ARTICLE
Alopecia areata (AA) is considered to be a T cell mediated autoimmune
disease of the hair follicle [1]. An infiltrate of CD4+ and
CD8+ T cells around the hair bulb is believed to cause reversible
patchy or total hair loss. Therefore, a therapeutic response would be
induced by immunosuppressive agents which act at the lower part of the
hair follicle. On the other hand, treatment of this psychologically disturbing
but not life-threatening disease should not bear serious side effects.
Hence, an immunosuppressive treatment should be performed topically to
avoid systemic side effects.
FK506 (tacrolimus) can be applied topically and suppresses activation
and proliferation of antigen-stimulated
T cells [2]. Topically applied FK506 is able to inhibit allergic contact
dermatitis [3, 4] and recent studies demonstrated that FK506 ointment
is effective and safe in the treatment of atopic dermatitis [5-7].
Hence, it seemed rational to test the efficacy of topically applied
FK506 ointment in AA. In a study using the Dundee experimental bald rat
(DEBR) model hair regrowth in AA could be achieved by treatment with a
topical FK506 solution [8]. The FK506 ointment, which has been tested
for safety in humans in clinical studies [5-7], has so far not been investigated
for its use in the treatment of human AA.
Because the C3H/HeJ mouse has been proven to be an appropriate animal
model for AA research by several studies concerning both pathogenesis
and treatment [9-13], we decided to test the efficacy of treatment with
0.1% FK506 ointment using this mouse model in a blinded, placebo controlled
study.
Materials and methods
Animals
C3H/HeJ mice with and without AA-like hair loss [9] were provided by
The Jackson Laboratory (Bar Harbor, ME). Because the number of mice that
develop AA spontaneously is very low, AA was induced in 10 unaffected
female C3H/HeJ mice as described previously [12]. In short, lesional skin
from an AA-affected C3H/HeJ mouse was grafted onto the back of unaffected
C3H/HeJ mice. Between 4-7 weeks after grafting the recipients had developed
initial hair loss. After they had developed AA on at least 50% of their
dorsal area, they were used for treatment studies. The extent of hair
loss on the back of the mice before treatment was assessed quantitatively
by means of an image analysis software (Sigma Scan Pro, Jandel Scientific,
San Rafael, CA) based on the photographs taken at the beginning of the
study. Briefly, photographs were scanned and areas of alopecic skin were
outlined using the software. The total area of alopecic skin on the back
of the mice was calculated and this was converted to a percentage of the
total back skin surface area visible in the photographs.
All mice were housed one per cage with controlled light cycles of 12-12h
light:dark and received acidified water (pH 2.8-3.2) and conventional
diet (altromin 1324, Altromin GmbH, Lage, Germany) ad libitum.
Treatment
0.1% FK506 ointment, and FK506-free ointment base, which served as placebo,
were kindly provided by Fujisawa GmbH (München, Germany). Six tubes
of 0.1% FK506 ointment and 4 tubes FK506-free ointment base were used
as study medication. Each tube was assigned to one mouse by random selection,
the study was performed double blind. All mice were treated once daily
5 days a week for 7 weeks. Two hundred and fifty mg of the study medication
was applied to the entire back skin of each mouse using a cotton applicator
over the course of the study.
Assessment of hair regrowth
Morphological changes were examined and documented daily. Photographs
were taken before treatment, and once per week during and after treatment.
By use of this photodocumentation, hair regrowth was assessed semiquantitatively
as complete, partial or no hair regrowth by two independent investigators.
Only mice with complete hair regrowth were included as hair regrowers
for statistical analysis. The one-sided exact Cochran-Armitage trend test
was applied to investigate the difference in complete hair regrowth between
the two treatment groups.
Histopathological examination and immunohistochemistry
After completion of treatment, all mice were anesthesized and their
dorsal skin was shaved. Afterwards, the animals were sacrificed by cervical
dislocation. Representative portions of both bald and hair-bearing skin
were removed, snap-frozen in liquid nitrogen and cut into 8-10 µm
vertical sections. For light microscopical examination, frozen sections
were stained with hematoxylin-eosin, and an Elastica-van-Gieson stain
for elastic fibers was likewise performed.
For immunohistochemical analysis, frozen skin sections were fixed in
acetone and air dried. Between all incubation steps, sections were washed
with Tris-buffered saline (TBS, 0.05 M, pH 7.6). Non-specific binding
was blocked by use of an avidin-biotin blocking kit solution (Vector Laboratories,
Burlingame, CA) and by 2% bovine normal serum and 2% goat normal serum
in TBS. Subsequently, the slides were incubated with the primary antibody,
diluted either 1:500 (anti-CD4, anti-CD8, anti-MHC class II) or 1:200
(anti-MHC class I) or 1:50 (anti-ICAM-1) in TBS containing 2% normal bovine
serum at room temperature for 1 hour. After washing, sections were incubated
with the secondary antibody, diluted 1:150 in TBS containing 4% mouse
normal serum and 2% goat normal serum for 30 minutes at room temperature.
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. Negative controls were performed
by replacement of primary antibody by normal rat IgG (Dianova, Hamburg,
Germany).
The following antibodies were used as primary antibodies: anti-CD4 (clone
RM4-5, rat IgG2a, Pharmingen, San Diego, CA), anti-CD8 (clone 53-6.7,
rat IgG2a, Southern Biotechnology, Birmingham, AL), anti-MHC class I (clone
ER MP 42, rat IgG2a, BMA, Augst, Switzerland), anti-MHC class II (clone
ER-TR 3, rat IgG2b, BMA, Augst, Switzerland), anti-ICAM-1 (clone KAT-1,
rat IgG2a, American Type Culture Collection, Manassas, VA). A biotinylated
goat anti-rat IgG (Dianova, Hamburg, Germany) was used as a secondary
antibody.
The number of infiltrating peri- and intrafollicular CD4+
and CD8+ cells and the aberrant expression of MHC class I,
MHC class II and ICAM-1 on mid and lower hair follicle epithelium was
assessed semi-quantitatively according to the following scoring system:
no positive cells/no aberrant expression (-); staining of isolated cells/involvement
of individual hair follicles (+); staining of a mild cell infiltrate/involvement
of multiple hair follicles (++); staining of an intense cell infiltrate/involvement
of most hair follicles (+++).
Results
Hair regrowth
The FK506-treated group consisted of 2 mice with large bald patches
(20% and 41% hair loss), and 4 mice with subtotal hair loss (48%, 70%,
79% and 85% hair loss) and 1 mouse with total hair loss. The FK506-free
base treated group comprised of 3 mice with large bald patches (17%, 30%
and 47% hair loss) and one mouse with diffuse hair loss, which cannot
be assessed quantitatively by the method used.
At the end of the study, 4/6 mice treated with 0.1% FK506 showed complete
hair regrowth on their backs with the exception of a bald ring around
the graft (Fig. 1, Table I). The remaining two FK506-treated mice
showed complete hair regrowth during the first 6 weeks of treatment with
the exception of a bald ring around the graft, but developed new patchy
hair loss during the last week of treatment. In one of these latter mice,
this process resulted in an increased pelage density compared with the
beginning of treatment (classified as partial hair regrowth, Table
I), whereas in the other mouse no improvement could be found (classified
as no hair regrowth, Table I).
In the vehicle-treated group, 2/4 mice showed no hair regrowth at any
time in the study (Table I). In 1/ 4 mice, continued hair loss
was observed during the first two weeks of treatment, followed by partial
hair regrowth for two weeks but subsequent hair loss during the last three
weeks of treatment. This resulted in no overall improvement in hair status
compared with the beginning of the study (classified as no hair regrowth,
Table I). The remaining 1/4 vehicle-treated mouse showed complete
hair regrowth on the back except for the graft and a ring around the graft
that remained bald (Table I).
The difference in complete hair regrowth is not significant at the 5%
test level. Nevertheless, there is a strong tendency towards preferential
hair regrowth in FK506-treated mice (p = 0.119).
Histopathology
In the three placebo-treated mice that had not shown hair regrowth,
dense peri- and intrafollicular mononuclear cell infiltrates around all
anagen hair follicles were observed. The infiltrates were located around
mid and lower regions of the hair follicles. Telogen hair follicles were
unaffected by the mononuclear cell infiltrate. Skin sections of the placebo-treated
mouse that had regrown hair mainly showed telogen hair follicles that
were unaffected by the mononuclear infiltrate. Anagen hair follicles could
only be found in the bald graft of this mouse, surrounded by a weak mononuclear
cell infiltrate.
In the mice showing hair regrowth after treatment with FK506, most anagen
hair follicles were unaffected by the mononuclear cell infiltrate, although
some anagen hair follicles showed a moderately dense peri- and intrafollicular
mononuclear cell infiltrate. Telogen hair follicles were not associated
with a perifollicular infiltrate. Pigmentary incontinence was found in
the dermis. By contrast, in those two mice which lost hair towards the
end of treatment, a dense peri- and intrafollicular mononuclear infiltrate
could be found in and around some (one mouse) or all (one mouse) anagen
hair follicles. Telogen hair follicles were unaffected by the inflammation.
In those mice that had regrown hair with the exception of a bald ring
around the graft, short parts of the skin sections showed rarefaction
of hair follicles. Elastica-van-Gieson stain showed reduction of
elastic fibers in these areas (data not shown).
Immunohistochemistry
A large number of peri- and intrafollicular CD4+ and CD8+
cells, with a predominance of the latter, were found in placebo-treated
mice without hair regrowth. CD8+ cells were located predominantly
at the center of the inflammatory infiltrate, partly within the hair follicle,
whereas CD4+ cells were more numerous at the periphery of the
infiltrate (Fig. 2a, b). The majority of hair follicles showed
an aberrant expression of MHC class I, MHC class II and ICAM-1 on mid
and lower hair follicle epithelium (data not shown). By contrast, in the
majority of mice showing hair regrowth under treatment with FK506, only
isolated peri- and intrafollicular CD4+ and CD8+
cells were found (Fig. 2c, d) and only occasional, individual hair
follicles showed an aberrant expression of MHC class I, MHC class II and
ICAM-1 on mid and lower hair follicle epithelium (Table II).
Discussion
Hair regrowth in 4/6 FK506-treated mice, when compared with hair regrowth
in only 1/4 placebo treated mouse, showed that topically applied 0.1%
FK506 ointment was able to induce hair regrowth in AA-affected C3H/HeJ
mice. These results are in line with the observations of McElwee et
al. [8], who successfully treated AA-affected DEBR rats with 0.25%
and 0.1% FK506 in solution. Differing from our results, McElwee et
al. reported subtotal hair regrowth in 2/6 and total hair regrowth
in 4/6 rats in the treated areas, suggesting that the 0.1% FK506 solution,
as used by this group, may be superior to the 0.1% FK506 ointment used
in the present study.
In contrast to topically applied FK506, systemic FK506 had no effect
on hair regrowth in the DEBR rat model of AA [14]. Although systemic resorption
of FK506 via the skin of C3H/HeJ mice or by licking off the ointment by
the mice cannot be excluded, it is unlikely that FK506 exerted a systemic
effect on hair regrowth in the present study.
The ring around the graft that remained bald in mice showing an otherwise
complete hair regrowth was most likely due to a scar-like tissue at the
junction between graft and host skin. Our histopathological examinations
showed a rarefication of hair follicles and elastic fibers in these areas
which can be explained as a consequence of grafting and wound-healing.
FK506 suppresses IL-2-production and IL-2-release in activated T cells
by inhibiting calcineurin. Subsequently, activation and proliferation
of T cells is inhibited [2, 15]. The resulting suppression of the T cell
mediated immune response is the most likely mechanism underlying the immunohistochemical
changes that accompanied hair regrowth in the present study: while placebo-treated
mice showed the typical immunohistochemical features of AA, i.e.
peri- and intrafollicular infiltrates of CD4+ and CD8+
T cells with a predominance of the latter and an aberrant expression of
MHC class I, MHC class II and ICAM-1 on mid and lower hair follicle epithelium,
the mice showing hair regrowth after treatment with FK506 had a marked
reduction of peri- and intrafollicular CD4+ and CD8+ T
cells. Furthermore these mice showed a reduction of aberrant MHC class
I, MHC class II and ICAM-1 expression on mid and lower hair follicle epithelium.
Whether this is caused by an additional direct action of FK506 on hair
follicle keratinocytes, or only secondary to the reduced activation of
keratinocytes by CD4+ and CD8+ T cells, remains
to be elucidated. FK506 has been shown to inhibit expression of HLA-DR,
but not of ICAM-1 on human keratinocytes in vitro [16]. Therefore
it cannot be ruled out that FK506 exerts an additional immunosuppressive
effect on hair follicle keratinocytes by suppressing antigen presenting
MHCs.
Topical application of FK506 induced anagen in telogen mouse skin and
stimulated hair growth in normal CD-1 mice and in SCID (severe combined
immunodeficiency) mice that lack both T and B cells [17-19]. These observations
account for a hair growth stimulatory effect of FK506 independent of its
T cell suppressive effect. In AA, induction of anagen would promote regrowth
of new hair fibers, but only if a new immune cell attack is prevented
would this lead to regrowth of visible healthy hair. Induction of anagen
might be an additional effect besides the immunosuppression of FK506 in
the treatment of AA. This would promote reentry of hair follicles into
anagen, which had been forced to premature catagen/telogen entry by the
immune attack of AA [20]. In cooperation with the immunosuppressive effect
of FK506, this would lead to hair regrowth.
In summary, the results of our study show that topically applied FK506
is able to induce hair regrowth in AA-affected C3H/HeJ mice, most likely
by suppressing the T cell mediated immune response against anagen hair
follicles. These results further suggest that topical application of FK506
could also be a safe and effective treatment of AA in humans. Further
studies are needed to test this hypothesis. *Acknowledgements
This work was supported by Fujisawa GmbH, Germany. KJM is supported
by the Ernst Schering Research Foundation and JPS by the National Institute
of Health (AR43801).
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