Asunto(s)
Epidermis/fisiopatología , Interleucinas/farmacología , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , beta-Ciclodextrinas/farmacología , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Citocinas/metabolismo , Dermatitis Atópica/inducido químicamente , Epidermis/patología , Proteínas Filagrina , Expresión Génica/efectos de los fármacos , Humanos , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/metabolismo , Interleucina-13/farmacología , Interleucina-17/farmacología , Interleucina-4/farmacología , Proteínas de Filamentos Intermediarios/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Linfopoyetina del Estroma TímicoRESUMEN
The heparin-binding EGF-like growth factor (HB-EGF) is an autocrine/paracrine keratinocyte growth factor, which binds to the epidermal growth factor (EGF) receptor family and plays a critical role during the re-epithelialization of cutaneous wound by stimulating the keratinocytes proliferation and migration. In this study, cellular stressing condition in autocrine cultures of human keratinocytes was induced by cholesterol depletion using methyl-beta-cyclodextrin (MßCD). MßCD treatment induces the expression and the release of HB-EGF. By analysis of the culture media, large amounts of cellular ATP were measured particularly after 1 h of MßCD treatment. To investigate whether ATP contributes to the expression of HB-EGF, the nonhydrolyzable ATP analogue, ATP-γ-S, was used to mimic the extracellular ATP released. We report that keratinocytes stimulated with ATP-γ-S induce HB-EGF expression and activate EGFR and ERK1/2. Using an antagonist of P2 purinergic receptors, we demonstrate that HB-EGF synthesis induced by lipid rafts disruption is dependent on ATP interaction with P2 purinergic receptors. Moreover, our data suggest that both MAPKs p38 and ERK1/2 are involved together or independently in the regulation of HB-EGF gene expression. These findings provide new insight into the signaling pathway by which HB-EGF is expressed after lipid rafts disruption. In summary, after lipid raft disruption, keratinocytes release large amount of extracellular ATP. ATP induces HB-EGF synthesis and release by interacting with the P2 purinergic receptor and through p38 and ERK1/2 signaling in response to a challenging environment. A release of ATP acts as an early stress response in keratinocytes.
Asunto(s)
Adenosina Trifosfato/metabolismo , Colesterol/deficiencia , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Queratinocitos/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Células Cultivadas , Colesterol/metabolismo , Células Epidérmicas , Receptores ErbB/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Queratinocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Receptores Purinérgicos/metabolismoRESUMEN
Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Burkholderia pseudomallei/inmunología , Melioidosis/inmunología , Linfocitos T/inmunología , Animales , Vacunas Bacterianas/administración & dosificación , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/fisiología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Células Cultivadas , Diabetes Mellitus Tipo 2/inmunología , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/microbiología , Masculino , Melioidosis/microbiología , Melioidosis/prevención & control , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T/metabolismo , Linfocitos T/microbiología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/microbiologíaRESUMEN
Transient cholesterol depletion from plasma membranes of human keratinocytes has been shown to reversibly activate signalling pathways in monolayer cultures. Consecutive changes in gene expression have been characterized in such conditions and were interestingly found to be similar to transcriptional changes observed in keratinocytes of atopic dermatitis (AD) patients. As an inflammatory skin disease, AD notably results in altered histology of the epidermis associated with a defective epidermal barrier. To further investigate whether the activation of keratinocytes obtained by cholesterol depletion could be responsible for some epidermal alterations reported in AD, this study was undertaken to analyse cholesterol depletion in stratified cultures of keratinocytes, i.e. a reconstructed human epidermis (RHE). RHE contains heterogeneous populations of keratinocytes, either proliferating or progressively differentiating and stratifying towards the creation of a cornified barrier. Cholesterol depletion induced in this model was found reversible and resulted in activation of signalling pathways similar to those previously identified in monolayers. In addition, selected changes in the expression of several genes suggested that keratinocytes in RHE respond to cholesterol depletion as monolayers. However, preserved histology and barrier function indicate that some additional activation, likely from the immune system, is required to obtain epidermal alterations such as the ones found in AD.
Asunto(s)
Colesterol/deficiencia , Epidermis/crecimiento & desarrollo , Queratinocitos/metabolismo , Western Blotting , Células Cultivadas , Dermatitis Atópica/genética , Humanos , Queratinocitos/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , beta-Ciclodextrinas/farmacologíaRESUMEN
This chapter provides methods suitable for the culture of primary human keratinocytes in serum-free culture conditions, starting from very small skin biopsies. It also explains procedures required for reconstruction of a stratified epidermis on polycarbonate filter, starting from keratinocytes cultured in serum-free conditions. Tissues reconstructed according to this method have been proven suitable for characterization of epidermal morphogenesis and for in vitro studies of the epidermal barrier. Utilization of the same method for successful isolation of keratinocytes from a patient suffering from Darier's disease and the reconstruction of a pathological epidermis which displays the same histological features as in vivo are also presented.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Epidérmicas , Epidermis/patología , Cemento de Policarboxilato/farmacología , Enfermedad de Darier/patología , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Proteínas/aislamiento & purificación , Proteínas/metabolismo , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
Epidermal homeostasis and repair of the skin barrier require that epidermal keratinocytes respond to alterations of their environment. We report that cellular stress with methyl-beta-cyclodextrin (MBCD), a molecule that extracts membrane cholesterol and thereby disrupts the structure of lipid rafts, strongly induces the synthesis of heparin-binding EGF-like growth factor (HB-EGF) in keratinocytes through the activation of p38 mitogen-activated protein kinase. Interesting parallels between lipid raft disruption and oxidative stress can be drawn as hydrogen peroxide induces p38 activation and HB-EGF synthesis in keratinocytes. Consistent with other studies, we show increased HB-EGF expression in keratinocytes located at the margin of wounded skin areas. Analyzing cultured keratinocytes exposed to rhHB-EGF, we report increased HB-EGF mRNA levels and alterations in the expression of differentiation markers. Interestingly, identical alterations in differentiation markers are shown to occur in vivo at the wound margin and in HB-EGF-treated cultures. In addition, in vitro sectioning of skin samples also induces the expression of HB-EGF at the border of the incisions. Altogether, our data suggest that expression of HB-EGF is a marker of the keratinocyte's response to a challenging environment and demonstrate that this growth factor alters the phenotype of keratinocytes in a manner similar to that observed during epidermal repair.