Yoko Karasawa

Elevated Levels of Cytokines Associated with Th2 and Th17 Cells in Vitreous Fluid of Proliferative Diabetic Retinopathy Patients

Macrophages are involved in low-grade inflammation in diabetes, and play pathogenic roles in proliferative diabetic retinopathy (PDR) by producing proinflammatory cytokines. T cells as well as other cells are also activated by proinflammatory cytokines, and infiltration into the vitreous of patients with PDR has been shown. In this study, we measured helper T (Th) cell-related cytokines in the vitreous of PDR patients to define the characteristics of Th-mediated immune responses associated with PDR. The study group consisted of 25 type 2 diabetic patients (25 eyes) with PDR. The control group consisted of 27 patients with epiretinal membrane (ERM), 26 patients with idiopathic macular hole (MH), and 26 patients with uveitis associated with sarcoidosis. Vitreous fluid was obtained at the beginning of vitrectomy, and centrifuging for cellular removals was not performed. Serum was also collected from PDR patients. IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, soluble sCD40L, and TNFα in the vitreous and serum samples were measured. Both percent detectable and levels of IL-4, IL-6, IL-17A, IL-21, IL-22, and TNFα in the vitreous were significantly higher than those in the serum in PDR patients. Vitreous levels of these cytokines and IL-31 were significantly higher in PDR than in ERM or MH patients. Vitreous levels of IL-4, IL-17A, IL-22, IL-31, and TNFα in PDR patients were also significantly higher than those of sarcoidosis patients. In PDR patients, vitreous IL-17A level correlated significantly with vitreous levels of IL-22 and IL-31, and especially with IL-4 and TNFα. Although it is unclear whether these cytokines play facilitative roles or inhibitory roles for the progression of PDR, the present study indicated that Th2- and Th17-related immune responses are involved in the pathogenesis of PDR.

Phototoxicity of indocyanine green under continuous fluorescent lamp illumination and its prevention by blocking red light on cultured Müller cells.

PURPOSE To investigate the phototoxicity of persistent indocyanine green (ICG) under continuous visible light illumination and to determine whether blocking peak absorbance wavelengths of ICG is cytoprotective. METHODS Cultured quail Müller cells were exposed to 0 to 5 mg/mL ICG for 30 seconds or 10 minutes and then were cultured in a colorless medium for 24 hours with or without continuous fluorescent lamp illumination. Cells exposed to 5 mg/mL ICG for 10 minutes were cultured under illumination filtered through a dichroic mirror that blocks red to near-infrared, green, or blue wavelengths. After microscopic observation, cell viability and cell death were evaluated. RESULTS ICG exposure followed by illuminated culture induced severe morphologic changes in cells, significant reductions in cell viability, and increases in cell death from apoptosis compared with exposure to ICG or illumination alone or with no exposure. Although ICG exposure at higher concentrations caused cell damage in a dose- and time-dependent manner, an increase in cell viability was noted for cells exposed to lower ICG concentrations. Blocking red to near-infrared wavelengths prevented the decrease in cell viability and the increase in cell death in the culture exposed to ICG followed by illuminated culture. CONCLUSIONS Continuous fluorescent lamp illumination enhanced the cytotoxicity of persistent ICG on Müller cells in a dose- and exposure time-dependent manner. Blocking peak absorbance wavelengths of ICG prevented photodynamic cytotoxicity of persistent ICG under continuous visible light illumination in vitro. This culture system could be used to study the mechanisms of prevention of unfavorable outcomes in ICG-assisted surgery.

Inhibition of Histone Deacetylation by Butyrate Induces Morphological Changes in Y79 Retinoblastoma Cells

PurposeExposure of Y79 cells, a retinoblastoma cell line, to sodium butyrate, a histone deacetylase inhibitor, induces neuronlike morphological changes and apoptosis. To determine whether the effect of butyrate results from an inhibition of histone deacetylation, we examined the morphological changes, cell viability, and histone acetylation levels of Y79 cells induced by butyrate and trichostatin A (TSA), a specific inhibitor of histone deacetylases.MethodsY79 cells cultured in a synthetic medium were exposed to butyrate or TSA, and the morphological changes and cell viability were followed. Actinomycin D was used to determine whether the morphological changes were transcription-dependent. The level of acetylated histone was determined by Western blotting and immunocytochemistry.ResultsButyrate and TSA induced morphological changes and apoptosis of Y79 retinoblastoma cells in a dose-dependent manner. The morphological changes were sustained and reversible with butyrate but were transient with TSA. Actinomycin D completely inhibited the morphological changes induced by butyrate and TSA. The elevation of histone H3 levels was sustained and reversible in butyrate but transient in TSA. The change in histone H3 acetylation levels preceded the morphological changes and apoptosis.ConclusionThe induction of morphological changes by butyrate results from an inhibition of histone deacetylation and transcription. Jpn J Ophthalmol 2004;48:542–551

Evaluation of mouse experimental autoimmune uveoretinitis by spectral domain optical coherence tomography

Aims To evaluate the efficacy of spectral domain optical coherence tomography (SD-OCT) in monitoring the development of mouse experimental autoimmune uveoretinitis (EAU) as an animal model of endogenous uveitis, and to develop an OCT-based grading system for EAU severity. Methods C57BL/6 mice were immunised with human interphotoreceptor retinoid-binding protein (amino acid sequence 1–20) peptide and complete Freunds adjuvant to induce EAU. The development of EAU was monitored by SD-OCT serially throughout the disease course, and the images were graded from 1 to 4 and compared with the clinical and histopathological grades. Results SD-OCT images depicted retinal lamella structures including the inner segment/outer segment (IS/OS) line in normal mice. Retinal structural changes were observed on SD-OCT images in mice that developed EAU clinically scored as grade 1 or higher, which precisely corresponded to the pathological findings. The SD-OCT images of EAU were graded as follows: grade 1, a few infiltrating cells in the vitreous and retina; grade 2, increased vitreous cells, retinal vasculitis, and granulomatous lesion; grade 3, cell infiltration into the whole retina, disappearance of IS/OS line, and destruction of the retinal layer structure; and grade 4, disappearance of the outer retina. The SD-OCT grade of EAU based on these criteria correlated significantly with both the clinical grade (R2=0.282, p<0.005) and histopathological grade (R2=0.846, p<0.0001). Conclusions SD-OCT is useful for evaluating the development and severity of mouse EAU. The SD-OCT scoring system we developed accurately reflects clinical and histopathological changes.

Effects of Superoxide Dismutase and Catalase on Growth of Retinal Pigment Epithelial Cells in vitro following Addition of Linoleic Acid or Linoleic Acid Hydroperoxide

The rod outer segments of the retina that are phagocytized by retinal pigment epithelial (RPE) cells are susceptible to lipid peroxidation because of their high content of polyunsaturated fatty acids. Linoleic hydroperoxides (LHP), synthesized by peroxidation of linoleic acids (LA), produce greater damage to retinal function than does LA. We compared the effects of LHP and LA on the growth of cultured chick embryonic RPE cells and analyzed a model of data sets using multiple linear regression for the number of cells as a function of time. The spectrum of LA had a sharp peak at 205 nm and a broad spectrum at 235 nm, while LHP had only a broad spectrum at 235 nm. Exposure to LA and LHP caused dose-dependent damage of chick embryonic RPE cells: they were significantly more affected by the addition of LHP than LA. The antioxidative enzymes catalase and superoxide dismutase minimized damage to the RPE cells caused by LHP in proportion to the enzyme concentration. However, RPE cells incubated with LA were more affected by the enzymes than when no enzymes were added.

Phototoxicity of Indocyanine Green and Brilliant Blue G under Continuous Fluorescent Illumination on Cultured Human Retinal Pigment Epithelial Cells

PURPOSE We compared the phototoxicity of indocyanine green (ICG) and Brilliant Blue G (BBG) in cultured RPE cells under fluorescent lamp illumination imitating ambient light. METHODS Cultured human RPE line cells were stained with ICG or BBG solution at concentrations of clinical use, and cultured in a colorless medium for 24 hours in the dark or under illumination from a fluorescent lamp. After culture, cell morphology and TUNEL-positive apoptotic cells were observed. Cell viability and cell death rate were evaluated. Absorption spectral changes of BBG before and after incubation were measured. RESULTS ICG-stained cells cultured under illumination changed to an oval morphology with increased number of apoptotic cells, whereas ICG-stained cells cultured in the dark, and BBG-stained cells cultured under illumination and dark conditions maintained a flat morphology without increase in apoptotic cells. Cell viability decreased and cell death rate increased only in cells stained by ICG followed by culture under illumination. Staining cells with ICG at one-tenth concentration of clinical usage induced no cytotoxicity after culture under illumination. Approximately 30% of total BBG retained in the stained cells was released into the culture supernatant after incubation for 24 hours. The absorption spectrum of BBG did not change after fluorescent light irradiation. CONCLUSIONS Illumination with a fluorescent lamp caused cell death via apoptosis in ICG-exposed, but not in BBG-exposed cultured RPE cells. BBG may be a safer dye than ICG because of low light-induced cytotoxicity and rapid elution from stained cells.

Purification and Characterization of Mouse Lacrimal Gland Epithelial Cells and Reconstruction of an Acinarlike Structure in Three-Dimensional Culture

PURPOSE To clarify which factors are involved in postnatal lacrimal gland development, the expressions of several growth factors and their receptors were analyzed in purified lacrimal gland epithelial and mesenchymal cells. Reconstruction of acinarlike structures in three-dimensional culture conditions from this epithelial cell population was attempted. METHODS Lacrimal gland epithelial and mesenchymal cells were isolated from newborn mice and purified using nylon mesh and collagenase. By real-time reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blotting, the expressions of several growth factors and their receptors were analyzed. Responses of epithelial cells to the growth factors were analyzed by the addition of these factors to the culture medium. RESULTS Fibroblast growth factor (FGF)10 and hepatocyte growth factor (HGF) were more intensely expressed in mesenchymal cells than in epithelial cells, and their receptors (FGFR2IIIb and c-Met, respectively) were less intensely expressed, whereas the expression of epidermal growth factor (EGF) and its receptor showed no significant difference between cell types. In the monolayer culture, cell viability was activated by the addition of EGF or HGF to epithelial cells, but no response was observed when FGF10 was added. The epithelial cells formed clusters with lumina when cultured on basement membrane matrix. These clusters contained secretion granules and showed positive immunostaining for aquaporin-5. CONCLUSIONS EGF and HGF were considered to act in an autocrine/paracrine manner in and around the postnatal lacrimal gland, whereas epithelial cells did not respond to FGF10. It was suggested that certain extracellular matrix conditions accommodate these epithelial cells to reconstruct functional acinarlike structures.

Apoptosis after butyrate-induced differentiation in retinoblastoma cell line Y-79.

PURPOSE To study the fate of Y-79 human retinoblastoma cells after induction of differentiation. METHODS Y-79 cells were cultured in a synthetic medium and were induced to neuronal differentiation by butyrate treatment. Neurofilaments, p53, and DNA-synthesizing nuclei labeled with 5-bromodeoxyuridine were immunostained, and apoptotic cells were labeled by in situ DNA nick end labeling (TUNEL). We combined these immunostaining and labeling methods to determine whether the cells expressed these markers at the same time. DNA fragmentation and p53 levels were also determined by electrophoresis. RESULTS Y-79 cells proliferated in the synthetic medium. After butyrate treatment, they extended protrusions and increased neurofilament immunoreactivity. The differentiated features were striking on day 7. Thereafter, differentiated cells decreased and apoptotic cells increased. DNA synthesis was detected in the cells expressing immunoreactivity for neurofilaments or p53. At day 7, most of the cells with p53-positive nuclei were alive and neurofilament-positive. However at day 10, the p53-positive cells were apoptotic and neurofilament-positive apoptotic cells accumulated. CONCLUSIONS We conclude that the Y-79 cells express p53 and undergo apoptosis after neuronal differentiation. There could be a p53-dependent apoptotic pathway in butyrate-induced differentiated Y-79 cells due to the inability to regulate cell cycling.

Association between aqueous humor and vitreous fluid levels of Th17 cell-related cytokines in patients with proliferative diabetic retinopathy

Inflammation is known to be involved in the progression of diabetic retinopathy. We have recently reported that vitreous levels of IL-4, IL-17A, IL-22, IL-31, and TNFα are higher than the respective serum levels in proliferative diabetic retinopathy (PDR) patients, and that vitreous levels of these cytokines are higher in PDR than in other non-inflammatory vitreoretinal diseases or uveitis associated with sarcoidosis. In the present study, we investigated inflammatory cytokines including Th17 cell-related cytokines in aqueous humor samples obtained from eyes with PDR, and analyzed the association between the aqueous humor and vitreous fluid levels of individual cytokines. The study group consisted of 31 consecutive type 2 diabetic patients with PDR who underwent cataract surgery and vitrectomy for vitreous hemorrhage and/or tractional retinal detachment. Undiluted aqueous humor was collected during cataract surgery, and then vitreous fluid was obtained using a 25G vitreous cutter inserted into the mid-vitreous cavity at the beginning of vitrectomy. IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, soluble CD40 ligand (sCD40L), and TNFα levels in the aqueous humor and vitreous fluid were measured using a beads-array system. Although IL-17A was detected in the aqueous humor of eyes with PDR and the level correlated with IL-17A level in the vitreous fluid, both percent detectable and level of IL-17A in the aqueous humor were significantly lower than those in the vitreous fluid. Vitreous IL-17A level was related significantly to IL-10, IL-22, and TNFα levels in aqueous humor as well as in vitreous fluid, On the other hand, aqueous IL-17A level was not related significantly to aqueous or vitreous levels of IL-10, IL-22 or TNFα level. The present study demonstrated that IL-17A level and detectable rate in the aqueous humor of patients with PDR are markedly lower than those in the vitreous fluid and aqueous IL-17A does not correlate with vitreous levels of other cytokines, and hence should not be used as a surrogate for IL-17A in the vitreous fluid.

Analysis of Th Cell-related Cytokine Production in Behçet Disease Patients with Uveitis Before and After Infliximab Treatment

ABSTRACT Purpose: To examine antigen-stimulated cytokine production by Behçet disease patients (BD) before and after infliximab infusion. Methods: PBMCs were obtained before and after infliximab infusion in BD patients with or without recurrent uveitis during at least 1 year of infliximab therapy, and from healthy subjects. PBMCs were cultured with IRBP, and Th-related cytokines in cultures were measured. Results: Levels of IL-4, IL-6, IL-10 IL-17A, IL-17F, IL-31, IFN-γ, and TNFα were higher in BD before infliximab infusion than in healthy subjects, and these levels were the highest in BD with recurrent uveitis. After infliximab infusion, these cytokine levels were reduced to a greater extent in BD without recurrent uveitis than in BD with recurrence. Conclusions: Th-related cytokines produced by IRBP-stimulated PBMCs were elevated in BD, and infliximab infusion suppressed these cytokines to a greater extent in BD without recurrent uveitis than in those with recurrence.