Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface.
OBJECTIVE
To evaluate whether experimentally induced dry eye in mice activates mitogen-activated protein kinase (MAPK) signaling pathways, c-Jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK), and p38 and stimulates ocular surface inflammation.


METHODS
129SvEv/CD-1 mixed mice aged 6 to 8 weeks were treated with systemic scopolamine and exposure to an air draft for different lengths of time, from 4 hours to 10 days. Untreated mice were used as the control. The concentrations of IL-1beta and TNF-alpha in tear fluid washings and in corneal and conjunctival epithelia were measured by ELISA. MMP-9 in tear washings was evaluated by zymography, and gelatinase activity in the cornea and conjunctiva was determined by in situ zymography. Corneal and conjunctival epithelia were lysed in RIPA buffer for Western blot with MAPK antibodies, or they were lysed in 4 M guanidium thiocyanate solution for extraction of total RNA, which was used to determine gene expression by semiquantitative RT-PCR, real-time PCR, and gene array.


RESULTS
Compared with those in age-matched control subjects, the concentrations of IL-1beta and MMP-9 in tear fluid washings and the concentrations of IL-1beta and TNF-alpha and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice receiving treatments to induce dry eye after 5 or 10 days. The expression of IL-1beta, TNF-alpha, and MMP-9 mRNA by the corneal and conjunctival epithelia was also stimulated in mice treated for 5 or 10 days. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were markedly increased as early as 4 hours after treatment, and they remained elevated up to 5 days.


CONCLUSIONS
Experimental dry eye stimulates expression and production of IL-1beta, TNF-alpha, and MMP-9 and activates MAPK signaling pathways on the ocular surface. MAPKs are known to stimulate the production of inflammatory cytokines and MMPs, and they could play an important role in the induction of these factors that have been implicated in the pathogenesis of dry eye disease.
Luo
Lihui
L
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, 6565 Fannin Street, Houston, TX 77030, USA.
Li
De-Quan
DQ
Doshi
Amish
A
Farley
William
W
Corrales
Rosa M
RM
Pflugfelder
Stephen C
SC
D000818:Animals; D003228:Conjunctiva / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis; D015352:Dry Eye Syndromes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004848:Epithelium / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism; D005123:Eye / Q000378:metabolism*; D018836:Inflammation Mediators / Q000378:metabolism; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013666:Tears / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D016207:Cytokines; D018836:Inflammation Mediators; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D020780:Matrix Metalloproteinase 9