ARTICLE
Auteur(s) : HJ Bovenschen, PCM Van De
Kerkhof, WJ Gerritsen, MMB Seyger
Department of Dermatology, Radboud University Nijmegen Medical
Center, Nijmegen, The Netherlands
accepté le 30 Juin 2005
Psoriasis is a disease characterized by the infiltration of
predominantly T cells, epidermal hyperproliferation and disturbed
keratinisation [1]. Secondly, psoriatic plaques contain elevated
levels of cytokines, chemokines and growth factors [2]. One of the
key players in the pathogenesis of psoriasis is one of these
cytokines: tumor necrosis factor alpha (TNF-α) [2, 3]. As in other
diseases such as rheumatoid arthritis and Crohn’s disease, elevated
levels of this cytokine have been associated with T-cell
infiltration in the diseased tissue [3]. With infliximab therapy
(anti-TNF-α) for plaque psoriasis, over 80% of patients reach ≥ 75%
PASI improvement in 10 weeks of treatment [4, 5]. We treated a
patient with severe psoriasis, recalcitrant to conventional
systemic therapies, with infliximab (Schering-Plough, Welwyn Garden
City, UK) and analyzed five serial skin biopsies for lesional T
cells, NK-T cells and epidermal growth and differentiation, using
standard immunohistochemistry [6].
Case report
A 53-year-old Dutch woman had suffered from generalized
recalcitrant plaque psoriasis for almost 20 years. As the psoriatic
lesions often became erythrodermic and as conventional systemic
treatments were either not successful or contraindicated, she was
treated at our in-patient clinic for several months each year.
Previously, she had been treated with numerous anti-psoriatic
agents, including topical corticosteroids and vitamin D3
derivatives, coal tar preparations and dithranol,
photo-(chemo)therapy: UVB and PUVA, systemic treatments, including
salazopyrin, acitretin, cyclosporin, fumaric acid, methotrexate and
various combinations of these therapies. All treatments had either
no effect, or were poorly tolerated due to severe side-effects. The
most recent treatment period was from August 2003 until April 2004
with methotrexate, which was still the only treatment option with
some success. Unfortunately, a liver biopsy performed in April 2004
revealed progress to grade IV on the Roenigk scale (liver
cirrhosis), forcing her to stop methotrexate treatment and leaving
no regular systemic treatment to treat her psoriasis.
Clinical examination
At this point, on examination she had widespread well-demarcated
and characteristic erythematosquamous plaques of the trunk and
limbs, the scalp and intertriginous areas, as well as nail
involvement and symptoms of psoriatic arthritis ( (figure 1A) ). The
Psoriasis Area and Severity Index (PASI) [7] was 25.4. The involved
“body surface area” was 18%. One plaque on the right lower arm was
selected as a “target lesion”, from where five serial biopsies were
taken. The SUM-score, ranging 0-12, is a cumulative clinical
severity measure for erythema (0-4), induration (0-4) and
desquamation (0-4) of one single target lesion [6]. Before the
start of therapy this score was 10.
Clinical results
It was decided to treat her with infliximab 500 mg (5 mg/kg)
intravenously over 2 hours, at week 0, 2, 6 and then with
maintenance infusions every 8 weeks. The treatment was well
tolerated, but the clinical results did not meet our high
expectations. During treatment the lesions enlarged to near
erythroderma. Involved “body surface area” was 18% at week 0,
increasing to 85% at week 22 ( (figure 1) ). ( Figure 2 ) illustrates the
PASI- and SUM-scores during infliximab treatment. The SUM-score
only decreased by 2 points and PASI increased from 25.4 to 38.8
with the most impressive aggravation during the first two weeks.
After the fourth infusion (week 14) the treatment with infliximab
was terminated due to the lack of efficacy. Follow-up was performed
up to week 22.
Immunohistochemistry and image analysis
Immunohistochemical staining
A non-lesional biopsy, taken at baseline, together with five
lesional biopsies, taken at week 0, 2, 6, 14 and 22 of infliximab
treatment, were embedded in Tissue Tek OCT compound (Miles
Scientific, Naperville, USA), instantly frozen in liquid nitrogen.
Staining of the following T-cell subsets was performed: CD4+, CD8+,
CD45RO+, CD45RA+, CD2+ and CD25+. Furthermore, cells expressing
NK-receptors CD94 and CD161 (NK-T cells), epidermal proliferation
(Ki67 positive nuclei) and epidermal differentiation (keratin 10)
were assessed.
Sections were sliced 7 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, they 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-CD4 (clone MT310) (1:200), anti-CD8 (clone DK25)
(1:200), anti-CD45RO (clone UCHL1) (1:100), anti-CD45RA (clone
4KB5) (1:200), anti-CD2 (1:200) (clone MT910), anti-CD25 (clone
ACT-1) (1:200), anti-CD94 (clone HP-3D9) (1:100), Ki67 (clone
MIB-1) (1:200), (all obtained from DAKO, Copenhagen Denmark),
keratin 10 (clone RKSE60) (1:100) (Monosan Laboratories Uden
Netherlands) and anti-CD161 (clone 191B8) (1:100) obtained from
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. The sections
were washed again 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, dried and mounted in glycerol gelatin (Sigma,
St Louis, USA).
From each biopsy, we performed a hematoxylin-eosin (HE)
staining. After dehydration in alcohol and histosafe, these
sections were mounted in Permount. With these HE-sections, we
verified that the histology of biopsies under study were
psoriasis-specific.
Quantification
Quantification of T-cell subsets was performed at 200X
magnification. In three sections at each time-point, CD4, CD8,
CD45RO, CD45RA, CD2, CD25, CD94 and CD161 positive cells in the
epidermis were counted from the basement membrane up to the stratum
corneum across the whole section (4 mm). Cells in the dermis
were counted from the basement membrane down to 100 μm under
the basement membrane also across the whole section. The mean cell
counts were expressed in “Positive cells per mm skin length” and
displayed with standard error of the mean (SEM).
Image analysis
In order to analyze Ki67 positive cells, three representative
digital photographs were made at 100× magnification. Each
photograph was analyzed using IP-lab software. A line, with known
length and following the stratum basale, was drawn after choosing a
representative “region of interest” (ROI). All positive cells above
this line were counted and expressed in the unit “Positive cells
per mm length of basement membrane” (mean ± SEM).
For quantification of K10 positive cells, digital photographs
were taken at 50× magnification. Using IP-lab software a defined
window (ROI) was set for the analysis of K10, in a representative
area of the analyzed sections. The ROI was chosen only in the
epidermal compartment and possible dermal tissue in the ROI was
subtracted. The total area of the epidermal compartment in the ROI
and the K10 positive area were measured using IP-lab software.
Epidermal differentiation was defined as: “% K10 positive epidermal
surface”. Epidermal thickness was measured in HE sections with the
help of IP-lab software, by calculating the means of the 3 smallest
and 3 largest vertical lengths of the epidermis, without the
stratum corneum.
Immunohistochemical results
T cells
The serial skin biopies revealed conflicting data on T cell
subsets. At baseline, all lesional T-cell subsets were more
abundantly present in contrast to their non-lesional counterparts.
Overall, lesional T cell subsets declined at 2 and 6 weeks after
start of treatment, but then rose again towards week 22. The
reduction of T cells in lesional skin never reached the values of
the non-lesional T-cell counts. The most marked temporary decrease
(72-74%) was observed for CD4+ T-helper cells in the dermis at week
2 and 6. At week 2 and 6 a less substantial, but clear reduction
was observed in the dermis and epidermis for CD8+ (cytotoxic) T
cells, CD45RO+ (memory effector) T cells, CD45RA+ (naive) T cells,
CD25+ (expressing the interleukin-2 receptor) T cells in the dermis
and CD2+ cells (all T cells, but more expression on activated T
cells) [1, 6-9].
NK-T cells
In the untreated lesions, cells expressing NK-receptors (NK-T
cells) [9-11] were present, whereas these were virtually absent in
the non-lesional skin. NK-T cells (CD94+) in the dermis showed a
transient decrease at weeks 2 and 6. In the epidermis, however,
these cells remarkably increased. The CD161+ cells in the dermis
were maximally reduced at week 14, whereas in the epidermis these
cells were reduced from week 2 onward ( (figure 3) ).
Epidermal growth and differentiation
Epidermal proliferation and differentiation, as measured by Ki67+
nuclei and keratin 10 positive epidermal area, never reached
non-lesional skin values during treatment. In the first two weeks
of treatment, an increase in Ki67+ nuclei was observed, together
with an increase in K10 expression. After that, these parameters
did not show a consistent change during the study. However, in
accordance with the reduction of T cells, epidermal thickness
substantially decreased from week 0 to week 2 (0.26-0.13 mm)
and then gradually increased again toward week 22 (0.21 mm) (
(figure 4) ).
Discussion
In the present case study we observed a clinical deterioration of
psoriasis, during 22 weeks of treatment with infliximab (5 mg/kg)
given as intravenous infusion at week 0, 2, 6, and 14. Grosso modo,
the SUM-score did not change during 22 weeks of treatment, whereas
the PASI and in particular the affected “body surface area”
increased up to near erythroderma, with the most marked increase
during the phase of decrease of T cell subsets. Several studies
have shown that as many as 82-88% of patients with
moderate-to-severe psoriasis reach ≥ 75% PASI improvement in 10
weeks of treatment [4, 5]. The clinical response of our patient is
in sharp contrast to the response of the vast majority of patients
in these clinical studies. This might be due to the facts that (I)
this patient has recalcitrant psoriasis of the utmost severity,
with poor responses to all treatments, (II) the discontinuation of
methotrexate 2 weeks before the institution of infliximab may have
accounted for the poor response to the treatment, with aggravation
up to near erythroderma, and (III) this patient might have created
antibodies against infliximab, neutralizing the infliximab effect.
In contrast to the poor clinical response, a decrease of
lesional T cell populations reached minimal levels after 2 and 6
weeks treatment and the most prominent decrease (72-74%) was
observed for the CD4+ T-helper subset. Remarkably, the number of
epidermal NK-T cells fairly correlated with the lack of clinical
efficacy. It was previously reported that anti-TNF-α therapies
target CD4+ cells, CD8+ cells, macrophages, dendritic cells and
natural killer cells [3]. In psoriasis, Gottlieb et al. described a
substantial decrease of epidermal CD3+ T cells and a subsequent
decrease in keratin 16, for infliximab responders [12]. Also in
rheumatoid arthritis, Crohn’s disease and ankylosing spondylitis
significant and fast reductions of CD8+, but mostly CD4+ T cells,
was observed in blood following a course of either infliximab or
etanercept [3]. In addition, in synovial and colonic biopsies of
these patients, infliximab was shown to reduce the (T cell)
infiltrate, mainly CD4+ T cells [13, 14]. The differential effects
of infliximab on T cells versus NK-T cells has never been studied
before.
Nickoloff et al. first described NK-T cells (CD94, CD161) in
psoriasis [10, 11]. The CD94 receptors are expressed on most
NK-cells and some T cells, whereas CD161 is an obligate NK-T cell
marker. It has been shown that a significantly higher number of
NK-T cells are present in the psoriatic lesion in comparison to the
uninvolved or normal skin. However, it is not clear whether these
cells (I) mainly initiate psoriasis, (II) have regulatory
properties that might in fact stabilize the psoriatic process, or
(III) are just bystander cells. This case report is an independent
confirmation that T cells bearing NK-receptors (e.g. NK-T cells)
such as CD94 and CD161 are in-fact present within both dermal and
epidermal compartments of active psoriatic plaques. The relative
impact of infliximab appears to have differential effects on
conventional CD4+ T cells and CD8+ T cells compared to
unconventional NK-T cells. The increase in epidermal CD94+ NK-T
cells suggest this subset has a pathogenic role, since this finding
is accompanied by a lack of clinical improvement. As the present
report is a single case, confirmation of our data by larger studies
is necessary. Furthermore, a confirmation of another target plaque
might be appropriate to interpret the role of lesional T cells.
Larger studies might also include the assessment of functional
tests and cytokine profiles, both in psoriatic plaques and in
peripheral blood. In conclusion, the discrepancy between the
clinical aggravation and marked reductions of T cells suggests that
a reduction of these cells in the psoriatic plaque might not be a
guarantee for positive clinical outcomes. Differences between
responders and non-responders to anti-psoriatic therapy, with
respect to lesional T cells, NK-T cells and epidermal parameters,
might contribute to clarify the ambiguous role of these cells in
plaque psoriasis.
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