Journal of Dermatological Science (2006) 44, 56—58

www.intl.elsevierhealth.com/journals/jods

LETTER TO THE EDITOR

Alterations of TGF-b/Smad mRNA expres-sion inatopicdermatitis followingnarrow-band ultraviolet B phototherapy: Resultsof a pilot study

KEYWORDSAtopic eczema;Signal transduction;TGF-beta;Smad proteins;TL-01;UVB

Dear Sir,

Defective skin barrier, immunological dysfunctions(types I and IV allergy), genetic disorders, and psy-chological factors contribute to the pathogenesis ofatopic dermatitis (AD). Among the aforementionedfactors, CD4+ Th cells are reported to play a parti-cularly crucial role in the pathogenesis of AD [1].Transforming growth factor b (TGF-b) and its signal-ing molecules are inhibitory for B and T cells, IgEproduction, and mast cell proliferation, and theycan induce apoptosis in eosinophils. Although TGF-bcan exert its effect on many different cell types,CD4+ Th cells may be the main targets since anti-CD4antibodies are protective in TGF-b1 knockout mice.Disruption of TGF-b signaling in T cells or a dimin-ished TGF-b/Smad expression may be involved inthe development of several immune disorders [2]. Inthe present pilot study, we monitored the mRNAexpression of TGF-b/Smad proteins in skin of ADpatients who underwent a course of narrowbandultraviolet B (NB UVB) phototherapy.

We studied 10 patients (seven men and threewomen; age range 26—55 years; mean age 39.6years) diagnosed with extrinsic AD [1]. In additionto the patient group, 10 healthy, non-atopic, gen-der/age-matched individuals were used as controlsubjects. The study followed a protocol approved by

0923-1811/$30.00 # 2006 Japanese Society for Investigative Dermadoi:10.1016/j.jdermsci.2006.06.004

our institutional review board. All subjects whoparticipated in the investigation signed an informedconsent. NB UVB phototherapy was performed usingthe Cosmedico GP-42 (Cosmedico MedizintechnikGmbH, VS-Schwenningen, Germany) cabin fittedwith ARIMED 311 fluorescent lamps. Irradiationwas performed thrice weekly for 6 weeks. Startingdose was 0.1 J/cm2 NB UVB for skin type II and 0.2 J/cm2 for skin type III. Depending on tolerability andskin type, NB UVB dosage was increased with 0.1—0.2 J/cm2. Maximum NB UVB dose was considered1.5 and 1.8 J/cm2, respectively [3]. Additional ther-apy was restricted to the use of emollients. Assess-ment of disease activity at baseline and afterphototherapy was performed using the Six Area,Six Sign, AD (SASSAD) score [4].

In AD patients, one 3-mm full-thickness skin spe-cimen was taken from lesional skin before the firstNB UVB irradiation and one specimen was recoveredfrom an area adjacent (about 2 cm) to the pre-treatment biopsy site 12 h following the last NBUVB exposure. Moreover, a 3-mm full-thickness skinspecimen was taken from healthy controls. In orderto perform quantitative analysis of real-time RT-PCRtotal cellular RNA was isolated from skin tissuesamples using RNeasy1 Lipid Tissue Kit (QIAGEN,Chatsworth, CA) following the manufacturer’s pro-tocol. Real-time PCR for TGF-b1, Smad3/4/7, andGAPDH was performed according to standard pro-cedures as previously described [5]. Data analysiswas performed using the statistical package Med-Calc Software (Mariakerke, Belgium). Non-normaldistribution of data was confirmed by the D’Agos-tino—Pearson test. Results were analysed usingpaired or independent non-parametric tests includ-ing theWilcoxon-rank test, Mann—Whitney test, andSpearman rank correlation procedure, respectively.We constrained experimentwise error rates due tomultiple comparisons to the standard alpha (P) levelof 0.05 by the Bonferroni method.

A significant (P < 0.05) reduction of the medianbaseline SASSAD score from 45 to 17 was observedafter phototherapy. Healthy skin of control subjectsshowed significantly (P < 0.05) higher Smad3/4

tology. Published by Elsevier Ireland Ltd. All rights reserved.

Letter to the Editor 57

Fig. 1 Diagram showing expression of TGF-b1/Smadproteins in lesional skin of 10 atopic dermatitis patientsassessed before (AD pre) and after (AD post) a 6-weekcourse of NB UVB phototherapy. For comparison, expres-sion of TGF-b1/Smad proteins in skin of 10 healthy sub-jects is also shown (Controls).

mRNA expression as compared to AD patients(Fig. 1). However, mRNA expression of Smad7 andTGF-b1 observed in controls did not significantly(P > 0.05) differ from AD patients. There was asignificant increase of mRNA expression ofSmad3/4 (P < 0.05) after NB UVB phototherapy.Nevertheless, post-treatment mRNA levels ofTGF-b1 and Smad7 did not significantly (P > 0.05)differ from baseline levels (Table 1). There was nolinear relationship between the relative SASSADscore reduction and relative increase of mRNAexpression of Smad3/4 as assessed by the Spearmanrank correlation procedure (r < 0.3; P > 0.05). In aprevious study, a significantly reduced transcriptionof TGF-b has been demonstrated in skin lesionsof canine AD [6]. Moreover, TGF-b1 appears tosuppress AD-like skin lesions in mice at least inpart through down-regulation of interferon-g [7].In a human study [8], the mRNA expressionpatterns of various cytokines were examined in

Table 1 Data of quantitative real-time RT-PCR of TGF-b/Smcontrols (n = 10) and in patients with atopic dermatitisphototherapy

Parameter AD pre A

TGF-b1 (PS) F 50-GGTACCTGAACCCGTGTTGCT-30

RT-PCR 0.014 (0.004—0.03) 0

Smad3 (PS) F 50-TGAGTTCGCCTTCAATATGAAGAART-PCR 0.035 (0.009—0.075) 0

Smad4 (PS) F 50-ACTGCAGAGTAATGCTCCATCAAGRT-PCR 0.067 (0.048—0.152) 0

Smad7 (PS) F 50-TAGCCGACTCTGCGAACTAGAGTRT-PCR 0.006 (0.003—0.011) 0

GAPDH (PS) F 50-CCTCAACTACATGGTTTACA-TGT

PS, primer sequence; GAPDH, Glyseraldehyde-3-phosphate dehydro

untreated lesional skin of patients with extrinsicAD, intrinsic AD, and healthy skin of normalsubjects. However, the authors did not observedifferences of TGF-b mRNA levels among threegroups. Toda et al. [9] evaluated the expressionof TGF-b1 bymeans of immunohistochemistry. Theyobserved that TGF-b1 expression was markedlyenhanced in both acute and particularly chroniclesions of AD.

Our study support data of previous studiesdemonstrating the high efficacy of NB UVB in thetreatment of AD [3]. In accordance with the findingsof Jeong et al. [7], we did not observe significantdifferences between TGF-b1 mRNA expressionfound in AD and healthy skin. However, we observedin skin of AD patients significantly decreased levelsof receptor-regulated (Smad3) and common media-tor (Smad4) intracellular signal transducers whichusually propagate TGF-b1 signals and regulateexpression of target genes [2]. After phototherapy,we observed a significant increase of mRNA expres-sion of Smad3/4 which was paralleled with a sig-nificant improvement of the patients’ skin status.Indeed, the design of the present pilot study doesnot allow to draw the conclusion that the improve-ment of AD was due to the increase of Smad3/4mRNA expression, the less the since we did notobserve a significant correlation between thechanges of the clinical score and the increase ofSmad mRNA expression. Quan et al. [10] have shownthat UVB blocks in vitro cellular responsiveness toTGF-b through down-regulation of TGF-b type IIreceptor and induction of Smad7. They also per-formed an in vivo study on healthy subjects, whowere exposed UVB, and observed an increase ofmRNA expression for TGF-b1 (48—72 h post-irradia-tion) and Smad7 (8 h post-irradiation). Quan et al.[10] did, however, not detect significant changes in

ad mRNA [median (range)] levelsa investigated in healthy(n = 10) before (AD pre) and after (AD post) NB UVB

D post Healthy controls

, R 50-TGTTGCTGTATTTCTGGTACAGCTC-30

.015 (0.008—0.027) 0.021 (0.005—0.390)

-30, R 50-CAGGAGGTAGAACTGGTGTCTCTACTCT-30

.082 (0.057—0.082) 0.321 (0.109—1.051)

T-30, R 50-GGATGGTTTGAATTGAATGTCCTT-30

.145 (0.104—0.227) 0.454 (0.356—2.102)

-30, R 50-GGACAGTCTGCAGTTGGTTTGA-30

.007 (0.004—0.012) 0.012 (0.006—0.027)

TCC-30, R 50-ATGGGATTTCCATTGA-TGA-CAAG-30

genase.

58 Letter to the Editor

Smad3/4 protein expression following short-termUVB exposure.

Taken together, we observed significantlyreduced mRNA expression of Smad3/4 in lesionalskin of AD patients as compared to healthy subjects.Following a 6-week course of NB UVB phototherapy,which resulted in a significant improvement of AD,the initially reduced Smad3/4 levels significantlyincreased. Our results underscore the significanceof the TGF-b/Smad signaling pathway in the etio-pathogenesis of AD. Possibly, phototherapy of ADworks through modulation of signal transducer pro-teins. Nevertheless, our results have to be substan-tiated by further studies including a larger samplesize, disease controls, such as psoriasis patients,and immunohistological studies.

References

[1] Abramovits W. Atopic dermatitis. J Am Acad Dermatol2005;53(Suppl. 1):86—93.

[2] Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Trans-forming growth factor-beta regulation of immune responses.Annu Rev Immunol 2006;24:99—146.

[3] Gambichler T, Breuckmann F, Boms S, Altmeyer P, Kreuter A.Narrowband UVB phototherapy in skin conditions beyondpsoriasis. J Am Acad Dermatol 2005;52:660—70.

[4] Berth-Jones J. Six Area, Six Sign Atopic Dermatitis (SASSAD)severity score: a simple system for monitoring diseaseactivity in atopic dermatitis. Br J Dermatol 1996;135:25—30.

[5] Giulietti A, Overbergh L, Valckx D, Decallone B, Bouillon R,Mathieu C. An Overview of real-time quantitative PCR:applications to quantify cytokine gene expression. Methods2001;25:386—401.

[6] Nuttall TJ, Knight PA, McAleese SM, Lamb JR, Hill PB.Expression of Th1, Th2 and immunosuppressive cytogenetranscripts in canine atopic dermatitis. Clin Exp Allergy2002;32:789—95.

[7] Jeong CW, Ahn KS, Rho NK, Park YD, Lee DY, Lee JH, et al.Differential in vivo cytokine mRNA expression in lesionalskin of intrinsic versus extrinsic atopic dermatitis patientsusing semiquantitative RT-PCR. Clin Exp Allergy 2003;33:1717—24.

[8] Sumiyoshi K, Nakao A, Ushio H, Mitsuishi K, Okumura K,Tsuboi R, et al. Transforming growth factor-beta1 suppressesatopic dermatitis-like skin lesions in NC/Nga mice. Clin ExpAllergy 2002;32:309—14.

[9] Toda M, Leung DY, Molet S, Boguniewicz M, Taha R, Chris-todoulopoulos P, et al. Polarized in vivo expression of IL-11and IL-17 between acute and chronic skin lesions. J AllergyClin Immunol 2003;111:875—81.

[10] Quan T, He T, Kang S, Voorhees JJ, Fisher GJ. Ultratvioletirradiation alters transforming growth factor b/Smadpathway in human skin in vivo. J Invest Dermatol 2002;119:499—506.

Thilo Gambichler*Nordwig S. TomiMarina SkryganPeter Altmeyer

Alexander KreuterDepartment of Dermatology, Ruhr-University

Bochum, Gudrunstr. 56, 44791 Bochum, Germany

*Corresponding author. Tel.: +49 234 5093458;fax: +49 234 5093409

E-mail address: t.gambichler@klinikum-bochum.de(T. Gambichler)

17 March 2006