Expression and function of toll-like receptor-3 and -9 in human corneal myofibroblasts.
OBJECTIVE
To investigate the expression and function of toll-like receptor (TLR)-3 and -9 in corneal myofibroblasts.


METHODS
Two types of human keratocytes were used, which were freshly isolated keratocytes from donor corneas and cultured keratocytes. Expression of the mRNAs for various molecular markers was analyzed in these cells by RT-PCR, and TLR-2, -3, -4, and -9 mRNAs were also analyzed by RT-PCR. Expression of TLR-3 and -9 at the protein level was assessed by flow cytometry. In addition, an antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of cultured keratocytes, with or without stimulation by poly inosine-polycytidylic acid (poly (I:C)) or CpG-DNA. Furthermore, a phagocytosis assay was performed to evaluate whether signaling via TLR-3 and -9 enhances phagocytosis.


RESULTS
Keratocytes cultured for three passages underwent differentiation into corneal myofibroblasts. TLR-3 and -9 were detected in corneal myofibroblasts at the mRNA and protein levels, but not in freshly isolated keratocytes. Stimulation of corneal myofibroblasts with poly (I:C) or CpG-DNA enhanced the production of IL-6, IL-8, GRO, ENA-78, and RANTES compared with that by untreated cells. Phagocytic activity of myofibroblasts was upregulated by signaling via TLR-3 and -9.


CONCLUSIONS
This is the first report on the in vitro expression and function of TLR-3 and -9 in corneal myofibroblasts. The findings suggest that the keratocyte phenotype determines the expression of TLR-3 and -9 and that corneal myofibroblasts may have an important role in bacterial and viral clearance.
Ebihara
Nobuyuki
N
Department of Ophthalmology, Juntendo University School of Medicine, Hongo, Tokyo, Japan. ebihara@med.juntendo.ac.jp
Yamagami
Satoru
S
Chen
Lizhong
L
Tokura
Tomoko
T
Iwatsu
Minoru
M
Ushio
Hiroko
H
Murakami
Akira
A
D000368:Aged; D018952:Antigens, CD34; D015415:Biomarkers; D002454:Cell Differentiation; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D008875:Middle Aged; D010587:Phagocytosis; D011070:Poly I-C / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases; D011509:Proteoglycans / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D051196:Toll-Like Receptor 3 / Q000235:genetics* / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000235:genetics* / Q000378:metabolism
D018952:Antigens, CD34; D015415:Biomarkers; D018925:Chemokines; C490948:KERA protein, human; D011509:Proteoglycans; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051196:Toll-Like Receptor 3; D051217:Toll-Like Receptor 9; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D011070:Poly I-C