Multi-parametric Human Cell-Based Inflammation Assay for Cytokines and Cell Surface Antigens

Abstract

Inflammation is a complex event in which cells respond to various endogenous and exogenous stimuli. Factors such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ activate signaling pathways leading to the expression of cytokines and cell-surface antigens. The ability to monitor up-regulation of these molecules provides an important physiological read-out for cell-based models of inflammation. We present results from a multiparametric cell-based assay that uses immunoassays for secreted cytokines and fluorescence read-outs of cell surface markers to evaluate the effect of different mediators on inflammatory response. Primary human umbilical vein endothelial cells (HUVEC) were stimulated with inflammation cytokines (TNF-α, IFN-γ, and IL-1β) for 20 hours. Then, a newly developed microfluidic-based assay system, PuMA, was used to quantify amount of MCP-1, IL-8, and IL-6 in cell supernatants. Expression of adhesion molecules VCAM and HLADR was quantified by measurement of total fluorescence intensity after staining cells with directly conjugated antibodies using an ImageXpress Nano Automated Imaging System. Concentration dependent inhibition of the inflammation responses by the compounds AG126 and MG132 were measured. Clear differences in cytokine expression were seen between the compounds consistent with their reported mechanisms of action. The combination of imaging and microfluidic-based assays provides an efficient multiparametric assay system that can be used to test the efficacy of anti-inflammatory compounds versus toxicity and also provide significant insight into the mechanism of action by selective inhibition of markers triggered by different signaling pathways.