RESUMEN
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.