ARTICLE
Auteur(s) : Verena Herbst1, Margot
Zöller2,3, Sabine Kissling1, Elke
Wenzel1, Nathalie Stutz1, Pia
Freyschmidt-Paul1
1Dept. of Dermatology, Philipp University,
Deutschhausstrasse 9, 35033 Marburg, Germany
2Dept of Tumor Progression and Immune Defence, German
Cancer Research Center, Heidelberg, Germany
3Department of Applied Genetics, University of
Karlsruhe, Germany
accepté le 1 Juin 2006
Alopecia areata (AA) is an autoimmune disease affecting anagen
stage hair follicles [1] and is mediated by peri- and
intrafollicular CD4+ and CD8+ T-cells [2-5].
Resistance to apoptosis of activated peripheral blood T-cells is
one of the hallmarks in human AA [6]. This resistance is associated
with downregulation of CD95L (FasLigand) and upregulation of CD44v7
that is known to be associated with anti-apoptotic gene expression
[7].At present the local application of a contact sensitizer like
diphencyprone or squaric acid dibutylester is the most effective
treatment of AA [8-11]. The mechanism underlying the therapeutic
effect of repeated elicitation of a contact dermatitis in AA is
still unknown. Several studies that focused on this topic revealed
a decrease in the CD4:CD8 ratio in the perifollicular infiltrate
[12]. Furthermore IFN-gamma expression was reduced in skin biopsies
of AA patients after DCP treatment, whereas mRNA expression of
IL-2, IL-8, IL-10, and TNF-alpha was increased, suggesting major
changes in the cytokine expression profile by treatment with a
contact sensitizer [13]. Recent studies in the C3H/HeJ mouse model
of AA provided evidence for unimpaired T-cell extravasation.
However, antigen presenting cell (APC) migration towards the
draining lymph node was impaired which could well account for
inefficient stimulation of AA-specific autoreactive T-cells [14].To
further elucidate the mechanisms underlying the therapeutic effect
of a contact sensitizer in AA, we analyzed skin biopsies of AA
patients before and after treatment with the contact sensitizer
diphenylcyclopropenone (diphencyprone, DCP). We focused on evidence
for impaired leukocyte extravasation or a hampered APC migration
and, in view of the recently reported apoptosis resistance of
autoimmune T-cells in AA [7], on apoptosis induction. We wanted to
obtain insight into the underlying mechanism by exploring Fas/Fas
Ligand (FasL) expression, which accounts for receptor-mediated
apoptosis, and CD44v7 expression, which supports upregulation of
anti-apoptotic proteins.
Material and methods
Patients and samples
A total of 23 persons were included in this study: 10 patients with
severe, untreated AA, 10 patients with initial hair regrowth after
DCP treatment and 3 volunteers with healthy scalps and no hair
loss. All AA-patients showed at least 25% hair loss for more than 3
months. 10 patients agreed to give a biopsy before treatment and 10
patients did this after successful treatment with DCP. Treatment
with the contact sensitizer DCP was performed as described
elsewhere [8, 11, 15]. Briefly, after sensitization with 2% DCP, an
individually chosen concentration of DCP was applied once weekly on
the scalp to elucidate a moderate contact dermatitis. To exclude
spontaneous hair regrowth the contact sensitizer was initially
applied unilaterally.
After written informed consent, an elliptical excision biopsy
was performed. The biopsy was taken from bald scalp in untreated
AA-patients, from DCP-treated areas showing initial hair regrowth
in successfully DCP-treated patients, and from normal unaffected
scalp skin in volunteers as a control. In treated patients, the
biopsy was performed one day after application of DCP. Patients
with untreated AA were observed for at least 3 months to confirm
that they do not show hair regrowth in the area of biopsy, and
DCP-treated patients were likewise observed during three months to
ensure that hair regrowth continued under treatment.
Immunohistochemistry
Immunohistochemistry was performed as previously described [9].
Briefly, 7-8 μm thick vertical cryostat sections were
air-dried and fixed in 1% paraformaldehyde. Between all incubation
steps, sections were washed with Phosphat-buffered saline (PBS).
Non-specific binding was blocked by the avidin-biotin-blocking-Kit,
10% normal goat serum and 10% low fat milk powder. Subsequently,
the slides were incubated for 1 hour at 37 °C with the first
antibody or unrelated control IgG-antibodies as negative control.
Sections were washed and incubated with biotinylated secondary
antibody for 30 minutes. After washing, a routine staining method
for avidin-biotin complex labeled with alkaline phosphatase was
used (Vector Laboratories, Burlingame, CA) and counterstained with
Mayer’s hematoxylin.
For TUNEL-double staining the Tunel ApopTag® Plus
Peroxidase In Situ Apoptosis Detection Kit, S7101 (Chemicon) was
used according to the manufacturer’s instructions. Afterwards the
slides were washed and subsequently incubated with the primary
antibodies for CD1a, CD4, CD8, CD68 and Fas at 4 °C overnight.
The staining was then continued as the single staining described
above.
For CD1a, CD4, CD8, CD68, CD44s, CD44v7, CD44v10, Fas and FasL
the number of peri- and intrafollicular infiltrating
CD4+ and CD8+ T cells was assessed
semi-quantitatively by use of three categories: no cells: –, single
cells: (+), mild infiltrate: +, moderately dense infiltrate: ++,
and dense infiltrate: +++. The peribulbar area of the hair
follicle, the mid part of the hair follicle between sebaceous gland
and hair bulb and the dermis were assessed separately. The average
per group was calculated for untreated AA-patients, DCP-treated
AA-patients and controls.
Antibodies
The following monoclonal antibodies were used as first antibodies:
mouse anti-CD1a (010), mouse anti-CD4 (MT310), mouse anti- CD8
(C8/144B), mouse anti-CD68 (M0814), all from DAKO; mouse anti-CD44s
(SFF-2), mouse anti-CD44v10 (VFF-14), mouse anti-44v7 (VFF-9), all
from Bender MedSystemsTM; mouse anti-CD49d (HP2/1) from
Abcam, mouse anti-Fas (DX2) from R&Dsystems, rat anti-FasL
(Mike-1) from Alexis. As secondary antibodies, biotinylated goat
anti rat (Code 112-065-167) or biotinylated goat anti mouse (Code
115-065-003) from Jackson ImmunoResearch were used.
Quantitative histomorphometry
This technique was performed to determine the number of
perifollicular and intrafollicular TUNEL-positive cells and
TUNEL-CD1a, TUNEL-CD4, TUNEL-CD8, TUNEL-CD68, TUNEL-Fas double
positive cells. Digital images were taken with a CC-12 digital
camera (Olympus) at 10-fold magnification. The number of positively
stained cells was assessed for each hair follicle by means of the
Cell software (Olympus), and the mean numbers of positive cells per
hair follicle were calculated for each patient/volunteer.
Statistical analysis
The differences between untreated AA patients and healthy
volunteers and between untreated and DCP-treated AA patients in the
number of peri- and intrafollicular TUNEL-positive and TUNEL-CD1a,
TUNEL-CD4, TUNEL-CD8, TUNEL-CD68, TUNEL-Fas double positive cells
were analyzed by the Mann-Whitney U test.
Results
Semiquantitative analysis of perifollicular infiltrates
Semiquantitative assessment of peri- and intrafollicular
infiltrating cells in the peri- and suprabulbar region of the hair
follicle showed a moderately dense infiltrate of CD4+
and CD68+ cells (macrophages) in untreated AA, the
infiltrate of CD8+ cells was mild to moderately dense.
Single CD1a+ (dendritic cells) could be observed, they
were located predominantly within the hair follicles ( (figure 1A) ). Healthy
controls showed single perifollicular CD68+ cells only,
whereas no CD1a+, CD4+ or CD8+
cells could be found. In successfully DCP-treated patients, the
number of infiltrating cells in the peri- and suprabulbar region
increased. Subtyping of infiltrates by staining for CD1a, CD4, CD8
and CD68 revealed a slight increase in the number of
CD1a+ and CD8+ cells while the number of
CD4+ and CD68+ cells stayed unchanged (table
1( Table 1 ), ( figure 1 )). CD1a
+ cells were found within the hair follicle as well as
in the perifollicular infiltrate. The dermal infiltrates showed a
striking increase in infiltrating CD1a+, CD4+
, CD8+ and CD68+ cells (data not shown).
To investigate the involvement of the Fas-FasL system in
hair-growth induction by DCP treatment, staining for the death
receptor Fas and its Ligand FasL was performed. The Fas receptor
was expressed mildly to moderately on single cells of the peri- and
intrafollicular infiltrate in untreated AA (table 1, ( figure 3 )), while it was
strongly expressed on the hair follicle epithelium ( (figure 3) ). The FasLigand
(FasL) was only seen on single cells in untreated AA. It was also
expressed on the hair follicle epithelium (( figure 1 ), table 1). In
DCP-treated patients, there was an increase in Fas expression and a
striking increase in FasL expression in the peri- and
intrafollicular infiltrate.
Staining for panCD44, CD44v10 and CD49d revealed only a slight
increase in biopsies from DCP-treated patients as compared to
untreated AA patients’ biopsies. Expression of CD44v7 remained
unaltered (table 1).
Table 1 Semiquantitative assessment of the peri- and
intrafollicular infiltrate in untreated and successfully
DCP-treated AA
|
Untreated AA
|
DCP-treated AA
|
Healthy controls
|
|
CD1a
|
(+)
|
+
|
-
|
|
CD4
|
++
|
++
|
-
|
|
CD8
|
+ / ++
|
++
|
-
|
|
CD68
|
++
|
++
|
- /(+)
|
|
CD49d
|
++
|
++ / +++
|
(+)
|
|
Fas
|
(+)
|
+ / ++
|
- / (+)
|
|
FasL
|
(+)
|
++
|
- / (+)
|
|
CD44std
|
++
|
++ / +++
|
+
|
|
CD44v7
|
+
|
+
|
- / (+)
|
|
CD44v10
|
(+)
|
+
|
- / (+)
|
Quantitative analysis of apoptotic cells within the
perifollicular infiltrates
To assess the number of apoptotic cells in the peri- and
intrafollicular infiltrate, quantitative histomorphometry of
TUNEL-single and double stainings was performed. Healthy controls
showed no perifollicular TUNEL-positive cells (data not shown).
However, there was a highly significant (p < 0.001) increase in
TUNEL-positive cells in the peri- and suprabulbar region of
DCP-treated compared to untreated AA biopsies (( figure 2A ) and ( figure 3 )).
To determine the involvement of the Fas-FasL system in
apoptosis-induction, TUNEL-Fas double staining was performed,
double expression of Fas and TUNEL providing evidence of
receptor-mediated apoptosis. In untreated AA, TUNEL-Fas double
positive cells were only rarely found and not in all biopsies,
whereas double stained cells were present in all DCP-treated
patients in the peri- and intrafollicular area. Although the
difference in the presence of TUNEL-Fas double positive cells
between treated and untreated patients was statistically highly
significant (p < 0.001), only 6.4 cells per perifollicular
infiltrate were TUNEL-Fas double positive (( figure 2B ) and ( figure 3 )).
To define the leukocyte subpopulation that undergoes apoptosis,
subtyping of TUNEL-positive cells by double staining with CD1a,
CD4, CD8 and CD68 was performed and showed a statistically
significant increase in all cell types of DCP-treated patients, but
with gradual differences (p = 0.005 for TUNEL-CD1a, p < 0.001
for TUNEL-CD4 and TUNEL-CD8, p < 0.05 for TUNEL-CD68). While 9.4
CD4+ and 4.7 CD8+ TUNEL-positive cells were
recovered per hair follicle ( (figure 2C) ), only a small
portion of CD68+ cells (mean 2 cells per hair follicle)
and of CD1a+ cells (0.8 cells per hair follicle) were
TUNEL-positive.
Apoptosis in hair follicle keratinocytes
In addition to apoptosis induction in the leukocyte infiltrate, DCP
treatment was accompanied by a striking decrease in TUNEL-positive
hair follicle keratinocytes, while TUNEL-positive hair follicle
keratinocytes were regularly found in untreated AA patients ((
figure 3A and B
)). Healthy controls showed no TUNEL-positive hair follicle
keratinocytes. Fas and FasL was expressed on hair follicle
keratinocytes in untreated as well as in DCP-treated AA-patients
and in healthy controls (( figure 3 ) E and F). Fas
and FasL expression on hair follicle epithelium did not differ
between the three groups. On the other hand, TUNEL-Fas double
positive keratinocytes were not detected in any group.
Discussion
Semiquantitative analysis of peri- and intrafollicular infiltrates
confirmed the observation of previous studies in human AA [12] and
AA of C3H/HeJ mice [9] that successful treatment of AA with a
contact sensitizer is accompanied by an increased number of
infiltrating leucocytes in the bulbar and suprabulbar area of the
hair follicle. The observed increase in perifollicular
CD8+ cells confirms the findings of Happle et al. [12]
who found that treatment with a contact sensitizer changes the
composition of the peribulbar infiltrate with a CD4/CD8 ratio of
4:1 in untreated progressive AA and a decreased CD4/CD8 ratio of
1:1 in AA, treated successfully with a contact sensitizer.
Furthermore, the elicitation of an allergic contact dermatitis by
application of DCP obviously does not only attract lymphocytes to
the dermis and epidermis but also to the peri- and suprabulbar area
of the hair follicle. The paradoxical fact that an increased
perifollicular lymphocytic infiltrate leads to hair regrowth in AA,
a disease characterized itself by perifollicular infiltrates,
obviously implies that newly recruited leukocytes suppress the
pathological activity of autoreactive hair-follicle specific
CD4+ and CD8+ cells by not yet defined
mechanisms. We considered induction of apoptosis as a likely
mechanism, because the elicitation of an allergic contact
dermatitis has to be repeated for months to observe hair regrowth.
Thus, induction of activation-induced cell death could possibly
become an important factor.
In fact, peribulbar CD4+ and CD8+ cells
undergo apoptosis. This can be concluded from TUNEL-staining which
revealed a striking increase in the number of apoptotic peri- and
suprabulbar CD4+ and CD8+ cells. Even though
the lymphocytes causing AA cannot be distinguished from
DCP-specific lymphocytes by immunohistochemistry, it is rather
likely that the AA-specific lymphocytes become apoptotic, because
biopsies were already taken one day after application of DCP when
recruitment of delayed type hypersensitivity effector cells is
still progressing [16].
Clues towards the DCP-induced mechanism of apoptosis were
obtained by Fas, TUNEL-Fas and CD44v7 staining. The increased
numbers of TUNEL-Fas double positive cells within the peri- and
suprabulbar part of the hair follicle in DCP-treated patients
suggest that apoptosis is mediated via the Fas receptor. The
increased expression of FasL on the perifollicular infiltrates in
DCP-treated individuals supports this hypothesis. Zoeller et al.
[7] have demonstrated that peripheral blood lymphocytes in AA show
a downregulation of FasL-expression, making them resistant to
apoptosis. Because the mediation of apoptosis in autoreactive
T-cells via the Fas-FasL system plays a crucial role in the
homeostasis of the immune system inducing the prevention of
autoimmune diseases [17-19], a downregulation of FasL on peripheral
blood lymphocytes may be a crucial factor in development of AA. On
the other hand, repeated antigenic stimulation of T-cells leads to
upregulation of Fas and FasL and subsequently to activation-induced
cell death (AICD) [17, 18]. Thus, the repeated activation of T
cells by weekly application of DCP could well promote
activation-induced cell death. This hypothesis is supported by a
recent study demonstrating that, in the immune response to the
contact sensitizer trinitrophenyl, CD8+ T-cells suppress
CD4+ T-cells in a Fas-dependent manner, resulting in
increased apoptosis of CD4+ T-cells in the presence of
CD8+ T-cells [20]. Whether this mechanism accounts
exclusively for CD4+ T-cells in DCP-treatment of AA
remains to be explored. So far, our findings suggest that apoptosis
may also become induced in CD8+ cells albeit to a lesser
degree. To further support our hypothesis it would be interesting
to assess whether patients who do not respond to treatment with a
contact sensitizer show impaired AICD via the Fas-FasL system.
Future studies should be performed to answer this question, but
they will bear the methodical difficulty that it cannot be excluded
in advance that patients who have not responded to treatment until
the time point of biopsy will respond to treatment if it is
continued for a longer time period. Whether the expression of FasL
on hair follicle keratinocytes is also involved in the induction of
apoptosis of perifollicular lymphocytes cannot be determined from
our data, because the expression of FasL on the hair follicle
epithelium does not differ between untreated and DCP-treated AA.
Because CD44v7 expression is of major importance in the resistance
to apoptosis of gut infiltrating leukocytes in chronic inflammatory
bowel disease [21], and because upregulated CD44v7 expression has
been observed in peripheral blood leukocytes of AA patients [7], we
also analysed CD44v7 expression in skin biopsies, which was
unchanged in DPC-treated as compared to untreated AA biopsies.
Thus, apoptosis induction by downregulation of CD44v7 is obviously
not involved.
A repeated elicitation of allergic contact dermatitis could also
interfere with leukocyte traffic, where CD44 and CD49d or, more
strongly, a CD44-CD49d complex, is known to be important in
leukocyte extravasation in autoimmune disease, as well as in
allergic contact eczema [22]. Moreover, there is strong evidence
that CD44v10, in particular, functions as a skin homing receptor
[23]. Antibody blocking studies in the mouse model of AA confirmed
the importance of CD44, particularly of CD44v10 and CD49d, for
leukocyte extravasation in AA and in the repeated elicitation of
allergic contact dermatitis [22, 24], but also provided evidence
that leukocyte extravasation in AA may not be hampered by allergen
treatment [14]. In line with the results of animals studies, an
increased expression of CD44s and CD44v10 within the perifollicular
infiltrates of DCP-treated AA patients, argues against a blockade
of T-cell extravasation by downregulation of CD44s or CD44v10 due
to DCP therapy. That T- cell extravasation is not hampered by DCP
treatment becomes also obvious from the increased number of
infiltrating CD4+ and CD8+ cells around the
hair follicles of DCP-treated AA-patients. Finally, and also in
line with the above mentioned study in allergen treated AA mice, we
noted an increased number of CD1a+ dendritic cells in
DCP-treated AA patients. In the murine AA model, migration of
dendritic cells towards the draining lymph node is severely
impaired in mice receiving topical applications of a contact
sensitzer. A reduced antigen transfer and presentation with the
consequence of insufficient T-cell activation could well contribute
to DTH-induced prevention of AA progression and to hair
regrowth.
In summary, we provide evidence that hair regrowth in AA
patients receiving repeated topical applications of a contact
sensitizer over a prolonged time period may at least in part be due
to induction of apoptosis in perifollicular CD4+ and
CD8+ cells, most likely mediated by the Fas-FasL system.
Moreover, our data support the concept that treatment with a
contact sensitizer has no impact on T-cell extravasation, but
rather impairs migration of dendritic cells towards the draining
lymph nodes [25].
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft,
grant Zo 40/9-1 (MZ) and FR 1509/1-2 (PFP) and by a grant of
Alopecia Areata Deutschland (AAD) to PFP.
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