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C o p yrig h t © M anksgaard ¡997
Contact Dermatitis, 1997, 37, 19-26
Printed in Denmark . A i l rights reserved
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CONTACT DERMATITIS
I S S N 0W5-ÌX7.1
Effect of a topical corticosteroid, a retinoid and a
vitamin D3 derivative on sodium dodecyl sulphate
induced skin irritation
T. K. M.
L e,
P.
de
M
o n
,
J.
Schalkw
ijk
and
P. G. M.
van
der
Valk
Department o f Dermatology, University Hospital Nijmegen, The Netherlands
Exposure of the skin to sodium dodecyl sulfate (SDS) leads to disruption of barrier and skin
irritation. We used repetitive short exposure to a low molarity SDS solution as an in vivo model to
mimic the development o f irritant contact dermatitis. In this model, we studied clinical (erythema),
functional (transepidermal water loss(TEWL)) and cell biological changes. 24 healthy volunteers
were patch tested with SDS (0.2%) for 4 h a day for 5 consecutive days. After removal of the
patches, the exposed sites were treated 1X daily either with a topical corticosteroid (triamcinolon
acetonide cream 0.05%), a retinoid (tretinoin cream 0.025%), or a vitamin D 3 derivative (calcipotriol ointment 50 microgram/g). Irritant reactions were assessed by erythema scoring and measure­
ment o f barrier function with TEWL up to 14 days after the first challenge. Skin biopsies were
taken for cell biological changes at day 4. Vehicle-treated sites served as controls. Repetitive ex­
posure of human skin to SDS resulted in a gradual increase in erythema scoring and TEWL
associated with the upregulation of proliferative cells as measured by the expression of Ki-67antigen and of differentiation markers, visualized by increased expression of involucrin and epider­
mal-fatty-acid binding protein (E-FABP). Skin irritation as assessed by erythema scoring and
TEWL was not significantly suppressed by triamcinolone cream. However, a significant reduction
o f the number of cycling keratinocytes and a decrease in involucrin positive cell layers was observed
in this group. Neither treatment with calcipotriol ointment nor with tretinoin cream induced im­
provement o f skin irritation as judged by visual scoring and TEWL. In contrast to steroid treat­
ment, no significant effect o f calcipotriol ointment or tretinoin cream treatment was observed with
regard to the number o f cycling cells and differentiation markers. Further studies are needed to
assess whether treatment with topical corticosteroids is an effective modality in skin irritation and
irritant contact dermatitis.
Key words: sodium dodecyl sulfate; irritant contact dermatitis; corticosteroids; retinoids; vitamin
D 3 derivatives; treatment; skin irritation. © Munksgaard, 1997.
Accepted for publication 18 March 1997
Irritant contact dermatitis (ICD) is characterized
by mild erythema,
itosis or dyskeratosis,
chapping and fissures (1) and is pathogenetically
thought to be the results of cumulative exposure to
irritants which may be o f various origin (2). Daily
exposure to these irritants cannot be standardized.
To study the pathogenesis of ICD, experimental
models in men are needed. In vivo models have
been used to investigate the effects o f the cumula­
tive exposure o f human skin to sodium dodecyl
sulfate (SDS) in terms of erythema, skin dryness
and transepidermal water loss (TEWL) (3). Reports on cell biological changes in SDS exposed
skin are scarce. Recently, we demonstrated that the
development of erythema and TEWL was associated with the upregulation o f the number of cyc-
ling cells and the epidermal differentiation markers
(involucrin, epidermal fatty acid binding protein
(E-FABP) ) in the irritant
reaction
single 4-h exposure to SDS (5%) (w/v) (4). An SDS
concentration of 0.2% caused a significant increase
TEWL
any change in
epidermal proliferation and different!
pared to the normal skin was seen (5) A 0.2%
centration of SDS was chosen in th
model to avoid severe damage to the skin and
mimic daily life exposure. In the model presented,
we studied the time course o f the erythematous re­
sponse, TEWL and epidermal growth in the irritant human skin induced by repeated daily 4-h ex­
posures to SDS (0.2%) for 5 consecutive days.
We postulate that drugs which can influence epi-
LE ET AL
20
A
3.5
DSDS+ Triamcinolon©
■ SDS+Vehicle
3
QSDS
2.5
2
C
H
UJ 1.5
l
0.5
0
ül
(12
d3
tl4
Ü5
d6
d7
dlO
d 14
B
3.5
QSDS+Tretlnoln
3
■SDS+Vehlcle
□ SDS
2.5
l
af 1.5
LU
1
0.5
0
dl
d2
d3
d4
(15
d6
d7
d 1U
d 14
c
inflammatory conditions (8, 9), is downregulated
to the normal levels in the steroid-treated epider­
mis (7).
The vitamin A metabolite, retinoic-acid, is es­
sential to normal differentiation of many tissues
including skin. Retinoic acid and synthetic retin­
oids are used in the treatment o f acne (10), pso­
riasis (11) and certain malignancies (12). Several
studies in mouse and man indicate that topical re­
tinoic acid speeds repair of skin damage due to
ultraviolet radiation (13, 14). Effects of retinoids
on the epidermis include modification of keratin
expression (15) and inhibition of cross-linked
cornified envelope synthesis (16). Retinoids can
either stimulate or inhibit cell growth (17).
Vitamin D 3 derivatives are the recent therapeutic
modalities in psoriasis.They are known to affect
the epidermal growth. Administration of vitamin
D 3 to cultured keratinocytes causes suppression of
cell proliferation and enhancement of cell differen­
tiation (18). Vitamin D 3 derivatives can modulate
inflammatory processes. When T-lymphocyte cul-
3.5
OSOS+Catcipotriol
y
■SDS+Vehicle
2.5
1
T
OSDS
uu
A
K 1.5
II»
1
40
35
n
M
ÿ
i
0.5
0
dl
d2
S) SDS‘►Triamcinolone
■ SDS+Vehicle
JC 30
OSDS
25
d3
d4
d5
Ü6
d7
dlO
d 14
□ Normal skin
20
15
Fig. 1. Time course of erythema from day 0 to day 14. 0, no
response; 1, slight, patchy redness; 2, diffuse mild redness; 3,
moderate redness; 4, intense redness; 5, intense redness with
edema: (A) o f the SDS-exposed skin and the triamcinolontreated group versus its vehicle; (B) of the SDS-exposed skin
and the tretinoin-treated group versus its vehicle; (C) of the
SDS-exposed skin and the calcipotriol-treated group versus its
vehicle.
10
1
5
0
dl
d2
d3
d4
d5
d7
d6
d 14
dlO
B
OSDS+Tretinoln
■ SDS+Vehfcle
□ SDS
□ Normal skin
dermal growth resulting in the improvement of the
skin barrier function, may be effective in the treat­
ment of ICD. Topical steroid, retinoic acid and vit­
amin D 3 derivative are the therapeutic modalities
used in dermatology to modulate epidermal
growth and differentiation. They are often used in
treatment of inflammatory skin disorder like pso­
riasis. Whether they can improve the disruption of
skin barrier and irritation by irritants is not
known.
Topical corticosteroids are frequently used in
ICD (6) due to their anti-inflammatory effects. A
previous study demonstrated a reduction o f mitotic activity of the keratinocytes in psoriatic
lesions associated with clinical improvement after
treatment by topical steroid (7). Furthermore, the
expression of skin-derived antileukoproteinase
(SKALP), an elastase inhibitor which is induced in
■ SOS+Calclpatrlol
■ SDS+Vehicle
Ü SDS
a Normal skin
10
5
V | V ', I
0
r, !•:
dl
d2
d3
d4
as
d6
JV.Vl
ï." I
V;
j : ': : '.I '
d7
dl°
dl4
Fig. 2. Time course of transepidermal water loss (TEWL) from
day 0 to day 14: (A) o f the SDS-exposed skin and the triamcinolon-treated group versus its vehicle; (B) o f the SDS-exposed
skin and the tretinoin-treated group versus its vehicle; (C) o f
the SDS-exposed skin and the calcipotriol-treated group versus
its vehicle.
EFFECTS ON SDS-INDUCED SKIN IRRITATION
'■à
.
titmatmL'.- -f
■■of .
■ :-V:
'
Sv^Kï
Fig. 3. H&E sections showing the difference between the triamcinolon-treated group versus its vehicle with respect to acanthosis,
hypergranulosis and perivascular infiltrate. (A) with triamcinolon treated; (B) with vehicle treated.
tures were incubated with la,25-(OH)2-D3, pro­
liferation o f T-lymphocytes was reduced (19). Calcipotriol is the first vitamin D 3-derivative registered in several European countries. Calcipotriol
entiation o f epidermal cells, resulting in improvement of psoriatic skin lesions (20, 21). A side-elTect
of psoriasis treatment with topical calcipotriol is
skin irritation. A study o f Serup et al. (22) demon­
strated that calcipotriol ointment was a mild irri­
tant of the non-corrosive type, i.e., with no influ­
ence on the skin barrier. The irritant effect of calci­
potriol ointment was also noted in some clinical
studies (23, 24). The vehicle o f calcipotriol ointin part responsible for
ment may be at
skin irritation.
In this study, we examined the effects of a topicetonide 0.05%
cream), a retinoid (tretinoin 0.025% cream) and a
vitamin D} derivative (calcipotriol ointment) in irritant reactions induced by repeated exposures of
the skin to SDS. The effects were studied clinically
(erythema), functionally (TEWL) and immunohistochemically using markers of epidermal
growth, known as Ki-67 antigen, present in cycling
cells: involucrin and E-FABP, two terminal differ­
entiation markers (25-27); skin-derived-anti leukoproteinase (SKALP) and cytokeratin 16, two molecules associated with hyperproliferation and inflammation (8, 28)
Materials and Methods
24 healthy volunteers with no history of skin diseases (14 male, 10 female, mean age 31, ranging
from 19 to 57 years) participated in the study,
After approval of Medical Ethic Committee, informed consent was obtained from all subjects,
200 //I of SDS (0.2%) (w/v), pipetted on the patches
consisting o f a 1.5 cm piece of absorbent, non2
on a 4 cm
wove n
, which was daily fixed at the same
site with tape (Fixomull® stretch, Beiersdorf AG,
Hamburg) to the back of the subjects for 4 h for 5
consecutive days, from day 0 to day 4. After re­
moval of the patches, 0.5 cm of the cream or ointment (about 0.35 0.40 g) was applied daily over
the area of previously SDS-exposed skin, dried at
room temperature for 30 min. The cream or ointment was applied for 7 consecutive days.
The following formulations were used: (i) triamcinolon acetonide (0.05%) cream; (11 ) tretinoin
(0.025%) cream; (iii) vehicle of triamcinolone and
tretinoin cream consisted of cetomacrogol cream;
(iv) calcipotriol ointment (Daivonex®, Leo, Danmark); (v) vehicle of calcipotriol ointment.
99
LE ET AL.
scoring and transepidermal water loss
(T E W L ) measurements
Number of cycling cells/mm
A
300
250
Visual grading and TEWL were assessed at: day 1
to day 7, day 10 and day 14. TEWL was measured
with a Tewameter™ 210 (Courage & Khazaka,
Cologne), according to standard guidelines (29).
Room temperature varied from 20°C to 25°C and
relative humidity from 40% to 60%. Clinical scor­
ing (erythema) was graded visually: 0, no response;
1, slight, patchy redness; 2, diffuse mild redness;
3, moderate redness; 4, intense redness; 5, intense
redness with edema.
200
150
100
50
s•/.;•;<,:.' ;•/Isss«*,VI^i;'/ >V^ J W - s'•
day*!
day2
Involucrin expression
B
60
©SDS+Tretlnoln
50
SSDS+Vahlcle
OSDS
A
0
30
Biopsy procedures
2Q
A total o f 40 punch biopsies (3 mm diameter)
were taken: 16 biopsies at day 2 to examine the
histology of the triamcinolon-treated group, the
tretinoin-treated group, the vehicle-treated group
I
10
0
day4
day2
E-FABP expression
c
ro
60
Number of cycling cells/mm
A
50
@SDS+Tretinoin
BSDS+Vehicle
□ SOS
40
300
jgSDS+Triamcmolone
250
30
GSlSDS+Vehlcle
20
a sas
200
10
150
0
day2
too
Fig. 5. Immunohistochemical stainings o f the tretinoin -treated
group versus its vehicle. The results of (B) and (C) are expressed
as a % o f the positive stained-cell layers of the total epidermal
cell layers. The values are mean±SEM: (A) number o f prolifer­
ative cells per mm length section; (B) involucrin expression; (C)
E-FABP expression.
50
0
day2
day4
involucrin expression
B
day4
60
ISJSDS^Trfflmdnolone
SO
0 SDS+Vöhicle
OSDS
and the SDS-exposed group; 24 biopsies at day
4 for these 4 groups and the calcipotriol-treated
group and its vehicle. Thus, 4 biopsies per treat­
ment/per timepoint. After 4 h fixation in form­
alin, the samples were embedded in paraffin, and
sectioned at 6 pm for immunohistochemical
staining.
30
20
I
10
day4
day2
E-FABP expression
c
70
V— .
‘ • -'ä
- w , V
- a n
r t i 1
¡ i —
i*~ —
i
|
ij
© S OS +Tnnmcinolona
ao
©SDS+Vehiclû
so
OSDS
Immunohistochemical methods
AO
30
20
10
''Vr :r'\ ;f;::!.Vf.r y,V;ò Ì-y-v:
. • ( I* r ! • V î i ^
: *> / : V
* f [: **.• V .• '
' ï ; C y : V r- r v
0
day 2
.V r ;•.
f1
îv
^ . 'ss s ^
day4
Fig, 4. Immunohistochemical stainings of the jriamcinolon_
.
(Q
expressed as a % of the positive stained-cell layers of the total
epidermal cell layers. The values are mean±SEM: (A) number
o f proliferative cells per mm length section; (B) involucrin ex­
pression; (C) E-FABP expression.
The biopsies were stained with the following
antibodies according to standard procedures, deies (4): (i) MIB-1 (ImmuA. Marseilles
Ki-67 antigen on formalin
material; (ii) M ON-150. which
volucrin, a structural precursor protein for the
formation of the cornified envelope, is described
and prepared previously (30); (iii) Anti E-FABP,
rabbit antiserum
mal
d binding pro te
which is associ-
EFFECTS ON SDS-INDUCED SKIN IRRITATION
ated with terminal
•entiation (kindly provided by Siegenthaler (26, 27); (iv) Anti SKALP/
elafin, a polyclonal antiserum directed against recombinant SKALP/elafin is prepared as we described previously (8, 9); (v) Ks8.12 (Sigma, St
Louis, MO, USA) was used to detect cytokeratin
16 which is expressed in suprabasal layers in the
hyperproliferative tissues (28); (vi) H&E accord­
ing to Mayer.
nim length of section. The expression o f involucrin
and E-FABP was similarly scored: at a representative interpapillary area, the ratio o f positively
stained cell layers and the total number of cell
layers, were calculated.
The Student /-test and the Wilcoxon test were used
when appropriate.
His to Iogi cal exam in a ti on
We quantified the number proliferative cells by
counting the number of M IB-1-positive nuclei per
A
Number of cycling colls/mm
200
150
100
50
H istology and immunohistochemistry
0
H & E staining. The exposed skin of day 4 showed
day 4
B
Involucrin expression
60
QSDS+Calcjpolrlol
0 S D S + Vehicle
50
40
30
20
10
0
day4
E-FABP expression
C
00
□ SDS+Calclpotriol
■ SDS+Vehlcle
40
30
20
10
0
Clinical grading and TEWL measurements
Repetitive exposure of human skin to SDS (0.2%)
induced an increase in erythematous reaction (Fig.
1) and in TEWL measurement (Fig. 2), enhancing
in intensity up to day 4, then gradually decreasing
to day 14. No significant difference in erythema
or in TEWL was found between the triamcinolon
acetonide, tretinoin or calcipotriol treated groups
versus their vehicles.
250
50
Results
s . ' S ; ;r-'i/>
:•'. ^
/>»•::■.•:-va-;; •r.<><;.->:r»y;^!
' o.v: Al•^
y:•■i* >v*vv.<- ?s
»*/fa>^ìUh.
'f.<\\S
day4
Fig. 6. immunohistochemical stainings of the calcipotrioltreated group versus its vehicle. The results of (B) and (C) are
expressed as a % of the positive stained-cell layers o f the total
epidermal cell layers. The values are m ean±SEM : (A) number
of proliferative cells per mm length section; (B) involucrin ex­
pression; (C) E-FABP expression.
mild to moderate parakeratosis, hypergranulosis,
acanthosis, exocytosis and moderate perivascular
infiltrate composed of mostly mononuclear cells
and some eosinophils. The sections of the triamci­
nolon acetonide cream treated skin demonstrated
a slightly milder perivascular infiltrate, less para­
keratosis and less acanthosis as compared to its
vehicle (Fig. 3). No difference between the tretinoin- or calcipotriol-treated skin and their vehicles
was observed with respect to hypergranulosis, pa­
rakeratosis, spongiosis, acanthosis and perivas­
cular infiltrate.
proliferation. The response of un­
treated skin, repetitively exposed to SDS, was
characterized by an increase in the number of
M IB-1-positive nuclei from 78±22 per mm
a maximum of 180±50
(mean
4 after stimulation. The
number of proliferative cells o f all
groups was also enhanced up to day 4. A
mar
cu it
(/;==().02) in
number of cycling cells/mm (72±14) of the tri­
amcinolon acetonide treated sections was ob­
served as compared to the other exposed sites
(Fig 4a). No significant difference in the number
of cycling cells/mm between the tretinoin- or calcipotriol-treated groups versus that with their vehides, was seen (Figs. 5a, 6a).
Epidermal differentiation. To study the effect o f
the verum drugs on epidermal differentiation in
our model, we investigated the expression of invol-
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Fig. 7. Histological series demonstrating the difference in the number of cycling cells/mm (A and B) and in the involucrin e x p r e s s io n
{A, C) with trimacinolon treated. (B, D) with vehicle tr e a te d .
respectively.
ucrin and E-FABP. Repetitive application of SDS
caused an increase in the expression of involucrin,
and an increase in the expression in E-FABP. At
day 4, a small decrease in involucrin expression of
borderline significance 09=0.09) and a non-sig­
nificant decrease in E-FABP expression (/;=0.25),
were found in the triamcinolon acetonide-treated
group versus its vehicle (Fig. 4b, c). There was no
difference in the expression of involucrin and E-
shown).
EFFECTS ON SDS-INDUCED SKIN IRRITATION
expression o f the triamcinolone-treated group versus its vehicle are illustrated in Fig. 7.
Discussion
The induction of irritant reactions by repetitive exposure to SDS and the repair with or without
treatment were studied using visual scoring,
TEWL and markers of epidermal proliferation
and differentiation. Repetitive exposure to SDS
causes an increase in erythema and in TEWL to a
maximum at day 4, then gradually decreases up
to day 14 after the first challenge. The repetitive
exposure to SDS not only induced histological
changes to the epidermal cell compartment (parakeratosis, perivascular mononuclear infiltrates)
but also upregulated the expression o f markers of
epidermal differentiation and proliferation, corresponding to a hyperproliferative skin. Treatment
of skin irritation by triamcinolon acetonide, retinoic acid or calcipotriol did not improve the clinical scores in this model. Neither significant de
crease in erythema scoring nor decrease in TEWL
was observed. In contrast to this clinical finding,
we found a statistically significant reduction of the
number of cycling cells in the triamcinolonetreated skin sites versus the other exposed sites. In
addition, the expression of involucrin and E-FABP
was also downregulated in the triamcinolontreated skin as compared to that treated with its
vehicle.
The fact that triamcinolone acetonide cream in­
duces a reduction of cycling cells may indicate that
triamcinolon acetonide cream causes less irritation
or delays repair. In contrast to other studies (31,
32) in which topical steroids are believed to be
diminish the erythematous response of SDS ex­
posed skin, we could neither demonstrate this ef­
fect in this model nor in a previous work (33).
Treatment with retinoic acid did not lead to any
change in visual scoring, TEWL and modulation
of the epidermal response to SDS, as compared to
its vehicle. Adding retinoic acid to keratinocytes in
vitro reduces transglutaminase activity and consequently may inhibit terminal
srentiation and
concomitant cor
envelope formation (34).
Retinoic acid can induce epidermal hyperprolifer­
ation and increase in transglutaminase immunoreactivity when applied topically for 4 months to
photoaged human skin (35). Furthermore, retinoic
acid can increase the expression of transglutamina­
se, loricrin, involucrin, filaggrin when applied to
normal human skin for 4 days under occlusion
(36). Here, we found no alterations in involucrinand E-FABP-expression at the tretinoin-treated
sites. The exact mechanism of retinoic acid in the
SDS-induced skin irritation remains unknown.
25
Mn
Calcipotriol did not lead to modulation o f the epidermal response after exposure to SDS with the
exception of an increase in TEWL. This may be
due to its vehicle, which is known to be irritant.
At day 5 after challenge, a small, non-significant
increase (/?=0.18) in TEWL of the vehicle of calcipotriol-treated sites versus the SDS exposed sites
was found. In our present study, repetitive exposure to SDS for 5 days induced an increase in
number of cycling cells and the expression of involucrin and E-FABP. Treatment with retinoic acid or
calcipotriol in this irritant skin did not show any
alteration with respect to these parameters.
Topical steroids may influence irritant reactions.
Skin may deal with repetitive irritant stimuli with
adaptation, regeneration or disease (irritant contact dermatitis). Intervention may influence the inflammatory response (7) and consequently may
prevent tissue damage as caused by the inflammation, but may also hamper repair processes by
its anti-mitotic activity. Therefore, it cannot be
concluded from our data that topical steroids are
beneficial to the skin.
Single exposure tests are inadequate for prediction o f the effect of treatment modalities. Cumulative exposure models or clinical studies with standardized exposure are needed.
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Address:
T.K.M. Le
P. O. Box 9101
6500 HB Nijmegen
The Netherlands