Neuroprotective effect of activated 5'-adenosine monophosphate-activated protein kinase on cone system function during retinal inflammation.

BACKGROUND: Retinal inflammation can cause retinal neural disorders. In particular, functional disorder in the cone photoreceptor system influences visual acuity. However, the underlying mechanism is not yet fully understood. In this study, we evaluated cone system function and the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) during retinal inflammation. RESULTS: Six to eight-week-old male C57BL/6 mice received an intraperitoneal injection of lipopolysaccharide (LPS) to induce retinal inflammation, and were treated with an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR; 250 mg/kg body weight) or phosphate-buffered saline as vehicle 3 h before the LPS injection. The b-wave of the photopic electroretinogram, which represents cone system function, was decreased 24 h after LPS injection and this reduction was suppressed by AICAR treatment. At this time point, there was no remarkable morphological change in the cone photoreceptor cells. At 1.5 h after LPS injection, the retina mRNA levels of an inflammatory cytokine, Tnf-α, were increased, and those of a regulator of mitochondrial biogenesis, Pgc1-α, were decreased. However, AICAR treatment suppressed these changes in mRNA expression. Immunohistochemistry showed that induction of glial fibrillary acidic protein expression was also suppressed by AICAR 24 h after LPS injection. Furthermore, the mouse cone photoreceptor-derived cell line 661W was treated with AICAR to increase the level of phosphorylated and activated AMPK. After 3 h of AICAR incubation, 661W cells showed decreased Tnf-α mRNA levels and increased Pgc1-α mRNA levels. CONCLUSION: AMPK activation has a neuroprotective effect on cone system function during inflammation, and the effect may, at least in part, involve the regulation of inflammatory cytokines and mitochondrial condition.

Roles of AMP-activated protein kinase in diabetes-induced retinal inflammation.

PURPOSE: AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. The purpose of the present study was to elucidate the roles of AMPK in the pathogenesis of diabetic retinopathy using the known AMPK activators resveratrol and AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) in a mouse model. METHODS: C57BL/6 mice with streptozotocin-induced diabetes were treated with resveratrol orally at 50 mg/kg for 7 days or with AICAR intraperitoneally at 100 mg/kg 24 hours before death. Retinal protein levels of phosphorylated and total AMPK, phosphorylated nuclear factor (NF)-κB p65, intercellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were evaluated by Western blot analysis or enzyme-linked immunosorbent assay. Retinal activity of sirtuin (SIRT)1 was measured by deacetylase fluorometric assay. Leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique. RESULTS: Induction of diabetes in mice led to retinal AMPK dephosphorylation, which was significantly reversed by either resveratrol or AICAR. Either resveratrol or AICAR significantly reversed SIRT1 deactivation and NF-κB phosphorylation, both of which were induced in the diabetic retina. Administration of resveratrol to diabetic mice significantly reduced diabetes-induced retinal leukocyte adhesion, together with retinal expression of ICAM-1 and VEGF. CONCLUSIONS: The present findings reveal that diabetes-induced retinal inflammation stems from downregulation of the AMPK pathway, leading subsequently to SIRT1 deactivation and NF-κB activation. The data also suggest the potential use of the AMPK activator resveratrol as a therapeutic agent for diabetic retinopathy.

Lack of TNF-alpha promotes caspase-3-independent apoptosis during murine cytomegalovirus retinitis.

PURPOSE: Both caspase-dependent and caspase-independent apoptosis contribute to retinal damage during murine cytomegalovirus (MCMV) retinitis, and TNF-α is among the inducers of apoptosis. The aim of this study was to determine the contribution of TNF-α by studying virus replication and apoptosis in immunosuppressed (IS) TNF-α(-/-) mice. METHODS: IS TNF-α(-/-) mice or wild-type mice were inoculated with MCMV by the supraciliary route. Injected eyes were examined by plaque assay, electron microscopy, Western blot analysis (caspase-3, caspase-8, caspase-12, Bid, NF-κB, cFlip, XIAP), staining for MCMV early antigen, and TUNEL assay. RESULTS: Although the titer of MCMV was similar in both groups, significantly more apoptotic cells were observed in the retinas of IS TNF-α(-/-) mice than in those of wild-type mice. The level of active caspase-3 was similar in both groups; however, more activated proteins for genes involved in the mitochondrial pathway (cleaved caspase-8, tBid) and endoplasmic reticulum (ER) stress (cleaved caspase-12) and, though less active, NF-κB subunits and antiapoptotic proteins (XIAP and cFlip) were detected in the TNF-α(-/-) eyes compared with wild-type mice. CONCLUSIONS: Although TNF-α is an inducer of apoptosis, the results of this study suggest that TNF-α is also antiapoptotic by the following mechanism: TNF-α activation of NF-κB promotes the production of the antiapoptosis genes, c-flip or XIAP, which, in turn, inhibit the activation of caspase-8 and the mitochondrial pathway or the activation of caspase-12 and ER stress.

Arginase activity mediates retinal inflammation in endotoxin-induced uveitis.

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

Leukocyte extravasation: an immunoregulatory role for alpha-L-fucosidase?

Fucosylated oligosaccharides and glycoconjugates have been implicated in several biological events, including the cell-cell adhesion processes that mediate inflammation. Alpha-L-fucosidase (ALF) is an exoglycosidase that is involved in the hydrolytic degradation of alpha-L-fucose from glycoconjugates. In this study, we investigated the potential role of ALF in regulation of leukocyte migration. Measurement of transendothelial migration in response to CCL5 demonstrated that pretreatment of monocytic cells with ALF reduced migration (p = 0.0004) to a greater extent than treatment of the endothelial monolayer (p = 0.0374). Treatment with ALF significantly reduced the adhesion of monocytic cells to immobilized P-selectin.Fc. A murine model of experimental autoimmune uveitis was then used to show that treatment of splenic cells with ALF produced an 8.6-fold decrease in rolling and a 3.2-fold decrease in cell migration across the retinal vasculature. Further in vitro studies demonstrated that treatment of monocytes with the chemokines CCL3 or CCL5 increased the level of mRNA encoding ALF; this was accompanied by the detection of significant increases in both the 51- and 56-kDa components of ALF by Western blotting. Treatment of monocytic cells with ALF for 2 h significantly reduced the cell surface expression of CD31, with a further decrease in expression observed after 5 h (p = 0.002). Thus, CD31 and fucosylated ligands of P-selectin seem to be the candidates through which ALF mediates its effect in vitro. These data identify a previously unrecognized immunoregulatory role for ALF in late stages of inflammation.

Evidence for extrathymic generation of regulatory T cells specific for a retinal antigen.

BACKGROUND: Thymic expression of a photoreceptor cell antigen, interphotoreceptor retinoid-binding protein, is known to generate regulatory T cells (T(reg)) that prevent spontaneous autoimmune disease of the retina. However, the contribution of other endogenous, tissue-specific antigens (Ags) expressed in the retina to the generation of T(reg) is uncertain. METHODS: Transgenic mice that express beta-galactosidase (beta-gal) in photoreceptor cells, together with beta-gal-specific T cell receptor transgenic mice, were used to study the induction of T(reg) in vivo. RESULTS: Transgenic expression of beta-gal on the arrestin promoter led to a spontaneous immunoregulatory response that inhibited the development of immune responses to beta-gal. The regulation was transferred by CD3+4+25+ T(reg). Several strategies were then used to show that beta-gal expressed in the retina supported spontaneous, thymus-independent T(reg) development. The endogenous T(reg) also differed from the T(reg) induced by Ag inoculation into the anterior chamber of the eye. CONCLUSION: These results demonstrate that retinal expression of very small amounts of a tissue-specific Ag can generate T(reg) in the periphery.

Lack of iNOS facilitates MCMV spread in the retina.

PURPOSE: The purposes of this study were to identify iNOS-producing retinal cells and to determine whether lack of iNOS facilitates MCMV spread and replication in the retina. METHODS: Immunosuppressed (IS) iNOS(-/-) mice or C57BL/6 (wild-type) mice were inoculated with 5 x 10(4) PFU of MCMV K181 strain (K181) via the supraciliary route. Injected eyes were collected at several times after inoculation and examined by plaque assay for replicating virus, RT-PCR for iNOS RNA, Western blot for iNOS protein and by staining for MCMV early antigen (EA), iNOS, and retinal cell antigens. RESULTS: iNOS mRNA and iNOS proteins were expressed in the MCMV-injected eye of wild-type mice. Most iNOS-producing cells were F4/80-positive, including macrophages, RPE-derived macrophages, and resident microglia. Significantly higher titers of virus were recovered from the injected eyes, and more infected cells were detected in the retina of IS iNOS(-/-) mice than in IS wild-type mice. Retinal necrosis and loss of retinal architecture throughout the retina were noted in IS iNOS(-/-) mice, whereas cytomegalic cells and retinitis were present only in the peripheral retina of IS wild-type mice. CONCLUSIONS: iNOS produced by macrophages, especially resident macrophages including microglia and RPE derived macrophages, plays an important role in limiting spread of MCMV in the retina.

Phospholipase D1 is up-regulated in the retina of Lewis rats with experimental autoimmune uveoretinitis.

To investigate whether phospholipase D1 (PLD1) is involved in autoimmune damage to the eyes, we used Western blotting and immunohistochemistry to examine the expression and distribution of PLD1 in the retinas of Lewis rats with experimental autoimmune uveoretinitis (EAU). Western blot analysis showed that the level of expression of PLD1 was significantly increased in EAU-affected retinas compared to that of control. Immunohistochemical analysis showed that ganglion cells and Muller cells, which express PLD1 weakly in the normal retina, showed increased expression of PLD1 in EAU-affected retinas; some ED1-immunopositive cells were also immunopositive for PLD1 in lesions at post-immunization days 14 and 21. These results suggest that the increased expression of PLD1 in inflammatory cells exacerbates an autoimmune response in EAU, while its expression in ganglion cells limits cell loss by activation of survival factors in eyes with autoimmune injury.

Elevated nitric oxide level in aqueous humor of AIDS patients with cytomegalovirus retinitis.

Human cytomegalovirus (HCMV) retinitis is the most common ocular opportunistic infection in AIDS. It often leads to blindness if left untreated. The questions as to how HCMV infection causes retinal immunopathogenesis and visual destruction in AIDS patients have not been completely established. Here we reported that the nitric oxide (NO) levels in aqueous humor samples in 10 AIDS patients with CMV retinitis (104.3 +/- 27.1 microM) were higher than the levels in 7 AIDS patients without CMV retinitis (36.1 +/- 10.4 micro M; p < 0.001). After ganciclovir treatment, the NO level in the vitreous body of 5 patients declined dramatically (53.4 +/- 11.8 micro M). By using immunohistochemistry assay, we found that the aggregates of macrophages infiltrated in the CMV-infected retina of 4 AIDS patients. Moreover, the expression of inducible-form NO synthase was detected in the infected retina of these patients. These results suggest that NO production in the eye may play a fundamental role in the immunopathogenesis of AIDS patients with CMV retinitis.

The matrix metalloproteinase inhibitor BB-1101 prevents experimental autoimmune uveoretinitis (EAU).

EAU is characterized by breakdown of the blood-retinal barrier and extravasation of leucocytes into retinal tissue leading to destruction of photoreceptor cells. Matrix metalloproteinases (MMP) have been implicated in trafficking of cells into tissues, but their role in inflammatory eye disease is unclear. A synthetic MMP inhibitor, BB-1101, was administered subcutaneously, from either day 0 or day 7, to Lewis rats challenged with bovine S-antigen to induce EAU. When given up to day 14, BB-1101 reduced the incidence of disease and delayed the day of onset of clinical disease. When administered from day 7 until day 21, EAU was completely abrogated. A quantitative polymerase chain reaction (PCR) assay showed an increase of both matrilysin (MMP-7), neutrophil collagenase (MMP-8) and macrophage metalloproteinase (MMP-12) in retinas from EAU animals compared with naive controls. These enzymes are produced by activated leucocytes and act on components of the basement membrane. These results therefore implicate these MMP as integral to the development of pathology in EAU.

Differential expression of nitric oxide synthase in experimental uveoretinitis.

PURPOSE: To investigate the site and the cellular source of inducible nitric oxide synthase (iNOS) expression in human S-antigen peptide-induced experimental autoimmune uveoretinitis (EAU). METHODS: Twenty-one Lewis rats were sensitized with human S-antigen peptides. Three rats were killed each consecutive day from day 6 through day 12 after sensitization. Frozen sections of the enucleated eyes were analyzed for iNOS by the dual immunohistochemical method. Primary antibodies included rabbit anti-mouse iNOS combined with anti-human endothelium NOS, anti-rat lysosomal protein (ED1), or anti-rat major histocompatibility complex class II molecule (OX6) monoclonal antibodies. Secondary antibodies were fluorescein-conjugated anti-mouse IgG and streptavidin rhodamine-labeled anti-rabbit IgG. The adjacent sections were separately stained with ED1, iNOS, and glial fibrillary acidic protein (GFAP). The mouse macrophage cell line RAW 264.7 was exposed to either interferon (IFN)gamma/lipopolysaccharide (LPS) or S-antigen and to interphotoreceptor retinoid-binding protein (IRBP), myelin basic protein, and bovine serum albumin for 12 hours. Cells were harvested for detection of iNOS expression by northern blot analysis hybridization and detection of protein by immunohistochemistry. RESULTS: In the retina of eyes with EAU, ED1+/iNOS+ and OX6+/iNOS+ cells were first detected on day 9 after sensitization. These iNOS+ cells increased in number on subsequent days in parallel with the increasing severity of retinal damage. Most of the cells localized around the outer retina. In contrast, a large number of ED1+ and OX6+ cells that were localized in the uvea and conjunctiva were negative for iNOS. Retinal pigment epithelial cells did not stain for iNOS. Macrophages exposed to IFNgamma/LPS, S-antigen, and IRBP showed expression of iNOS mRNA and the protein. CONCLUSIONS: Macrophages are an important source of NO production in eyes with EAU. These macrophages preferentially express iNOS in the retina. Such a differential expression of iNOS by the macrophages appears to be related to retinal soluble proteins.

Retinal expression of a neo-self antigen, beta-galactosidase, is not tolerogenic and creates a target for autoimmune uveoretinitis.

Recent studies revealing active mechanisms of immune privilege in neural tissues have diminished the putative role of passive tolerance. To examine the significance of Ag localization in the retina on immune privilege, the immune responses of transgenic mice expressing high and low levels of beta-galactosidase (beta-gal) in the photoreceptor cells of the retina were compared with those of normal mice and those of mice expressing moderate levels of beta-gal systemically. Immunization with beta-gal induced experimental autoimmune uveoretinitis indistinguishable from that induced by known photoreceptor cell autoantigens, including destruction of photoreceptor cells, in transgenic mice with high level retinal expression. Retinal expression had no apparent effect on the immune responses to beta-gal, showing that tolerance was not elicited by levels of retinal beta-gal sufficient to serve as a target for autoimmune disease. Mice with systemic expression exhibited reduced lymphoproliferative responses following immunization with beta-gal and did not develop autoimmune disease. T cells prepared from normal mice immunized with beta-gal transferred experimental autoimmune uveoretinitis to the transgenic mice with high level retinal beta-gal expression, but no disease was found in mice with systemic transgene expression under these conditions. The results of our experiments are most consistent with sequestration being the primary mechanism of retinal immune privilege. The results also show that beta-gal can serve as an immunopathogenic neural autoantigen, and that T cells raised by immunization of normal mice with a foreign Ag can be immunopathogenic in certain transgenic recipients.

Systemic murine cytomegalovirus infection of mice with retrovirus-induced immunodeficiency results in ocular infection but not retinitis.

Experiments were performed to explore the ability of murine cytomegalovirus (MCMV) to disseminate to the eye following intravenous inoculation and to cause infection of ocular tissues and necrotizing retinitis in C57BL/6 mice with retrovirus-induced immunodeficiency syndrome (MAIDS). Although infectious virus could be detected in whole eye homogenates of mice with MAIDS at 10 days after intravenous MCMV inoculation, a recombinant MCMV (RM461) that carries an MCMV IE1 promoter-LacZ insert was used as a tracer virus to confirm direct infection of ocular tissues. Evidence for MCMV replication (determined by RM461-induced expression of beta-galactosidase) was consistently observed in the nonpigmented epithelium of the ciliary body of the eyes of MAIDS animals at 14 days after infection. In sharp contrast, however, the neurosensory retina was spared and necrotizing retinitis failed to develop. These findings demonstrate that systemic MCMV infection of mice with MAIDS results in ocular MCMV infection without development of ocular MCMV disease. Conversion of occult subclinical MCMV infection of ocular tissues to overt clinical MCMV retinitis may require as yet unidentified cofactor(s). Identification of these cofactors could lead to more innovative therapeutic approaches for prevention and/or treatment of CMV retinitis in patients with AIDS.

Nitric oxide accelerates the onset and increases the severity of experimental autoimmune uveoretinitis through an IFN-gamma-dependent mechanism.

The production of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been described as a double-edged sword eliciting pro- or anti-inflammatory effects in different immune situations. Our aim, therefore, was to investigate its role in experimental autoimmune uveoretinitis (EAU), a model of ocular inflammation, induced in the Lewis rat following a single footpad injection of retinal Ags. iNOS enzyme was not detected in the normal Lewis rat eye, but was strongly expressed by infiltrating ED1+ macrophages during the acute inflammatory stages of EAU. Treating immunized animals with L-arginine increased urinary NO metabolite (NOx) levels, accelerated the inflammatory response, and increased disease severity, whereas treatment with the NOS inhibitor, N(G)-nitro-L-arginine methyl ester, reduced NOx excretion, delayed the onset, and reduced the clinical signs of EAU. Reverse transcription-PCR analysis of ocular tissue from untreated and treated animals detected iNOS mRNA at all stages of disease, and expression was up-regulated during peak disease. L-arginine treatment enhanced cytokine mRNA expression, particularly of IFN-gamma, which was detected earlier than in control animals, corresponding with the more rapid onset of disease and increased disease severity observed in this group. N(G)-nitro-L-arginine methyl ester had little or no effect on iNOS or inflammatory cytokine mRNA expression. These results suggest NO is central to the pathogenesis of EAU and highlight the importance of the macrophage as an effector cell in what is considered a CD4+ T cell-dependent disease. Furthermore, this study demonstrates the therapeutic potential of NOS inhibitors in the treatment of inflammatory and autoimmune mediated disease.

Localisation of macrophage lysosomal enzyme in experimental toxoplasma retinitis.

Rabbit retinal tissue experimentally infected with Toxoplasma gondii was processed for the lysosomal enzyme aryl sulphatase. Abundant lysosomal activity was found in lysosomal bodies of the infected macrophages. There appeared to be a lack of fusion of the lysosomal bodies with the phagosomes containing the organisms. Examination of the majority of macrophage vacuoles containing trophozoites failed to show consistently lead sulphide deposition for aryl sulphatase activity. By light microscopy 83% of 115 macrophage vacuoles containing the trophozoites of T. gondii showed an absence of lysosomal enzyme activity; 7% of the vacuole containing the trophozoites were found to contain lysosomal enzyme activity. In the remaining 10% of the vacuoles containing the trophozoites of T. gondii the presence or absence of lysosomal enzyme activity could not be determined with certainty. The frequent absence of lysosomal enzyme activity within the phagosomes containing T. gondii organisms may be related to the parasite's ability to multiply and encyst in an intracellular locus. The abundant lysosomal enzyme activity in the lysosomal bodies within the cytoplasm of the infected macrophages may contribute to the cellular destruction of surrounding tissues when infected macrophages burst open owing to proliferation of the trophozoites.