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2 Arch Dermatol Res (1996) 288: Springer-Verlag 1996 C. J. M. van der Vleuten E. M. G. J. de Jong P. C. M. van de Kerkhof «Epidermal differentiation characteristics of the psoriatic plaque during treatment with calcipotriol Received; I August 1995 Abstract Treatment of psoriasis with vitamin D3 analogues is well established in present dermatological practice. One of the clinical signs of the psoriatic plaque that reduces early and markedly during treatment with the vitamin D3 analogue calcipotriol is scaling. Since scaling is the clinical manifestation of disordered epidermal differentiation, early changes in im- lmmohistochemical markers for differentiation (transglutaminase, involucrin and filaggrin) were studied in patients who had been treated with calcipotriol for 4 weeks. Markers for proliferation (Ki-67 antigen) and inflammation (polymorphonuclear leucocytes and T lymphocytes) were also studied and correlated with the differentiation characteristics. Clinically, a major improvement was seen in all patients. A significant decrease in the percentage of transglutaminase-positive cell layers was observed during the first week of treatment. In contrast, an increase in transglutaminase activity in epidermal cell cultures following incubation with calcipotriol has been reported. Involucrin expression was only slightly modulated in vivo. However, a major restoration of the filaggrin-positive cell layer and significant reduction in the recruitment of cycling epidermal cells characterized the epidermal response to calcipotriol treatment. Markers for inflammation (Til-positive cells and elastase-positive cells) were also reduced substantially during the first week of treatment with calcipotriol. From this study it may be concluded that inhibition of epidermal growth and recovery of the filaggrin-positive cell layer are among the in vivo effects of calcipotriol. Key words Immunohistochemistry Psoriasis Calcipotriol Epidermal differentiation C. J. M, van der Vleuten (El) *E. M. G. J. de Jong P. C. M. van cle Kerkhof Department of Dermatology, University Hospital Nijmegen, P.O. Box 911, 65 HB Nijmegen, The Netherlands Tel ; Fax +3 I Introduction Vitamin D3 and its analogues have been shown to exert an important antipsoriatic effect [1]. In vitro and in vivo studies have shown a marked antiproliferative and keratinization-enhancing activity [2-6]. Inflammation is also affected by active vitamin D3 derivatives [6, 7]. Calcipotriol is one of the first line treatments for psoriasis. While treating patients with this therapy, it is our impression that scaling is one of the clinical signs that diminishes early and obviously. So one might speculate that normalization of epidermal differentiation is one of the important antipsoriatic effects of active vitamin D3. Indeed, in vitro studies have shown that proliferation of keratinocytes is inhibited and that the formation of the cornified envelope, transcription of involucrin and transglutaminase activity are enhanced by calcipotriol [2, 3, 7 12J. In vivo, inhibition of Ki-67 expression and a reduction in polymorphonuclear leucocytes (PMN) are early effects, whereas a reduction in suprabasal expression of keratin 16 and T lymphocyte accumulation are late effects [6. The aim of the present study was to elucidate the behaviour of the suprabasal compartment of the epidermis during treatment of psoriatic plaques with calcipotriol The following questions were addressed; (1) Does calcipotriol ointment have a substantial effect on markers of epidermal differentiation during the treatment of chronic plaque psoriasis? (2) What is the relationship between the effect on differentiation markers and other immunohistochemical effects of calcipotriol? A 4-week clinical study with calcipotriol in patients with psoriasis was carried out to answer these questions. Cryostat sections of skin biopsies were studied immunohistochemically using a panel of monoclonal antibodies.

3 * Materials and methods Patients Six patients with chronic plaque psoriasis, five males and one female, participated in this study. Their ages ranged from 33 to 61 years and their average duration of psoriasis was 17 ±,8 (mean ± SEM) years. They had used no systemic treatment for at least 2 months and no topical treatment for at least 2 weeks. In fact, the patients had hardly used any antipsoriatic treatment for periods of months or years before starting the present study. Patients were instructed to apply calcipotriol ointment (5 jig/g; Daivonex, LEO Pharma, Denmark) twice daily to lesionai skin with a maximum of 1 g ointment weekly. No additional topical therapy was allowed. This study was approved by the local ethical committee. AI! patients gave their written informed consent prior' to inclusion in the 367 munoglobulin conjugated with peroxidase (1:5, RAM-PO; Dakopatts) diluted in PB containing 5% human ÁB serum for 3 min. After washing with PB and demineralized water, staining was visualized using a 3-amino-9-ethylcarbazo.le (AEC) solution. Staining with T il was done with an indirect peroxidase-antiperoxidase technique (PAP). The slides were put in PB for 1 min and preincubated with 5% normal rabbit serum in PB for 2 min. After washing with PB the slides were incubated with the primary monoclonal antibody in a Miele Microwave at 8 W for 9 min. The slides were then washed again in PB and incubated with rabbit antimouse immunoglobulin (1:25, RAM-Ig; Dakopatts) in the microwave at 8 W for 9 min. After washing in PB the slides were incubated with PAP complexes (I: 1, peroxidase monoclonal mouse antiperoxidase complexes; Dakopatts) in the microwave at 8 W for 8 min. After washing, this cycle was repeated. The complexes were visualized using the AEC solution. All slides were counters tai ned with Mayer's haeniatoxylin (Sigma, SL Louis, Mo.f USA) and mounted in glyeerol-gelatine. Clinical assessments efore therapy and 1, 2 and 4 weeks after therapy the severity of one target lesion was scored for each of the following clinical signs: erythema, induration and scaling. Each parameter was scored using a 5-point scale: = complete lack of cutaneous involvement, I = slight involvement, 2 = moderate involvement, 3 = severe involvement, 4 = severest possible involvement. Biopsy procedure Punch biopsies of 3 mm were taken from the previously determined target lesion before therapy and 1, 2 and 4 weeks after therapy from all patients after local anaesthesia with 1% Xylocaine and adrenaline. The biopsies were embedded in Tissue Tek OCT compound (Miles, Scientific, Naperville, Ul., USA), snap frozen in liquid nitrogen and stored at -8 C until use. Sections of 7 um were cut, air dried and fixed for 1 min in acetone/ether (6/4%) (MlB-1 staining) or in acetone (other staining«) and again stored at -8 C. Misto logica l examìnalio n The histological examination was performed blinded. Epidermal proliferation was measured by counting the number of MlB-1 -positive nuclei per millimetre length of section. Involucrin and transglutaminase expression were assessed by calculai ion of the ratio of positive cell layers to total cell layers of the viable epidermis. This was done at two sites: above the dermal papilla and between two dermal papillae, Filaggrin expression was assessed by measuring the percentage of the length of the stratum corneum and stratum granulosum which was stained. Inflammation (PMN and T lymphocytes) was assessed separately for the dermis and epidermis. Dermal inflammation was semiquantitatively evaluated by expressing the number of positively stained cells as a percentage of the total number of infiltrate cells [14];, no positive cells; I, sporadic positive cells; 2, 1-25%; 3, 26-5%; 4, 51-75%; 5, 76-99%; 6, 1%. Epidermal inflammation was assessed using a five-point scale:» no staining; 1, sporadic staining; 2, minimal staining; 3, moderate staining; 4, pronounced staining. M onoc Ion a I an t i bod ies A panel of monoclonal antibodies was used. To assess epidermal differentiation monoclonal antibodies against involucrin (MON- 15 [13J, I :25), antihuman keratinocyte transglutaminase (1:25, Mouse Monoclonal Antibody, IgG^; BT621; Biomedical Technologies, Stoughton, Mass., USA) and against filaggrin and profilaggrin (I :5, antifilaggrin, BT576; Biomedical Technologies) were used. To estimate the number of cycling epidermal cells in the basal layer an antibody directed against the Ki-67 antigen was used (MIB-1, I :5; Immunoleeh, Marseilles, France). The inflammatory infiltrate was analysed by assessing Ihe T lymphocytes and PMN using, respectively, the monoclonal antibodies DAKO-T11 (1:1; Dakopatts, Copenhagen, Denmark) and DAKO-elastase (1 : 1; Dakopatts). 8 a (O 1 c tm m o *' Staining procedure For all monoclonal antibodies, except T il, an indirect immunoperoxidase technique was used. For 1 min the.slides were fixed in acetone/ether (6/4%) in the ease of MIB-l or in acetone for the other stainings. The slides were air dried and put in a phosphate buffer (PB; 72 mm Na2HP4 and 28 maf NaH2PO(I). Only the slides stained with antielastase were prei neu bated with mcthanol/.1% H2 2 (3%) for 2 min. All antibodies were diluted in PB, The slides were incubated with the different primary monoclonal antibodies for 3 min except for the antibody against transglutaminase (6 min). After washing with PB the slides were incubated with the secondary antibody, rabbit anlhnouse im-.5 ~ 1 weeks Fig. 1. Erythema ( ), induration (------) and scaling ( -) before and during t rca I meni with calcipotriol expressed as means ± SEM

4 368 % TGase + cell layers J 5 -i í!^!j M m SI M u ll; % involucrin + cell layers ämgfösiiimfjslm b o % filaggrin positive o 4 d o 1 F ig.2 a-c a Transglutaminase above ihe dermal papilla before and during treatment with calcipotriol; b transglutaminase be tween two dermal papillae before and during treatment with cal cipotriol; c Involucrin above the dermal papilla before and tim ing treatment with calcipotriol; d Involucrin between two der mal papillae before and during treatment with calcipotriol; c fiiaggrin in the stratum corneum ( ) and stratum granulos uni ( ) before and during treatment with calcipotriol, Values are means ± SEM. The shaded areas (a-d ) represent the mean ± 2 x SD of the value for normal skin [ 15, 33]

5 369 w J i t i n l i Vmi 35 l For statistical analysis the Mest lor paired values was used. A twotailed hypothesis was employed to interpret the data. Results 3 Q> O =1 c > > Clinical response <n o The course of the clinical scores of the target lesions is shown in Fig. 1. Transglutaminase expression (Fig. 2 a) above the dermal papilla showed a significant decrease during the first week of therapy (P =.3), but during subsequent weeks no significant changes were seen, in the interpapillary epidermis transglutaminase expression (Fig. 2 b) slightly diminished resulting in a significant difference alter 4 weeks of therapy (P =. 1). After 4 weeks o f therapy normal values were reached [15], Involucrin expression crease (P =.4) after 4 weeks of therapy, but involucrin... ;; -.i- ' a j Ï «; Q. K. 2 weeks Fig.4 M I ß - 1 staining before and during treatment with cal ci potriol expressed as means ± SEM (the shaded area represents the mean ± 2 x SD of the value for normal human skin [16]) expression in the interpapillary epidermis (F ig.2d) did not show significant changes. Filaggrin staining (Fig.2e) in the stratum corneum significantly increased after 2 =.4). In the stratum granulosum a significant increase was seen as early as after I week of treatment (P =.1). Recovery of the stratum corneum was only partial, but recovery of the stratum granulosum was nearly com plete. Immunohistochemical staining for filaggrin before and after therapy is shown in Fig. 3. The num ber of Ki-67-positive nuclei per millimetre of section (Fig. 4) significantly decreased during the first week of therapy (P no significant changes were seen. The number of Ki-67positive nuclei approached, but remained above, the nor mal range 116 ]. The staining of T lymphocytes in the dermis (Fig. 5 a) altered significantly after the first week of therapy (P =.3), but subsequently no significant changes, were ob served. T i l staining in the epidermis (Fig.5b) showed a to decrease (P =.8) during the first week, but during subsequent weeks no significant changes were seen. Antielastase staining (PMN) in =.1 ), but no further significant changes were observed. In the epidermis no changes in eliistase binding were ob served. Epidermal T lymphocyte accumulation and PMN accumulation remained above the range for normal skin ft íá "r.pßffi. -M. : i."*... / 4» * i i b VX<S}' \ * *» T j..% :!.< } S-=. or? ii 1* S -H % Fig.3 Anlifilaggrin staining in the same patient before (a) and ai ter (b) 4 weeks of therapy Discussion In the present study, a major clinical i observed during a 4-week treatment cale ipotriol treatment since the as with cal reduction in

6 37 V> E ai TD Oú, 3 -- O U V) «*m + d) Í â Vtn o o Z S a 2-1 * -I &*l!» liffliilélilliiiilsibimllâilislâ'iiiisiftfii-ïsslâiliiîliiitail i >/<; vn i í x ^ í r W ' T» i??. ÿ?.- «jijhii f à ÿ^-a-5 j ^ f-j! i.'ñ i tay? -* i í ; j -:i *: j L?? ì í í- >? / v-í-i I - i tv-ïï w -i» 2<f-ÂÎ ' f? s weeks Fig,5 a c a, b T1I positivity before and during treatment with calcipotriol. a Dermis (the shaded area represents the mean ± 2 x SD of the value for normal human skin [15]); b epidermis, c AntieJastase in the dermis (------) and epidermis ( ) before and during treatment with calcipotriol, Values are means ± SEM severity scores is reached after an 8-week treatment period [17]. Most studies agree that calcipotriol induces pronounced changes in epidermal behaviour, leaving the mononuclear infiltrate relatively unaffected [6, 18-2], The present results are in line with these studies and clinically, an early and relatively pronounced effect of calcipotriol on scaling, the clinical manifestation of epidermal differentiation, was found. Epidermal differentiation involves the formation of cornified envelopes by crosslinking (formation of the e- (y-glutamyl)lysine bonds) involucrin, ioricrin and keratolinin involving the calcium-dependent enzyme mem- brane-bound transglutaminase (TGase 1) [21-24]. In normal skin, staining with antibodies against TGase 1 and involucrin shows a band-like pattern in the upper part of the epidermis representing the upper stratum spinosum and the stratum granulosum [25]. In situ hybridization techniques have demonstrated involucrin and TGase 1 mrna at the transition to the stratum granulosum as a very early event in differentiation [26]. Activity of TGase 1 has been demonstrated in vivo in mice in the transition zone (one or at most two cell layers) from the stratum granulosum to the stratum corneum [25]. At this point the actual formation of the cornified envelopes takes place resulting in the stratum corneum in which antigenicity of involucrin is lost 4 [25]. In psoriatic skin, the keratinization process is disturbed. Large, clinically visible, histologically parakera- totic squames are formed in lesionai skin [26]. Staining with monoclonal antibodies against involucrin and TGase 1 demonstrates far more positive cell layers, relatively as well as absolutely [27]. The activity of TGase 1 and the number of cornified envelopes produced are also greatly enhanced [28]. Messenger RNA of involucrin and TGase 1 is localized early in the lower cell layers of the suprabasal epidermis [26]. This is in contrast to the situation found in nomi al skin [26]. Increased expression of TGase 1 protein and mrna in psoriasis may partly explain the characteristic hyperkeratosis. The formation of a more highly crosslinked stratum corneum may lead to a greater retention of the squames observed in lesionai skin [29]. The present in vivo observations on epidermal differentiation during treatment with calcipotriol indicate that modulation of the process of keratinization is a relatively early effect of this compound. However, these observations are in several respects at variance with in vitro observations, TGase 1 activity in cultured keratinocytes is enhanced by calcipotriol [2]. In the present in vivo study, however, TGase 1-positive cells decreased during treatment. In vivo, a similar decrease has been observed during treatment of psoriatic plaques with the vitamin D* analogues l,25-(oh)2-d3 and l,24-(oh)2~d3 Whereas vitamin D3 analogues in vitro enhance the transcription of involucrin [ I I, 12], only a minor modulation of the number of invoiucrin-positive cell layers was observed in the present in vivo study. Treatment with 1,25- (OH)2-D:î and l,24-(oh)2-d3 reduces the number of in- volucrin-positive cell layers [4, 5. Despite the decrease in anti-tgase 1 binding, TGase 1 as well as involucrin remained high during this investigation. The newly formed keratinocytes in psoriatic lesions keep on expressing TGase 1 and involucrin mrna too early in their maturation process. So far, there is no convincing evidence that calcipotriol modulates epidermal differentiation in vivo via DNA transcription of involucrin or TGase 1. A remarkable and consistent effect of calcipotriol (present study) and of l,25-(oh)2-d3 and l,24-(oh)2-d3 [4, 5] is the pronounced reformation of the filaggrin-positive cell layer. Filaggrin plays a major role in the aggregation of keratin filaments, which results in the keratin configuration as can be seen in the lower stratum corneum [3].

7 371 The present study demonstrates a potent reduction in the recruitment of cycling epidermal cells (Ki-67-positive nuclei). This result confirms the antiproliferative activity as observed in earlier studies [6, 18]. The expression of keratin 16 and keratin 17 in the suprabasal compartment is considered to be related to epidermal recovery. During treatment with calcipotriol these hyperproliferation-associated keratins reduce [6, 18, 31]. The antiproliferative action of calcipotriol has proved to be consistent in the vitro and the in vivo situation. Vitamin D3 analogues interfere with several elements of cutaneous inflammation [32]. Modification of T-cell subpopulations and cytokine pattern and a reduction in PMN accumulation have been observed previously [32]. The results of the present study are at variance with those of a previous study [6] with respect to T lymphocytes. In the present study the early reduction in the number of T lymphocytes together with the reduction in PMN accumulation might be the result of the long periods (months or even years) that patients had been untreated. In the previous study [6], which showed only a modest effect on T lymphocytes, the patients had already been treated with calcipotriol or topical steroids up to 2 weeks before treatment. From this study it may be concluded that the in vivo effects of calcipotriol on epidermal differentiation include inhibition of epidermal growth, recovery of the filaggrin- positive cell layer and a decrease in TGase 1- and invoiucrin-positive cells. Acknowledgements Dr. J. van Duijnhoven is acknowledged for providing the monoclonal antibody MON-15. The authors would also like to thank Mrs. E. de Bakker for her amiable technical assistance. References 1. Berth Jones J, Hutchinson PE (1992) Vitamin D analogues and psoriasis. Br J Dermatol Î 27: Kragballe K, Wild! an g IL (199) Calcipotriol (MC 93), a novel vitamin D3 analogue stimulâtes terminal differentiation and inhibits proliferation of cultured human keratinocytes. Arch Dermatol Res 282: Smith EL, Walworth NC, Holiek MF ( 1986) Effect of 1 alpha, 25-dihydroxyvitamin D3 on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions. J Invest Dermatol 86: Gerritsen MJ, Boezeman JB, Vlijmen Willems 1M van, Kerkhof PC van de (1994) The effect of tnealcitol ( 1,24(11 )2D3) on cutaneous inflammation, epidermal proliferation and keratinization in psoriasis: a placebo-controlled, double-blind sludy. Br J Dermatol 131: Gerritsen MJ, Rulo HF, Vlijmen Willems I van, Erp PE van, Kerkhof PC van de (1993) Topical treatment of psoriatic plaques with 1,25-dihydroxyvitamin D3: a cell biological study. Br J Dermatol 128 : Jong EM de, Kerkhof PC van de (1991) Simultaneous assessment of inflammation and epidermal proliferation in psoriatic plaques during long-term treatment with the vitamin D3 analogue MC93: modulations and interrelations. 13r J Dermatol 124: Bagot M, Chame D, Lescs MC, Pamphile RPt Revu/. J (1994) Immunosuppressive effects of 1,25-dihydroxy vitamin D3 and its analogue calcipotriol on epidermal cells. Br J Dermatol 13: H.Hosomi J, Hosoi J* Abe E, Suda T, Kuroki T (1983) Regulation of terminal differentiation of cultured mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3. Endocrinology 113: Regnier M, Darmon M (1991) 1,25-Dihydroxy vitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes. Differentiation 47 : Lee SC, Ikai K, Ando Y, Imamura S (1989) Effects of I alpha,25-dihydroxyvitamin D3 on the transglutaminase activity of transformed mouse epidermal cells in culture. 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8 Gerritsen MJ, Erp PE van, Kerkhof PC van de (1995) Transglutaminase positive cells in psoriatic epidermis during treatment with calcitriol ( la,25 dihydroxy vitamin D3) and tacalcitol (la,24 dihydroxy vitamin D3). Br J Dermatol 133 : Es man n J, Voorhees JJ, Fisher GJ (1989) Increased membraneassociated transglutaminase activity in psoriasis. Biochem Biophys Res Commun 164: Schroeder WT, Thacher SM, Stewart Galetka S, Ann are lia M, Chema D, Siciliano MJ, Davies PJ, Tang HY, Sowa BA, Duvic M (1992) Type I keratinocyte transglutaminase: expression in human skin and psoriasis, J Invest Dermatol 99: Dale BA, Holbrook KA, Steinert PM (1978) Assembly of stratum corneum basic protein and keratin filaments in macrofibrils. Nature 276: Jong EM de, Vlijmen IM van, Erp PE van, Ramaekers FC, Troyanovski SM, Kerkhof PC van de (1991) Keratin 17: a uscirli marker in anti-psoriatic therapies. Arch Dermatol Res 283 : Kerkhof PC van de (1995) Biological activity of vitamin D analogues in the skin, with special reference to antipsoriatic mechanisms. BrJ Dermatol 132: Gerritsen MJ, Eibers M, Jong EM de, Kerkhof PC van de (1996) Recruitment of cycling epidermal cells and expression of filaggrin, involucrin and tenascin in the margin of the active psoriatic plaque, in the uninvolved skin of psoriatic patients and in the normal healthy skin. J Dermatol (in press)