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.
Transcript
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
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 (2-4).
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.
INTRODUCTION
To investigate the potential Immunomodulatory effects of AZM on corneal inflammation.
PURPOSE
Amelioration of Corneal Inflammation by Topical Azithromycin
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.
Naïve
Cauterized Cornea Vehicle (BID)
Cauterized Cornea AZM (Bid)
Real Time PCR ICAM-1
Flow cytometry CD45, CD11c, Gr1+, CD11b
Immunohistochemistry ICAM-1
Days 1, 3, 7, 10, 14
METHODS
Amelioration of Corneal Inflammation by Topical Azithromycin
CD45+
0
10000
20000
30000
40000
Naive Day 1 Day 3 Day 7 Day 10 Day 14
CD45+
0
10000
20000
30000
40000
Naive Day 1 Day 3 Day 7 Day 10 Day 14
Gr-1+
0
2000
4000
6000
8000
10000
Naive Day 1 Day 3 Day 7 Day 10 Day 14
Amelioration of Corneal Inflammation by Topical Azithromycin (AZM)
Ce
ll n
um
be
rs p
er
corn
ea
Ce
ll n
um
be
rs p
er
corn
ea
RESULTS
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).
Vehicle
AZM
Vehicle
AZM
A. B.
*
Amelioration of Corneal Inflammation by Topical Azithromycin
CD11c+
0
2000
4000
6000
Naive Day 1 Day 3 Day 7 Day 10 Day 14
Ce
ll n
um
be
rs p
er
corn
ea
*
Vehicle
AZM
CD11b+
0
5000
10000
15000
20000
25000
Naive Day 1 Day 3 Day 7 Day 10 Day 14
Ce
ll n
um
be
rs p
er
corn
ea
Vehicle
AZM
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).
A. B.
Amelioration of Corneal Inflammation by Topical Azithromycin
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).
Corneal Epithelium
Vehicle-treated
LimbalVessel
AZM-treated
ICAM
CD31Corneal
Epithelium
LimbalVessel
ICAM
CD31
CD31ICAM DAPI
CD31ICAM DAPI
Effect of AZM on Intercellular Adhesion Molecule-1
Amelioration of Corneal Inflammation by Topical Azithromycin
0
5
10
15
20
25
30
35
40
Day 1 Day 3 Day 7 Day 10 Day 14
Vehicle
AZM
P = .01
ICAM-1F
old
incr
ease
com
pare
d to
nai
ve
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
1. Delves PJ, Roitt IM. The immune system. First of two parts. N Engl J Med. 2000; 343:37-49.
2. Biswas PS, Banerjee K, Kim B, Rouse BT: Mice transgenic for IL-1 receptor antagonist protein are resistant to herpetic stromal keratitis: possible role for IL-1 in herpetic stromal keratitis pathogenesis. J Immunol 2004, 172:3736–3744.
3. Muller WA, Weigl SA, Deng XH, Phillips DM. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Mecl. 1993; 178:449-460.
4. Vaporciyan AA, DeLisser HM, Yan HC, et al. Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo. Science. 1993; 262:1580 -1582.
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 and ketolides: focus on respiratory tract infections, Drugs 61 2001; 61:443–498.
6. O. Culic, V. Erakovic and M.J. Parnham, Anti-inflammatory effects of macrolide antibiotics, Eur. J. Pharmacol. 2001; 429: 209–229.
7. M.T. Labro, Cellular and molecular effects of macrolides on leukocyte function, Curr. Pharm. 2004; 3067–3080.
