RESUMEN
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.
Asunto(s)
Selectina E/genética , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos , Molécula 1 de Adhesión Celular Vascular/genética , Vasculitis/prevención & control , alfa-MSH/farmacología , Animales , Adhesión Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Lipopolisacáridos/farmacología , Linfocitos/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Microcirculación , FN-kappa B/fisiología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología , Vasculitis/inducido químicamenteRESUMEN
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
Asunto(s)
Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Proopiomelanocortina/biosíntesis , Hormona Adrenocorticotrópica/metabolismo , Ácido Aspártico Endopeptidasas/genética , Western Blotting , Línea Celular , Expresión Génica , Humanos , Interleucina-1/farmacología , Masculino , Microcirculación , Proteínas del Tejido Nervioso/genética , Proteína 7B2 Secretora Neuroendocrina , Hormonas Hipofisarias/genética , Proopiomelanocortina/genética , Proproteína Convertasas , ARN Mensajero/metabolismo , Rayos Ultravioleta , Regulación hacia Arriba/efectos de la radiación , alfa-MSH/metabolismoRESUMEN
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.
Asunto(s)
Endotelio Vascular/fisiología , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Receptores de Corticotropina/genética , Piel/irrigación sanguínea , Animales , Quimiocinas/genética , Endotelio Vascular/inmunología , Endotelio Vascular/efectos de la radiación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-1/farmacología , Microcirculación , Receptores de Corticotropina/fisiología , Receptores de Melanocortina , Rayos Ultravioleta , alfa-MSH/farmacologíaRESUMEN
Exposure of the skin to ultraviolet (UV) light causes DNA damage, inflammation, and impairment of local as well as systemic immune responses. Dermal microvascular endothelial cells are key elements for the recruitment of inflammatory cells during the pathogenesis of inflammatory skin diseases via the expression of adhesion molecules and the release of cytokines. Because UVB may directly affect the function of dermal cells it was investigated whether UVB irradiation alters the production of proinflammatory and chemotactic cytokines by endothelial cells. UVB exposure of transformed human microvascular endothelial cells (HMEC-1) resulted in a dose dependently increased mRNA expression as well as release of interleukin (IL)-1beta, IL-6, IL-8, and growth-regulated oncogene alpha (GROalpha). Maximum cytokine production was observed 16-24 h after irradiation when 7.5-12.5 mJ UVB per cm2 were used. In addition, it was examined whether IL-10, which is upregulated in keratinocytes following UVB irradiation and accounts for UV mediated immunosuppression such as inhibition of contact hypersensitivity, also affects endothelial cell cytokine production. Treatment of HMEC-1 with IL-10 significantly enhanced IL-6 and IL-8 release and further upregulated UVB-induced IL-6 and IL-8 mRNA expression. These findings demonstrate that UVB both directly and indirectly via the release of IL-10 stimulates microvascular endothelial cells to produce proinflammatory cytokines and chemokines that are required for the migration and activation of inflammatory cells in UV-mediated inflammatory skin reactions.