ARTICLE
Auteur(s) :, Aki Sakai, Yuki Yamamoto, Koji Uede, Fukumi
Furukawa*
Department of Dermatology, Wakayama Medical University, 811-1
Kimiidera, Wakayama, Japan 641-0012
accepté le 16 Mars 2005
Chemical peeling is used to exfoliate the surface of the skin.
Various chemical agents peel the skin at specific depths of the
skin, and the subsequent inflammation and wound healing advance the
skin regeneration process [1]. Chemical peeling is one of the
dermatological treatments available for acne, solar lentigo and so
on [2-4].Chemical agents injure the corneum, and damage the
function of the skin barrier. The mechanisms responsible for the
actions of chemical peeling are not understood completely. However,
our recent studies revealed that chemical peeling involved several
biological mechanisms, such as allergic or non-allergic contact
dermatitis, as well as wound healing [5-8].Since it is not clear
how these agents affect the skin immune system, we studied the
Langerhans cells (LCs), which play a central role in the immune
surveillance system as an antigen presenting cell [9], in epidermis
which was treated with trichloroacetic acid (TCA) and liquid
nitrogen.
Materials and methods
40% and 60% TCA (w/v)-in distilled water were applied once on the
inside of the upper arm of 3 healthy Japanese adults, and the
peeled sites were left open, as has been described in our recent
reports [5-7]. For the control, the upper arm near the TCA
application site received a single freeze-thaw cryosurgery using a
cotton ball (5 mm in diameter) with liquid nitrogen for 5
seconds. After the peeling, 4 mm biopsy specimens were obtained at
2, 6, and 12 hours, and at 1, 2 and 7 days. They were embedded in
Tissue-Tek OCT compound (Sakura Finetechnical Co. LTD., Tokyo,
Japan), snap-frozen in liquid nitrogen and stored at
– 80 °C. Cryostat sections, 6 μm thick, were
air-dried at room temperature for 1 hour, fixed in acetone for 5
minutes, rinsed in phosphate-buffered saline (PBS, pH 7.2), and
then incubated immediately with murine monoclonal antibodies (mAbs)
to clone 010 (human CD1a, DAKO, Glostrup, Denmark), clone TAL.1B5
(human HLA-DR, DAKO, Glostrup, Denmark), and Lag (from Kyoto
University). The mAb designated Lag specifically reacts to Birbeck
granules (BGs) and their related structures in human LCs [10]. The
specimens were rinsed again in PBS, and incubated with goat
anti-mouse immunoglobulins conjugated to a peroxidase labeled
dextran polymer (DAKO Envision plus HRP system, DAKO Cytomation,
Carpinteria, Ca, USA) for 30 minutes. The reactions were visualized
with 0.2 mg/ml of 3,3’-diaminobenzidine tetrahydrochloride
(Dojin Chemical, Kumamoto, Japan) and 0.005% hydrogen peroxide. The
sections were then counterstained with Mayer’s hematoxylin for 20
seconds. For hematoxylin and eosin (HE) staining, they were fixed
with 10% paraformaldehyde and embedded in paraffin by a standard
protocol.
Positive (CD1a, HLA-DR and Lag) cells were counted at high
magnification (× 400) at four different fields according to our
previous report [11]. The number of positive cells was averaged
over three independent observations (AS, YY, KU). The average
number of positive cells per field and standard deviations were
calculated. Significance was calculated using the two-tailed
Student’s t-test, and a p-value < 0.05 was considered to be a
statistically significant difference.
Results
HE staining
There were no observable differences between the 40% TCA and 60%
TCA groups from 2 to 12 hours after treatment (figures 1 and 2).
Although the degree of tissue damage was dose-dependent, complete
epithelization was induced within 7 days in both the 40% and 60%
TCA groups.
When compared with the TCA peelings, liquid nitrogen induced the
appearance of round epidermal cells with marked nuclei from 2 hours
to 1 day post-treatment ( (figure 3) ). Few
vacuolated epidermal cells were present at day 2, and
re-epithelization was almost finished by day 7.
Immunohistochemical staining
The time-dependent changes in the LCs are summarized in ( figure 4 ).
In the 40% TCA group, the number of CD1a-positive cells
gradually decreased until day 7 ( (figure 5) ), whereas both
HLA-DR- and Lag-positive cells decreased until 12 hours, increased
until day 2and then decreased thereafter. On day 7, the score for
all 3 markers was significantly lower than that of the non-treated
skin (time 0). In the 60% TCA group, the number of CD1a-, HLA-DR-
and Lag-positive cells decreased gradually until day 1, increased
temporarily until day 2, and then decreased again until day 7. On
day 7, the LC numbers were very low, like those of the 40% TCA
group. When compared with CD1a-positive cells in 40% TCA treated
skin ( (figure
5) ), dendritic cells were found in greater numbers at 2hr,
6hr and 2 days after 60% peeling ( (figure 6) ).
In contrast, LCs in the liquid nitrogen-treated skin as a
control decreased gradually and slightly until day 2, but the
number of CD1a- and HLA-DR-positive cells on day 7 were
statistically similar to those at time 0. Phenotypic changes of
CD1a-positive cells were much less than those of TCA treated skins
( (figure 7)
).
Taken together, TCA reduced the number of epidermal LCs when
compared with liquid nitrogen, which suggests a temporary
impairment of the skin defense system following TCA treatment.
Discussion
LCs were originally described by by Steinmen and Cohn as a novel
type of immune cell in the peripheral lymphoid tissue of mice [12].
LCs are antigen presenting cells, which are capable of uptaking
exogeneous antigens, processing them and presenting them to naïve T
cells, where they are considered immature, becoming mature after
contact with the antigen [13]. LCs are of critical importance in
cutaneous biology, such as wound healing [14], allergic contact
dermatitis [11, 15, 16], cutaneous photobiology [17-19] and so on.
Any dysfunction of the LCs might induce epidermal abnormalities and
epidermal neoplasms. Experimental models have revealed that a
defect in local antigen presentation, caused by a decrease in the
number or activity of LCs, is a determining factor in the escape of
newly transformed cells from immune surveillance, and thus in its
ability to develop into a tumor [20, 21].
In this study, the epidermis was damaged by TCA peeling and
liquid nitrogen, and completed re-epithelization of the degenerated
skin was obtained by day 7. However, in both cases, the number of
epidermal LCs on day 7 was statistically lower than before
treatment. These changes resemble those of LCs in human allergic
contact dermatitis [22]. And when compared with liquid nitrogen,
which is a standard and safe tool for dermatological treatment, the
number of CD1a-positive-, HLA-DR-positive- and Lag-positive cells
in the TCA-treated skin did not recover to the normal range by day
7. Bartosik J et al. [23] found that two dendritic cell types of
LCs and melanocytes appeared to react differently to photodynamic
therapy, and they found morphological nuclear changes in some LCs
in skin 3 hours after tape-stripping and 5-aminolevulinic acid
hydrochloride-treatment. These changes look like those of LCs after
TCA peeling.
TCA is one of the most common agents for chemical peeling but a
long-term evaluation of its safety has not been performed.
Recently, paradoxical effects for TCA were reported by Dainichi et
al. [24]. They reported that chemical peeling with TCA might
increase the risk of mutations leading to tumorigenesis while it
might also reduce tumorigenesis by destroying atypical cells. They
showed a relatively increased number of squamous cell carcinomas
formed at the peripheral sites of TCA painting in ultraviolet
B-irradiated mouse models. Non-carcinogenic antigens like 2, 4,
6-trinitrochlorobenzene induce LC depletion and might have a role
in tumor promotion by establishing an immunosuppressive environment
[21]. Thus, we cannot deny the possibility that TCA may promote
hidden or subclinical tumors.
We found that the number of LCs with TCA peeling was
statistically lower than before treatment and than with liquid
nitrogen. Taken together, it is likely that TCA induced a temporary
impairment of the skin defense system. Therefore, long-term and
frequent TCA peeling will need special attention for unexpected
potential carcinogenesis.
Acknowledgments
This study was supported in part by the Japanese Ministry of
Education, Science, Culture, Technology and Sports, and the Segawa
Skin Research Grant.
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