TLR4 mediates human retinal pigment epithelial endotoxin binding and cytokine expression.
OBJECTIVE
We previously demonstrated toll-like receptor 4 (TLR4) to be involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classic role of TLR4 in HRPE endotoxin (lipopolysaccharide [LPS]) binding leading to HRPE proinflammatory cytokine secretion.


METHODS
Cultured human HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). HRPE cells labeled with fluorescent monoclonal antibodies (mAb) to TLR4 and its associated adhesion molecule, CD14, were analyzed by real-time microscopy and resonance energy transfer (RET), determining associations of fluorescent LPS, TLR4, and CD14. LPS-induced HRPE secretion of interleukin-8 (IL-8) was measured with and without specific blocking mAb to TLR4 and CD14. HRPE TLR4 expression was measured after LPS exposure in the presence and absence of blocking antibodies to TLR4 and CD14.


RESULTS
All three HRPE cell lines demonstrated constitutive TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. Examination of HRPE cells labeled with fluorescent mAb to TLR4, CD14, and LPS demonstrated RET among the three molecules, indicating that LPS-CD14 binding preceded LPS-TLR4 binding and the close association of CD14 and TLR4. LPS-induced IL-8 was significantly reduced (P < .05) in the presence of blocking mAb to TLR4 or CD14. Up-regulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14.


CONCLUSIONS
HRPE TLR4 is a multifunctional molecule that participates in photoreceptor outer segment membrane recognition, oxidant production, LPS recognition, and cytokine production. These roles indicate potential involvement in retinal degenerative and inflammatory processes.
Elner
Susan G
SG
Department of Ophthalmology, University of Michigan, Kellogg Eye Center, Ann Arbor, Michigan, USA.
Petty
Howard R
HR
Elner
Victor M
VM
Yoshida
Ayako
A
Bian
Zong-Mei
ZM
Yang
Dongli
D
Kindezelskii
Andrei L
AL
D015153:Blotting, Western; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005470:Fluorometry; D005786:Gene Expression Regulation / Q000187:drug effects; D006706:Homeostasis; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism* / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014018:Tissue Distribution; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000502:physiology*
D016207:Cytokines; D016209:Interleukin-8; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4