Cantharidin-induced inflammation in mouse ear model

for translational research of novel anti-inflammatories

VANESA IVETIC TKALCEVIC, BOSKA HRVACIC, MARTINA BOSNAR, SNJEZANA CUZIC,
BERISLAV BOSNJAK, VESNA ERAKOVIC HABER, and INES GLOJNARIC
ZAGREB, CROATIA

The murine model of cantharidin-induced ear inflammation was profiled in detail

for its alignment with the human model and to explore the mechanism of antiinflammatory activity of the macrolide antibiotics, clarithromycin and azithromycin.
Ear swelling in CD1 mice persisted for 7 days, with peak intensity at 16 h after inflammation induction. As in humans, cantharidin (12.5 mg/ear) generated macrophageinflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1,
keratinocyte-derived chemokine (KC), interleukin (IL)-6, IL-1b, and myeloperoxidase
(MPO) production, as well as neutrophil accumulation in mouse ear tissue. The tested
macrolides, clarithromycin and azithromycin, administered orally (2 3 150 mg/kg)
0.5 h before and 5 h after cantharidin challenge, reduced MIP-2, MCP-1, KC, and
MPO concentrations and thereby decreased ear swelling. Our results suggest
that cantharidin-induced acute inflammation represents an excellent model for
translational research of novel anti-inflammatories. (Translational Research
2012;160:137145)
Abbreviations: ELISA enzyme-linked immunosorbent assay; ENA-78 epithelial cell-derived
neutrophil attractant-78; GM-CSF granulocyte macrophage-colony stimulating factor;
GRO-a growth-related oncogene-a; IL interleukin; KC keratinocyte-derived chemokine;
LPS lipopolysaccharide; MCP-1 monocyte chemoattractant protein-1; MIP-2
macrophage-inflammatory protein-2; MPO myeloperoxidase; OXA oxazolone; PBS
phosphate buffered saline; TNF-a tumor necrosis factor-a

n drug development, screening for the most promising candidate compound is based on numerous
in vitro and in vivo experiments and, after rigorous
safety and pharmacokinetic tests, the compound is
selected for further clinical investigations. However,
detailed preclinical testing of drug candidates does not
assure their successful development in clinical studies.1
One of the reasons for failure is the lack of direct translation of data from animal models to disease character-

istics in humans.2 The challenge for translation research

(bench-to-bedside) is to correlate more efficiently
preclinical investigations with clinical situations,
thereby increasing the chance of success of the compound during the clinical stage of drug development.3,4
Improved correlation between animal models and
human diseases should enhance the prediction value
of animal studies for the efficacy of treatment
strategies in clinical trials.2,3,5

From the GSK Research Centre Zagreb Ltd., Zagreb, Croatia.

Reprint requests: Vanesa Ivetic Tkalcevic, Galapagos Research Centre

Ltd., Prilaz baruna Filipovica 29, 10000 Zagreb, Croatia; e-mail:
Vanesa.IveticTkalcevic@glpg.com.

This work was supported by GlaxoSmithKline Research Centre

Zagreb Ltd.
Conflict of interest: At the time of study, the authors were all employees of GlaxoSmithKline Research Centre Zagreb Ltd. All authors
have read the journals policy on disclosure of potential conflicts of interest.

1931-5244/$ - see front matter

2012 Mosby, Inc. All rights reserved.
doi:10.1016/j.trsl.2012.02.001

Submitted for publication November 4, 2011; revision submitted

February 1, 2012; accepted for publication February 2, 2012.

137

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Ivetic Tkalcevic et al

AT A GLANCE COMMENTARY
Ivetic Tkalcevic V, et al.
Background

Cantharidin-induced skin blisters represent a

simple model for investigating leukocyte trafficking and cytokine production at sites of inflammation in humans. In contrast to clinical studies,
cantharidin-induced inflammation in animal
models remains obscure. We aimed this study
to more efficiently correlate pre-clinical investigation to clinical situation, which would assure
better success of compound testing in clinical
stage of drug development.
Translational Significance

We profiled in more detail murine model of

cantharidin-induced ear inflammation, investigated its alignment with human model, and tested
the anti-inflammatory effects of macrolides on extended panel of cantharidin-induced inflammatory
markers. Our results suggest that mouse model
of cantharidin-induced inflammation can be exploited as a part of translational studies dealing
with novel anti-inflammatory agents progressing
toward testing in humans.

The cantharidin-induced skin blister represents

a simple model for investigating leukocyte trafficking
and cytokine production at sites of inflammation in
humans, which has already been applied successfully
for testing of novel anti-inflammatory agents.6-8
Cantharidin is a protein phosphatase inhibitor that
causes atraumatic acantholysis and blister formation
by detachment of desmosomes.7 The pathophysiology
of cantharidin-induced blister formation in human
skin has been well described, and includes predominately neutrophil inflammation driven by mediators including interleukin (IL)-8, epithelial cell-derived
neutrophil attractant (ENA)-78, tumor necrosis factor
(TNF)-a, and IL-1b.6,7 Neutrophils are among the
first inflammatory cells to migrate into the site of
inflammation. Primed by cytokines and mobilized by
chemokines, neutrophils secrete a variety of
proinflammatory cytokines and express MHC class II
molecules in a manner that allows activation of T
cells.9 A widely recognized enzyme marker for tissue
neutrophil content is myeloperoxidase (MPO),
a heme-containing peroxidase highly expressed by neutrophils.10 It is frequently used as a quantitative marker
of cutaneous neutrophil accumulation.11

In contrast to clinical studies, cantharidin-induced inflammation in experimental animal models in vivo is

less clearly characterized. Mouse ear inflammation induced by different irritants, such as croton oil, phorbol
12-myristate 13-acetate, oxazolone (OXA), arachidonic
acid, zymosan, or carrageenin, provides a range of skin
inflammation models suitable for the evaluation of both
topical and systemic anti-inflammatory agents.12,13 The
global reaction can be easily quantified by measurement
of ear weight and thickness and is, therefore, appropriate
for the screening of new drugs.14 Previous reports
showed that cantharidin application to mouse ears induces inflammation that is localized to the epidermis,
leading to epidermal hypertrophy, which can be quantified as ear swelling.14,15 However, the molecular and
cellular mechanisms of inflammation in this animal
model remain unexplored. Detailed understanding of
cantharidin-induced ear swelling in mice might allow
better correlation of this in vivo model to cantharidininduced skin inflammation in humans, and provide further
data for successful translational research of novel drugs.
Our objective was to profile in more detail the murine
model of cantharidin-induced ear inflammation and
attempt to align it with the human model by assessing
potentially relevant pharmacodynamic biomarkers for
human studies with novel anti-inflammatory compounds. To this end, we tested the effects of the macrolide antibiotics (macrolides) clarithromycin and
azithromycin on an extended panel of inflammatory
markers in cantharidin-induced ear edema. Literature
data indicate that, in addition to their antimicrobial
activity, macrolides also posses anti-inflammatory
and immunomodulatory activity.16,17 They accumulate
in inflammatory cells, especially neutrophils and
macrophages,18 inhibiting the synthesis of reactive
oxygen species and / or secretion of cytokines and chemokines.17,19,20 Several reports have demonstrated
anti-inflammatory activity of macrolides in models of
skin inflammation in vivo. Applied orally and topically,
macrolides inhibited ear and paw edema induced by various inflammatory agents.13,21-23
MATERIALS AND METHODS
Animals. In accordance with previously published reports describing cantharidin-induced ear inflammation,
all experiments were performed on male mice. 12,14,23,24
Male CD1 mice, 8- to 10-weeks-old, were obtained from
Charles River, France. Animals were kept on wire mesh
floors with irradiated maize granulate bedding (Scobis
Due, Mucedola s. r. l., Settimo Milanese, Italy) and
maintained under standard laboratory conditions
(temperature 2324 C, relative humidity 60% 6 5%,
approx. 15 air changes per h, artificial lighting with

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circadian cycle of 12 h). Food (Standard food for mice

and rats, 4R21; Mucedola, Italy) and tap water were
provided ad libitum. Mice were allowed to acclimate
for 10 days before the beginning of each experiment.
Experimental groups were composed of 8 animals.
All procedures on animals were performed in accordance with (1) the Revised Directive 2010/63/EU on
the approximation of laws, regulations and administrative provisions of the EU Member States regarding the
protection of animals used for experimental and other
scientific purposes; and (b) Statute of Republic Croatia, Animal Welfare Law, NN 135 of 13th December 2006.
Materials. Cantharidin was obtained from Sigma, St
Louis, Mo. Clarithromycin was from Spectrum Chemical Mfg Corp, Gardena, Calif and azithromycin was
from PLIVA Inc., Zagreb, Croatia. The inhalation anesthetic, isoflourane, used in all experiments was obtained
from Forane, Abbott Laboratories Ltd., UK. The Luminex kit for cytokine determination was purchased
from R&D Systems Inc., Minneapolis, Minn, and the
enzyme-linked immunosorbent assay (ELISA) kit for
determination of MPO was from Hycult Biotechnology
b.v., Uden, The Netherlands. All other reagents were
from Sigma, St Louis, Mo.
Experimental methods. Cantharidin-induced inflammation in mouse ear. Prior to cantharidin application,

animals were anesthetized by inhalation of isoflurane

delivered in an anesthesia induction chamber (Stoelting Co., Dublin, Ireland). Gas scavenging was provided using the Fluovac 240V system (International
Market Supply, Cheshire, England). In all experiments, cantharidin was dissolved in acetone and topically applied once by pipette (50 mL, Eppendorf),
using disposable pipette tips, onto the inner surface
of the left ear (10 mL/ear).
To determine the time-course of the ear edema, cantharidin at a concentration of 25 mg/10 mL acetone
was applied. Ear edema and histologic evaluation of
the inflammatory reaction were assessed at 6 h, 16 h,
24 h, 48 h, 120 h, and 168 h after cantharidin challenge.
For ear tissue sampling, animals were euthanized by exposure to 100% CO2 atmosphere. To evaluate ear edema
weight, an 8 mm diameter circle was cut out of the left
and right ear of each animal using a disposable biopsy
punch (Stiefel Laboratories Ltd., Berkshire, Ireland)
and weighed on a precise analytical balance. Ear edema
was calculated as the difference between left (treated)
and right (not treated) ear weight. Sampled ears were
fixed in 10% buffered neutral formalin fixative for histopathologic assessment.
To obtain the most suitable dose of cantharidin, both
ears were treated with cantharidin at 12.5, 18, and 25
mg/10 mL acetone/ear. Ears were sampled as previously

Ivetic Tkalcevic et al

139

described, 16 h after inflammation induction. Ear

weights were determined by weighing on a precise
analytical balance and stored at 80 C for future MPO
concentration analysis by ELISA. In ear tissue treated
with cantharidin at 12.5 and 18.5 mg/10 mL acetone/
ear, concentrations of macrophage-inflammatory protein
(MIP)-2, monocyte chemoattractant protein (MCP)-1,
keratinocyte-derived chemokine (KC), IL-6, IL-1b,
IL-12p70, granulocyte macrophage-colony stimulating
factor (GM-CSF), and TNF-a levels were also measured by Luminex (R&D Systems Inc., Minneapolis,
Minn).
Macrolide treatment. Clarithromycin and azithromycin (2 3 150 mg/kg) or vehicle (5% [v/v] dimethyl
sulfoxide and 0.5% [w/v] methylcellulose; 10 mL/kg)
were administered by oral gavage 0.5 h before and
5 h after cantharidin application to both ears (12.5 mg/
10 mL acetone/ear). Their anti-inflammatory effects on
ear edema and ear MPO, MIP-2, MCP-1, KC, IL-6,
and IL-1b production were tested 16 h after the
challenge.
MPO and cytokine concentration analysis. Prior to
analysis, ear samples (both ears from each animal in
those bilaterally treated with cantharidin) were homogenized in 1 mL of phosphate buffered saline (PBS) in
which protease inhibitors were added (pepstatin A, phenylmethanesulfonyl fluoride, leupeptin, and aprotinin).
Homogenate was centrifuged (10,000 3 g) for 10 min
at 4 C, and the resulting supernatant used for further
analysis. Total protein concentration in the ear tissue
was determined by biochemical analyzer (Olympus
AU 400, London, UK).
Concentration of MPO in ear tissue was analyzed
by sandwich ELISA, using capture and detection antibodies according to the manufacturers recommendations. Tissue homogenate samples were diluted 4
times with the supplied dilution buffer. A volume of
100 mL was transferred into appropriate wells, incubated for 1 h at room temperature and washed 4 times
with wash buffer. The subsequent steps included addition of 100 mL of diluted tracer, 100 mL of diluted
streptavidin-peroxidase, and 100 mL of tetramethylbenzidine (TMB) to each well. Before adding each
reagent, the plate was incubated for 1 h at room temperature and washed 4 times with wash buffer. After
TMB addition, the plate was incubated for 25 min at
room temperature and the reaction stopped by adding
100 mL of stop solution. Fifteen minutes after addition of stop solution, the optical density of each
well was determined using a microplate reader
(EnVision Multilabel Plate Reader; PerkinElmer,
Branford, Conn) set to 450 nm. The resulting concentration was expressed as mg/g of total protein in the
ear tissue.

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August 2012

Fig 1. A, Ear edema weight at indicated time points after cantharidin (25 mg/10 mL acetone/ear) induced inflammation. Data are presented as group means 6 SEM. *Indicates a significant difference from vehicle-treated
animals (P , 0.05, Mann-Whitney test, n 5 8). B, Representative photomicrographs of mouse ear tissue (hematoxylin/eosin) in vehicle treated (10 mL acetone/ear) animals (a) and cantharidin (25 mg/ear) treated animals (bg);
b, Six hours after application, cantharidin induced an acute inflammatory reaction observable as dermal edema
(square) and intraepidermal blister filled with neutrophils (ellipse); c, The square indicates dermal edema and neutrophil accumulation which was pronounced up until 16 h at which ear swelling was maximal; d, Twenty-four
hours after cantharidin application, intraepidermal blisters filled with neutrophils and necrotic debris were
observed (arrow); e, After 48 h, cantharidin induced necrotic ulcers (ellipse) covered with crust (arrow) and surrounded by neutrophils; f, The square indicates re-epithelialization with newly formed, thick epidermis that was
complete after 120 h, designating the onset of inflammatory resolution; g, Structured granulation tissue (ellipse)
appeared 168 h after cantharidin application. (Color version of figure is available online.)

Concentrations of MIP-2, MCP-1, KC, IL-6, IL-1b,

IL-12p70, GM-CSF, and TNF-a in ear tissue were determined by Fluorokine MAP MultyAnalyte Profiling

kit (R&D Systems) according to the manufacturers

recommendations. Tissue homogenate samples were
diluted 4 times with the supplied dilution buffer and

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141

aliquots of 50 mL transferred into appropriate wells.

Samples were analyzed with the Luminex 200 and STarStation software v2.3 (Applied Cytometry Systems,
Sheffield, UK) using five-parameter logistic-curve fitting and expressed as pg/mL.
Histologic analysis. Formalin-fixed ear tissue was embedded in toto, cut into 3 mm sections and stained routinely with hematoxylin/eosin. Inflammatory markers
were investigated observer-blind in four sections from
each ear specimen. Neutrophil accumulation was
graded in a semiquantitative manner according to the
following criteria:
0
1
2
3

No neutrophils
Few scattered neutrophils (mild accumulation)
Larger aggregates of neutrophils (moderate accumulation)
Marked accumulation of neutrophils (severe accumulation)

Statistical analysis. Differences between observed

values were analyzed statistically using the MannWhitney test. Statistical analyses were conducted
using the GraphPad InStat v. 4.0 (GraphPad Software
Inc., San Diego, Calif). The level of significance was
set at P , 0.05 in all cases.

RESULTS
Time-course experiment. In an initial experiment,
we investigated the time-course of the inflammatory
response to topical cantharidin application, determined
by ear swelling and histologic analysis of ear tissue
(Fig 1).
In contrast to vehicle, topical administration of cantharidin at 25 mg/ear induced ear swelling, measured
as an increase in ear weight, by 6 h after administration.
Swelling was observable for 168 h (7 days), with peak
intensity at 16 h after inflammation induction (Fig 1, A).
Since, for technical reasons, blister fluid could not be
collected in mice for evaluation of inflammatory intensity and dynamics, histologic analysis of ear tissue was
performed instead (Fig 1, B). Cantharidin (25 mg/ear),
6 h after application, induced acute inflammation, observed as dermal edema and intraepidermal blisters
moderately filled with neutrophils (Fig 1, B-b). Dermal
edema and neutrophil accumulation peaked at 16 h after
cantharidin challenge, also resulting in maximal ear
swelling at this time-point (Fig 1, A and B-c). Twentyfour hours after cantharidin application, intraepidermal
blisters, which were intensely filled with neutrophils
and necrotic debris, were observed (Fig 1, B-d), while
at 48 h, necrotic ulcers covered with crust and surrounded with neutrophils were present (Fig 1, B-e).
Re-epithelialization with newly formed thick epidermis

Fig 2. Ear weight (A) and myeloperoxidase (MPO) concentration in

mouse ear tissue (B) at 16 h after vehicle treatment (10 mL acetone/
ear) or induction of inflammation with different doses of cantharidin.
Data are presented as group means 6 SEM. * Indicates a significant
difference from vehicle-treated animals; 1 indicates a significant difference from animals treated with cantharidin at 25 mg/ear (P , 0.05,
Mann-Whitney test, n 5 8).

was complete 120 h (5 days) after inflammation induction, designating the onset of inflammatory resolution
(Fig 1, B-f). Structured granulation tissue appeared
168 h (7 days) after cantharidin application (Fig 1, B-g).
Cantharidin dose-dependency. To select the optimal
dose of cantharidin, different cantharidin doses (12.5,
18.5, and 25 mg/ear) were applied (Fig 2). The dose
range was selected in accordance with published
reports demonstrating cantharidin-induced inflammation
in both humans and in mice.7,14,25 The effects of
cantharidin were assessed at 16 h after cantharidin
application, when the inflammatory reaction was at
a peak, as shown by the time-course experiment.
In comparison to vehicle treatment, the cantharidin
doses applied caused significant increases in ear
weight (Fig 2, A). However, there was no significant
weight difference between ears treated with 12.5,
18.5, and 25 mg/ear cantharidin. The same was observed with ear MPO concentration, which was significantly enhanced at all cantharidin doses applied.

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Fig 3. Monocyte chemoattractant protein (MCP)-1 (A), macrophage-inflammatory protein (MIP)-2 (B),
keratinocyte-derived chemokine (KC) (C), interleukin (IL)-6 (D) and IL-1b (E) concentrations in mouse ear tissue
at 16 h after vehicle treatment (10 mL acetone/ear) or inflammation induced with different doses of cantharidin.
Data are presented as group means 6 SEM. * Indicates a significant difference from the corresponding
vehicle-treated, healthy control (P , 0.05, Mann-Whitney test, n 5 8).

Nevertheless, in comparison to 12.5 and 18.5 mg/ear

cantharidin, in ears treated with 25 mg/ear cantharidin, MPO concentration was significantly lower
(Fig 2, B).
In addition to ear weight and ear MPO concentration,
MIP-2, MCP-1, KC, IL-1b, IL-6, IL-12p70, GM-CSF,
and TNF-a in ear tissue were investigated to more profoundly compare cantharidin model between humans
and mice (Fig 3). Since cantharidin at 12.5 and 18.5
mg/ear induced a more intense inflammatory reaction
than 25 mg/ear cantharidin, only ear tissue treated with
the lower two doses was further analyzed. Cantharidin
at 12.5 and 18.5 mg/ear significantly increased concentrations of MCP-1, MIP-2, KC, IL-6, and IL-1b in ear
tissue (Fig 3, AE), while the concentrations of
IL-12p70, GM-CSF, and TNF-a were below the limit
of detection (data not shown). There was no significant
difference in concentrations of tested mediators between the two cantharidin doses applied.
Macrolide treatment. To validate our model for preclinical testing of novel anti-inflammatories, the anti-

inflammatory activity of the macrolide antibiotics,

azithromycin and clarithromycin, was investigated
(Figs 4 and 5). Based on the previous results, a
cantharidin dose of 12.5 mg/ear and determination of
ear edema, MPO and inflammatory mediators at 16 h
after inflammation induction were selected for further
experimentation.
Clarithromycin and azithromycin administered orally
(2 3 150 mg/kg) 0.5 h before and 5 h after cantharidin
administration significantly reduced ear edema by 63%
and 33%, respectively (Fig 4, A). MPO concentration
in ear tissue also was significantly decreased by clarithromycin (50%) and azithromycin (47%) treatment
(Fig 4, B). Both tested macrolides significantly inhibited KC, MIP-2, and MCP-1 generation in ear tissue
(Fig 5, AC), while showing no effect on IL-1b and
IL-6 concentrations (data not shown).
DISCUSSION

Cantharidin blister is well-established model of acute

inflammation in humans6,7 used for monitoring novel

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143

Fig 4. Effects of oral clarithromycin (CLA) and azithromycin (AZI)

(2 3 150 mg/kg) or vehicle [VEH; 5% (v/v) dimethyl sulfoxide and
0.5% (w/v) methylcellulose; 10 mL/kg] administered 0.5 h before
and 5 h after cantharidin (12.5 mg/10 mL acetone/ear) induced inflammation, on ear edema weight (A) and myeloperoxidase (MPO) concentration in ear tissue (B) at 16 h after inflammation induction.
Data are presented as group means 6 SEM. Numbers in brackets represent percent inhibition in comparison to vehicle treated, healthy control. * Indicates a significant difference from the corresponding
vehicle and cantharidin treated group (P , 0.05, Mann-Whitney
test, n 5 8).

treatment strategies.8 Animal models of cantharidininduced ear edema have been investigated in less detail,
ear swelling being the basic experimental output.14,24
We, therefore, investigated the molecular and cellular
mechanisms of inflammation after cantharidin
application to the mouse ear to correlate these
characteristics with those during cantharidin blister in
humans. Histologic and ELISA analyses of ear tissue
were performed, since collection of blister fluid in
mice is technically not possible.
In our experimental model, cantharidin-induced ear
swelling occurred with severe neutrophilic skin inflammation, dermal edema, and epidermal blister formation.
The time-course of cantharidin-induced inflammation
was in accordance with published reports, showing
that in Swiss mice and CD rats, topical cantharidin-

Fig 5. Effects of oral clarithromycin (CLA) and azithromycin (AZI)

(2 3 150 mg/kg) or vehicle [VEH; 5% (v/v) dimethyl sulfoxide and
0.5% (w/v) methylcellulose; 10 mL/kg] administered 0.5 h before
and 5 h after cantharidin (12.5 mg/10 mL acetone/ear) induced inflammation, on keratinocyte-derived chemokine (KC) (A), macrophageinflammatory protein (MIP)-2 (B) and monocyte chemoattractant
protein (MCP)-1 (C) in ear tissue at 16 h after inflammation induction.
Data are presented as group means 6 SEM. * Indicates a significant
difference from the corresponding vehicle and cantharidin treated
group (P , 0.05, Mann-Whitney test, n 5 8).

induced ear swelling is detectable after 6 h, maximal

at 24 h, and sustained for 7 days.14,24 A slight delay in
the peak of the inflammatory response may be
attributable to differences in animal species used.
Although Tarayre et al14 previously indicated that the
application of 2.5, 6.25, 25, and 62.5 mg cantharidin

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Ivetic Tkalcevic et al

induces a dose-dependent increase in ear weight, in our

experimental model all cantharidin doses applied (12.5,
18.5, and 25 mg/ear) increased ear weight to an equal extent. Again, these differences might have been to the
choice of mouse strain, as also observed with OXA,
which induces distinctive inflammatory reactions in various mice strains.26
For comparison of cantharidin-induced inflammatory
reactions between humans and mice, inflammatory
markers were selected based on results from cantharidininduced human blisters. Neutrophil accumulation in the
skin was confirmed by histology and quantified by measurement of MPO concentration. MPO is a neutrophilspecific enzyme which comprises approximately 5% of
neutrophilic mass and is used as a marker for tissue neutrophil content.11 Its major function is generation of hypochlorous acid as part of the neutrophils antimicrobial
armory by which it potentiates neutrophilic activity.10 In
addition to neutrophil accumulation, increased concentrations of MIP-2, MCP-1, KC, IL-1b, and IL-6 in ear
tissue were also detected. In the mouse, the CXC chemokines, MIP-2 and KC, functional homologs of human
IL-8,27 are known to selectively target neutrophils and
are expressed at sites of tissue inflammation. Armstrong
et al28 reported that, in skin, MIP-2 production is
restricted to infiltrating leukocytes, including neutrophils and monocytes, which appear later in the inflammatory response. Therefore, it is likely that high
MIP-2 and KC concentrations in cantharidin-treated
ear tissue contributed to dermal and epidermal neutrophil accumulation. Recruitment of monocytes and macrophages to sites of tissue injury is mediated, among
others, by the CC chemokine MCP-1.29,30
Our results in the mouse are in close agreement with
data from previous clinical studies. Day et al7 reported
granulocyte accumulation within blisters in humans at
24 h post cantharidin application and the presence of
the chemokines IL-8, ENA78, and growth-related oncogene (GRO)-a, vasoactive factors C3a, C5a, and
histamine, and cytokines TNF-a, IL-4, IL-1b, and
IL-12. The same finding was subsequently confirmed
by Harbord et al6 who showed that besides the inflammatory mediators mentioned above, cantharidin also
increases IL-5 and MCP-1 concentrations in human
blisters.
To validate our model for preclinical testing of
novel anti-inflammatory agents, we tested the antiinflammatory activity of azithromycin and clarithromycin on cantharidin-induced inflammatory markers also
present in human blisters. Based on our previous investigations, which indicated that clarithromycin has no
effect on cantharidin-induced skin inflammation after
single oral administration at different time-points prior

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August 2012

to inflammation induction (data not shown), clarithromycin and azithromycin were administered orally (2 3 150
mg/kg) 0.5 h before and 5 h after cantharidin application.
Both macrolides effectively inhibited the cantharidininduced inflammatory response, including ear edema
and MPO, MIP-2, MCP-1, and KC production. The ability of macrolides to inhibit the production of MPO, KC,
IL-1b, GM-CSF, and TNF-a as well as neutrophil accumulation and ear edema in various in vivo models, independently of direct antibacterial actions, has been well
documented by our group and others.13,31-35 Since
clarithromycin and azithromycin, in our experimental
model, reduced ear MPO concentration, we can
conclude that the macrolides inhibited cantharidininduced neutrophil accumulation in ear tissue and
thereby decreased ear swelling. The mechanism of the
observed anti-inflammatory activity probably involves
reduction of the neutrophil chemoattractants, MIP-2
and KC, as well as MCP-1 concentrations. A similar
finding was previously reported by Uriarte et al36 who
showed that azithromycin and roxithromycin reduce
neutrophil and monocyte transendothelial migration
in vitro, possibly due to inhibition of IL-8 and MCP-1
production. Although earlier investigations showed that
azithromycin and clarithromycin inhibit IL-1b secretion
in vitro17 and in vivo,32 the macrolides had no effect on
cantharidin-induced IL-1b production in mouse ear tissue. Azithromycin and clarithromycin also had no effect
on cantharidin-induced IL-6 production in mouse ear tissue. IL-6 is generally regarded as an inflammation modulating or even anti-inflammatory cytokine17 and both,
stimulatory and inhibitory effects of macrolides on its
production have been observed.17 It appears, therefore,
that macrolide anti-inflammatory activity varies between
different models. These discrepancies probably depend
on the inflammatory agent involved.
In conclusion, we have defined more precisely the
molecular and cellular mechanisms of cantharidininduced ear swelling in mice, aligning the murine model
more closely with the human model of cantharidininduced blister. As in humans, cantharidin induced production of MIP-2, MCP-1, KC, IL-6, and IL-1b, thereby
stimulating neutrophil accumulation and MPO activity
in mouse ear tissue. We further validated the model by
studying the potential mechanisms for the antiinflammatory activity of clarithromycin and azithromycin. These macrolides reduced ear MIP-2, KC, MCP-1,
and MPO concentrations and consequently decreased
ear swelling. Altogether, these results suggest that the
mouse model of cantharidin-induced inflammation can
be exploited for translational studies on novel antiinflammatory agents to be progressed toward testing
in humans.

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The authors wish to thank Prof. M. J. Parnham for critical reading of

the manuscript and Ms. S. Skender, M. Horvatincic, R. Povrzenic, and

I. Cubela,
and Mr. H. Poduska and V. Vrban for their excellent technical assistance.
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