Multiplex Immunoassays as an Effective Method to Simultaneously Analyze Inflammatory Mediators in Central Nervous System (CNS) Cells: Human Astrocytes Stimulated With Sarin (GB)

摘要

The central nervous system (CNS) is an immuneprivileged site where the role of immune cells and mediators in brain injury caused by organophosphates (OP) is poorly understood. Many mediators have been identified in nervous system tissue. For instance, interleukin-6 (IL-6), a cytokine that acts on a wide range of tissue influencing cell growth and differentiation, is an agonist for vascular endothelial growth factor (VEGF). CNS cells producing IL-6 include astrocytes, macrophages, microglia, neurons, and brain endothelial cells. Here we describe the response of five different mediators, human interleukin-1beta (hIL-1b), hIL-6, hIL-8, tumor necrosis factor alpha (hTNF-a), and human granulocyte macrophage-colony stimulating factor (hGM-CSF) associated with human astrocytes incubated with an OP, sarin (GB). Human astrocytes ((caret) 106 cell density) were stimulated with a high concentration of GB (0.8 mM) for 48 hours at 37 degrees C. The expressed mediators in human astrocytes were detected by using the Luminex100 trademark protein multiplex immunoassay. Constitutive non-stimulated human astrocytes secreted hGM-CSF (0.59 0.03 ng/mL), hIL- 1b (0.33 0.05 ng/mL), hIL-6 (1.43 0.02 ng/mL) and hIL-8 (0.39 0.02 ng/mL). hTNF-a secretion was not detected or observed. GB decreased the endogenous secretion of these mediators as follows: hGM-CSF (0.51 0.02 ng/mL), hIL-1b (0.28 0.03 ng/mL), hIL-6 (1.00 0.02 ng/mL). Meanwhile, the induction chemokine hIL-8 was increased 0.42 0.03 ng/mL by GB as measured by Luminex100 trademark. Up and down-regulation of these mediators in human astrocytes promises a nonintrusive mechanism for assessment of the role of individual mediators in brain cell development, function and response to insult, such as that caused by the OP. Many neurodegenerative disorders are associated with inflammatory processes in the central nervous system (CNS).