Secreted and intracellular cytokines are complementary measures for human monocytes treated with Toll-like receptor agonists

Cytokine production by human peripheral blood mononuclear cells including monocytes, is frequently assessed by measuring secreted cytokines using enzyme linked immunosorbent assay (ELISA), whereby the total concentration of one cytokine of interest is obtained without information regarding the cell type responsible for making the cytokine. Cytokines can be retained inside the cell using protein transport inhibitors. Subsequent analysis by flow cytometry not only identifies the cell type producing the cytokine but can semi-quantitate the amount of cytokine produced by measuring the geometric mean fluorescence intensity (gMFI) and is amenable to analyzing more than one protein associated with the same cell (multiplexing). We hypothesized that a more comprehensive and biologically meaningful cytokine profile could be acquired by measuring both secreted and the retained intracellular cytokines in parallel cultures of magnetic-sorted CD14+ monocytes. Peripheral monocytes were isolated from 18 healthy donors and treated with standardized molecules that stimulate cytokine production; Toll-like receptor (TLR)4 agonist (lipopolysaccharide, LPS) or TLR7/8 agonist (R848). Pro-inflammatory cytokines (interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)) secreted into the culture medium were measured by ELISA. Parallel cultures were treated with LPS and R848 in the presence of brefeldin A (protein transport inhibitor) and the accumulated intracellular cytokines measured by flow cytometry. Each cytokine (IL-6/IL-8/TNF) gave a unique general pattern when secreted versus intracellular cytokine measurements (frequency and gMFI) were plotted to determine correlation. For monocytes treated with the TLR4 agonist, secreted IL-8 correlated with the frequency of IL-8 positive cells (R = 0.559,p = .016) and not with the amount (gMFI) of IL-8 per cell. In contrast, monocytes treated with the TLR7/8 agonist showed no correlation of secreted IL-8 with the frequency of IL-8 positive cells, but with this treatment secreted IL-6 was correlated with an increase in the frequency of IL-6 positive cells (R = 0.501, p = .034). TNF secretion from monocytes treated with either the TLR4 or TLR7/8 agonist did not correlate with the frequency or gMFI of TNF positive cells. However, there were significant correlations between the TLR4 and TLR7/8 induced TNF response (secreted and gMFI). We conclude that there are fundamental differences in secreted and intracellular IL-6/IL-8/TNF production after monocytes are treated with TLR agonists. Furthermore, secreted and intracellular cytokine analyses are complementary measures that should be used in parallel to explore inflammatory response and cytokine biology.