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Macrophage migration inhibitory factor ameliorates UV-induced photokeratitis in mice
Experimental Eye Research 86 (2008) 929–935
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Macrophage migration inhibitory factor ameliorates UV-induced photokeratitis in mice Nobuyoshi Kitaichi a, *, Tadamichi Shimizu b, Kazuhiko Yoshida a, Ayumi Honda b, Yoko Yoshihisa b, Satoru Kase a, Kazuhiro Ohgami a, Osamu Norisugi b, Teruhiko Makino b, Jun Nishihira c, Sho-ichi Yamagishi d, Shigeaki Ohno a a
Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, Japan Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan Department of Medical Information, Hokkaido Information University, Ebetsu, Japan d Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2007 Accepted in revised form 6 March 2008 Available online 16 March 2008
Acute ultraviolet (UV) exposure causes photokeratitis, and induces apoptosis in corneal cells of the eye. Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. Today, MIF is considered as an integral component of the host antimicrobial alarm system and stress response that promotes the proinflammatory functions of immune cells. Also, MIF is considered to contribute the wound healing process. The aim of the present study is to determine the effects of MIF expression on UV irradiated corneal damage. MIF transgenic (MIF-Tg), wild type (WT), and MIF deficient (MIF KO) mice were UVB-irradiated of 400 mJ/cm2 to induce acute UV-photokeratitis. MIF Tg mice constitutively produce high levels of MIF. Morphological changes were most severe in MIF KO mice, and WT and MIF Tg mice were following. Corneal basement membrane of MIF-Tg was well preserved. Prominent higher level of MIF was observed in MIF-Tg than WT after UVB irradiation in cornea. TUNEL staining showed a significantly smaller number of TUNEL positive nuclei in MIF-Tgm (6.2 4.3 cells/section, p < 0.01 compared with WT) than WT (30.7 9.1) and MIF KO mice (32.1 12.7) 24 h after UV exposure. The number of c-Jun positive nuclei was significantly higher in MIF Tg (p < 0.01) than in WT and MIF KO mice. Serial observation revealed that BrdU incorporation was significantly upregulated in MIF Tg (p < 0.01), but downregulated in MIF KO (p < 0.01) than WT mice. MIF expression may thus be related to the amelioration of UVB-caused corneal injury, and this association was attributable to the upregulation of cell proliferation after acute UV-induced corneal damage, which involves the c-Jun dependent pathway. In conclusion, UV-damaged cornea is recoverable without MIF, however it takes longer time than normal condition. Cornea is less damaged and can make a quick recovery when ocular tissue is enough supplied with MIF. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: apoptosis BrdU c-Jun cornea keratitis MIF ultraviolet
1. Introduction Ultraviolet (UV) irradiation represents a significant environmental and occupational hazard that can cause acute and chronic inflammatory changes in the exposed cornea and lens. The acute exposure of artificial sources, such as tanning lamps, can result in severe pain and inflammation in the cornea. In addition, since the main UV source is the sun in nature, the solar UV dose received on a sunny day while skiing or sailing can often cause acute
* Corresponding author. Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan. Tel.: þ81 11 706 5944; fax: þ81 11 736 0952. E-mail address: [email protected] (N. Kitaichi). 0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.03.009
photokeratitis when the eyes are unprotected. In addition, the recent enhancement of UV on the surface of the earth due to stratospheric ozone depletion may have also increased the risk of photochemically induced ocular damage (Charman, 1990). The action spectrum for UV irradiation-induced keratitis has been studied in rabbits. Early studies have indicated that exposure of the cornea to excessive UV irradiation leads to damage at both cellular and molecular levels (Kennedy et al., 1997). Acute UVB exposure causes corneal edema and photokeratitis, and it is associated with an inflammatory reaction in the cornea (Schein, 1992). Recent studies have shown that such damage goes deeper than the epithelium, in fact, the response involves the full corneal thickness (Bergmanson et al., 1987; Cullen et al., 1984; Doughty and Cullen, 1989; Doughty and Cullen, 1990; Pitts et al., 1987; Riley et al., 1987; Ringvold and Davanger, 1985; Ringvold et al., 1982). UV induces
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apoptosis in corneal cells (Podskochy et al., 2000), and recent studies have described the influences of UVB for the transduction pathway of corneal cells; UV-induced apoptosis of corneal epithelial cells associated with Kþ channel activity (Lu et al., 2003; Wang et al., 2005), and produced matrix metalloproteinase (MMP)-2 from corneal fibroblasts after UVB exposure (Kozak et al., 2003). Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine that concentrates macrophages at inflammatory loci, and it is a potent activator of macrophages in vivo to play an important role in cell-mediated immunity (Bloom and Bennett, 1966; David, 1966). Today, MIF is considered as an integral component of the host antimicrobial alarm system and stress response that promotes the proinflammatory functions of immune cells (Calandra and Roger, 2003). In ocular inflammatory disorders, the serum MIF levels have been shown to be elevated in uveitis patients with Behçet disease, sarcoidosis, and iridocyclitis (Kitaichi et al., 1999, 2000a; Kotake et al., 2002), and anti-MIF antibody ameliorated experimental autoimmune uveoretinitis (EAU), an Th1-mediated animal model for human endogenous uveitis (Kitaichi et al., 2000b). MIF is also associated with Th2-mediated allergic condition in human (Yanagi et al., 2006). We recently detected quite high MIF levels in tears as well as the sera of patients with severe atopic dermatitis (Kitaichi et al., 2006). In addition, purified recombinant MIF exhibits several proinflammatory functions which induce TNF-a release from macrophages, act together with IFN-g to promote release of nitric oxide, and augment the macrophage killing of intracellular pathogens (Calandra et al., 1994). Another study showed that MIF was also upregulated in keratitis induced by Pseudomonas aeruginosa in mice (Thakur et al., 2001). Since the mechanism of wound healing is complex; namely, consisting of inflammation, granulation, and remodeling of the tissue as described in skin (Martin, 1997), several growth factors and cytokines alone or in combination play important roles during tissue repair and enhance normal wound healing (Zhao et al., 2005). It was recently reported that MIF could contribute to the inflammatory phase of wound healing process (Shimizu et al., 2004). In fact, MIF has been immunohistochemically detected in the cornea, iris, ciliary body, and retina in human (Matsuda et al., 1996, 1997b), and the MIF expression increased when the cornea healed after surgical wounds in rats (Matsuda et al., 1997a). UV irradiation induces the production of multiple proinflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-a from human corneal cells (Kennedy et al., 1997). Another study demonstrated an upregulation of the same cytokines in corneal epithelium after exposure to UV radiation, and their data suggested this induction to be mediated by nuclear factor (NF)-kB. The particular role of MIF in the eye and the effects of UV on the MIF expression of the cornea still remain unclear. In the present study, we induced UV-photokeratitis in transgenic mice overexpressing MIF, wild type (WT), and MIF knockout (KO) mice. 2. Materials and methods 2.1. Animals The MIF-over expressed transgenic mice (H-2b) were established following cDNA microinjection, and general manifestations and several biochemical markers in MIF transgenic (Tg) mice, including body weight, blood pressure, serum levels of cholesterol, and blood sugar were normal as reported elsewhere (Sasaki et al., 2004). The expression of the transgene was regulated by a hybrid promoter composed of the cytomegalovirus (CMV) enhancer and bactin/b-globin promoter, as reported previously (Akagi et al., 1997). MIF deficient mice (H-2b) were established and described previously (Honma et al., 2000). MIF gene was obtained from l-phage
DNA clone isolated from a 129/SVJ mouse genomic library. A genetargeting vector was generated using a 6.0 kb XbaI fragment that contains all of the MIF exons subcloned. A 201 bp SacI fragment consisting of 30 region of exon 1 and 50 region of intron 1 was replaced with pMC1-neo poly (A) cassette in a forward orientation relative to MIF gene transcription. A DT-A cassette was also introduced at 30 flanking region for negative selection. R1 embryonic stem (ES) cells were cultured, transfected, and subjected to positive selection using G418. Resistant colonies were selected, replaced individually, and subjected to genotype analysis using PCR. BamHIdesigned DNA from PCR-positive clones was subjected to Southern analysis using an external probe to confirm that the MIF gene had undergone homologous recombination. C57BL/6 mice of 8–10 weeks old were obtained from Clear Japan (Tokyo, Japan). Transgenic, wild type, and knockout mice (H-2b) were maintained under specific-pathogen-free conditions at the Institute for Animal Experiments of Hokkaido University School of Medicine. All procedures involving the animals were performed in accordance with the ARVO Resolution on the Use of Animal in Research. 2.2. Materials The following materials were obtained from commercial sources. The Isogen RNA extraction kit was taken from Nippon Gene (Tokyo, Japan). Moloney murine leukemia virus (M-MLV) reverse transcriptase were from Invitrogen (Invitrogen Japan, Tokyo), Taq DNA polymerase was from Perkin-Elmer Life and Analytical Sciences (Boston, MA), horseradish peroxidase-conjugated goatrabbit antibody was from Bio-Rad (Hercules, CA). The Cell Death Detection Kit was provided by Roche Molecular Biochemicals (Indianapolis, IN). 2.3. UV irradiation MIF Tg, WT, and MIF KO mice were UVB irradiated of 400 mJ/ cm2 (0.8 mJ/cm2/s for 500 s) under anesthesia. Other WT mice were not irradiated as negative controls. The corneas were collected at 24 h following irradiation for the histopathological study (H&E). Histological slides were made by Sapporo General Pathology Laboratory Co. (Sapporo, Japan) after the eyeballs were fixed with 10% formalin. For RT-PCR, the eyes were collected at 16 h after UV exposure, and the collected corneas were stored at 80 C until use. 2.4. RT-PCR analysis Mouse corneas were surgically obtained from irradiated MIF Tg and WT mice. The total RNAs were extracted with Isogen RNA extraction kit. The reverse transcription of RNAs was carried out with Moloney murine leukemia virus (M-MLV) reverse transcriptase using oligo-dT primer. Subsequent PCR amplification was performed essentially as described using a thermal cycler (Model 480, ABI). MIF primers used in the present study were 50 -GTTTCT GTCGGAGCTCAC-30 (55–72) (forward) and 50 -AGCGAAGGTGGAA CCGTTCCA-30 (215–236) (reverse). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was utilized as a positive control, and the primers were 50 -GAAGGTCGGTGTGAACGGATTTG-30 (6–28) (forward) and 50 -GTCCACCACCCTGTTGCTGTAGC-30 (949–971) (reverse). After PCR, the amplified products were analyzed by 2% agarose gel electrophoresis following PCR (Zhao et al., 2005). 2.5. Morphological properties of corneal epithelium Eyes were collected from mice 24 h after UVB irradiation. Some of collected eyes were stained with hematoxylin-eosin. Other eyeballs were paraffin-embedded. Then, the slides were dried for
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1 h, rinsed twice in phosphate-buffered saline, and incubated with antibodies to cytokeratin 5 (cK5) (BAbCO; Richmond, CA), which stained the epithelium of the cornea (Yoshida et al., 2000). Binding of the primary antibody was localized by Cy3-conjugated antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:200. Nuclei were then stained with YO-pro-1 for 5 min. The slides were examined with laser scanning confocal microscopy (MRC-1024; Bio-Rad, Hercules, CA; and LSM 510; Carl Zeiss Meditec, Dublin, CA). 2.6. Immunohistochemistry for MIF, c-Jun and apoptosis analysis The eyes were dissected from irradiated mice at 24 h after UVB exposure. The dissected eyes were washed in saline and fixed in ice-cold 4% Para formaldehyde (PFA) in 0.1 M borate buffer (pH 9.5) for 2 h, and then were processed for paraffin sectioning. To detect MIF proteins, sections were dewaxed and dehydrated and then rinsed twice in phosphate-buffered saline (PBS). The slides were incubated with normal goat serum, and then with anti-human MIF rabbit polyclonal antibody (Shimizu et al., 1996). Binding of the primary antibody was localized by fluorescence microscopy. To detect c-Jun reactivity, the slides were dried for an hour, rinsed in PBS twice, and incubated with anti-c-Jun antibody (rabbit polyclonal, H79; Santa Cruz Biotechnology, Santa Cruz, CA), at 4 C for 12 h. The binding of the primary antiserum was localized using an avidin-biotin immunoperoxidase kit (Vector Laboratories, Burlingame, CA) (Yoshida et al., 2002). Apoptotic cells were detected with a Cell Death Detection Kit (Roche Molecular Biochemicals), containing all necessary reagents for staining. 2.7. BrdU incorporation BrdU (Bromodeoxyuridine) is a non-radioactive alternative for labeling cells entering the S-phase. For BrdU labeling, the mice were injected with BrdU (Sigma Chemical, St. Louis, MO) peritoneally at a dosage of 30 mg/kg body weight 30 min before sampling. Anti-BrdU staining was performed as described elsewhere (Yoshiki et al., 1991). Briefly, the sections were immersed in
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0.4 mg/ml pepsin in 0.1 N HCl at 30 C for a minute and then in 2N HCl at 40 C for one hour. After the slides were washed with PBS, they were incubated with normal goat serum and then with antiBrdU antibody (BD Biosciences, San Jose, CA) at a dilution of 1:1000. 2.8. Statistical analysis All values were expressed as the means standard deviation (SD) of the respective test or control group. Statistical significances between the control and irradiated groups were evaluated by the non-parametric Mann–Whitney U-test, and all data were representative of three independent experiments or more. 3. Results 3.1. Morphological properties We, first, examined the morphological properties of UVB irradiated corneas with H&E staining. Twenty-four hours after 400 mJ/ cm2 UVB irradiation, the corneal basement membrane was destroyed, and corneal epithelial defect and thinning of the epithelium were observed in wild-type C57BL/6 mice irradiated by UVB (Fig. 1a,b). Corneas collected from MIF KO mice were more damaged than those of WT mice (Fig. 1e,f). On the contrary, corneal basement membrane of MIF-Tgm was well preserved, and the thickness of epithelium looked nearly normal (Fig. 1c,d,g,h). 3.2. MIF protein and mRNA expression in the corneal epithelial layers MIF Tg mice showed increase in MIF mRNA expression compared with non-transgenic WT mice at cornea before irradiation (Fig. 2). At 16 h after UV exposure, prominent higher level of MIF mRNA was observed in MIF Tgm than that of WT mice in cornea (Fig. 2). These results indicate that UVB irradiation induces MIF expression in a larger amount in corneas of MIF Tgm than WT mice. Influence of UVB in mRNA levels was confirmed by using immunohistochemcal study on the corneas collected from WT mice
Fig. 1. H&E staining (a, c, e, g) and immunodetection of cK5 (b, d, f, h) of UVB irradiated (a–f) and non-irradiated (g and h) cornea of WT mice (a, b, g, h), MIF Tgm (c, d) and MIF KO mice (e and f). In WT mice, corneal basement membrane was destroyed (a, b). In contrast, corneal basement membrane was preserved well through the basement membrane in MIF Tgm. The cornea collected from MIF Tg mice were less damaged than WT mice by UVB exposure (c, d). MIF KO mice had most severe damage of cornea among these three strains of mice, and the cornea lacks surface layer of epithelium (e, f). Non-irradiated WT mouse cornea was shown as control (g, h). Scale bar indicates 50 mm. Original magnification; 1000.
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Fig. 2. Expression of MIF mRNA was examined. Total RNA was isolated at 16 h after UVB, and analyzed by RT-PCR to determine MIF mRNA and GAPDH expression. The corneas collected from MIF Tg mice showed higher levels of MIF mRNA even before irradiation. After UVB exposure, the MIF mRNA expression dramatically increased in comparison to that of WT mice.
(6.2 4.3 mean SD cells/section, p < 0.01 compared with WT and MIF KO) than those of WT (30.7 9.1) and MIF KO (32.1 12.7) mice 24 h following irradiation (Fig. 4). MIF activates Jun-activation domain-binding protein 1 (JAB1) that activates JUN N-terminal kinase (JNK) to phosphorylate c-JUN and functions as a co-activator of activator protein 1 (AP1), a transcription factor that is implicated in cell growth (Kleemann et al., 2000). To examine the influences of MIF on JNK signal pathway after UV exposure, we stained the corneal tissue with c-Jun immunohistochemically. The number of c-Jun positive nuclei was significantly higher in MIF Tg (271.4 31.0 mean SD cells/section, p < 0.01 compared with WT and MIF KO) than those in WT (101 17.0), and MIF KO (66.9 15.3) mice at 16 h after UV exposure (Fig. 5). 3.4. BrdU incorporation
and MIF Tg mice, irradiated or non-irradiated with UVB. We performed immunohistochemistry by using anti-MIF polyclonal antibody to examine the influence of UVB to induce MIF protein. Before irradiation, MIF protein was hardly detected in WT mice, however positive MIF staining was observed in MIF Tg mice in corneal epithelium (Fig. 3a,b). The difference was remarkable after UVB exposure. MIF expression was enhanced by UVB more in MIF Tg than WT mice (Fig. 3c,d). The camera could detect no positive luminescence in MIF KO mice both before and after UV exposure (data not shown). 3.3. TUNEL staining and c-Jun expression We next determined that TUNEL labeling in the corneas of MIF Tg, WT, and MIF KO mice 24 h after UV exposure. A significantly fewer TUNEL positive nuclei were detected in MIF Tg mice
To evaluate the proliferative activity of the corneal cells, we examined BrdU incorporation, which should be incorporated into cells only on the S phase. UVB irradiated MIF Tg, WT, and MIF KO mice were injected with BrdU a half hour before euthanasing. Eyes were collected at 6, 24, and 72 h after UV exposure. At 6 h after irradiation, BrdU positive cells were detected in the corneal epithelial of MIF Tg mice (17.3 6.98, mean SD cells/section, p < 0.01 compared with WT and KO), although only a few corneal epithelial cells were considered as positive in WT (6.4 5.1) and MIF KO (3.0 0.9) mice at the time (Fig. 6). At 24 h after irradiation, the numbers of BrdU positive cells were detected as following: 51.7 19.2 in MIF Tg, 41.4 15.5 in WT, 27.0 4.3 in MIF KO mice. At 72 h after UV exposure, the numbers of BrdU positive nuclei continued to increase as following: 109.5 26.1 in MIF Tg, 65.8 27.1 in WT, 35.0 16.7 cells/section in MIF KO mice,
Fig. 3. Expression of MIF protein in cornea was determined. The mice were exposed to 400 mJ/cm2 UVB. MIF protein was induced in the central corneas of WT (a, c) and MIFtransgenic mice (b, d) without irradiation (a, b) or 24 h after the irradiation (c, d).
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Fig. 4. The UVB exposed corneas were stained with TUNEL for apoptosis. The numbers of TUNEL positive nuclei were compared among the three strains of mice. MIF Tgm showed a significantly smaller number of apoptotic cells than WT and MIF KO mice (p < 0.01).
respectively. Proliferation of corneal cells was significantly higher in MIF Tg (p < 0.05) but less in MIF KO (p < 0.05) compared with WT mice (Fig. 6). 4. Discussion In this study, we showed that MIF-overexpressed Tg mice suffered from less damage from UV radiation to the cornea than WT. And MIF KO mice had most severe corneal injury morphologically. The corneal epithelium serves to protect the corneal structures against UV damage, probably by absorbing a substantial amount of the UV energy applied to the eye (Podskochy, 2004). UVC irradiation also induced apoptosis of corneal epithelial cells, and it was mediated through the activation of the SEK/JUK signaling pathway by increases in the Kþ channel activity in cell membrane in vitro (Lu et al., 2003). We showed here a prominent increase of mRNA and protein expressions, as well as c-Jun positive cells in the cornea following UVB exposure. Therefore, our present results are considered to be consistent with the previous in vitro data. Also, we recently reported that in cultured human dermal fibroblasts, UV irradiation up-regulated the MIF production. And such increased
Fig. 5. Irradiated corneas were stained with c-Jun immunohistochemically. The number of c-Jun positive nuclei was compared among the three strains of mice. MIF Tg mice showed a significantly larger number of c-Jun positive cells than that in WT and MIF KO mice (p < 0.01).
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Fig. 6. Serial observations of BrdU incorporation were determined after UVB exposure. At 6 h after irradiation, only a few BrdU positive cells were observed in WT and MIF KO mice, however significantly more BrdU positive cells were detected in MIF Tg mice than other two strains (p < 0.01). The number of BrdU positive cells was significantly elevated in the cornea of MIF Tg mice in comparison with WT mice 6 (p < 0.01), 24 and 72 (p < 0.05) hours after irradiation. In contrast, MIF KO mice had significantly fewer positive cells of BrdU than WT mice (p < 0.05).
MIF plays some role in the upregulation of MMP-1 thorough some pathways, and c-Jun-dependent pathway may be one of them (Watanabe et al., 2004). Another study reported that UVB irradiation induced c-Jun and c-fos genes as well as MMP-1 on cultured conjunctival epithelial cells of the eye in vitro (Di Girolamo et al., 2005). MMP-1 involves the activation of AP-1, a protein dimer consisting of c-Jun and c-fos, and AP-1 activation is directed by the MAP kinase pathway (Tower et al., 2002). It is thought that MIF expression at the corneal basal membrane may mediate cell growth and differentiation (Matsuda et al., 1996; Shimizu et al., 1996; Wistow et al., 1993). And MIF was highly expressed during corneal wound healing in rats (Matsuda et al., 1997a). As reported previously, MIF activates a cascade of events consisting of the phosphorylation of extra cellular signal-regulated kinase (ERK) 1/ERK2, the induction of cytoplasmic phospholipase A2 (PLA2), arachidonic acid, Jun N-terminal kinase (JNK) activity and prostaglandin E2 (PGE2) (Calandra and Roger, 2003). PGE2 is associated with UVB-induced skin inflammation (Kabashima et al., 2007). The transcription of Jun is autoregulated by c-Jun protein (Angel et al., 1988). The c-Jun is an important component of the transcription factor AP-1, and this is suggested to play a role in cell proliferation, cell differentiation, and apoptosis (Karin et al., 1997). Also, AP1 is part of stress response and has wide ranging effects. UV sometimes injures the lens as well as cornea. When we irradiated WT mice with UVB, their lens epithelial cells were never detected in anterior to, but posterior to the equator area. Since one of the initial morphological findings in many cataracts is the posterior migration of the lens epithelium, it is possible that irradiated WT mice may suffer from cataracts caused by UVB irradiation. However, most lens epithelial cells were located in the anterior area in the irradiated MIF Tgm (data not shown). Further studies are planned to examine whether MIF plays some role in UV-associated cataracts as well as keratitis. MIF was found to be upregulated by UVB irradiation in mouse cornea. MIF was originally discovered as a proinflammatory cytokine thus, we first expected that MIF overexpression might worsen damages from UV radiation. However, that may be too simplistic a view to be true in our model. MIF may have functions in cell protection in the eye as suggested previously. MIF overexpression may thus be related to the amelioration of UVB-caused corneal
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injury, and this association was attributable to the upregulation of cell protection successfully, which involved the c-Jun dependent pathway. According to the recent studies, corneal nuclear factorkappa B (NF-kB) activation was necessary for the retention of transparency in the cornea of UVB-exposed mice (Alexander et al., 2006), and MIF can up-regulate NF-kB (Amin et al., 2006). Although it has long been speculated that proinflammatory cytokines may play an important role in the wound repair of ocular tissue, this mechanism has never been fully understood. This newly identified mechanism may contribute to our understanding of photo-induced ocular damage. These findings are promising for elucidating the potential of MIF enhancement for therapeutic use in the case of ocular photo damage-related disorders in the near future. In this study, we determined morphological corneal damage, expression of MIF protein and mRNA, apoptosis, signal transduction, and cell proliferation of corneal epithelial cells following UVB exposure by using H&E, TUNEL, and c-Jun, and BrdU incorporation. As shown in the present study, MIF Tg mice had milder corneal damages and less apoptotic cells, and more c-Jun positive cells than the WT mice. And the results from MIF KO mice were opposite. Thus, in conclusion, corneal tissue can recover from acute UV photokeratitis without MIF, however cornea can be resolved sooner than normal condition if the ocular surface is enough supplied with MIF. Acknowledgments This work was supported by grants from Akiyama Foundation (Sapporo, Japan), The Japan Society for the Promotion of Science, The Ministry of Education, Culture, Sports, Science, and Technology (MEXT) Japan. References Akagi, Y., Isaka, Y., Akagi, A., Ikawa, M., Takenaka, M., Moriyama, T., Yamauchi, A., Horio, M., Ueda, N., Okabe, M., Imai, E., 1997. Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and beta-actin/beta-globin gene in glomerular epithelial cells in vivo. Kidney Int. 51, 1265–1269. Alexander, G., Carlsen, H., Blomhoff, R., 2006. Corneal NF-kappaB activity is necessary for the retention of transparency in the cornea of UV-B-exposed transgenic reporter mice. Exp. Eye Res. 82, 700–709. Amin, M., Haas, C., Zhu, K., Mansfield, P., Kim, M., Lackowski, N., Koch, A., 2006. Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NFkappaB. Blood 107, 2252–2261. Angel, P., Hattori, K., Smeal, T., Karin, M., 1988. The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell 55, 875–885. Bergmanson, J.P., Pitts, D.G., Chu, L.W., 1987. The efficacy of a UV-blocking soft contact lens in protecting cornea against UV radiation. Acta Ophthalmol. (Copenh.) 65, 279–286. Bloom, B.R., Bennett, B., 1966. Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science 153, 80–82. Calandra, T., Roger, T., 2003. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat. Rev. Immunol. 3, 791–800. Calandra, T., Bernhagen, J., Mitchell, R.A., Bucala, R., 1994. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J. Exp. Med. 179, 1895–1902. Charman, W.N., 1990. Ocular hazards arising from depletion of the natural atmospheric ozone layer: a review. Ophthalmic Physiol. Opt. 10, 333–341. Cullen, A.P., Chou, B.R., Hall, M.G., Jany, S.E., 1984. Ultraviolet-B damages corneal endothelium. Am. J. Optom. Physiol. Opt. 61, 473–478. David, J.R., 1966. Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction. Proc. Natl. Acad. Sci. USA 56, 72–77. Di Girolamo, N., Coroneo, M., Wakefield, D., 2005. Epidermal growth factor receptor signaling is partially responsible for the increased matrix metalloproteinase-1 expression in ocular epithelial cells after UVB radiation. Am. J. Pathol. 167, 489–503. Doughty, M.J., Cullen, A.P., 1989. Long-term effects of a single dose of ultraviolet-B on albino rabbit corneadI. in vivo analyses. Photochem. Photobiol. 49, 185–196. Doughty, M.J., Cullen, A.P., 1990. Long-term effects of a single dose of ultraviolet-B on albino rabbit corneadII. Deturgescence and fluid pump assessed in vitro. Photochem. Photobiol. 51, 439–449.
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The effects of UV-B irradiation on the corneal endothelium. Curr. Eye Res. 6, 1021–1033. Ringvold, A., Davanger, M., 1985. Changes in the rabbit corneal stroma caused by UV-radiation. Acta Ophthalmol. (Copenh.) 63, 601–606. Ringvold, A., Davanger, M., Olsen, E.G., 1982. Changes of the cornea endothelium after ultraviolet radiation. Acta Ophthalmol. (Copenh.) 60, 41–53. Sasaki, S., Nishihira, J., Ishibashi, T., Yamasaki, Y., Obikane, K., Echigoya, M., Sado, Y., Ninomiya, Y., Kobayashi, K., 2004. Transgene of MIF induces podocyte injury and progressive mesangial sclerosis in the mouse kidney. Kidney Int. 65, 469–481. Schein, O.D., 1992. Phototoxicity and the cornea. J. Natl. Med. Assoc. 84, 579–583. Shimizu, T., Ohkawara, A., Nishihira, J., Sakamoto, W., 1996. Identification of macrophage migration inhibitory factor (MIF) in human skin and its immunohistochemical localization. FEBS Lett. 381, 199–202. Shimizu, T., Nishihira, J., Watanabe, H., Abe, R., Honda, A., Ishibashi, T., Shimizu, H., 2004. Macrophage migration inhibitory factor is induced by thrombin and factor Xa in endothelial cells. J. Biol. Chem. 279, 13729–13737. Thakur, A., Xue, M., Wang, W., Lloyd, A., Wakefield, D., Willcox, M., 2001. Expression of macrophage migration inhibitory factor during Pseudomonas keratitis. Clin. Exp. Ophthalmol. 29, 179–182. Tower, G.B., Coon, C.C., Benbow, U., Vincenti, M.P., Brinckerhoff, C.E., 2002. Erk 1/2 differentially regulates the expression from the 1G/2G single nucleotide polymorphism in the MMP-1 promoter in melanoma cells. Biochim. Biophys. Acta 1586, 265–274. Wang, L., Dai, W., Lu, L., 2005. Ultraviolet irradiation-induced K(þ) channel activity involving p53 activation in corneal epithelial cells. Oncogene 24, 3020–3027. Watanabe, H., Shimizu, T., Nishihira, J., Abe, R., Nakayama, T., Taniguchi, M., Sabe, H., Ishibashi, T., Shimizu, H., 2004. Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts. J. Biol. Chem. 279, 1676–1683.
N. Kitaichi et al. / Experimental Eye Research 86 (2008) 929–935 Wistow, G.J., Shaughnessy, M.P., Lee, D.C., Hodin, J., Zelenka, P.S., 1993. A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc. Natl. Acad. Sci. USA 90, 1272–1275. Yanagi, T., Kodama, K., Yoshihisa, Y., Shimizu, H., Shimizu, T., 2006. Macrophage migration inhibitory factor in zinc-allergic systemic contact dermatitis. Cytokine 35, 270–274. Yoshida, K., Hu, Y., Karin, M., 2000. IkappaB kinase alpha is essential for development of the mammalian cornea and conjunctiva. Invest. Ophthalmol. Vis. Sci. 41, 3665–3669. Yoshida, K., Behrens, A., Le-Niculescu, H., Wagner, E.F., Harada, T., Imaki, J., Ohno, S., Karin, M., 2002. Amino-terminal phosphorylation of c-Jun regulates apoptosis
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in the retinal ganglion cells by optic nerve transection. Invest. Ophthalmol. Vis. Sci. 43, 1631–1635. Yoshiki, A., Hanazono, M., Oda, S., Wakasugi, N., Sakakura, T., Kusakabe, M., 1991. Developmental analysis of the eye lens obsolescence (Elo) gene in the mouse: cell proliferation and Elo gene expression in the aggregation chimera. Development 113, 1293–1304. Zhao, Y., Shimizu, T., Nishihira, J., Koyama, Y., Kushibiki, T., Honda, A., Watanabe, H., Abe, R., Tabata, Y., Shimizu, H., 2005. Tissue regeneration using macrophage migration inhibitory factor-impregnated gelatin microbeads in cutaneous wounds. Am. J. Pathol. 167, 1519–1529.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Macrophage migration inhibitory factor ameliorates UV-induced photokeratitis in mice Nobuyoshi Kitaichi a, *, Tadamichi Shimizu b, Kazuhiko Yoshida a, Ayumi Honda b, Yoko Yoshihisa b, Satoru Kase a, Kazuhiro Ohgami a, Osamu Norisugi b, Teruhiko Makino b, Jun Nishihira c, Sho-ichi Yamagishi d, Shigeaki Ohno a a
Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, Japan Department of Dermatology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan Department of Medical Information, Hokkaido Information University, Ebetsu, Japan d Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2007 Accepted in revised form 6 March 2008 Available online 16 March 2008
Acute ultraviolet (UV) exposure causes photokeratitis, and induces apoptosis in corneal cells of the eye. Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. Today, MIF is considered as an integral component of the host antimicrobial alarm system and stress response that promotes the proinflammatory functions of immune cells. Also, MIF is considered to contribute the wound healing process. The aim of the present study is to determine the effects of MIF expression on UV irradiated corneal damage. MIF transgenic (MIF-Tg), wild type (WT), and MIF deficient (MIF KO) mice were UVB-irradiated of 400 mJ/cm2 to induce acute UV-photokeratitis. MIF Tg mice constitutively produce high levels of MIF. Morphological changes were most severe in MIF KO mice, and WT and MIF Tg mice were following. Corneal basement membrane of MIF-Tg was well preserved. Prominent higher level of MIF was observed in MIF-Tg than WT after UVB irradiation in cornea. TUNEL staining showed a significantly smaller number of TUNEL positive nuclei in MIF-Tgm (6.2 4.3 cells/section, p < 0.01 compared with WT) than WT (30.7 9.1) and MIF KO mice (32.1 12.7) 24 h after UV exposure. The number of c-Jun positive nuclei was significantly higher in MIF Tg (p < 0.01) than in WT and MIF KO mice. Serial observation revealed that BrdU incorporation was significantly upregulated in MIF Tg (p < 0.01), but downregulated in MIF KO (p < 0.01) than WT mice. MIF expression may thus be related to the amelioration of UVB-caused corneal injury, and this association was attributable to the upregulation of cell proliferation after acute UV-induced corneal damage, which involves the c-Jun dependent pathway. In conclusion, UV-damaged cornea is recoverable without MIF, however it takes longer time than normal condition. Cornea is less damaged and can make a quick recovery when ocular tissue is enough supplied with MIF. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: apoptosis BrdU c-Jun cornea keratitis MIF ultraviolet
1. Introduction Ultraviolet (UV) irradiation represents a significant environmental and occupational hazard that can cause acute and chronic inflammatory changes in the exposed cornea and lens. The acute exposure of artificial sources, such as tanning lamps, can result in severe pain and inflammation in the cornea. In addition, since the main UV source is the sun in nature, the solar UV dose received on a sunny day while skiing or sailing can often cause acute
* Corresponding author. Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan. Tel.: þ81 11 706 5944; fax: þ81 11 736 0952. E-mail address: [email protected] (N. Kitaichi). 0014-4835/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2008.03.009
photokeratitis when the eyes are unprotected. In addition, the recent enhancement of UV on the surface of the earth due to stratospheric ozone depletion may have also increased the risk of photochemically induced ocular damage (Charman, 1990). The action spectrum for UV irradiation-induced keratitis has been studied in rabbits. Early studies have indicated that exposure of the cornea to excessive UV irradiation leads to damage at both cellular and molecular levels (Kennedy et al., 1997). Acute UVB exposure causes corneal edema and photokeratitis, and it is associated with an inflammatory reaction in the cornea (Schein, 1992). Recent studies have shown that such damage goes deeper than the epithelium, in fact, the response involves the full corneal thickness (Bergmanson et al., 1987; Cullen et al., 1984; Doughty and Cullen, 1989; Doughty and Cullen, 1990; Pitts et al., 1987; Riley et al., 1987; Ringvold and Davanger, 1985; Ringvold et al., 1982). UV induces
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apoptosis in corneal cells (Podskochy et al., 2000), and recent studies have described the influences of UVB for the transduction pathway of corneal cells; UV-induced apoptosis of corneal epithelial cells associated with Kþ channel activity (Lu et al., 2003; Wang et al., 2005), and produced matrix metalloproteinase (MMP)-2 from corneal fibroblasts after UVB exposure (Kozak et al., 2003). Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine that concentrates macrophages at inflammatory loci, and it is a potent activator of macrophages in vivo to play an important role in cell-mediated immunity (Bloom and Bennett, 1966; David, 1966). Today, MIF is considered as an integral component of the host antimicrobial alarm system and stress response that promotes the proinflammatory functions of immune cells (Calandra and Roger, 2003). In ocular inflammatory disorders, the serum MIF levels have been shown to be elevated in uveitis patients with Behçet disease, sarcoidosis, and iridocyclitis (Kitaichi et al., 1999, 2000a; Kotake et al., 2002), and anti-MIF antibody ameliorated experimental autoimmune uveoretinitis (EAU), an Th1-mediated animal model for human endogenous uveitis (Kitaichi et al., 2000b). MIF is also associated with Th2-mediated allergic condition in human (Yanagi et al., 2006). We recently detected quite high MIF levels in tears as well as the sera of patients with severe atopic dermatitis (Kitaichi et al., 2006). In addition, purified recombinant MIF exhibits several proinflammatory functions which induce TNF-a release from macrophages, act together with IFN-g to promote release of nitric oxide, and augment the macrophage killing of intracellular pathogens (Calandra et al., 1994). Another study showed that MIF was also upregulated in keratitis induced by Pseudomonas aeruginosa in mice (Thakur et al., 2001). Since the mechanism of wound healing is complex; namely, consisting of inflammation, granulation, and remodeling of the tissue as described in skin (Martin, 1997), several growth factors and cytokines alone or in combination play important roles during tissue repair and enhance normal wound healing (Zhao et al., 2005). It was recently reported that MIF could contribute to the inflammatory phase of wound healing process (Shimizu et al., 2004). In fact, MIF has been immunohistochemically detected in the cornea, iris, ciliary body, and retina in human (Matsuda et al., 1996, 1997b), and the MIF expression increased when the cornea healed after surgical wounds in rats (Matsuda et al., 1997a). UV irradiation induces the production of multiple proinflammatory cytokines, such as IL-1, IL-6, IL-8, and TNF-a from human corneal cells (Kennedy et al., 1997). Another study demonstrated an upregulation of the same cytokines in corneal epithelium after exposure to UV radiation, and their data suggested this induction to be mediated by nuclear factor (NF)-kB. The particular role of MIF in the eye and the effects of UV on the MIF expression of the cornea still remain unclear. In the present study, we induced UV-photokeratitis in transgenic mice overexpressing MIF, wild type (WT), and MIF knockout (KO) mice. 2. Materials and methods 2.1. Animals The MIF-over expressed transgenic mice (H-2b) were established following cDNA microinjection, and general manifestations and several biochemical markers in MIF transgenic (Tg) mice, including body weight, blood pressure, serum levels of cholesterol, and blood sugar were normal as reported elsewhere (Sasaki et al., 2004). The expression of the transgene was regulated by a hybrid promoter composed of the cytomegalovirus (CMV) enhancer and bactin/b-globin promoter, as reported previously (Akagi et al., 1997). MIF deficient mice (H-2b) were established and described previously (Honma et al., 2000). MIF gene was obtained from l-phage
DNA clone isolated from a 129/SVJ mouse genomic library. A genetargeting vector was generated using a 6.0 kb XbaI fragment that contains all of the MIF exons subcloned. A 201 bp SacI fragment consisting of 30 region of exon 1 and 50 region of intron 1 was replaced with pMC1-neo poly (A) cassette in a forward orientation relative to MIF gene transcription. A DT-A cassette was also introduced at 30 flanking region for negative selection. R1 embryonic stem (ES) cells were cultured, transfected, and subjected to positive selection using G418. Resistant colonies were selected, replaced individually, and subjected to genotype analysis using PCR. BamHIdesigned DNA from PCR-positive clones was subjected to Southern analysis using an external probe to confirm that the MIF gene had undergone homologous recombination. C57BL/6 mice of 8–10 weeks old were obtained from Clear Japan (Tokyo, Japan). Transgenic, wild type, and knockout mice (H-2b) were maintained under specific-pathogen-free conditions at the Institute for Animal Experiments of Hokkaido University School of Medicine. All procedures involving the animals were performed in accordance with the ARVO Resolution on the Use of Animal in Research. 2.2. Materials The following materials were obtained from commercial sources. The Isogen RNA extraction kit was taken from Nippon Gene (Tokyo, Japan). Moloney murine leukemia virus (M-MLV) reverse transcriptase were from Invitrogen (Invitrogen Japan, Tokyo), Taq DNA polymerase was from Perkin-Elmer Life and Analytical Sciences (Boston, MA), horseradish peroxidase-conjugated goatrabbit antibody was from Bio-Rad (Hercules, CA). The Cell Death Detection Kit was provided by Roche Molecular Biochemicals (Indianapolis, IN). 2.3. UV irradiation MIF Tg, WT, and MIF KO mice were UVB irradiated of 400 mJ/ cm2 (0.8 mJ/cm2/s for 500 s) under anesthesia. Other WT mice were not irradiated as negative controls. The corneas were collected at 24 h following irradiation for the histopathological study (H&E). Histological slides were made by Sapporo General Pathology Laboratory Co. (Sapporo, Japan) after the eyeballs were fixed with 10% formalin. For RT-PCR, the eyes were collected at 16 h after UV exposure, and the collected corneas were stored at 80 C until use. 2.4. RT-PCR analysis Mouse corneas were surgically obtained from irradiated MIF Tg and WT mice. The total RNAs were extracted with Isogen RNA extraction kit. The reverse transcription of RNAs was carried out with Moloney murine leukemia virus (M-MLV) reverse transcriptase using oligo-dT primer. Subsequent PCR amplification was performed essentially as described using a thermal cycler (Model 480, ABI). MIF primers used in the present study were 50 -GTTTCT GTCGGAGCTCAC-30 (55–72) (forward) and 50 -AGCGAAGGTGGAA CCGTTCCA-30 (215–236) (reverse). Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was utilized as a positive control, and the primers were 50 -GAAGGTCGGTGTGAACGGATTTG-30 (6–28) (forward) and 50 -GTCCACCACCCTGTTGCTGTAGC-30 (949–971) (reverse). After PCR, the amplified products were analyzed by 2% agarose gel electrophoresis following PCR (Zhao et al., 2005). 2.5. Morphological properties of corneal epithelium Eyes were collected from mice 24 h after UVB irradiation. Some of collected eyes were stained with hematoxylin-eosin. Other eyeballs were paraffin-embedded. Then, the slides were dried for
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1 h, rinsed twice in phosphate-buffered saline, and incubated with antibodies to cytokeratin 5 (cK5) (BAbCO; Richmond, CA), which stained the epithelium of the cornea (Yoshida et al., 2000). Binding of the primary antibody was localized by Cy3-conjugated antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:200. Nuclei were then stained with YO-pro-1 for 5 min. The slides were examined with laser scanning confocal microscopy (MRC-1024; Bio-Rad, Hercules, CA; and LSM 510; Carl Zeiss Meditec, Dublin, CA). 2.6. Immunohistochemistry for MIF, c-Jun and apoptosis analysis The eyes were dissected from irradiated mice at 24 h after UVB exposure. The dissected eyes were washed in saline and fixed in ice-cold 4% Para formaldehyde (PFA) in 0.1 M borate buffer (pH 9.5) for 2 h, and then were processed for paraffin sectioning. To detect MIF proteins, sections were dewaxed and dehydrated and then rinsed twice in phosphate-buffered saline (PBS). The slides were incubated with normal goat serum, and then with anti-human MIF rabbit polyclonal antibody (Shimizu et al., 1996). Binding of the primary antibody was localized by fluorescence microscopy. To detect c-Jun reactivity, the slides were dried for an hour, rinsed in PBS twice, and incubated with anti-c-Jun antibody (rabbit polyclonal, H79; Santa Cruz Biotechnology, Santa Cruz, CA), at 4 C for 12 h. The binding of the primary antiserum was localized using an avidin-biotin immunoperoxidase kit (Vector Laboratories, Burlingame, CA) (Yoshida et al., 2002). Apoptotic cells were detected with a Cell Death Detection Kit (Roche Molecular Biochemicals), containing all necessary reagents for staining. 2.7. BrdU incorporation BrdU (Bromodeoxyuridine) is a non-radioactive alternative for labeling cells entering the S-phase. For BrdU labeling, the mice were injected with BrdU (Sigma Chemical, St. Louis, MO) peritoneally at a dosage of 30 mg/kg body weight 30 min before sampling. Anti-BrdU staining was performed as described elsewhere (Yoshiki et al., 1991). Briefly, the sections were immersed in
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0.4 mg/ml pepsin in 0.1 N HCl at 30 C for a minute and then in 2N HCl at 40 C for one hour. After the slides were washed with PBS, they were incubated with normal goat serum and then with antiBrdU antibody (BD Biosciences, San Jose, CA) at a dilution of 1:1000. 2.8. Statistical analysis All values were expressed as the means standard deviation (SD) of the respective test or control group. Statistical significances between the control and irradiated groups were evaluated by the non-parametric Mann–Whitney U-test, and all data were representative of three independent experiments or more. 3. Results 3.1. Morphological properties We, first, examined the morphological properties of UVB irradiated corneas with H&E staining. Twenty-four hours after 400 mJ/ cm2 UVB irradiation, the corneal basement membrane was destroyed, and corneal epithelial defect and thinning of the epithelium were observed in wild-type C57BL/6 mice irradiated by UVB (Fig. 1a,b). Corneas collected from MIF KO mice were more damaged than those of WT mice (Fig. 1e,f). On the contrary, corneal basement membrane of MIF-Tgm was well preserved, and the thickness of epithelium looked nearly normal (Fig. 1c,d,g,h). 3.2. MIF protein and mRNA expression in the corneal epithelial layers MIF Tg mice showed increase in MIF mRNA expression compared with non-transgenic WT mice at cornea before irradiation (Fig. 2). At 16 h after UV exposure, prominent higher level of MIF mRNA was observed in MIF Tgm than that of WT mice in cornea (Fig. 2). These results indicate that UVB irradiation induces MIF expression in a larger amount in corneas of MIF Tgm than WT mice. Influence of UVB in mRNA levels was confirmed by using immunohistochemcal study on the corneas collected from WT mice
Fig. 1. H&E staining (a, c, e, g) and immunodetection of cK5 (b, d, f, h) of UVB irradiated (a–f) and non-irradiated (g and h) cornea of WT mice (a, b, g, h), MIF Tgm (c, d) and MIF KO mice (e and f). In WT mice, corneal basement membrane was destroyed (a, b). In contrast, corneal basement membrane was preserved well through the basement membrane in MIF Tgm. The cornea collected from MIF Tg mice were less damaged than WT mice by UVB exposure (c, d). MIF KO mice had most severe damage of cornea among these three strains of mice, and the cornea lacks surface layer of epithelium (e, f). Non-irradiated WT mouse cornea was shown as control (g, h). Scale bar indicates 50 mm. Original magnification; 1000.
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Fig. 2. Expression of MIF mRNA was examined. Total RNA was isolated at 16 h after UVB, and analyzed by RT-PCR to determine MIF mRNA and GAPDH expression. The corneas collected from MIF Tg mice showed higher levels of MIF mRNA even before irradiation. After UVB exposure, the MIF mRNA expression dramatically increased in comparison to that of WT mice.
(6.2 4.3 mean SD cells/section, p < 0.01 compared with WT and MIF KO) than those of WT (30.7 9.1) and MIF KO (32.1 12.7) mice 24 h following irradiation (Fig. 4). MIF activates Jun-activation domain-binding protein 1 (JAB1) that activates JUN N-terminal kinase (JNK) to phosphorylate c-JUN and functions as a co-activator of activator protein 1 (AP1), a transcription factor that is implicated in cell growth (Kleemann et al., 2000). To examine the influences of MIF on JNK signal pathway after UV exposure, we stained the corneal tissue with c-Jun immunohistochemically. The number of c-Jun positive nuclei was significantly higher in MIF Tg (271.4 31.0 mean SD cells/section, p < 0.01 compared with WT and MIF KO) than those in WT (101 17.0), and MIF KO (66.9 15.3) mice at 16 h after UV exposure (Fig. 5). 3.4. BrdU incorporation
and MIF Tg mice, irradiated or non-irradiated with UVB. We performed immunohistochemistry by using anti-MIF polyclonal antibody to examine the influence of UVB to induce MIF protein. Before irradiation, MIF protein was hardly detected in WT mice, however positive MIF staining was observed in MIF Tg mice in corneal epithelium (Fig. 3a,b). The difference was remarkable after UVB exposure. MIF expression was enhanced by UVB more in MIF Tg than WT mice (Fig. 3c,d). The camera could detect no positive luminescence in MIF KO mice both before and after UV exposure (data not shown). 3.3. TUNEL staining and c-Jun expression We next determined that TUNEL labeling in the corneas of MIF Tg, WT, and MIF KO mice 24 h after UV exposure. A significantly fewer TUNEL positive nuclei were detected in MIF Tg mice
To evaluate the proliferative activity of the corneal cells, we examined BrdU incorporation, which should be incorporated into cells only on the S phase. UVB irradiated MIF Tg, WT, and MIF KO mice were injected with BrdU a half hour before euthanasing. Eyes were collected at 6, 24, and 72 h after UV exposure. At 6 h after irradiation, BrdU positive cells were detected in the corneal epithelial of MIF Tg mice (17.3 6.98, mean SD cells/section, p < 0.01 compared with WT and KO), although only a few corneal epithelial cells were considered as positive in WT (6.4 5.1) and MIF KO (3.0 0.9) mice at the time (Fig. 6). At 24 h after irradiation, the numbers of BrdU positive cells were detected as following: 51.7 19.2 in MIF Tg, 41.4 15.5 in WT, 27.0 4.3 in MIF KO mice. At 72 h after UV exposure, the numbers of BrdU positive nuclei continued to increase as following: 109.5 26.1 in MIF Tg, 65.8 27.1 in WT, 35.0 16.7 cells/section in MIF KO mice,
Fig. 3. Expression of MIF protein in cornea was determined. The mice were exposed to 400 mJ/cm2 UVB. MIF protein was induced in the central corneas of WT (a, c) and MIFtransgenic mice (b, d) without irradiation (a, b) or 24 h after the irradiation (c, d).
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Fig. 4. The UVB exposed corneas were stained with TUNEL for apoptosis. The numbers of TUNEL positive nuclei were compared among the three strains of mice. MIF Tgm showed a significantly smaller number of apoptotic cells than WT and MIF KO mice (p < 0.01).
respectively. Proliferation of corneal cells was significantly higher in MIF Tg (p < 0.05) but less in MIF KO (p < 0.05) compared with WT mice (Fig. 6). 4. Discussion In this study, we showed that MIF-overexpressed Tg mice suffered from less damage from UV radiation to the cornea than WT. And MIF KO mice had most severe corneal injury morphologically. The corneal epithelium serves to protect the corneal structures against UV damage, probably by absorbing a substantial amount of the UV energy applied to the eye (Podskochy, 2004). UVC irradiation also induced apoptosis of corneal epithelial cells, and it was mediated through the activation of the SEK/JUK signaling pathway by increases in the Kþ channel activity in cell membrane in vitro (Lu et al., 2003). We showed here a prominent increase of mRNA and protein expressions, as well as c-Jun positive cells in the cornea following UVB exposure. Therefore, our present results are considered to be consistent with the previous in vitro data. Also, we recently reported that in cultured human dermal fibroblasts, UV irradiation up-regulated the MIF production. And such increased
Fig. 5. Irradiated corneas were stained with c-Jun immunohistochemically. The number of c-Jun positive nuclei was compared among the three strains of mice. MIF Tg mice showed a significantly larger number of c-Jun positive cells than that in WT and MIF KO mice (p < 0.01).
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Fig. 6. Serial observations of BrdU incorporation were determined after UVB exposure. At 6 h after irradiation, only a few BrdU positive cells were observed in WT and MIF KO mice, however significantly more BrdU positive cells were detected in MIF Tg mice than other two strains (p < 0.01). The number of BrdU positive cells was significantly elevated in the cornea of MIF Tg mice in comparison with WT mice 6 (p < 0.01), 24 and 72 (p < 0.05) hours after irradiation. In contrast, MIF KO mice had significantly fewer positive cells of BrdU than WT mice (p < 0.05).
MIF plays some role in the upregulation of MMP-1 thorough some pathways, and c-Jun-dependent pathway may be one of them (Watanabe et al., 2004). Another study reported that UVB irradiation induced c-Jun and c-fos genes as well as MMP-1 on cultured conjunctival epithelial cells of the eye in vitro (Di Girolamo et al., 2005). MMP-1 involves the activation of AP-1, a protein dimer consisting of c-Jun and c-fos, and AP-1 activation is directed by the MAP kinase pathway (Tower et al., 2002). It is thought that MIF expression at the corneal basal membrane may mediate cell growth and differentiation (Matsuda et al., 1996; Shimizu et al., 1996; Wistow et al., 1993). And MIF was highly expressed during corneal wound healing in rats (Matsuda et al., 1997a). As reported previously, MIF activates a cascade of events consisting of the phosphorylation of extra cellular signal-regulated kinase (ERK) 1/ERK2, the induction of cytoplasmic phospholipase A2 (PLA2), arachidonic acid, Jun N-terminal kinase (JNK) activity and prostaglandin E2 (PGE2) (Calandra and Roger, 2003). PGE2 is associated with UVB-induced skin inflammation (Kabashima et al., 2007). The transcription of Jun is autoregulated by c-Jun protein (Angel et al., 1988). The c-Jun is an important component of the transcription factor AP-1, and this is suggested to play a role in cell proliferation, cell differentiation, and apoptosis (Karin et al., 1997). Also, AP1 is part of stress response and has wide ranging effects. UV sometimes injures the lens as well as cornea. When we irradiated WT mice with UVB, their lens epithelial cells were never detected in anterior to, but posterior to the equator area. Since one of the initial morphological findings in many cataracts is the posterior migration of the lens epithelium, it is possible that irradiated WT mice may suffer from cataracts caused by UVB irradiation. However, most lens epithelial cells were located in the anterior area in the irradiated MIF Tgm (data not shown). Further studies are planned to examine whether MIF plays some role in UV-associated cataracts as well as keratitis. MIF was found to be upregulated by UVB irradiation in mouse cornea. MIF was originally discovered as a proinflammatory cytokine thus, we first expected that MIF overexpression might worsen damages from UV radiation. However, that may be too simplistic a view to be true in our model. MIF may have functions in cell protection in the eye as suggested previously. MIF overexpression may thus be related to the amelioration of UVB-caused corneal
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injury, and this association was attributable to the upregulation of cell protection successfully, which involved the c-Jun dependent pathway. According to the recent studies, corneal nuclear factorkappa B (NF-kB) activation was necessary for the retention of transparency in the cornea of UVB-exposed mice (Alexander et al., 2006), and MIF can up-regulate NF-kB (Amin et al., 2006). Although it has long been speculated that proinflammatory cytokines may play an important role in the wound repair of ocular tissue, this mechanism has never been fully understood. This newly identified mechanism may contribute to our understanding of photo-induced ocular damage. These findings are promising for elucidating the potential of MIF enhancement for therapeutic use in the case of ocular photo damage-related disorders in the near future. In this study, we determined morphological corneal damage, expression of MIF protein and mRNA, apoptosis, signal transduction, and cell proliferation of corneal epithelial cells following UVB exposure by using H&E, TUNEL, and c-Jun, and BrdU incorporation. As shown in the present study, MIF Tg mice had milder corneal damages and less apoptotic cells, and more c-Jun positive cells than the WT mice. And the results from MIF KO mice were opposite. Thus, in conclusion, corneal tissue can recover from acute UV photokeratitis without MIF, however cornea can be resolved sooner than normal condition if the ocular surface is enough supplied with MIF. Acknowledgments This work was supported by grants from Akiyama Foundation (Sapporo, Japan), The Japan Society for the Promotion of Science, The Ministry of Education, Culture, Sports, Science, and Technology (MEXT) Japan. References Akagi, Y., Isaka, Y., Akagi, A., Ikawa, M., Takenaka, M., Moriyama, T., Yamauchi, A., Horio, M., Ueda, N., Okabe, M., Imai, E., 1997. Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and beta-actin/beta-globin gene in glomerular epithelial cells in vivo. Kidney Int. 51, 1265–1269. Alexander, G., Carlsen, H., Blomhoff, R., 2006. Corneal NF-kappaB activity is necessary for the retention of transparency in the cornea of UV-B-exposed transgenic reporter mice. Exp. Eye Res. 82, 700–709. Amin, M., Haas, C., Zhu, K., Mansfield, P., Kim, M., Lackowski, N., Koch, A., 2006. Migration inhibitory factor up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 via Src, PI3 kinase, and NFkappaB. Blood 107, 2252–2261. Angel, P., Hattori, K., Smeal, T., Karin, M., 1988. The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell 55, 875–885. Bergmanson, J.P., Pitts, D.G., Chu, L.W., 1987. The efficacy of a UV-blocking soft contact lens in protecting cornea against UV radiation. Acta Ophthalmol. (Copenh.) 65, 279–286. Bloom, B.R., Bennett, B., 1966. Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science 153, 80–82. Calandra, T., Roger, T., 2003. Macrophage migration inhibitory factor: a regulator of innate immunity. Nat. Rev. Immunol. 3, 791–800. Calandra, T., Bernhagen, J., Mitchell, R.A., Bucala, R., 1994. The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor. J. Exp. Med. 179, 1895–1902. Charman, W.N., 1990. Ocular hazards arising from depletion of the natural atmospheric ozone layer: a review. Ophthalmic Physiol. Opt. 10, 333–341. Cullen, A.P., Chou, B.R., Hall, M.G., Jany, S.E., 1984. Ultraviolet-B damages corneal endothelium. Am. J. Optom. Physiol. Opt. 61, 473–478. David, J.R., 1966. Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction. Proc. Natl. Acad. Sci. USA 56, 72–77. Di Girolamo, N., Coroneo, M., Wakefield, D., 2005. Epidermal growth factor receptor signaling is partially responsible for the increased matrix metalloproteinase-1 expression in ocular epithelial cells after UVB radiation. Am. J. Pathol. 167, 489–503. Doughty, M.J., Cullen, A.P., 1989. Long-term effects of a single dose of ultraviolet-B on albino rabbit corneadI. in vivo analyses. Photochem. Photobiol. 49, 185–196. Doughty, M.J., Cullen, A.P., 1990. Long-term effects of a single dose of ultraviolet-B on albino rabbit corneadII. Deturgescence and fluid pump assessed in vitro. Photochem. Photobiol. 51, 439–449.
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