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Narrowband ultraviolet B and medium-dose ultraviolet A1 are equally effective in the treatment of moderate to severe atopic dermatitis
Narrowband ultraviolet B and medium-dose ultraviolet A1 are equally effective in the treatment of moderate to severe atopic dermatitis I. M. Leonie Majoie, MD,a* J. Marja Oldhoff, MD, PhD,a* Huib van Weelden, MSc,a Marloes Laaper-Ertmann,a Mente T. Bousema, MD,b Vigfus Sigurdsson, MD, PhD,a Edward F. Knol, PhD,a Carla A. F. Bruijnzeel-Koomen,MD, PhD,a and Marjolein S. de Bruin-Weller, MD, PhDa Utrecht and Amersfoort, The Netherlands Background: Phototherapy may be effective in atopic dermatitis (AD). Medium-dose (MD) ultraviolet (UV) A1 was introduced for the treatment of AD. Few immunohistochemical data are available pertaining to phototherapy in AD. Regulatory T cells may play a role in clearing AD. Objectives: We sought to compare the clinical and immunohistochemical effects of narrowband (NB) UVB and MD UVA1 treatment in patients with AD. Methods: Thirteen adult patients with AD were included in this randomized investigator-blinded halfsided comparison study between NB UVB and MD UVA1. Disease activity was measured using the Leicester sign score. Skin biopsy specimens were taken before and after phototherapy. Regulatory T cells were stained with the forkhead box protein P3 (FoxP3). Results: NB UVB and MD UVA1 both significantly decreased AD severity (P \.01) and the dermal cellular infiltrate. The percentage of FoxP31CD31 T cells did not change after NB UVB or MD UVA1 treatment. Limitation: MD UVA1 therapy was given 3 times per week instead of the preferred regimen of 5 times per week. This was necessary to achieve good blinding of the study. Conclusions: NB UVB and MD UVA1 seem equally effective in the treatment of patients with moderate to severe AD. Neither MD UVA1 nor NB UVB had an effect on the percentage of FoxP31CD31 T cells. ( J Am Acad Dermatol 2009;60:77-84.)
A
topic dermatitis (AD) is an inflammatory skin disease with a chronic relapsing course. Phototherapy may be effective in AD. Ultraviolet (UV) lamps that have been studied include broadband UVB (280-315 nm), narrowband (NB) UVB (311 nm), UVA (315-400 nm), UVA1
From the Department of Dermatology and Allergology, University Medical Center, Utrecht,a and Department of Dermatology, Meander Medical Center, Amersfoort.b *These authors contributed equally to this study. Funding sources: None. Conflicts of interest: None declared. Accepted for publication August 29, 2008. Reprint requests: J. Marja Oldhoff, MD, PhD, University Medical Center Utrecht, Heidelberglaan 100, Department of Dermatology, G02.124, 3508 GA Utrecht, the Netherlands. E-mail: [email protected]. 0190-9622/$36.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2008.08.048
Abbreviations used: AD: FoxP3: LSS: MD: NB: NHuS: PBS: Tregs: UV: UVAB: VAS:
atopic dermatitis forkhead box protein P3 Leicester sign score medium dose narrowband normal human serum phosphate-buffered saline regulatory T cells ultraviolet combinations of ultraviolet A and ultraviolet B visual analog scale
(340-400 nm), photochemotherapy (psoralen plus UVA), bath psoralen plus UVA, combinations of UVA and UVB (UVAB), and extracorporal photochemotherapy.1,2 NB UVB phototherapy for AD was first studied in 1993 by George et al.3 Since then, several opendesign studies have been published that show its 77
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Fig 1. Trial flow chart. During the treatment period phototherapy was administered 3 times/wk; patients were randomly assigned to receive narrowband ultraviolet (UV) B to one side of the body and medium-dose UVA1 to the other side. LSS, Leicester sign score; VAS, visual analog scale.
efficacy in AD.4-8 Clinical assessment of this mode of phototherapy in psoriasis suggested a number of advantages over conventional broadband UVB. These include a reduced incidence of burning episodes, increased efficacy, and longer remission periods. A study performed by Jekler and Larko9 shows that UVAB is superior to conventional broadband UVB or UVA in the management of AD. Another study comparing UVAB with NB UVB showed a preference for NB UVB.10 The therapeutic effectiveness of UVA1 radiation in AD was first studied by Krutmann et al11 in an open study in which 15 patients with acute, severe AD were exposed to high-dose UVA1 (130 J/cm2) and compared with 10 patients receiving UVAB phototherapy. In comparison with UVAB therapy, significant improvement in clinical score was found in favor of high-dose UVA1.12 During the course of the following years, these observations have been confirmed by several reports, which mainly represent uncontrolled, open, and sometimes even noncomparative studies.12-14 Because of concerns about possible long-term side effects of high-dose UVA1 therapy (eg, photodamage and carcinogenesis), the effect of lower doses of UVA1 was studied.15 The results of this study demonstrated that both high-dose (maximum 130 J/cm2) and medium-dose (MD) (maximum 65 J/cm2) UVA1 are effective, whereas low-dose (maximum 20 J/cm2) UVA1 is not.15 The clinical effect of MD UVA1 phototherapy in AD has been studied by several authors.16,17 In a bilateral comparison study in 10 patients with AD, Tzaneva et al18 showed that MD UVA1 is as effective as high-dose UVA1. In a comparative study by von Kobyletzki et al,16 MD UVA1 treatment showed a significantly greater improvement in clinical scores for AD than did UVAB. Phototherapy is thought to improve chronic skin diseases such as AD and psoriasis by immunosuppressive action.19 The main mechanisms by which phototherapy induces immune suppression include the induction of apoptosis (cell death) in infiltrating T cells, induction of immunomodulatory cytokines, and reduction in the number of Langerhans cells.
The effect of phototherapy on regulatory T cells (Tregs) in human beings has not been studied so far. Human Tregs express the forkhead box protein P3 (FoxP3) and act as suppressor cells.20 NB UVB is the first choice of phototherapy to treat patients with AD in the Netherlands and MD UVA1 is a rather new phototherapeutic modality. Therefore, we compared the clinical effect of MD UVA1 and NB UVB phototherapy in patients with AD. Furthermore, we compared the immunohistochemical effects of both treatment modalities, including the number of Tregs, by measuring FoxP3 expression.
METHODS Study design The study was done in a randomized, investigator-blinded, and half-sided comparison design. Fig 1 shows the trial flow chart. Half-sided irradiation with threshold erythematic doses of NB UVB and MD UVA1 was performed 3 times weekly during a period of 8 weeks with a follow-up period of 4 weeks. Patients Thirteen patients with AD were enrolled, of whom 8 were female, median age 25 years (range 20-56). All patients fulfilled the criteria of Hanifin and Rajka21 and all had symmetric distribution of their eczema. Exclusion criteria included local treatment with corticosteroids or other medical topical agents within the last 2 weeks or systemic treatment with antibiotics, corticosteroids, or oral immunosuppressive drugs within the last 4 weeks. Clinical scoring Severity of the eczema was evaluated by the Leicester sign score (LSS) (range 0-108) by a blinded investigator. Severity is scored by 6 clinical features (erythema, purulence, excoriation or crusting, dryness or scaling, cracking or fissuring, and lichenification) graded at 6 defined body sites on a scale of 0 (none) to 3 (severe).22 Patients were asked to complete a visual analog scale (VAS) for pruritus, where the level of their itch is reflected on a scale of 0 to 10
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(0 = no itch and 10 = most intense itch imaginable). Both LSS and VAS-pruritus scoring were done before, during, and after the treatment period (Fig 1). Each scoring was done just before the next phototherapy session, so that erythema caused by phototherapy could not influence scoring. Before the treatment period, at the moment of inclusion, the median LSS for MD UVA1 was 20 (range 8-31). The median LSS for NB UVB was 19 (range 9-29). The baseline characteristics are shown in Table I. All participants in this study gave their informed consent. This study was approved by the local medical ethical committee, which follows the Declaration of Helsinki protocol.
Table I. Baseline characteristics of patients with atopic dermatitis and a symmetric distribution of their disease
Phototherapy and dosimetry The NB UVB phototherapy unit consisted of a light cabin (Waldmann, Schwenningen, Germany) with 20 311-nm lamps (TL-01, Philips, Eindhoven, The Netherlands). The minimal erythemal dose for NB UVB for each patient was determined before treatment. UVB treatment was started with an initial dose of 70% of the minimal erythemal dose. Subsequent dose increments were given on the basis of erythemic reactions of the skin. The intention was for each exposure to induce slight erythema. If the previous exposure failed to induce any reaction the dose was increased by 20%. If the resulting erythema was slight the dose was increased by 10%. This dose regimen is slightly less intensive than normally used in the treatment of psoriasis.23 The MD UVA1 phototherapy unit consisted of a Waldmann light cabin with 40 lamps (TL-10R, Philips) emitting wavelengths of 350 to 400 nm only, with a maximum of 6370 nm. The first dose was 30 J/cm2. In two steps the dose was increased to 45 J/cm2. In 3 of the 13 patients the dose had to be decreased because the reaction (erythema and/or papules) was too strong. The average dose of UVA1 was more than 40 J/cm2. Throughout the experiments the irradiation of the skin was monitored with a UVA/UVB detection device (Waldmann). Spectral distribution measurements were taken with a calibrated standard UVvisible spectoradiometer (model 752, Optronic Laboratories Inc, Orlando, FL). Phototherapy took place 3 times a week for 8 weeks, with a follow-up period of 4 weeks. Patients were randomly assigned to receive NB UVB to one body side and MD UVA1 to the other body side. The nonexposed body sides were covered with a halfsided overall. During the treatment period no topical treatment apart from emollients was allowed. During the follow-up period topical corticosteroids were allowed if needed. The face was excluded from halfsided comparison and analysis. It was only treated
with MD UVA1 and, if necessary, mild topical corticosteroids (European class I or II).
Patients with AD, n = 13
Age, y Male:female Half-sided comparison LSS VAS-pruritus
25 (20-56) 5:8 MD UVA1 NB UVB 20 (8-31) 19 (9-29) 7.5 (3.5-10) 7.5 (3.5-10)
Baseline characteristics were same for both body sides before half-sided phototherapy. Data are expressed as median values (minimum-maximum values). AD, Atopic dermatitis; LSS, Leicester sign score; MD, medium dose; NB, narrowband; UV, ultraviolet; VAS, visual analog scale.
Biopsy specimens Skin biopsy specimens of 9 patients taken from lesional skin on both body sides were analyzed before phototherapy. Two days after the last treatment, skin biopsy specimens were taken from healed lesional AD skin at approximately the same locations as the prephototherapy biopsy specimens. Skin biopsy specimens (4 mm) were taken under local anesthesia (lidocaine), snap-frozen in liquid nitrogen, embedded in Tissuetek (Sakura, Torrance, CA), and stored at e708C until further handling. Sections (5 m) were cut from the Tissuetek (Sakura)-embedded skin tissue and mounted on 3aminopropyl tri-ethoxy silaneecoated (Sigma, St Louis, MO) glass slides. Immunohistochemistry Antibodies. Mouse antibodies recognizing the following antigens were used as markers for immunohistochemical staining of the frozen sections: CD3 (Leu-4, clone SK7, Becton Dickinson, San Jose, CA); eosinophilic cationic protein (clone EG2, Phadia, Uppsala, Sweden); mast cell tryptase (clone AA1, DAKO, Glostrup, Denmark); neutrophil elastase (clone NP57, DAKO); FoxP3 (rat antihuman, clone PCH101, eBioscience, San Diego, CA); and CD1a (fluorescein isothiocyanate conjugated, clone NA 1/34, DAKO). Immunostaining. Staining for CD3, mast cell tryptase, eosinophil cationic protein, and neutrophil elastase was combined with a biotinylated horse antimouse IgG (Vector Laboratories Inc, Burlingame, CA) as described by Thepen et al.24 The fluorescein isothiocyanateeconjugated CD1a antibody sections were fixed with dry acetone, air dried, and preincubated for 20 minutes in 10% normal human serum
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by a blinded observer. Before evaluation, sections were compared with the isotype-controlestained sections or were compared with nonstained sections. Positive cells in the dermis were counted in 3 different sections of 1 to 1.5 mm2/section and calculated as cells/mm2. In fields containing sweat ducts and hair shafts, only the intervening dermal regions were counted. For EG2, CD3, FoxP3, elastase, and CD1a, dermal and epidermal compartments were examined separately. The number of epidermal CD1a1 cells was graded as: no, or hardly any positive cells present (0), presence of scattered positive cells (1), abundant presence of positive cells (2), or closed maze of positive cells (3). A second independent observer checked one of every 5 analyses. The mean interobserver coefficient of variation was within 10%.
Fig 2. Clinical results. Mean reduction in Leicester sign score (LSS ) (A) and visual analog scale (VAS ) pruritus (B). No significant difference in favor of narrowband (NB) ultraviolet (UV) B. MD, Medium dose; X-axes, time in weeks; Y-axes, mean score. Vertical lines display SD.
(NHuS) in phosphate-buffered saline (PBS). Primary antibodies were diluted in 1% NHuS and incubated with the sections for 60 minutes. Slides were washed 3 times for 5 minutes with PBS plus 0.05% Tween 20 (P-1379, Sigma) and were subsequently incubated with alkaline phosphatase conjugated sheep antifluorescein (Fab fragments, Boehringer Mannheim, Mannheim, Germany) in PBS for 30 minutes. After incubation, slides were washed with Tris-HCl (0.1 mol/L, pH 8.5). Alkaline phosphatase reactivity was demonstrated as previously described.24 For FoxP3 single staining, slides were preincubated with 10% NHuS and 10% normal rabbit serum in PBS. The primary antibody was diluted in PBS with 1% NHuS and 1% bovine serum albumin and incubated with the sections for 60 minutes. The staining was then combined with biotinylated rabbit antirat immunoglobulin (DAKO) for 30 minutes at room temperature, followed by an avidin-biotin-peroxidase complex (Vectastain Elitekit, Vector Laboratories Inc) for another 30 minutes at room temperature. Peroxidase activity was visualized using 3-amino-9-ethylcarbazole (Sigma). Quantification of staining. Skin sections were examined by light microscopy at 3400 magnification
Statistical analysis Statistical analysis was performed using software (SPSS for Windows, Version 10.0.5, SPSS Inc, Chicago, IL). The Wilcoxon signed rank test was used for all paired comparisons. A P value of .05 or less was considered to be significant. Adjustments for multiple comparisons were not done.
RESULTS Clinical results Thirteen patients fulfilled the entry criteria and were randomly assigned to phototherapy. All patients completed the trial. Only data obtained from the phototherapy period (0-8 weeks) were used for statistical analysis because during this period, all patients exclusively received phototherapy with no other treatment. During the follow-up period (8-12 weeks) patients were allowed to use local corticosteroids, which most patients did. Patients received median cumulative doses of 10.5 J/cm2 of NB UVB (range 9.9-11.5, average increment 10%/exposure) to one body side and 930.6 J/cm2 of MD UVA1 (range 717.1-1067.4) to the other body side. Fig 2 shows the clinical results assessed for both body sides separately. Both the NB UVB and MD UVA1 treatments resulted in significantly decreased LSS scores: median reduction for NB UVB from 18 to 10 points (P \.01); and for MD UVA1 from 19 to 12 points (P \.01). The patients’ self-assessment, measured by the VAS-pruritus, was also significantly decreased after both treatment modalities: median reduction for NB UVB from 7 to 1.8 points (P \.01); and for MD UVA1 from 7 to 4.1 points (P \ .01). When the improvement in the NB UVBetreated side was compared with that in the MD UVA1etreated side, no significant difference was observed (measured by LSS and VAS-pruritus).
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Fig 3. Immunohistochemical results (n = 9). Cellular infiltrate before (t = 0) and after (t = 8) phototherapy in patients with atopic dermatitis. Treatment of one body side was with narrowband (NB) ultraviolet (UV ) B and other body side with medium-dose (MD) UVA1. AA1, Mast cell; elastase, neutrophil; CD1a, dendritic cell; CD3, T cell; EG2, eosinophil; X-axes, time: 0 = lesional skin, 8 = after 8 weeks of phototherapy; Y-axes, cells/mm2. *Significantly different decrease in cells compared with t = 0. No significant differences were observed between NB UVB and MD UVA1 sides.
Immunohistochemical results Epidermis. The numbers of T cells, dendritic cells, and neutrophils in the epidermis were significantly decreased after both treatment modalities. No
change was observed in the number of epidermal eosinophils (data not shown). Between-treatment comparison did not show significant differences in the composition of the epidermal cell infiltrate.
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Fig 4. Immunohistochemical results of forkhead box protein P3 (n = 9). Cellular infiltrate before (t = 0) and after (t = 8) phototherapy in patients with atopic dermatitis. Treatment of one body side was with narrowband (NB) ultraviolet (UV) B and other body side with medium-dose (MD) UVA1. CD3, T cell; FoxP3, positive T cell; X-axes, time: 0 = lesional skin, 8 = after 8 weeks of phototherapy; Y-axes, cells/mm2. *Significant decrease in cells compared with t = 0. No significant differences were observed between NB UVB and MD UVA1 sides.
Dermis. The dermal cellular infiltrate was scored for CD3 (T cell), FoxP3 (Treg), EG2 (eosinophil), CD1a (dendritic cell), AA1 (mast cell), and elastase (neutrophil) counts. The distribution of these cell types was the same on both body sides before phototherapy. Figs 3 and 4 show the immunohistochemical results. A significant decrease in the number of dermal T cells (P \ .01), eosinophils (P \ .01), and neutrophils (P \ .05) was observed after MD UVA1 and after NB UVB. The number of dermal dendritic cells was significantly decreased after NB UVB (P \ .05), but not after MD UVA1. Between-treatment comparison did not show a significant difference in dermal dendritic cells. The number of mast cells was not significantly changed after either treatment. Although the number of CD31 T cells significantly
decreased after both NB UVB and MD UVA1, the percentage of FoxP31CD31 T cells did not change. Between-treatment comparison did not show significant differences in the composition of the dermal cell infiltrate.
DISCUSSION The clinical effect of NB UVB and MD UVA1 is similar in the studied population of patients with AD. This is supported by a comparable decrease in inflammatory cell infiltrate after both treatments. The follow-up period was confounded by the use of dermatocorticosteroids. Therefore, the differential effect of both treatments 4 weeks after phototherapy is not useful for evaluation. Our data are in accordance with the results found in a study by Legat et al.25 In their study, with a design similar to ours,
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NB UVB was compared with MD UVA1 in patients with AD using half-sided irradiations. Although they could not demonstrate a significant difference between NB UVB and MD UVA1 treatment, NB UVB was preferred over MD UVA1 phototherapy for AD. The total amount of irradiance needed for effective phototherapy is less for NB UVB than for MD UVA1. Because of this, the exposure time is short and less heat is produced during NB UVB treatment compared with MD UVA1. This aspect makes NB UVB more comfortable in particular for patients with AD, where heat can be an itch trigger. The design of our study, which used half-sided irradiation, allows within-patient comparisons. The possibility of systemic effects from phototherapy in half-sided irradiation studies is still a matter of discussion in the literature. However, a study by Jekler and Larko26 demonstrated that UVB clears AD through local effects. Statistically we could not demonstrate any difference between NB UVB and MD UVA1 phototherapy. Based on the current results, power calculations indicate that at least 800 patients are needed to prove a difference of 20% in therapeutic effect between NB UVB and MD UVA1. We now demonstrate that the lack of difference in the effects of NB UVB and MD UVA1 is accompanied by a lack of difference in reduction of the local cellular infiltrate. We demonstrate that both types of phototherapy result in a significant decrease of not only epidermal T cells, neutrophils, and Langerhans cells, but also in a significant decrease in dermal T cells, eosinophils, and neutrophils. Interestingly, we were able to confirm the presence of FoxP31CD31 T cells in lesional AD skin before phototherapy, which was shown recently by Caproni et al.27 However, we could not show any change in the percentage of FoxP31CD31 T cells before and after phototherapy. Recently, de Boer et al28 demonstrated that there are no differences in the percentage of FoxP31CD31 T cells in chronic dermatoses compared with healthy skin. Our results indicate, in addition to the results of de Boer et al,28 no difference in the percentage of FoxP31CD31 T cells in chronic AD compared with successfully treated skin after phototherapy. Because UVB is assumed to be less able to penetrate human skin than UVA, it may be hypothesized that MD UVA1 would be superior in reducing the dermal cellular infiltrate compared with NB UVB. Our results, however, show that NB UVB has at least the same capacity to decrease the number of inflammatory cells in the dermis as MD UVA1. Summarizing, we found no significant differences in the therapeutic effects of NB UVB and MD UVA1. This was supported by a lack of difference in the
cellular infiltrates. NB UVB has some practical advantages over MD UVA1; less heat load and shorter duration of phototherapy. For this reason, we suggest NB UVB to be the phototherapy treatment of choice for patients with AD.
REFERENCES 1. Larko O. Phototherapy of eczema. Photodermatol Photoimmunol Photomed 1996;12:91-4. 2. Krutmann J. Phototherapy for atopic dermatitis. Dermatol Ther 1996;1:24-31. 3. George SA, Bilsland DJ, Johnson BE, Ferguson J. Narrow-band (TL-01) UVB air-conditioned phototherapy for chronic severe adult atopic dermatitis. Br J Dermatol 1993;128:49-56. 4. Collins P, Ferguson J. Narrowband (TL-01) UVB air-conditioned phototherapy for atopic eczema in children. Br J Dermatol 1995;133:653-5. 5. Hudson-Peacock MJ, Diffey BL, Farr PM. Narrow-band UVB phototherapy for severe atopic dermatitis. Br J Dermatol 1996; 135:332. 6. Grundmann-Kollmann M, Behrens S, Podda M, Peter RU, Kaufmann R, Kerscher M. Phototherapy for atopic eczema with narrow-band UVB. J Am Acad Dermatol 1999;40:995-7. 7. Der-Petrossian M, Seeber A, Honigsmann H, Tanew A. Halfside comparison study on the efficacy of 8-methoxypsoralen bath-PUVA versus narrow-band ultraviolet B phototherapy in patients with severe chronic atopic dermatitis. Br J Dermatol 2000;142:39-43. 8. Berneburg M, Rocken M, Benedix F. Phototherapy with narrowband vs broadband UVB. Acta Derm Venereol 2005; 85:98-108. 9. Jekler J, Larko O. Combined UVA-UVB versus UVB phototherapy for atopic dermatitis: a paired-comparison study. J Am Acad Dermatol 1990;22:49-53. 10. Hjerppe M, Hasan T, Saksala I, Reunala T. Narrow-band UVB treatment in atopic dermatitis. Acta Derm Venereol 2001;81: 439-40. 11. Krutmann J, Czech W, Diepgen T, Niedner R, Kapp A, Schopf E. High-dose UVA1 therapy in the treatment of patients with atopic dermatitis. J Am Acad Dermatol 1992;26:225-30. 12. Krutmann J, Diepgen TL, Luger TA, Grabbe S, Meffert H, Sonnichsen N, et al. High-dose UVA1 therapy for atopic dermatitis: results of a multicenter trial. J Am Acad Dermatol 1998;38:589-93. 13. Kowalzick L, Kleinheinz A, Weichenthal M, Neuber K, Kohler I, Grosch J, et al. Low dose versus medium dose UV-A1 treatment in severe atopic eczema. Acta Derm Venereol 1995;75:43-5. 14. Meffert H, Sonnichsen N, Herzog M, Hutschenreuther A. UVA1-Kaltlichttherapie des akut exazerbierten, schweren atopischen Ekzems. Dermatologische Monatsschrift 1992;178:291-6. 15. Dittmar HC, Pflieger D, Schopf E, Simon JC. UVA1 phototherapy: pilot study of dose finding in acute exacerbated atopic dermatitis [in German]. Hautarzt 2001;52:423-7. 16. von Kobyletzki G, Pieck C, Hoffmann K, Freitag M, Altmeyer P. Medium-dose UVA1 cold-light phototherapy in the treatment of severe atopic dermatitis. J Am Acad Dermatol 1999;41:931-7. 17. Abeck D, Schmidt T, Fesq H, Strom K, Mempel M, Brockow K, et al. Long-term efficacy of medium-dose UVA1 phototherapy in atopic dermatitis. J Am Acad Dermatol 2000;42:254-7. 18. Tzaneva S, Seeber A, Schwaiger M, Honigsmann H, Tanew A. High-dose versus medium-dose UVA1 phototherapy for patients with severe generalized atopic dermatitis. J Am Acad Dermatol 2001;45:503-7.
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19. Krutmann J, Honigsmann H, Elmets CA, Bergstresser PR. Mechanisms of photo(chemo)therapy. In: Krutmann J, editor. Dermatological phototherapy and photodiagnostic methods. Heidelberg, Germany: Springer-Verlag; 2001. pp. 54-109. 20. Ziegler SF. FOXP3: of mice and men. Annu Rev Immunol 2006; 24:209-26. 21. Hanifin JM, Rajka G. diagnostic features of atopic dermatitis. Acta Derm Venereol Suppl (Stockh) 1980;92:44-7. 22. Sowden JM, Berth-Jones J, Ross JS, Motley RJ, Marks R, Finlay AY, et al. Double-blind, controlled, crossover study of cyclosporin in adults with severe refractory atopic dermatitis. Lancet 1991;338:137-40. 23. van Weelden H, De La Faille HB, Young E, van der Leun JC. A new development in UVB phototherapy of psoriasis. Br J Dermatol 1988;119:11-9. 24. Thepen T, Langeveld-Wildschut EG, Bihari IC, Van Wichen DF, Van Reijsen FC, Mudde GC, et al. Biphasic response against
25.
26. 27.
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aeroallergen in atopic dermatitis showing a switch from an initial TH2 response to a TH1 response in situ: an immunocytochemical study. J Allergy Clin Immunol 1996;97:828-37. Legat FJ, Hofer A, Brabek E, Quehenberger F, Kerl H, Wolf P. Narrowband UV-B vs medium-dose UV-A1 phototherapy in chronic atopic dermatitis. Arch Dermatol 2003;139:223-4. Jekler J, Larko O. UVB phototherapy of atopic dermatitis. Br J Dermatol 1988;119:697-705. Caproni M, Torchia D, Antiga E, Volpi W, del Bianco E, Fabbri P. The effects of tacrolimus ointment on regulatory T lymphocytes in atopic dermatitis. J Clin Immunol 2006;26: 370-5. de Boer OJ, van der Loos CM, Teeling P, van der Wal AC, Teunissen MB. Immunohistochemical analysis of regulatory T cell markers FOXP3 and GITR on CD41CD251 T cells in normal skin and inflammatory dermatoses. J Histochem Cytochem 2007;55:891-8.
A
topic dermatitis (AD) is an inflammatory skin disease with a chronic relapsing course. Phototherapy may be effective in AD. Ultraviolet (UV) lamps that have been studied include broadband UVB (280-315 nm), narrowband (NB) UVB (311 nm), UVA (315-400 nm), UVA1
From the Department of Dermatology and Allergology, University Medical Center, Utrecht,a and Department of Dermatology, Meander Medical Center, Amersfoort.b *These authors contributed equally to this study. Funding sources: None. Conflicts of interest: None declared. Accepted for publication August 29, 2008. Reprint requests: J. Marja Oldhoff, MD, PhD, University Medical Center Utrecht, Heidelberglaan 100, Department of Dermatology, G02.124, 3508 GA Utrecht, the Netherlands. E-mail: [email protected]. 0190-9622/$36.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2008.08.048
Abbreviations used: AD: FoxP3: LSS: MD: NB: NHuS: PBS: Tregs: UV: UVAB: VAS:
atopic dermatitis forkhead box protein P3 Leicester sign score medium dose narrowband normal human serum phosphate-buffered saline regulatory T cells ultraviolet combinations of ultraviolet A and ultraviolet B visual analog scale
(340-400 nm), photochemotherapy (psoralen plus UVA), bath psoralen plus UVA, combinations of UVA and UVB (UVAB), and extracorporal photochemotherapy.1,2 NB UVB phototherapy for AD was first studied in 1993 by George et al.3 Since then, several opendesign studies have been published that show its 77
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Fig 1. Trial flow chart. During the treatment period phototherapy was administered 3 times/wk; patients were randomly assigned to receive narrowband ultraviolet (UV) B to one side of the body and medium-dose UVA1 to the other side. LSS, Leicester sign score; VAS, visual analog scale.
efficacy in AD.4-8 Clinical assessment of this mode of phototherapy in psoriasis suggested a number of advantages over conventional broadband UVB. These include a reduced incidence of burning episodes, increased efficacy, and longer remission periods. A study performed by Jekler and Larko9 shows that UVAB is superior to conventional broadband UVB or UVA in the management of AD. Another study comparing UVAB with NB UVB showed a preference for NB UVB.10 The therapeutic effectiveness of UVA1 radiation in AD was first studied by Krutmann et al11 in an open study in which 15 patients with acute, severe AD were exposed to high-dose UVA1 (130 J/cm2) and compared with 10 patients receiving UVAB phototherapy. In comparison with UVAB therapy, significant improvement in clinical score was found in favor of high-dose UVA1.12 During the course of the following years, these observations have been confirmed by several reports, which mainly represent uncontrolled, open, and sometimes even noncomparative studies.12-14 Because of concerns about possible long-term side effects of high-dose UVA1 therapy (eg, photodamage and carcinogenesis), the effect of lower doses of UVA1 was studied.15 The results of this study demonstrated that both high-dose (maximum 130 J/cm2) and medium-dose (MD) (maximum 65 J/cm2) UVA1 are effective, whereas low-dose (maximum 20 J/cm2) UVA1 is not.15 The clinical effect of MD UVA1 phototherapy in AD has been studied by several authors.16,17 In a bilateral comparison study in 10 patients with AD, Tzaneva et al18 showed that MD UVA1 is as effective as high-dose UVA1. In a comparative study by von Kobyletzki et al,16 MD UVA1 treatment showed a significantly greater improvement in clinical scores for AD than did UVAB. Phototherapy is thought to improve chronic skin diseases such as AD and psoriasis by immunosuppressive action.19 The main mechanisms by which phototherapy induces immune suppression include the induction of apoptosis (cell death) in infiltrating T cells, induction of immunomodulatory cytokines, and reduction in the number of Langerhans cells.
The effect of phototherapy on regulatory T cells (Tregs) in human beings has not been studied so far. Human Tregs express the forkhead box protein P3 (FoxP3) and act as suppressor cells.20 NB UVB is the first choice of phototherapy to treat patients with AD in the Netherlands and MD UVA1 is a rather new phototherapeutic modality. Therefore, we compared the clinical effect of MD UVA1 and NB UVB phototherapy in patients with AD. Furthermore, we compared the immunohistochemical effects of both treatment modalities, including the number of Tregs, by measuring FoxP3 expression.
METHODS Study design The study was done in a randomized, investigator-blinded, and half-sided comparison design. Fig 1 shows the trial flow chart. Half-sided irradiation with threshold erythematic doses of NB UVB and MD UVA1 was performed 3 times weekly during a period of 8 weeks with a follow-up period of 4 weeks. Patients Thirteen patients with AD were enrolled, of whom 8 were female, median age 25 years (range 20-56). All patients fulfilled the criteria of Hanifin and Rajka21 and all had symmetric distribution of their eczema. Exclusion criteria included local treatment with corticosteroids or other medical topical agents within the last 2 weeks or systemic treatment with antibiotics, corticosteroids, or oral immunosuppressive drugs within the last 4 weeks. Clinical scoring Severity of the eczema was evaluated by the Leicester sign score (LSS) (range 0-108) by a blinded investigator. Severity is scored by 6 clinical features (erythema, purulence, excoriation or crusting, dryness or scaling, cracking or fissuring, and lichenification) graded at 6 defined body sites on a scale of 0 (none) to 3 (severe).22 Patients were asked to complete a visual analog scale (VAS) for pruritus, where the level of their itch is reflected on a scale of 0 to 10
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(0 = no itch and 10 = most intense itch imaginable). Both LSS and VAS-pruritus scoring were done before, during, and after the treatment period (Fig 1). Each scoring was done just before the next phototherapy session, so that erythema caused by phototherapy could not influence scoring. Before the treatment period, at the moment of inclusion, the median LSS for MD UVA1 was 20 (range 8-31). The median LSS for NB UVB was 19 (range 9-29). The baseline characteristics are shown in Table I. All participants in this study gave their informed consent. This study was approved by the local medical ethical committee, which follows the Declaration of Helsinki protocol.
Table I. Baseline characteristics of patients with atopic dermatitis and a symmetric distribution of their disease
Phototherapy and dosimetry The NB UVB phototherapy unit consisted of a light cabin (Waldmann, Schwenningen, Germany) with 20 311-nm lamps (TL-01, Philips, Eindhoven, The Netherlands). The minimal erythemal dose for NB UVB for each patient was determined before treatment. UVB treatment was started with an initial dose of 70% of the minimal erythemal dose. Subsequent dose increments were given on the basis of erythemic reactions of the skin. The intention was for each exposure to induce slight erythema. If the previous exposure failed to induce any reaction the dose was increased by 20%. If the resulting erythema was slight the dose was increased by 10%. This dose regimen is slightly less intensive than normally used in the treatment of psoriasis.23 The MD UVA1 phototherapy unit consisted of a Waldmann light cabin with 40 lamps (TL-10R, Philips) emitting wavelengths of 350 to 400 nm only, with a maximum of 6370 nm. The first dose was 30 J/cm2. In two steps the dose was increased to 45 J/cm2. In 3 of the 13 patients the dose had to be decreased because the reaction (erythema and/or papules) was too strong. The average dose of UVA1 was more than 40 J/cm2. Throughout the experiments the irradiation of the skin was monitored with a UVA/UVB detection device (Waldmann). Spectral distribution measurements were taken with a calibrated standard UVvisible spectoradiometer (model 752, Optronic Laboratories Inc, Orlando, FL). Phototherapy took place 3 times a week for 8 weeks, with a follow-up period of 4 weeks. Patients were randomly assigned to receive NB UVB to one body side and MD UVA1 to the other body side. The nonexposed body sides were covered with a halfsided overall. During the treatment period no topical treatment apart from emollients was allowed. During the follow-up period topical corticosteroids were allowed if needed. The face was excluded from halfsided comparison and analysis. It was only treated
with MD UVA1 and, if necessary, mild topical corticosteroids (European class I or II).
Patients with AD, n = 13
Age, y Male:female Half-sided comparison LSS VAS-pruritus
25 (20-56) 5:8 MD UVA1 NB UVB 20 (8-31) 19 (9-29) 7.5 (3.5-10) 7.5 (3.5-10)
Baseline characteristics were same for both body sides before half-sided phototherapy. Data are expressed as median values (minimum-maximum values). AD, Atopic dermatitis; LSS, Leicester sign score; MD, medium dose; NB, narrowband; UV, ultraviolet; VAS, visual analog scale.
Biopsy specimens Skin biopsy specimens of 9 patients taken from lesional skin on both body sides were analyzed before phototherapy. Two days after the last treatment, skin biopsy specimens were taken from healed lesional AD skin at approximately the same locations as the prephototherapy biopsy specimens. Skin biopsy specimens (4 mm) were taken under local anesthesia (lidocaine), snap-frozen in liquid nitrogen, embedded in Tissuetek (Sakura, Torrance, CA), and stored at e708C until further handling. Sections (5 m) were cut from the Tissuetek (Sakura)-embedded skin tissue and mounted on 3aminopropyl tri-ethoxy silaneecoated (Sigma, St Louis, MO) glass slides. Immunohistochemistry Antibodies. Mouse antibodies recognizing the following antigens were used as markers for immunohistochemical staining of the frozen sections: CD3 (Leu-4, clone SK7, Becton Dickinson, San Jose, CA); eosinophilic cationic protein (clone EG2, Phadia, Uppsala, Sweden); mast cell tryptase (clone AA1, DAKO, Glostrup, Denmark); neutrophil elastase (clone NP57, DAKO); FoxP3 (rat antihuman, clone PCH101, eBioscience, San Diego, CA); and CD1a (fluorescein isothiocyanate conjugated, clone NA 1/34, DAKO). Immunostaining. Staining for CD3, mast cell tryptase, eosinophil cationic protein, and neutrophil elastase was combined with a biotinylated horse antimouse IgG (Vector Laboratories Inc, Burlingame, CA) as described by Thepen et al.24 The fluorescein isothiocyanateeconjugated CD1a antibody sections were fixed with dry acetone, air dried, and preincubated for 20 minutes in 10% normal human serum
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by a blinded observer. Before evaluation, sections were compared with the isotype-controlestained sections or were compared with nonstained sections. Positive cells in the dermis were counted in 3 different sections of 1 to 1.5 mm2/section and calculated as cells/mm2. In fields containing sweat ducts and hair shafts, only the intervening dermal regions were counted. For EG2, CD3, FoxP3, elastase, and CD1a, dermal and epidermal compartments were examined separately. The number of epidermal CD1a1 cells was graded as: no, or hardly any positive cells present (0), presence of scattered positive cells (1), abundant presence of positive cells (2), or closed maze of positive cells (3). A second independent observer checked one of every 5 analyses. The mean interobserver coefficient of variation was within 10%.
Fig 2. Clinical results. Mean reduction in Leicester sign score (LSS ) (A) and visual analog scale (VAS ) pruritus (B). No significant difference in favor of narrowband (NB) ultraviolet (UV) B. MD, Medium dose; X-axes, time in weeks; Y-axes, mean score. Vertical lines display SD.
(NHuS) in phosphate-buffered saline (PBS). Primary antibodies were diluted in 1% NHuS and incubated with the sections for 60 minutes. Slides were washed 3 times for 5 minutes with PBS plus 0.05% Tween 20 (P-1379, Sigma) and were subsequently incubated with alkaline phosphatase conjugated sheep antifluorescein (Fab fragments, Boehringer Mannheim, Mannheim, Germany) in PBS for 30 minutes. After incubation, slides were washed with Tris-HCl (0.1 mol/L, pH 8.5). Alkaline phosphatase reactivity was demonstrated as previously described.24 For FoxP3 single staining, slides were preincubated with 10% NHuS and 10% normal rabbit serum in PBS. The primary antibody was diluted in PBS with 1% NHuS and 1% bovine serum albumin and incubated with the sections for 60 minutes. The staining was then combined with biotinylated rabbit antirat immunoglobulin (DAKO) for 30 minutes at room temperature, followed by an avidin-biotin-peroxidase complex (Vectastain Elitekit, Vector Laboratories Inc) for another 30 minutes at room temperature. Peroxidase activity was visualized using 3-amino-9-ethylcarbazole (Sigma). Quantification of staining. Skin sections were examined by light microscopy at 3400 magnification
Statistical analysis Statistical analysis was performed using software (SPSS for Windows, Version 10.0.5, SPSS Inc, Chicago, IL). The Wilcoxon signed rank test was used for all paired comparisons. A P value of .05 or less was considered to be significant. Adjustments for multiple comparisons were not done.
RESULTS Clinical results Thirteen patients fulfilled the entry criteria and were randomly assigned to phototherapy. All patients completed the trial. Only data obtained from the phototherapy period (0-8 weeks) were used for statistical analysis because during this period, all patients exclusively received phototherapy with no other treatment. During the follow-up period (8-12 weeks) patients were allowed to use local corticosteroids, which most patients did. Patients received median cumulative doses of 10.5 J/cm2 of NB UVB (range 9.9-11.5, average increment 10%/exposure) to one body side and 930.6 J/cm2 of MD UVA1 (range 717.1-1067.4) to the other body side. Fig 2 shows the clinical results assessed for both body sides separately. Both the NB UVB and MD UVA1 treatments resulted in significantly decreased LSS scores: median reduction for NB UVB from 18 to 10 points (P \.01); and for MD UVA1 from 19 to 12 points (P \.01). The patients’ self-assessment, measured by the VAS-pruritus, was also significantly decreased after both treatment modalities: median reduction for NB UVB from 7 to 1.8 points (P \.01); and for MD UVA1 from 7 to 4.1 points (P \ .01). When the improvement in the NB UVBetreated side was compared with that in the MD UVA1etreated side, no significant difference was observed (measured by LSS and VAS-pruritus).
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Fig 3. Immunohistochemical results (n = 9). Cellular infiltrate before (t = 0) and after (t = 8) phototherapy in patients with atopic dermatitis. Treatment of one body side was with narrowband (NB) ultraviolet (UV ) B and other body side with medium-dose (MD) UVA1. AA1, Mast cell; elastase, neutrophil; CD1a, dendritic cell; CD3, T cell; EG2, eosinophil; X-axes, time: 0 = lesional skin, 8 = after 8 weeks of phototherapy; Y-axes, cells/mm2. *Significantly different decrease in cells compared with t = 0. No significant differences were observed between NB UVB and MD UVA1 sides.
Immunohistochemical results Epidermis. The numbers of T cells, dendritic cells, and neutrophils in the epidermis were significantly decreased after both treatment modalities. No
change was observed in the number of epidermal eosinophils (data not shown). Between-treatment comparison did not show significant differences in the composition of the epidermal cell infiltrate.
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Fig 4. Immunohistochemical results of forkhead box protein P3 (n = 9). Cellular infiltrate before (t = 0) and after (t = 8) phototherapy in patients with atopic dermatitis. Treatment of one body side was with narrowband (NB) ultraviolet (UV) B and other body side with medium-dose (MD) UVA1. CD3, T cell; FoxP3, positive T cell; X-axes, time: 0 = lesional skin, 8 = after 8 weeks of phototherapy; Y-axes, cells/mm2. *Significant decrease in cells compared with t = 0. No significant differences were observed between NB UVB and MD UVA1 sides.
Dermis. The dermal cellular infiltrate was scored for CD3 (T cell), FoxP3 (Treg), EG2 (eosinophil), CD1a (dendritic cell), AA1 (mast cell), and elastase (neutrophil) counts. The distribution of these cell types was the same on both body sides before phototherapy. Figs 3 and 4 show the immunohistochemical results. A significant decrease in the number of dermal T cells (P \ .01), eosinophils (P \ .01), and neutrophils (P \ .05) was observed after MD UVA1 and after NB UVB. The number of dermal dendritic cells was significantly decreased after NB UVB (P \ .05), but not after MD UVA1. Between-treatment comparison did not show a significant difference in dermal dendritic cells. The number of mast cells was not significantly changed after either treatment. Although the number of CD31 T cells significantly
decreased after both NB UVB and MD UVA1, the percentage of FoxP31CD31 T cells did not change. Between-treatment comparison did not show significant differences in the composition of the dermal cell infiltrate.
DISCUSSION The clinical effect of NB UVB and MD UVA1 is similar in the studied population of patients with AD. This is supported by a comparable decrease in inflammatory cell infiltrate after both treatments. The follow-up period was confounded by the use of dermatocorticosteroids. Therefore, the differential effect of both treatments 4 weeks after phototherapy is not useful for evaluation. Our data are in accordance with the results found in a study by Legat et al.25 In their study, with a design similar to ours,
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NB UVB was compared with MD UVA1 in patients with AD using half-sided irradiations. Although they could not demonstrate a significant difference between NB UVB and MD UVA1 treatment, NB UVB was preferred over MD UVA1 phototherapy for AD. The total amount of irradiance needed for effective phototherapy is less for NB UVB than for MD UVA1. Because of this, the exposure time is short and less heat is produced during NB UVB treatment compared with MD UVA1. This aspect makes NB UVB more comfortable in particular for patients with AD, where heat can be an itch trigger. The design of our study, which used half-sided irradiation, allows within-patient comparisons. The possibility of systemic effects from phototherapy in half-sided irradiation studies is still a matter of discussion in the literature. However, a study by Jekler and Larko26 demonstrated that UVB clears AD through local effects. Statistically we could not demonstrate any difference between NB UVB and MD UVA1 phototherapy. Based on the current results, power calculations indicate that at least 800 patients are needed to prove a difference of 20% in therapeutic effect between NB UVB and MD UVA1. We now demonstrate that the lack of difference in the effects of NB UVB and MD UVA1 is accompanied by a lack of difference in reduction of the local cellular infiltrate. We demonstrate that both types of phototherapy result in a significant decrease of not only epidermal T cells, neutrophils, and Langerhans cells, but also in a significant decrease in dermal T cells, eosinophils, and neutrophils. Interestingly, we were able to confirm the presence of FoxP31CD31 T cells in lesional AD skin before phototherapy, which was shown recently by Caproni et al.27 However, we could not show any change in the percentage of FoxP31CD31 T cells before and after phototherapy. Recently, de Boer et al28 demonstrated that there are no differences in the percentage of FoxP31CD31 T cells in chronic dermatoses compared with healthy skin. Our results indicate, in addition to the results of de Boer et al,28 no difference in the percentage of FoxP31CD31 T cells in chronic AD compared with successfully treated skin after phototherapy. Because UVB is assumed to be less able to penetrate human skin than UVA, it may be hypothesized that MD UVA1 would be superior in reducing the dermal cellular infiltrate compared with NB UVB. Our results, however, show that NB UVB has at least the same capacity to decrease the number of inflammatory cells in the dermis as MD UVA1. Summarizing, we found no significant differences in the therapeutic effects of NB UVB and MD UVA1. This was supported by a lack of difference in the
cellular infiltrates. NB UVB has some practical advantages over MD UVA1; less heat load and shorter duration of phototherapy. For this reason, we suggest NB UVB to be the phototherapy treatment of choice for patients with AD.
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