Defensin expression by the cornea: multiple signalling pathways mediate IL-1beta stimulation of hBD-2 expression by human corneal epithelial cells.
OBJECTIVE
To investigate the expression of human beta-defensins (hBDs) by human corneal epithelium and determine the effects of proinflammatory cytokines on expression of human beta-defensin (hBD)-2 by human corneal epithelial cells (HCECs) in culture.


METHODS
RNA was extracted from corneal epithelial cells scraped from cadaveric corneas and from cultured HCECs, and RT-PCR was performed to detect hBD-1, -2, and -3 mRNA. To study the effects of proinflammatory cytokines on expression of defensin, HCECs were cultured and then exposed to interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha for up to 36 hours, with a range of concentrations (0.01-100 ng/mL). In some experiments, cells were pretreated with various cell signaling pathway inhibitors before the addition of IL-1beta. At the end of the incubations, the cells were harvested for RT-PCR and the culture media collected for the detection by immunoblot analysis of secreted defensin peptide.


RESULTS
All epithelial tissue collected from cadaveric corneas expressed mRNA for hBD-1. hBD-2 was detectable in two of eight donors corneas, whereas hBD-3 was detected in five. All primary cultures of HCECs expressed hBD-1 and -3. A faint band for hBD-2 was detectable in three of eight cultures. Cultures of simian virus (SV)40-transformed HCECs always expressed hBD-1 and -3, but did not express hBD-2 under control conditions. IL-1beta and TNFalpha each stimulated the expression of hBD-2 in HCECs and were more effective in combination than alone. The effects of IL-1beta were concentration- (maximal at 10 ng/mL) and time-dependent (maximal at 12 hours and 24 hours for hBD-2 mRNA expression and protein secretion, respectively). The upregulation of hBD-2 mRNA persisted for at least 24 hours after removal of IL-1beta. The NFkappaB inhibitors pyrrolidinedithiocarbamate (PDTC; 100 microM), caffeic acid phenethyl ester (CAPE; 90 microM), and MG-132 (25 microM), blocked IL-1beta-stimulated expression of hBD-2. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (5 microM) and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (25 microM) partially blocked (by 47% and 59%, respectively) the effect of IL-1beta. However, PD98059, an ERK inhibitor, had no effect. Genistein (50 microM) and dexamethasone (1 microM) also partially blocked (by 26% and 28%, respectively) the effect of IL-1beta.


CONCLUSIONS
Human corneal epithelium expresses hBD-1 and -3. hBD-2 is not typically present, but its expression can be stimulated by proinflammatory cytokines such as IL-1beta, acting through mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB pathways. Because IL-1 is known to be increased at the ocular surface after injury, the current observations provide a mechanism to explain the previous finding that hBD-2 is upregulated in regenerating corneal epithelium. Cytokine stimulation of hBD-2 expression most likely provides additional protection against infection and raises the possibility that this defensin in particular may be involved in the wound-healing response, per se.
McDermott
Alison M
AM
College of Optometry, University of Houston, Houston, Texas 77204-2020, USA. amcdernott@popmail.opt.uh.edu
Redfern
Rachel L
RL
Zhang
Bei
B
Pei
Ying
Y
Huang
Ling
L
Proske
Rita J
RJ
D000368:Aged; D000890:Anti-Infective Agents / Q000378:metabolism*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation; D023083:beta-Defensins / Q000235:genetics* / Q000378:metabolism
D000890:Anti-Infective Agents; C406407:DEFB1 protein, human; C422950:DEFB103A protein, human; C106641:DEFB4A protein, human; D007375:Interleukin-1; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D023083:beta-Defensins; D012313:RNA; D020928:Mitogen-Activated Protein Kinases