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
PURPOSE: Gram-negative bacterial infections of the eye can lead to corneal bacterial keratitis, visual impairment, and blindness. Many of these pathologic changes may be mediated by bacterially derived products such as lipopolysaccharide (LPS). In this investigation, it has been established for the first time that human corneal cells are capable of expressing the functional LPS receptor complex proteins, CD14 and Toll-like receptor 4 (TLR4). METHODS: CD14 and TLR4 mRNA expression in human corneal cells was determined by RT-PCR and Northern blot analysis, and cell surface expression of these proteins was measured by flow cytometry. LPS-mediated corneal cell activation was determined by measuring intracellular calcium mobilization. Cellular cytokine and chemokine secretion in response to LPS was measured by ELISA. The expression and localization of CD14 in whole human cornea was determined by immunohistochemistry. RESULTS: Human corneal epithelial, stromal, and endothelial cells expressed CD14 mRNA and cell surface CD14. LPS binding to cornea CD14 resulted in a rapid intracellular calcium response and the secretion of multiple proinflammatory cytokines and chemokines. CD14 mRNA expression in corneal epithelial cells was upregulated by LPS. In addition to CD14, corneal epithelial cells expressed the functional LPS receptor-signaling protein TLR4, which was also augmented by LPS. CONCLUSIONS: The cornea expresses functional CD14 and TLR4 LPS receptor proteins. Understanding the function and biology of the corneal LPS receptor complex may lead to novel therapies for the management of ocular Gram-negative bacterial infections.
Asunto(s)
Córnea/efectos de los fármacos , Proteínas de Drosophila , Receptores de Lipopolisacáridos/genética , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Pseudomonas aeruginosa , Receptores de Superficie Celular/genética , Secuencia de Bases , Northern Blotting , Calcio/metabolismo , Córnea/citología , Córnea/metabolismo , Citocinas/metabolismo , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Receptores de Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4 , Receptores Toll-Like , Regulación hacia ArribaRESUMEN
Nerve growth factor is an essential neurotrophic factor required for the growth and maintenance of cutaneous sensory nerves. In the skin, keratinocytes are a significant source of nerve growth factor; however, the regulation of cutaneous nerve growth factor production still remains to be fully understood. In this study we tested the hypothesis that neuropeptides released by cutaneous sensory nerves have the capacity to modulate directly the expression of keratinocyte nerve growth factor, which would have important implications for the maintenance and repair of nerves in the skin. In order to address this question experimentally we examined the effect of the neuropeptides, substance P and neurokinin A, on nerve growth factor expression in human keratinocytes and the murine keratinocyte PAM 212 cell line by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and the PC-12 nerve growth factor bioassay. The results of these studies indicated that substance P and neurokinin A can directly induce nerve growth factor mRNA expression and the secretion of bioactive nerve growth factor protein in both human and murine keratinocytes. The specificity of these responses was demonstrated using neuropeptide receptor antagonists and nerve growth factor blocking antibodies. Additional studies also demonstrated a significant in vivo upregulation of keratinocyte nerve growth factor expression in murine epidermis after the topical application of the neuropeptide releasing agent capsaicin. This is the first report demonstrating the induction of cutaneous nerve growth factor by sensory nerve-derived neuropeptides such as substance P and neurokinin A. This direct effect of the neurosensory system on keratinocyte nerve growth factor production may have important consequences for the maintenance and regeneration of cutaneous nerves in normal skin and during inflammation and wound healing.
Asunto(s)
Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuroquinina A/farmacología , Sustancia P/farmacología , Animales , Capsaicina/farmacología , Línea Celular Transformada , Epidermis/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/fisiología , Neuropéptidos/metabolismo , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
Improved treatment options for patients with high-risk melanoma are of great importance for clinicians who participate in the care of these patients. There remains an overall lack of response to existing treatment options, which continues to fuel the efforts of basic scientists and clinicians to pursue other approaches for the treatment of melanoma that is no longer limited to the skin. Continued investigation into the innovative and concurrent use of surgery, chemotherapy, immunotherapy, and radiation therapy holds significant promise for improved outcomes in the management of patients with this devastating disease.
Asunto(s)
Melanoma/terapia , Neoplasias Cutáneas/terapia , Vacunas contra el Cáncer/uso terapéutico , Terapia Genética , Humanos , Melanoma/diagnóstico , Pronóstico , Factores de Riesgo , Biopsia del Ganglio Linfático Centinela , Neoplasias Cutáneas/diagnósticoAsunto(s)
Antipruriginosos/uso terapéutico , Dermatitis Atópica/complicaciones , Prurito/fisiopatología , Corticoesteroides/uso terapéutico , Citocinas/inmunología , Citocinas/farmacología , Dermatitis Atópica/fisiopatología , Histamina/inmunología , Histamina/farmacología , Humanos , Inmunosupresores/uso terapéutico , Inflamación , Neuropéptidos/inmunología , Neuropéptidos/farmacología , Prurito/etiologíaRESUMEN
Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Dermatitis Alérgica por Contacto/patología , Dermatitis Alérgica por Contacto/prevención & control , Neprilisina/fisiología , Sustancia P/toxicidad , Administración Cutánea , Animales , Antiinflamatorios no Esteroideos/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/metabolismo , Permeabilidad Capilar/genética , Permeabilidad Capilar/inmunología , Capsaicina/administración & dosificación , Aceite de Crotón/toxicidad , Dermatitis Alérgica por Contacto/enzimología , Dermatitis Alérgica por Contacto/genética , Dermatitis Irritante/enzimología , Dermatitis Irritante/genética , Dermatitis Irritante/patología , Dermatitis Irritante/prevención & control , Inhibidores Enzimáticos/administración & dosificación , Femenino , Glicopéptidos/administración & dosificación , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neprilisina/antagonistas & inhibidores , Neprilisina/deficiencia , Neprilisina/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Piperidinas/administración & dosificación , Quinuclidinas/administración & dosificación , Piel/irrigación sanguínea , Piel/enzimología , Piel/patologíaRESUMEN
PURPOSE: The interferon-gamma-inducing factor Interleukin-18 (IL-18) is a recently described cytokine that appears to have multiple important pro-inflammatory effects including the induction of interferon-gamma (IFN-gamma) by activated T-cells. The expression of IL-18 by human cornea has not been previously reported. In the present study, we examine the possibility that human corneal epithelial cells are capable of producing this leukocyte-activating factor which may play an important role in IFN-gamma-dependent inflammation responses in the cornea. METHODS: Northern blot analysis and ELISA were used to investigate the in vitro expression of IL-18 mRNA and protein respectively in primary (HCEC) and transformed human corneal epithelial cells (HCET). To determine if IL-18 expression was modulated by pro-inflammatory mediators, cells were treated with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA) or synthetic double stranded RNA (poly dI : dC). IL-18 bioactivity was determined in a leukocyte interferon-gamma induction assay and IL-18 was immunolocalized in whole human cornea by immunohistochemistry using a specific anti-IL-18 antibody. RESULTS: IL-18 mRNA and bioactive protein was constitutively expressed by human corneal epithelial cells and upregulated by PMA, LPS and poly dI : dC. The constitutive expression of IL-18 protein immunoreactivity was also demonstrated in the epithelial cells of whole human cornea tissue. CONCLUSIONS: This is the first study demonstrating that corneal epithelial cells are capable of producing the IFN-gamma inducing factor IL-18. Increased bioactive corneal IL-18 production can be induced by a number of pro-inflammatory agents and may play an important role in initiating gamma-interferon-mediated inflammatory responses in the cornea.
Asunto(s)
Epitelio Corneal/metabolismo , Interleucina-18/biosíntesis , Northern Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Interleucina-18/genética , Lipopolisacáridos/farmacología , Polidesoxirribonucleótidos/farmacología , ARN Mensajero/biosíntesis , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
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
The neurological system plays an important role in modulating some inflammatory skin diseases. Neuro-cutaneous interactions may be mediated by the release of neuropeptides such as substance P (SP) which activate immunocompetent cells in the skin by binding to high affinity neurokinin receptors (NKR). Since epidermal keratinocytes produce a variety of cytokines and are intimately associated with cutaneous sensory fibers, we tested the ability of these cells to participate in the cutaneous neuroimmune system by the secretion of potent cytokines such as interleukin 1 (IL-1) in response to released SP. RT-PCR studies demonstrated that cultured PAM 212 murine keratinocytes expressed mRNA for NK-2R but not NK-1R. Correspondingly, the addition of SP to these cells resulted in a rapid increase in intracellular Ca2+ levels that could be specifically blocked by an NK-2R antagonist. NK-2R was also shown in normal mouse epidermis by immunohistochemistry. SP augmented the expression of PAM 212 keratinocyte IL-1alpha mRNA in a dose and time dependent manner and this induction was inhibited by an NK-2R antagonist. Secretion of bioactive IL-1alpha by the PAM 212 keratinocytes was likewise stimulated by SP in a dose dependent manner. These data support the hypothesis that SP released from cutaneous sensory nerves contributes to neuroimmune inflammatory responses in the skin by modulating the expression and release of cytokines from epidermal keratinocytes.
Asunto(s)
Interleucina-1/biosíntesis , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Receptores de Neuroquinina-2/metabolismo , Sustancia P/farmacología , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular Transformada , Cartilla de ADN/genética , Expresión Génica/efectos de los fármacos , Interleucina-1/genética , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Queratinocitos/metabolismo , Cinética , Ratones , Neuroinmunomodulación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/antagonistas & inhibidores , Receptores de Neuroquinina-2/genética , Piel/inervación , Piel/metabolismoRESUMEN
There is increasing evidence that the cutaneous neurosensory system can directly modulate inflammatory responses in the skin by the release of neuropeptides such as substance P (SP). Dermal microvascular endothelial cell (DMEC) cellular adhesion molecule (CAM) expression plays a key role in directing leukocyte trafficking during cutaneous inflammatory responses. In recent studies, our laboratory examined the direct effect of SP on DMEC CAM expression and function in vitro and in vivo. Our studies indicate that DMEC express high affinity functional receptors for SP. After exposure to SP, DMEC expressed significant levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which was accompanied by increased binding to leukocytes expressing the appropriate integrin counter receptors for these CAM. We then determined the in vivo effect of released neuropeptides on DMEC CAM expression. Our results indicate that the topical cutaneous application of the neuropeptide-releasing agent capsaicin resulted in increased ICAM-1 and VCAM-1 immunostaining of microvascular cells in the skin of human volunteers. Little is known regarding the cellular regulatory events by which SP modulates DMEC CAM expression. Our studies indicate that SP-induced cellular Ca+2 signals led to the activation of the NF-kappaB pathway, resulting in nuclear translocation of p65/p50 heterodimers that bind to high-affinity tandem kappaB sites on the VCAM-1 promoter, whereas SP activation induced NF-AT activation and ICAM-1 DNA binding. Thus, these studies further support the role of the cutaneous neurologic system in modulating inflammatory processes in the skin.
Asunto(s)
Dermatitis/inmunología , Endotelio Vascular/inmunología , Endotelio Vascular/inervación , Piel , Animales , Dermatitis/fisiopatología , Humanos , Neuroinmunomodulación/fisiología , Piel/irrigación sanguínea , Piel/inmunología , Piel/inervaciónRESUMEN
Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.
Asunto(s)
Dinitroclorobenceno/farmacología , Mediadores de Inflamación/farmacología , Interleucina-18/genética , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Interferón gamma/farmacología , Interleucina-18/metabolismo , Lipopolisacáridos/farmacología , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Psoriasis/metabolismo , ARN Mensajero/metabolismo , Piel/química , Linfocitos T/fisiología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Upon stimulation, cutaneous sensory nerves release neuropeptides such as substance P (SP), which modulate responses in the skin by activating a number of target cells via neurokinin receptors. We have demonstrated that SP preferentially binds to the NK-1R on human dermal microvascular cells, resulting in increased intracellular Ca2+ and induction of ICAM-1 and VCAM-1 expression. In the current studies, we identify specific elements in the regulatory regions of ICAM-1 and VCAM-1 genes as necessary and sufficient for SP-dependent transcriptional activation. SP treatment of human dermal microvascular endothelial cells leads to coincident activation and binding of the transcription factor NF-AT to the -191/-170 region of the ICAM-1 gene (a region bound by activated p65/p65 homodimers in response to TNF-alpha), and NF-kappa B (p65/p50) to tandem NF-kappa B binding sites at -76/-52 of the VCAM-1 gene. The SP-elicited intracellular Ca2+ signal was required for activation and subsequent binding of both NF-AT and NF-kappa B. The transacting factor induction by SP was specific, since a selective NK-1R antagonist blocked SP activation and subsequent NF-AT and NF-kappa B activation and binding. These data demonstrate coincident activation of NF-AT and NF-kappa B via SP-induced intracellular Ca2+ mobilization and indicate a crucial role for neuropeptides in modulating localized cutaneous inflammatory responses.
Asunto(s)
Señalización del Calcio/fisiología , Moléculas de Adhesión Celular/genética , Proteínas de Unión al ADN/fisiología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Líquido Intracelular/metabolismo , FN-kappa B/fisiología , Proteínas Nucleares , Sustancia P/fisiología , Factores de Transcripción/fisiología , Regiones no Traducidas 5'/genética , Membrana Celular/metabolismo , Células Cultivadas , Ciclosporina/farmacología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Dimerización , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B , Factores de Transcripción NFATC , Proteínas Proto-Oncogénicas c-rel/metabolismo , Elementos de Respuesta/efectos de los fármacos , Eliminación de Secuencia , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
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.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neuropéptidos/metabolismo , Sistemas Neurosecretores/efectos de la radiación , Trastornos por Fotosensibilidad/etiología , Piel/efectos de la radiación , Rayos Ultravioleta , Animales , Reparación del ADN , Humanos , Factores de Crecimiento Nervioso , Piel/inmunología , Piel/inervaciónRESUMEN
Proteinase-activated receptor-2 (PAR-2) is a G-protein coupled receptor. Tryptic proteases cleave PAR-2 exposing a tethered ligand (SLIGKV), which binds and activates the receptor. Although PAR-2 is highly expressed by cultured keratinocytes and is an inflammatory mediator, its precise localization in the normal and inflamed human skin is unknown, and the proteases that activate PAR-2 in the skin have not been identified. We localized PAR-2 in human skin by immunohistochemistry, examined PAR-2 expression by RT-PCR and RNA blotting, and investigated PAR-2 activation by mast cell tryptase. PAR-2 was localized to keratinocytes, especially in the granular layer, to endothelial cells, hair follicles, myoepithelial cells of sweat glands, and dermal dendritic-like cells. PAR-2 was also highly expressed in keratinocytes and endothelial cells of inflamed skin. PAR-2 mRNA was detected in normal human skin by RT-PCR, and in cultured human keratinocytes and dermal microvascular endothelial cells by Northern hybridization. Trypsin, tryptase and a peptide corresponding to the tethered ligand (SLIGKVNH2) increased [Ca2+]i in keratinocytes, measured using Fura-2/AM. Although tryptase-containing mast cells were sparsely scattered in the normal dermis, they were numerous in the dermis in atopic dermatitis, and in the dermis, dermal-epidermal border, and occasionally within the lower epidermis in psoriasis. Tryptase may activate PAR-2 on keratinocytes and endothelial cells during inflammation.
Asunto(s)
Queratinocitos/fisiología , Mastocitos/enzimología , Receptores de Trombina/fisiología , Serina Endopeptidasas/fisiología , Piel/metabolismo , Transporte Biológico/fisiología , Northern Blotting , Calcio/metabolismo , Células Cultivadas , Quimasas , Dermatitis Atópica/enzimología , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Inmunohistoquímica , Microcirculación/fisiología , Receptor PAR-2 , Receptores de Trombina/metabolismo , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/metabolismo , Piel/irrigación sanguínea , Piel/patología , Distribución Tisular/fisiología , TriptasasRESUMEN
Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.
Asunto(s)
Neprilisina/biosíntesis , Piel/enzimología , Heridas y Lesiones/enzimología , Anciano , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Colorantes , Contaminación de Medicamentos , Endotelio Vascular/citología , Femenino , Fibroblastos/enzimología , Humanos , Inmunohistoquímica , Queratinocitos/enzimología , Queratinocitos/metabolismo , Queratinas/inmunología , Masculino , Microcirculación , Persona de Mediana Edad , Neprilisina/genética , Neprilisina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/químicaRESUMEN
Sensory nerves in skin are capable of releasing multiple neuropeptides, which modulate inflammatory responses by activating specific cutaneous target cells. Extravasation of particular subsets of leukocytes depends upon the regulated expression of cellular adhesion molecules such as VCAM-1 on microvascular endothelial cells. We examined the direct effect of cutaneous neuropeptides on the expression and function of human dermal microvascular endothelial cell (HDMEC) VCAM-1. A significant increase in VCAM-1 immunostaining of microvascular endothelium was observed in vivo following capsaicin application to human skin. Multiple cutaneous sensory C-fiber-released neuropeptides were evaluated for their ability to induce VCAM-1 cell surface expression on HDMEC. Only substance P (SP) was found to be capable of inducing HDMEC VCAM-1 expression. This SP-mediated VCAM-1 induction appeared to be a direct effect that did not require the release of other HDMEC-derived soluble factors. Increased HDMEC VCAM-1 mRNA expression was detected 1 h after the addition of SP, with peak mRNA increase at 6-9 h postinduction. FACS studies demonstrated a 6.5-fold increase in endothelial cell surface VCAM-1 expression detectable 16 h after addition of SP, which was specifically blocked by a neurokinin-1 receptor antagonist. Increased VCAM-1 cell surface expression on SP-treated HDMEC resulted in a 4-fold increase in the functional binding of 51Cr-labeled MOLT-4 T cells. These data indicate that SP is capable of directly and specifically up-regulating functional endothelial VCAM-1 expression and thus may play a key role in modulating certain inflammatory responses in the skin.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Sustancia P/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Capsaicina/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Humanos , Inflamación/etiología , Neuroinmunomodulación/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sustancia P/fisiología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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/farmacologíaAsunto(s)
Hormona Adrenocorticotrópica/genética , Endotelio Vascular/metabolismo , Proopiomelanocortina/genética , Piel/irrigación sanguínea , Subtilisinas/genética , Transcripción Genética , alfa-MSH/genética , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de la radiación , Furina , Humanos , Cinética , Microcirculación , Proopiomelanocortina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Rayos UltravioletaRESUMEN
There is increasing evidence that sensory nerves may participate in cutaneous inflammatory responses by the release of neuropeptides such as substance P (SP). We examined the direct effect of SP on human dermal microvascular endothelial cell (HDMEC) intercellular adhesion molecule 1 (ICAM-1) expression and function. Our results indicated that, although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMEC intracellular Ca2+ levels, primarily by the activation of NK-1R. Immunohistochemistry studies likewise demonstrated that HDMEC predominantly expressed NK-1R. The addition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1 mRNA levels, followed by a fivefold increase in ICAM-1 cell surface expression. This functionally resulted in a threefold increase in 51Cr-labeled binding of J-Y lymphoblastoid cells to HDMEC. In vivo studies demonstrated a marked increase in microvascular ICAM-1 immunostaining 24 and 48 h after application of capsaicin to the skin. These results indicate that neuropeptides such as SP are capable of directly activating HDMEC to express increased levels of functional ICAM-1 and further support the role of the cutaneous neurological system in modulating inflammatory processes in the skin.