Defensin gene expression in the cornea.
OBJECTIVE
To determine whether defensin genes are expressed in human corneas and bovine corneal keratocytes.


METHODS
In situ hybridization and immunohistochemistry were used to localize defensin mRNA and protein in normal and diseased human corneas. Cultured bovine keratocytes were stimulated with IL-1alpha or TNFalpha to determine whether defensin mRNA production occurred. Reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify defensin cDNA from cytokine-induced keratocytes, and Southern blots were used to verify the specificity of RT-PCR amplification products.


RESULTS
Defensin mRNA and protein were not detected in normal human corneal stroma, but were readily detectable in the corneal stroma in cases of rejected transplants and postinfectious keratitis. IL-1alpha was a potent inducer of defensin gene expression in keratocytes, which began 12 h after challenge and peaked at 18 to 24 h. TNFalpha weakly induced defensin mRNA in keratocytes at about 18 h. Southern blots of the RT-PCR products probed with an oligonucleotide complementary to internal sequences of defensin demonstrated the appropriately sized products (198 bp) specific for defensin.


CONCLUSIONS
This report demonstrates the presence of defensin in the human cornea and the capacity of corneal keratocytes to produce defensin mRNA in response to IL-1alpha and TNFalpha. Release of defensins by keratocytes in response to cytokines elaborated in corneal inflammation may contribute to the host defense response in microbial keratitis.
Gottsch
J D
JD
Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Li
Q
Q
Ashraf
M F
MF
O'Brien
T P
TP
Stark
W J
WJ
Liu
S H
SH
D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism*; D015139:Blotting, Southern; D002417:Cattle; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism*; D003316:Corneal Diseases / Q000378:metabolism; D003319:Corneal Stroma / Q000187:drug effects / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D023082:Defensins; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D007375:Interleukin-1 / Q000494:pharmacology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000890:Anti-Infective Agents; D017931:DNA Primers; D023082:Defensins; D007375:Interleukin-1; D011506:Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha