CCL22 were purchased from R & D Systems (Minneapolis, MN).
Mice
Female BALB/c mice, 6 weeks old, were purchased from Nippon Kurea (Osaka, Japan) and kept in specific pathogen-free conditions for 1 week before experiments. Mice were treated with depilatory cream (Kanebo, Tokyo, Japan) on the belly to remove hair. After 24 h, mice were anaesthetized by diethyl ether and injected intradermally with 100 µl of PBS alone or containing 10 ng of murine IFN-
. After various time points, mice were sacrificed by cervical dislocation and skin tissues of 6-mm diameter were punched out at injected sites.
RT-PCR
Total RNA was prepared from skin biopsies and cultured keratinocytes using Trizol reagent (Invitrogen Corp., Carlsbad, CA). RNA samples were further purified using RNeasy (Qiagen, Hilden, Germany). Reverse transcription of total RNA (1 µg) was carried out using oligo(dT)18 primer and SuperScript II reverse transcriptase (Invitrogen Corp.). First-strand DNA (20 ng total RNA equivalent) and original total RNA (20 ng) were amplified in a final volume of 20 µl containing 10 pmol of each primer and 1 U of Ex-Taq polymerase (Takara, Kyoto, Japan). The primers used were: +5'-ACTGCTCCAGGGATGCCATCGTTTTT-3' and 5'-ACAAGGGGATGGGATCTCCCTCACTG-3' for TARC; +5'-AGGACAGAG CATGGCTCGCCTACAGA-3' and 5'-TAATGGCAGGGAGGTAGGGCTCCTGA-3' for MDC; +5'-AAGAAGAACAAGGCGGTGAAGATG-3' and 5'-AGGCCCCTGCAGGTTTTGAAG-3 for CCR4; +5'-TGAGGTCACTTCAGATGCTGC-3' and 5'-ACCAATCTGATGGCCTTCTTC-3' for mouse TARC; +5'-TCTGATGCAGGTCCCTATGGT-3' and 5'-TTATGGAGTAGCTTCTTCAC-3' for mouse MDC; +5'-GCCAAGGTCATCCATGACAACTTTGG-3' and 5'-GCCTGCTTCACCACCTTCTT GATGTC-3' for G3PDH. Amplification conditions were denaturation at 94°C for 30 s (5 min for the first cycle), annealing at 60°C for 30 s and extension at 72°C for 30 s (5 min for the last cycle) for 32 cycles for TARC and MDC, 37 cycles for CCR4, 35 cycles for mouse TARC and mouse MDC, and 27 cycles for human and mouse G3PDH. Amplification products (10 µl each) were subjected to electrophoresis on 2% agarose and stained with ethidium bromide.
Immunohistochemistry
Skin biopsy specimens were snap frozen using liquid nitrogen. Cryostat sections were reacted with a mouse anti-TARC mAb 5F12 (Morita et al., in preparation) or a mouse anti-MDC mAb 252Y (9). After washing, tissue sections were incubated with biotin-conjugated anti-mouse IgG (Dako, Kyoto, Japan). After washing, tissue sections were incubated with ultra-avidinhorseradish peroxidase (Sigma). After washing, sections were treated with diaminobenzidine and counter-stained with methylgreen or hematoxylin.
ELISA
A sandwich-type ELISA specific for MDC with a detection limit of 15 pg/ml was described previously (9). A sandwich-type ELISA specific for TARC with a detection limit of 0.6 pg/ml was developed by using two newly generated mouse anti-TARC mAb, 4A3 and 5F12, and will be described elsewhere (Morita et al., in preparation). A sandwich-type ELISA for human IFN-
with a detection limit of 8 pg/ml was purchased from American Research Products (Belmont, MA).
Statistical analysis
Data on plasma contents of TARC and MDC were expressed as geometric means since logarithmically transformed values of the data followed normal distribution. Differences were analyzed with unpaired t-test. Pearsons correlation coefficient was calculated between two parameters.
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Results
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Elevated plasma TARC and MDC levels in patients with AD
Using plasma samples obtained from eight normal subjects and 52 AD patients, we examined circulating contents of TARC and MDC in the blood. The patients were divided into three groups according to their EASI scores (20): mild <10, moderate 1020 and severe >20. As shown in Fig. 1, plasma TARC and MDC levels were elevated in most AD patients and significantly correlated with the disease severity. The average values of TARC in normal subjects and AD patients with mild, moderate and severe disease groups were 37 ± 12, 101 ± 67, 369 ± 230 and 3769 ± 4712 pg/ml respectively. Similarly, those of MDC in normal subjects and AD patients with mild, moderate and severe disease groups were 671 ± 159, 692 ± 331, 1531 ± 756 and 1969 ± 1316 pg/ml respectively.

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Fig. 1. Elevated plasma levels of TARC and MDC in patients with AD. Plasma samples were obtained from eight control and 52 AD subjects. Plasma contents of TARC and MDC were measured by ELISA. All measurements were done in duplicate and mean values were obtained. The AD subjects were grouped into three groups according to their EASI scores: mild (<10), moderate (1020) and severe (>20). (A) TARC; (B) MDC.
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We next analyzed correlation of plasma TARC and MDC levels with other clinical parameters known to be elevated in AD patients. As shown in Fig. 2, the logarithmic values of plasma TARC levels strongly correlated with those of MDC levels (r = 0.775, P < 0.001) and the clinical scores (r = 0.791, P < 0.001). Plasma TARC levels also correlated strongly with serum LDH levels (r = 0.717, P < 0.001), and weakly with serum IgE levels (r = 0.393, P < 0.01) and blood eosinophilia (r = 0.398, P < 0.01). Similarly, the logarithmic values of plasma MDC levels correlated with serum LDH levels (r = 0.735, P < 0.001), serum IgE levels (r = 0.632, P < 0.001) and blood eosinophil counts (r = 0.567, P < 0.001) (not shown). Thus, plasma MDC levels appeared to correlate with serum IgE levels and eosinophil counts slightly better than plasma TARC levels.

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Fig. 2. Correlation analysis. Correlations of plasma TARC levels with plasma MDC levels, EASI scores, serum LDH levels, serum total IgE and blood eosinophil counts were analyzed for the AD patients.
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Elevated expression of TARC and MDC in AD skin lesions
Elevated levels of circulating TARC and MDC in AD patients are likely to be due in part to elevated production of these chemokines in AD skin lesions. To test this possibility, we first carried out RT-PCR analysis on expression of TARC and MDC in skin tissues from normal donors (n = 3) and AD patients (n = 3). As shown in Fig. 3, TARC and MDC were clearly detected in skin tissues from all AD patients, but not in those from normal donors. Furthermore, CCR4 was also detected in lesional skin of two AD patients who had strong signals for TARC and MDC, suggesting infiltration of CCR4-expressing T cells by locally produced TARC and MDC in these patients. To determine the cells expressing TARC and MDC in AD lesional skin, we next carried out immunohistochemical staining of these chemokines in skin tissues obtained from normal donors (n = 3), AD patients (n = 3) and psoriatic patients (n = 3), using specific mAb. Typical results are shown in Fig. 4. We observed TARC immunoreactivity in epidermal keratinocytes, mainly those in the basal layers (Fig. 4A and B), and also in dermal vascular endothelial cells (Fig. 4C) in AD lesional skin (n = 3). Furthermore, we observed MDC immunoreactivity in epidermal keratinocytes (Fig. 4E and F), which was even stronger and more widely distributed in the epidermal layers than TARC immunoreactivity. We also observed MDC+ cells in the dermis of AD lesional skin (Fig. 4G), which were likely to be mostly dendritic cells as reported previously (6,21). On the other hand, we hardly observed immunoreactivity for TARC and MDC in normal skin (n = 3) (not shown) or psoriatic skin (n = 3) (Fig. 4D and H).

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Fig. 3. RT-PCR analysis on expression of TARC, MDC and CCR4 in human skin tissues. Total RNA samples were prepared from peripheral blood mononuclear cells treated with phytohemagglutinin for 24 h (PHA-treated PBMC), skin biopsies of three normal donors (normal) and those from three atopic patients (AD). RT-PCR analysis was carried out for MDC, TARC, CCR4 and G3PDH as described in Methods.
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Fig. 4. Immunohistochemical staining of TARC and MDC in lesional skin of AD and psoriasis. Indirect staining of TARC and MDC with monoclonal anti-TARC and anti-MDC antibodies was carried out as described in Methods. (AD) Staining with anti-TARC; (EH) staining with anti-MDC. (AC and EG) AD skin; (D and H) psoriasis skin. Horizontal bars indicate 50 µm (AF and H) or 20 µm (G).
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Inducible co-expression of TARC and MDC in primary human epidermal keratinocytes in vitro
Positive TARC and MDC immunoreactivity in epidermal keratinocytes in AD skin lesions led us to examine expression of TARC and MDC by primary human epidermal keratinocytes in vitro. As shown in Fig. 5, TARC and MDC were both induced in primary epidermal keratinocytes primarily by IFN-
. MDC was also weakly induced by the combination of IL-1
and TNF-
. On the other hand, IL-4, the major Th2-type cytokine, was totally incapable of inducing TARC or MDC alone or in combination with other cytokines. As shown in Fig. 6(A), MDC was dose-dependently secreted from primary keratinocytes upon treatment with IFN-
. By using the same culture supernatants, however, we hardly detected TARC (<0.6 pg/ml). We also examined production of TARC and MDC by a human keratinocytic cell line HaCaT. As shown in Fig. 6(B), HaCaT constitutively produced MDC and its production was moderately augmented by IFN-
. HaCaT also constitutively produced TARC, but at a level much lower than that of MDC and again its production was moderately elevated by IFN-
.

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Fig. 6. Secretion of MDC and TARC by epidermal keratinocytes. Primary human epidermal keratinocytes (A) and an immortalized human epidermal keratinocytic cell line HaCaT (B) were cultured in six-well plates to confluency and mock-treated or treated with indicated concentrations of IFN- for 24 h. The contents of MDC (filled bars) and TARC (dotted bars) in the culture supernatants were determined with ELISA. Representative results from two separate experiments are shown.
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In vivo induction of TARC and MDC in mouse skin by IFN-
To test whether IFN-
was also capable of inducing TARC and MDC in vivo, we injected IFN-
into mouse skin, and examined tissue expression of TARC and MDC by RT-PCR. As shown in Fig. 7, a low level expression of TARC, but not MDC, was seen in control mouse skin tissues. Upon injection with IFN-
, both TARC and MDC were strongly induced with a peak at 6 h.
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Discussion
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Previously, highly elevated serum levels of TARC and MDC were reported in AD patients (9,10). However, chemokine contents in serum samples could be quite different from those in the circulating blood, since (i) some chemokines are known to be actively released from platelets during clotting (14,15), (ii) many chemokines are also passively released from DARC on red blood cells during clotting (17) and (iii) some chemokines are adsorbed to newly formed blood clots (18). In fact, we have recently demonstrated that platelets contain a substantial amount of TARC and release it upon coagulation (Fujisawa et al., submitted). Therefore, in the present study, we re-evaluated circulating levels of TARC and MDC in normal and AD subjects by using plasma samples. We have shown for the first time that plasma levels of TARC and MDC are significantly elevated in AD patients and correlate well with disease severity and other clinical parameters known to be elevated in AD patients (Figs 1 and 2). Thus, plasma levels of TARC and MDC are quite useful for clinical evaluation of AD patients. These results also strongly support that TARC and MDC play important roles in AD pathogenesis.
By RT-PCR analysis, we showed that signals of TARC, MDC and their shared receptor CCR4 were strongly up-regulated in AD lesional skin tissues (Fig. 3). This supports that local production of TARC and MDC in AD skin lesions promoted infiltration of T cells expressing CCR4, which are known to be mostly Th2-polarized memory T cells and/or those expressing cutaneous lymphocyte antigen (5,22). Recently, Nakatani et al. as well as Wakugawa et al. indeed demonstrated prominent dermal infiltration of CCR4+ memory T cells in AD lesional skin tissues by immunohistochemical staining (11,12). Previously, Vestergaard et al. also demonstrated TARC immunoreactivity in the epidermal basal layers of AD skin lesions (13). They did not, however, observe TARC immunoreactivity in any other cells in the dermis including vascular endothelial cells. On the other hand, Kakinuma et al. detected TARC immunoreactivity in epidermal keratinocytes, dermal vascular endothelial cells, infiltrating T cells and dermal dendritic cells in AD skin lesions (10). In the case of MDC, Galli et al. reported MDC-immunoreactivity in dermal-infiltrating cells in AD skin, which were identified as T cells and dendritic cells by double-staining techniques (9). They did not, however, mention MDC immunoreactivity in epidermal keratinocytes. In the present study, we showed by immunohistochemical staining using specific mAb that not only TARC but also MDC was produced in the epidermal layer of AD skin lesions (Fig. 4). We also detected TARC immunoreactivity in vascular endothelial cells, while that of MDC in dermal dendritic cells (Fig. 4). Thus, our results on TARC immunoreactivity were very similar to those by Kakinuma et al. (10). Furthermore, the present paper is the first to demonstrate a prominent MDC production by epidermal keratinocytes in AD lesional skin. The discrepancies among previous and present studies concerning the staining patterns of TARC and MDC in AD lesional skin may be due to different staining conditions, including use of different antibodies. We did not observe any immunoreactivity for TARC and MDC in normal skin as reported previously (9,10,13) or in psoriasis skin (Fig. 4).
To prove the intrinsic capability of epidermal keratinocytes to produce MDC as well as TARC, we first examined expression of MDC and TARC mRNA in primary epidermal keratinocytes. We demonstrated that not only TARC mRNA, but also that of MDC was induced in primary epidermal keratinocytes upon treatment with IFN-
(Fig. 5). We also demonstrated in vivo induction of MDC and TARC mRNA in mouse skin upon local injection of IFN-
(Fig. 7). Accordingly, primary epidermal keratinocytes and an immortalized human epidermal keratinocytic cell line HaCaT efficiently secreted MDC upon treatment with IFN-
(Fig. 6). This is the first report to show the ability of epidermal keratinocytes to produce MDC. Unexpectedly, however, we hardly detected secretion of TARC by IFN-
-stimulated primary epidermal keratinocytes (Fig. 6). We were also unable to detect TARC protein in IFN-
-stimulated primary epidermal keratinocytes by immunofluorescent staining (not shown). Previously, Vestergaard et al. reported vigorous secretion of TARC by a human keratinocytic cell line HaCaT upon stimulation with cytokines such as IFN-
and TNF-
(13). We found that HaCaT indeed secreted TARC upon treatment with IFN-
, but at levels much lower that those of MDC (Fig. 6). Since both TARC and MDC transcripts were induced at more or less similar levels in primary keratinocytes and HaCaT upon stimulation with IFN-
, production of TARC protein by epidermal keratinocytes may require further conditions such as particular stages of differentiation and/or some other signals from the local milieu. Consistently, TARC immunoreactivity in epidermal keratinocytes in AD skin lesions was much more restricted than that of MDC (Fig. 4).
It is notable that IFN-
(the Th1-type cytokine), but not IL-4 (the Th2-type cytokine), induces epidermal keratinocytes to express MDC and TARC, which are the pair of Th2-type chemokines acting on CCR4 (Fig. 5). IFN-
is also the potent inducer of the trio of Th1-type CXC chemokines Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in various types of cells including human keratinocytes (23). Mig, IP-10 and I-TAC commonly act on CXCR3 and selectively attract Th1 cells (3). Previous studies on the cytokine pattern of AD skin lesions have demonstrated that a Th2 cytokine profile (IL-4, IL-5 and IL-13) is predominant during the initial phase of skin inflammation, but both Th2 and Th1 cytokines (IL-5 and IFN-
) are up-regulated in chronic lesions (24). Even though most AD patients had undetectable levels of IFN-
in their plasma samples as control subjects (<8 pg/ml, data not shown), IFN-
may be the key factor in the chronic phase of AD because of its unique ability to simultaneously induce both Th1- and Th2-attracting chemokines from epidermal keratinocytes in AD skin. Indeed, the critical role of IFN-
in AD pathogenesis has been amply demonstrated (24). Our findings that IFN-
is a potent inducer of MDC in epidermal keratinocytes may now provide a new clue for its pivotal role in the AD pathogenesis.
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Acknowledgements
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This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Abbreviations
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ADatopic dermatitis
EASIeczema area and severity index
LDHlactate dehydrogenase
MDCmacrophage-derived chemokine
TARCthymus and activation-regulated chemokine
TNFtumor necrosis factor
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