Neuropeptide α-Melanocyte-Stimulating Hormone Promotes Neurological Recovery and Repairs Cerebral Ischemia/Reperfusion Injury in Type 1 Diabetes.

Persons with type 1 diabetes have an increased risk of stroke compared with the general population. α-Melanocyte-stimulating hormone (α-MSH) is a neuropeptide that has protective effects against ischemia/reperfusion (I/R) induced organ damages. In this study, we aimed to investigate the neuroprotective role of this peptide on I/R induced brain damage after experimental stroke associated with hyperglycemia using C57BL/6J Ins2Akita/+ mice. Experimental stroke was induced by blocking the right middle cerebral artery for 2 h with reperfusion for 2 and 22 h, respectively using the intraluminal method. Animals were treated intraperitoneally with or without α-MSH at 1 h after ischemia and 1 h after reperfusion. Significantly higher survival rate and lower neurological scores were recorded in animals injected with α-MSH. Similarly, neuron death, glial cells activation as well as oxidative and nitrosative stress were significantly decreased in α-MSH treated group. Relative intensities of matrix metallopeptidases 9, cyclooxygenase 2 and nuclear factor-κB were significantly decreased while intensities of Akt, heme oxygenase (HO) 1, HO-2 and B-cell lymphoma 2 were significantly increased after α-MSH treatment. In addition, gene expressions of monocarboxylate transporter (MCT) 1, MCT-2 and activity-regulated cytoskeleton-associated protein were significantly higher in brain samples treated with α-MSH, suggesting this peptide may have role in neuron survival by an involvement of lactate metabolism. In conclusion, α-MSH is neuroprotective under hyperglycemic condition against I/R induced brain damage by its anti-inflammatory, anti-oxidative and anti-apoptotic properties. The use of α-MSH analogues may be potential therapeutic agents for diabetic stroke.

The Role of Retinal Pigment Epithelial Cells in Regulation of Macrophages/Microglial Cells in Retinal Immunobiology.

The ocular tissue microenvironment is immune privileged and uses several mechanisms of immunosuppression to prevent the induction of inflammation. Besides being a blood-barrier and source of photoreceptor nutrients, the retinal pigment epithelial cells (RPE) regulate the activity of immune cells within the retina. These mechanisms involve the expression of immunomodulating molecules that make macrophages and microglial cells suppress inflammation and promote immune tolerance. The RPE have an important role in ocular immune privilege to regulate the behavior of immune cells within the retina. Reviewed is the current understanding of how RPE mediate this regulation and the changes seen under pathological conditions.

Extracellular Soluble Membranes from Retinal Pigment Epithelial Cells Mediate Apoptosis in Macrophages.

A central characterization of retinal immunobiology is the prevention of proinflammatory activity by macrophages. The retinal pigment epithelial cells (RPEs) are a major source of soluble anti-inflammatory factors. This includes a soluble factor that induces macrophage apoptosis when the activity of the immunomodulating neuropeptide alpha-melanocyte-stimulating hormone (α-MSH) is neutralized. In this manuscript, isolated extracellular soluble membranes (ESMs) from primary RPE were assayed to see if they could be the soluble mediator of apoptosis. Our results demonstrated that RPE ESMs mediated the induction of macrophage apoptosis that was suppressed by α-MSH. In contrast, the RPE line ARPE-19, cultured under conditions that induce similar anti-inflammatory activity to primary RPEs, did not activate apoptosis in the macrophages. Moreover, only the ESMs from primary RPE cultures, and not those from the ARPE-19 cell cultures, expressed mFasL. The results demonstrate that RPE ESMs are a soluble mediator of apoptosis and that this may be a mechanism by which the RPEs select for the survival of α-MSH-induced suppressor cells.

Association of α-Melanocyte-Stimulating Hormone With Corneal Endothelial Cell Survival During Oxidative Stress and Inflammation-Induced Cell Loss in Donor Tissue.

Importance: Corneal endothelial cell (CEnC) damage and loss are major issues in eye banking and transplantation. The underlying mechanisms for CEnC loss are incompletely understood, and cytoprotective strategies that enhance CEnC viability could have a major effect on donor tissue quality and graft survival. Objective: To investigate the cytoprotective role of neuropeptide α-melanocyte-stimulating hormone (α-MSH) in preventing CEnC loss in eye bank cold-stored corneas under oxidative and inflammatory cytokine-induced stress. Design, Setting, and Participants: This single-center comparative research study conducted ex vivo experiments using 16 pairs of research-grade human donor corneas (courtesy of Eversight Eye Bank). Data were collected from June 2018 to November 2019, and data were analyzed from December 2019 to January 2020. Exposures: Two corneas from the same donor were randomized to either control or 0.1 mmol/L of α-MSH treatment and then subjected to oxidative stress (1.4 mmol/L of hydrogen peroxide-phosphate-buffered saline for 15 minutes at 37 °C; n = 8 pairs) or cytokine-induced stress (100 ng/mL of tumor necrosis factor-α and 100 ng/mL of interferon γ for 18 hours at 37 °C; n = 8 pairs). Corneas were then stored at 4 °C. Specular images were taken at baseline and repeated twice per week using a calibrated wide-field specular microscope. CEnC viability was assessed using a fluorescent live/dead viability assay. Main Outcome and Measures: Endothelial morphometry analysis, central corneal thickness measurements, and percentage of dead cells at day 11. Results: Of 16 donors who provided corneas, 9 (56%) were male, and the mean (SD) age was 57.9 (12.4) years. Corneas were paired, and baseline parameters were comparable between all groups. At all time points, CEnC loss was lower in the α-MSH groups compared with the control groups. This difference was statistically significant after cytokine-induced stress (20.2% vs 35.2%; sample estimate of median, -14.9; 95% CI, -23.6 to -6.3; P = .008). Compared with the control group, α-MSH treatment resulted in a smaller increase in central corneal thickness (cytokine-induced stress: 89.3 µm vs 169.8 µm; sample estimate of median, -84.9; 95% CI, -131.5 to -41.6; P = .008; oxidative stress: 43.6 µm vs 111.9 µm; sample estimate of median, -68.8; 95% CI, -100.0 to -34.5; P = .008) and a smaller proportion of cell death (cytokine-induced stress: 2.7% vs 10.4%; difference, -7.7; 95% CI, -13.1 to -2.4; P = .01; oxidative stress: 2.9% vs 12.4%; difference, 9.5; 95% CI, 5.1 to 13.9; P = .006). Conclusions and Relevance: In this study, α-MSH treatment attenuated CEnC loss during cold storage after acute oxidative and cytokine-induced stress in human eye bank cold-stored corneas. These data suggest that supplementation of corneal storage solution with α-MSH may positively affect CEnC survival after transplant and protect the endothelium from proinflammatory cytokines and oxidative stress after full-thickness or endothelial keratoplasty, which is particularly valuable in patients at high risk of graft failure.

Probing the Role of Melanocortin Type 1 Receptor Agonists in Diverse Immunological Diseases.

Background: The melanocortin α-melanocyte stimulating hormone (α-MSH), an endogenous peptide with high affinity for the melanocortin 1 receptor (MC1r), has demonstrated prevention and reversal of intestinal and ocular inflammation in animal models. Preclinical studies were performed to determine whether two MC1r receptor agonists, PL-8177 and PL-8331, exhibit actions and efficacy similar to α-MSH in preventing and reversing intestinal and ocular inflammation. Methods: Both PL-8177 and PL-8331 were assessed in a Eurofins LeadProfilingScreen selectivity panel including 72 in vitro assays. PL-8177 and PL-8331 were evaluated in an in vitro assay using human whole blood stimulated by lipopolysaccharide to determine inhibition of tumor necrosis factor alpha (TNF-α); for comparison, adrenocorticotropic hormone (ACTH) and α-MSH were used as positive controls. PL-8177, dosed at 0.5, 1.5, and 5.0 µg, was assessed in a cannulated rat model of dinitrobenzene sulfonic acid (DNBS)-induced bowel inflammation versus vehicle and oral sulfasalazine. PL-8177 was also dosed at 0.3 mg/kg/mouse injected intraperitoneally versus untreated controls and α-MSH treatment in mice with experimental autoimmune uveitis (EAU). PL-8331 at 3 doses, 3 times daily, was evaluated in a murine model of scopolamine-induced dry eye disease (SiccaSystemTM model), versus twice-daily Restasis® and Xiidra®. Results: Both PL-8177 and PL-8331 demonstrated no significant activity at the 1 µm concentration in any of the 72 in vitro assays. PL-8177 and PL-8331 inhibited lipopolysaccharide-induced TNF-α to a similar degree as ACTH and α-MSH. In the DNBS rat model of bowel inflammation, PL-8177 was significantly superior to untreated controls at all 3 doses (P < 0.05) in reducing bowel inflammation parameters, with effects similar to sulfasalazine. In the murine EAU model, PL-8177 significantly reduced retinal inflammation scores versus untreated controls (P = 0.0001) over 3-5 weeks, and to a similar degree as α-MSH. In the murine scopolamine-induced model of dry eye disease, PL-8331 reduced corneal fluorescein staining scores at all doses, significantly (P = 0.02) for the highest dose (1 × 10-5 mg⋅mL-1), and similarly to Restasis®; Xiidra® demonstrated no effect. Conclusion: The MC1r receptor agonists PL-8177 and PL-8331 exhibited actions similar to those of α-MSH in preventing and reversing intestinal and ocular inflammation in preclinical disease models.

Retinal Pigment Epithelial Cells Suppress Phagolysosome Activation in Macrophages.

Purpose: The eye is an immune-privileged microenvironment that has adapted several mechanisms of immune regulation to prevent inflammation. One of these potential mechanisms is retinal pigment epithelial cells (RPE) altering phagocytosis in macrophages. Methods: The conditioned media of RPE eyecups from eyes of healthy mice and mice with experimental autoimmune uveitis (EAU) were used to treat primary macrophage phagocytizing pHrodo bacterial bioparticles. In addition, the neuropeptides were depleted from the conditioned media of healthy RPE eyecups and used to treat phagocytizing macrophages. The conditioned media from healthy and EAU RPE eyecups were assayed for IL-6, and IL-6 was added to the healthy conditioned media, and neutralized in the EAU conditioned media. The macrophages were treated with the conditioned media and assayed for fluorescence. The macrophages were imaged, and the fluorescence intensity, relative to active phagolysosomes, was measured. Also, the macrophages were assayed using fluorescent viability dye staining. Results: The conditioned media from healthy, but not from EAU RPE eyecups suppressed phagolysosome activation. Depletion of the neuropeptides alpha-melanocyte-stimulating hormone and neuropeptide Y from the healthy RPE eyecup conditioned media resulted in macrophage death. In the EAU RPE eyecup conditioned media was 0.96 ± 0.18 ng/mL of IL-6, and when neutralized the conditioned media suppressed phagolysosome activation. Conclusions: The healthy RPE through soluble molecules, including alpha-melanocyte-stimulating hormone and neuropeptide Y, suppresses the activation of the phagolysosome in macrophages. In EAU, the IL-6 produced by the RPE promotes the activation of phagolysosomes in macrophages. These results demonstrate that under healthy conditions, RPE promotes an altered pathway of phagocytized material in macrophages with implications on antigen processing and clearance.

MC5r and A2Ar Deficiencies During Experimental Autoimmune Uveitis Identifies Distinct T cell Polarization Programs and a Biphasic Regulatory Response.

Autoantigen-specific regulatory immunity emerges in the spleen of mice recovering from experimental autoimmune uveitis (EAU), a murine model for human autoimmune uveoretinitis. This regulatory immunity provides induced tolerance to ocular autoantigen, and requires melanocortin 5 receptor (MC5r) expression on antigen presenting cells with adenosine 2 A receptor (A2Ar) expression on T cells. During EAU it is not well understood what roles MC5r and A2Ar have on promoting regulatory immunity. Cytokine profile analysis during EAU revealed MC5r and A2Ar each mediate distinct T cell responses, and are responsible for a functional regulatory immune response in the spleen. A2Ar stimulation at EAU onset did not augment this regulatory response, nor bypass the MC5r requirement to induce regulatory immunity. The importance of this pathway in human autoimmune uveitis was assayed. PBMC from uveitis patients were assayed for MC5r expression on monocytes and A2Ar on T cells, and comparison between uveitis patients and healthy controls had no significant difference. The importance for MC5r and A2Ar expression in EAU to promote the induction of protective regulatory immunity, and the expression of MC5r and A2Ar on human immune cells, suggests that it may be possible to utilize the melanocortin-adenosinergic pathways to induce protective immunity in uveitic patients.

Recovery from experimental autoimmune uveitis promotes induction of antiuveitic inducible Tregs.

The recovery of EAU, a mouse model of endogenous human autoimmune uveitis, is marked with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. This regulatory immunity is mediated by a melanocortin-driven suppressor APC that presents autoantigen and uses adenosine to activate an antigen-specific CD4(+) Tregs through the A2Ar. These cells are highly effective in suppressing uveitis, and they appear to be inducible Tregs. In this study, we determined whether they are inducible or natural Tregs and identified the dependent mechanism for the function of these post-EAU Tregs. The post-EAU spleen CD25(+)CD4(+) T cells were sorted for NRP-1 expression and transferred to recipient mice immunized for EAU. The sorted NRP-1(-), but not the NRP-1(+), Tregs suppressed EAU. These NRP-1(-) Tregs coexpress PD-1 and PD-L1. Treatment of naive APCs with α-MSH promoted a regulatory APC that induced CD25(+) CD4(+) Tregs in a CD73-dependent manner. These Tregs were PD-L1(+) PD-1(+) NRP-1(-) FOXP3(+) HELIOS(-) and suppressed EAU when transferred to recipient mice. In contrast, PD-1(-) T cells did not suppress EAU, indicating that PD-1 is necessary for the suppressive activity of iTregs. Moreover, these Tregs did not suppress effector T cells when the PD/-1/PD-L1 pathway was blocked. These results demonstrate that post-EAU Tregs are inducible Tregs, which use a PD-1/PD-L1 mechanism to suppress disease.

Influence of subretinal fluid in advanced stage retinopathy of prematurity on proangiogenic response and cell proliferation.

PURPOSE: The clinical phenotype of advanced stage retinopathy of prematurity (ROP, stages 4 and 5) cannot be replicated in an animal model. To dissect the molecular events that can lead up to advanced ROP, we examined subretinal fluid (SRF) and surgically dissected retrolental membranes from patients with advanced ROP to evaluate its influences on cell proliferation, angiogenic properties, and macrophage polarity. METHODS: We compared our findings to SRF collected from patients with uncomplicated rhegmatogenous retinal detachment (RD) without proliferative vitreoretinopathy and surgically dissected epiretinal membrane from eyes with macular pucker. All subretinal fluid samples were equalized for protein. The angiogenic potential of SRF from ROP eyes was measured using a combination of capillary cord formation in a fibrin clot assay, and its proliferative effect was tested with a DNA synthesis of human retinal microvascular endothelial cells. Findings were compared with SRF collected from participants with uncomplicated rhegmatogenous RD without proliferative vitreoretinopathy. The ability of SRF to induce nitric oxide production was measured in vitro using murine J774A.1 macrophages. Cytokine profiles of SRF from ROP and RD eyes were measured using a multienzyme-linked immunosorbent assay (ELISA). Fluorescent immunohistochemistry of retrolental membranes from ROP was performed to detect the presence of leukocytes and the composition of tissue macrophages using markers for M1 and M2 differentiation. RESULTS: The cytokine composition in SRF revealed that in ROP, not only were several proangiogenic factors were preferentially elevated but also the profile of proinflammatory factors was also increased compared to the RD eyes. SRF from ROP eyes supported cell proliferation and endothelial cord formation while SRF from RD eyes had inhibitory effects. SRF from eyes with ROP but not RD robustly induced nitric oxide production in macrophages. Furthermore, fluorescent immunostaining revealed a preponderance of M1 over M2 macrophages in retrolental fibrous membranes from ROP eyes. The cytokine profile and biologic properties of SRF in ROP promote a proangiogenic environment, which supports the maintenance and proliferation of fibrous membranes associated with advanced stages of ROP. In contrast, SRF from RD eyes exhibits a suppressive environment for endothelial cell proliferation and angiogenesis. CONCLUSIONS: Our investigation demonstrates that the microenvironment in advanced ROP eyes is proangiogenic and proinflammatory. These findings suggest that management of advanced ROP should not be limited to the surgical removal of the fibrovascular membranes and antiangiogenic therapy but also directed to anti-inflammatory therapy and to promote M2 activation over M1 activity.

Both MC5r and A2Ar are required for protective regulatory immunity in the spleen of post-experimental autoimmune uveitis in mice.

The ocular microenvironment uses a poorly defined mela5 receptor (MC5r)-dependent pathway to recover immune tolerance following intraocular inflammation. This dependency is seen in experimental autoimmune uveoretinitis (EAU), a mouse model of endogenous human autoimmune uveitis, with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. In this study, we found that the MC5r-dependent regulatory immunity increased CD11b(+)F4/80(+)Ly-6C(low)Ly-6G(+)CD39(+)CD73(+) APCs in the spleen of post-EAU mice. These MC5r-dependent APCs require adenosine 2A receptor expression on T cells to activate EAU-suppressing CD25(+)CD4(+)Foxp3(+) regulatory T cells. Therefore, in the recovery from autoimmune disease, the ocular microenvironment induces tolerance through a melanocortin-mediated expansion of Ly-6G(+) regulatory APCs in the spleen that use the adenosinergic pathway to promote activation of autoantigen-specific regulatory T cells.

Alpha-melanocyte stimulating hormone (α-MSH) is a post-caspase suppressor of apoptosis in RAW 264.7 macrophages.

The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.

The neuropeptides α-MSH and NPY modulate phagocytosis and phagolysosome activation in RAW 264.7 cells.

Within the immunosuppressive ocular microenvironment, there are constitutively present the immunomodulating neuropeptides alpha-melanocyte stimulating hormone (α-MSH) and neuropeptide Y (NPY) that promote suppressor functionality in macrophages. In this study, we examined the possibility that α-MSH and NPY modulate phagocytic activity in macrophages. The macrophages treated with α-MSH and NPY were significantly suppressed in their capacity to phagocytize unopsonized Escherichia coli and Staphylococcus aureus bioparticles, but not antibody-opsonized bioparticles. The neuropeptides significantly suppressed phagolysosome activation, and the FcR-associated generation of reactive oxidative species as well. This suppression corresponds to neuropeptide modulation of macrophage functionality within the ocular microenvironment to suppress the activation of immunogenic inflammation.

Localized retinal neuropeptide regulation of macrophage and microglial cell functionality.

The functionality of immune cells is manipulated within the ocular microenvironment to protect the sensitive and non-regenerating light-gathering tissue from the collateral damage of inflammation. This is mediated partly by the constitutive presence of immunomodulating neuropeptides. Treating primary resting macrophages with soluble factors produced by the posterior eye induced co-expression of Arginase1 and NOS2. The neuropeptides alpha-melanocyte stimulating hormone and Neuropeptide Y alternatively activated the macrophages to co-express Arginase1 and NOS2 like myeloid suppressor cells. Similar co-expressing cells were found within healthy, but not in wounded retinas. Therefore, the healthy retina regulates macrophage functionality to the benefit of ocular immune privilege.

Inflammatory cytokines in eyes with uveal melanoma and relation with macrophage infiltration.

PURPOSE: The presence of an inflammatory phenotype, characterized by an increased expression of HLA antigens and an immunologic infiltrate, carries a bad prognosis in uveal melanoma. This study was conducted to determine whether the aqueous humor (AqH) from eyes with uveal melanoma contains inflammatory cytokines and whether their presence is associated with inflammation. METHODS: Immediately after enucleation, AqH was obtained from 37 eyes containing uveal melanoma. Samples were stored at -80°C until use. Fifteen different cytokines were measured with a multiplex bead array. Intratumoral macrophages were analyzed by immunohistochemistry and immunofluorescence staining. The presence of specific cytokines was compared with histopathologic, genetic, and clinical tumor characteristics, as well as patient survival. RESULTS: Several cytokines showed significantly higher expression in the AqH of uveal melanoma-containing eyes than in the AqH of eyes undergoing cataract surgery. MCP-3 was associated with the presence of CD68(+) macrophages. Correlations were found between some cytokine levels and a few known prognostic factors of uveal melanoma, but cytokine levels were not of predictive value for survival. CONCLUSIONS: Uveal melanoma-containing eyes often carry increased levels of inflammation-related cytokines in their AqH. However, the presence of most specific cytokines was not related to the presence of macrophages, clinical or histopathologic parameters, or prognosis.

The immune privileged retina mediates an alternative activation of J774A.1 cells.

PURPOSE: We have previously found that retinal pigment epithelial (RPE) cells suppressed endotoxin-stimulated macrophages; moreover, it induced expression of anti-inflammatory cytokines. We further assessed the possibility that the RPE is alternatively activating macrophages. METHODS: J774A.1 cells were stimulated with endotoxin and treated with the conditioned media (CM) of RPE, or neuroretinal eyecups from healthy mouse eyes. The supernatant was assayed for IL-1 beta, TNF-alpha, IL-6, IL-12(p70), and IL-10, and for nitric-oxide generation. The RPE conditioned media (RPE CM) was absorbed of known soluble factors to identify the factor that augments nitric-oxide generation. RESULTS: We found the RPE CM suppressed all cytokine production except IL-10, and augmented nitric-oxide generation. The augmented nitric-oxide levels were mediated by RPE derived alpha-melanocyte stimulating hormone (alpha-MSH). CONCLUSIONS: Healthy RPE not only suppresses inflammatory activity, it promotes an alternative activation of macrophages that can further promote immune privilege.

Diminishment of alpha-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages.

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is a powerful suppressor of inflammation mediated by macrophages, which express at least two receptors, melanocortin 1 and 3 receptors (MC1r and MC3r) that bind alpha-MSH. Albeit, the anti-inflammatory activity of alpha-MSH has been well documented in macrophages, the mechanisms of alpha-MSH activity in macrophages are not clearly understood. This study is to investigate which of the MCr expressed on macrophages is associated with the immunosuppressive activities of alpha-MSH on LPS-stimulated macrophages. To address this question, we transfected RAW264.7 macrophage cells with MC1r small interfering (si)RNA, which specifically targets mouse MC1r mRNA. The diminution of MC1r mRNA expression was 82% at 24 h and 67% at 48 h after transfection. There was a significant loss in alpha-MSH suppression of NO generation and TNF-alpha production by MC1r siRNA-transfected macrophages stimulated with LPS. There was an equally diminished alpha-MSH suppression of LPS-stimulated intracellular activation of NF-kappaB and p38 phosphorylation. In addition, the diminishment of MC1r expression by siRNA transfection had no influence on MC3r expression and function in the macrophages. These findings demonstrate that alpha-MSH suppression of LPS-induced inflammatory activity in macrophages requires expression of MC1r. The results imply that although all of the MCr are G-coupled proteins, they may not necessarily function through the same intracellular pathways in macrophages.

Effect of the ocular microenvironment in regulating corneal dendritic cell maturation.

OBJECTIVE: To determine whether the ocular anterior segment (aqueous humor and cornea) actively inhibits dendritic cell (DC) maturation. METHODS: Dendritic cells were injected into syngeneic corneas or conjunctivae, and their surface major histocompatibility complex class II expression in response to the local milieu was assessed using confocal microscopy. Immature DCs were cocultured with corneal supernatant or with aqueous humor to evaluate their regulation of DC phenotypic and functional maturity. RESULTS: In contrast to conjunctivally injected DCs, DCs injected into the cornea resisted up-regulation in expression of surface major histocompatibility complex class II. Corneal supernatant-treated and aqueous humor-treated DCs retained their immaturity, as reflected by high antigen uptake but low costimulatory molecule (CD80 and CD86) expression and poor T-cell stimulation. Anti-transforming growth factor beta(2) treatment of aqueous humor and of corneal supernatant led to complete and partial blockade of their inhibition of DC maturation, respectively. However, alpha-melanocyte-stimulating hormone and calcitonin gene-related peptide had no demonstrable effect on DC maturation. CONCLUSION: Cornea and aqueous humor, principally through transforming growth factor beta(2,) promote generation of phenotypically and functionally immature DCs. Clinical Relevance Our results indicate that relative immune quiescence in the cornea and in the anterior segment is actively maintained in part by the inhibitory effect of transforming growth factor beta(2) on resident DCs and by their suppression of T-cell-mediated immune and inflammatory responses.

Creating an immune-privileged site using retinal progenitor cells and biodegradable polymers.

We describe the creation of local immune privilege (IP) using retinal progenitor cells (RPCs) and biodegradable polymers. Murine RPCs were seeded on poly(lactic-coglycolic acid) polymers to generate composite grafts. Composites or RPCs alone were transplanted into allogeneic kidney capsules. Grafts survived at all time points, differentiating into neurons and astrocytes. Upon treatment with interferon gamma (IFNgamma), major histocompatibility complex antigens were upregulated. Although 10% of IFNgamma-treated RPC grafts survived 14 days, 66% of the IFNgamma-treated composites survived in part by producing immune suppressive factors transforming growth factor-beta2, Fas ligand, and indoleamine 2,3-dioxygenase. The composites were assayed for delayed-type hypersensitivity (DTH) by seeding composites with antigen-presenting cells incubated with ovalbumin. This resulted in suppression of ovalbumin-specific DTH, indicating that composite grafts consisting of biodegradable polymers and central nervous system progenitor cells can be used to generate local IP. This technology may be used to promote the survival of nonprivileged grafts (e.g., pancreas, liver, or skin). Disclosure of potential conflicts of interest is found at the end of this article.

Pigment epithelial growth factor suppresses inflammation by modulating macrophage activation.

PURPOSE: To study the contribution of murine retinal pigment epithelial (RPE) cells to the innate immune-privilege status of the subretinal space as determined by the ability of pigment epithelial-derived factor (PEDF) and somatostatin (SOM), produced by RPE, to regulate macrophage-mediated inflammation. METHODS: Serum-free medium was added to RPE eyecups (a healthy monolayer of RPE resting on choroid and sclera) and the supernatants were removed after 24 hours (RPE SN). The RPE SN was assayed for the presence of PEDF and SOM and for its ability to regulate interleukin (IL)-12, IL-10, and nitric oxide (NO) production by resting and activated macrophages. A group of mice received intradermal injection of lipopolysaccharide (LPS) and PEDF in one ear and LPS alone in the other ear. Ear thickness was measured before- and 24 hours after ear injections. RESULTS: Soluble factors present in the RPE SN inhibited IL-12 production and substantially increased IL-10 while having minimal effects on NO production by activated macrophages. The message for PEDF, SOM, and IL-10 was detected in RPE cells, and the protein for these factors was found in the RPE SN. The stimulation of IL-10 and suppression of IL-12 production by RPE-SN-treated macrophages was neutralized by anti-PEDF antibodies. Neutralization of SOM in the RPE SN, suppressed NO production by activated macrophages. Intradermal injection of PEDF substantially inhibited LPS-induced inflammatory response. CONCLUSIONS: PEDF inhibits LPS-driven macrophage activation in vitro and in vivo. By producing PEDF, the RPE contributes to innate immune privilege of the eye.

Thrombospondin plays a vital role in the immune privilege of the eye.

PURPOSE: The role of thrombospondin (TSP)-1 in TGF-beta activation and T-cell suppression was studied in the retinal pigment epithelial (RPE) cells, a monolayer of pigmented cells that line the subretinal space, an immune-privileged site in the eye. METHODS: Posterior eyecups were prepared by excising the anterior segment, lens, and retina from enucleated eyes of C57BL/6, thrombospondin-1 knockout (TSP-1KO), and TGF-beta2 receptor II double-negative (TGF-beta2 RII DN) mice, leaving behind a healthy monolayer of RPE resting on choroid and sclera. Serum-free medium was added to these RPE eyecups, and, after various time intervals, supernatants (SNs) were removed and tested. RESULTS: SNs of an ex vivo culture of RPE cells from C57BL/6 mice were shown to inhibit both antigen and anti-CD3 activation of T cells, partially due to constitutive production of TGF-beta and to the ability of RPE to activate the latent form of TGF-beta. Activation of TGF-beta was entirely dependent on TSP-1, also produced by RPE. SNs of RPE from TSP-1KO mice failed to inhibit T-cell activation. Ovalbumin (OVA)-specific delayed hypersensitivity (DH) was not impaired when OVA was injected either into the subretinal space or into the anterior chamber of TSP-1KO mice before OVA immunization. Moreover, experimental autoimmune uveoretinitis was significantly more intense in eyes of TSP-1KO mice and failed to undergo spontaneous resolution unlike wild-type mice. CONCLUSIONS: Production of both TSP-1 and active TGF-beta by RPE is essential to the creation and maintenance of immune privilege in the subretinal space and that the immune privilege limits the severity and duration of retinal inflammation due to autoimmunity.