Mast Cells Limit the Exacerbation of Chronic

Allergic Contact Dermatitis in Response to

This information is current as
of January 22, 2019.
Repeated Allergen Exposure
Vladimir-Andrey Gimenez-Rivera, Frank Siebenhaar,
Carolin Zimmermann, Hanna Siiskonen, Martin Metz and
Marcus Maurer
J Immunol published online 2 November 2016
http://www.jimmunol.org/content/early/2016/11/01/jimmun
ol.1600236

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Supplementary http://www.jimmunol.org/content/suppl/2016/11/01/jimmunol.160023
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Published November 2, 2016, doi:10.4049/jimmunol.1600236
The Journal of Immunology

Mast Cells Limit the Exacerbation of Chronic Allergic

Contact Dermatitis in Response to Repeated
Allergen Exposure

Vladimir-Andrey Gimenez-Rivera,1 Frank Siebenhaar,1 Carolin Zimmermann,

Hanna Siiskonen, Martin Metz, and Marcus Maurer
Allergic contact dermatitis is a chronic T cell–driven inflammatory skin disease that is caused by repeated exposure to contact
allergens. Based on murine studies of acute contact hypersensitivity, mast cells (MCs) are believed to play a role in its patho-
genesis. The role of MCs in chronic allergic contact dermatitis has not been investigated, in part because of the lack of murine
models for chronic contact hypersensitivity. We developed and used a chronic contact hypersensitivity model in wild-type and
MC-deficient mice and assessed skin inflammatory responses to identify and characterize the role of MCs in chronic allergic

Downloaded from http://www.jimmunol.org/ by guest on January 22, 2019

contact dermatitis. Ear swelling chronic contact hypersensitivity responses increased markedly, up to 4-fold, in MC-deficient
KitW-sh/W-sh (Sash) and MCPT5-Cre+iDTR+ mice compared with wild-type mice. Local engraftment with MCs protected Sash mice from
exacerbated ear swelling after repeated oxazolone challenge. Chronic contact hypersensitivity skin of Sash mice exhibited elevated
levels of IFN-g, IL-17a, and IL-23, as well as increased accumulation of Ag-specific IFN-g–producing CD8+ tissue-resident
memory T (TRM) cells. The CD8+ T cell mitogen IL-15, which was increased in oxazolone-challenged skin of Sash mice during
the accumulation of cutaneous TRM cells, was efficiently degraded by MCs in vitro. MCs protect from the exacerbated allergic skin
inflammation induced by repeated allergen challenge, at least in part, via effects on CD8+ TRM cells. MCs may notably influence
the course of chronic allergic contact dermatitis. A better understanding of their role and the underlying mechanisms may
lead to better approaches for the treatment of this common, disabling, and costly condition. The Journal of Immunology,
2016, 197: 000–000.

A
llergic contact dermatitis (ACD) is a chronic inflam-
matory skin disease caused by the sensitization and re-
peated exposure to contact allergens. ACD is characterized
by itchy eczematous lesions that typically appear between 24 and 72 h
after acute allergen contact. With subsequent exposures to the contact
allergen, patients show more pronounced episodic and persistent
eczema (i.e., skin redness, papules, vesicles, scaling, skin thickening,
and fissures). ACD often starts at a young age, with a prevalence of
15% in 12–16-y olds, represents ∼20% of occupational skin disor-
ders, and frequently persists for life (1). Avoidance of relevant al-
Department of Dermatology and Allergy, Charité - Universitätsmedizin Berlin, D-10405
Berlin, Germany
1 allergen challenge (2).
V.-A.G.-R. and F.S. contributed equally to this work.
ORCID: 0000-0002-4070-9976 (M. Metz).
Received for publication February 9, 2016. Accepted for publication October 5,
2016.
This work was supported by grants from The Paulo Foundation and the Finnish
Society of Dermatology (to H.S.) and by Deutsche Forschungsgemeinschaft Grant
ME 2668/3-2 (to M. Metz) and SPP1394. V.-A.G.-R. was the recipient of a fellow-
ship from the European Academy of Allergy and Clinical Immunology. This work
was supported by the Corporation in Science and Technology Action BM1007 (Mast
Cells and Basophils – Targets for Innovative Therapies) of the European Community.
Address correspondence and reprint requests to Prof. Marcus Maurer, Department of
Dermatology and Allergy, Allergie-Centrum-Charité, Charite-Universitätsmedizin
Berlin, Charitéplatz 1, D-10117 Berlin, Germany. E-mail address: marcus.maurer@
charite.de
The online version of this article contains supplemental material.
Abbreviations used in this article: ACD, allergic contact dermatitis; BMCMC, bone
marrow–derived cultured MC; CCHS, chronic CHS; CHS, contact hypersensitivity;
CPA, carboxypeptidase A; Cre+, MCPT5-Cre+iDTR+; dLN, draining lymph node;
DNFB, dinitrofluorobenzene; DT, diphtheria toxin; MC, mast cell; OXA, oxazolone;
Sash, KitW-sh/ W-sh; TRM, tissue-resident memory T; WT, wild-type.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
lergens results in remission but is difficult to achieve for many
allergens (e.g., nickel); better approaches for avoiding ACD recur-
rence and episodic exacerbation are needed.
Our understanding of the mechanisms that underlie the sensi-
tization to allergens, the response to allergen exposure, and the
resolution of inflammation in ACD patients is largely shaped by
the results of mouse studies of contact hypersensitivity (CHS). In
these CHS studies, mice are sensitized and then challenged with
an experimental allergen (e.g., oxazolone [OXA] or dinitro-
fluorobenzene [DNFB]) by topical application to the skin. For
example, CHS studies revealed the role of TLR signaling in the
sensitization phase and the importance of several T cell subsets, as
well as dermal dendritic cells, for the inflammatory response to
Importantly, the vast majority of CHS studies are acute CHS
studies (i.e., limited to investigations of the sensitization phase and
the acute inflammatory response to a single allergen challenge). A
few CHS studies used continued repeated allergen exposure to
investigate the mechanisms of subchronic CHS (3–6), and all of
them were limited to a few weeks of allergen exposure. There are
no models or studies of chronic CHS (CCHS; i.e., repeated
chronic allergen challenge over several months); as a conse-
quence, the mechanisms of CCHS and ACD remain ill-defined.
Mast cells (MCs) are tissue-resident effector cells of innate
immune responses to pathogens and IgE-mediated allergic in-
flammatory reactions. Two independent lines of evidence suggest
that MCs may play important roles in the episodic exacerbation of
chronic ACD and CCHS. First, MCs are predominantly located in
organs that border our environment (e.g., airways, intestine, and
skin). Large numbers of MCs are present in skin areas that are
frequently exposed to contact allergens and that are predilection
sites of ACD, such as the hands and the face (7). Also, chronically

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600236
2 MAST CELLS IN CHRONIC ALLERGIC DERMATITIS
affected skin sites of ACD patients show markedly increased
numbers of MCs (8). Second, MCs were reported to promote acute
CHS responses to a single allergen challenge in some studies (9–
12), although not all (13, 14). MCs also were demonstrated to
limit and reduce acute CHS responses to a single allergen chal-
lenge in one study (15) but not another (12).
The aim of our study was to better characterize the mechanisms
that drive and control CCHS to better understand chronic responses
in ACD. To this end, we developed and used a murine model of
CCHS, which mimics the recurrent sporadic allergen encounters
that occur in ACD patients and lead to pronounced episodic ex-
acerbation and persistence of their condition. This CCHS model,
which involves repeated OXA challenges in sensitized mice every
30 d over several months, was applied to genetically MC-deficient
mice and normal control mice to identify and characterize the
relevance and role of MCs in CCHS.
Materials and Methods
Mice
All mice used in the experiments were 6–8-wk-old females on a C57BL/6
background. Wild-type (WT) C57BL/6-Kit+/+ and C57BL/6-KitW-Sh/W-Sh
(Sash) mice were bred and housed at our facilities, and MCPT5-Cre 3
iDTR (Cre+ or Cre2) mice were kindly provided by Dr. Axel Roers
(University of Dresden, Dresden, Germany). Ly5.1 mice were purchased
from the Jackson Laboratory. All mice were kept under specific pathogen–
free conditions, and all experiments were conducted according to institu-
tional regulations.
CCHS
Animals were sensitized on day 25 with 20 ml of 6% w/v OXA (Sigma-
Aldrich, Steinheim, Germany) in acetone by topical application to shaved
abdominal skin. Inflammation was elicited at selected skin sites by topical
application of 20 ml of 0.6% w/v OXA in acetone at 30-d intervals. We
designated the inflammatory response after the first OXA challenge as
acute CHS. Inflammatory responses induced by the third or subsequent
challenges with OXA were designated as chronic CHS. The OXA-
challenged sites and the number of challenges for each group are speci-
fied in the figure legends. For cross-sensitization experiments using DNFB
(Sigma-Aldrich), animals were sensitized on day 85 with 0.5% v/v DNFB
in acetone by topical application on shaved abdominal skin. On day 90,
inflammation was elicited with 20 ml of 0.1% v/v DNFB in acetone and
0.6% w/v OXA in acetone on the right and left ears, respectively. Ear
thickness was assessed with a micrometer caliper (Mitutoyo) before and
for several days after each challenge.
In vivo anti-CD8 Ab treatment
On days 5 and 6 after the induction of acute CHS, 250 mg of anti-CD8 Ab
clone 2.43 (2, 16) in 500 ml of PBS or PBS alone was injected i.p.
Depletion of MCs with diphtheria toxin
MCPT5-Cre 3 iDTR mice were injected weekly for 4 wk with diphtheria
toxin (DT, i.p., 25 ng/g bodyweight in saline; Sigma-Aldrich) before al-
lergen sensitization. Two days before the induction of CCHS, mice were
injected intradermally in the ear pinnae with 40 ml of DT (5 ng/g body-
weight in saline). To maintain the MC deficiency, i.p. injections of DT
(25 ng/g bodyweight in saline) were administered weekly from day 0
(OXA sensitization day) until day 120.
Adoptive transfer of MCs or CD8+ tissue-resident memory
T cells
For engraftment of MCs in the ears of Sash mice (Sash-Ear+BMCMCs), 4-wk-
old bone marrow–derived cultured MCs (BMCMCs, 2 3 106 cells per
mouse) were cultured as previously described (17) and injected into the ear
pinnae of mice (40 ml per ear). Only BMCMCs that exhibited .95%
purity, as determined by the coexpression of CD117/FcεRI, were used for
reconstitution experiments. Mice were used for experiments 4–6 wk after
the engraftment of BMCMCs.
For the adoptive transfer of CD8+ tissue-resident memory T (TRM) cells,
donor Ly5.1 mice (WT animals expressing the CD45.1 haplotype) and re-
cipient Sash and WT mice were sensitized (day 25) and challenged (day 0)
with OXA. On day 5, the draining lymph nodes (dLNs) and total blood
of donor animals were collected, passed through a 70-mm cell strainer, and
washed with PBS to produce a cell suspension. Blood was depleted of
erythrocytes using an erythrocyte lysis buffer (eBioscience, Frankfurt,
Germany) and a Lymphoprep centrifugation gradient (STEMCELL Tech-
nologies, Cologne, Germany). The cell suspensions from both organs were
pooled, stained with anti-CD8b–PE Ab (1:400; BioLegend, London, U.K.)
and purified (.90% purity) by FACS. The positive fraction was injected
into the tail vein of recipient mice (2 3 107 cells per mouse). Three days
after the adoptive transfer of CD45.1+CD8+ T cells, the ears of WT and
Sash recipient mice were harvested, and the composition and numbers of
CD45.1+CD8+ T cells expressing TRM cell markers were analyzed by
FACS.
Cytokine array
A total of 200 ml of protein lysate was extracted from ear skin, purified
as described by Lan et al. (18), and analyzed using a Multi-Analyte
ELISArray Kit (QIAGEN, Hilden, Germany).
Whole mount of ear skin and confocal microscopy
Ears were perfused with 80 ml of TBS, harvested, fixed in 4% PFA for
20 min, split in ventral and dorsal halves, and digested with proteinase
K (1:1000 in TBS; DAKO, Hamburg, Germany) for 1 h. For quantification
of skin MCs, the ear halves were incubated with a mixture of DAPI (1:100)
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and Avidin–Texas Red (1:500; both from Roche, Mannheim, Germany) for
15 min at 37˚C. For identification of CD8+IFN-g+ double-positive cells,
the ear halves were incubated with a mixture containing anti-CD8b–PE Ab
(1:100) and anti-IFN-g–Biotin Ab (1:50; both from BioLegend) for 3 h at
37˚C. The tissue was blocked with Biotin and Avidin block for 15 min at
37˚C (DAKO) and then incubated with FITC-streptavidin (1:200; eBio-
science) in TBS and 0.5% BSA. After separating the epidermal layer from
the dermis, the dermal layer was washed in TBS and 0.5% BSA, mounted,
and prepared for confocal imaging. Images were taken with Olympus
FV1000MPE and Zeiss LSM 510 microscopes in single-photon settings.
For visualization, 50 mm of the z-axis was scanned at 5-mm intervals
and merged in the z-axis to construct the z-stacks. T cells and MCs in
every stack were analyzed in terms of their numbers, localization, and
distribution.
Cell preparations and flow cytometry
The ear halves were split by separating the ventral half from the dorsal
half, cut into small pieces, and incubated in a vial containing 0.2 mg/ml
of Liberase TL (Roche) diluted in 1% streptomycin/penicillin in RPMI
1640. The reaction was stopped by adding RPMI 1640 with 10% FCS
v/v. Cell sorting was performed with a FACS Aria II (BD Bioscience,
Heidelberg, Germany). The phenotypic characterization of MCs and
T cells was performed with a MACSQuant Analyzer (Miltenyi Biotec,
Bergisch Gladbach, Germany). The following Abs (all from BioLegend)
were used for the identification of T cell populations: CD45 (clone
30-F11), CD103 (clone 2E7), CD8b (clone YTS156.7.7), CD44
(clone IM7), TCRb (clone H57-597), CD3 (clone 145-2C11), and CD4
(clone GK1.5).
In vitro experiments on the degradation of IL-15
Peritoneal lavage from WT mice (three to five animals per experiment, three
experiments) was collected, and the total cell number was adjusted to 1.5 3
107 cells per milliliter in PBS with Ca2+ and Mg2+. The cells were stim-
ulated with 2 mM ionomycin for 30 min at 37˚C, after which the super-
natant was isolated, diluted if appropriate, and treated with 100 mM
chymostatin, 100 mM leupeptin, 400 mg/ml carboxypeptidase A (CPA)
inhibitor from potato tuber (all from Sigma-Aldrich), or vehicle in addition
to 8 ng of recombinant mouse IL-15 in PBS as a substrate for 2 h at 37˚C.
Samples were kept in 280˚C until analysis of IL-15 levels with a mouse
IL-15 ELISA (R&D Systems, Minneapolis, MN), according to the man-
ufacturer’s instructions.
Statistical analysis
For all of the experiments, the data are shown as means of pooled values of
independent experiments with error bars representing 6 SEM, unless
otherwise indicated. The Mann–Whitney U test was used to analyze ex-
periments in Figs. 1A, 2A, 2B, 5A, and 5B. The nonparametric Wilcoxon
test was used to analyze experiments in Figs. 1C and 4A. The Student t test
was used to analyze experiments in Figs. 3A, 6A, 6B, and 7A. For data
shown in Fig. 5A, linear regression between left/right ear and strain
was performed, whereas for data in Fig. 7B and 7C, ANOVA with the
Tukey post test was performed. All statistical tests were performed with
GraphPad Prism version 5.0b (GraphPad, La Jolla, CA).
The Journal of Immunology
Results
MCs control CCHS responses
CCHS responses to the third monthly challenge with OXA were
stronger than acute CHS responses to a single OXA challenge in the
ears of WT mice (Fig. 1A). Unexpectedly, CCHS responses were
even more pronounced and of longer duration in genetically MC-
deficient C57BL/6-KitW-sh/W-sh (Sash) mice. Compared with WT
mice, Sash mice showed up to 4-fold increased ear swelling, as
well as skin scaling and necrosis (Fig. 1A, 1B). In contrast, acute
CHS responses were similar in Sash and WT mice (Fig. 1A, 1C).
MC-depleted MCPT5-Cre+iDTR+ (Cre+) mice also developed
more pronounced and persistent CCHS responses compared with
controls (i.e., MCPT5-Cre2iDTR+ mice) (Fig. 2A), despite having
not developed skin scaling or necrosis as it was observed in Sash
mice. CCHS in Sash mice that had been locally repaired of their
MC deficiency (Sash-Ear+BMCMCs) was less severe and of shorter
duration than that in MC-deficient control ears, did not show
scaling or necrosis, and was similar to CCHS in WT mice
(Fig. 2B). Taken together, these findings suggested that local skin
MCs control CCHS responses.
Th1 cytokine levels and numbers of CD8+ T cells are increased
in CCHS in the absence of MCs
To better understand how MCs control CCHS responses, we analyzed
the skin cytokine profile and the cellular infiltrate of MC-deficient
CCHS ears. We found higher levels of IFN-g, IL-17a, and IL-23,
but not IL-4, IL-6, or IL-10, in Sash mice compared with WT mice
24 h after induction of CCHS responses (Fig. 3A). In CCHS ears, the
CD8+ T cells appeared to be a prominent source of IFN-g, indicating
that these cells were proinflammatory (Fig. 3B). We also found higher
numbers of CD8+ T cells, but not CD4+ T cells, in the CCHS ears of
Sash mice and MC-depleted Cre+ mice (Fig. 4A). The majority of
these CD8+ T cells were TRM cells, as determined by their coex-
pression of CD44high and CD103 (19) (Fig. 4B).
The control of CCHS by skin MCs is local, not systemic,
involves CD8+ TRM cells, and is Ag specific
Do MCs control CCHS by controlling cutaneous CD8+ TRM cell
numbers? To address this question, we sensitized mice to OXA,
challenged them multiple times, and induced and compared CHS
responses at skin sites that had not previously been challenged
with OXA (naive skin sites) with skin sites that had been re-
peatedly challenged. MC-deficient mice showed CHS responses
comparable to those of their WT controls in their naive skin sites
but exacerbated CHS responses in their repeatedly challenged skin
FIGURE 1. CCHS reactions are more pronounced and persistent in the absence of MCs. Animals were sensitized to OXA (day 25, abdominal skin) and
challenged with OXA (day 0, left ears; day 30, right ears; and day 60, left ears) or treated with vehicle (acetone) as control (day 0, right ears; day 30, left
ears; and day 60, right ears). (A) MC-deficient Sash mice show uncontrolled ear swelling after chronic CHS to OXA (right panel), but not after acute CHS
(left panel). Data are shown as means of pooled values of n = 4 experiments, and error bars indicate SEM of 20 animals per group. Dagger symbols (†)
indicate the number of mice that showed signs of scaling or necrosis on the ears at the specified points in time. (B) Representative pictures showing the
macroscopic state of the skin on day 68. A total of 12 of 35 Sash mice developed scaling and necrosis (†) at CCHS sites. (C) The area under the curve (AUC)
was calculated for acute CHS (days 0–8) and chronic CHS (days 60–68). **p , 0.01, ***p , 0.001.
3
sites (Fig. 5A), indicating that local, and not systemic, effects are
critical for MC-mediated CCHS control.
Next, we subjected skin sites in Sash mice to acute CHS and then
depleted them of CD8+ cells (Sash2CD8), which reduced the numbers
of cutaneous CD8+ TRM cells at these sites to levels similar to those
observed in WT mice (Fig. 5B). Subsequent CCHS responses at
these sites were normal and similar to those in WT mice (Fig. 5C),
indicating that CD8+ T cells and their local increase are important
for uncontrolled CCHS responses in the absence of MCs.
Finally, we tested whether MCs control CCHS responses via
effects on Ag-specific cells. To this end, we sensitized and repeatedly
challenged Sash mice with OXA, sensitized them to DNFB, which
does not cross-react with OXA, and then challenged them, at sites of
previous OXA challenges, with DNFB or OXA (Fig. 5D). The
DNFB-challenged sites showed controlled CHS responses compa-
rable to those in WT and Sash2CD8 mice (Fig. 5D). In contrast, the
OXA-challenged sites showed uncontrolled inflammation, suggest-
ing that MCs control CCHS by effects on Ag-specific CD8+ T cells
(i.e., OXA-specific CD8+ TRM cells).
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Skin MCs control CD8+ TRM cell accumulation at sites of
allergen challenge
How do skin MCs control the increase in CD8+ TRM cell numbers
at CHS sites? The naive skin of Sash and WT mice contained no
CD8+ TRM cells (Fig. 6A). During acute CHS, these cells gradu-
ally increased in both mice, more so in Sash mice than in WT
mice (Fig. 6A). In parallel, CD8+ TRM cell numbers also increased
in the dLNs of both mice, again more so in Sash mice than in WT
mice (Fig. 6A). This suggested that MCs may control the increase
in CD8+ TRM cells at CHS sites by controlling their recruitment to
the skin and/or by controlling their generation in lymph nodes.
To clarify this, we adoptively transferred CD45.1+CD8+ TRM cells to
CD45.12 recipient Sash mice and WT controls and subjected
these mice to CHS. We found significantly more donor-derived
CD45.1+CD8+ TRM cells in the CHS ears of Sash mice compared
with WT mice (Fig. 6B), indicating that MCs, through local
mechanisms, restrict the recruitment of CD8+ TRM cells to allergen-
challenged skin. Because Sash CHS ears also had higher numbers
of endogenously generated TRM cells (i.e., CD45.12CD8+ TRM cells)
(Fig. 6B), it suggests that MCs may also control the development of
CD8+ TRM cells in CHS in dLNs. Next, we analyzed the ear skin
levels of TGF-b, IL-7, and IL-15, which are known to be important
for cutaneous CD8+ TRM cells (20, 21). Our results showed higher
levels of IL-15 (Fig. 7A), but not of TGF-b and IL-7 (data not shown),
in the challenged skin of Sash mice compared with WT mice.
4 MAST CELLS IN CHRONIC ALLERGIC DERMATITIS
FIGURE 2. Skin MCs control CCHS responses.
(A) MC-depleted Cre+ mice show increased ear
swelling after CCHS to OXA compared with
MCPT5-Cre2iDTR+ control mice. Data are shown
as means of pooled values of n = 3 experiments,
and error bars indicate SEM of 12 animals per
group. (B) Sash mice reconstituted locally with
BMCMCs in the ears (Sash-Ear+BMCMCs) show
normal inflammatory responses after CCHS to
OXA. Data are shown as means of pooled values of
n = 4 experiments, and error bars indicate SEM of
15 animals per group. **p , 0.01. Dagger symbols
(†) indicate the number of mice that showed signs
of scaling or necrosis on the ears at the specified
points in time.
To investigate further the possible mechanism how MCs could
control the accumulation of TRM cells by modulating IL-15 levels
during CHS in the skin, we incubated supernatants from ionomycin-
activated peritoneal lavage cells including MCs, which closely re-
semble skin MCs, with IL-15 ex vivo and found a significant
concentration-dependent decrease in IL-15 levels (Fig. 7B). The
reduction in IL-15 levels was inhibited by the addition of MC pro-
tease inhibitors, such as chymostatin (inhibitor of chymase) and CPA
inhibitor, to the supernatant before the substrate (Fig. 7C), suggest-
ing that chymase and CPA are capable of degrading IL-15.
Discussion
Our results show that MCs control allergic skin inflammation in a
murine CCHS model of human ACD.
To investigate the role of MCs in CCHS, we first had to develop a
new and suitable murine model. Unlike acute CHS models (single
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allergen challenge) and subchronic CHS models (multiple chal-
lenges for a few weeks), as used in previous studies, our CCHS
model entails once-monthly allergen challenges over several
months. This mimics the course of ACD in many patients, with
chronic recurrent allergic skin inflammation triggered by episodic
skin contact with relevant allergens. Our CCHS model shows many
features of ACD, including increasingly more pronounced in-
flammatory responses upon repeated allergen challenges, Th1
skewing, and increased expression of IFN-g at sites of inflam-
mation (22), as well as long-lasting accumulation of immune cells
(e.g., MCs and T cells) at involved skin sites (23).
Our results complement those of a previous study by Hershko
et al. (4), who found that MCs contribute to subchronic CHS. In
that study, sensitized mice were challenged with OXA three times
per week for up to 10 times. MCs were found to control CHS by
migrating to the dLNs and to the spleen and by controlling CD4+
FIGURE 3. In CCHS, Th1 cytokine levels are increased
in the absence of MCs. The ears of mice were harvested
24 h after the induction of CCHS and processed for cyto-
kine analysis (A) or for whole-mount immunohistochem-
istry (B). (A) Cutaneous levels of cytokines in Sash and
WT mice during CCHS to OXA (day 61). Data are shown
as the mean of pooled values of n = 3 experiments, and
error bars indicate SEM of nine animals per group. (B)
Sash skin CD8+ T cells are a prominent source of IFN-g in
CCHS. Immunofluorescence staining (original magnifica-
tion 340). Pictures from ear whole mount. The CD81IFNg
panel is a merge of the CD8 and IFNg panels. DAPI (blue;
nuclei), anti-CD8b Ab (red), anti–IFN-g Ab (green), or iso-
type Abs (Control). ***p , 0.001, *p , 0.05.
The Journal of Immunology 5

FIGURE 4. In CCHS, TRM cells are increased in the

absence of MCs. Ears of mice were harvested 30 d after
the induction of CCHS (on day 90), when they were no
longer macroscopically inflamed, and analyzed by FACS.
(A) MC-deficient Sash mice contain more CD8+ cells, but
not CD4+ cells, at sites of previous CCHS responses. The
bar graphs show fold increases in CD8+ cells and CD4+
cells in relation to naive skin of the respective genotypes,
in which cell numbers were low. Data are shown as means
of pooled values of n = 3 experiments, and error bars in-
dicate SEM of nine animals per group. (B) Most CD8+
T cells in the skin of MC-deficient Sash and Cre+ mice
4 wk after induction of CCHS are TRM cells (red dots). In
the FACS gating strategy, the CD45+CD42TCRb+CD8b+
CD103+CD44high cells represented the CD8+ TRM cells.
##
p , 0.01, #p , 0.05 versus naive skin; **p , 0.01
versus WT controls.

T cells via IL-2. In our chronic CHS study, cutaneous MCs at sites more pronounced than in WT mice, but only Sash mice, and not
of allergen challenge control CHS by effects on TRM cells. In both Cre+ mice, showed skin scaling and necrosis at CCHS sites. The

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studies, MCs protect from allergic inflammation induced by re-
peated allergen challenge. The differences in the mechanisms
involved may reflect differences in the models used or point to the
involvement of multiple mechanisms in MC-mediated suppression
of allergic skin inflammation, which may change in relevance as
CHS progresses from acute to subchronic to chronic.
To our knowledge, this is the first CHS study to use two inde-
pendent MC-deficient mouse models showing very similar results.
In both MC-deficient mice, CCHS inflammation was significantly
most likely explanation for this is that the MC-depleted Cre+ mice
contain more skin MCs than Sash mice. Under noninflammatory
conditions, the depletion efficiency of skin MCs in Cre+ mice is
.90% (12) (Supplemental Fig. 1A). However, in CCHS we found
only a 75% reduction in skin MCs in Cre+ mice compared with a
.90% reduction in Sash mice (Supplemental Fig. 1B). These
results suggest that the depleting effect of DT is counteracted by
the accumulation of local skin MCs, probably triggered by the
induction of local inflammation. In line with this, WT mice

FIGURE 5. The control of CCHS by skin MCs is local, not systemic, involves CD8+ T cells, and is Ag specific. (A) The control of CCHS by skin MCs is
local. All mice were sensitized to OXA and challenged multiple times. CHS was induced at skin sites that had been repeatedly challenged with OXA or at
sites that had never been challenged with OXA. Data are shown as means of pooled values of n = 3 experiments, and error bars indicate SEM of 12 animals
per group. ##p , 0.01 versus naive skin; **p , 0.01 versus WT controls. (B) Numbers of skin CD8+ T cells after treatment with anti-CD8 Ab. Eight days
after the first challenge with OXA, the ear skin was harvested, and the numbers of CD8+ T cells were assessed by flow cytometry. One representative
experiment of n = 3 independent repetitions. **p , 0.01 versus Sash. (C) The control of CCHS by skin MCs involves CD8+ T cells. Mice were sensitized
(day 25, abdominal skin) and challenged (day 0, both ears; day 30, abdominal skin; and day 60, both ears) with OXA. Additionally, on days 5 and 6 after
the first OXA challenge, some Sash mice were injected with anti-CD8 Ab (Sash2CD8). Control mice were injected with saline (Sash and WT). **p , 0.01
versus controls. (D) The control of CCHS by skin MCs is Ag specific. The mice in (B) were sensitized to a different allergen (DNFB) on the abdominal skin
(on day 85) and subsequently challenged (on day 90) with DNFB on the right ears (left panel) and with OXA on the left ears (right panel). Sash, Sash2CD8,
and WT mice show comparable ear swelling responses after CHS to DNFB (day 90–98) but exacerbated CCHS responses to OXA. ##p ,0.01, Sash versus
Sash2CD8 mice. Data in (B and C) are shown as means of pooled values of n = 3 experiments, and error bars indicate SEM of 10 animals per group. Dagger
symbols (†) indicate the number of mice that showed signs of scaling or necrosis on the challenged ears at the specified points in time. n.s., not significant.
6 MAST CELLS IN CHRONIC ALLERGIC DERMATITIS

FIGURE 6. MCs control the recruitment of

CD8+ TRM cells to sites of CCHS responses.
(A) Sash mice show increased levels of CD8+
TRM cells in the ears (left panel) and dLNs
(right panel) as early as 6 d after acute CHS.
(B) Sash mice contain more donor and recip-
ient CD8+ TRM cells than do WT mice after
acute CHS. Statistical significance was calcu-
lated with the Student t test. For all experi-
ments, data are shown as the means of pooled
values of n = 3 experiments, and error bars
indicate SEM of nine animals per group. *p ,
0.05, **p , 0.01, ***p , 0.001. n.s., not

Downloaded from http://www.jimmunol.org/ by guest on January 22, 2019

significant.

showed MC activation (degranulation) and MC accumulation at
CCHS skin sites (Supplemental Fig. 2).
The key findings that led us to explore the importance of CD8+
TRM cells for MC-mediated suppression of CCHS were that Th1
cytokine levels and numbers of CD8+ T cells at CCHS skin sites
were increased in the absence of MCs. The critical role of these
Ag-specific TRM cells is underlined by the outcome of our ex-
tensive characterization of CCHS skin infiltrates and our depletion
experiments that, taken together, indicate that MCs control CCHS by
controlling CD8+ TRM cells at sites of allergen challenge. MC-deficient
mice showed higher numbers of adoptively transferred and endog-
enously generated CD8+ TRM cells. This suggests that MCs may
limit the recruitment of CD8+ TRM cells to the skin sites of CCHS
and/or limit their generation in the dLNs. Gaide et al. (24) recently
demonstrated the relevance of Ag-specific expansion of TRM cells in
ACD, as well as in DNFB-induced CHS, in mice. Because TGF-b,
IL-7, and IL-15 are known to be secreted locally in the skin and
required for the maintenance of cutaneous CD8+ TRM cells (20, 21),
we analyzed these cytokines in our CCHS model and found higher
IL-15 levels in the ear skin of MC-deficient Sash mice compared

FIGURE 7. MC-deficient Sash mice have increased cutaneous levels of IL-15 at the time of CD8+ TRM cell accumulation in their skin, and MC enzymes
are capable of degrading IL-15 in vitro. (A) Cutaneous levels of IL-15 7 d after acute CHS in Sash and WT mice. *p , 0.05 versus WT mice. (B) Su-
pernatant from ionomycin-activated peritoneal lavage cells degrades IL-15 in a concentration-dependent manner. ***p , 0.001, vehicle versus supernatant.
(C) Reduction in IL-15 levels is inhibited by chymostatin and CPA inhibitor but not by leupeptin. ***p , 0.001, **p , 0.01, vehicle versus supernatant or
between supernatant with and without inhibitors. n.s., not significant.
The Journal of Immunology
with controls. Furthermore, we found that chymase and CPAs are
able to degrade IL-15 in vitro, as also shown earlier for several other
cytokines (25). MCs are the sole source of chymase and CPA (26),
but the effects of other CPA-secreting cells cannot be ruled out.
However, it seems that MCs may impair the survival or proliferation
of skin CD8+ TRM cells in CCHS by contributing to the reduced
local levels of IL-15, which is believed to be the mechanism that
decreases the availability of IL-15 in situ (27, 28). Interestingly, our
present findings and the ability of IL-15 to suppress chymase activity
that was reported earlier (29) point to a novel feedback loop that
regulates the levels of IL-15 and chymase. In addition to the possible
enzymatic degradation of IL-15, MCs have a high expression of IL-
15Rs (30, 31), are highly responsive to IL-15 in vitro (32) and, under
in vitro conditions, consume large amounts of IL-15 (33). However,
the effects of MCs on other cells producing IL-15 (e.g., mononuclear
cells) (34) cannot be ruled out.
In conclusion, we show that the increased accumulation of CD8+
TRM cells in the skin of MC-deficient mice results in exacerbated
inflammation upon repeated allergen challenge. This supports an
immunoregulatory function for cutaneous MCs and encourages
the further study of this role of MCs in TRM cell–mediated dis-
eases, such as ACD or psoriasis (24, 35). Approaches that increase
local skin MC numbers or enhance their functions could limit or
reduce the exacerbation of symptoms in ACD patients by reducing
pathogenic TRM cells in the skin.
Acknowledgments
We thank Dr. Axel Roers for providing MCPT5-Cre 3 iDTR mice. We also
thank the staff of the microscope facility at the Max-Delbr€uck Center
(Berlin, Germany) and the Danish Molecular Biomedical Imaging Center
(Odense, Denmark) for technical support and Sina Heydrich and Mariella
Bothke for excellent technical assistance.
Disclosures
The authors have no financial conflicts of interest.
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