ARTICLE
Auteur(s) : Martin KUCHAREKOVA, Wynand H.P.M. VISSERS,
Joost J. SCHALKWIJK, Peter C.M. van de KERKHOF, Peter G.M. van der
VALK
Department of Dermatology, University Medical Centre Nijmegen,
P.O. Box 9101, 6500 HB Nijmegen The Netherlands
Reprints: M. Kucharekova Fax: (+31) 24 3541184 E-mail:
m.kucharekovaderma.umcn.nl
Article accepted on 17/3/03
E-FABP has been characterised as a molecule with a molecular weight
of 15 kDA. It is a member of the mammalian intracellular lipid
binding protein family (LBP), which plays a variety of roles in
intracellular fatty acid, retinoid, bile acid and sterol
trafficking.[1] The function of E-FABP in skin has not been
elucidated. It is assumed that this protein is involved in the
transport of fatty acids (FA) which are the most important
substrates for (1) energy production, (2) formation of
phospholipids, i.e the maintenance of membrane lipid structures and
(3) participation in signal transduction pathways [2]. The
upregulation of E-FABP expression is associated with a continuous
high fatty acid transport, needed for the restoration of barrier
function [3]. A study on the cell biological alterations during
irritant contact dermatitis strongly suggested that E-FABP
expression is upregulated after barrier disruption [4]. However,
only its function with the respect to skin barrier has been
discussed [5, 6]. The upregulation of the E-FABP gene expression in
response to oxidative stress and the generation of reactive oxygen
species has been shown in a variety of cell types [1].
The expression of E-FABP in dithranol irritation has not been
studied before. Dithranol-induced irritation is a unique type of
skin irritation. It is caused by anthralin free radicals and oxygen
radicals [7]. Remarkably, a single application of dithranol which
induces skin irritation, does not compromise the skin barrier as
assessed by measurement of transepidermal water loss [8].
The aim of the present study was to investigate the expression
of E-FABP in a well-established model of free radical induced skin
irritation in order to contribute to the elucidation of the
function of this interesting protein in skin disorders.
Material and methods
Subjects
Nine patients with chronic stable psoriasis participated in the
study (5 females, 4 males, age range 50-70 years,
the mean of age 55.7 years). They had no history of other skin
disease. None of the patients used any systemic medication.
Permission of the Ethical Committee and written informed consent
from all volunteers were obtained. The experiment was conducted
from May to June 2002.
Application of dithranol
The dithranol 2% cream (dithranol 69 g, cetiol V
660 g, cetomacrogol wax 450 g, liquid petrolatum
450 g, salicylic acid 30 g, sorbic acid 4.5 g,
ascorbic acid 1.5 g, demineralised water up to 3000 g)
was applied once on one selected site of 2 cm Ø of uninvolved
skin of lower back of patients with psoriasis. The application time
was 1 hour, after which the dithranol cream was removed with
water. This model of dithranol irritation has already been
described and used [9]. Patients were not allowed to use any
topical medication on the investigated site during the study.
Immunohistochemical assessment
A total of 18 punch biopsies (3 mm diameter) were
taken from the investigated sites of nine patients after visual
reading and TEWL measurements at 48 hours and at day 10. After
4 h fixation in formalin the samples were stored in alcohol
70% until embedding in paraffin was done. Then the samples were
sectioned at 6 μm for immunohistochemical staining. The slides
were deparaffinized by incubation in a histosafe for 2 times
15 minutes, followed by dipping in an ethanol series from 100%
to 50%. After washing in phosphate-buffer saline (PBS) the sections
were pre-incubated with normal goat serum (20%) for
15 minutes. Then all sections were incubated for
60 minutes with the anti-E-FABP (kindly provided by Dr.
Siegenthaler) [10] at a dilution of 1:400. After washing with PBS,
sections were incubated for 30 minutes with biotinylated
goat-anti-rabbit IgG (dilution 1:200). Another washing step with
PBS was carried out, and then complexes were formed between biotin
and avidin using the Vectastain Elite ABC peroxidase kit, according
to the manufacturer’s instructions (Vector Laboratories,
Burlingame, USA). Sections were developed with amino-ethylcarbazol.
All sections were subsequently counterstained with hematoxylin
1%.
Evaluation
Visual scoring. The clinical changes at the exposure
sites were scored after 48 hours and after 10 days and
rated as follows: 0 = no erythema, 1 = minimal
erythema, 2 = moderate erythema, 3 = marked
erythema and 4 = marked erythema with oedema.
Transepidermal water loss. TEWL measurements were performed
using an evaporimeter (Tewameter TM 210, Courage and Khazaka, Koln,
Germany). Measurements were performed according to the guidelines
described by the Standardisation Group of the European Society of
Contact Dermatitis [11]. Room temperature was kept between
20 and 22 °C and relative humidity between 40 and
56%. TEWL measurements were performed before exposure on day
0 and at 48 hours and at 10 days after exposure to
dithranol.
Results
Visual assessment and transepidermal water loss
Table I summarises the visual scores
(mean ± SEM) and the TEWL (mean ± SEM)
measurements following dithranol application (n = 9). All
patients clearly developed erythema after 48 hours of
dithranol application which was also observed at day 10. In none of
the patients oedema or vesicle formation was seen and no complains
of pain or itching were reported. The baseline values of TEWL of
the uninvolved skin of psoriatic patients were comparable to the
values of the healthy skin as reported in the literature [12].
There was no significant increase in TEWL at 48 hours and at
10 days compared to baseline.
Table I. Transepidermal water loss (g/m²/h;
mean ± SEM) en erythema score (mean ± SEM)
following a single application of dithranol 2% after 48 hours
and 10 days. (n = 9)
|
TEWL
(mean ± SEM) |
Erythema
(mean ± SEM) |
| Baseline |
6.7 ± 1.3 |
0 |
| 48 hours |
6.1 ± 0,7 |
1.7 ± 0.2 |
| 10 days |
6.6 ± 0.9 |
1.4 ± 0.2 |
The expression of E-FABP
Following single application of dithranol on uninvolved
psoriatic skin (n = 9), strong cytoplasmic staining in
the stratum granulosum and the upper spinous layer was observed at
48 hours and at 10 days.(Fig. 1a) This pattern of
E-FABP expression is the same as in normal skin (Fig. 1b) [5]. The
staining of the untreated lesional psoriatic skin that we used as a
positive control, showed the expression of this protein in all
suprabasal cells (Fig.
1c) as previously observed [3]. No staining was observed
with the pre-immune serum in normal human skin.
Discussion
The expression of epidermal fatty acid binding protein (E-FABP)
was studied in dithranol induced irritation of uninvolved skin of
psoriasis patients using immunohistochemical methods. In the
present study, after a single application of dithranol, we observed
a pronounced erythema after 48 hours in line with other
studies [13-15]. As observed also in a previous study [8],
dithranol did not increase TEWL indicating that single application
of dithranol does not compromise the skin barrier. After dithranol
application, we observed epidermal expression of E-FABP equal to
normal human skin. These results show that the expression of E-FABP
in dithranol irritation correlates with a normal function of the
skin barrier indicating a role for E-FABP in skin barrier
formation. The upregulation of E-FABP has been observed in skin
conditions associated with the impairment of skin barrier like
sodium dodecyl sulphate induced skin irritation [4], psoriasis [3]
and atopic dermatitis (unpublished data). Furthermore, delayed
recovery of TEWL after acetone application to the skin of
E-FABP-deficient mice has been reported [16]. All these studies
demonstrate that the role of E-FABP is associated with maintaining
of the skin barrier function although the exact mechanism for this
function of E-FABP remains to be further elucidated. In contrast,
the presence of E-FABP in the non-keratinized oral mucosa
epithelium and its expression in tissues other than skin suggests
that the role of E-FABP is not restricted to the skin barrier
alone.
Recently, a new function of E-FABP as an antioxidant protein has
been reported [1]. E-FABP was identified as a molecular target for
4-Hydroxynonenal (4-HNE). 4-HNE is the most cytotoxic unsaturated
aldehyde that is produced by lipid peroxidation that occurs in
response to oxidative stress [17]. The identification of E-FABP as
a target molecule for a well known end-product of lipid
peroxidation could indicate that the E-FABP functions as an
antioxidant protein by scavenging reactive lipids [1]. E-FABP is
found in cells exposed to high oxidative stress, including the
retina, lens, lung and tongue [18]. The skin is also particularly
vulnerable to the oxidative stress due to (i) its constant exposure
to high oxygen tension, and (ii) the presence of considerable
amounts of polyunsaturated fatty acid [19, 20]. Indeed, E-FABP is
observed to be upregulated in the skin conditions associated with
oxidative stress [21-24]. Also dithranol-induced skin irritation is
strongly suggested to be caused by the generation of dithranol free
radicals and oxygen radicals [25]. There is some evidence that the
release of active oxygen species from dithranol could initiate
lipid peroxidation in cellular membranes of the skin. Firstly,
dithranol slightly elevates lipid peroxidation in mouse skin after
topical application, demonstrated by the exhalation of ethane,
which is produced from gamma-3 polyunsaturated fatty acids[26]
and by malondialdehyde production [27]. In addition, singlet oxygen
can accelerate lipid peroxidation by directly reacting with
unsaturated fatty acyl moieties to give hydroperoxides, which has
also already been demonstrated for dithranol [28]. Thus the
peroxidative damage to membrane lipids after dithranol application
is generally believed to be related to its antipsoriatic action as
well as induction of skin irritation [25, 29]. Interestingly, the
presumed generation of free radicals following dithranol
application does not cause the induction of a protective endogenous
mechanism such as enhanced expression of E-FABP.
In the context of oxidative stress, the lack of upregulation of
E-FABP observed in this study may be due to several reasons.
Although 4-HNE was found to be responsible for the damaging effects
associated with lipid peroxidation in various tissues [17]
including skin, [30-33] there are no studies demonstrating that
4-HNE is generated by dithranol-induced lipid peroxidation and
therefore the enhanced expression of E-FABP as a target for 4-HNE
is not observed. The scavenging of the 4-HNE by other skin
antioxidant systems like glutathione S-transferase[22] or
superoxide dismutase [34, 35], both observed to be modified
after dithranol application, should be considered. In conclusion,
in this study the lack of upregulation of the expression of E-FABP
in dithranol irritation correlates with the lack of skin barrier
dysfunction as assessed by measurements of TEWL, indicating the
role for E-FABP in the maintaining of skin barrier. n
Acknowledgements. We thank Prof. Dr. Klaus
Mller (Institute of Pharmaceutical Chemistry, University of Mnster)
for the critical reading of this manuscript.
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