Nerve growth factor modulates toll-like receptor (TLR) 4 and 9 expression in cultured primary VKC conjunctival epithelial cells.
OBJECTIVE
To investigate if nerve growth factor (NGF) might modulate toll-like receptor (TLR) 4 and 9 expression in primary cultures of vernal keratoconjunctivitis (VKC)-derived conjunctival epithelial cells (VKC-ECs).


METHODS
Primary cultures of ECs were established from VKC (n=7) and healthy-control (n=5) conjunctival specimens. Primary untouched and short-term NGF-exposed VKC-ECs were analyzed for molecular (relative real-time PCR) and biochemical (confocal and fluorescence-activated cell sorting analysis of TLR4 and TLR9 expression. Data were compared to their untreated as well as stimulated healthy-control counterparts. Conditioned media were analyzed for interferon (IFN)-gamma, interleukin (IL)-4, IL-10, and IL-12 p40 secretion.


RESULTS
Primary untouched VKC-ECs showed TLR4 increase and TLR9 decrease compared to their healthy-control counterparts. NGF exposure resulted in a strong upregulation of TLR4 and a moderate upregulation of TLR9 in few passages VKC-ECs. Both TLR4 and TLR9 upregulation occurred in a dose-dependent fashion and were supported by biochemical analysis. NGF triggered a dose-response release of IL-10 in VKC-ECs conditioned media, an effect not detected for IL-4, IL-12 p40, and IFN-gamma.


CONCLUSIONS
Our data indicate that NGF is able to induce TLR4/TLR9 overexpression in VKC-ECs. These cells exhibited poor IL-4, IL-12 p40, and IFN-gamma responses to NGF, while a significant IL-10 decreased secretion was detected. The different NGF-induced TLR response between VKC and healthy-control conjunctival ECs as well as the different cytokine response might reflect a different pattern of cell activation according to the state of VKC.
Micera
Alessandra
A
CIR, Laboratory of Ophthalmology, University Campus Bio-Medico, Rome, Italy.
Stampachiacchiere
Barbara
B
Normando
Eduardo Maria
EM
Lambiase
Alessandro
A
Bonini
Sergio
S
Bonini
Stefano
S
D000293:Adolescent; D000818:Animals; D002478:Cells, Cultured; D003228:Conjunctiva / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D003233:Conjunctivitis, Allergic / Q000378:metabolism* / Q000473:pathology; D016207:Cytokines / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D051379:Mice; D020932:Nerve Growth Factor / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020800:Receptor, Nerve Growth Factor / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D051217:Toll-Like Receptor 9 / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D012333:RNA, Messenger; D020800:Receptor, Nerve Growth Factor; D051197:Toll-Like Receptor 4; D051217:Toll-Like Receptor 9; D020932:Nerve Growth Factor