The alpha-melanocyte stimulating hormone-related tripeptide K(D)PT stimulates human hair follicle pigmentation in situ under proinflammatory conditions.

BACKGROUND: Alpha-melanocyte stimulating hormone (alpha-MSH) is a well-tolerated immunomodulator with cytoprotective and anti-inflammatory effects that is known to stimulate melanogenesis and proliferation of follicular melanocytes. As human hair follicles (HFs) locally synthesize alpha-MSH, pharmacologically more easily handled alpha-MSH-related tripeptides, such as K(D)PT, may imitate this endogenous regulation, and may show a favourable side-effect profile on clinical use. OBJECTIVES: To investigate the effect of the synthetic, alpha-MSH-related peptide K(D)PT [which is identical to interleukin (IL)-1beta(193-195)] on melanogenesis in human anagen HFs, under normal and proinflammatory growth conditions. METHODS: Normal human anagen VI scalp HFs were microdissected and organ cultured with different concentrations of K(D)PT with or without coadministration of a proinflammatory, catagen-inducing stimulus, interferon (INF)-gamma. Masson-Fontana histochemistry and NKI/beteb immunohistochemistry were employed to assess changes in the degree of human HF pigmentation and melanocyte dendricity. RESULTS: As confirmed by quantitative (immuno-)histomorphometry, compared with controls, K(D)PT alone did not affect human HF pigmentation in organ culture. However, in the presence of a strong, prototypic proinflammatory stimulus (IFN-gamma), K(D)PT significantly stimulated HF melanin content and melanocyte dendrite formation in situ. CONCLUSIONS: The IL-1beta- and alpha-MSH-related tripeptide, K(D)PT, displays interesting hair pigmentation-stimulatory activities under proinflammatory conditions. These might become exploitable for innovative antigreying strategies, notably in postinflammatory poliosis (regrowth of white hair, e.g. during recovery from alopecia areata), where no effective clinical therapy is yet available.

Crucial role of the melanocortin receptor MC1R in experimental colitis.

BACKGROUND AND AIMS: alpha-Melanocyte stimulating hormone (alpha MSH) is known to exert anti-inflammatory effects, for example in murine DSS (dextran sodium sulphate induced) colitis. The anti-inflammatory functions of alpha MSH are mediated by the melanocortin1-receptor (MC1R) in an autoregulatory loop. The aim of this study was therefore to determine whether a breakdown of the alpha MSH-MC1R pathway leads to worsening of disease. METHODS: Experimental colitis was induced in mice with a frameshift mutation in the MC1R gene (MC1Re/e), C57BL/6 wild type mice, and MC1Re/e-C57BL/6 bone marrow chimeras. The course of inflammation was monitored by weight loss, histological changes in the colon, and myeloperoxidase activity. In addition, MC1R expression was analysed in intestinal epithelial cells. RESULTS: While the colon of untreated MC1Re/e appeared normal, the course of DSS-colitis in MC1Re/e mice was dramatically aggravated, with a significantly higher weight loss and marked histological changes compared to C57BL/6WT. The inflammation eventually led to death in all MC1Re/e, while all C57BL/6WT survived. Similar observations were detected in a transmissible murine colitis model induced by Citrobacter rodentium. Infected MC1Re/e showed delayed clearance of infection. To determine whether missing haematopoietic cell expressed MC1R was responsible, DSS colitis was induced in MC1Re/e-C57BL/6 bone marrow chimeras. MC1Re/e mice receiving MC1R+ bone marrow showed a similar course of inflammation to non-transplanted MC1Re/e. Likewise, transplantation of MC1R bone marrow into C57BL/6WT mice did not lead to any worsening of disease. CONCLUSIONS: This is the first study to show a functional role of MC1R in intestinal inflammation. The data suggest a pivotal role of non-haematopoietic cell expressed MC1R in the host's response to pathogenic stimuli.

Alpha-melanocyte stimulating hormone prevents lipopolysaccharide-induced vasculitis by down-regulating endothelial cell adhesion molecule expression.

The neuroendocrine hormone alpha-melanocyte stimulating hormone (MSH) has profound antiinflammatory and immunomodulating properties. Here we have examined the possibility that alpha-MSH may interfere with the expression and function of cell adhesion molecules (CAMs) expressed by human dermal microvascular endothelial cells (HDMECs) in response to lipopolysaccharide (LPS) or TNFalpha in vitro and in vivo. In HDMEC, alpha-MSH (10(-8)/10(-12) M) profoundly reduced the mRNA and protein expression of E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 induced by LPS or TNFalpha as determined by semiquantitative RT-PCR, ELISA, and fluorescence-activated cell sorter analysis. In addition, alpha-MSH significantly impaired the LPS-induced ICAM-1 and VCAM-1-mediated adhesion of lymphocytes to HDMEC monolayer in a functional adhesion assay. Likewise, alpha-MSH effectively inhibited the transcription factor nuclear factor-kappaB activation in HDMEC, which is required for CAM gene expression. Importantly in vivo, in murine LPS-induced cutaneous vasculitis (local Shwartzman reaction), a single ip injection of alpha-MSH significantly suppressed the deleterious vascular damage and hemorrhage by inhibiting the sustained expression of vascular E-selectin and VCAM-1. This persistent expression has been implicated in the dysregulation of diapedesis and activation of leukocytes, which subsequently leads to hemorrhagic vascular damage. Our findings indicate that alpha-MSH may have an important therapeutical potential for the treatment of vasculitis, sepsis, and inflammatory diseases.

Failure of alpha-melanocyte stimulating hormone to attenuate cerebral complications in experimental pneumococcal meningitis.

Alpha-melanocyte-stimulating hormone (alpha-MSH) is an endogenous neuroimmunomodulatory peptide that can inhibit a broad range of inflammatory mediators known to be involved in the pathophysiology of bacterial meningitis. We evaluated the effect of alpha-MSH in a rat model of pneumococcal meningitis. Rats were intracisternally infected with Streptococcus pneumoniae and treatment was started 6 h after infection. Both systemic and intracisternal alpha-MSH failed to influence blood-brain barrier disruption, increased intracranial pressure, brain cytokine concentrations (IL-1beta, IL-6, TNF-alpha, MIP-2, and IL-10), CSF bacterial titers, and clinical parameters of disease severity (weight loss, body temperature, and blood pressure), although the treatment strongly increased the CNS concentrations of alpha-MSH. However, systemic but not intracisternal alpha-MSH slightly reduced the CNS leukocyte accumulation, indicating that leukocyte extravasation is inhibited by alpha-MSH from the blood side. Our results show that alpha-MSH reduces the CNS leukocyte accumulation by its systemic action, but does not attenuate meningitis-associated intracranial complications.

Keratinocytes in epidermal immune responses.

Recent studies have advanced our understanding that the regulation of immune responses is not only confined to immunocompetent cells. Upon stimulation, keratinocytes are capable of releasing various factors and expressing receptors that are significantly involved in immunoregulation. Dysregulation and the abnormal production of cytokines and other inflammatory mediators as well as their receptors can be observed in various skin diseases. An understanding of keratinocytes as important regulators of the skin immune system opens new opportunities for the treatment of various skin diseases.

Expression of proopiomelanocortin peptides in human dermal microvascular endothelial cells: evidence for a regulation by ultraviolet light and interleukin-1.

Proopiomelanocortin peptides such as alpha-melanocyte-stimulating hormone and adrenocorticotropin are expressed in the epidermal and dermal compartment of the skin after noxious stimuli and are recognized as modulators of immune and inflammatory reactions. Human dermal microvascular endothelial cells mediate leukocyte-endothelial interactions during cutaneous inflammation by the expression of cellular adhesion molecules and cytokines such as interleukin-1. This study addresses the hypothesis that human dermal microvascular endothelial cells express both proopiomelanocortin and prohormone convertases, which are required to generate proopiomelanocortin peptides. Semiquantitative reverse transcriptase polymerase chain reaction and northern blot studies revealed a constitutive expression of proopiomelanocortin mRNA by human dermal microvascular endothelial cells in vitro that was time- and concentration-dependently upregulated by interleukin-1 beta. Furthermore, irradiation of human dermal microvascular endothelial cells with ultraviolet A1 (30J per cm(2)) or ultraviolet B (12.5 mJ per cm(2)) enhanced proopiomelanocortin expression as well as the production and release of the proopiomelanocortin peptides adrenocorticotropin and alpha-melanocyte-stimulating hormone. In addition to proopiomelanocortin, prohormone convertase 1 mRNA expression was detected by reverse transcriptase polymerase chain reaction in unstimulated human dermal microvascular endothelial cells and was augmented after exposure to alpha-melanocyte- stimulating hormone, interleukin-1 beta, or irradiation with ultraviolet. These findings demonstrate that human dermal microvascular endothelial cells express proopiomelanocortin and prohormone convertase 1 required for the generation of adrenocorticotropin. Additionally, human dermal microvascular endothelial cells express mRNA for the prohormone convertase 2 binding protein 7B2. Taken together these findings indicate that human dermal microvascular endothelial cells upon stimulation express both proopiomelanocortin and prohormone convertases required for the generation of alpha-melanocyte-stimulating hormone. As proopiomelanocortin peptides were found to regulate the production of human dermal microvascular endothelial cell cytokines and adhesion molecules and to have a variety of anti-inflammatory properties these peptides may significantly contribute to the modulation of skin inflammation. J Invest Dermatol 115:1021-1028 2000

Vitamin E reduces platelet adhesion to human endothelial cells in vitro.

Although it has been reported that vitamin E (alpha-tocopherol) can reduce platelet adhesiveness and aggregation in vivo, the mechanism is still unknown. Therefore, the aim of the present study was to determine whether incubations of platelet-rich plasma (PRP) with vitamin E influence platelet adhesion to cultured endothelial cells. To exclude blood plasma involvement, also washed platelets were pretreated with alpha-tocopherol. Vitamin E (0.5-1.0 mM) was added to PRP or washed platelets. Endothelial cells in monolayer were incubated with thrombin-activated platelets (1 or 2 U/ml). After 1 hr of incubation, non-adhered platelets were removed and counted. Treating of PRP with alpha-tocopherol inhibited platelet adhesion to endothelial cell monolayer. This effect was dose dependent on concentrations of alpha-tocopherol and thrombin. In our experiments PRP was treated with alpha-tocopherol and endothelial cell monolayer was used as test surface. These findings agree with previous observations on the adhesivity of platelets to synthetic surfaces after dietary vitamin E in healthy volunteers. When washed platelets were incubated with alpha-tocopherol, no significant reduction of adhesion was detectable. As preincubation of washed platelets with alpha-tocopherol does not inhibit platelet adhesion, it may be supposed that the effect of vitamin E does not occur in a directly cellular mechanism. The data suggest that alpha-tocopherol may reduce platelet adhesiveness probably after incorporation by plasma lipoproteins.

The role of alpha-MSH as a modulator of cutaneous inflammation.

Among various neuropeptides such as substance P, calcitonin gene-related peptide and others, alpha-melanocyte-stimulating hormone (alpha-MSH) was found to be produced in the skin. Moreover, melanocortin receptor 1 (MC-1R), which is specific for alpha-MSH and ACTH, is expressed in the skin on keratinocytes, dendritic cells, macrophages and endothelial cells. In monocytes, macrophages and dendritic cells alpha-MSH inhibits the production and activity of immunoregulatory and proinflammatory cytokines such as IL-2, IFN-gamma, TNF-alpha and IL-1. It downregulates the expression of costimulatory molecules such as CD86 and CD40 and induces the production of suppressor factors such as the cytokine synthesis inhibitory factor IL-10. On endothelial cells alpha-MSH is capable of downregulating the LPS-induced expression of adhesion molecules such as vascular cell adhesion molecule (VCAM) and E-selectin. Moreover, the LPS-induced activation of transcription factors such as NF kappa B is downregulated by alpha-MSH. In a mouse model i.v. or topical application of alpha-MSH was found to inhibit the induction phase as well as the effector phase of contact hypersensitivity (CHS) reactions and to induce hapten-specific tolerance. These findings indicate that the production of immunosuppressing neuropeptides such as alpha-MSH by epidermal cells may play an essential role during the pathogenesis of immune and inflammatory reactions in the skin.

Detection of melanocortin-1 receptor antigenicity on human skin cells in culture and in situ.

The proopiomelanocortin (POMC) products alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) bind to specific receptors known as the melanocortin (MC) receptors. There is increasing evidence that the MC receptor subtype 1 (MC-1R) is expressed in vitro by several other cutaneous cell types besides melanocytes and keratinocytes. Our knowledge on the MC-1R expression in skin, however, remains fragmentary. In order to examine the expression of MC-1R in human skin cells in vitro and In situ, we made use of a recently described antibody directed against the amino acids 2-18 of the human MC-1R. Flow cytometry analysis revealed the highest MC-1R antigenicity in normal melanocytes and keratinocytes, followed by dermal fibroblasts, microvascular endothelial cells and WM35 melanoma cells. Little or no expression was detected in KB carcinoma cells and Fs4 fibroblasts. In normal human skin, immunoreactivity for the anti-MC-1R antibody was detected in hair follicle epithelia, sebocytes, secretory and ductal epithelia of sweat glands, and periadnexal mesenchymal cells. Interfollicular epidermis was largely unreactive in adult skin as opposed to undifferentiated keratinocytes of fetal skin. Our findings form a framework within which MC-1 receptor expression can be studied in various skin diseases.

Effect of ultraviolet light on the release of neuropeptides and neuroendocrine hormones in the skin: mediators of photodermatitis and cutaneous inflammation.

Ultraviolet (UV) irradiation of the skin causes both inflammation and alterations in the skin immune system. There is increasing experimental evidence that UV-induced skin inflammation is influenced by the sensory nervous system and the neuroendocrine system in the skin. The resulting complex network of cytokines, chemokines, neuropeptides, neuropeptide-degrading enzymes, neurohormones, and other inflammatory mediators mediate photodermatitis and cutaneous inflammation. Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) are released from sensory nerves innervating the skin upon UV exposure. In addition, a variety of cells in the skin produce increased neuroendocrine hormones such as proopiomelanocortin (POMC) peptides and their receptors as well as neurotrophins after UV exposure. Neuropeptides and neurohormones are capable of directly or indirectly mediating UV-induced cutaneous neurogenic inflammation by the induction of vasodilatation, plasma extravasation, and augmentation of UV-induced cytokine, chemokine, or cellular adhesion molecule expression required for activation and trafficking of inflammatory cells into the inflamed tissue. Neuropeptides and neurotrophins may also play a role in the repair of cutaneous UV injury. In addition to proinflammatory effects, UV-induced neuropeptides and neurohormones such as CGRP and alpha-melanocyte-stimulating hormone may have immunosuppressive effects in the skin. This review will focus on the role that SP, CGRP, POMC peptides, and their receptors may play in modulating UV-induced inflammation in the skin.

Human peripheral blood-derived dendritic cells express functional melanocortin receptor MC-1R.

The neuropeptide, alpha-melanocyte-stimulating hormone (alpha-MSH) is well known for its immunomodulating capabilities. alpha-MSH antagonizes the activity of numerous proinflammatory mediators; for example, Interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNF alpha), and bacterial endotoxin. In vivo alpha-MSH has been shown to suppress a contact hypersensitivity reaction in mice, and to induce hapten-specific tolerance. Since antigen presenting cells (APC) represent key elements for tolerance induction, the effect of alpha-MSH, and the expression of its receptor-melanocortin receptor-1 (MC-1R), on human peripheral blood-derived monocytes and dendritic cells (DC), was investigated. Semiquantitative RT-PCR demonstrated that monocytes and DC express MC-1R, but none of the other members of the MC-receptor family. Moreover, the extent of MC-1R expression correlated with the state of activation of these cells. Since the major ligand of MC-1R is alpha-MSH the question of whether alpha-MSH affects the function of monocyte derived DC was further investigated. We found that the expression of the costimulatory molecules CD 86 and CD 40 was downregulated on DC in the presence of alpha-MSH. Thus, alpha-MSH may exert its immunosuppressive effects by altering the function of APC.

Role of epidermal cell-derived alpha-melanocyte stimulating hormone in ultraviolet light mediated local immunosuppression.

Irradiation of the skin with ultraviolet light (UV) results in profound alterations of both local and systemic immune responses. These effects are largely mediated by soluble mediators released from epidermal cells in response to UV. It is well known that keratinocytes release increased amounts of cytokines upon UV-irradiation. UV-light also induces the release of the proopiomelanocortin (POMC)-derived peptide, alpha-melanocyte-stimulating hormone (alpha MSH), from keratinocytes, and upregulates the expression of POMC mRNA. alpha MSH exerts a variety of immunomodulating and antiinflammatory effects, mainly by virtue of its capacity to alter the function of antigen presenting cells and vascular endothelial cells. Within an in vivo mouse-model, both intravenous and topical application of alpha MSH resulted in inhibiting the induction, eliciting a contact hypersensitivity reaction, and inducing hapten-specific tolerance. These findings indicate that alpha MSH, released in the epidermis after UV irradiation, may contribute to UV-mediated immunosuppression. The therapeutic application of alpha MSH or alpha MSH-derived peptides may prove to be a useful approach for treating inflammatory skin diseases.

Molecular basis of the alpha-MSH/IL-1 antagonism.

The neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) is recognized as a potent mediator of immune and inflammatory reactions. Accordingly, alpha-MSH in vitro, as well as in vivo, antagonizes the proinflammatory activities of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF alpha). Since the molecular basis of these antiinflammatory effects is not well known, the influence of alpha-MSH on IL-1 beta-induced chemokine production and transcription factor activation was investigated in human keratinocytes. alpha-MSH, in a dose-dependent manner, after 48 h, significantly reduced the IL-1 beta mediated secretion of the C-X-C chemokines IL-8 and Gro alpha. This was confirmed by semiquantitative RT-PCR, which revealed a marked down-regulation in IL-8 and Gro alpha mRNA expression. Furthermore, we determined the effect of alpha-MSH on the IL-1 beta-induced activation of the nuclear factor kappa B (NF kappa B)--a major transcription factor for chemokine genes. Electrophoretic mobility-shift-assays showed that alpha-MSH, in a dose range from 10(-6) to 10(-12) M, significantly downregulated the IL-1 beta-induced activation of NF kappa B 10 minutes after stimulation. Therefore, NF kappa B inactivation by alpha-MSH appears to be a crucial event, one that is responsible for the downregulation of cytokine gene transcription.

Expression of functional melanocortin receptors and proopiomelanocortin peptides by human dermal microvascular endothelial cells.

Human dermal microvascular endothelial cells (HDMEC) are capable of mediating leukocyte-endothelial interactions by the expression of cellular adhesion molecules and the release of proinflammatory cytokines and chemokines during cutaneous inflammation. Recent studies support the important role for proopiomelanocortin (POMC) peptides, such as alpha-melanocyte stimulating hormone (alpha-MSH), as immunomodulators in the cutaneous immune system. The purpose of the studies described here was to determine whether HDMEC serves as both target and source for POMC peptides. RT-PCR and Northern blot studies demonstrated the constitutive expression of mRNA for the adrenocorticotropin (ACTH) and alpha-MSH-specific melanocortin receptor 1 (MC-1R) in HDMEC, and the microvascular endothelial cell line HMEC-1 that could be upregulated by stimulation with IL-1 beta and alpha-MSH. HDMEC responded to stimulation by alpha-MSH with a dose- and time-dependent synthesis and release of the CXC chemokines, IL-8 and GRO alpha. Likewise, alpha-MSH augmented HDMEC chemokine release induced by TNF or IL-1. HD-MEC were found to constitutively express POMC and prohormone convertase 1 (PC-1); the latter being required to generate ACTH from the POMC prohormone. POMC and PC-1 mRNA expression are increased as a result of stimulation with UVB and UVA1 radiation, IL-1, and alpha-MSH. In addition, UV-radiation is capable of inducing the release of HDMEC, ACTH, and alpha-MSH in a time- and dose-dependent fashion. Thus, these data provide evidence that HDMEC are capable of expressing functional MC-1R, POMC, and PC-1 mRNA; and of releasing POMC peptides with UV light, IL-1, and alpha-MSH as regulatory factors. The expression and regulation of these peptides may be of importance, not only for the autocrine or paracrine regulation of physiologic functions of dermal endothelial cells, but also for the regulation of certain microvascular-mediated cutaneous or systemic inflammatory responses.

Mechanisms of the antiinflammatory effects of alpha-MSH. Role of transcription factor NF-kappa B and adhesion molecule expression.

The recruitment of leukocytes from the circulation to inflamed tissue is regulated by the expression of adhesion molecules on both leukocytes and endothelial cells. The proopiomelanocortin-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) is known to modulate inflammation. Thus, we investigated the influence of alpha-MSH on the LPS-induced expression of the adhesion molecules E-selectin and VCAM-1 on endothelial cells. Human microvascular endothelial cells (HMEC-1) were treated with LPS (100 ng/ml) alone or in the presence of alpha-MSH (10(-8) to 10(-16) M). RT-PCR analysis showed that alpha-MSH significantly reduced LPS-induced expression of VCAM-1 and E-selectin. Since many adhesion molecules contain regulatory NF-kappa B sites in their promoter region, the role of alpha-MSH in the activation of the transcription factor NF-alpha B was also investigated. alpha-MSH significantly downregulated the LPS-mediated activation of NF-kappa B, in a dose-dependent manner. These findings indicate that modulation of the transcription factor NF-kappa B is a crucial molecular event, one that seems to be responsible for the antiinflammatory effects of alpha-MSH.

Characterization of a polyclonal antibody raised against the human melanocortin-1 receptor.

We have generated a polyclonal antibody raised against a synthetic peptide corresponding to the amino acids 2-18 of the extracellular, N-terminal domain of the human melanocortin-1 receptor (MC-1R). Specificity of the affinity-purified anti-MC-1R antibody was confirmed by dot blot analysis with the antigenic peptide. The antibody detected MC-1R antigenicity on the surface of normal human melanocytes and WM35 melanoma cells, as shown by FACS and immunofluorescence analysis. The antibody was suitable for immunoperoxidase staining of deparaffinized skin sections, revealing prominent MC-1R staining of a cutaneous melanoma as opposed to undiseased skin in which normal melanocytes were only occasionally immunoreactive. Distinct adnexal structures in normal skin also displayed MC-1R immunostaining. Specificity of the MC-1R immunoreactivity in each technique was confirmed by preabsorption with the immunogenic peptide, omission, or substitution of the primary antibody with preimmune serum. Our results provide a baseline for future studies on MC-1R expression in diseased human skin.