The endocannabinoid system in cells of the ocular surface

IRIBARNE M; TORBIDONI V; PRESTIFILIPPO JP; RETTORI V; SINHA D; BERRA A; SUBURO AM

Buenos Aires

Congreso; XVII International Congress of Eye Research (ICER); 2006

Internationational Society of Eye Research

Purpose: The endocannabinoid system is involved in several immunomodulatory mechanisms, including mucosal immunity. Epithelial cells of the ocular surfaces are known to play an integral role in the ocular defense mechanism both as mechanical barriers and as active participants of immunological processes. Moreover, these epithelial cells are involved in allergic inflammation of the eye. Therefore we have investigated the presence of the endocannabinoid receptors CB1 and CB2 in mouse tissues and in a cell line derived from human conjunctiva. Methods: Eyes were enucleated from deeply anesthetized BALB-c mice and cryosections were obtained both after 4% paraformaldehyde perfusion or from fresh frozen material. The human conjunctival cell line IOBA-NHC was also used. Cryosections and monolayers seeded on glass coverslips were incubated with rabbit antibodies against CB-1 and CB-2. Specimens were developed using an immunoenzymatic procedure with nickel enhancement. Changes in the levels of intracellular cAMP were assayed by radioimmunoassay after ethanol extraction in confluent IOBA-NHC cells, incubated with 0.5 M forskolin and 0.5mM 3-isobutyl-1-methylxanthine (IBMX) in presence or absence of the endocannabinoid anandamide (AEA). Results: Strong immunostaining of the conjunctiva and cornea was obtained with two different antisera against CB-1 and two different antisera against CB-2. Immunoreactive cells were found both in corneal and conjunctival cells. CB-1 and CB-2 immunoreactivities were also observed in IOBA-NHC monolayers. Immunoreactivity showed a granular distribution, suggesting the presence of receptors in cytoplasmic vesicles and membrane domains. Addition of AEA (0.5 M) to the culture medium AEA reduced intracellular levels cAMP. Conclusions: The two cannabinoid receptors, CB1 and CB2, were detected in corneal and conjunctival epithelia using antibodies directed to different epitopes of each receptor. They were also present in a human conjunctiva-derived cell line. AEA-induced reduction of intracellular cAMP levels indicates that these receptors are functional.