Cannabinoid signaling and stress markers in the conjunctival epithelium

M IRIBARNE, V TORBIDONI, M CASTANEDA, J PRESTIFILIPPO, V RETTORI, A BERRA, AM SUBURO.

Washington DC, United States

Congreso; American Society for Cell Biology (ASCB), Annual Meeting, 2007.; 2007

American Society for Cell Biology

Purpose: The endocannabinoid system is involved in several immunomodulatory mechanisms, including mucosal immunity. Since epithelial cells of the ocular surface are involved in ocular defense mechanisms, we investigated the presence of the endocannabinoid receptors CB1 and CB2 in mouse and human conjunctival cells and tested their interaction with intracellular inflammatory responses.Methods: Immunohistochemical detection of CB1 and CB2 was done on mouse conjunctival cryosections, human conjunctival exfoliated cells and IOBA-NHC monolayers, a cell line derived from human conjunctiva. The presence of CB1 and CB2 mRNAs was evaluated by RT-PCR, and changes in the levels of cellular cAMP were assayed by radioimmunoassay after stimulation of IOBA-NHC cells, a cell line derived from human conjunctiva, with the endocannabinoid anandamide (AEA) or the cannabinoid analogue CP55,940. Stress signaling cascades (NF-kappaB p-65 and Phospho-SAPK/JNK kinase) were studied in IOBA-NHC cells activated with TNF-a with or without preincubation with CP55,940.Results: Strong immunostaining of the conjunctival epithelium was obtained with different antisera against CB1 and CB2. CB1 and CB2 immunoreactivities were also observed in human exfoliated conjunctival cells and IOBA-NHC monolayers. Immunoreactivity showed a granular distribution, suggesting the presence of receptors in cytoplasmic vesicles and membrane domains. RT-PCR demonstrated the presence of mRNA messenger for both receptors. Additionally, both AEA and CP55,940 reduced intracellular levels of cAMP in IOBA-NHC cells.In these cells, TNF-a induced nuclear translocation of NF-kappaB and increased immunoreactivity of JNK. Both responses disappeared when cells were simultaneously incubated with CP54,940.Conclusions: The conjunctival epithelium expresses both cannabinoid receptors. As expected for these receptors, activation by specific ligands reduced cAMP intracellular levels. Most importantly, these receptors appear to be involved in stress signaling mechanisms, suggesting that their regulation could be involved in diseases affecting the ocular surface.