Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 821967, 8 pages
Attenuates the Developmentof AtopicDermatitis-Like
SkinLesionsby SuppressingSerumIgE Levels,IFN-γ, and
SunminPark,1,2JungBok Lee,1and SunaKang1
1Department of Food and Nutrition, Hoseo University, 165 Sechul-Ri, BaeBang-Yup, Asan-Si,
ChungNam-Do 336-795, Republic of Korea
2Department of Mechatronics Engineering, Hoseo University, Asan 336-795, Republic of Korea
Correspondence should be addressed to Sunmin Park, email@example.com
Received 22 September 2011; Revised 25 October 2011; Accepted 1 November 2011
Academic Editor: Shrikant Anant
Copyright © 2012 Sunmin Park et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chrysanthemum indicum L. (CIL) is widely used as an anti-inflammatory agent in Asia and our preliminary study revealed that
CIL reduced interleukin (IL)-4 and IL-13 in 2,4-dinitrochlorobenzene (DNCB)-treated HaCaT cells, a human keratinocyte cell
line. We investigated the atopic dermatitis (AD) effect of topically applied CIL in mice with AD-like symptoms. After topical
application of 1,3-butylen glycol (control), CIL-Low (5%), CIL-High (30%), or 0.1% hydrocortisone (HC) on the AD-like skin
lesions in DNCB-treated NC/Nga mice for 5 weeks, the ear thickness, mast cell infiltration, and serum immunoglobulin E (IgE),
IgG1, IL-4 and interferon (IFN)-γ were measured. The gene expressions of IL-4, IL-13, and IFN-γ in the dorsal skin were assayed.
CIL treatment dosedependently reduced severity of clinical symptoms of dorsal skin, ear thickness, and the number of mast cells
and eosinophils. CIL-High significantly decreased serum IgE, IgG1, IL-4, and IFN-γ levels and reduced mRNA levels of IFN-γ,
IL-4, and IL-13 in dorsal skin lesion. The improvement by CIL-High was similar to HC, but without its adverse effects such as skin
atrophy maceration, and secondary infection. In conclusion, CIL may be an effective alternative substance for the management of
Atopic dermatitis (AD) is a common skin disease charac-
terized by chronic and relapsing inflammatory dermatitis
with immunological disturbances . The incidence of AD
is continuously increasing worldwide with a prevalence rate
of approximately 10–20% and is more common among
infants and children. Most patients with AD have increased
circulating eosinophils and immunoglobulin (Ig) E due to
elevated interleukin (IL)-4, IL-5, and IL-13 produced by T-
helper (Th) 2 cells [2, 3]. Th2 immune responses are known
to play an important role in the pathogenic mechanism of
gamma (IFN-γ) is also associated with the pathophysiology
skin disease, provoked by Th1/Th2 immune responses .
Acute AD skin lesions exhibit Th2 type inflammatory
cytokine profiles, whereas the chronic phase is characterized
by Th1 type immune responses releasing Th1 cytokines such
as IFN-γ and IL-12 and delayed-type hypersensitivity reac-
es plays important roles in the development of AD [6, 7].
NC/Nga mice are the most extensively studied animal
model of AD since the inbred strain was established in 1957
. These mice spontaneously develop AD-like eczematous
skin lesions when kept in air-uncontrolled conventional
housing, but not when maintained under specific pathogen-
free (SPF) conditions . However, when these mice are
maintained under conventional conditions, the low inci-
ical hapten, 2,4-dinitrofluorobenzene (DNFB), or 2,4-
dinitrochlorobenzene (DNCB) application produces 100%
2Evidence-Based Complementary and Alternative Medicine
reproducible AD-like lesions in NC/Nga mice [8–10]. Both
DNCB- and DNFB-induced contact hypersensitivity is a T-
cell-mediated inflammatory skin reaction that is believed to
be associated with Th1 activation [11, 12]. Since the clin-
ical symptoms displayed in the NC/Nga mice treated with
chemicals and humans with AD are similar, the mice are
considered to be AD animal model suitable for understand-
ing and evaluating effective therapeutic strategies for AD
. Since topical hydrocortisone exerts anti-inflammatory,
antipruritic, and vasoconstrictive actions, it is widely ac-
cepted that topical hydrocortisone therapy is crucial for the
management of AD. However, it cannot be used for long
periods, and adverse side effects are frequently observed
chosis, acneiform eruptions, hypopigmentation, perioral
dermatitis, allergic contact dermatitis, maceration of the
skin, secondary infection, skin atrophy, and striae.
Accordingly, a wide variety of natural products are cur-
rently being evaluated for alternative AD therapies, with the
ideal agent possessing potent efficacy with a minimal side
ly used for anti-inflammatory agents in Southeast Asian folk
medicine. It was also found to have anti-microbial, anti-
oxidant, and anti-inflammatory action [15, 16]. Chrysanthe-
mum indicum L. inhibits inflammatory mediators, such as
NO, PGE2, TNF-α, and IL-1β, via suppression of MAPKs
and NF-κB-dependent pathways . In addition, our pre-
liminary study revealed that the 1,3-butylene glycol extract
of dried flowers of Chrysanthemum indicum L. decreases
the expression of IL-4 and IL-13 the most in DNCB-treated
HaCaT cells, human keratinocytes, when the extracts of
23 herbs including Chrysanthemum indicum L. were com-
pared. These effects may be due to the sesquiterpene com-
pounds such as kikkanol A, B, and C and flavone gly-
cones such as eriodictyol 7-O-β-D-glucopyranosiduronic
acid, acaciin, and luteolin 7-O-β-D-glucopyranoside [17,
18]. Thus, Chrysanthemum indicum L. may relieve AD-like
symptoms. However, it has not yet been determined whether
Chrysanthemum indicum L. suppresses the development and
progression of AD. In this study, we used the DNCB-treated
NC/Nga murine model to examine the inhibitory effect
of Chrysanthemum indicum L. on the development and
progression of AD-like skin lesions.
2.1. Preparation of Chrysanthemum indicum L. Extracts.
Dried flowers of Chrysanthemum indicum L. were purchased
in Kyung-Dong Herb market (Seoul, Korea) in 2008, iden-
tified by Dr. Joo YS (Department of Herbology, Woosuk
University, Wanju-gun, Korea), and a voucher specimen
(No. 2008-05 and 2008-06) deposited at the herbarium of
Department of Food & Nutrition, Hoseo University. Since
1,3-butylene glycol is a good solvent for making skin lotion,
dried flowers of Chrysanthemum indicum L. (1kg) were
extracted at room temperature for 12 hours with 20 or 3.3
L of 1,3-butylene glycol, filtered, and the filtrates centrifuged
at 450×g to make 5 or 30% extracts. When more than 30%
Chrysanthemum indicum L. was extracted with 1,3-butylene
glycol, the extract formed a precipitate. The supernatants
were used for topical application.
2.2. Animals. Twenty female 6-week-old NC/Nga mice were
purchased from Charles River Japan (Yokohama, Japan) and
maintained under conventional conditions: a 12h light/12h
dark cycle, room temperature of 22-23◦C, and humidity of
55 ± 15%. The mice had free access to food and water.
All surgical and experimental procedures were performed
according to the guidelines of the Animal Care and Use
Review Committee at Hoseo University, Korea.
2.3. Induction of AD-Like Skin Lesion. Mice were anes-
thetized with a mixture of ketamine and xylazine (100 and
10mg/kg body weight), after which the animal’s back hair
and right ear were shaved 1 day prior to sensitization. In the
first day, 1% DNCB in acetone/olive oil (3:1) was applied to
the dorsal skin and right ear then 0.2% DNCB was applied
every other day . The same volume of acetone/olive oil
vehicle was applied instead of DNCB solution to controls.
2.4. Topical Application of 1,3-Butylene Glycol Extracts of
Chrysanthemum indicum L. To determine the AD effect
of Chrysanthemum indicum L., two dosages were assigned
based on the preliminary cell-based study and the maximum
dosage to extract. A preliminary study showed that 50μg/mL
of Chrysanthemum indicum L. was effective against an AD
model in HaCaT cells, a human keratinocyte cell line, but
not 5μg/mL. After the induction of the AD-like skin lesions,
animals were divided into four groups of 10 mice each.
These groups were then treated topically in the dorsal skin
and right ear for 5 weeks with one of four agents: 1,3-
butylene glycol (BG; control), 5 or 30% dried flower of
hydrocortisone butyrate (HC; positive control) twice a day.
Mice with no induction of AD-like skin lesion were treated
with 1,3-butylene glycol as a normal control.
severity was assessed macroscopically using the following
scoring procedure. The total skin severity score was defined
as the sum of the individual scores for each of the following
four signs: (1) erythema and hemorrhage, (2) edema, (3)
erosion (excoriation), and (4) scaling (dryness) . In
this system, 0 was defined as exhibiting no symptoms, 1 as
mild symptoms, 2 as moderate symptoms, and 3 as severe
symptoms. Additionally, the mice were photographed once
Ear thickness was measured before and after induction of
the inflammatory response using a digital micrometer (MSI-
Viking, Duncan, SC). The micrometer was applied near the
tip of the ear just distal to the cartilaginous ridges, and
the thickness was recorded in micrometers. To minimize
technique variations, a single investigator performed the
measurements throughout each experiment.
2.6. Measurement of Serum IgG1 and IgE Levels and
Iterleukin-4 (IL-4) and Interferon-γ (IFN-γ) Cytokine Levels.
The total serum IgG1 and IgE was quantified by sandwich
Evidence-Based Complementary and Alternative Medicine3
enzyme-linked immunosorbent assay (ELISA) Quantitation
Kit (R&D Systems, Minneapolis, MN, USA) according to
the manufacturer’s protocol. The serum concentrations of
the cytokines (IL-4 and IFN-γ) were also quantified using a
mouse cytokine enzyme immunoassay kit (R&D Systems).
2.7. Real-Time Quantitative Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR). The dorsal skin tissues from five
mice from each group were collected at the end of treat-
ment. Total RNA was isolated from the skin tissues using
a monophasic solution of phenol and guanidine isothiocy-
anate (Trizol reagent, Gibco-BRL, Rockville, MD), followed
by extraction and precipitation with isopropyl alcohol. The
cDNA was synthesized from equal amounts of total RNA
with superscript III reverse transcriptase, and polymerase
chain reaction (PCR) was performed with high-fidelity Taq
DNA polymerase. Equal amounts of cDNA were mixed
with SYBR Green supermix (Bio-Rad) mix and were ana-
lyzed using a realtime PCR machine (BioRad Laboratories,
Hercules, CA). The expression level of the gene of interest
was corrected for that of the house keeping gene, β-actin.
The following primers were used for PCR reactions (5?-3?),
mouse IFN-γ, sense 5?-CGGCACAGTCATTGAAAGCCTA-
3?, and antisense 5?-GTTGCTGATGGCCTGATTGTC-
3?; IL-4, sense 5?-TCTCGAATGTACCAGGAGCCATATC-
3?, and antisense 5?-AGCACCTTGGAAGCCCTACAGA-
3?; mouse IL-13, sense 5?-cagctccctggttctctcac-3?, and anti-
sense 5?-ccacactccataccatgctg-3?; mouse β-actin, sense 5?-
CATCCGTAAAGACCTCTATGCCAAC-3?, and antisense 5?-
ATGGAGCCACCGATCCACA-3?. The primers were de-
signed to sandwich at least one intron in order to distinguish
between the products derived from mRNA and genomic
2.8. Histological Analysis. Dorsal samples were taken 24h
after final DNCB administration on day 35 and they were
fixed in 10% buffered neutral formaldehyde and embedded
in paraffin wax. Histological sections were 6μm thick and
were stained with haematoxylin and eosin for counting
number of eosinophils. The sections were also stained with
0.5% toluidine blue for investigating the number of mast
cells. The cell counts were performed in six consecutive
microscopic fields at 400x magnification.
2.9. Statistical Analysis. Statistical analysis was performed
mean ± standard deviation. The biological and metabolic
effects of CIL-Low, CIL-High, HC (positive control), and
vehicle (a negative control) were compared by one-way
ANOVA. Significant differences in the main effects among
thegroupswereidentifiedbyTukey’stestatP < 0.05.Thesig-
nificance of differences between the mice with and without
AD-like skin lesion was determined by two-sample t test.
3.1. Severity of Skin Lesion. The NC/Nga mice developed
as evidenced by skin lesion scores, whereas normal controls
Treatment periods (weeks)
Scores of skin lesion
Ear thickness (mm)
Treatment periods (weeks)
Figure 1: Changes in severity scores and ear thickness of atopic
dermatitis (AD) in NC/Nga mice. AD was induced in Nc/Nga
mice by topical application of 2,4-dinitrochlorobenzene (DNCB)
in the dorsal skin, and a right ear was topically treated with 1,3-
Low), 30% Chrysanthemum indicum L. (CIL-High), and 0.1%
hydrocortisone (HC) on the lesions for 5 weeks. Normal controls
did not have DCNB-induced AD. CIL decreased clinical severity of
atopic dermatitis symptoms and ear thickness in a dose-dependent
manner, and CIL-High had a statistically similar effect on the
severity as HC. (a) Changes in severity scores. (b) Changes of ear
thickness. Each value represents the mean ± SD of 10 mice in each
group.∗Significantly different among the different treatments in
Nc/Nga mice at P < 0.05. a, b, c Values with different superscripts
were significantly different among Nc/Nga mice by Tukey test at
P < 0.05.†Significantly different between control (BG) and normal
control at P < 0.05.
did not exhibit any skin lesions. The total scores were
calculated from sums of the scores for erythema, edema,
erosion, and dryness; with a score of 12 indicating the most
severe state. The normal controls exhibited no changes in
skin lesion scoresover time (Figure 1(a)).Inaccordancewith
control group increased gradually depending on the times of
challenge with DNCB. All mice in the control group treated
with BG (control) exhibited AD-like skin lesions including
erythema, edema, erosion, and dryness in the dorsal skin
(Figure 1(a)). However, topical application of CIL dose-
dependently lowered the skin lesion scores below that of the
controls: CIL-Low significantly decreased the clinical scores
4Evidence-Based Complementary and Alternative Medicine
at4and 5weeksfromtheDNCBsensitization in comparison
to the control, and CIL-High lowered the scores more than
CIL-Low (Figure 1(a)). The decreased clinical scores in CIL-
High were similar to those observed with HC treatment.
These results indicated that CIL-Low and CIL-High reduced
the progression of AD-like skin lesions in comparison to the
control, and CIL-High exhibited the similar improvement
to HC. The scores increased after topical application and
began to be lowered from day 21 in CIL-Low and CIL-High,
whereas they reached a maximum at day 28 in the BG group
3.2. Ear Thickness. Ear thickness gradually increased after
topical application of DNCB in BG and CIL-Low groups up
to the 4th week but in CIL-High and HC groups, only up to
toms were alleviated after the peak of ear thickness. Ear
thickness in the CIL-Low group significantly lowered only
at the 4th week while CIL-High suppressed its increase in
comparison to the BG group from the 2nd week of sen-
sitization (Figure 1(b)). Ear thickness of the CIL-Low and
CIL-High groups was significantly different at the 4th and
5th weeks. The decrease in the CIL-High group was not sig-
nificantly different from HC (Figure 1(b)). Ear thickness did
not change in the normal control group during the exper-
3.3. Serum IgG1, IgE, IL-4, and INF-γ Levels. At day 35 after
the sensitization, serum IgG1 and IgE levels were higher in
the control group than in the normal control group, but the
levels were dosedependently reduced by CIL treatments in
DNCB-treated mice (Figure 2(a)). CIL-Low and CIL-High
decreased circulating levels of IgG1 and IgE, but it was
statistically significant only for CIL-High which had levels
close to those of HC. IL-4 produced by Th2 cells was much
greater in the control group than the normal control group
whereas only CIL-High significantly suppressed the increase
in DNCB-treated mice, and the suppression was as much as
in HC, a positive control (Figure 2(b)). The control group
also exhibited higher levels of INF-γ produced by Th1 cells
than the normal control group, but CIL-High lowered the
levels in DNCB-treated mice, but it did not reach levels of
the normal control group (Figure 2(b)). The reduction of
serum INF-γ levels was statistically similar to HC. The ratio
of IL-4 and IFN-γ was higher in control group than normal
control, whereas the ratio was dosedependently lowered by
CIL in DNCB-treated mice, but only CIL-High significantly
CIL-High and HC (data not shown).
Skin. The dorsal skin of the mice in the control groups
exhibited hypertrophy, hyperkeratosis, intercellular edema,
the liquefaction degeneration of the basal layer, and infiltra-
tion of inflammatory cells such as mast cells and eosinophils
in contrast to the normal control group (Figure 3). The
symptoms seen in dorsal skin such as hypertrophy, hyper-
keratosis, intercellular edema, and liquefaction degeneration
of the basal layer were relieved by the treatments with CIL
IL-4 (pg/mL)IFN-γ (ng/mL)
Figure 2: Serum levels of IgG1, IgE, IL-4, and IFN-γ levels at
the end of experimental periods. AD was induced in Nc/Nga
mice by topical application of 2,4-dinitrochlorobenzene (DNCB)
in the dorsal skin, and a right ear was topically treated with
1,3-butylen glycol (BG; control), 5% Chrysanthemum indicum L.
(CIL-Low), 30% Chrysanthemum indicum L. (CIL-High), and 0.1%
hydrocortisone (HC) on the lesions for 5 weeks. Normal controls
did not have DCNB-induced AD. After 5 weeks of treatments,
serum was separated to measure immunoglobulins and cytokines.
CIL-High significantly reduced circulating levels of cytokines and
immunoglobulins in comparison to the control, and CIL-High
exhibited a statistically similar amelioration to HC. (a) Serum levels
of IgG1 and IgE. (b) Serum levels of IL-4 and INF-γ. Each value
represents the mean ± SD of 10 mice in each group.∗Significantly
different among the different treatments in Nc/Nga mice at P <
0.05. a, b, c Means on the bars with different superscripts were
significantlydifferentamongNc/NgamicebyTukeytestatP < 0.05.
†Significantly different between control (BG) and normal control at
P < 0.05.
in a dose-dependent manner. As seen in Figure 3(a), hyper-
trophy, hyperkeratosis, intercellular edema, and liquefaction
degeneration of the basal layer were ameliorated with CIL-
Low and CIL-High treatment in comparison to the control
group (Figure 3(a)). The improvement of the symptoms in
CIL-High was similar to the improvements in the HC group.
An increased number of mast cells and eosinophils were
observed in the skin lesions of the control group compared
to the normal control group (Figures 3(a) and 3(b)).
The increase in mast cell and eosinophil counts was dose
dependently suppressed by CIL treatment in DNCB-treated
Evidence-Based Complementary and Alternative Medicine5
HC Normal control
Figure 3: Continued.
6 Evidence-Based Complementary and Alternative Medicine
Number of eosinophil cells/mm2section
Number of mast cells/mm2section
Figure 3: The number of mast cells and eosinophils of the dorsal skin with histopathological analysis. AD induced in Nc/Nga mice by
topical application of 2,4-dinitrochlorobenzene (DNCB) in the dorsal skin, and a right ear was topically treated with 1,3-butylen glycol
(BG; control), 5% Chrysanthemum indicum L. (CIL-Low), 30% Chrysanthemum indicum L. (CIL-High), and 0.1% hydrocortisone (HC) on
the lesions for 5 weeks. Normal controls did not have DCNB-induced AD. After 5 weeks of treatments, the dorsal skin was fixed with 10%
formaldehyde, embedded in paraffin, and then sections were made. The skin sections were stained with hematoxylin and eosin and toluidine
blue staining. CIL-High significantly elevated mast cells and eosinophils in the lesion of the dorsal skin in comparison to the control and
as determined by hematoxylin and eosin staining. Eosinophils exhibited as blue dots. (b) The number of mast cells determined by toluidine
blue staining. Mast cells appear as blue dots.∗Significantly different among the different treatments in Nc/Nga mice at P < 0.05. a, b, c
Means on the bars with different superscripts were significantly different by Tukey test at P < 0.05.†Significantly different between control
(BG) and normal control at P < 0.05.
mice. CIL-Low decreased the mast cells and eosinophils
in comparison to the control, but it was not significantly
different. CIL-High significantly lowered them, and the
3.5. Cytokine mRNA Expression Levels. To determine cytok-
ine production in the inflamed dorsal skin lesions, mRNA
expression of IL-4, IL-13, and INF-γ cytokines was analyzed.
The dorsal skin tissues of DNCB-treated control mice
exhibited much higher expression levels of IL-4, IL-13,
and IFN-γ than those of the normal controls (Figure 4).
CIL suppressed the mRNA expression of IL-4 and IL-13
produced by Th2 cells in a dose-dependent manner, while
CIL treatment (Figure 4). However, the decreases in mRNA
levels were significantly different only in CIL-High, not in
CIL-Low, and the reduction of INF-γ was less than that
of IL-4 and IL-13 in CIL treatment. The decreased expres-
sions of IL-4, IL-13, and IFN-γ by CIL-High were similar
to those following treatment with HC, a positive control
Atopic dermatitis is a biphasic inflammatory skin disease,
provoked by an imbalance between Th1 and Th2 immune
responses . Th2 immune responses are mediated by
interleukin (IL)-4, IL-5, and IL-13 while Th1 immune
responses are modulated by IFN-γ . In particular, Th2
responses are key elements to the pathogenesis of atopic
disorders. NC/Nga mice were the first mouse model of AD
reported by Matsuda et al. , and the mice treated with
DFBN, DNCB, or picryl chloride have also been used as an
animal model for human AD . Elevated levels of serum
total IgE have been reported to correlate with the appearance
of the AD-like lesions in NC/Nga mice, with massive
infiltration of IL-4- and IL-13-producing Th2 cells and the
degranulation of mast cells and eosinophils . HC is a
potent topical corticosteroid used to alleviate rash, eczema,
effects such as facial hypertrichosis, folliculitis, miliaria, and
genital ulcers , which has inspired to investigations of
have reported that Herbal Oriental Medicine therapy may
be effective in AD patients . The present study showed
that CIL dose dependently reduced the severity of AD-like
skin lesions by decreasing serum IgE and IgG1 levels and
infiltration of mast cells and eosinophils in DNCB-treated
NC/Nga mice. The results also indicate that the effects of CIL
were probably due to a decrease in IFN-γ, IL-4, and IL-13
production by activated Th1 and Th2 cells. CIL-High was
found to be as effective as HC for alleviating AD.
Mast cells are known as key effector cells in IgE-mediated
allergic disorders and are activated by cross-linking of a high
Evidence-Based Complementary and Alternative Medicine7
Relative intensity of mRNA
Relative intensity of mRNA
Figure 4: IL-4, IL-13, and IFN-γ expression in the dorsal skin.
AD induced in Nc/Nga mice by topical application of 2,4-
dinitrochlorobenzene (DNCB) in the dorsal skin, and a right
ear was topically treated with 1,3-butylen glycol (BG; control),
5% Chrysanthemum indicum L. (CIL-Low), 30% Chrysanthemum
indicum L. (CIL-High), and 0.1% hydrocortisone (HC) on the
lesions for 5 weeks. Normal controls did not have DCNB-induced
AD. After 5 weeks of treatments, total RNA was extracted from the
dorsal skin, and cDNA was generated. The mRNA expression of
IL-4, IL-13, and INF-γ was measured by realtime PCR, and their
relative expression was standardized according to respective β-actin
4, IL-13, and INF-γ in the lesion of the dorsal skin in comparison
to the control and exhibited a statistically similar decrease as HC.
Each value represents the mean ± SD of 5 mice in each group.
∗Significantly different among the different treatments in Nc/Nga
mice at P < 0.05. a, b, c Means on the bars with different super-
scripts were significantly different by Tukey test at P < 0.05.†Sig-
nificantly different between control (BG) and normal control at
P < 0.05.
affinity IgE receptor . Upon activation, mast cells undergo
degranulation and release a variety of biologically active
substances, which play an important role in host defense
and allergic reactions including AD. Infiltration of mast cells
into the dermis is a necessary characteristic for defining an
appropriate animal model for AD . We investigated the
efficacy of CIL for preventing AD-like skin lesions in DNCB
challenged NC/Nga mice and its mechanism for preventing
and alleviating AD was explored since CIL has been widely
used as an anti-inflammatory agent in Southeast Asian folk
medicine. We found that CIL-High significantly suppressed
the numbers of mast cells infiltrating in the skin lesions of
the atopic dermatitis mice, suggesting that the activation and
migration of mast cells may be an immunopharmacological
target of CIL-High.
It is known that mast cell activation is tightly modulated
by IgE from B cells, and increased total serum IgE levels are a
hallmark of AD . AD induced by DNFB application acti-
vates B cells in NC/Nga mice by a Th2 reaction through IL-4
that elevates IgE production . IgE expression is known
to cause both acute and chronic phase skin symptoms.
Consistent with these reports, we found that serum IgE levels
NC/Nga mice, as was IL-4 and IL-13 production by activated
Th2 cells. These cytokines are known to play important roles
in the inflammation and hypertrophy of the skin in AD .
We also found that higher IgE, IL-4, and IL-13 levels were
associated with more severe skin lesions. Topical application
of CIL-High significantly improved the severity of AD-like
skin lesions by suppressing serum IgE, IL-4, and IL-13 levels.
The improvement by CIL-High was as effective as with HC.
In addition to cytokines released from Th2 cells, Th1
cytokines such as INF-γ are involved in AD development
. Under normal conditions, the differentiation of naive
T cells to Th1 and Th2 lineages is regulated by cytokines
that are secreted from various cells, including themselves,
and the Th1/Th2 balance is maintained [6, 7]. However, in
atopic dermatitis, the balance shifts to Th2 dominance; this
eventually leads to excessive Th2 cytokine production .
Some studies have found that increased INF-γ levels alleviate
AD-like symptoms, but those results remain controversial.
In the present study, repeated DNCB application increased
both IFN-γ and IL-4 production from activated CD4+T
cells of draining lymph nodes in NC/Nga mice. CIL dose
dependently decreased both Th1 (INF-γ) and Th2 cytokines
(IL-4 and IL-13) as effectively as HC. Furthermore, the ratio
of Th1 to Th2 cytokines was increased by CIL treatment.
In conclusion, CIL-High reduced the development of
AD-like skin lesions resulting from repeated DNCB applica-
tion in NC/Nga mice by suppressing total serum IgE levels
and IFN-γ and IL-4 production by activated CD4+T cells.
CIL-High ameliorated AD symptoms as effectively as HC.
Sesquiterpenes and flavonoids in CIL may be the active
components exerting anti-AD-like effects; further study is
needed to identify the primary active components.
This work was supported by Business for Cooperative R&D
between Industry, Academy, and Research Institute funded
Korea Small and Medium Business Administration in 2009.
All authors have no conflict of interests. The data in this
paper, or parts thereof, has not been submitted or published
elsewhere for publication. All authors listed in the paper
submitted to this journal have contributed substantially to
8 Evidence-Based Complementary and Alternative Medicine
the work, participated in the writing of the paper and seen
and approved the submitted version.
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