ARTICLE
Psoriasis is a common inflammatory skin disease which is characterized
by abnormal keratinocyte proliferation and differentiation, in association
with leukocyte infiltration and alterations of cytokine production. It
has become apparent that keratinocyte hyperproliferation and inflammation
are strictly correlated in psoriasis. A large body of evidence indicates
that interactions between activated T lymphocytes and keratinocytes, via
cytokines, play a crucial role in the pathogenesis of this disease [1].
A variety of cytokines released by keratinocytes and inflammatory cells,
in fact, appears implicated in the induction or persistence of the inflammatory
and hyperproliferative process in psoriasis. An increased expression of
transforming growth factor-alpha (TGF-alpha), interleukin (IL)-6, IL-8
and a dysregulation of IL-1 metabolism have been described in psoriatic
epidermis as factors involved in both the epidermal hyperplasia and the
accumulation of inflammatory cells seen in psoriatic lesions [2-5].
Topical corticoids remain the mainstay of treatment for moderate psoriasis.
These therapeutic compounds are most useful for reducing inflammation
and controlling itching. The therapeutic effect of corticosteroids in
skin may be partially explained by their varying ability to inhibit cytokine
production. Recently, in vitro studies have demonstrated that corticosteroids
block the synthesis of IL-1alpha, IL-6, IL-8 and tumor necrosis factor
(TNF)-alpha by keratinocytes [6, 7].
In the present study we investigated the different effects of a 0.1%
mometasone furoate cream on cell proliferation, inflammatory infiltrate,
adhesion molecules and the cytokine system in psoriasis. For this purpose
we analysed immunohistochemically, skin biopsies from ten patients before
treatment and after 1 and 3 weeks of treatment, employing a broad panel
of monoclonal and polyclonal antibodies specific for cytokines and their
receptors, adhesion molecules, proliferation-associated antigens and lympho-monocytic-specific
antigens.
Materials and methods
Patients
Ten patients, 5 women and 5 men aged 22 to 72 years, suffering from
moderate to severe chronic plaque psoriasis, with a duration of disease
from 1-30 years (mean 13 years), were included in this study. All topical
psoriasis preparations and systemic treatment had to be discontinued 2
weeks to 2 months, respectively, prior to the study. Informed consent
was obtained and the study protocol was approved by the Medical Ethical
Commitee of the University of Milan and the IRCCS Ospedale Maggiore of
Milan. Upon entry into the study, each patient was provided with mometasone
furoate cream 0.1% and shown how to apply the cream to the designated
side of the body for the entire period of the study. Patients applied
mometasone furoate cream 0.1% (Elocon, Schering-Plough) to the psoriatic
lesions once daily for 3 weeks. Activity and severity of disease were
evaluated before treatment, after 1 week and after 3 weeks of treatment
of various test sites and expressed as scores of mean values in each patient.
Erythema, infiltration and scaling of the lesions were assessed using
a 4-point semiquantitative scale: 0, no cutaneous involvement; 1, slight;
2, moderate; 3, severe.
Tissue studies were performed on punch biopsies taken from lesional
skin before treatment, after 1 week and after 3 weeks of treatment. The
specimens were embedded in Tissue-Tek OCT Compound (Miles Scientific,
Elkhart, USA), snap frozen in freon 22, cooled in liquid nitrogen and
stored at 80° until ready for sectioning. Normal skin, used
as control, was obtained from discarded surgical specimens.
Antibodies
The primary antibodies used are listed in Table
I.
Immunohistochemistry
Four µm cryostat sections, air dried for 10 min at room temperature
and acetone-fixed for 10 min, were dried for 1 to 2 hrs before the incubation
with primary antibodies in the proper dilutions. Sometimes for the cytokine
staining, the specimens were postfixed with Formalin and treated for 5-10
min with a detergent such as NP40 0.02% or Triton X-100 0.05%. The specimens
were processed using the alkaline-phosphatase anti-alkaline phosphatase
(APAAP) method according to the standard procedure [8], using commercially
available reagents, rabbit anti-mouse IgG(H+L) and APAAP purchase from
Dakopatts Glostrup, Denmark. For polyclonal antibodies, a swine anti-rabbit
Ig was used as a secondary antibody. The immunological reaction was revealed
using New-Fuchsin, naftol AS-BI phosphate sodium salt, and 1 mM levamisole
(all from Sigma Chemicals, Saint Louis, Missouri USA) for 20 min. The
samples were counterstained with Mayer hematoxylin, dried at room temperature,
mounted in DPX and observed using a microscope.
Stained sections were evaluated by two independent observers, using
a semiquantitative method. The level of staining was scored on the following
4-point scale: no staining (grade 0), moderate focal/faint diffuse staining
(grade 1), strong focal/moderate diffuse staining (grade 2), and strong
diffuse staining (grade 3). All results were expressed as the mean. A
separate graded score system was used to evaluate the expression of lymphocytes,
monocytes/macrophages and polymorphonuclear leukocytes markers in the
dermal infiltrates. The percentage of positively stained cells was scored
as follows: 0, no stained cells; 1, 1-25% of the infiltrate cells stained;
2, 26-50%; 3, 51-75%, 4, > 75% of the infiltrate cells stained. Ki-67
and PCNA positive nuclei were counted/mm length of epidermis.
Statistical analysis
The data were analyzed with the Wilcoxon Sign Ranks Test (WSRT). A p
value less than 0.05 was considered statistically significant.
Results
Clinical response
A semiquantitative evaluation of the principal clinical parameters is
presented in Table II.
All but two patients enrolled in the study showed a moderate improvement
of the test sites with respect to the clinical parameters evaluated after
1 week of treatment. The other two weeks of therapy led to a significant
reduction of erythema, infiltration and scaling without complete clearing
of psoriatic lesions. In two remaining cases, a pronounced reduction of
psoriatic erythema, infiltration and scaling was observed within the first
7 days of treatment and was followed by a steady decline to almost complete
blanching on day 22 (Table II).
Immunohistochemical findings before treatment
Epidermis. The alpha2(CD49b), alpha3(CD49c) alpha6(CD49f), ß4(CD104)
and ß1(CD29) integrins were overexpressed in the epidermis of psoriatic
skin lesions before treatment (Table
III). In particular, alpha3 integrin showed the most intense diffuse
staining extending up to the stratum spinosum (Fig.1a).
In contrast, alpha6/ß1 and ß4(CD104) integrins, which in normal
skin stained the dermal pole of the basal keratinocytes, showed a focal
overexpression on suprabasal keratinocytes above the papillary rete ridges.
The alpha1, alpha4 and alpha5 integrins were undetectable on psoriatic
keratinocytes. Moreover, an aberrant expression of HLA-DR and intercellular
adhesion molecule-1 (ICAM-1;CD54) molecules was observed in the epidermis.
These antigens normally found on Langerhans cells only, displayed a focal
expression in groups of basal and suprabasal keratinocytes overlying the
elongated rete ridges.
Evidence of significant proliferative activity within the epidermis
was manifested by an increased number of keratinocytes in the basal and
suprabasal layer reacting with monoclonal antibodies (mAbs) to Ki-67 antigen,
proliferating cell associated antigens (PCNA) and epidermal growth factor
receptor (EGF-R).
The number of dendritic cells CD1a+ in the epidermis was
increased particularly in the papillary area, and the cells showed very
long dendrites, indicating a strong activation as demonstrated by the
HLA-DR/CD1a staining.
Keratinocytes of the untreated psoriatic skin reacted with various cytokine
and cytokine receptor mAbs in all cases, only a slight interindividual
variation being seen with respect to the expression of these molecules.
A particularly strong basal and suprabasal staining with anti-IL-1alpha,
TNF-alpha and interferon gamma (IFN-gamma) mAbs was detected (Fig.
2a). A less marked expression of IL-1 receptor (IL-1R), and IFN-gamma
receptor (IFN-gammaR) was also demonstrated. Furthermore, the keratinocyte
labelling of IL-8, monocyte chemotactic and activating factor (MCAF/MCP-1)
and granulocyte monocyte-colony stimulating factor (GM-CSF), was very
intense in psoriatic plaques before treatment. Interestingly, the major
overexpression of GM-CSF appeared in the epithelial cells composing the
elongated rete ridges (Fig. 3a).
A weak positivity with anti-IL-6 was observed especially in basal and
suprabasal keratinocytes. Finally, one of the most noteworthy findings
characterizing the untreated psoriatic epidermis was represented by the
slight suprabasal reactivity with anti-IL-4 and IL-4 receptor (IL-4R)
mAbs.
Dermis. The vessels of the papillary dermis were strongly stained
with anti-alpha1 and ß1 integrin mAbs. An increased expression of
E-selectin (CD62E), P-selectin (CD62P), ICAM-1, ICAM-2(CD102) and vascular
cell adhesion molecule-1 (VCAM-1;CD106) antigens was detected on psoriatic
dermal endothelium, particularly in the areas of maximal neutrophil and
lymphocyte intra- and perivascular accumulation. Vessels of the papillary
dermis were strongly stained by mAbs directed against IL-1alpha, TNF-alpha,
IFN-gamma and GM-CSF.
The infiltrate was predominantly composed of T lymphocytes expressing
CD3, CD4, CD11a, CD28, ICAM-3(CD50), and CD69 molecules. A notable proportion
of T lymphocytes expressed the CD25 and HLA-DR activation antigens and
the HML-1(CD103) homing/activation molecule. Few CTLA-4+ cells
were observed. Only a small population of CD8+ T lymphocytes
was observed. Few polymorphonuclear leukocytes were demonstrated with
anti-CD11b, CD15, and CD66 mAbs. In the superficial dermis, the number
of macrophages and particularly of dendritic cells was greatly increased.
Dendritic cells expressed in addition to the classical markers of the
dendritic subset (HLA-DR, CD1a, CD1c), CD11a, CD11c antigen, alpha5 and
ß1 integrins, GM-CSF receptor (GM-CSF-R) and ICAM-1 molecules. The
cytokine expression on the upper dermal infiltrate is summarised in Table
IV. We would emphasize the overexpression of several cytokines,
such as IL-1alpha, TNF-alpha, and IFN-gamma.
Immunohistochemical findings
after treatment
Epidermis. After topical treatment with mometasone furoate, we
observed an altered expression of integrin adhesion receptors by keratinocytes
(Table III). The alpha2,
alpha3 (Fig. 1b), alpha6,
and ß1 integrins staining appeared less intense after one week of
treatment and further diminished and was absent in some areas after three
weeks, particularly in two of the ten cases in which a complete clinical
remission was obtained. A remarkable reduction in the number of Ki-67
and PCNA stained nuclei was demonstrated, EGF-R expression was also decreased.
The number of CD1a+ dendritic cells in the epidermis was
diminished in the majority of the cases examined. In most of the cases
(8 out of 10), no variation of cytokine and cytokine receptor staining
was observed in either the epithelium or in the infiltrating cells after
one week of treatment. At the end of the treatment, we documented several
changes with respect to the expression of these substances. Epidermal
reactivity with mAbs directed against the major pro-inflammatory cytokines,
such as IL-1alpha, TNF-alpha and IFN-gamma, appeared consistently reduced.
A marked decrease of labeling was noted in two patients that had showed
complete clinical remission (Fig.
2b). The receptors of the above mentioned cytokines displayed
very similar behaviour. The immunohistochemical overexpression of the
chemotactic cytokines and GM-CSF on psoriatic epidermal cells also diminished
only slightly after one week, but continued to decline between 1 and 3
weeks of application of mometasone furoate, and thus were significantly
(p < 0.05) reduced in the majority of patients at the conclusion
of treatment (Fig. 3b).
By contrast, chemokine staining had already decreased significantly after
one week of topical steroid therapy in the two cases characterized by
early and marked clinical improvement. Interestingly, the weak epidermal
reactivity with anti-IL-6 and IL-4 mAbs remained substantially unchanged
in all cases.
Dermis. In the vascular endothelium of involved psoriatic skin
after treatment, we noted a significantly (p < 0.05) decreased
expression of ICAM-1, ICAM-2. A weak and patchy alpha1 integrin and HLA-DR
labeling on endothelium was seen in a small percentage of vessels. The
lympho-monocytic infiltrate was clearly reduced. Few lymphocytes CD3+,
CD4+ were detected in the perivascular areas. Some of these
cells expressed CD25 activation antigen. However, several monocytic HLA-DR+,
CD11c+, and dendritic cells CD1c+ were recognized
in the perivascular areas. The immunohistochemical cytokine pattern in
the dermis showed some moderate changes resulting from a reduction in
the percentage of inflammatory cells composing the infiltrate (Table IV).
Discussion
The efficacy of topical steroid treatment in several inflammatory diseases
such as psoriasis, which depends upon the anti-proliferative and anti-inflammatory
properties of these drugs, is well known [9, 10]. However, the biological
mechanisms of these effects remain still incompletely elucidated. In this
study, we carried out a comprehensive examination of the effects of mometasone
furoate treatment on the complex network of mediators involved in the
pathophysiology of psoriasis, including cytokines, cytokine receptors,
and adhesion molecules. Our results seem to suggest a role for mometasone
furoate in the induction of a down-regulation of keratinocyte hyperproliferation.
In fact, after treatment, a significant reduction in the number of keratinocytes
expressing PCNA and Ki-67 proliferation antigens, and EGF-R was observed.
The inhibition of IL-1, IL-8 and TNF-alpha, which are implicated in the
triggering of keratinocyte hyperproliferation, may represent a possible
explanation of the antiproliferative properties of corticosteroids. However,
in our study only minimal effects of mometasone furoate on cytokine expression
were found. More interestingly, we demonstrated that mometasone furoate
therapy had not only reduced the expression of alpha2, alpha3, alpha6,
and ß1 integrins in all treated patients but had also restored the
integrin expression pattern to that observed in normal skin. Considering
that an altered epidermal network of integrins could contribute to the
keratinocyte hyperproliferation, it may be regarded as a complementary
target for the antiproliferative action of corticosteroids. On the other
hand, it has been well established that steroids also possess an anti-inflammatory
activity, which represents the other important component of their therapeutic
action [9]. Recently, various corticosteroids have been found to block
the production of IL-1alpha, IL-6, IL-8 and TNF-alpha by keratinocytes
in vitro [6], suggesting that the effects on the cytokine system
may be a relevant part of their anti-inflammatory action. In particular,
mometasone has proven to be the most potent inhibitor of IL-6, IL-1, and
TNF-alpha production as compared with other classic topical steroids [11,
12]. Epidermal reactivity with mAbs directed against IL-1, TNF-alpha,
and IFN-gamma were only slightly decreased after three weeks of treatment
in 8 out of our 10 patients. In addition, a more pronounced reduction
of the epidermal expression of GM-CSF, the most important member of the
colony-stimulating factor family, was observed in the same cases. Mometasone
furoate induced restoring to normal of the above cytokine epidermal network
in two patients, who showed almost complete clinical remission after therapy.
These findings seem to indicate that clinical improvement is closely related
to steroid-induced, down-regulation of cytokines.
A significant reduction of ICAM-1 and ICAM-2 expression on dermal vascular
endothelial cells of all treated patients was also seen, suggesting that
the inhibitory action of mometasone furoate on leucocyte recruitment into
skin depends in part on the down modulation of endothelial adhesion molecules.
By contrast, we demonstrated in the majority of patients treated, some
persistence of LFA-1+ dendritic cells, which are reported to
be crucial for T-cell activation [13]. This finding, in association with
the lack of disappearance of the inflammatory infiltrate, may provide
an explanation for the occurrence of the relatively rapid relapse following
cessation of treatment with corticosteroids.
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