|Molecular Vision 2007;
Received 14 May 2007 | Accepted 14 August 2007 | Published 14 August 2007
TNFα suppression of corneal epithelium migration
Winston W.-Y. Kao,2
1Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan; 2Department of Anatomy, Graduate School of Medicine, Osaka City University, Osaka, Japan; 3Department of Ophthalmology, University of Cincinnati Medical Center, Cincinnati, OH
Correspondence to: Shizuya Saika, M.D., Ph.D., Department of Ophthalmology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-0012, Japan; Phone: 81-73-447-2300; FAX: 81-73-448-1991; email: firstname.lastname@example.org
Purpose: To evaluate the role of tumor necrosis factor α (TNFα) in regulation of corneal epithelial cell migration.
Methods: Cell culture of immortalized corneal epithelial cell line was employed to examine the role of transforming growth factor β1 (TGFβ1) and TNFα on cell migration and cell signaling. Healing of central epithelial defect was also observed in organ culture in the presence or absence of neutralizing antibody against either TNFα or TGFβ1.
Results: In cell cultures of corneal epithelial cell line, adding TNFα suppresses activation of p38 signal and cell migration, but not Smad2 activation, upon TGFβ1 exposure. In an organ culture system, healing of an epithelial defect was promoted by the loss of TNFα. A neutralizing antibody against TNFα also promoted closure of an epithelial defect of organ cultured WT mouse corneas. Anti-TGFβ neutralizing antibody reversed facilitation of epithelial healing in KO corneas in organ culture.
Conclusions: TNFα inhibits migration of corneal epithelial cells.
Epithelial defects in the cornea must be rapidly resurfaced to avoid microbial infection and further damage to the underlying stroma. It is of interest to note that two cellular responses, i.e., cell migration and cessation of cell proliferation, take place concomitantly at the early phase of corneal epithelial debridement wound healing [1-5].Various cytokines and growth factors are believed to regulate cell migration and other healing-related cell behavior [1-5]. Among them, transforming growth factor β (TGFβ) is known to be one of the major factors which promote epithelial cell migration in the cornea. We previously reported that signal of p38 mitogen-activated protein kinase (MAPK) activated by TGFβ has an important role in cell migration promotion, while TGFβ/Smad pathway has minimum role in it [6,7].
A proinflammatory cytokine, tumor necrosis factor α (TNFα), is also considered to be involved in the healing process of an injured corena. TNFα is expressed by corneal epithelium and inflammatory cells [8,9]. We previously reported that endogenous TNFα counteracts TGFβ in alkali-burned mouse corneas, resulting in marked inflammation, tissue destruction, and fibrosis of the stroma . TNFα is known to be upregulated in tear fluid in eyes with dry eye or allergic ocular surface disorders [11,12]. However, the role of endogenous TNFα in homeostasis maintenance or healing of corneal epithelium is not fully understood, although TNFα's antagonistic effects on TGFβ1 [13-19] suggest an unfavorable effect of this cytokine on healing of corneal epithelial disorders.
In the present study, we examined the role of TNFα in epithelial debridement healing of mouse cornea by using TNFα-deficient mice and a cultured corneal epithelial cell line. We showed here that endogenous TNFα counteracts TGFβ/p38MAPK signal and therefore slows migration of corneal epithelium.
Experiments were approved by the DNA Recombination Experiment Committee and the Animal Care and Use Committee of Wakayama Medical University, and conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Epithelial defect in mouse cornea
Four-week-old TNFα+/+ (wild type, WT, n=16) and TNFα-/- (knockout, KO, n=16) mice of the C57BL/6 background were used. Under general anesthesia and topical anesthesia, a round epithelial debridement (1.6 mm in diameter) was produced in the central cornea of the right eye [6-20]. In the current studies, after different periods of injury (4, 10, and 22 h), the animals were administered BrdU (120 μg/g body weight; Sigma, St. Louis, MI) and sacrificed 2 h later by CO2 asphaxia and cervical dislocation as previously reported [6-20]. For specimens immediately after epithelial debridement, central corneal epithelium debridement was created 2 h after BrdU i.p. administration, and the experimental animals were sacrificed as described above. Affected eyes from 4 animals at each time point were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 48 h. Specimens were dehydrated through a graded ethanol series and paraffin embedded.
Organ-culture of epithelium-debrided corneas of WT or KO mice
To examine the role of endogenous TNFα in epithelial healing, we performed an organ-culture experiment as previously reported . Our previous study showed that anti-TGFβ neutralizing antibody retarded epithelial migration in this organ-culture system. Under general anesthesia and topical anesthesia as previously reported , a round epithelial debridement (1.6 mm in diameter) was produced in the central cornea of eyes of WT or KO mice as previously reported . The animals were killed without reawaking and the eye globe was enucleated. Epithelium-debrided WT or KO mouse eyes were then organ cultured in Eagle's medium supplemented with 2.0% fetal bovine serum either with mouse monoclonal anti-TGF beta-neutralizing antibody (20 mg/ml, R & D system) or nonimmune IgG at the same concentration. The closure of the epithelial defect was determined by fluorescein staining following a 2 h labeling with BrdU after 12, 24, 36, and 48 h of culture. Four eyes were prepared and analyzed for each experimental condition at each time point. The eye was then fixed and embedded in paraffin for histology.
Paraffin sections 5 μm thick were processed for immunohistochemistry for phosphorylated p38MAPK, F4/80 macrophagic antigen, and BrdU . After blocking with 3% dry milk and 5% bovine serum in PBS, the sections were incubated with mouse monoclonal IgM anti-phospho-p38MAPK antibody (1:100 dilution in phosphate-buffered saline [PBS]; Santa Cruz Biotechnology, Santa Cruz, CA) or rat monoclonal F4/80 antibody (1:100 dilution in PBS; Clone A3-1, 1:400, BMA Biomedicals, August, Switzerland) for 12 h at 4 °C. After three washes with PBS, the specimens were then treated with FITC-conjugated antibodies against each immunoglobulin for 4 h at 4 °C. After another wash in PBS, FITC-conjugated secondary antibodies were detected under fluorescent microscopy following embedded in a medium containing nuclear staining dye.
For BrdU immunostaining, paraffin sections were treated with 2 N HCl for 1 h at 37 °C and then washed in PBS prior to the application of anti-BrdU antibody (11X in PBS; Boehringer Mannheim, Germany) . These slides were then processed for secondary antibody reaction, DAB reaction, and observed under light microscopy.
Effect of TNFα and TGFβ1 on migration of human corneal epithelial cells
Recombinant TGFβ1 (2.0 ng/ml) and/or recombinant TNFα (10.0 ng/ml) was added to confluent cultures of the SV40-immortalized Araki-Sasaki human corneal epithelial cell line in serum-free medium. Then the monolayer in noncoated 2 well chamber glass slides was wounded by a silicone rubber needle as previously reported . Three wounds were prepared in one chamber. Closure of defect was monitered at 0, 14, and 28 h post-injury. The cells were then stained with hematoxylin and eosin. Three wounds were prepared for data at each timepoint. The width of the remaining defect was measured at 3 independent points in each wound .
Effect of TNFα and TGFβ1 on cytoplasmic signaling in human corneal epithelial cells
For examination of cell signaling, confluent cultures of Araki-Sasaki corneal epithelial cell were treated with recombinant TGFβ1 (2.0 ng/ml) or TGFβ1 (2.0 ng/ml) and TNFα (10.0 ng/ml). The cells were lysed in cell lysis buffer and processed for western blotting. Subconflunet cells in wells of 8 chamber glass slides were also treated in the same way as above with TGFβ1 and TNFα. Cells were fixed with 4.0% paraformaldehyde at 0, 1, 3, and 6 h, and processed for immunostaining for phospho-p38MAPK and phospho-Smad2 .
Data were statistically analyzed by using ANOVA and a p<0.05 was considered significant.
Loss of TNFα promotes reepithelialization of debridement in central corneal epithelium
To examine the role of TNFα in reepithelialization of debrided cornea, we first evaluated healing of debridement in central corneas of TNFα KO or WT mice (Figure 1). At each time point the defect remaining was larger in WT (Figure 1A-D) mice as compared with KO (Figure 1E-H) mice. Finally at 24 h the epithelial defect was resurfaced in 3 of 4 KO corneas examined, while all 4 corneas of WT mice exhibited a significant remaining defect in its epithelium. Histology by hematoxylin and eosin staining and F4/80 immunohistochemistry (not shown) showed no difference of inflammation by polymorphonuclear leukocytes and macrophages in the epithelium-debrided stroma between a WT and a KO mouse at and before 12 h post-debridement. At 24 h, post-epithelial defect stromal inflammation was more marked beneath the defected epithelium in a WT mouse (Figure 2A,C) as compared with a KO mouse (Figure 2B,D). Expression of phospho-p38MAPK was detected in migrating epithelial cells of both WT (Figure 2E,G) and KO corneas (Figure 2F,H).
As for cell proliferation activity in the healing epithelium, no difference of distribution of BrdU-labeled epithelial cells between WT and KO mice was observed throughout the healing interval  (data not shown).
To rule out the contribution of blood-derived inflammatory cell invasion on epithelial healing, we observed epithelial healing in an organ-cultured mouse eye with a circular epithelial defect. As shown in our previous report anti-TGFβ neutralizing antibody retarded epithelial healing in WT corneas (Figure 3A-H), and this was the case also in KO eyes (Figure 3I-P).
Effect of TNFα and TGFβ1 on migration of human corneal epithelial cells
To further examine the role of TNFα in migration of corneal epithelial cells, the in vitro wound healing model of Araki-Sasaki human corneal epithelial cell line was used. Adding recombinant TGFβ1 promoted closure of defect, while addition of TNFα did not alter the closure rate of the defect. Closre rate was similar between a culture with TNFα alone and a culture with TGFβ1 and TNFα (Figure 4).
Effect of TNFα and TGFβ1 on cytoplasmic signaling in human corneal epithelial cells
Western blotting showed that p38MAPK is activated upon exposure to exogenous TGFβ1 at 5.0 ng/ml as early as 0.5 h with a peak at 1.0 h and the activation lasted over 5.0 h (Figure 5A). The presence of exogenous TNFα at 10.0 ng/ml did not activation level of p38MAPK at 0.5 h but thereafter suppressed it (Figure 5A). Immunocytochemistry coincided with the finding obtained by western blotting; nuclear accumulation of phospho-p38MAPK was seen in both cultures with TGFβ1 alone or TGFβ1 and TNFα (Figure 5B-G). It was still seen in a TGFβ1-plus culture at 3 h (Figure 5D), but not in a culture with TNFα and TGFβ1 (Figure 5G).
Smad signal is also a major signaling cascade downstream of the TGFβ receptor. Both western (Figure 6A) blotting and immunostaining (Figure 6B-G) showed that the presence of TNFα did not affect Smad2 activation.
In the present study we show that loss of TNFα promotes healing of an epithelial defect in a mouse cornea. We previously reported that TGFβ promotes cell migration of an injured corneal epithelium [6,7] and it is also well known that TNFα counteracts the TGFβ signal in certain conditions, i. e., in cell culture [10,14-19]. Thus, it is conceivable that the absence of endogenous TNFα enhances reepithelialization of an epithelial defect by acceleration of epithelial cell migration in a cornea. BrdU immunohistochemistry did not show a difference in cell proliferation activity in in vivo healing corneal epithelium between WT and KO mice (data not shown). Also, no difference of cell proliferation was observed in organ-cultured WT and KO healing epithelium (data not shown). However, the organ culture system used for epithelial cell migration required 2% serum for epithelial cell survival and thus the cytokine(s) in the serum might mask the effect of blocking cytokines on cell proliferation in the healing epithelium. This is understandable since we have reported that one of the two major signaling limbs from TNFα receptor, nuclear factor-κB, counteracts cell proliferation promotion in a healing corneal epithelium mediated by another major TNFα signal, c-Jun NH2-terminal kinase . Both TNFα and TGFβ1 observed in the injured stroma is considered to be secreted not only by resident corneal cells but also by inflammatory cells, i.e., invaded macrophages. We reported that loss of TNFα results in marked inflammation in a healing alkali-burned cornea in mice as compared with WT mice. In this report we concluded that such marked inflammation by macrophages were due to over-activation of the TGFβ signal in the absence of TNFα's counteraction. However, in the current project we did not histologically observe a difference of inflammation in the epithelium-debrided stroma between a WT and a KO mouse at and before 12 h post-debridement. At 24 h post-epithelial defect, stromal inflammation was more marked beneath the defected epithelium in a WT mouse as compared with a KO mouse. This difference might be due to the invasion of tear fluid-derived inflammatory cells through the defect in the central epithelium, but might not to the direct effect of the absence of TNFα on epithelial cell locomoation. Therefore, acceleration of epithelial healing in KO mouse corneas might not due to the difference of levels of cytokine(s) secreted by inflammatory cells in the stroma, but might be due to a direct effect of lacking TNFα on epithelial-migration-enhancing signal from ligand(s), i.e., TGFβ. We further confirmed this notion by performing an organ-culture experiment. Organ-culture of an eye with a corneal epithelial defect does not allow inflammatory cells to invade into the healing cornea from blood vessels. The organ-culture also showed that loss of TNFα again facilitates resurfacing of the epithelial defect and this promotion was abolished by adding anti-TGFβ neutralizing antibody.
It has been reported that migration of corneal epithelium depends on TGFβ/p38MAPK signal rather than Smad2/3 signal . Migrating corneal epithelium in the in vivo specimen showed upregulation of phosphorylated p38MAPK. The counteraction of TNFα on TGFβ-driven enhancement of epithelial cell migration was well reproduced in a culture of SV40-immortalized Araki-Sasaki human corneal epithelial cell line. Therefore, we used this cell line to examine the role of TNFα on TGFβ1/p38MAPK in corneal epithelial cells. Specifically, we conducted western blot analysis and immunocytochemistry for p38 and Smad2 in a culture of this cell line. The result showed that the presence of TNFα reduced the phosphorylation/activation level of p38MAPK in the cells, while it did not affect the activation of Smad2.
As in synovial fluid in a rheumatoid arthritis patient [23,24], TNFα concentration increases in tear fluid of ocular surface inflammations, i.e., Sjogren's syndrome or allergic conjunctivitis [11,12]. However, the role of TNFα in corneal wound healing needs to be further investigated. In a mouse skin lacking TNFα receptor healing is promoted . Because our present study shows that TNFα performs an unfavorable effect on healing of corneal epithelium, suppression of TNFα in tear fluid, i.e., blocking by topical application of neutralizing antibody against TNFα, might have a therapeutic effect on epithelial disorders with those diseases.
Cross-talk between TNFα signaling and TGFβ/Smad signaling has been reported [13-17]. TNFα signaling inhibits the TGFβ/Smad pathway by multiple mechanisms, including induction of Smad7, inhibition of Smad3 by c-Jun N-terminal kinase activation of AP-1, and down-regulation of TGFβ receptor expression [13-17]. As previously reported in dermal fibroblasts , the present study showed that TNFα counteracted induction of connective tissue growth factor by TGFβ1 in cultured ocular fibroblasts and this might also occur in the healing cornea in vivo. Unfortunately, the present study could not reveal the detailed mechanism of suppression of TGFβ/p38MAPK by TNFα signal. Nevertheless, the unfavorable effect of TNFα on epithelial healing as revealed here indicates the need for development of a new strategy in the treatment of persistent epithelial disorders associated with dry eye or allergic conjunctival diseases.
This study was supported by a Grant from the Ministry of Education, Science, Sports and Culture of Japan (C11591871 to S.S. and C16590150 to K.I.), NIH grant EY 13755, Research to Prevent Blindness, and Ohio Lions Eye Research Foundation (to W.W.-Y.K.).
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