High glucose may decrease the innate immune through TLRs in cornea epithelium.
OBJECTIVE
The purpose of this study was to investigate the high potential of glucose in inhibiting the innate immune in cultured human cornea epithelial cells (HCEC) and try to determine whether the role of high glucose on the HCEC relate to toll-like receptor (TLR)2 and TLR4.


METHODS
Cells were cultured for 3 days in 5 mmol/l (normal glucose). Then high glucose (25 mmol/l) was added along with normal glucose with daily changes in media for 24 h. The cells were also treated with mannitol as an osmotic control. The cellular abundance of the mRNAs for TLR2 and TLR4 was determined by real-time polymerase chain reaction analysis. The proteins of TLR2 and TLR4 were also compared by immunofluorescent staining and western blot. The release of interleukin 6 (IL-6) and IL-8 from cultured HCEC was measured using enzyme-linked immunosorbent assays (ELISA) in the presence and absence of specific blocking antibodies to TLR2 and TLR4.


RESULTS
Incubation of HCEC with high glucose showed that the mRNA expression of TLR2 and TLR4 was markedly inhibited. Immunofluorescent staining and western blot analysis confirmed that the protein expression of TLR2 and TLR4 was downregulated in response to high glucose. The result of ELISA also showed that the release of IL-6 and IL-8 can be inhibited by high glucose, but these inhibitions were partly counteracted after pretreatment with anti-TLR2 and/or anti-TLR4 monoclonal antibody. The results also showed that the osmotic control did not affect the expression of TLR2, TLR4, and IL-6, 8.


CONCLUSIONS
High glucose may decrease the innate immune through TLRs in cornea epithelium.
Eye Center, Affiliated Second Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Hailong
H
Ni
Xiaoran
X
Yan
Zhenyun
Z
Lin
Xiuming
X
Jin
D057134:Antibodies, Neutralizing / Q000494:pharmacology; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D015870:Gene Expression; D005947:Glucose / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D007113:Immunity, Innate; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D008353:Mannitol / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D060888:Real-Time Polymerase Chain Reaction; D051195:Toll-Like Receptor 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology
D057134:Antibodies, Neutralizing; D015850:Interleukin-6; D016209:Interleukin-8; D012333:RNA, Messenger; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D008353:Mannitol; D005947:Glucose