Hong Qiao

The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age‐related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular‐infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild‐type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein‐1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular‐infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7‐1 and B7‐2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor α, was also observed. Collectively, we conclude that ocular‐infiltrating macrophages play an important role in CNV generation.

Clearance of apoptotic photoreceptors: Elimination of apoptotic debris into the subretinal space and macrophage-mediated phagocytosis via phosphatidylserine receptor and integrin αvβ3

The effective phagocytotic clearance of apoptotic debris is fundamental to the maintenance of neural tissues during apoptosis. Retinal photoreceptors undergo apoptosis after retinal detachment. Although their induction phase of apoptosis has been well discussed, their phagocytotic process remains quite unclear. We herein demonstrate that apoptotic photoreceptors are selectively eliminated from their physiological localization, the outer nuclear layer, to the subretinal space, and then phagocytosed by monocyte-derived macrophages. This could be shown by an ultrastructural and immunophenotypic analysis. Moreover, in chimera mice expressing transgenic green fluorescent protein in bone marrow-derived cells, the local infiltration of macrophages could be detected after retinal detachment-induced photoreceptor apoptosis. The local injection of an antibody blocking the phosphatidylserine receptor (PSR) or a peptide (GRGDSP)-blocking integrin αvβ3 revealed that phagocytotic clearance involves the PSR as well as integrin αvβ3 in vivo. Importantly, the level of blockade obtained with these reagents was different. Although anti-PSR increased the frequency of apoptotic cells that fail to bind to macrophages, GRGDSP prevented the engulfment (but not the recognition) of apoptotic photoreceptor cells by macrophages. To our knowledge, this is the first report describing the mechanisms through which apoptotic photoreceptors are selectively eliminated via a directional process in the subretinal space.

The characterisation of hyalocytes: the origin, phenotype, and turnover

Aim: To determine the characterisation of hyalocytes: the origin, phenotype, and turnover in the rodent. Methods: To characterise the ultrastructure and distribution of hyalocytes, transmission and scanning electron microscopy was performed in rat eyes. Immunophenotypical analysis was performed by either anti-ED1 or ED2 antibodies. To examine the origin of the hyalocytes, the chimeric mice were created and were used to transplant the bone marrow (BM) cells from enhanced green fluorescent protein (EGFP) transgenic mice. The turnover of hyalocytes was examined at 0, 4, 6, 7, and 12 months after BM transplantation. Results: Hyalocytes were distributed especially in the vitreous cortex and had an irregular shape with a spherical granule. Immunophenotypical studies demonstrated that most of the hyalocytes in rat eyes expressed ED2 but not ED1. In the chimeric mice, the hyalocytes were GFP negative right after BM transplantation. Interestingly, more than 60% of hyalocytes were replaced within 4 months and approximately 90% within 7 months after BM transplantation. Conclusions: The rodent hyalocytes were shown to express tissue macrophage marker, were derived from BM, and totally replaced within 7 months. These data provide the characterisation of hyalocytes in physiological conditions, especially their origin, distribution, and turnover, and may contribute to the better understanding of the pathogenesis of vitreoretinal disease.

The relative contributions of each subset of ocular infiltrated cells in experimental choroidal neovascularisation

Aim: Choroidal neovascularisation (CNV) is a major cause of blindness in adults. The aim of this study was to investigate the role of infiltrating cells in the development of experimental CNV. Methods: CNV was induced in C57BL/6 (B6) mice by laser photocoagulation (PC). After PC, the numbers of each subset of infiltrated cells were analysed by flow cytometry at multiple time points. Each subset (except for macrophages) was depleted by the specific antibodies in vivo. Thereafter, the area of CNV was compared between the control B6 mice and the specific antibody treated mice 7 days after PC. The CNV formation in neutrophil depleted CC chemokine receptor-2 (CCR2) knockout mice was also examined to minimise the effects of macrophages. Results: In the early phase of CNV formation, a large number of neutrophils and macrophages infiltrated to the eyes. Natural killer (NK) cells and T lymphocytes were barely detected while no B lymphocytes were detected. The CNV areas did not significantly change compared between the control B6 mice and the specific antibody treated mice. However, the neutrophil depleted CCR2KO mice resulted in a reduction of CNV. Conclusion: Although lymphocytes and NK cells had little effect on CNV formation, neutrophils partially contributed to CNV in the absence of macrophages.

The analysis of systemic tolerance elicited by antigen inoculation into the vitreous cavity: vitreous cavity-associated immune deviation

The immune privilege that exists in the eye is maintained by various mechanisms. One of the best studied is a form of systemic tolerance termed anterior chamber‐associated immune deviation (ACAID). We have investigated the mechanisms by which ocular inflammation associated with the vitreous cavity (VC) is reduced, by injecting either ovalbumin (OVA) or allogeneic splenocytes into the VCs of mice, and assessed the effect of this on delayed type hypersensitivity (DTH) responses. After antigen inoculation into the VC, antigen‐specific DTH responses were significantly impaired and we named this phenomenon ‘vitreous cavity‐associated immune deviation’ (VCAID). VCAID could also be induced by inoculating antigen‐pulsed macrophages into the VC. However, VCAID did not develop either in mice with inflamed eyes, whether as a result of experimental autoimmune uveitis or coadministration of interleukin (IL)‐6 in the VC, or in knockout mice deficient for natural killer T (NKT) cells. Finally, we found that so‐called ‘hyalocytes’ are the only cells present in the VCs of normal mice, uniformly distributed on the retinal surface. Interestingly, they express F4/80, suggesting that hyalocytes are candidate antigen‐presenting cells (APCs) responsible for mediating VCAID. As for the anterior chamber model, systemic tolerance can be induced in the VC in non‐inflamed eyes and in the presence of invariant NKT cells.

Immunoregulatory Role of Ocular Macrophages: The Macrophages Produce RANTES to Suppress Experimental Autoimmune Uveitis

Murine experimental autoimmune uveitis (EAU) is a model of human uveitis. Ocular-infiltrating macrophages play a crucial role in the generation of tissue damage in EAU. In fact, several chemokines are actually produced in the inflamed eye. The aim of this study was to elucidate the role of ocular macrophage-derived chemokines in EAU. C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein peptide 1–20, and the EAU severity was scored at multiple time points based on microscopic fundus observations (retinal vascular dilatation and exudates) and histological examinations. The peak inflammatory response was observed 1 wk (day 16) after the beginning of macrophage infiltration to the eye (day 9). Ocular-infiltrating cells were enriched or depleted of macrophages by magnetic beads and analyzed by real-time RT-PCR for chemokine mRNA production. We found that only the macrophage-enriched cells from the eye produced RANTES, and thus proposed that macrophage-derived RANTES facilitated the ocular inflammations. In contrast to our postulate, neutralization of RANTES by specific Ab in vivo on days 9 and 13 exacerbated EAU. We also found that the ratio of ocular CD4/CD8 T cells was markedly increased after treatment. As a result, RANTES neutralization might exacerbate EAU by modulating the type of T cell subsets recruited to the eye. In conclusion, our data provide insight into the immunoregulatory role of macrophages and RANTES in the pathogenesis of ocular inflammation. Not all macrophage-derived chemokines cause local inflammation, since RANTES produced by ocular macrophages appears to suppress EAU.

Interleukin‐18 regulates pathological intraocular neovascularization

Recently, the proinflammatory cytokine IL‐18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen‐induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL‐18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL‐18 reduction in oxygen‐treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL‐18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL‐18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL‐18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL‐18‐binding protein was administered during the OIR recovery phase to neutralize endogenous IL‐18, OIR was still apparent on Day 24. We therefore concluded that IL‐18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans.

Abnormal retinal vascular development in IL-18 knockout mice

Recent studies have indicated that interleukin 18 (IL-18) might act as either an angiogenic or an angiostatic factor, but the true function of this protein in vascular development is unclear. We therefore investigated the role of IL-18 in the formation of retinal vessels. Development of the retinal vasculature was compared in IL-18 knockout (KO) and wild-type (WT) mice at several different time points. The formation of vessels was evaluated using angiography of flat-mounted retinal samples after inoculation with fluorescein dextran. Retinal samples from both groups were also evaluated through histological examinations, and the expression of angiogenic factors was examined using the reverse-transcription-polymerase chain reaction. The capillary retinal vessels in both WT and IL-18 KO mice had reached the peripheral retina by postnatal day (P) 7. However, IL-18 KO mice showed angiectasis and vascular leakage at P7, especially in the mid-peripheral retina. These symptoms were not observed in WT mice at any stage. Histopathological analysis confirmed abnormal vascular formation in IL-18 KO mice at P14. Interestingly, these abnormalities regressed over time and had disappeared by P84. Several angiogenesis-associated factors, including vascular endothelial growth factor (VEGF), basic fibroblast-growth factor (bFGF), platelet-derived growth factor (PDGF) and pigment epithelium-derived factor (PEDF), were overexpressed in the retinas of IL-18 KO mice compared with those of WT mice at P14. Interferon-γ was detected only in WT mouse retinas at P14. These results provide new evidence for the role of IL-18 in retinal vascular development.

Development of a New Mouse Model of Branch Retinal Vein Occlusion and Retinal Neovascularization

PurposeRetinal neovascularization (NV) is associated with various disorders, such as retinal vein occlusion, diabetic retinopathy, and retinopathy of prematurity, and often causes severe loss of vision. To determine the mechanism of retinal NV and develop new therapy, we developed a mouse model using a photodynamic method.MethodsC57BL/6 mice were injected with rose bengal via the tail vein, and then selected venous points were photocoagulated.ResultsAll eyes demonstrated venous occlusion on day 1, and capillary nonperfusion areas were observed until day 3. Twenty of 33 eyes (60.6%) developed retinal NV on day 14, confirmed by fluorescein isothiocyanate-perfused retinal flat-mounts and immunochemical and histopathological analyses. Reverse transcriptase-polymerase chain reaction showed an increase in the expression of vascular endothelial growth factor at the retina on day 7.ConclusionsBecause of the simplicity, low cost, and feasibility of genetic manipulations, our model is believed to represent an advance in investigating molecular mechanisms and establishing therapy for retinal NV. Jpn J Ophthalmol 2007;51:251–257 @ Japanese Ophthalmological Society 2007

Analysis of corneal inflammation induced by cauterisation in CCR2 and MCP-1 knockout mice.

Aim: To elucidate the role of CCR2/MCP-1 in corneal inflammation. Methods: A cauterisation induced corneal inflammation model was used. The corneas were cauterised with silver nitrate in CCR2 knockout (KO) mice, MCP-1 KO mice, and control mice. Clinical signs such as corneal oedema and opacity were examined 96 hours after cauterisation and the phenotypes of the cells infiltrating the cornea were analysed by flow cytometry. Corneal inflammation in neutrophil depleted mice was also analysed. Results: After cauterisation both CCR2 KO and MCP-1 KO mice showed the same levels of corneal oedema and opacity as control mice. Flow cytometry revealed that in control mice most of the infiltrating cells were neutrophils and macrophages, whereas in both CCR2 KO mice and MCP-1 KO mice, the number of macrophages infiltrating the cornea were markedly reduced. However, prominent infiltrates of neutrophils were still observed in the cornea in CCR2 KO mice and MCP-1 KO mice. The depletion of neutrophils significantly reduced the oedema and opacity induced in the cornea by cauterisation. Conclusion: The CCR2 and MCP-1 molecules are not essential for cauterisation induced corneal inflammation. Neutrophils, rather than migrated macrophages, are the final effector cells involved in inducing inflammation in this model.