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

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.

Methotrexate-associated orbital lymphoproliferative disorder in a patient with rheumatoid arthritis: a case report.

PURPOSE: Lymphoproliferative disorders (LPDs) can develop in patients treated with methotrexate (MTX) and usually respond well to MTX withdrawal. Mucosa-associated lymphoid tissue (MALT) lymphoma is a relatively rare type of MTX-LPD. The development of MTX-LPD in the orbit has not been previously described. We here report a case of orbital MALT lymphoma that disappeared after MTX withdrawal in a patient treated with MTX for rheumatoid arthritis. CASE: A 78-year-old woman who complained of swelling of the left upper eyelid had been treated with MTX for >8 years for rheumatoid arthritis. Slit-lamp examination revealed a temporal subconjunctival mass, salmon pink in color, in the left eye. Fundus photographs also suggested the presence of a temporal tumor in the left orbit. [(18)F]Fluorodeoxyglucose positron emission tomography-computed tomography revealed highly integrated lesions in the left inferotemporal orbit and a left external iliac lymph node, a left obturator lymph node, and an inguinal lymph node. Pathologic analysis of a tumor biopsy specimen showed small- and medium-sized lymphocytes positive for CD20, MIB-1, and bcl-2 and negative for CD10, CD3, bcl-1, IgG4, and EBV-ISH. On the basis of these findings, we diagnosed the tumor as MTX-induced MALT lymphoma. The subconjunctival and orbital masses disappeared gradually over 10 months after MTX withdrawal and did not recur within 2 years. CONCLUSION: This case of orbital MTX-LPD suggests that the possibility of MTX-LPD should be considered even for ocular tumors in patients treated with MTX.

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 17ß-estradiol, progesterone, or dehydro-epiandrosterone. Collagen contraction induced by transforming growth factor-ß2 (TGF-ß2) 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 17ß-estradiol and progesterone inhibited TGF-ß2-induced collagen contraction mediated by RPE cells, whereas the male sex hormone dehydro-epiandrosterone had no such effect. The TGF-ß2-induced release of MMP-2 and MMP-9 from RPE cells was also inhibited by 17ß-estradiol and progesterone, and the MMP inhibitor GM6001 attenuated TGF-ß2-induced collagen contraction. Expression of the mesenchymal markers α-SMA and fibronectin, interleukin-6 release, and Smad2 and MLC phosphorylation induced by TGF-ß2 were all inhibited by 17ß-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-ß2-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.

Autoantibodies to transient receptor potential cation channel, subfamily M, member 1 in a Japanese patient with melanoma-associated retinopathy.

PURPOSE: To report a case of melanoma-associated retinopathy (MAR) in a Japanese patient found to have autoantibodies to transient receptor potential cation channel, subfamily M, member 1 (TRPM1). CASE: An 82-year-old man presented with blurred vision OS as well as night blindness and photopsia OU. Fundus photography, fluorescein angiography, and spectral domain-optical coherence tomography findings were essentially normal. Goldmann perimetry revealed a relative central scotoma, including the blind spot in the right eye, as well as a relative scotoma around a blind spot OS. The full-field scotopic electroretinograms showed a "negative-type" pattern OU, suggestive of extensive bipolar cell dysfunction. Systemic examination revealed that the patient had malignant melanoma of the anus with lung metastasis. Autoantibodies to TRPM1 were detected in the serum of the patient by immunoblot analysis. Vitreous opacity developed during follow-up. The visual symptoms and vitreous opacity of the patient were markedly improved after oral prednisolone therapy. The patient died as a result of widespread metastasis of the melanoma at 11 months after his first visit. CONCLUSION: The present case is the first reported instance of MAR positive for autoantibodies to TRPM1 in an Asian patient.

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-1ß, 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.

[Remodeling in ocular allergic diseases].

Vernal keratoconjunctivitis (VKC), a chronic and severe form of ocular allergic disease, is characterized by tissue remodeling such as the formation of giant papillae at the upper tarsal conjunctiva and the development of corneal plaques. Giant papillae develop as a result of infiltration of inflammatory cells, changes in the epithelial layer, increased deposition of extracellular matrix molecules, proliferation of conjunctival fibroblasts, and an increase in the number of blood vessels. Corneal plaques form subsequent to corneal epithelial defects, and their surface remains uncovered by the corneal epithelium; consequensly, corneal epithelial cells are not able to attach to or migrate over the plaques. These remodeling lesions not only affect tissue structure but also contribute to amplification of allergic inflammation in the conjunctiva and cornea. Recent evidence from in vitro studies indicates that activated fibroblasts play a central role in the induction and amplification of ocular allergic inflammation and the consequent development of giant papillae and corneal disorders in individuals with VKC. Tissue remodeling thus represents a potential therapeutic target for treatment of VKC.

Fibroblasts as local immune modulators in ocular allergic disease.

Vernal keratoconjunctivitis (VKC), a severe form of ocular allergic disease, is characterized by the formation of giant papillae at the upper tarsal conjunctiva and corneal lesions that threaten vision. Recent evidence indicates that resident fibroblasts function as immune modulators in the pathogenesis of the chronic allergic inflammation associated with VKC. The T helper 2 (Th2) cell-derived cytokines interleukin (IL)-4 and IL-13 stimulate the migration and proliferation of conjunctival fibroblasts as well as protecting these cells from apoptotic cell death, effects that likely underlie the hyperplasia of fibroblasts that contributes to the formation of giant papillae. Conjunctival fibroblasts also synthesize extracellular matrix proteins and tissue inhibitors of metalloproteinases as well as down-regulate the expression of matrix metalloproteinases in response to these cytokines, effects that likely contribute to the excessive deposition of extracellular matrix that is characteristic of giant papillae. Stimulation of fibroblasts in the corneal stroma with the combination of a proinflammatory cytokine and either IL-4 or IL-13 results in up-regulation of the expression of the chemokine eotaxin and thymus- and activation-regulated chemokine as well as of vascular cell adhesion molecule-1, which together mediate the infiltration and activation of eosinophils and Th2 cells. Fibroblasts therefore appear to play a central role in the induction and amplification of ocular allergic inflammation and the consequent development of giant papillae and corneal disorders in individuals with VKC. Fibroblasts and fibroblast-derived factors thus represent new and potentially important therapeutic targets for treatment of the giant papillae and corneal disorders associated with VKC.

Inhibition of matrix metalloproteinase-3 synthesis in human conjunctival fibroblasts by interleukin-4 or interleukin-13.

PURPOSE: Fibroproliferative lesions of the conjunctiva known as giant papillae are a characteristic of vernal keratoconjunctivitis (VKC). The abundance of T helper 2 (Th2) cells and cytokines is increased in the giant papillae and tear fluid of individuals with VKC, and the Th2 cytokines interleukin (IL)-4 and IL-13 each stimulate the production of extracellular matrix (ECM) proteins by conjunctival fibroblasts. The role of Th2 cytokines in the development of giant papillae was further examined by determination of the effects of these molecules on the production by conjunctival fibroblasts of matrix metalloproteinase (MMP)-3, a key enzyme in ECM degradation. METHODS: The amount of MMP-3 released into the culture medium by human conjunctival fibroblasts was determined by enzyme-linked immunosorbent assay, and the intracellular abundance of MMP-3 mRNA was quantitated by reverse transcription and real-time polymerase chain reaction analysis. Signaling by the transcription factors NF-kappaB and AP-1 was evaluated by immunoblot and immunofluorescence analyses. RESULTS: Of the Th2 cytokines tested, only IL-4 and -13 inhibited both the basal and IL-1beta-induced release of MMP-3 by conjunctival fibroblasts. These effects of IL-4 and -13 were inhibited by neutralizing antibodies to the IL-4 receptor complex. IL-4 and -13 also each reduced the basal abundance, as well as inhibited the IL-1beta-induced upregulation, of MMP-3 mRNA in these cells. Neither IL-4 nor -13 affected the IL-1beta-induced activation of NF-kappaB or the AP-1 component c-Jun. CONCLUSIONS: IL-4 and -13 each inhibit MMP-3 synthesis in human conjunctival fibroblasts, suggesting that these Th2 cytokines may contribute to the excessive deposition of ECM in giant papillae by preventing matrix degradation mediated by this enzyme.

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.

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.

Potentiation of lipopolysaccharide-induced chemokine and adhesion molecule expression in corneal fibroblasts by soluble CD14 or LPS-binding protein.

PURPOSE: The detection of bacterial lipopolysaccharide (LPS) by human cells is facilitated by LPS-binding protein (LBP) and soluble (s)CD14. The effects of these proteins on chemokine release and adhesion molecule expression in cultured human corneal fibroblasts were examined. METHODS: The release of chemokines into culture supernatants and the expression of the intercellular adhesion molecule (ICAM)-1 on the cell surface were determined by enzyme-linked immunosorbent assays. The intracellular abundance of chemokine and ICAM-1 mRNAs was quantitated by reverse transcription and real-time polymerase chain reaction analyses. The phosphorylation and degradation of IkappaB-alpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively. RESULTS: Neither sCD14 nor LBP alone affected the expression of chemokines or ICAM-1 in cultured human corneal fibroblasts. However, sCD14 or LBP enhanced the LPS-induced upregulation of ICAM-1 and the chemokines interleukin-8 and monocyte chemoattractant protein (MCP)-1 in these cells at the protein and mRNA levels. Combined stimulation with LPS and either sCD14 or LBP also induced the phosphorylation and degradation of IkappaB-alpha and the translocation of NF-kappaB from the cytoplasm to the nucleus of corneal fibroblasts. CONCLUSIONS: LBP and sCD14 may play important roles in the defense of the cornea against bacterial infection, by facilitating the detection of LPS by corneal fibroblasts.

Protection of human conjunctival fibroblasts from NO-induced apoptosis by interleukin-4 or interleukin-13.

PURPOSE: To examine the possible roles of T helper type 2 (Th2) cell-derived cytokines in formation of the giant papillae characteristic in individuals with vernal keratoconjunctivitis, the effects of these cytokines on the proliferation and apoptosis of cultured human conjunctival fibroblasts were investigated. METHODS: Apoptosis was induced by the NO donor sodium nitroprusside. Cell viability was determined by measurement of mitochondrial metabolic activity, and apoptotic cells were identified on the basis either of nuclear morphology after staining with 4',6-diamidino-2-phenylindole or of TUNEL staining. The activation of antiapoptotic signaling mediated by the protein kinase Akt was assessed by immunoblot analysis and by an in vitro kinase assay. Expression of interleukin (IL)-4 and IL-13 receptor subunits was examined by reverse transcription and polymerase chain reaction analysis and by flow cytometry. RESULTS: IL-4 and IL-13, but not IL-5, IL-9, or IL-10, induced the proliferation of conjunctival fibroblasts as well as protecting these cells from NO-induced apoptosis. Both IL-4 and IL-13 induced the phosphorylation of Akt and increased the kinase activity of this enzyme in a manner that was sensitive to the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. These inhibitors also blocked the antiapoptotic effects of IL-4 and IL-13. Transcripts encoding IL-4 and IL-13 receptor components were detected in conjunctival fibroblasts, and the proteins were expressed at the cell surface. CONCLUSIONS: Among the various Th2 cytokines tested, only IL-4 and IL-13 induced the proliferation of human conjunctival fibroblasts and protected these cells from apoptosis. These effects may contribute to the formation of giant papillae in individuals with vernal keratoconjunctivitis.

Lipopolysaccharide-induced expression of intercellular adhesion molecule-1 and chemokines in cultured human corneal fibroblasts.

PURPOSE: Invasion of bacteria into the corneal stroma induces the infiltration of leukocytes and subsequent corneal ulceration. The role of corneal fibroblasts in the detection of bacterial invasion into the stroma was investigated by examining the in vitro expression of the receptor complex for lipopolysaccharide (LPS), a common component of Gram-negative bacteria, as well as the possible effects of LPS on both the expression of adhesion molecules and the release of chemokines in cultured human corneal fibroblasts. METHODS: Expression of the LPS receptor complex, intercellular adhesion molecule (ICAM)-1, and the chemokines interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 was examined by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, or immunofluorescence analysis. RESULTS: Corneal fibroblasts were found to contain transcripts encoding toll-like receptor-4, CD14, and MD-2, all of which are components of the LPS receptor complex. The expression of ICAM-1 at the surface of corneal fibroblasts and the amount of ICAM-1 mRNA in the cells were both increased by LPS. Similarly, LPS increased both the release of IL-8 and MCP-1 by corneal fibroblasts as well as the amounts of the corresponding mRNAs in these cells. These various effects of LPS were potentiated by the presence of a low concentration of human serum. CONCLUSIONS: Corneal fibroblasts may play an important role in the defense system of the cornea by recognizing the presence of LPS and subsequently expressing adhesion molecules and chemokines that promote leukocyte infiltration.

Synergistic effect of TNF-alpha 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)-alpha, 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-alpha, 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-alpha, 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-alpha 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-alpha together with either IL-4 or IL-13. CONCLUSION: Stimulation of human corneal fibroblasts with the combination of TNF-alpha 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.

Expression of functional ICAM-1 on cultured human keratocytes induced by tumor necrosis factor-alpha.

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.

Differential expression of thymus- and activation-regulated chemokine (CCL17) and macrophage-derived chemokine (CCL22) by human fibroblasts from cornea, skin, and lung.

BACKGROUND: Allergic diseases of the ocular surface, skin, and lung are triggered by T(H)2 cells, which are recruited by thymus- and activation-regulated chemokine (TARC; CCL17) and macrophage-derived chemokine (MDC; CCL22). Resident fibroblasts are thought to contribute to inflammatory cell infiltration through chemokine production. OBJECTIVE: We sought to provide insight into the clinical differences apparent among these allergic diseases of the eye, skin, and lung, and we compared the abilities of corneal, dermal, and lung fibroblasts to produce TARC and MDC. METHODS: The amounts of chemokines released into the culture supernatant were determined by means of ELISA, and the intracellular abundance of chemokine mRNAs was quantitated by means of reverse transcription and real-time PCR analysis. RESULTS: Neither TNF-alpha, IFN-gamma, IL-4, nor IL-13 alone induced the release of TARC from or affected the amount of TARC mRNA in corneal, dermal, or lung fibroblasts. The combination of TNF-alpha with either IL-4 or IL-13, however, markedly increased both TARC release and the abundance of TARC mRNA in corneal and dermal fibroblasts, but not in lung fibroblasts. Neither MDC release nor MDC mRNA was detected in any of the 3 types of fibroblasts stimulated with any of the cytokines examined. CONCLUSION: These results indicate that cytokine regulation of TARC expression differs among fibroblasts derived from the cornea, skin, or lung. Corneal and dermal fibroblasts might thus be important sources of TARC during allergic inflammation.

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.

Inhibition of eotaxin expression in human corneal fibroblasts by interferon-gamma.

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-gamma (IFN-gamma), 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-gamma inhibited in a dose-dependent manner the release of eotaxin induced by each of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and IL-1 beta in corneal fibroblasts. IFN-gamma 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-alpha and the T helper cell 2-derived cytokine IL-4 were also both markedly inhibited by the treatment of cells with IFN-gamma. CONCLUSIONS: IFN-gamma inhibited eotaxin expression at both the protein and mRNA levels in cultured human corneal fibroblasts. This effect of IFN-gamma may contribute to the inhibition of eosinophil infiltration into the cornea. Exogenous IFN-gamma thus represents a potential new therapeutic agent for the treatment of corneal disorders associated with inflammatory ocular diseases such as vernal keratoconjunctivitis.