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
|
|
–
|
|
M
|
60
|
24
|
12.4
|
|
–
|
|
M
|
42
|
8
|
7.5
|
|
–
|
|
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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