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Amelioration of Corneal Inflammation by Topical
Azithromycin
Zahra Sadrai MD, Amir Reza Hajrasouliha MD, Sunil Chauhan PhD, Daniel
Saban PhD, Reza Dana MD, MPH, MSc.
Schepens Eye Research Institute,
Massachusetts Eye & Ear Infirmary,
Department of Ophthalmology,
Harvard Medical School, Boston, MA
Financial Disclosure: The authors have no financial interest in the product mentioned in this poster.
Funding Sources: Inspire Pharmaceuticals provided research support for this study.
Amelioration of Corneal Inflammation by Topical
Azithromycin
INTRODUCTION
Disease or injury to the cornea results in inflammation within the corneal epithelium and underlying stroma.
Adhesion molecules and cytokines coordinate leukocyte migration in inflammation (1) which can amplify
leukocyte infiltration and induce scarring. Intercellular adhesion molecule-1 (ICAM-1) on vascular endothelial
cells (VEC) binds to the integrin leukocyte function-associated antigen-1 (LFA-1) on leukocyte surfaces to
arrest the motion of rolling leukocytes and facilitate leukocyte endothelial transmigration into the cornea (24).
Macrolides are broad-spectrum antibiotics that are widely used to treat bacterial infections (5). There is also
some in vitro evidence that macrolides have anti-inflammatory and immunomodulatory activities. Studies
have demonstrated that macrolides suppress the activation of NF-kB and the release of pro-inflammatory
cytokines in vivo (6, 7).
We hypothesized here that Azithromycin (AZM), a broad-spectrum macrolide antibiotic recently approved for
ocular infection, has endogenous anti-inflammatory properties.
Amelioration of Corneal Inflammation by Topical
Azithromycin
PURPOSE
To investigate the potential Immunomodulatory effects of AZM on corneal
inflammation.
Amelioration of Corneal Inflammation by Topical
Azithromycin
METHODS
Cornea of 6-8 week old BALB/c mice underwent thermal cautery to induce inflammation and leukocyte influx.
Ten corneas were randomly divided into two groups treated topically either with AZM ophthalmic solution 1%
(AzaSite®; Inspire Pharmaceuticals, Inc, NC, USA) or the relevant vehicle (DuraSite®; Inspire
Pharmaceuticals, Inc, NC, USA) twice per day. Corneas were harvested at various time-points to characterize
the inflammatory infiltrate via FACS analysis, and to quantitate intercellular adhesion molecule expression via
real time PCR. All experiments were approved by the IACUC of the Schepens Eye Research Institute and
adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Days 1, 3, 7, 10, 14
Flow cytometry CD45,
CD11c, Gr1+, CD11b
Naïve
Real Time PCR
ICAM-1
Cauterized Cornea
Vehicle (BID)
Immunohistochemistry
ICAM-1
Cauterized Cornea
AZM (Bid)
Amelioration
Amelioration of
of Corneal
Corneal Inflammation
Inflammation by
by Topical
Topical
Azithromycin
Azithromycin
(AZM)
RESULTS
Vehicle
40000
AZM
30000
*
20000
10000
0
B.
Cell numbers per cornea
A.
Cell numbers per cornea
Gr-1+
CD45+
Vehicle
10000
AZM
8000
6000
4000
2000
0
Naive
Day 1
Day 3
Day 7
Day 10
Day 14
Naive
Day 1
Day 3
Day 7
Day 10
Figure 1. Within 24 hours of cauterization, leukocyte (CD45+ cell) infiltration into the vehicle-treated
corneas was apparent and peaked by day 7. Corneas treated with AZM, led to a 30% reduction in
leukocyte infiltration at day 1 and 40% reduction at day 7 (A). The reduction of infiltration at day 1
was predominantly comprised of neutrophils [(Gr-1+ cells) (25% reduction)] (B).
Day 14
Amelioration of Corneal Inflammation by Topical
Azithromycin
Vehicle
CD11c+
AZM
6000
4000
*
2000
0
Naive
Day 1
Day 3
Day 7
Day 10 Day 14
B.
Cell numbers per cornea
Cell numbers per cornea
A.
CD11b+
Vehicle
AZM
25000
20000
15000
10000
5000
0
Naive Day 1 Day 3 Day 7 Day 10 Day 14
Figure 2. Dendritic cell (CD11c+ cells, the most efficient antigen-presenting cells) infiltration
was greatest at day 7 post cautery in the vehicle-treated group. In the AZM-treated group the
reduction of infiltration at day 7 was comprised primarily of CD11c+ cells (35%) (A). CD11b+
macrophages peaked at day 3 in all groups. There was no statistically significant difference
between the AZM-treated eyes compared to the vehicle control group, though the level was
lower in the AZM-treated group at day 7 (B).
Amelioration of Corneal Inflammation by Topical
Azithromycin
Effect of AZM on Intercellular Adhesion Molecule-1
CD31
ICAM
DAPI
Corneal
Epithelium
CD31
Limbal
Vessel
CD31
ICAM
DAPI
ICAM
Corneal
Epithelium
ICAM
CD31
Limbal
Vessel
Vehicle-treated
AZM-treated
Figure 3. Topical application of AZM suppressed the up-regulation of ICAM-1 induced in cautery
model as compared to the vehicle treatment (day 7).
Amelioration of Corneal Inflammation by Topical
Azithromycin
ICAM-1
Vehicle
Fold increase compared to naive
40
AZM
P = .01
35
30
25
20
15
10
5
0
Day 1
Day 3
Day 7
Day 10
Day 14
Figure 4. The real-time PCR showed a decreased expression of ICAM-1 at all time
points, though statistical significance was only achieved on day 7.
Amelioration of Corneal Inflammation by Topical
Azithromycin
CONCLUSIONS
Following an inflammatory insult, topical AZM significantly reduced the leukocyte
infiltration into the cornea. This was further supported by an associated decrease
in expression of ICAM-1 in the cornea. This indicates AZM may have a potential
anti-inflammatory effect on corneal inflammation.
Amelioration of Corneal Inflammation by Topical
Azithromycin
References
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5.
G.G. Zhanel, M. Dueck, D.J. Hoban, L.M. Vercaigne, J.M. Embil, A.S. Gin and J.A. Karlowsky, Review of macrolides
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O. Culic, V. Erakovic and M.J. Parnham, Anti-inflammatory effects of macrolide antibiotics, Eur. J. Pharmacol. 2001;
429: 209–229.
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M.T. Labro, Cellular and molecular effects of macrolides on leukocyte function, Curr. Pharm. 2004; 3067–3080.
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