The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critical for Corneal Endothelial Cell Protection and Graft Survival after Transplantation.

Corneal transplantation is the most common form of tissue transplantation. The success of corneal transplantation mainly relies on the integrity of corneal endothelial cells (CEnCs), which maintain tissue transparency by pumping out excess water from the cornea. After transplantation, the rate of CEnC loss far exceeds that seen with normal aging, which can threaten sight. The underlying mechanisms are poorly understood. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide that is constitutively found in the aqueous humor with both cytoprotective and immunomodulatory effects. The curent study found high expression of melanocortin 1 receptor (MC1R), the receptor for α-MSH, on CEnCs. The effect of α-MSH/MC1R signaling on endothelial function and allograft survival in vitro and in vivo was investigated using MC1R signaling-deficient mice (Mc1re/e mice with a nonfunctional MC1R). Herein, the results indicate that in addition to its well-known immunomodulatory effect, α-MSH has cytoprotective effects on CEnCs after corneal transplantation, and the loss of MC1R signaling significantly decreases long-term graft survival in vivo. In conclusion, α-MSH/MC1R signaling is critical for CEnC function and graft survival after corneal transplantation.

Stimulating the Melanocortin System in Uveitis and Diabetes Preserves the Structure and Anti-Inflammatory Activity of the Retina.

The endogenous neuropeptide α-Melanocyte Stimulating Hormone (α-MSH) is a potent suppressor of inflammation and has an essential role in maintaining the normal anti-inflammatory microenvironment of the retina. While the therapeutic use of α-MSH peptide in uveitis and diabetic retinopathy models has been demonstrated, its short half-life and instability limit its use as a therapeutic drug. A comparable analog, PL-8331, which has a stronger affinity to melanocortin receptors, longer half-life, and, so far, is functionally identical to α-MSH, has the potential to deliver melanocortin-based therapy. We examined the effects of PL-8331 on two mouse models of retinal disease, Experimental Autoimmune Uveoretinitis (EAU) and Diabetic Retinopathy (DR). PL-8331 therapy applied to mice with EAU suppressed EAU and preserved retinal structures. In diabetic mice, PL-8331 enhanced the survival of retinal cells and suppressed VEGF production in the retina. In addition, retinal pigment epithelial cells (RPE) from PL-8331-treated diabetic mice retained normal anti-inflammatory activity. The results demonstrated that the pan-melanocortin receptor agonist PL-8331 is a potent therapeutic drug to suppress inflammation, prevent retinal degeneration, and preserve the normal anti-inflammatory activity of RPE.

Melanocortin receptor agonists suppress experimental autoimmune uveitis.

The melanocortin system plays an essential role in the regulation of immune activity. The anti-inflammatory microenvironment of the eye is dependent on the expression of the melanocortin-neuropeptide alpha-melanocyte stimulating hormone (α-MSH). In addition, the melanocortin system may have a role in retinal development and retinal cell survival under conditions of retinal degeneration. We have found that treating experimental autoimmune uveitis (EAU) with α-MSH suppresses retinal inflammation. Also, this augmentation of the melanocortin system promotes immune tolerance and protection of the retinal structure. The benefit of α-MSH-therapy appears to be dependent on different melanocortin receptors. Therefore, we treated EAU mice with α-MSH-analogs with different melanocortin-receptor targets. This approach demonstrated which melanocortin-receptors suppress inflammation, preserve retinal structure, and induce immune tolerance in uveitis. At the chronic stage of EAU the mice were injected twice 1 day apart with 50 µg of α-MSH or an α-MSH-analog. The α-MSH-analogs were a pan-agonist PL8331, PL8177 (potent MC1r-only agonist), PL5000 (a pan-agonist with no MC5r functional activity), MT-II (same as PL5000) and PG901 (MC5r agonist, but also an antagonist to MC3r, and MC4r). Clinical EAU scores were measured until resolution in the α-MSH-treated mice, when the eyes were collected for histology, and spleen cells collected for retinal-antigen-stimulated cytokine production. Significant suppression of EAU was seen with α-MSH or PL8331 treatment. This was accompanied with significant preservation of retinal structure. A similar effect was seen in EAU-mice that were treated with PL8177, except the suppression of EAU was temporary. In EAU mice treated with PL5000, MTII, or PG901, there was no suppression of EAU with a significant loss in whole retina and outer-nuclear layer thickness. There was significant suppression of IL-17 with induction of IL-10 by retinal-antigen stimulated spleen T cells from EAU mice treated with α-MSH, PL8331, PL8177, or PL5000, but not from EAU mice treated with MT-II, or PG901. Our previous studies show the melanocortin system's importance in maintaining ocular immune privilege and that α-MSH-treatment accelerates recovery and induces retinal-antigen-specific regulatory immunity in EAU. Our current results show that this activity is centered around MC1r and MC5r. In addition, the results suggest that a therapeutic potential to target MC1r and MC5r together to suppress uveitis induces regulatory immunity with potentially maintaining a normal retinal structure.

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.

Re-evaluating the treatment of acute optic neuritis.

Clinical case reports and prospective trials have demonstrated a reproducible benefit of hypothalamic-pituitary-adrenal (HPA) axis modulation on the rate of recovery from acute inflammatory central nervous system (CNS) demyelination. As a result, corticosteroid preparations and adrenocorticotrophic hormones are the current mainstays of therapy for the treatment of acute optic neuritis (AON) and acute demyelination in multiple sclerosis.Despite facilitating the pace of recovery, HPA axis modulation and corticosteroids have failed to demonstrate long-term benefit on functional recovery. After AON, patients frequently report visual problems, motion perception difficulties and abnormal depth perception despite 'normal' (20/20) vision. In light of this disparity, the efficacy of these and other therapies for acute demyelination require re-evaluation using modern, high-precision paraclinical tools capable of monitoring tissue injury.In no arena is this more amenable than AON, where a new array of tools in retinal imaging and electrophysiology has advanced our ability to measure the anatomic and functional consequences of optic nerve injury. As a result, AON provides a unique clinical model for evaluating the treatment response of the derivative elements of acute inflammatory CNS injury: demyelination, axonal injury and neuronal degeneration.In this article, we examine current thinking on the mechanisms of immune injury in AON, discuss novel technologies for the assessment of optic nerve structure and function, and assess current and future treatment modalities. The primary aim is to develop a framework for rigorously evaluating interventions in AON and to assess their ability to preserve tissue architecture, re-establish normal physiology and restore optimal neurological function.

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.

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.

Alpha-Melanocyte-Stimulating Hormone Maintains Retinal Homeostasis after Ischemia/Reperfusion.

Augmenting the natural melanocortin pathway in mouse eyes with uveitis or diabetes protects the retinas from degeneration. The retinal cells are protected from oxidative and apoptotic signals of death. Therefore, we investigated the effects of a therapeutic application of the melanocortin alpha-melanocyte-stimulating hormone (α-MSH) on an ischemia and reperfusion (I/R) model of retinal degenerative disease. Eyes were subjected to an I/R procedure and were treated with α-MSH. Retinal sections were histopathologically scored. Also, the retinal sections were immunostained for viable ganglion cells, activated Muller cells, microglial cells, and apoptosis. The I/R caused retinal deformation and ganglion cell loss that was significantly reduced in I/R eyes treated with α-MSH. While α-MSH treatment marginally reduced the number of GFAP-positive Muller cells, it significantly suppressed the density of Iba1-positive microglial cells in the I/R retinas. Within one hour after I/R, there was apoptosis in the ganglion cell layer, and by 48 h, there was apoptosis in all layers of the neuroretina. The α-MSH treatment significantly reduced and delayed the onset of apoptosis in the retinas of I/R eyes. The results demonstrate that therapeutically augmenting the melanocortin pathways preserves retinal structure and cell survival in eyes with progressive neuroretinal degenerative disease.

The central melanocortin system as a treatment target for obesity and diabetes: A brief overview.

The melanocortins are derived from proopiomelanocortin (POMC) and include three forms of melanocyte-stimulating hormone (α-, ß-, γ-, MSH) and adrenocorticotropic hormone. α-MSH, a potent POMC-derived neuropeptide, binds to melanocortin 4 receptor (MC4R) in the brain to reduce food intake (via appetite suppression) and increase energy expenditure (via sympathetic nervous system) after integration of central neuronal signal (e.g. serotonin, glutamate) and peripheral signals such as anorexigenic hormones (e.g. leptin, insulin) and nutrient (e.g. glucose). Mutations in POMC or MC4R can cause increase in food intake and body weight. Weight gain and obesity in turn result in a phenotypic switch of white adipose tissue, which then secretes proinflammatory cytokines that play a role in the development of insulin resistance and type 2 diabetes. Besides α-MSH's effects in decreasing food intake and body weight, α-MSH also carries protective anti-inflammatory properties in both immune cells and non-immune cells (e.g. adipocyte) that express melanocortin receptors. Since type 2 diabetic patients who have overweight or obese are recommended to lose body weight while current available anti-obesity drugs have various side effects, α-MSH-based therapeutics might be hopeful for the management of both obesity and type 2 diabetes.

Anti-inflammatory α-Melanocyte-Stimulating Hormone Protects Retina After Ischemia/Reperfusion Injury in Type I Diabetes.

Retinal ischemia/reperfusion (I/R) injury is a major cause of vision loss in many ocular diseases. Retinal I/R injury is common in diabetic retinopathy, which as a result of hyperglycemia damages the retina and can cause blindness if left untreated. Inflammation is a major contributing factor in the pathogenesis of I/R injury. α-Melanocyte-stimulating hormone (α-MSH) is an anti-inflammatory peptide hormone that has displayed protective effects against I/R-induced organ damages. Here, we aimed to investigate the protective role of α-MSH on I/R-induced diabetic retinal damage using hyperglycemic C57BL/6J Ins2Akita/+ mice. Experimental I/R injury was induced by blocking the right middle cerebral artery (MCA) for 2 h followed by 2 h or 22 h of reperfusion using the intraluminal method. Since ophthalmic artery originates proximal to the origin of the MCA, the filament also blocked blood supply to the retina. Upon treatment with α-MSH at 1 h after ischemia and 1 h after reperfusion, animals displayed significant improvement in amplitudes of b-wave and oscillatory potentials during electroretinography. α-MSH also prevented I/R-induced histological alterations and inhibited the development of retinal swelling. Loss of retinal ganglion cells as well as oxidative stress were significantly attenuated in the α-MSH-treated retinae. Level of interleukin 10 was significantly increased after α-MSH treatment. Moreover, gene expression of glutamate aspartate transporter 1, monocarboxylate transporter (MCT) 1 and MCT-2 were significantly higher after α-MSH administration. In conclusion, α-MSH mitigates the severity of I/R-induced retinal damage under hyperglycemic condition. These beneficial effects of α-MSH may have important therapeutic implications against retinal I/R injury under hyperglycemic condition.

Melanocortin 5 Receptor Expression and Recovery of Ocular Immune Privilege after Uveitis.

PURPOSE: The therapeutic use of the RPE-neuropeptide α-MSH suppresses experimental autoimmune uveitis (EAU). This suppression is partially through the α-MSH melanocortin 5 receptor (MC5r). Therefore, we examined the possible role of MC5r-expression in the recovery of RPE suppression of phagolysosome-activation in macrophages following α-MSH-treatment of EAU. METHODS: The conditioned media of cultured in situ RPE-eyecup from α-MSH-treated EAU wild-type and MC5r(-/-) mice were used to treat macrophages to assay for phagolysosome activation. RESULTS: MC5r(-/-) mice treated with α-MSH recovered from EAU, but with greater retinal damage, and the RPE suppressed phagolysosome activation in wild type but not in MC5r(-/-) macrophages. In addition, α-MSH did not suppress phagolysosome activation in MC5r(-/-) macrophages, and resting-MC5r(-/-) macrophages had augmented phagocytic activity. CONCLUSION: α-MSH treatment of EAU mediates a MC5r-dependent recovery of RPE suppression of phagolysosome activation in macrophages possibly altering antigen processing and presentation. Also, MC5r-expression helps protect the retina from inflammatory damage. In addition, MC5r-expression is important in the homeostatic maintenance of phagosome-maturation within macrophages.

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.

Draining lymph nodes of corneal transplant hosts exhibit evidence for donor major histocompatibility complex (MHC) class II-positive dendritic cells derived from MHC class II-negative grafts.

To examine the widely accepted dogmas that corneal grafts lack passenger leukocytes or cells capable of migrating directly to lymph nodes (LNs), we tracked the migration of corneal graft-derived transgenic green fluorescent protein (GFP; Ia(b)) cells into the draining LNs of allogeneic (Ia(d)) recipients. GFP(+) cells were identified in cervical LNs several hours after transplantation, and this traffic was significantly enhanced when grafts were placed in inflamed recipient beds. Draining cells were Ia(b+), CD45(+), and CD11c(+), and examination of ungrafted corneas revealed numerous similarly CD45(+)CD11c(+)CD3(-)CD8alpha(-) cells that uniformly lacked major histocompatibility complex (MHC) class II expression; transmission electron microscopy confirmed the presence of morphologically similar cells. After transplantation, or placement in culture, these CD11c(+) cells became class II(+) in a time-dependent manner and were capable of allostimulatory function. However, the stimulatory capacity of these cornea-derived dendritic cells (DCs) was suppressed compared with splenic controls. These results demonstrate for the first time that the cornea is endowed with resident DCs that are universally MHC class II(-) but that are capable of expressing class II antigen after surgery and migrating to draining LNs of allografted hosts. These data refute the tenet that the cornea is immune privileged due to lack of resident lymphoreticular cells or due to antigenic sequestration from systemic immunity.

Review of the activation of TGF-beta in immunity.

The evolutionarily conserved TGF-beta proteins are distributed ubiquitously throughout the body and have a role in almost every biological process. In immunity, TGF-beta has an important role in modulating immunity. Much is understood about the process of TGF-beta production as a latent molecule and of the consequences and the intercellular signaling of active TGF-beta binding to its receptors; however, there is little discussed between the production and activation of TGF-beta. This review focuses on what is understood about the biochemical and physiological processes of TGF-beta activation and identifies the gaps in understanding immune cell activation of TGF-beta. A mechanistic understanding of the process activating TGF-beta can lead to regulating multiple biological systems by enhancing or inhibiting TGF-beta activation.

Applications of the role of α-MSH in ocular immune privilege.

There is an important role for α-MSH and the melanocortin receptors in ocular immunity, development and health. This chapter will cover what is known about how α-MSH is part of the mechanisms of ocular immune privilege, about the expression of melanocortin receptors and the implications of these findings on the role of α-MSH in ocular physiology and its potential use to treat ocular pathologies.

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.

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.

The diminishment of experimental autoimmune encephalomyelitis (EAE) by neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) therapy.

The neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) plays an important role in immune privilege by its suppression of inflammation, and its induction of regulatory T cells. This finding led us to test the possibility that we can use alpha-MSH to suppress autoimmune diseases, and promote re-establishment of immune tolerance to autoantigens. To test this possibility, SJL mice with experimental autoimmune encephalomyelitis (EAE) were injected with alpha-MSH at the first signs of paralysis. The alpha-MSH-treated mice in comparison with untreated EAE mice had a profound diminishment in the severity and tempo of EAE. The spleen cells in alpha-MSH-treated EAE produced TGF-beta in response to PLP-antigen stimulation in contrast to untreated mice spleen cells that produced IFN-gamma. When the alpha-MSH-treated EAE mice were reimmunized there was a delay of a week before the second episode of EAE. Although this delay maybe because of the induction of TGF-beta-producing spleen cells by the alpha-MSH-treatment, it was not adequate to suppress IFN-gamma-production by PLP-antigen stimulated spleen cells from untreated mice, nor able to suppress the eventual second episode of EAE. Therefore, the injection of alpha-MSH at the onset of paralysis is extremely effective in diminishing the severity and tempo of EAE, and the subsequent induction of potential PLP-specific Treg cells suggests that an alpha-MSH therapy could be attempted as part of a therapeutic regiment to impose immunoregulation and immunosuppression on an autoimmune disease of the central nervous system.