The inflammatory milieu associated with conjunctivalized cornea and its alteration with IL-1 RA gene therapy.
OBJECTIVE
This study was designed to gain an insight into the inflammatory milieu into which a donor limbal graft is routinely introduced. The objective of this study was to modulate this environment by gene therapy with the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1 RA).


METHODS
In a mouse model, the ocular surface cytokine environment associated with a conjunctivalized cornea was assessed 4 weeks after injury. Total corneal epithelial and limbal debridement was performed with a combination of alkali and scrape injury. The cytokines and adhesion molecules measured included IL-1alpha, IL-1beta, IL-6, VEGF, intercellular adhesion molecule (ICAM)-1, and vascular adhesion molecule (VCAM)-1, by real-time PCR or ELISA. Injured corneas were transfected with IL-1 RA by injection of naked plasmid vector pIRES-EGFP-IL-1 RA immediately after injury. Corneas transfected with pIRES-EGFP served as the control. Expression of corneal IL-1 RA after transfection with pIRES-EGFP-IL1-RA was assessed over a 2-week period by real-time PCR and Western blot analysis. In addition, limbal stem cell grafts transfected with IL-1 RA were assessed for leukocyte influx.


RESULTS
Conjunctivalized corneas showed increased expression of IL-1alpha, IL-1beta, IL-1 RA, IL-6, VEGF, ICAM-1, and VCAM-1, compared with normal cornea. Transfection-efficiency experiments indicated that corneal expression of IL-1 RA peaked between 12 and 24 hours and lasted up to 2 weeks after the initial transfection. IL-1 RA corneal gene therapy resulted in a downregulation of IL-1beta and VCAM-1 expression at 4 weeks after injury, whereas downregulation of IL-6 was evident only at 1 week after injury. Corneal neovascularization was also reduced. In addition, corneal limbal stem cell grafts transfected with IL-1 RA showed a decreased leukocyte influx compared with control grafts.


CONCLUSIONS
Transfection of a cornea with IL-1 RA immediately after epithelial injury selectively altered the cytokine profile of the resultant conjunctivalized cornea and suppressed corneal neovascularization. Transfection of corneal limbal donor tissue with IL-1 RA before engraftment can reduce leukocyte influx into the graft. The findings demonstrate the feasibility of using transient cytokine gene expression, either in donor or recipient corneal tissue, to alter the ocular surface environment beneficially.
Department of Ophthalmology, Royal Victoria Hospital, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom.
Jonathan E
JE
Moore
Tara C B
TC
McMullen
Iain L
IL
Campbell
Richard
R
Rohan
Yuichi
Y
Kaji
Natalie A
NA
Afshari
Tomo
T
Usui
Desmond B
DB
Archer
Anthony P
AP
Adamis
D000818:Animals; D015153:Blotting, Western; D002464:Cell Migration Inhibition; D003228:Conjunctiva / Q000276:immunology / Q000473:pathology; D016510:Corneal Neovascularization / Q000517:prevention & control; D003646:Debridement; D016228:Endothelial Growth Factors / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004848:Epithelium / Q000276:immunology / Q000473:pathology; D019573:Epithelium, Corneal / Q000293:injuries / Q000378:metabolism* / Q000473:pathology; D005131:Eye Injuries / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D015316:Genetic Therapy; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007962:Leukocytes / Q000502:physiology; D008222:Lymphokines / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000235:genetics*; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; C507764:Il1rn protein, mouse; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D015850:Interleukin-6; D008222:Lymphokines; D017472:Receptors, Interleukin-1; D012795:Sialoglycoproteins; D019010:Vascular Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D018799:Intercellular Adhesion Molecule-1