RESUMEN
OBJECTIVE: To evaluate the effect of tumour necrosis factor alpha (TNFalpha), interleukin (IL) 1beta, and their respective inhibitors the p75 TNFalpha soluble receptor (sTNFR) and the type II sIL1betaR (sIL1RII) on whole muscle and isolated myoblast activation. METHODS: Normal muscle samples were stimulated for 7 days with TNFalpha alone or in combination with IL1beta, and myoblasts from these samples for 48 hours. IL6 production was measured by ELISA. Nuclear translocation of NF-kappaB was analysed by immunofluorescent staining and class I MHC expression by FACS. RESULTS: TNFalpha and IL1beta induced IL6 production by normal muscle samples and myoblasts, the action of TNFalpha being more potent on muscle samples. Their soluble receptors (1 microg/ml) decreased this production. Suboptimal concentrations of TNFalpha and IL1beta induced NF-kappaB translocation. sTNFR markedly down regulated TNFalpha-induced translocation while sIL1RII was less potent on IL1beta-induced activation. NF-kappaB translocation induced by the combination of optimal concentrations of TNFalpha and IL1beta was completely inhibited by their soluble receptors. TNFalpha and to a lesser extent IL1beta induced class I MHC expression by myoblasts and this effect was completely inhibited by their respective soluble receptors. CONCLUSION: These results suggest that TNFalpha and IL1beta should be targeted for myositis treatment.
Asunto(s)
Interleucina-1/farmacología , Interleucina-6/biosíntesis , Músculo Esquelético/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Interacciones Farmacológicas , Etanercept , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulina G/farmacología , Interleucina-1/antagonistas & inhibidores , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , Receptores de Interleucina-1 , Receptores Tipo II de Interleucina-1 , Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes/farmacología , Técnicas de Cultivo de Tejidos , Translocación Genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
We studied the production of interleukin (IL)-6 and CCL20/macrophage inflammatory protein-3 alpha (MIP-3 alpha) by human myoblasts and muscle samples in response to IL-17 alone or in combination with IL-1 beta. Both IL-17 and IL-1 beta induced IL-6 production by normal myoblasts and muscle samples. IL-17 had no effect on CCL20 production by myoblasts. Combination of IL-17 and IL-1 beta further increased IL-6 and CCL20 production by muscle samples but not that of CK. IL-17 induced also HLA class I, C-Fos, nuclear factor kappa B (NF-kappa B) and C-Jun expression by myoblasts but not that of HLA class II, CD40, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Finally, immunostaining of dermatomyositis (DM) and polymyositis (PM) muscle biopsies showed IL-17 and CCL20 expression. Our study shows that low levels of cytokines produced by T cells (IL-17) and monocytes (IL-1 beta) can act in combination on skeletal myoblasts and muscle tissue.
Asunto(s)
Interleucina-17/fisiología , Interleucina-1/fisiología , Músculo Esquelético/inmunología , Miositis/inmunología , Linfocitos T/inmunología , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/fisiología , Citocinas/biosíntesis , Combinación de Medicamentos , Sinergismo Farmacológico , Humanos , Interleucina-1/farmacología , Interleucina-17/farmacología , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/inmunología , Mioblastos Esqueléticos/metabolismo , Miositis/metabolismo , Linfocitos T/metabolismoRESUMEN
Bone loss occurs in microgravity whereas an increase in bone mass is observed after skeletal loading. This tissue adaptation involves changes in osteoblastic proliferation and differentiation whose mechanisms remain largely unknown. In this context, we investigated the expression and the nuclear translocation of Egr-1 and NF-kappa B, in a simulated microgravity model (clinostat) and in a model of mechanical strain (Flexcell). We performed RT-PCR and immunocytochemistry analyses at baseline and up to 2 h after stimulation (a mitogenic regimen, 1% stretch, 0.05 Hz, 10 min, or clinorotation 50 rpm, 10 min) in osteoblastic ROS17/2.8 cells. Egr-1 induction as well as NF-kappa B nuclear translocation were activated by mechanical changes. PKC downregulation and COX1/2 inhibition did not alter these inductions. In contrast, ERK1/2, p38(MAPK) and src-kinases pathways were differentially involved in both models. Thus, we demonstrated that changes in the mechanical environment induced an activation of Egr-1 and NF-kappa B with specific kinetics and involved various transduction pathways including MAPKs and src-kinases. These could partially explain the later alterations of proliferation observed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , FN-kappa B/metabolismo , Osteoblastos/metabolismo , Factores de Transcripción/metabolismo , Familia-src Quinasas/fisiología , Animales , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Humanos , Cinética , Proteína Quinasa 3 Activada por Mitógenos , FN-kappa B/genética , Transporte de Proteínas , ARN Mensajero/biosíntesis , Ratas , Estrés Mecánico , Factores de Transcripción/genética , Activación Transcripcional , Células Tumorales Cultivadas , Simulación de IngravidezRESUMEN
Osteoblastic cells cultured on microcarriers in bioreactors are a potentially useful tool to reproduce the in vivo three-dimensional (3D) bone network. The aim is to compare different types of 3D and two-dimensional (2D) osteoblastic culture. ROS17/2.8 cells are cultured in a bioreactor (rotating-wall vessel) or in two kinds of control (3D petri dish, 3D Percoll) and on two types of microcarrier (Cytodex 3 and Biosilon). Growth and morphology are determined by cell count and SEM, and differentiation is determined by dosage of alkaline phosphatase (ALP) activity and northern blots (ALP and osteocalcin (OC)). SEM shows that Biosilon microcarriers are the best substrate. Proliferation in the RWV and 3D petri dish is still in the exponential phase, whereas growth in the 2D culture reaches a plateau after eight days of culture. ALP activity and the ALP and OC mRNA levels are similar at day 8 for both the RWV and 3D petri dish. However, at day 10, cells are more differentiated in the RWV. The study shows that osteoblasts are both proliferate and differentiate in 3D structures. A BrDU immunocytochemical approach shows that only the cells in the periphery of the aggregates proliferate. Therefore the bioreactor may be a suitable tissue culture model for investigation of growth and differentiation processes in tissue engineering.