Crosstalk between TGF-beta and MAPK signaling during corneal wound healing.

PURPOSE: The aim of this study was to elucidate the mechanisms governing epithelial cell migration and proliferation during wound healing. METHODS: The authors used wound healing of mouse corneal epithelium to examine the role TGF-ß signaling plays during the healing process. To achieve this goal, they used transgenic mice in which the TGF-ß receptor type II (Tbr2) was conditionally ablated from the corneal epithelium. Epithelium debridement wounds were made, followed by the assessment of cell migration, proliferation, and immunostaining of various signaling pathway components. RESULTS: The authors showed that in the absence of TGF-ß signaling corneal epithelial wound healing is delayed by 48 hours; this corresponds to a delay in p38MAPK activation. Despite the delayed p38MAPK activation, ATF2, a substrate of p38MAPK, is still phosphorylated, leading to the suppression of cell proliferation at the leading edge of the wound. These data provide evidence that in the absence of TGF-ß signaling, the suppression of cell proliferation during the early stages of wound healing is maintained through the JNK activation of ATF2. CONCLUSIONS; Together the data presented here demonstrate the importance of the TGF-ß and MAPK signaling pathways in corneal epithelial wound healing.

MHC-matched corneal allograft rejection in an IFN-gamma/IL-17-independent manner in C57BL/6 mice.

PURPOSE: It has been widely accepted that Th1- and IFN-gamma-mediated immune responses are indispensable for corneal allograft rejection in BALB/c hosts. The present study was designed to determine the role of IFN-gamma and IL-17 in the rejection by C57BL/6 hosts, which display high rejection rates. METHODS: MHC-matched or -mismatched corneal allografts were grafted onto IFN-gamma-knockout (GKO), IFN-gamma-receptor-knockout (GRKO), IL-17-knockout (IL-17KO), or wild-type (WT) C57BL/6 hosts. Graft fates were assessed clinically and histologically. At appropriate time intervals after allografting, RNA was isolated from corneal graft parenchymal and stromal tissues and cervical lymph nodes. The cytokine mRNA levels of Th1, -2, and -17 type were analyzed by real-time PCR. RESULTS: No significantly prolonged allograft survival was observed in any combinations. The rejected MHC-mismatched corneas in GKO elicited intensive infiltration of eosinophils, CD11b(+) macrophages, and B cells, but few Gr-1(+)CD11c(-) neutrophils. In contrast, rejected MHC-matched corneas in GKO hosts, as well as GRKO and WT hosts, elicited intensive infiltration of CD11b(+) macrophages and Gr-1(+)CD11c(-) neutrophils, but no B220(+) B cells and eosinophils. At 1 week after MHC-matched allografting, mRNA levels of IL-6 and IL-17A in the lymph node were extensively upregulated in GKO hosts. It is of interest that anti-IFN-gamma treatment did not improve the allograft survival in IL-17KO hosts. CONCLUSIONS: IFN-gamma and IL-17 play no critical role in the development of minor-specific allograft rejection in C57BL/6 mice. This indicates the presence of sophisticated rejection mechanisms that are still elusive and cannot be ascribed simply to Th1, -2, or -17.

Major histocompatibility complex semi-matching improves murine corneal allograft survival under oxidative macrophage dominancy.

BACKGROUND: Corneal allograft rejection is mediated by a Th1-type response. Because major histocompatibility complex (MHC)-compatible allografts are the prerequisite for efficient graft survival by reducing the Th1 response, we explored a new strategy for the acceptance of (MHC+minor H)-disparate allografts. METHODS: N,N'-diacetyl-L-cystine dimethylester (NM2) reduces the intracellular glutathione content (icGSH) in antigen-presenting cells (APCs) and down-regulates Th1 responses. Therefore, we subconjunctivally (scj) injected NM2 into donor- and/or recipient mice on days 1, 4, and 7 before penetrating keratoplasty. C57BL/10, B10.D2, or CBF1 corneal allografts were then placed on neovascularized graft beds of BALB/c mice. Corneal grafting was also performed between CBF1 and B6C3F1 mice. RESULTS: The saline-injected controls rejected all allografts within 3 weeks. Minor H-only-disparate B10.D2 allografts survived indefinitely after the intraperitoneal (ip) (65.0%) and scj (71.4%) injection of NM2 (P<0.0001). MHC+minor H-disparate C57BL/10 allograft survival was prolonged only by scj injection of NM2 (median survival time [MST]=43.6+/-1.5, P<0.001); there was no indefinite allograft survival. Allelic-gene semi-matched CBF1 grafts survived indefinitely after ip (75.0%) or scj (86.6%) delivery of NM2 and 50% of the B6C3F1 grafts survived indefinitely only after scl NM2 injection; no control grafts survived (MST=18.9+/-1.1, P<0.0001). CONCLUSIONS: The local application of NM2 improved the survival of MHC-disparate allografts. Allelic-gene semi-matching, combined with the local induction of APCs with reduced icGSH, resulted in allograft survival comparable to the survival of MHC-matched grafts.

Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth.

PURPOSE: To determine the kinetics of corneal epithelial maturation during embryonic development and postnatal growth. METHODS: Expression patterns of keratin (K)12 and K14 were determined in mouse embryos (embryonic days [E]15.5-19.5), corneas of postnatal day (P)0 to 10 months, and healing corneas after epithelial debridement in P30 and P90 mice. The expression of alkaline phosphatase (AP) was determined during postnatal growth and healing of epithelial debridement of Krt12(Cre/Cre)/ZAP bitransgenic mice. RESULTS: During embryonic development, K12 expression by corneal peridermal epithelium commenced at E15.5. In the period from E15.5 to P10, the expression of K12 was restricted to the suprabasal and/or superficial cells of the corneal epithelium, whereas the K14 expression was restricted to the basal cells. After P30, K12 expression was sporadically detected in the basal corneal epithelium, and the number of K12-positive basal cells increased as the mice grew older. The number of K14-positive cells that coexpressed K12 increased with age and reached a plateau after P180. Healing of the debrided epithelium facilitated the increase in K14-positive cells that coexpressed K12. Many basal cells of Krt12(Cre/Cre)/ZAP mice remained undifferentiated and expressed LacZ at P15, and they then differentiated to express Cre, which leads to excision of LacZ and AP expression. CONCLUSIONS: In the mouse, the corneal epithelium does not become fully mature until 3 to 6 months after birth, in that a significant number of corneal basal epithelial cells of young mice (

Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice.

PURPOSE: To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. METHODS: A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. RESULTS: Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. CONCLUSIONS: A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.

Effects of dexamethasone and cyclosporin A on human beta-defensin in corneal epithelial cells.

Human beta-defensins (hBDs), which are mainly expressed in epithelial tissues, may contribute to infection-protective mechanisms in the ocular surface. We examined hBD1 and hBD2 expression in the ocular surface by RT-PCR and immunohistochemistry and studied the effects of immunosuppressive agents on their expression in human corneal epithelial cells in vitro. mRNA expression of hBD1 and hBD2 was confirmed in corneal epithelial cells. While the hBD1 peptide was immunohistochemically detected in corneal, limbal, and conjunctival epithelium, the level of expression was stronger in limbal- and conjunctival- than in corneal epithelium. Very weak expression of the hBD2 peptide was detected only in corneal epithelium. After 48-h culture of human corneal epithelial cells in the presence of 10(-5) M dexamethasone or 1 mg l(-1) cyclosporin A, total RNA was extracted and hBD1 and hBD2 mRNA expressions compared using semi-quantitative RT-PCR and introduced amplified fragment length polymorphism (iAFLP) assay. The two methods yielded almost identical results. hBD1 mRNA expression was not changed by dexamethasone but was down-regulated by cyclosporin A. hBD2 mRNA expression was up-regulated by dexamethasone and down-regulated by cyclosporin A. Our findings suggest that hBD1 and hBD2 are regulated by different mechanisms and that hBD1 may contribute to infection-protective mechanisms in the relatively immunosuppressed status induced by dexamethasone.

The association of HLA with young-onset keratoconus in Japan.

PURPOSE: To report the association of HLA antigens with keratoconus in Japanese patients. DESIGN: Observational consecutive case series. METHODS: In 90 consecutive Japanese keratoconus patients, HLA class I (HLA-A, -B, -C) and class II (HLA-DR, -DQ) were analyzed. RESULTS: Compared with control frequencies, based on mean gene frequencies for the Japanese population, higher frequencies of HLA-A26, B40, and DR9 antigens were found in patients whose conditions were diagnosed before 20 years of age (chi(2) = 6.45, P =.01; chi(2) = 6.78, P =.01; chi(2) =3.99, P =.05, respectively), but were not found in patients whose conditions were diagnosed later in life. Men were significantly younger at diagnosis than were women. No obvious relation was found between HLA antigens and other clinical data. CONCLUSION: HLA-A26, B40, and DR9, which were found relatively frequently in the ancient Japanese population, seem to be associated with keratoconus in younger individuals.