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Flexural versus plaque lesions in psoriasis: an immunohistochemical differentiation

European Journal of Dermatology. Volume 15, Number 1, 13-7, January-February 2005, Investigative report

Summary

Author(s) : WHPM Vissers, J Roelofzen, EMGJ De Jong, PEJ Van Erp, PCM Van de Kerkhof , Department of Dermatology, University Medical Centre St Radboud, René Descartesdreef 1, 6500 HB Nijmegen, The Netherlands Fax: (+31)24 35 41184..

Summary : Clinical presentation, therapeutic options, micro-environment and HLA-typing have been reported to be different in flexural psoriasis as compared to plaque psoriasis. We were interested in any difference concerning the pathogenesis of both conditions. By analysing T-cell subsets, NK-T cells and proliferation and differentiation markers, insight into the pathogenesis of both subtypes was obtained. Quantitative studies of T-cell subsets, cells expressing NK-receptors and markers of proliferation and differentiation in flexural and plaque psoriasis were investigated. Biopsies from 6 patients with both flexural and plaque lesions were obtained and processed for immunohistochemistry. Several T-cell subsets were stained: CD4+, CD8+, CD2+, CD25+, CD45RO+ and CD45RA+ T-cells. In addition cells expressing NK receptors were stained: CD94+ cells and CD161+ cells. T-cell subsets and cells expressing NK-receptors were analysed by immunohistochemical scoring. The proliferation marker Ki-67 and differentiation marker keratin-10 were revealed immunohistochemically by MIB-1 and RKSE60 respectively. Both markers were analysed using quantitative image analyses. The number of Ki-67 positive nuclei and the percentage of keratin-10 positive epidermal cells in flexural and plaque psoriasis were comparable. There is no difference between flexural and plaque psoriasis concerning other T-cell subsets. However a highly significant reduction is seen in flexural psoriasis with respect to CD161+ cells in the dermis. The similarity of chronic plaque psoriasis compared to flexural psoriasis with respect to T-cell subsets, epidermal proliferation and keratinization suggests that both conditions are pathogenetically identical. We propose the decreased quantity of lesional CD161+ cells in the dermis of flexural psoriatic lesions may result from chronic microbial challenge in flexural psoriasis.

Keywords : flexural psoriasis, T-lymphocyte subsets, NK-T cells, immunohistochemistry

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ARTICLE

Auteur(s) :, WHPM Vissers^*, J Roelofzen, EMGJ De Jong, PEJ Van Erp, PCM Van de Kerkhof

Department of Dermatology, University Medical Centre St Radboud, René Descartesdreef 1, 6500 HB Nijmegen, The Netherlands Fax: (+31)24 35 41184.

accepté le 30 Août 2004

Chronic plaque psoriasis is the most frequent manifestation of psoriasis. The well-known predilection sites are the extensor surfaces of the elbows and knees, the scalp and the regio sacralis. Flexural psoriasis is another manifestation of psoriasis. Its prevalence is about 2-6% of the patients with psoriasis and this condition is more common in adults than in children [1-3]. Areas involved are the axillae, groins, gluteal clefts and submammary folds. Scaling, a typical sign of plaque psoriasis, is mostly absent in flexural psoriasis. Lesions of inverse psoriasis tend to be more erythematous as compared to chronique plaque psoriasis. Patients with flexural psoriasis have a higher frequency of palmar involvement as compared to patients with chronic plaque psoriasis [4].Karvonen et al. postulated that flexural psoriasis is genetically distinct from plaque psoriasis. They found different HLA types in the group of patients with flexural psoriasis as compared to plaque psoriasis [5].In the pathogenesis of psoriasis, bacterial overgrowth and mycotic infections have been indicated as triggering factors [6]. It was shown by Kanda et al. that several fungi influence cytokine production by peripheral blood mononuclear cells in psoriatic patients. They also showed that Malassezia furfur was able to induce a Th1 skewed response in psoriatic patients [7]. Several studies showed the immunomodulatory effects of fungi [8-10]. Bacterial overgrowth is a well established feature of flexural psoriasis, however the association between mycotic infections and flexural psoriasis is less convincing [11-13]. Furthermore it is hypothesized by some investigators that patients with psoriasis are protected against microbial super-infection by overexpression of human β-defensins [14-16].In addition to the clinical, genetic and pathogenetic differences, the therapeutic responsiveness of flexural and chronic plaque psoriasis is also different. Topical corticosteroids, and especially the more potent ones, are less well tolerated in flexural psoriasis as compared to chronic plaque psoriasis. Local side effects such as athrophy, telangiectasia and striae formation are often more pronounced in intertriginous areas because these areas are more sensitive to corticosteroid penetration [17, 18]. If a corticosteroid is prescribed in flexural psoriasis, a mild corticosteroid should be selected only for a short period and preferably this treatment should be combined with another therapy, for example a vitamin D3 analogue [19]. Other topical therapies for psoriasis are vitamin D3 analogues. Several studies have demonstrated that the naturally occurring, hormonally active form of vitamin D3, calcitriol, is better tolerated in flexural psoriasis than the vitamin D3 derivative calcipotriol [20]. The recently introduced calcineurin inhibitors tacrolimus and pimecrolimus have been shown to be effective in the treatment of flexural psoriasis [21, 22]. Freeman et al. demonstrated that topical tacrolimus ointment is a safe and effective nonsteroidal treatment of psoriasis in the facial and intertriginous areas [22]. Treatment with imidazoles and antibacterial treatments are suggested to be effective in flexural psoriasis. However solid evidence that antimycotic and antibacterial treatments are effective in flexural psoriasis has not been provided so far.Because of differences between flexural psoriasis and chronic plaque psoriasis with respect to clinical presentation, microbial colonization and therapeutic responsiveness, we posed the question whether flexural psoriasis is pathogenetically different from chronic plaque psoriasis. In order to answer this question we carried out an inventory of T-cell subsets, cells expressing NK receptors and markers for epidermal growth and differentiation in flexural and chronic plaque psoriasis.

Material and methods

Patients

Six patients with a combination of plaque psoriasis and inverse psoriasis participated in this study. Demographic data are shown in table 1( Table 1 ). Furthermore, patients had stopped topical treatment 2 weeks before the investigation. Systemic treatment or UV-radiation had been stopped at least one month before. They did not use any medication that could interfere with the severity of psoriasis. They had not used any “biological” medication in the past.
Table 1 Demographic findings of patients. Treatments were all stopped before the start of the investigations, according to inclusion and exclusion criteria

Sex	Age	Duration of disease (years)	PASI	Former treatment modalities	Koebner
M	45	31	13.4	Anthralin/ Vit.D3/cortico’s	+
F	53	34	6.9	Cortico’s/ Vit. D3	–
M	60	24	12.4	Cortico’s/ Vit. D3	–
M	42	8	7.5	Cortico’s/ Vit. D3	–
M	47	unknown	5.8	Cortico’s	+
M	52	20	3.8	Vit. D3	–

Clinical score and biopsy procedure

After receiving informed consent, the SUM-scores of two target lesions, one plaque lesion and one flexural lesion, were assessed. Three millimetre punch biopsies were taken from the plaque lesion and from the inverse lesion. The SUM-score is a clinical score indicating the severity of a psoriatic plaque. The SUM-score comprises of three clinical parameters for erythema (0-4), scaling (0-4) and induration (0-4). Punch biopsies 4 mm in diameter were taken from the chronic plaque lesion and flexural lesion after local anaesthesia. Biopsy specimens were stored at –80 °C before use.

Immunohistochemistry

The following markers were investigated: (i) T-cell subsets: CD4, CD8, CD45RO, CD45RA, CD2 and CD25. (ii) Cells expressing NK receptors: CD94, CD161. (iii) Epidermal proliferation: Ki-67 positive keratinocytes. (iiii) Epidermal differentiation: Keratine-10 positive epidermis.

Biopsies were embedded in Tissue Tek OCT compound (Miles Scientific, Naperville, USA), snap frozen in liquid nitrogen and stored at –80 °C until use.

Sections were sliced 6 μm thick and were air-dried for 30 minutes. Then the sections were fixed in cold acetone for 10 minutes. After blocking 5 minutes for endogenous peroxidase, using 0.2% sodium azide, they were washed in PBS for 10 minutes. Subsequently sections were incubated with the primary antibodies for 1 hour. The following primary antibodies (mouse anti-human) were used, diluted in 1% bovine serum albumin (Sigma, St Louis, USA)/PBS: anti-CD2 (1:50) (clone MT910), anti-CD4 (clone MT310) (1:25), anti-CD8 (clone DK25) (1:25), anti-CD45RO (clone UCHL1) (1:25), anti-CD45RA (clone 4KB5) (1:25), anti-CD94 (clone HP-3D9) (1:25), anti-CD25 (clone ACT-1) (1:25), Ki67 (clone MIB-1) (1:100), (all obtained from DAKO, Copenhagen, Denmark), keratine-10 (clone RKSE60) (1:100) (Monosan Laboratories, Uden, Netherlands), anti-CD161 (clone 191B8) (1:25) (Immunotech, Marseille, France).

Sections were washed in PBS for 15 minutes. Secondary IgG labeled polymer, HRP anti-mouse EnVision+ (DAKO Copenhagen Denmark) was added for 30 minutes [23]. The sections were washed for 15 minutes in PBS. To visualize the staining we used AEC + High Sensitivity Substrate Chromogen for 10 minutes (DAKO, Copenhagen, Denmark). Counterstaining was performed with Mayer’s Haematoxylin (Sigma, St Louis, USA). The sections were washed in tap water and dried. Sections were finally mounted in glycerol gelatin (Sigma, St Louis, USA). Furthermore, from each patient we performed a hematoxiline-eosine staining. After dehydration in alcohol and histosafe, these sections were mounted in Permount.

Immunohistochemical scoring

For the quantitative analysis of the T-lymphocyte markers we used the following procedure: One section of the slide was chosen and all immunocytes with 200x magnification in the epidermal compartment were counted. Epidermal immunocytes in the stratum corneum were not counted. The immunocytes in the dermal compartment were also counted. Positive dermal T-lymphocytes were counted between the basement membrane and 100 μm under the basement membrane.

Image analysis

For analysing Ki-67 positive keratinocytes three representative digital photographs were made at 100x magnification. Each photograph was analysed using IP-lab software. A line was drawn, with a known length, following the stratum basale, and all positive cells above this line were counted. Quantification was done in the unit: positive cells per mm length of basement membrane.

For quantification of keratine-10 positive cells, photographs were taken at 50x magnification. For counting the keratine-10 positive epidermal surface, only the epidermal compartment was used as region of interest (ROI). Using IP-lab software a defined window was set for the analysis of the markers from an area which was representative for the section. This window was designated as Region of Interest (ROI). The dermal surface was substracted if it was present in the epidermal compartment. Quantification was measured as a % of epidermal surface as unit.

Statistical analysis

Student’s t-test was employed to determine that two populations (or representative measures from it) are equal. If the probability is < 0.05 one may assume that the populations are not equal.

Results

The SUM scores of the chronic plaque lesions were 7.83 ± 0.31 (mean ± SEM) and for the flexural lesions 6.17 ± 0.31 (mean ± SEM). The p-value was 0.011, when comparing the clinical scores in plaque and flexural psoriasis. The differences between these scores were largely due to the differences in scores for scaling (table 2( Table 2 )).

The number of Ki-67 positive nuclei and the percentage of keratin 10 positive keratinocytes in flexural and plaque psoriasis are shown in figures 1 and 2. The differences between flexural and plaque psoriasis with respect to the number of ki-67 positive nuclei and percentage keratin 10 positive cells were at the level of p = 0.487 and p = 0.933 respectively, which implies no statistical difference.

T-cell subsets in flexural and chronic plaque psoriasis are shown in figures 3 and 4. It can be seen that the number of CD8+ T-cells in flexural and plaque psoriasis are higher as compared to the number of CD4+ T-cells in the epidermis, and that the reverse is true for the dermis. The number of CD45RO+ T-cells is higher as compared to the number of CD45RA+ T-cells in epidermis as well as dermis. The p-values for differences between flexural and plaque psoriasis are shown in table 3( Table 3 ). It can be seen that there is not even a tendency for a difference between flexural and plaque psoriasis with respect to the T-cell subsets investigated.

Cells expressing NK receptors (CD94 and CD161) are shown in figures 3 and 4. Epidermal CD94+ cells show a tendency to an increase in flexural psoriasis as compared to plaque psoriasis. A highly significant difference between flexural and plaque psoriasis is observed with respect to CD161+ cells in the dermis. CD161+ cells in the dermis of flexural psoriasis are markedly decreased as compared to plaque psoriasis (p < 0.001).
Table 2 Clinical scores for flexural and chronic plaque lesions (mean ± SEM). p-value is 0.011 for the difference in clinical SUM-score between flexural and plaque psoriasis

	Plaque lesion	Flexural lesion
SUM-score	7.83 ± 0.31	6.17 ± 0.31
Erythema	2.83 ± 0.17	3 ± 0
Induration	2.33 ± 0.21	1.67 ± 0.21
Scaling	2.67 ± 0.21	1.50 ± 0.22

Table 3 p-values for differences between plaque psoriasis and flexural psoriasis for T-cell subsets and cells expressing NK-receptors in the epidermis and dermis

p-value	CD2	CD4	CD8	CD25	CD45RO	CD45RA	CD94	CD161
Epidermis	0.6432	0.8965	0.3208	0.6468	0.6563	0.7975	0.0486	0.7762
Dermis	0.6424	0.7621	0.9042	0.4915	0.4909	0.5271	0.3519	0.0098

Discussion

The present study shows that markers for epidermal proliferation and keratinization in flexural- and plaque psoriasis are comparable in both conditions. Also T-cell subsets did not even show a tendency to a difference between flexural and plaque psoriasis. The fact that CD45RO+ T-cells outnumber CD45RA+ T-cells and the dominancy of CD8+ T-cells in epidermis and CD4+ T-cells in the dermis in both conditions is totally in line with other studies on T-cell subpopulations in psoriasis [24-26].

In the light of the similarity between plaque psoriasis and flexural psoriasis the differences in therapeutic responsiveness are most likely to be the result of differences in pharmaco-dynamics of the flexural skin with respect to transepidermal penetration and the semi-occlusive effect of the flexures.

It is a well-established fact that bacterial overgrowth characterizes flexural psoriasis. Microbial infections may act as a triggering factor [7], although such a triggering effect may be restricted to some episodes of the initiation of flexural lesions and may be of limited value as target for anti-psoriatic treatment. Due to up-regulation of β-defensins in psoriatic skin, the role of micro-organisms in the initiation and persistence of lesions in flexural areas may be of less importance than in case of, for instance, atopic dermatitis, that lacks such up-regulation. This also explains the similarity in quantity and quality of the T-cell subsets in plaque and flexural psoriasis, because β-defensins may counteract the microbial challenge in flexural areas.

NK-T-cells are an interphase between innate and adaptive immunity. In the present study, the presence of cells expressing NK receptors in the epidermis was comparable in plaque versus flexural psoriasis. An unexpected finding was the highly significant and substantial decrease of CD 161+ cells in the dermis of flexural versus plaque psoriasis. It is attractive to speculate that chronic microbial challenge in flexural psoriasis is counteracted by NK-T cells, resulting in a depletion of the dermal reservoir of these cells.

Additional studies on the regulation of NK-T cells in flexural psoriasis may further elucidate the interaction of microbes with the immunopathogenesis of flexural psoriasis.

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