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Purpose: The expression of Fas ligand (FasL) in ocular tissues is thought to play a critical role in maintaining immune privilege in the eye. In this study, to clarify the involvement of the Fas-FasL system in inflammatory processes of the eye,we examined soluble FasL (sFasL) in ocular inflammation., Methods: Using ELISA systems recently developed, sFasL concentrations in aqueous humor (AH) and/or vitreous fluid (VF) were measured. AH was obtained from 17 eyes of 17 uveitis patients and from 12 eyes of 12 non-uveitis (cataract) patients. VF was obtained from 22 eyes of 22 uveitis patients and 7 eyes of 7 non-uveitis (macular hole) patients. Serum levels of sFasL were also determined., Results: sFasL in AH and VF was below the detection limit of the ELISA systems in all non-uveitis eyes. On the other hand, sFasL was detected in AH from uveitis patients where it measured 367.0 +/- 154.7 pg/ml (mean +/- SEM). sFasL was also detected in VF from uveitis patients where it measured 1132.2 +/- 281.7 pg/ml. None of the sera from patients with or without uveitis contained a detectable level of sFas L., Conclusions: sFasL levels in AH and VF are elevated in the eye during ocular inflammation. Fas-FasL mediated apoptosis may play an important role in the regulation of inflammation during uveitis.

The mechanism of corneal wound healing has not been clarified yet. However, evidence has accumulated that various kinds of growth factor such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) play a key role in corneal wound healing. For example, these growth factors are expressed in the corneal epithelial cells, keratocytes and endothelial cells, and their receptors are expressed in the corneal cells. Furthermore, these growth factors promote the proliferation of corneal cells and induce the migration of corneal cells. In addition to the growth factors, inflammatory cytokines such as interleukin (IL)-1, IL-6 and TNF-alpha are involved in corneal wound healing. These cytokines are expressed in the normal and inflammatory cornea after infections, alkaliburn, etc. where they control the growth of corneal cells and induce the migration of corneal cells. Thus, a number of growth factors and cytokines function in the regulation of corneal cell proliferation and in the maintenance of corneal transparency.

Purpose: Interleukin (IL)-1alpha and IL-6 levels in the cornea are greatly elevated during the early stages after an alkali burn in mice. The authors investigated the effect of synthetic inhibitor of matrix metalloproteinase (SIMP) on the expression of inflammatory cytokines in alkali-burned murine corneas and evaluated the clinical appearance of the eyes., Methods: After 0.5N NaOH-alkali burns to 400 corneas of ICR mice, 200 received 400 microg/ml of SIMP topically 4 times a day while 200 corneas were similarly treated with vehicle only. At days 4, 7 and 14 after injury, each cornea was assigned a clinical score for corneal opacity, corneal epithelial defect, hyphema and cataract. Extracts of injured corneas in each group were then assayed for cytokine production using ELISA systems for IL-1alpha, IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha)., Results: The levels of IL-1alpha, IL-1beta and IL-6 were significantly lower in the SIMP-treated group than in the vehicle-treated group 7 days after the burn. However, levels of these cytokines were similar in the SIMP and non-SIMP groups at days 4 and 14. Levels of TNF-alpha did not differ between both groups at any postinjury time. In the SIMP-treated corneas, there was less opacification and hyphema formation and epithelial regeneration was faster., Conclusions: Topical application of SIMP in alkali-burned murine corneas reduced the expression of IL-1alpha, IL-1beta, and IL-6 and lessened the severity of the injury.

Aim: To study herpes simplex virus (HSV) DNA in tears from patients with atypical epithelial keratitis of unknown aetiology., Methods: Tear samples were collected from 17 affected eyes of 17 consecutive patients suffering from epithelial keratitis in whom HSV keratitis was suspected but whose diagnosis was difficult on the basis of clinical manifestations alone. Using reduced sensitivity polymerase chain reaction (PCR), tear samples were tested for HSV DNA. Tears from the unaffected eyes of the 17 patients were also examined, along with 38 tear samples from 19 normal volunteers. Southern blot analysis was performed to confirm that amplified DNA bands were specific for HSV. Clinical correlation with photographs of corneal lesions was also investigated., Results: HSV DNA was detected in tears from the affected eyes of eight of the 17 patients with suspected HSV keratitis. Tears from the affected eyes of the other patients were PCR negative, as were tears from the unaffected eyes of all 17 patients, and from the 38 normal eyes. There was no correlation between PCR results and clinical manifestation of keratitis., Conclusions: Based on the sensitivity of the PCR system, eight of 17 suspected HSV keratitis patients were confirmed as suffering from HSV keratitis. HSV keratitis should therefore be considered as a possible diagnosis in atypical epithelial keratitis.

To investigate the role of cytokines in the pathogenesis of acute herpetic keratitis (HK), we examined the kinetics of cytokine expression in the corneas and the trigeminal ganglia (TG) of C57BL/6Cr (B6) mice after herpes simplex virus type 1 (HSV-1) infection and observed the influence of the targeted disruption of interferon-gamma (IFN-gamma) gene on the clinical course of HK and/or viral clearance. Following corneal infection with HSV-1 Amakata strain, all corneas developed a typical dendritic keratitis. Quantitative analysis using enzyme-linked immunosorbent assay (ELISA) revealed that the expression of interleukin-1alpha (IL-1alpha), IL-5, IL-6, and IFN-gamma in corneas and TGs significantly elevated immediately after infection, peaked between days 2 and 7 postinfection (p.i.), and then diminished. One exception was IFN-gamma, whose expression significantly persisted in the TGs until day 30 p.i. An additional experiment using IFN-gamma-/- (gko) mice revealed that there was no significant difference in the peak level of viral replication in corneas and TGs between gko and B6 mice, although gko mice showed a significant delay of virus clearance in both corneas and TGs (p < 0.005) and higher mortality rate than B6 mice after HSV-1 infection (p < 0.01). These data suggest that the production of proinflammatory cytokines closely correlates with the pathogenesis of HK, and that IFN-gamma plays an important role in enhancing viral clearance from the cornea and TG.

Purpose: To examine whether peripheral lamellar keratoplasty (LKP) using preserved cornea was effective for the treatment of corneoscleral cysts., Methods: Three patients with corneoscleral cysts underwent peripheral lamellar keratoplasty. Two patients had no history of trauma or ocular surgery and were considered to have congenital cysts. The other patient had a history of strabismus surgery that had been performed 7 years previously. The anterior wall of the cysts was removed by trephination, and the epithelial membrane lining the posterior wall was peeled off. Lamellar corneal buttons obtained from preserved corneas then were put in place and secured with 8-10 interrupted sutures. In one case, because the cyst was large and extended to the pupillary axis, peripheral LKP was performed for removal of the scleral and peripheral corneal cyst, and the inner wall of the central corneal cyst was removed with vigorous irrigation and a spatula., Results: Histologic examination showed that all of the cysts were lined with nonkeratinizing epithelial cells. In all three cases, cysts have not reformed after a 1-5-year follow-up., Conclusions: The cysts were lined in epithelial cells, and removal of these epithelial cells was considered to be important for the prevention of recurrence. Peripheral LKP is effective for the treatment of corneoscleral cysts, since this procedure removes displaced epithelial cells and reconstructs the thin part of the cornea and sclera.

Purpose: The acquisition of cell-mediated immunity against donor antigens has been shown to be associated with rejection of orthotopic corneal allografts, but the mechanisms that cause corneal allograft destruction in grafted tissue remain obscure. To determine which T-cell subsets infiltrate graft tissue and cause graft rejection, cytokine expression was examined in corneal tissue after orthotopic corneal allograft., Methods: BALB/c mice received orthotopic corneal allografts from either syngeneic BALB/c or allogeneic C57BL/6 donors. At 1 or 4 weeks after grafting, the mice were euthanatized, and their corneas were removed. Corneal tissue was frozen, homogenized, and placed in phosphate-buffered saline (PBS). Each sample consisted of five corneas in 500 ml PBS. After centrifugation, the supernatant was collected, and the concentration of the following cytokines was measured by enzyme-linked immunosorbent assay: interleukin (IL)-1alpha, IL-2, IL-4, IL-10, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha., Results: Significantly increased amounts of proinflammatory cytokines (IL-1alpha and TNF-alpha) were detected in supernatants from all grafted corneas (both syngeneic and allogeneic) at 1 week after grafting. At 4 weeks after grafting, supernatants from normal corneas, corneas with syngeneic grafts, and corneas with accepted corneal allografts contained undetectable amounts of IL-2 and IFN-gamma, whereas supernatants from corneas with rejected corneal allografts contained significant amounts of IL-2 and IFN-gamma. There were no significant differences in the amounts of IL-4 or IL-10 among all samples. Histologic examination confirmed the expression of IL-2 and IFN-gamma in rejected corneal allografts., Conclusions: Because IL-2 and IFN-gamma are secreted primarily by T-helper type 1 (Th 1) cells, whereas IL-4 and IL-10 are secreted by T-helper type 2 (Th 2) cells, these results indicate that Th 1-type cytokines, rather than Th 2-type cytokines, contribute to the rejection of orthotopic corneal allografts in graft tissue.

Purpose: To elucidate inflammatory cytokine levels in normal human tears, we investigated the concentrations of interleukin-1alpha (IL-1alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and interleukin-8 (IL-8) both in basal tears and reflex tears of normal healthy eyes., Methods: Tear samples were collected from 270 healthy, normal volunteers with no past history of ocular surface disease; the total number of eyes was 295. All tear samples were collected in graduated disposable microcapillaries from the conjunctival cul-de-sac. Reflex tear stimulation was carried out by inserting an applicator into each volunteer's nose. Cytokine levels in basal tears and reflex tears were determined by ELISA. Tears were not diluted, and pooled samples were used for the cytokine assays., Results: In basal tears, the concentrations of IL-1alpha, -1beta, -6 and -8 were 10.9 +/- 1.2, 12.9 +/- 2.3, 226.2 +/- 29.6 and 731.4 +/- 116.2 pg/ml, respectively. In reflex tears, IL-1beta was not detectable, and the concentrations of IL-1alpha, -6 and -8 were 9.3 +/- 2.7, 11.6 +/- 1.6 and 276.1 +/- 47.5 pg/ml respectively. IL-1beta, -6 and -8 levels in reflex tears were significantly lower than those in basal tears, except for IL-1alpha., Conclusions: In the present study, the normal concentration range of inflammatory cytokines in human basal tears was clarified, as a baseline for further studies to examine the levels of these cytokines in tears during disease states.

Objective: To evaluate the hypothesis that transforming growth factor beta2 (TGF-beta2) is involved in the cause of proliferative diabetic retinopathy (PDR)., Methods: We assayed TGF-beta2 levels in the vitreous of patients with PDR and other vitreoretinal disorders. Forty-nine vitreous specimens were obtained from eyes of patients with PDR undergoing vitrectomy, and 19 vitreous specimens from nondiabetic subjects served as controls. We assessed TGF-beta2 levels using an enzyme-linked immunosorbent assay. Both mature and total TGF-beta2 levels were quantified., Results: The mean (+/- SD) total levels of TGF-beta2 were 2634 (+/- 1652) pg/mL in the patients with PDR and 1305 (+/- 972) pg/mL in controls. The mean (+/- SD) levels of mature TGF-beta2 were 244 (+/- 316) pg/mL in patients with PDR and 79 (+/- 81) pg/mL in controls. Total and mature TGF-beta2 levels were significantly greater in patients with PDR (total TGF-beta2, P <.001; mature TGF-beta2, P <.01). Mature TGF-beta2 levels were higher in the vitreous of patients who had severe fibrous proliferation., Conclusion: The results indicate increased levels of both total and mature TGF-beta2 in the vitreous of patients with PDR, suggesting that TGF-beta2 plays an important role in the pathogenesis of PDR.

Purpose: To help test the hypothesis that transforming growth factor beta (TGF-beta) may serve an autocrine function in the retina, we asked whether human Müller (glial) cells in culture express TGF-beta receptors, contain transcripts for various isoforms of this cytokine, and release TGF-beta s into the medium., Methods: Using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with specific primers for TGF-beta 1, -beta 2 and -beta 3 precursors and for TGF-beta type I and type II receptors, we searched for mRNA transcripts expressed by cultured human Müller cells. Also, an ELISA assay allowed quantification of the levels of various TGF-beta s in medium exposed to these glial cells., Results: Human Müller cells in culture express transcripts for both type I and type II TGF-beta receptors and also for TGF-beta 1 and TGF-beta 2. In conditioned medium, the concentration of TGF-beta 1 in the mature form was below detectable levels, and the total TGF-beta 1 was relatively low (mean = 252 pg/ml in confluent cultures). In contrast, the mean levels of mature (55 pg/ml) and total (2530 pg/ml) TGF-beta 2 were markedly higher., Conclusions: Our observations that cultured Müller cells contain mRNA coding for the TGF-beta 2 precursor, release TGF-beta 2 into the medium and express transcripts for both type I and type II TGF-beta receptors are consistent with the idea that this cytokine serves an autocrine function for these glia in the retina.

The kinetics of inflammatory cytokines in sensory retina of mice with corneal alkali-burned eyes were examined. Retinal samples were collected from ICR mice with unburned and alkali-burned eyes from day 3 to day 14. Enzyme-linked immunosorbent assay was used to quantitate the expression levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta and IL-6. Concentrations of IL-1 beta and IL-6 were significantly elevated at day 3 after alkali burn, and reduced to normal level by day 7. The concentration of IL-1 alpha was below assay sensitivity at all times. A significant elevation of IL-1 beta and IL-6 in the sensory retina may contribute to the retinal inflammatory response in the alkali-burned eyes. This is the first report suggesting that the retina is involved in the damage process of alkali burn.

Corneal epithelial wound healing is intimately controlled by a variety of growth factors, such as epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and transforming growth factor-betas (TGF-betas). In this study, we investigate the effects of TGF-beta1 and TGF-beta2 on cultured rabbit corneal epithelial cell proliferation promoted by EGF, KGF, or HGF. Both TGF-beta1 and -beta2 dose-dependently inhibited corneal epithelial cell proliferation promoted by KGF (40 ng ml-1) and HGF (40 ng ml-1), and weakly inhibited cell proliferation promoted by EGF (4 ng ml-1). Furthermore, the inhibitory effect tended to be stronger with TGF-beta2 than TGF-beta1. These findings suggest that TGF-beta1 and -beta2 play important roles as negative modulators against the cell proliferation effect of EGF, KGF and HGF., (Copyright 1997 Academic Press Limited.)

Purpose: This study investigated the cytokine expression profile in alkali-burned mouse corneas, in order to elucidate the mechanisms of corneal damage and repair., Methods: The cytokines expressed in alkali-burned corneas were identified by polymerase chain reaction (RT-PCR), then quantitated using ELISA. Based on the ELISA results, immunohistochemical analyses were performed to localize cytokine expression., Results: Among the ten cytokines examined, IL-1 (IL-1 beta), IL-6, IL-10 and TNF-alpha mRNA were expressed in alkali-burned corneas. Quantitation revealed that IL-alpha and IL-6 were strongly induced in the early stages of alkali burn, peak production of IL-1 alpha (53.2 pg/cornea) and IL-6 (23.6 pg/cornea) occurring at days 3 and 7, respectively. The production of IL-10 and TNF-alpha was not significantly elevated during the 42 day period after burn. Immunohistochemical analyses revealed that both IL-1 alpha and IL-6 were mainly localized in regenerating epithelial basal cells., Conclusions: IL-1 alpha and IL-6 levels in the cornea are dramatically elevated in the regenerated epithelium during the early stages of alkali burn, and may play an important role in associated corneal damage and repair.

Mature and total TGF-beta 2 levels were determined in normal human tears, from 37 volunteers, using a quantitative "sandwich' enzyme immunoassay technique. Since this ELISA method recognizes only mature TGF-beta 2, total TGF-beta 2 levels in tears were measured after acid activation. Aqueous humor samples from 9 patients with cataract were also tested as controls. Mature and total TGF-beta 2 in aqueous humor (AH) averaged 182 pg/ml and 737 pg/ml, respectively. The levels of mature TGF-beta 2 in normal human tears ranged from 36 to 71 pg/ml (mean: 55 pg/ml). And the levels of total TGF-beta 2 ranged from 3446 to 14910 pg/ml (mean: 7854 Pg/ml), a very high level. Percentage of mature TGF-beta 2 ranged 0.4 to 1.3% (mean: 0.9%). It is reported for the first time that there are very high levels of total TGF-beta 2 in normal human tears.

Purpose: To examine whether the topical application of keratinocyte growth factor (KGF) can enhance corneal epithelial healing in vivo. In addition, the distribution of S-phase cells in KGF-treated and control corneas was investigated during regeneration and under normal conditions., Methods: A 10-mm diameter epithelial defect was made in the center of rabbit corneas. A 50-microliters aliquot of 10 micrograms/ml human keratinocyte growth factor (hKGF) was then applied topically five times a day. The same volume of phosphate-buffered saline (PBS) vehicle was applied to the contralateral eye as a control. Each corneal epithelial defect was subsequently photographed every 12 hours and was measured by a computer-assisted digitizer. For the S-phase cell analysis, entire corneas were labeled with 3H-thymidine and were subjected to autoradiography at 24 hours after wounding or in the normal cornea at 24 hours after the application of KGF or PBS., Results: Topical application of 10 micrograms/ml hKGF significantly accelerated corneal epithelial wound healing when compared with controls. In the S-phase cell analysis, KGF did not have any effect on normal corneal epithelial cells. However, in the regenerating cornea, the number of S-phase cells in the KGF-treated limbal epithelium was twofold higher than in the controls., Conclusions: Topical application of KGF accelerated corneal epithelial wound healing in vivo and increased cell proliferation in the limbal epithelium of the regenerating cornea.

To investigate the role of platelet-derived growth factor (PDGF) in corneal wound healing, we examined the effect of human natural PDGF, recombinant PDGF-BB, and PDGF-AA on the migration of rabbit corneal endothelial cells. In a modified Boyden chamber in fetal bovine serum-free conditions, natural PDGF and PDGF-BB, at a concentration of 1-3 or 10 ng/ml, enhanced the migration of endothelial cells, whereas at a higher concentration (10 or 30 ng/ml), this enhanced migration was suppressed; the optimal concentration range for enhancing migration was 3-10 ng/ml. PDGF-AA did not enhance the migration. Natural PDGF and PDGF-BB activity was found for up to 6 h after the beginning of culture, and was completely blocked by anti-PDGF neutralizing antibodies. A checkerboard assay demonstrated that PDGF-BB had a chemotactic effect on the corneal endothelial cell migration. These results suggest that natural PDGF and PDGF-BB, but not PDGF-AA, are involved in corneal wound healing by stimulating the migration of corneal endothelial cells.

The effect of platelet-derived growth factor (PDGF) on the growth of rabbit corneal endothelial cells and the expression of PDGF receptor mRNA in these cells were examined in the present study. When rabbit corneal endothelial cells were cultured in the presence of natural PDGF, recombinant PDGF-BB, and PDGF-AA for 1, 3, and 5 days, natural PDGF and recombinant PDGF-BB enhanced the growth of the endothelial cells, but PDGF-AA did not. This growth promotion was suppressed by anti-PDGF antibody. PDGF enhanced also the incorporation of 3H-thymidine into the cells. When PDGF was combined with epidermal growth factor (EGF), the additive effect was recognized. The expression of the PDGF receptor mRNA was found in the endothelial cells by the polymerase chain reaction (PCR) method. It was concluded from these results that PDGF enhanced the growth of the corneal endothelial cells and that the PDGF receptor mRNA was expressed in these cells. These results suggest that PDGF may regulate corneal endothelial cell growth.

The expression of TGF-beta 1, TGF-beta 2 and TGF-beta 3 precursors mRNA transcripts in in vivo human corneal cells were studied. Complementary DNA (cDNA) was generated from poly A+RNA extracted from in vivo human corneal epithelial cells, stromal keratocytes and endothelial cells. With the cDNAs as template, polymerase chain reaction (PCR) was carried out using specific primers of TGF-beta 1, TGF-beta 2 and TGF-beta 3 precursors, synthesized by choosing specific nucleotide sequences in the latency-associated peptide region of each precursor. Southern blot analysis of the PCR products was carried out. In corneal epithelial cells, TGF-beta 2 mRNA transcript was strongly expressed; TGF-beta 1 and -beta 3 mRNA transcripts were also expressed; stromal keratocyte samples expressed TGF-beta 1 and -beta 2 mRNA transcripts but not TGF-beta 3 mRNA transcript. Endothelial cells expressed all three transcripts. The present study, together with the authors' previous immunohistochemical study demonstrates that both protein and mRNA of TGF-beta 2-LAP are present in human corneal epithelium, indicating that TGF-beta 2 may play a crucial role in corneal epithelial cell layers.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is expressed in stromal fibroblasts and acts specifically on cells of epithelial origin as a paracrine mediator. In this study, we examined KGF and KGF receptor (KGF-R) mRNA expression in rabbit corneal epithelial cells and keratocytes, using a reverse transcription polymerase chain reaction (RT-PCR) method. We also examined the effects of KGF on the proliferation of rabbit corneal cells in vitro. mRNA from cultured rabbit skin fibroblasts, conjunctival fibroblasts, corneal epithelial cells and keratocytes was first reverse-transcribed, and then amplified by PCR using primers specific for KGF and KGF-R. KGF mRNA was expressed in skin fibroblasts, conjunctival fibroblasts and keratocytes, but not in corneal epithelial cells. In contrast, KGF receptor mRNA was expressed in corneal epithelial cells, but not in skin fibroblasts, conjunctival fibroblasts or keratocytes. The results of Southern hybridization experiments of PCR products also showed that KGF mRNA was expressed in skin fibroblasts, conjunctival fibroblasts and keratocytes, whereas KGF receptor mRNA was expressed in corneal epithelial cells. Nucleotide sequencing of the PCR products revealed that their sequences were identical to the expected nucleotide sequences. Using the methylene blue method, we found that KGF enhanced the growth of cultured rabbit corneal epithelial cells, but not keratocytes, at a concentration of 0.01 to 100 ng ml-1. Furthermore, KGF also augmented 3H-thymidine incorporation into the corneal epithelial cells. These results suggest that KGF is a paracrine mediator of the proliferation of corneal epithelial cells as well as of keratinocytes in the skin.

The expression of keratinocyte growth factor (KGF) messenger RNA (mRNA) was examined in both ex vivo and in vitro specimens of human skin, conjunctiva and cornea. Total cellular mRNA was extracted from human skin, conjunctiva, cornea, cultured skin fibroblasts, cultured conjunctival fibroblasts and cultured keratocytes. Oligo dT-primed complementary DNA (cDNA) was synthesized using each RNA sample as a template. Polymerase chain reaction (PCR) was used to amplify the DNA sequence of KGF using each cDNA sample as a template. PCR products were then digested with restriction enzymes in order to determine the KGF prototype. In all samples, PCR products of the expected size for KGF were detected. The resulting restriction pattern proved that these products were identical to KGF prototype I, ie, authentic KGF. In conclusion, human keratocytes and conjunctival fibroblasts express mRNA coding for KGF prototype I, ie, authentic KGF, in both ex vivo and in vitro specimens.