Alpha-melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma cell lines

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.     

Prostaglandin production by melanocytic cells and the effect of alpha-melanocyte stimulating hormone

Prostaglandins are potent mediators of the inflammatory response and are also involved in cancer development. In this study, we show that human melanocytes and FM55 melanoma cells express cyclooxygenase-1 and -2 (COX-1 and -2) and thus have the capability to produce prostaglandins. The FM55 cells produced predominantly PGE2 and PGF2alpha, whereas the HaCaT keratinocyte cell line produced mainly PGE2. The anti-inflammatory peptide, alpha-melanocyte stimulating hormone (alpha-MSH), reduced prostaglandin production in FM55 and HaCaT cells and reversed the effect of the pro-inflammatory cytokine TNF-alpha in the former. These results indicate that melanocytes produce prostaglandins and that alpha-MSH, by inhibiting this response, may play an important role in regulating inflammatory responses in the skin.     

Ultraviolet light induced injury: immunological and inflammatory effects

This article reviews many of the complex events that occur after cutaneous ultraviolet (UV) exposure. The inflammatory changes of acute exposure of the skin include erythema (sunburn), the production of inflammatory mediators, alteration of vascular responses and an inflammatory cell infiltrate. Damage to proteins and DNA accumulates within skin cells and characteristic morphological changes occur in keratinocytes and other skin cells. When a cell becomes damaged irreparably by UV exposure, cell death follows via apoptotic mechanisms. Alterations in cutaneous and systemic immunity occur as a result of the UV-induced inflammation and damage, including changes in the production of cytokines by keratinocytes and other skin-associated cells, alteration of adhesion molecule expression and the loss of APC function within the skin. These changes lead to the generation of suppressor T cells, the induction of antigen-specific immunosuppression and a lowering of cell-mediated immunity. These events impair the immune system's capacity to reject highly antigenic skin cancers. This review gives an overview of the acute inflammatory and immunological events associated with cutaneous UV exposure, which are important to consider before dealing with the complex interactions that occur with chronic UV exposure, leading to photocarcinogenesis.     

Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.     

Neuropeptides and neuroendocrine hormones in ultraviolet radiation-induced immunosuppression

Exposure of the skin to ultraviolet radiation (UVR) can lead to deleterious effects such as sunburn, photoaging, and the development of skin cancer. UVR has also been shown to reduce local and systemic immune responses in humans and animals. In the recent past it has become clear that neuropeptides mediate some of the effects of UVR-induced immunosuppression. Among the neuropeptides released from cutaneous nerves after exposure to UVR, calcitonin gene-related peptide (CGRP) has been examined most extensively. It appears to lead to a reduction of contact hypersensitivity by inducing mast cells to degranulate and thus release tumor necrosis factor alpha (TNF-alpha) and, most likely, interleukin (IL)-10. Nitric oxide, which is coreleased with CGRP, seems to also play a role in immunosuppression through a yet undiscovered mechanism of action, while substance P may have counterregulatory effects. New evidence suggests that the release of neuropeptides from cutaneous sensory c-fibers after UVR is induced by keratinocyte-derived nerve growth factor. UVR can also induce epidermal and some dermal cells, such as melanocytes, keratinocytes, and dermal microvascular epithelial cells, to produce proopiomelanocortin (POMC) and its derivatives. The POMC product alpha-melanocyte-stimulating hormone (alpha-MSH) has been implicated in suppression of contact hypersensitivity and induction of hapten-specific tolerance, most likely by inducing keratinocytes and monocytes to produce the anti-inflammatory cytokine IL-10. Other POMC derivatives have not yet been investigated with regard to a possible role in UVR-induced effects on immunity.     

Alpha-melanocyte stimulating hormone cytoprotective biology in human dermal fibroblast cells

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory peptide effective in various tissues including skin. It acts by inhibiting the production and action of several pro-inflammatory stimuli including TNF-alpha, IL-1beta and LPS in a number of cell types. The role of such stimuli in inducing cellular apoptosis is also well described; however the precise role of alpha-MSH in apoptosis is presently unclear, with studies reporting both anti- and pro-apoptotic activity. The present study demonstrates that cultured human dermal fibroblasts respond to serum depletion and TNF-alpha, IL-1beta and LPS with an increase in membrane permeability, a decrease in viability and an increase in phosphatidylserine externalization (indicative of apoptosis) over 48-96 h. alpha-MSH (at 10(-6) M, but not 10(-9) M) was found to inhibit the serum free and pro-inflammatory mediated reduction in membrane permeability and cellular viability and also inhibited increases in apoptosis. In conclusion, data support a cytoprotective and anti-apoptotic role of the alpha-MSH peptide in human dermal fibroblast cells.     

2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, enhances the adhesion of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes

2-Arachidonoylglycerol (2-AG), an endogenous cannabionoid receptor (CB1 and CB2) ligand, enhanced the adhesion of HL-60 cells differentiated into macrophage-like cells to fibronectin and the vascular cell adhesion molecule-1. The CB2 receptor, Gi/Go, intracellular free Ca(2+) and phosphatidylinositol 3-kinase were shown to be involved in 2-AG-induced augmented cell adhesion. 2-AG also enhanced the adhesion of human monocytic leukemia U937 cells and peripheral blood monocytes. These results strongly suggest that 2-AG plays some essential role in inflammatory reactions and immune responses by inducing robust adhesion to extracellular matrix proteins and adhesion molecules in several types of inflammatory cells and immune-competent cells.     

Stimulation of adhesion molecule expression in human endothelial cells (HUVEC) by adrenomedullin and corticotrophin

Adrenomedullin (AM) and corticotrophin (ACTH) are both vasoactive peptides produced by a variety of cell types, including endothelial cells. Although AM and ACTH are considered to be important in the control of blood pressure and the response to stress, respectively, their role in inflammation and the immune response has not been clarified. This study shows, with the use of a cell-based ELISA, that AM and ACTH induce cell surface expression of the adhesion molecules E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVEC). Furthermore, this effect appears to be mediated in part via elevation of cAMP, given that both peptides elevate cAMP, the cell-permeable cAMP analog dibutyryl cAMP is able to mimic induction of all three cell adhesion molecules and the effect of AM and ACTH is inhibited by the adenylyl cyclase inhibitor SQ-22536. These findings demonstrate a role for AM and ACTH in the regulation of the immune and inflammatory response. E-selectin; intercellular adhesion molecule-1; vascular cell adhesion molecule-1; adrenomedullin; adrenocorticotropic hormone; human umbilical vein endothelial cells     

Cell model of inflammation

Chemical and physical stimuli trigger a cutaneous response by first inducing the main epidermal cells, keratinocytes, to produce specific mediators that are responsible for the initiation of skin inflammation. Activation modulates cell communication, namely leucocyte recruitment and blood-to-skin extravasation through the selective barrier of the vascular ECs (endothelial cells). In the present study, we describe an in vitro model which takes into account the various steps of human skin inflammation, from keratinocyte activation to the adhesion of leucocytes to dermal capillary ECs. Human adult keratinocytes were subjected to stress by exposure to UV irradiation or neuropeptides, then the conditioned culture medium was used to mimic the natural micro-environmental conditions for dermal ECs. A relevant in vitro model must include appropriate cells from the skin. This is shown in the present study by the selective reaction of dermal ECs compared with EC lines from distinct origins, in terms of leucocyte recruitment, sensitivity to stress and nature of the stress-induced secreted mediators. This simplified model is suitable for the screening of anti-inflammatory molecules whose activity requires the presence of various skin cells.     

Resveratrol Couples Apoptosis with Autophagy in UVB-Irradiated HaCaT Cells

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm²). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.     

The role of cytokines in the pathogenesis of cutaneous lupus erythematosus

Cutaneous lupus erythematosus (CLE) is an inflammatory disease with a broad range of cutaneous manifestations that may be accompanied by systemic symptoms. The pathogenesis of CLE is complex, multifactorial and incompletely defined. Below we review the current understanding of the cytokines involved in these processes. Ultraviolet (UV) light plays a central role in the pathogenesis of CLE, triggering keratinocyte apoptosis, transport of nucleoprotein autoantigens to the keratinocyte cell surface and the release of inflammatory cytokines (including interferons (IFNs), tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-8, IL-10 and IL-17). Increased IFN, particularly type I IFN, is central to the development of CLE lesions. In CLE, type I IFN is produced in response to nuclear antigens, immune complexes and UV light. Type I IFN increases leukocyte recruitment to the skin via inflammatory cytokines, chemokines, and adhesion molecules, thereby inducing a cycle of cutaneous inflammation. Increased TNFα in CLE may also cause inflammation. However, decreasing TNFα with an anti-TNFα agent can induce CLE-like lesions. TNFα regulates B cells, increases the production of inflammatory molecules and inhibits the production of IFN-α. An increase in the inflammatory cytokines IL-1, IL-6, IL-10, IL-17 and IL-18 and a decrease in the anti-inflammatory cytokine IL-12 also act to amplify inflammation in CLE. Specific gene mutations may increase the levels of these inflammatory cytokines in some CLE patients. New drugs targeting various aspects of these cytokine pathways are being developed to treat CLE and systemic lupus erythematosus (SLE).     

Alpha-melanocyte-stimulating hormone inhibits allergic airway inflammation

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuropeptide controlling melanogenesis in pigmentary cells. In addition, its potent immunomodulatory and immunosuppressive activity has been recently described in cutaneous inflammatory disorders. Whether alpha-MSH is also produced in the lung and might play a role in the pathogenesis of inflammatory lung conditions, including allergic bronchial asthma, is unknown. Production and functional role of alpha-MSH were investigated in a murine model of allergic airway inflammation. alpha-MSH production was detected in bronchoalveolar lavage fluids. Although aerosol challenges stimulate alpha-MSH production in nonsensitized mice, this rapid and marked stimulation was absent in allergic animals. Treatment of allergic mice with alpha-MSH resulted in suppression of airway inflammation. These effects were mediated via IL-10 production, because IL-10 knockout mice were resistant to alpha-MSH treatment. This study provides evidence for a novel function of alpha-MSH linking neuroimmune functions in allergic airway inflammation.     

Alpha-melanocyte-stimulating hormone immunoreactivity in human melanoma metastases extracts

Seven human melanoma metastases were extracted in order to check the possible presence of any alpha-melanocyte stimulating hormone (MSH) immunoreactivity. The aim of that study was to provide some explanation for, mainly, two observations that we have already made and reported: 1) increased plasma alpha-MSH levels in melanoma of tumour-bearing patients as compared with tumour-free patients; 2) the presence of specific alpha-MSH receptors on human melanoma cells in culture. We could measure large amounts of immunoreactive alpha-MSH in all tumours ranging from 0.31 to 4.27 pmoles per g of wet tissue. Further identification of the extracted material by high-performance liquid chromatography revealed compounds of higher molecular weight and more hydrophobic than synthetic alpha-MSH. In addition, purified extracts could also displace 125I-labelled alpha-MSH from its cellular binding sites in an alpha-MSH specific radio-receptor binding assay. Our findings would suggest a possible presence of some hormone precursor(s) inside the melanoma tumours.     

Homologous regulation of melanocortin-1 receptor (MC1R) expression in melanoma tumor cells in vivo

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist alpha-melanocyte-stimulating hormone (alpha-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B 16F1 cells, a single injection of 50 to 500 microg alpha-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16-24 h. By continuous administration of alpha-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.     

The challenges of UV-induced immunomodulation for children's health

Exposure to solar ultraviolet radiation (UVR) is recognised to have both beneficial and harmful effects on human health. With regard to immune responses, it can lead to suppression of immunity and to the synthesis of vitamin D, a hormone that can alter both innate and adaptive immunity. The consequences in children of such UV-induced changes are considerable: first there are positive outcomes including protection against some photoallergic (for example polymorphic light eruption) and T cell-mediated autoimmune diseases (for example multiple sclerosis) and asthma, and secondly there are negative outcomes including an increased risk of skin cancer (squamous cell carcinoma, basal cell carcinoma and cutaneous malignant melanoma) and less effective control of several infectious diseases. Many uncertainties remain regarding the amount of sun exposure that would provide children with the most effective responses against the variety of immunological challenges that they are likely to experience.     

Rate limiting factors in melanocortin 1 receptor signalling through the cAMP pathway

The melanotropic actions of alpha-melanocyte-stimulating hormone (alpha-MSH) and other melanocortins are mediated by activation of the melanocortin 1 receptor (MC1R). This G protein-coupled receptor is positively coupled to Gs and triggers the cyclic adenosine mono-phosphate (cAMP) pathway. Mutations of the MC1R gene are associated with skin type and pigmentation phenotypes, and with increased risk of skin cancers. Genetic studies have demonstrated an heterozygote carrier effect for these associations, suggesting the importance of variant allele dosage. This could be accounted for, at least partially, if the number of MC1R molecules, rather than the Gs protein or the effector enzyme, adenylyl cyclase, is limiting for the activation of the signalling pathway. However, the nature of the limiting factor(s) in MC1R signalling has not been investigated. We addressed this question by comparing the cAMP output of clones of human melanoma cell lines enriched in MC1R by stable transfection. We also analysed heterologous cell systems widely used for functional studies of MC1R. We show that cAMP production in clones of Chinese hamster ovary cells stably expressing the MC1R is a linear function of receptor number up to high, supraphysiological levels of approximately 50,000 alpha-MSH binding sites per cell. Enrichment of human melanoma cell lines with MC1R also results in increased cAMP levels, with a small leftward shift of the agonist dose-response curves. Therefore, at physiological expression levels second-messenger generation is dependent on receptor density. Within melanoma cells and also likely in normal melanocytes, MC1R appears the limiting factor controlling the output of the cAMP signalling pathway.     

Stimulation of Cloudman melanoma tyrosinase activity occurs predominantly in G2 phase of the cell cycle

A widely accepted notion is that an increasing cellular cyclic AMP (cAMP) concentration is prerequisite for increasing tyrosinase activity and melanin synthesis and for regulating proliferation of pigment cells. alpha-Melanocyte stimulating hormone (alpha-MSH) increases cAMP and tyrosinase activity in Cloudman melanoma cells. Prostaglandins (PGs) E1 and E2 increase melanoma cell tyrosinase activity and inhibit proliferation. Both PGs, but not alpha-MSH, block the progression of Cloudman melanoma cells from G2 phase of the cell cycle into M or G1. Only PGE1 and not PGE2 causes an elevation of cellular cAMP concentrations. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) at 5 x 10(-4) M effectively blocks the increased cAMP synthesis by cells treated with 10 micrograms/ml PGE1. The addition of DDA, however, enhances the melanogenic response of melanoma cells to 10 micrograms/ml PGE1 or PGE2, 10(-7) M alpha-MSH, 10(-4) M isobutylmethylxanthine, 10(-4) M dibutyryl cyclic AMP. DDA also augments the effects of PGE1 or PGE2 on the melanoma cell cycle. Moreover, when DDA is added concomitantly with alpha-MSH, more cells are recruited into G2 than observed in untreated controls. Neither alpha-MSH nor DDA alone has any effect on the cell cycle. These findings undermine the role of cAMP in the melanogenic process and suggest that blocking melanoma cells in G2 may be required for the remarkable stimulation of tyrosinase activity observed with PGE1 or PGE2 alone or in combination with DDA. The observed block in G2 may be essential for the synthesis of sufficient mRNA, which is required for stimulation of tyrosinase activity.     

Expression of adhesion molecules and ligands for activating and costimulatory receptors involved in cell-mediated cytotoxicity in a large panel of human melanoma cell lines

Knowledge of the interactions between MHC-unrestricted cytotoxic effector cells and solid tumour cells is essential for introducing more effective NK cell-based immunotherapy protocols into clinical practise. Here, to begin to obtain an overview of the possible universe of molecules that could be involved in the interactions between immune effector cells and melanoma, we analyse the surface expression of adhesion and costimulatory molecules and of ligands for NK-activating receptors on a large panel of cell lines from the “European Searchable Tumour Cell Line and Data Bank” (ESTDAB, http://www.ebi.ac.uk/ipd/estdab/) and discuss their potential role in the immune response against this tumour. We show that most melanoma cell lines express not only adhesion molecules that are likely to favour their interaction with cells of the immune system, but also their interaction with endothelial cells potentially increasing their invasiveness and metastatic capacity. A high percentage of melanoma cell lines also express ligands for the NK-activating receptor NKG2D; whereas, the majority express MICA/B molecules, ULBP expression, however, was rarely found. In addition to these molecules, we also found that CD155 (poliovirus receptor, PVR) is expressed by the majority of melanoma cell lines, whereas CD112 (Nectin-2) expression was rare. These molecules are DNAM-1 ligands, a costimulatory molecule involved in NK cell-mediated cytotoxicity and cytokine production that also mediates costimulatory signals for triggering naïve T cell differentiation. The phenotypical characterisation of adhesion molecules and ligands for receptors involved in cell cytotoxicity on a large series of melanoma cell lines will contribute to the identification of markers useful for the development of new immunotherapy strategies.     

Adhesion molecules: Key players in Mesenchymal stem cell-mediated immunosuppression

Adhesion molecules are known to -be important components of an active T-cell mediated immune response. Signals generated at a site of inflammation cause circulating T cells to respond by rolling, arrest and then transmigration through the endothelium, all of which are mediated by adhesion molecules. Consequently, strategies have been developed to treat immune disorders with specific antibodies that block the interaction of adhesion molecules. However, the therapeutic effects of such remedies are not always achieved. Our recent investigations have revealed that intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) work together with chemokines to induce immunosuppression mediated by Mesenchymal stem cells (MSCs), thus demonstrating the dual role of adhesion molecules in immune responses. Since MSCs represent an important component of the stromal cells in an inflammatory microenvironment, our findings provide novel information for understanding the regulation of immune responses and for designing new strategies to treat immune disorders.Adhesion molecules are cell surface proteins that mediate the interaction between cells, or between cells and the extracellular matrix (ECM). There are four families of adhesion molecules: immunoglobulin-like adhesion molecules, integrins, cadherins and selectins. Most of them are typical transmembrane proteins that have cytoplasmic, transmembrane and extracellular domains. In the immune system, cell adhesion plays a critical role in initiating and sustaining an effective immune response against foreign pathogens.¹ Based on our recent data, we discuss herein the role of immunoglobulin superfamily cell adhesion molecules, ICAM1 and VCAM-1, in the immunosuppression mediated by Mesenchymal stem cells.