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Role of tumour necrosis factor-α (TNFα) in the functional properties of hyalocytes Yasuaki Hata, Shintaro Nakao, Ri-ichiro Kohno, et al. Br J Ophthalmol 2011 95: 261-265 originally published online October 28, 2010
doi: 10.1136/bjo.2010.190322
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Laboratory science
Role of tumour necrosis factor-a (TNFa) in the functional properties of hyalocytes Yasuaki Hata,1 Shintaro Nakao,1 Ri-ichiro Kohno,1 Kumiyo Oba,1 Takeshi Kita,1 Muneki Miura,1 Yukio Sassa,1 Alexander Schering,2 Tatsuro Ishibashi1 1
Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Higashi-Ku, Fukuoka, Japan 2 Angiogenesis Laboratory, Massachusetts Eye & Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA Correspondence to Dr Tatsuro Ishibashi, Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan;
[email protected] Accepted 12 September 2010 Published Online First 28 October 2010
ABSTRACT Background/aim Tumour necrosis factor-a (TNFa) is an inflammatory cytokine that is upregulated in various vitreoretinal diseases including uveitis and diabetic retinopathy. Recently, our studies have indicated that hyalocytes contribute to the pathogenesis of these diseases. However, the impact of TNFa on the functional properties of hyalocytes is unknown. Methods Hyalocytes were isolated from bovine eyes. Cellular proliferation, migration and gel contraction in response to TNFa and the other inflammatory cytokines were analysed by thymidine uptake, Boyden’s chamber assay and collagen gel contraction assay, respectively. Furthermore, we estimated the effect of dexamethasone on these properties of hyalocytes. Results TNFa promoted proliferation, migration and gel contraction by hyalocytes. Dexamethasone inhibited TNFa-induced proliferation but not migration. Dexamethasone did not inhibit TNFa-induced gel contraction but further increased contraction. Furthermore, dexamethasone inhibited TNFa-induced extracellular signal-related kinase (ERK)1/2 phosphorylation in hyalocytes. Conclusion This study indicates that TNFa in vitreous and retina causes activation of hyalocytes, and the activated hyalocytes contribute to the pathogenesis of inflammatory vitreoretinal diseases. Steroid treatment appears to inhibit the activation of hyalocytes in the early stages of the diseases, but might have adverse effects in the late stage through membrane contraction.
INTRODUCTION The relationship between cytokines and vitreoretinal diseases has been widely examined.1 In particular, increased vitreous levels of cytokines, such as platelet-derived growth factor (PDGF), transforming growth factor-b (TGFb), tumour necrosis factor-a (TNFa) and interleukin-1b (IL-1b) have been shown to cause vitreoretinal diseases.2e4 Among many factors promoting vitreoretinal diseases, TNFa is known to be one of the key regulators that are intimately involved in the pathogenesis of uveitis and intraocular angiogenic diseases such as diabetic retinopathy5 and agerelated macular degeneration.6 TNFa is a proinflammatory cytokine produced by activated monocytes, glial cells7 and retinal pigment epithelium (RPE).8 TNFa also acts as an angiogenic and permeability factor in various mechanisms.9 Recently, drugs that target TNFa have been used in patients with ocular inflammation.10 Hyalocytes have been described as occurring in the peripheral or cortical region of the vitreous close Br J Ophthalmol 2011;95:261e265. doi:10.1136/bjo.2010.190322
to the inner surface of the retina.11 Although hyalocytes were discovered more than a 100 years ago, the molecular and cellular biological characteristics of hyalocytes have yet to be fully elucidated.12 We have reported that hyalocytes are resident macrophages and originate from the blood monocytes.13 Hyalocytes have rare mitotic activity under physiological conditions.14 However, under pathological conditions our studies demonstrated that hyalocytes play important roles in the pathogenesis of proliferative vitreoretinal diseases and that they are a candidate for cellular targeting therapy.2 15e17 PDGF-BB or TGFb induced proliferation of hyalocytes and gel contraction in an in vitro wound-healing assay embedding hyalocytes.2 3 18 However, it is not known how hyalocytes contribute to inflammatory proliferative vitreoretinal diseases. Steroids have been used for the treatment of ocular inflammation. Intravitreal injection of steroids is currently used in the management of intravitreal inflammation in various vitreoretinal diseases, including uveitis and diabetic retinopathy.19 20 Recently, we have shown that vascular epithelial growth factor (VEGF)-A expression by hyalocytes is inhibited by dexamethasone.21 However, the effects of dexamethasone on the functional properties of hyalocytes in inflammationassociated diseases have not been clarified. In this study, to reveal the possible role of hyalocytes in inflammatory vitreoretinal diseases, we investigated the impact of TNFa on the properties of hyalocytes and the effects of steroids on hyalocytes under TNFa stimulation.
MATERIALS AND METHODS Materials
Recombinant human TNFa (T6674), TGFb2 (T2815) and dexamethasone (D2915) were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Recombinant human VEGF-A165 (293-VE) and monocyte chemoattractant protein 1 (MCP-1) (279MC) were obtained from R&D Systems (Minneapolis, Minnesota, USA).
Cell isolation and identification Bovine hyalocytes were isolated and cultivated as previously reported.3 Hyalocytes obtained between passages 4 and 7 were used in experiments.
[3H]-Thymidine uptake The hyalocytes were seeded into 24-well plates at a density of 13104 cells/well. After 24 h starvation, the cells were stimulated with each cytokine for 18 h. 261
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Laboratory science [3H]-Thymidine was then added (9.253103 Bq/well) for an additional 5 h. Incorporated [3H]-thymidine was determined by scintillation counting.
mAb (1:1000, #9106; Cell Signaling Technology, Beverly, California, USA) or p44/42 (1:200, SC-94; Santa Cruz, California, USA).
Chemotaxis assay
Statistical analysis
The assay was performed using a 24-well Boyden’s chemotaxis chamber (Chemotaxicell (Cosmo Bio, Tokyo, Japan); 8 mm pores) with a slight modification to the previously described method.9 After incubation for 4 h at 378C, the migrated cells were observed. Three random objective fields of stained cells were counted for each well, and the mean number of migrating cells per one field was calculated. The number of migrated cells, obtained from six independent wells for each group, was compared using analysis of variance (ANOVA) and Dunnett’s tests.
The experimental data are expressed as mean6SD. Statistical significance was assumed when p was
