The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells.
OBJECTIVE
Gram-negative bacterial infections of the eye can lead to corneal bacterial keratitis, visual impairment, and blindness. Many of these pathologic changes may be mediated by bacterially derived products such as lipopolysaccharide (LPS). In this investigation, it has been established for the first time that human corneal cells are capable of expressing the functional LPS receptor complex proteins, CD14 and Toll-like receptor 4 (TLR4).


METHODS
CD14 and TLR4 mRNA expression in human corneal cells was determined by RT-PCR and Northern blot analysis, and cell surface expression of these proteins was measured by flow cytometry. LPS-mediated corneal cell activation was determined by measuring intracellular calcium mobilization. Cellular cytokine and chemokine secretion in response to LPS was measured by ELISA. The expression and localization of CD14 in whole human cornea was determined by immunohistochemistry.


RESULTS
Human corneal epithelial, stromal, and endothelial cells expressed CD14 mRNA and cell surface CD14. LPS binding to cornea CD14 resulted in a rapid intracellular calcium response and the secretion of multiple proinflammatory cytokines and chemokines. CD14 mRNA expression in corneal epithelial cells was upregulated by LPS. In addition to CD14, corneal epithelial cells expressed the functional LPS receptor-signaling protein TLR4, which was also augmented by LPS.


CONCLUSIONS
The cornea expresses functional CD14 and TLR4 LPS receptor proteins. Understanding the function and biology of the corneal LPS receptor complex may lead to novel therapies for the management of ocular Gram-negative bacterial infections.
Song
P I
PI
Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Abraham
T A
TA
Park
Y
Y
Zivony
A S
AS
Harten
B
B
Edelhauser
H F
HF
Ward
S L
SL
Armstrong
C A
CA
Ansel
J C
JC
D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D029721:Drosophila Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D018950:Lipopolysaccharide Receptors / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D015854:Up-Regulation
D016207:Cytokines; D017931:DNA Primers; D029721:Drosophila Proteins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D002118:Calcium