Youichiro Fujitsu

Role of structural cells of the cornea and conjunctiva in the pathogenesis of vernal keratoconjunctivitis.

Vernal keratoconjunctivitis (VKC) is a severe type of allergic conjunctival disease characterized by the presence both of various corneal epithelial and stromal lesions as well as of conjunctival proliferative changes such as giant papillae of the upper tarsal conjunctiva and limbal lesions. These clinical findings as well as various pathophysiological characteristics of VKC are distinct from those of other types of ocular allergy and allergic diseases of other organs. The outer eye possesses specific allergological characteristics, one of which is communication between the cornea and conjunctiva through a thin layer of tear fluid. Fibroblasts of the cornea and the conjunctiva are activated by proinflammatory and T helper 2 (Th2) cell-derived cytokines. Corneal fibroblasts enhance ocular allergic reactions as a result of their activation-induced expression both of chemokines such as eotaxin and TARC as well as of adhesion molecules such as ICAM-1 and VCAM-1, all of which together promote the activation and infiltration of eosinophils and Th2 lymphocytes. In contrast, corneal epithelial cells suppress such reactions by physically separating corneal fibroblasts from bioactive substances in tear fluid. Exaggerated proliferation of and deposition of extracellular matrix by conjunctival fibroblasts likely exacerbate conjunctival inflammation. Restoration of an intact corneal epithelium and inhibition of the activities of corneal and conjunctival fibroblasts may provide a basis for the development of new treatments for severe ocular allergic diseases such as VKC.

IL-4-induced cell proliferation and production of extracellular matrix proteins in human conjunctival fibroblasts

Giant papillae, characteristic lesions of vernal keratoconjunctivitis, are formed as a result of the proliferation of conjunctival fibroblasts, the deposition of extracellular matrix, and the infiltration of inflammatory cells. The concentration of interleukin (IL)-4 is also increased in the tear fluid of individuals with ocular allergic diseases. The possible role of IL-4 in the development of giant papillae was investigated by examining the effects of this cytokine on cultured human conjunctival fibroblasts. Reverse transcription and polymerase chain reaction analysis revealed the presence of transcripts encoding the IL-4 receptor alpha chain in these cells, and flow cytometry demonstrated the expression of this protein on the cell surface. IL-4 induced the proliferation of conjunctival fibroblasts in a concentration-dependent manner, and this effect was inhibited by neutralizing antibodies to the IL-4 receptor. Enzyme immunoassays revealed that IL-4 also increased in a concentration-dependent manner the amounts of procollagen type I C-peptide and fibronectin released into the culture supernatant by conjunctival fibroblasts. A whole-cell enzyme-linked immunosorbent assay showed that IL-4 increased the deposition of collagen type III by conjunctival fibroblasts. Furthermore, reverse transcription combined with real-time polymerase chain reaction analysis revealed that IL-4 increased the abundance of collagen type III mRNA in these cells. These results demonstrate that human conjunctival fibroblasts express receptors for IL-4, and that IL-4 stimulates both the proliferation of and the production of extracellular matrix proteins by these cells. These effects of IL-4 might contribute to the formation of giant papillae in individuals with vernal keratoconjunctivitis.

Expression of Functional ICAM-1 on Cultured Human Keratocytes Induced by Tumor Necrosis Factor-α

PURPOSE Leukocytes such as neutrophils contribute to the pathogenesis of corneal ulcer. The effect of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha on the expression of intercellular adhesion molecule (ICAM)-1 by cultured human keratocytes was investigated because the interaction of leukocytes with ICAM-1 expressed on the surface of structural cells mediates leukocyte infiltration into tissue at sites of inflammation. METHODS Cultured human keratocytes were incubated with various concentrations of TNF-alpha. The surface expression of ICAM-1 was evaluated by whole-cell enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. The abundance of ICAM-1 mRNA in cell lysate was determined by quantitative reverse transcription and polymerase chain reaction analysis. Adhesion of neutrophils to corneal fibroblasts was assayed by measuring the fluorescence of Calcein-AM-labeled neutrophils. RESULTS Incubation of keratocytes with TNF-alpha induced increased expression of ICAM-1 on the surface of keratocytes in a dose- and time-dependent manner. The abundance of ICAM-1 mRNA in keratocytes was increased by the incubation of cells with TNF-alpha. Exposure of keratocytes to TNF-alpha increased the adherence of human neutrophils to these cells. CONCLUSIONS Stimulation of keratocytes with TNF-alpha resulted in an increase in the abundance of ICAM-1 mRNA, the cell surface expression of ICAM-1 protein, and enhanced adhesion of neutrophils to these cells. The expression of ICAM-1 on human keratocytes may thus contribute to leukocyte infiltration into the corneal stroma of individuals with inflammatory ocular diseases.

Synergistic effect of TNF-α and either IL-4 or IL-13 on VCAM-1 expression by cultured human corneal fibroblasts

Purpose. To examine the role of corneal fibroblasts in the pathogenesis of vernal keratoconjunctivitis, we investigated the effects of tumor necrosis factor (TNF)–&agr;, interleukin (IL)–4, and IL-13 on the expression of vascular cell adhesion molecule (VCAM)–1 by cultured human corneal fibroblasts. Methods. Cultured human corneal fibroblasts were incubated with various combinations and concentrations of TNF-&agr;, IL-4, and IL-13. The cell surface expression of VCAM-1 was subsequently evaluated by whole-cell enzyme-linked immunosorbent assay and immunocytochemistry, and the abundance of VCAM-1 mRNA in cell lysates was determined by quantitative reverse transcription and polymerase chain reaction analysis. Results. Corneal fibroblasts incubated in the absence of cytokines exhibited minimal expression of VCAM-1. Whereas incubation of the cells with TNF-&agr;, IL-4, or IL-13 alone, or with the combination of IL-4 and IL-13, induced only a small increase in VCAM-1 expression, exposure of the cells to TNF-&agr; in combination with either IL-4 or IL-13 resulted in a marked synergistic increase in expression of this adhesion molecule that was both time and dose dependent. The abundance of VCAM-1 mRNA in corneal fibroblasts was also increased in a synergistic manner by incubation of the cells with TNF-&agr; together with either IL-4 or IL-13. Conclusion. Stimulation of human corneal fibroblasts with the combination of TNF-&agr; and either IL-4 or IL-13 resulted in synergistic increases in both the abundance of VCAM-1 mRNA and the cell surface expression of VCAM-1 protein. This cytokine-induced increase in VCAM-1 expression by corneal fibroblasts may contribute to eosinophil infiltration in corneal lesions associated with vernal keratoconjunctivitis.

Inhibition of Eotaxin Expression in Human Corneal Fibroblasts by Interferon-γ

Background: The chemokine eotaxin is a potent and selective chemoattractant for eosinophils. The production of eotaxin by corneal fibroblasts likely contributes to eosinophil infiltration into the corneal stroma. The regulation of eotaxin synthesis in these cells was investigated by examining the effect of interferon-γ (IFN-γ), a T helper cell 1-derived cytokine, on eotaxin expression in cultured human corneal fibroblasts. Methods: The release of eotaxin from cultured corneal fibroblasts was measured by enzyme-linked immunosorbent assay, and the abundance of eotaxin mRNA in these cells was determined by reverse transcription combined with real-time polymerase chain reaction analysis. Results: IFN-γ inhibited in a dose-dependent manner the release of eotaxin induced by each of the proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α) and IL-1β in corneal fibroblasts. IFN-γ also inhibited the increase in the abundance of eotaxin mRNA induced by each of these cytokines. The synergistic increases in eotaxin release and in eotaxin mRNA abundance induced by the combination of TNF-α and the T helper cell 2-derived cytokine IL-4 were also both markedly inhibited by the treatment of cells with IFN-γ. Conclusions: IFN-γ inhibited eotaxin expression at both the protein and mRNA levels in cultured human corneal fibroblasts. This effect of IFN-γ may contribute to the inhibition of eosinophil infiltration into the cornea. Exogenous IFN-γ thus represents a potential new therapeutic agent for the treatment of corneal disorders associated with inflammatory ocular diseases such as vernal keratoconjunctivitis.

Inhibition by Female Sex Hormones of Collagen Gel Contraction Mediated by Retinal Pigment Epithelial Cells

PURPOSE Collagen contraction mediated by retinal pigment epithelial (RPE) cells contributes to the pathogenesis of proliferative vitreoretinopathy (PVR). We examined the effects of sex hormones on this process. METHODS Mouse RPE cells were cultured in a type I collagen gel and exposed to 17β-estradiol, progesterone, or dehydro-epiandrosterone. Collagen contraction induced by transforming growth factor-β2 (TGF-β2) was determined by measurement of gel diameter. Expression of α-smooth muscle actin (α-SMA), as well as phosphorylation of Smad2 and myosin light chain (MLC), was examined by immunoblot analysis. Matrix metalloproteinase (MMP) release was evaluated by gelatin zymography. Fibronectin and interleukin-6 secretion was measured with immunoassays. RESULTS The female sex hormones 17β-estradiol and progesterone inhibited TGF-β2-induced collagen contraction mediated by RPE cells, whereas the male sex hormone dehydro-epiandrosterone had no such effect. The TGF-β2-induced release of MMP-2 and MMP-9 from RPE cells was also inhibited by 17β-estradiol and progesterone, and the MMP inhibitor GM6001 attenuated TGF-β2-induced collagen contraction. Expression of the mesenchymal markers α-SMA and fibronectin, interleukin-6 release, and Smad2 and MLC phosphorylation induced by TGF-β2 were all inhibited by 17β-estradiol and progesterone. Immunohistochemical analysis also detected nuclear immunoreactivity for estrogen and progesterone receptors in proliferative fibrocellular membranes of PVR patients. CONCLUSIONS Female sex hormones inhibited TGF-β2-induced collagen contraction mediated by RPE cells. This action appeared to be mediated through inhibition both of MMP, α-SMA, and fibronectin expression as well as of Smad2 and MLC phosphorylation. Female sex hormones might thus prove effective for the treatment of PVR.

Activation of Corneal Fibroblast–Derived Matrix Metalloproteinase-2 by Tryptase

Purpose: The presence of the active form of matrix metalloproteinase (MMP)-2 and an increased concentration of tryptase are characteristics of tear fluid of individuals with vernal keratoconjunctivitis. Although tryptase does not mediate the activation of purified MMP-2, we have now examined whether it might activate MMP-2 in the presence of cultured human corneal fibroblasts. Methods: Corneal fibroblasts were cultured in the absence or presence of tryptase, and the activation status of MMP-2 was determined by gelatin zymography. Results: MMP-2 released from corneal fibroblasts was activated by exogenous tryptase. This effect was not mediated by protease-activated receptor 2 or the plasmin-plasminogen system, and it was not apparent on incubation of tryptase with medium conditioned by corneal fibroblasts. It was inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 but not by TIMP-1. Conclusions: Tryptase activates MMP-2 released from corneal fibroblasts. This action requires the presence of the cells themselves and might be responsible for the presence of activated MMP-2 in tear fluid of individuals with vernal keratoconjunctivitis.

Identification of common secreted factors in human corneal fibroblasts exposed to LPS, poly(I:C), or zymosan.

Infection of the cornea with bacteria, viruses, or fungi can result in corneal ulceration. Corneal stromal cells participate in the immune and inflammatory responses to such infection in part by producing various cytokines and chemokines. The effects of lipopolysaccharide (LPS), polyinosinic-polycytidylic acid [poly(I:C)], and zymosan as surrogates for bacteria, viruses, and fungi, respectively, on the release of cytokines and chemokines from cultured human corneal fibroblasts were examined in order to identify common factors in infectious corneal keratitis. The secretion of various cytokines and chemokines by human corneal fibroblasts exposed to LPS, poly(I:C), or zymosan was measured with a multiplex assay system. LPS induced the release of interleukin (IL)-6, IL-8, MCP-1, RANTES, IP-10, eotaxin, and IL-12 from corneal fibroblasts. Poly(I:C) stimulated the secretion of IL-6, IL-8, MCP-1, RANTES, IP-10, eotaxin, MIP-1β, and interferon-γ, whereas zymosan triggered the production of IL-6, IL-8, and MCP-1. LPS, poly(I:C), and zymosan thus each induced a distinct pattern of cytokine and chemokine release from human corneal fibroblasts, with the release of IL-6, IL-8, and MCP-1 being commonly elicited by all three agents. Our results suggest that IL-6, IL-8, and MCP-1 may therefore play a key role in the inflammatory response to corneal infection.

Device for cataract analysis: Development and relevance to cataract surgery

PURPOSE: To evaluate the relationship between World Health Organization (WHO) cataract grade determined with a new device and (1) preoperative visual acuity and (2) the difficulty of specific steps in cataract surgery. SETTING: Yamaguchi University Hospital, Yamaguchi, Japan. METHODS: Patients who had cataract surgery between January 2006 and September 2008 were enrolled in this prospective study. Preoperatively, the Konan Anterior Segment Tri Camera System 1000 cataract analysis device was used to evaluate the WHO cataract grade in each eye. The main outcome measures were preoperative visual acuity, the time required for continuous curvilinear capsulorhexis (CCC) and for irrigation/aspiration (I/A), and the total effective phaco time (EPT). RESULTS: Sixty‐four eyes (53 patients) were evaluated. Preoperative visual acuity decreased significantly as the posterior subcapsular cataract (PSC) grade increased (P<.01). Preoperative logMAR values also differed significantly between cataracts classified as mild (score 1 to 3), moderate (score 4 to 6), and severe (score 7 to 9) on the basis of the total nuclear (NUC) + cortical (COR) + PSC score. The CCC and I/A times increased with increasing COR grade, whereas the total EPT increased with increasing NUC grade. CONCLUSIONS: Evaluation of lens opacity based on the WHO grading system using the new cataract analysis device indicated which surgical procedures are likely to be problematic. The device may also be useful in training residents in cataract surgery. Financial Disclosure: Mr. Araki is an employee of Konan Medical, Inc. No other author has a financial or proprietary interest in any material or method mentioned.

Combination management by C-arm fluoroscopy and extraocular muscle severance for penetrating ocular trauma with a retrobulbar foreign body.

ABSTRACT We report here the successful removal of a retrobulbar metallic foreign body in a patient with penetrating ocular trauma by a transconjunctival approach and combination management with C-arm fluoroscopy and extraocular muscle severance. A 37-year-old man sustained a penetrating injury to the right eye while using an iron hammer. Initial slitlamp examination revealed a corneoscleral laceration, iridocele, anterior chamber collapse, and a traumatic cataract. Visual acuity in the right eye was limited to the perception of hand motion. Computed tomography revealed an orbital foreign body in the retrobulbar area. The patient underwent corneoscleral suturing, severance of extraocular muscles, removal of the foreign body with guidance by C-arm fluoroscopy, pars plana lensectomy, and pars plana vitrectomy. Combination management with C-arm fluoroscopy and extraocular muscle severance may thus be a suitable approach to the removal of a retrobulbar metallic foreign body.