イヌのアトピー性皮膚炎病変部および非病変部におけるCCL27およびCCL28 mRNAの発現解析

誌名誌名 The journal of veterinary medical science

ISSNISSN 09167250

巻/号巻/号 701

掲載ページ掲載ページ p. 51-55

発行年月発行年月 2008年1月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

FULL PAPER lnternal Medicine

Expression Analysis of CCL27 and CCL28 mRNA in Lesional and Non-Lesional Skin

of Dogs with Atopic Dermatitis

Sadatoshi MAEDA1l， Hiromi TSUCHIDA1l， Sanae SHIBATA1l， Tetsuji KAWAKAMI1l， ToshihiroTSUKUI2l，

Yasunori OHBA1l， Tsuneo FUKATA1l and Hitoshi KITAGAWA1l

I)Department ofVeterinary lnternal Medicine， Facul砂ofApptied Biological Sciences， G抑 Univers町， G抑 50/-1193and2!Animal Life Science Laboratory， Nippon Zenyaku Kogyo Co.， Ltd.， 1-1 Tairanoue Sasagawa， Asaka， Koriyama， Fukushima 963--0196， Japan

(Received 8 August 2007/Accept巴d11 September 2007)

ABSTRACT. Chemokines are important regulators of the selective recruitment of inflammatory cells into sites of allergic inflammation. Since canine atopic dermatitis (AD) shares many clinical features of human AD， patterns of chemokine production in dogs may also be sirnilar with those in humans. The aim of this study was to exarnine mRNA expression of CCL27 and CCL28 in lesional skin of dogs with AD to demonstrat巴sirnilarityof chemokine production with human counte叩arts.RNA was extracted from skin biopsy specimens of 12 dogs with AD. The mRNA expression of CC chemokines (CCL4， CCL19， CCL20， CCL21， CCL24， CCL27 and CCL28) was analyzed by quantitative real-time PCR and was compared between lesional and non-lesional skin. S巴ventypes of chemokines exarnined were constitutively express巴din both lesional and non-lesional skin. It was found that mRNA expression levels of CCL27 and CCL28 among the chemokines were significantly different b巴tweenlesional and non-Iesional skin (P<0.05). Expression level of CCL27 mRNA in lesional skin was significantly lower than that in non-Iesional skin. On the other hand， CCL28 mRNA expr巴ssionin lesional skin was found to be higher than that in non-Iesional skin. Thes巴 resultssuggest that CCL28 but not CCL27 may play important roles in immunopathogenesis of canine AD， indicating that experimental canine study may provide additional information that can be ex位apo-lated to human AD KEY WORDS: atopic dermatitis， canine， chemokines， real-time RT-PCR.

Chemokine r芭ceptors紅宅 expressedon various types of leukocytes and regulate specific or selective cell trafficking

[35]. Specific expression profiles of chemokine receptors

have been demonstrated in two different subsets of helper T-

cells， Thl and Th2 cells [1]. Regarding specific chemokine

rec巴ptors，it has been shown that Th 1 cells selectively

express CXCR3 [23] and CCR5 [11] whereas Th2 cells express CCR3 [24] and CCR4 [5]. Thus， the difference of expression patterns of the chemokine receptors between

Thl and Th2 cells may cause selective infiltration or migra-

tion of the cells into inflammatory sites， depending on the ch巴mokinesproduced in allergic lesions. In human AD， it

was found that CCR4 expr巴ssionon CD4+ cells was up-reg-

ulated [21] and correlated with disease severity [32]. In

lesional skin of human AD， CCR4+ cells were found to be markedly infiltrated [18]， and thus may be involved in the

production of a functional ligand for CCR4， thymus and activation問 gulatedchemokine (CCL17 /T ARC)， in the

lesion. The plasma CCL17 level was increased in human

AD and correlates with disease severity [9]. In 1巴sionalskin

of human AD， keratinocytes we印 themajor cell source of

CCL17 production [29]. In HaCaT cells， CCL17 production

was up-regulated by stimulation with inflammatory cytok-

ines including IL-l s， IFN-yand TNF-α[31]. These r芭sults

suggest that CCR4-CCL17 interaction plays an essential role in the immunopathogenesis of AD in humans.

*CORR邸内NDENCETO: MAEDA， S.， Department of Veterinary Inter-nal Medicine， Faculty of Applied Biological Sciences， Gifu Uni-versity， 1-1 Yanagido， Gifu 501-1193， Japan. e-mail: sadat@gifu-u.ac.jp

よVet.Med. Sci. 70(1): 51-55， 2008

It has been shown出atcutaneous lymphocyte-associated antigen (CLA) was preferentially expressed in skin homing

T cells [2]. Since CLA is a ligand for E-selectin， which is prominently expressed on inflamed vascular endothelium

[25]， interaction of CLA and E-selectin most likely mediates

the selective infiltration of skin homing T cells. Rec巴nt

studies demonstrated that CLA + cells exclusively expressed on CCRlO [3] and CCRlo+ cells were predominantly infil-trat巴din humans with AD [3]. Cytokine profile of CCRlo+ cells has not been fully characterized; however， these are

less likely categorized as Th2 cells because only 27% of CCRlO+ cells co-expressed CCR4 in atopic lesion [30]. To

date， cutaneous T cell attracting chemokine (CCL27/ CTACK) [4] and mucosae-associated epith巴lialchemokine

(CCL28ル1EC)[33] have been identified for the ligands of

CCRlO. Previous studies indicated that both CCL27 [3] and CCL28 [8] wer巴 stronglyexpressed in atopic lesions in

humans. The plasma CCL27 [10] and CCL28 [8] levels were increased in human AD and correlated with diseas巴

severity. These results indicated that interaction of CCR 1 0-

CCL27 or CCL28 may be important to regulate chemotaxis

of skin-homing T cells. Canine AD sbares a number of clinical features with

human AD [16]. House dust mite all巴rgen，which is the most common environmental allergen in humans， has been identified as the most important allergen in canine AD [17]. It has been speculated that a predisposing genetic factor may

exist in dogs with AD， since there is a high incidence of the

disease in certain breeds and families [28]. Histopathologi-cal analysis revealed that CD4+ cells were markedly infil-

52 S. MAEDA ET AL.

trated in lesional skin of canjne AD [26]. A r巴centstudy indicated that IL-4 mRNA was preferentially expressed in lesional skin of dogs with AD [20]. In lesional skjn of canine AD， expression of CCL17 [12] and CCR4 rr武NA[14] was detected in conjunction with the expression of inflammatory cytokjnes including IL-Is， IFN-y and TNF-α [12]. Furthermore， it was also found that the number of CCR4+ cells was incr巴asedin peripheral CD4+ cells from

dogs with AD [13]. A recent study also demonstrated that keratinocytes were m勾orCCL 17 -producing cells in lesional skin of dogs with AD [15]. These studies indicate that出eimmunopathogenesis of carune AD may be similar to出atofhuman AD， suggesting that canine AD can be us巴das an animal model for spontaneous allergy rather than a mice model

In this study， the possible association between allergic reaction and CCL27 and CCL28 was investigated to further clarify similarities in immunopathogenesis between canjne

and human AD.

MATERIALS AND METHODS

Dog cases with AD: This study was approved by the Institutional Animal Care and Use Committee at Gifu Uni-versity. Twelve dogs referred to the Veterinary Medical Teaching Hospital of Gifu University were diagnosed with AD according to Willemse's criteria for clinical diagnosis of carune AD [34]. All of the dogs with AD had compatible historical and clirucal fmdings of AD including seasonal or recurrent and chronic pruritus. Dogs with other skin dis-eases causing pruritus such as infestation of parasites and infection of bacteria or fungi were excluded based on rou-tine dermatologic exarillnations and a therapeutic trial of antibiotics. Food hypersensitivity was excluded based on the negative result by food elimination test， which was per-formed with commercial prescription diets for 8 weeks. In 白epresent study出erewere no dogs showing improvement of clinical signs by the food elirillnation. All dogs showed positive reaction to Dermatophagoidesfarinae加 dD. pter-onyssinus based on intradermal skin testing， wruch are the most common environmental allergens for AD in Japan [17].

Skin biopsies and isolation of total RNA: Skin biopsies wer巴 carriedout using a 6・mmdisposable punch (DER-MAPUNCH~， Nipro Medical Industri巴sLtd.， Tokyo， Japan) under local analgesia with 2% lidocaine hydrochloride

(Xylocaine@， Fujisawa Pharmaceutical Co.， Ltd.， Osaka， Japan). If necessary， dogs were sedated by intramuscular injection of medetomidine (Domitor@， Orion Corporation，

Espoo， Finland) at a dose of 0.04 mglkg and midazolam (Dormicum@， Roch巴， Basel， Switzerland) at a dose of 0.3

mglkg. A pair of skin biopsy samples was obtained from sites of both lesional and non-lesional skin in each dog with AD. Gross findings of白elesional skin were generally con-sistent among the dogs and included lichenification and hair loss showing characteristic chronic lesion rather出anacute lesion. All skin samples were immediately submerged in RNA later (Qiagen Inc.， Valencia， CA， U.S.A.) and stored at 200C until the巴xtractionoftotal RNA. Following homog-

enization of skin samples， total RNA was extracted using a commercially available kit (RNeasy Plus Mini Kit， Qiagen Inc.， Valencia， CA， U.S.A.) and stored at -80oC until expression analysis.

Quantitative Real-Time PCR: Transcription of chemok-ine mRNA was quantified by one-step real-time PCR (OneStep RT-PCR Kit， Qiagen Inc.， Valencia， CA， U.S.A.) according to manufacturer's instructions. Template amplifi-cation and detection were performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems， Foster City， CA， U.S.AふSequencesof the primer and probe pairs (QuantiTect Probe RT-PCR Kit， Qiagen Inc.， Valencia， CA， U.S.A.) for canine CCL4， 19，20，21，24，27，28 and GAPDH are listed in Table 1. A comparative CT method was used for quantiiication of chemokine mRNA expression as previ-ously report巴d[12]. For each sample， the CT values for the targ巴tamplicon (chemokines) and the calibrator (GAPDH) were deterrillned to report the relative transcription of the amplicon cDNA against calibrator cDNA， respectively. The CT value of出巴 calibratorwas subtracted from the CT value of th巴 targetchemokine (delta CT) to normalize for differences in the amount of total nucleic acid add巴dto each reaction and the efficiency of the reverse transcription. All samples were exarillned in duplicate and the mean value of delta CT was calculated. The amounts of chemokine mRNA were calculated by 2・deJla口， resulting in the evalua-

tion of the samples as n-fold difference relative to出atof GAPDHmRNA.

Statistical analysis: Paired t-test was used for statistical analysis of chemokjne expression between lesional and non-lesional skin. A value of P<0.05 was considered to be sta-tistical

Table 1. Sequences of prIm巴rsand probes for quantitative real-time sequence detection system

Chemokines Forward (5'-3') Reverse (5'-3') Probe (5'-3')

CCL4lMipls TCCTTTCTCTCCTTGTGCTA GCTGCTGGTCTCAAAGTAATC TCCTCCTACTGCCTGCT CCLl9lMip3s TGGTGCCAATGATGCTGA CAGTCTACAGCCATCCTTGA CCTTTCACTACCTCCTC CCL20ILARC ATGTGCAGTAGCAAGAAT GTATATCGAAGACAGCAA CAAGTCAGAAGCAGCAA CCL21/SLC GCTGTAAAAAGGACAAGGG GGTCTGTGGCTGTTCAGT GGCAAGAAGGGAAAGG CCL24/eotaxin2 TGGGTCCAGAAGTTGA TG TTAGATGGAGGTGCTGTTG CCAGGAGGAAGAAGGTGT CCL27/CTACK TGCATCCACCCTCAGAAT TTCCTTGTCAGCCCCAAA GGGACTCTACCTAACCTG CCL28IMEC AAGGCTTCTGGAGAGAGT AGAGTCTTCTGCGCCTGA TTGTGACTTGGCTGCTG GAPDH CTGGAGAAAGCTGCCAAA TGTTGAAGTCACAGGAGA AGAAGGTAGTGAAGCAG

CHEMOK町EEXPRESSION町 CAN町EATOPIC DERMA TITIS 53

Table 2. Means and standard errors of 2-deltaσof chemokine rnRNA expressed in lesional and non-lesional skin samples of dogs with AD

2-d山口

(Mean :t SEM) P value

Chemokines Lesional skin

CCL4lMip1s 1.6土0.5X 10-3

CCL19IMip3s 2.9:t 0.8 x 10-3

CCL20ILARC 8.1 :t 2.6 x 10-3

CCL211SLC 4.3 :t 2.2 x 10-2

CCL24/eotaxin2 6.0士3.2X 10-3

CCL27/CTACK 6.4 :t 2.3 x 10-2

CCL28IMEC 1.8土0.5X 10-2

NC， U.S.A.).

RESULTS

Dogs with AD: Twelve dogs were diagnosed with AD

based on the clinical diagnostic criteria proposed by Willemse [34]. The dogs with AD consisted of 4 mal回 (1

neutered) and 8 females whose age ranged from 2 to 11

years old (6.1 :t 4.0 years old). All of the dogs with AD had

various degrees of pruritic skin lesions with signs such as

erythema， hair loss， papules， and lichenification on the ven-traI abdomen. Lesional skin samples were originat巴dfrom

ventral abdomen， whereas non-Iesional skin samples were obtained from lat巴ralthorax， which gross findings did not show any abnormality.

Quantitative Rea/-Time PCR: Quantitative Real-Time PCR was perfoロnedto evaluate mRNA expression of CC

chemokines， using total RNA samples from both lesional

and non-Iesional skin samples. Expression of mRNA for the chemokines examined was observed in all samples. Values of 2-de)t. cr of the chemokines are summarized in Table 2.

Statistical analysis revealed白紙nodifference of the expres-

sion levels ofCCL4， 19，20，21 and 24 between lesional and

non-Iesional skin samples. Of出巴 12cases， eight cases indi-cated lower expression of CCL27 in lesional compared to

non-Iesional skin (Fig. lA)， which was statistically signifi-cant (P=0.027). On the other hand， expression levels of CCL28 in lesional skin were statistically higher than those

in non-Iesional skin from most cases except for one case (P=0.024) (Fig. lB).

DISCUSSION

It was reported出atskin-infiltrating Iymphocytes express

CCRlO in patients with psoriasis， atopic or contact allergic dermatitis [3]. Effecter function of CCRlO+ cells has not been determined based on cytokine production because

CCR 1 0+ cells showed mixed IL骨 21IL-4expression pattern

and minor proportion expressed CCR4 [30]. Since its ligands， CCL27 and CCL28， were preferentially expressed in inf1amed skin [3， 8] and their serum levels were increased

in humans with AD [8， 10]， CCRlO-CCL27 or CCRI0-

Non-Iesional skin (Iesional vs non-Iesional)

0.9 :t 0.2 x 10-3 0.1961 2.6:t 0.7 x 10-3 0.5572 14.8士7.0x10-3 0.4034 2.2:t 0.7 x 10-2 0.3807 7.8士2.9X 10-3 0.7053 22.9:t 0.5 x 1σ2 0.0266 0.4士0.1X 10-2 0.0242

(A)

0.7 p=O.0266

0.6

~ 0.5

E5E、F¥3 J 、0.4

N」 03 o 00.2

0.1

。L NL

但)

0.07

0.06 p=0.0242

0.05

出0.04

L=3 己z0.03

o 0.02 o

0.01

。一0.01

L NL

Fi沼g.1ト 2_de由e山i

leω悶s剖ional(Lυ) and non-l巴s剖IOn】al(NLυ) skin s鈎amplesof 12 dogs with AD.

CCL28 may play an important role on T-cell skin homing.

Further investigation demonstrated that CCRlo+ cells did not express CD27 and CCR7， markers of naive cells [27]. Thus， CCRI0+ cells could be associated with effecter but not trafficking phenotypes. InitiaIly， CCRlO would be nec-essary for cutaneous homing ofT cells; however， CCRlo+ T

54 S.恥1AEDAET AL.

celIs were determined to have no advantage ov巴rCCR4+ T

celIs in T celI homing [27]. A recent study also suggested

that CCRlO may play more important roles for the migra-

tion of keratinocyte precursor celIs from bone marrow [6].

In the present study， we confirmed lower expression of

CCL27 mRNA compared with norトlesionalskin. This

result is consistent with a human study reporting lower

expression of CCL27 in chronic skin lesion compared with

normal skin [19]. Taken together with previous results in

humans， CCL27 may be less associated with chemoattrac-

tion for T -ceIls infiltrating chronic lesional skin.

CCL28 is the latest CC chemokine that was identified by

searching the Human Genome Science and GenBank

dbEST databases [33]. Expression ofCCL28 has been con-

firmed in mucosal epithelial surfaces such as skin and colon

[33]. Like CCL27， CCL28 was initially determined as a

ligand for CCRI0， and a later study demonstrated that

CCL28 could also induce chemotaxis of eosinophils

expressing CCR3 [22]. CCL28 was strongly expressed in

epidermal keratinocytes of humans with AD [8]， suggesting

an involvement of CCL28 in the pathog巴nesisof human

AD. However， no study was conducted to compare levels of mRNA expression between lesional and non-lesional skin.

The present study is the first to analyze expression 1己velsin

skin of the dogs with AD， indicating high巴rexpression of

CCL28 mRNA in lesional skin compared to non-lesional

skin. Although little is known about which CCRlO or

CCR3 can preferentiaIly bind to CCL28， a recent study indi-

cated that CCL28 regulated eoshinophil recruitment via

CCR3 in allergic inflammation [7]. In this study， histo-pathological examination was carried out in all cases， show-

ing infiltration of eosinophils in lesional skin (data not

shown); nevertheless， no difference was found on mRNA

expression level of CCL24/eotaxin2， which is one of the

chemokine recruiting eosinophils. It may be possible that

CCL28 may play an essential role for infiltration of eosino-

phils， not T-cells， in atopic skin lesions. To prove this

hypothesis， expression analysis of not only other chemok-ines for eosinophils such as CCLll/eotaxin， CCL26/ 巴otaxin3and CCL5/lミANTES，but also CCR3 and CCRlO，

should be investigated in future prospective studies.

In conc¥usion， expression analysis of ligands for CCR 1 0

suggests that CCL28 but not CCL27 may play important

roles in immunopathogenesis of A

ACKNOWLEDGMENT. This work was supported by a

Ministry of Education， Science， Sports and Culture Grant-in-Aid for Scientific Research

REFERENCES

1. Bonecchi， R.， Bianchi， G.， Bordignon， P. P.， D' Ambrosio， D.，

Lang， R.， Borsatti， A.， Sozzani， S.， Allavena， P.， Gray， P. A.，

Mantovani， A. and Sinigaglia， F. 1998. Differential expression of chemokine receptors and chemotactic responsiven~ss of type 1 T he1per cells (Th1s) and Th2s. J. Exp. Med. 187: 129-

134 2. C1ark， R. A.， Chong， B.， Mirchandani， N.， Brinster， N. K.，

Yamanaka， K.， Dowgiert， R. K. and Kupper， T. S. 2006. The vast majority of CLA+ T cells are resident in normal sk.in. J

lmmunol. 176: 4431-4439. 3. Homey， B.， Alenius， H.， Muller， A.， Soto， H.， Bowman， E. P.，

Yuan， W.， McEvoy， L.， Lauerma， A. 1.， Assmann， T.， Bune-mann， E.， Lehto， M.， Wolff， H.， Y巴n，D.， Marxhausen， H.， To，

W.， Sedgwick， J.， Ruzicka， T.， Lehmann， P. and Zlotnik， A. 2002. CCL27-CCRlO interactions regulate T cell-mediated sk.in inflammation. Nat. Med.8: 157-165

4. Homey， B.， Wang， W.， Soto， H.， Buch加加，M. E.， Wiesen-born， A.， Catron， D.， Muller， A.， McClanahan， T. K.， Dieu-Nosjean， M. C.， Orozco， R.， Ruzicka， T.， Lehmann， P.， Old-ham， E目 andZlotnik， A. 2000. Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 (GPR-2， CCR 1 0) binds the skin-associated chemokine CCL27 (CT ACK/ ALPIILC)よlmmunol.164: 3465-3470.

5. Imai， T.， Nagira， M.， Takagi， S.， Kakizaki， M.， Nishimura， M.，

Wang， J.， Gray， P. W.， Matsushima， K. and Yoshie， O. 1999. Selective recruitment of CCR4-bearing Th2 cells toward anti-gen-presenting dells by出eCC chemok.ines thymus and ac-tiva-tion-regulated chemok.ine and macrophage-derived chemok.ine. lnt. lmmunol. 11: 81-88.

6. lnokuma， D.， Abe， R.， Fujita， Y.， Sasaki， M.， Shib誌i，A.，

Nakamura， H.， McMillan， J. R.， Shimizu， T. and Shimizu， H.

2006. CT ACK/CCL27 accelerates sk.in regeneratio日 vlaaccu-mulation of bone marrow-d巴rivedkeratinocytes. Stem Cells 24: 28Hト2816

7. John， A. E.， Thomas， M. S.， Berlin， A. A. and Lukacs， N. W 2005. Temporal production of CCL28 corresponds to eosino-phi1 accumulation and airway hyperreactivity in allergic airway inflarnmation. Am. J. Pathol. 166: 345-353.

8. Kagami， S.， Kakinuma， T.， Saek.i， H.， Tsunemi， Y.， F町ita，H.，

Sas広i，K.， Nakamura， K.， Takekoshi， T.， Kishimoto， M.， Mit-sui， H目，Komine， M.， Asahina， A. and Tamaki， K. 2005. Increased serum CCL28 levels in patients with atopic dermatト

tis， psoriasis vulgaris and bullous pemphigoid. J. lnvest. Der-

matol. 124: 1088-1090. 9. Kakinuma， T.， Nakamura， K.， Wakugawa， M.， Mitsui， H.，

Tada， Y.， Sa巴k.i，H.， Torii， H.， Asahina， A.， Onai， N.， Matsush ima， K. and Tamaki， K. 2001. Thymus and activation-regu1ated chemok.ine in atopic dermatitis: Serum thymus and activation-regulat巴dchemok.ine level is closely related wi出 diseaseactiv-ity. J. Allergy Clin. lmmunol. 107: 535-541.

10. Kakinuma， T.， Saek.i， H.， Tsunemi， Y.， Fujita， H.， Asano， N.，

Mitsui， H.， Tada， Y.， Wakugawa， M.， Wa

CHEMOKllぜEEXPRESSION IN CAN別EATOPIC DERMA TITIS 55

tized to Japanese cedar pollen. Clin. Exp. Allergy. 34: 1467 1473

14. Maeda， S.， Okayama， T.， Omori， K.， Masuda， K.， Sakaguchi，

M.， Ohno， K. and Tsujimoto， H. 2002. Expression of CC

chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion. Vet. lmmunol. Immunopathol. 90: 145-154.

15. Maeda， S.， Tsukui， T.， Saze， K.， Masuda， K.， Ohno， K.，

Tsujimoto， H. and Iwabuchi， S. 2005. Production of a mono-

clonal antibody to canine thymus and activation-regu1ated chemokine (TARC) and detection of TARC in lesional skin

from dogs with atopic d巴rmatitis.Vet. lmmunol. lmmuno-pathol. 103: 83-92.

16. Marsella， R.， Olivry， T. 2003. Animal mod巴Isof atopic derma-

titis. Clin. Dermatol. 21: 122-133. 17. Masuda， K.， Sakaguchi， M.， Fujiwara， S.， Kurata， K.， Yamash-

ita， K.， Odagiri， T.， Nakao， Y.， Matsuki， N.， Ono， K.， Watari，

T.， Hasegawa， A. and Tsujimoto， H. 2000. Positive reactions to common allerg巴nsin 42 atopic dogs in Japan. Vet. lmmunol.

lmmunopathol. 73: 193-204.

18. Nakatani， T.， Kaburagi， Y.， Shimada， Y.，lnaoki， M.， Takehara，

K.， Mukaida， N. and Sato， S. 2001. CCR4 memory CD4+ T Iymphocytes are increased in peripheral blood and lesional skin

from patients with atopic dermatitis. J. Allergy Clin. lmmunol 107・353-358.

19. Nomura， 1.， Gao， B.， Boguniewicz， M.， Darst， M. A.， Travers，

J. B. and Leung， D. Y. 2003. Distinct pattems of gene expres-sion in the skin lesions of atopic dermatitis and psoriasis: a

gene microarray ana1ysis. J. Allergy Clin. lmmunol. 112・1195-1202

20. Nuttall， T. J.， Knight， P. A.， McAleese， S. M.， Lamb， J. R. and

Hill， P. B. 2002. Expression ofTh1， Th2 and immunosuppres-sive cytokine gene transcripts in canine atopic dermatitis. Clin Exp. Allergy 32: 789-795.

21. Okazaki， H.， Kakurお， M.， Hirata， D.， Sato， H.， Kamimura， T.，

Onai， N.， Matsushima， K.， Nakagawa， H.， Kano， S. and

Minota， S. 2002. Characterization of chemokin巴 receptor

expression and cytokine production in circulating CD4+ T cells from patients with atopic dermatitis: up-regulation of C-C ch巴mokinereceptor 4 in atopic dermatitis. Clin. Exp. Allergy 32: 1236-1242.

22. Pan， J.， Kunkel， E. J.， Gosslar， U.， Lazarus， N.， Langdon， P.，

Broadwell， K.， Vie町a，M. A.， Genovese， M. C.， Butcher， E. C. and Soler， D. 2000. A novel chemokine ligand for CCR10 and

CCR3 expressed by巴pithelialcells in mucosal tissues. J.

lmmunol. 165: 2943-2949.

23. Sallusto， F.， Lenig， D.， Mackay， C. R. and Lanzavecchia， A

1998. Flexible programs of chemokine rec巴ptorexpression on

human polarized T h巴Iper1 and 2 Iymphocyte

tive expression of the eotaxin receptor CCR3 by human T

helper 2 cells. Science 277: 2005-2007. 25. Santamaria， Babi， L. F.， Picker， L. J.， Perez， Soler， M. T.， Drzi-

malla， K.， Flohr， P.， Blaser， K. and Hauser， C. 1995. Circulat-ing allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-

S巴lectivehoming receptor， the cutaneous Iymphocyte-associ-

ated antigen. J. Exp. Med. 181: 1935-1940

26. Sinke， J. D.， Thepen， T.， Bihari， 1. C.， Rutten， V. P. and Willemse， T. 1997. Immunophenotyping of skin-infiltrating T-

cell subsets in dogs with atopic dermatitis. Vet. lmmunol. lmmunopathol. 57・13-23.

27. Soler， D.， Humphreys， T. L.， Spinola， S. M. and Campbell， J. J 2003. CCR4 versus CCRIO in human cutaneous TH Iympho-

cyte trafficking. Blood 101: 1677-1682. 28. Sousa， C. A.， Marsella， R. 2001. The ACVD task force on

canine atopic dermatitis (II): g巴neticfactors目 Vet.lmmunol. lmmunopathol. 81: 153-157.

29. Vestergaard， C.， Bang， K.， Gesser， B.， Yoneyama， H.， Mat-

sushima， K. and Larsen， C. G. 2000. A Th2 chemokine， TARC，

produced by keratinocytes may recruit CLA+CCR4+ Iympho-cytes into lesional atopic dermatitis skin. J. lnvest. Dermatol.

115: 640-646. 30. Vestergaard， c.， Deleuran， M.， Gess巴r，B. and Gronhoj， Larsen，

C. 2003. Expression of the T-helper 2-specific chemokine

re氾eptorCCR4 on CCRlO-positive Iymphocytes in atopic der-

matitis skin but not in psoriasis skin. Br. J. Dermatol. 149: 457-463.

31. Vestergaard， c.， Kirst司n，N.， Gesser， B.， Mortensen， J. T.，

島1atsushima，K. and Larsen， C. G. 2001. IL-10 augments the

IFN-gamma and TNF-alpha induced TARC production in HaCaT cells: a possible mechanism in the inflammatory reac-

tion of atopic dermatitis. J. Dermatol. Sci. 26: 46-54.

32. Wakugawa， M.， Nakamura， K.， Kakinuma， T.， Onai， N.， Mat-sushima， K. and Tamaki， K. 2001. CC chemokine receptor 4

expression on peripheral blood CD4+ T cells reflects disease

activity of atopic dermatitis. J. lnvest. Dermatol. 117: 188-

196. 33. Wang， W.， Soto， H.， Oldham， E. R.， Buchanan， M. E.， Homey，

B.， Catron， D.， Jenkins， N.， Copeland， N. G.， Gilbert， D. J.，

Nguyen， N.， Abrams， J.， Kershenovich， D.， Smith， K.，

McClanahan， T.， Vicari， A. P. and Zlotnik， A. 2000. Identifica-tion of a nov巴1chemokine (CCL28)， which binds CCRlO

(GPR2). J. Biol. Chem. 275・22313-22323.34. Willemse， T. 1986. Atopic skin disease: a review and a recon-

sideration of diagnostic criteria. J. Small Anim. Pract. 27: 771-778.

35. Zlotnik， A. a