RESUMEN

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/biosíntesis

RESUMEN

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 fármacos

RESUMEN

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.

Asunto(s)

Endotelio Vascular/metabolismo , Molécula 1 de Adhesión Intercelular/fisiología , Neuropéptidos/fisiología , Piel/irrigación sanguínea , Capsaicina/farmacología , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Senescencia Celular/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Microcirculación/fisiología , ARN Mensajero/metabolismo , Receptores de Neuroquinina-1/metabolismo , Receptores de Taquicininas/genética , Piel/efectos de los fármacos , Sustancia P/farmacología

RESUMEN

There is increasing experimental evidence that the neurologic system can directly participate in cutaneous inflammation and wound healing. Recent studies indicate that neuropeptides released by cutaneous nerves such as c-fibers can activate a number of target cells including keratinocytes, Langerhans cells, mast cells, and endothelial cells. One such neuropeptide, substance P (SP), is able to specifically bind to murine and human keratinocytes and induce the release of cytokines such as interleukin 1 (IL-1). Other studies demonstrate that SP can also activate mast cells to produce the potent pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha). More recently, we examined the effect of cutaneous neuropeptides on human dermal microvascular endothelial cell (HDMEC) activities. Our studies indicate that the c-fiber-derived calcitonin gene-related peptide (CGRP) is capable of stimulating HDMEC to secrete the neutrophil chemotactic factor interleukin 8 (IL-8). In addition, SP is able to directly activate HDMEC to express high levels of the important cellular adhesion molecule vascular cellular adhesion molecule 1 (VCAM-1). Thus, these studies support the role that the neurologic system may play in mediating the biologic processes that occur during inflammation and wound healing in the skin.

Asunto(s)

Dermatitis/fisiopatología , Fenómenos Fisiológicos del Sistema Nervioso , Piel/inervación , Cicatrización de Heridas/fisiología , Animales , Endopeptidasas/metabolismo , Humanos , Neuropéptidos/farmacología , Receptores de Neuropéptido/fisiología , Piel/efectos de los fármacos

RESUMEN

Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the epidermis plays a critical role in the regulation of cutaneous inflammation, immunologic reactions, and tissue repair. Transcriptional upregulation of ICAM-1 in response to interferon-gamma (IFNgamma) occurs through a palindromic response element pIgammaRE. pIgammaRE is homologous to IFNgamma-activated sequences, which bind to tyrosine phosphorylated members of the transcription factor family known as signal transducers and activators of transcription (STAT). The importance of tyrosine phosphorylation events in the STAT pathway led us to investigate the effect of the protein tyrosine phosphatase inhibitor, pervanadate, on ICAM-1 expression. We show that treatment of A431 cells and human keratinocytes with pervanadate stimulates protein complex formation on pIgammaRE in a time- and concentration-dependent manner. As demonstrated by mobility supershift assays, the pervanadate-stimulated complex is similar to the IFNgamma-stimulated complex and contains Stat1. Pervanadate treatment also led to an increase in overall protein tyrosine phosphorylation and phosphorylation of Stat1, as well as the subsequent increase in ICAM-1 mRNA and cell surface protein levels. These data show that pervanadate can mimic each step in the IFNgamma-mediated pathway leading to ICAM-1 expression, demonstrate the ability of a pharmacologic agent to bypass the standard cytokine-receptor interaction required for increased ICAM-1 expression, and emphasize the importance of protein tyrosine phosphatases and protein tyrosine kinases in mediating inflammatory responses in the skin.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Inhibidores Enzimáticos/farmacología , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/farmacología , Transactivadores/fisiología , Vanadatos/farmacología , Expresión Génica , Humanos , Queratinocitos/química , Proteínas de la Membrana/genética , Proteínas Tirosina Fosfatasas/fisiología , ARN Mensajero/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Células Tumorales Cultivadas

RESUMEN

In response to interferon gamma (IFNgamma), intercellular adhesion molecule-1 (ICAM-1) is expressed on human keratinocytes, a cell type that is critically involved in cutaneous inflammation. An ICAM-1 5' regulatory region palindromic response element, pIgammaRE, has been shown to confer IFNgamma-dependent transcription enhancement. By electrophoretic mobility shift assays (EMSA), pIgammaRE forms a distinct complex with proteins from IFNgamma-treated human keratinocytes, termed gamma response factor (GRF). Binding of GRF is tyrosine phosphorylation-dependent, and mutations of pIgammaRE that disrupt the palindromic sequence or alter its spatial relationship abrogate GRF binding. Supershift EMSAs using antibodies to characterized STAT proteins suggest that GRF contains a Stat1alpha-like protein; however, non-ICAM-1 IFNgamma-responsive elements (REs) known to bind Stat1alpha homodimers fail to compete for GRF binding in EMSA, and pIgammaRE does not cross-compete with these REs that complex with homodimeric stat1alpha. The pIgammaRE x GRF complex also displays a distinctly different electrophoretic mobility compared to that of IFNgammaREs complexed to homodimeric Stat1alpha. These findings indicate that a distinct complex containing a Stat1alpha-like protein mediates IFNgamma-induced ICAM-1 gene transcription and identifies a subset of IFNgamma-responsive genes that appear to be regulated by this complex.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/farmacología , Transactivadores/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Humanos , Queratinocitos/metabolismo , Fosforilación , Factor de Transcripción STAT1 , Tirosina/metabolismo