(B) Quantification of the percentage of IL\6\positive cells from panel A following LPS stimulation over time in presence (green) or absence (blue) of BrefA. large spread among cells/donors. Moreover, IL\6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL\6 production. IL\6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs. ytyt 0.05 was considered statistically significant (* 0.05, ** 0.01, *** 0.001). Results We first measured the secretion of IL\6 from dendritic cells derived from monocytes isolated from blood of healthy volunteers. In resting conditions, these dendritic cells secreted on average about 0.05 fgcell?1h?1 of IL\6 (Fig. ?(Fig.1).1). Given that the molecular weight of the main form of IL\6 is 23.7 kDa 16, this equals about 1300 IL\6 moleculescell?1h?1. We then stimulated the cells with LPS for 24 h. During this period, cells secrete more IL\6 which accumulates in medium in an almost linear fashion for over 24 h SNF5L1 3. Overnight stimulation of the DCs with LPS increased the IL\6 secretion on average roughly 40\fold to ~ 46 000 moleculescell?1h?1 (Fig. ?(Fig.1).1). Thus, a resting dendritic cell on average releases an IL\6 molecule approximately every 3 s. Upon LPS stimulation, this number increases to about 13 IL\6 molecules per second. Open in a separate window Figure 1 IL\6 secretion by dendritic cells. (A) The total cellular secretion of IL\6 by dendritic cells measured by ELISA with and without overnight stimulation with LPS. Data points: individual donors. (B) Same as panel A, but now divided through the total number of cells and the time to calculate the average IL\6 secretion per cell per hour. Student’s 0.01. Next, we determined cellular heterogeneity in our dendritic cell populations by determining the number of cells that produced IL\6. It is well established that even within one cell type, major differences in protein (R)-BAY1238097 expression and cytokine secretion can be present 17, 18, 19. Therefore, we estimated the percentage of IL\6 producing dendritic cells upon LPS stimulation by flow cytometry combined with immunolabeling of intracellular IL\6. Only a (R)-BAY1238097 minor population of ~ 10% of the dendritic cells showed intracellular pools of IL\6, and this accumulation was low and only observable 4C6 h after LPS stimulation (Fig. ?(Fig.2A,B),2A,B), indicating that most IL\6 was secreted rapidly after synthesis. Based on immunofluorescence staining of endogenous IL\6, IL\6 accumulated at the Golgi region and (less) at REs (Fig. ?(Fig.2C),2C), as reported previously 6. To prevent the (R)-BAY1238097 secretion and accumulate (R)-BAY1238097 all produced IL\6 within the cells, we repeated the flow cytometry experiments in presence of Brefeldin A (BrefA) which prevents cytokine secretion by disrupting ER\Golgi trafficking 20. This enabled us to (R)-BAY1238097 estimate the percentage of cells within our population that is capable of IL\6 secretion. BrefA treatment resulted in both a higher IL\6 signal and a higher fraction of ~ 42% of all cells showing intracellular pools of IL\6 (for 6 h LPS stimulation; Fig. ?Fig.2).2). After 6 h, the intracellular accumulation decreased, possibly due to degradation. When we correct the average IL\6 secretion rate for the fraction of IL\6\producing cells (~ 42% of the population; BrefA condition in Fig. ?Fig.2B),2B), we find that LPS\stimulated cells on average secrete roughly 30 IL\6 molecules per second. However, based on the spread of intensities of the IL\6 signals in the flow cytometry experiments (Fig. ?(Fig.2A),2A), the variation in IL\6 production among the cell population is large and ranges for over an order of magnitude. Open in a separate window Figure 2 IL\6 production by dendritic cells is heterogeneous. (A) Representative flow cytometry plots showing the distribution of intracellular IL\6 in unstimulated cells or cells stimulated with LPS for 6 h with or without BrefA. SSC, side scatter. (B) Quantification of the percentage of IL\6\positive cells from panel A following LPS stimulation over time in presence (green) or absence (blue) of BrefA. Shown is mean SEM ( 3 donors). (C) Confocal images of LPS\activated dendritic cells immunostained for the SNARE VAMP3 (green in merge) and IL\6 (magenta). Arrowhead: cell positive for IL\6. Yellow regions in intensity distributions: overlap of VAMP3 and IL\6 intensities. Scale bar, 10 m. To obtain a rough estimate of the number of IL\6 molecules per secretory vesicle, we estimated the rate of exocytotic events from dendritic.