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A. Micera, A. Lambiase, B. Stampachiacchiere, E. Normando, L. Mastrella, S. Bonini, S. Bonini; Nerve Growth Factor Modulates Human Conjunctival Epithelial Cell Line. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5302.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate if the Nerve Growth Factor, a neurotrophin with pleiotrophic effects among nervous, immune and visual systems, might modulate conjunctival epithelial cell growth, differentiation and immune properties.
Conjunctival epithelial cell line (JECs; a kind gift from Prof. Gipson) were expanded in KSFM and switched to stratification medium (dF12 plus 10% FCS, 10ng/mL EGF). JECs were subject to specific trkANGFR and p75NTR expression (FACS/confocal/qPCR). At confluence, JECs were cultured in dF12 without FCS and EGF, but with increasing NGF concentrations (1-250ng/mL). Monolayers were analysed for quantify cell growth (MTT/Ki76/BrdU) and goblet cell differentiation (periodic acid Schiff/muc5ac confocal staining). Conditioned media were collected from cultures (24hrs) to quantify IL1ß, IL10, TGFß1 and muc5ac. Total RNA and proteins were isolated from cultures (6hrs/24hrs) to analyze differentiation (muc1,4,5ac) as well as immune related (TLR-2,4,9) markers, by qPCR and FACS/Western blot respectively.
JECs express both trkANGFR and p75NTR. NGF-treated JECs showed a significant increase in goblet cell number and PAS positivity, with no effect on proliferation. qPCR analysis revealed a 7-fold increase expression of muc5ac (p<.05), while muc1 and muc4 were not significantly affected (-1.78 and 2.45 respectively; p>.05). Neither IL1ß or IL10, were modulated by NGF treatment (p>.05). NGF induced a decrease of TGFß1, TLR2 and TLR4 mRNAs and proteins, while TLR9 did not change (p<.05).
These findings suggest that NGF might play a role in the complex mechanism leading to JEC differentiation, by increasing goblet cell/muc5ac expression. By modulating both TLR2 and TLR4, NGF might also influence local immune response via TLR expression
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