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ATP-Induced IL-1β Specific Secretion: True Under Stringent Conditions.

Stoffels M, Zaal R, Kok N, van der Meer JW, Dinarello CA, Simon A - Front Immunol (2015)

Bottom Line: Subsequently, adenosine triphosphate (ATP) is added to the cells in order to trigger the P2X7 receptor resulting in processing and secretion of mature IL-1β.However, it is often reported that secretion is due to cytotoxic effects of ATP with P2X7 receptor-activation-related cell death.More importantly we show that these conclusions can only be drawn under stringent experimental conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Radboud University Medical Center, Nijmegen Institute for Infection, Inflammation and Immunity (N4i) , Nijmegen , Netherlands.

ABSTRACT
Interleukin-1β is a potent proinflammatory cytokine, of which processing and secretion are tightly regulated. After exposure to various stimuli, mononuclear phagocytes synthesize the inactive precursor (pro-IL-1β), which is then cleaved intracellularly by caspase-1 and secreted. A widely used method for in vitro secretion of IL-1β employs LPS-primed human peripheral blood monocytes. Subsequently, adenosine triphosphate (ATP) is added to the cells in order to trigger the P2X7 receptor resulting in processing and secretion of mature IL-1β. However, it is often reported that secretion is due to cytotoxic effects of ATP with P2X7 receptor-activation-related cell death. We have challenged this concept and demonstrate IL-1β specific secretion, since there is no increase in cell death and IL-1α and IL-18 are not released in the same cultures. More importantly we show that these conclusions can only be drawn under stringent experimental conditions.

No MeSH data available.


Related in: MedlinePlus

Effect of ATP preparations on IL-1β secretion. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or different concentrations of ATP for 15 min. We observed a dose dependent increase in IL-1β secretion regardless of the time in between the ATP preparation and the stimulation, with a maximum response at a dose of 3 mM ATP. IL-1β secretion was higher when fresh ATP preparations were used (A), as compared to older preparations, e.g., 10 min (B), and 20 min old (C). Results from two independent experiments with a total of six donors were pooled and represented as mean + SEM.
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Figure 2: Effect of ATP preparations on IL-1β secretion. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or different concentrations of ATP for 15 min. We observed a dose dependent increase in IL-1β secretion regardless of the time in between the ATP preparation and the stimulation, with a maximum response at a dose of 3 mM ATP. IL-1β secretion was higher when fresh ATP preparations were used (A), as compared to older preparations, e.g., 10 min (B), and 20 min old (C). Results from two independent experiments with a total of six donors were pooled and represented as mean + SEM.

Mentions: Next, we studied whether the ATP concentration could overcome the diminished stimulatory capacity of the “older” ATP preparation. As shown (Figure 2), cells stimulated with LPS and subsequently with ATP that was prepared 2 min (Figure 2A), 10 min (Figure 2B), or 20 min (Figure 2C) before addition, all secrete IL-1β. However, IL-1β secretion decreased with increasing time in between the preparation and the stimulation with ATP. Also, at each time point 3 mM ATP induces a greater IL-1β secretion than 1 mM ATP. However, the 5 and 10 mM concentrations do not increase the response further. Thus, the maximum response in this cell model is observed at 3 mM ATP. Nevertheless, even this response is not able to overcome the preparation effect; with a 20 min 3 mM ATP preparation IL-1β secretion is still lower than with a 2 min 1 mM ATP preparation.


ATP-Induced IL-1β Specific Secretion: True Under Stringent Conditions.

Stoffels M, Zaal R, Kok N, van der Meer JW, Dinarello CA, Simon A - Front Immunol (2015)

Effect of ATP preparations on IL-1β secretion. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or different concentrations of ATP for 15 min. We observed a dose dependent increase in IL-1β secretion regardless of the time in between the ATP preparation and the stimulation, with a maximum response at a dose of 3 mM ATP. IL-1β secretion was higher when fresh ATP preparations were used (A), as compared to older preparations, e.g., 10 min (B), and 20 min old (C). Results from two independent experiments with a total of six donors were pooled and represented as mean + SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325933&req=5

Figure 2: Effect of ATP preparations on IL-1β secretion. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or different concentrations of ATP for 15 min. We observed a dose dependent increase in IL-1β secretion regardless of the time in between the ATP preparation and the stimulation, with a maximum response at a dose of 3 mM ATP. IL-1β secretion was higher when fresh ATP preparations were used (A), as compared to older preparations, e.g., 10 min (B), and 20 min old (C). Results from two independent experiments with a total of six donors were pooled and represented as mean + SEM.
Mentions: Next, we studied whether the ATP concentration could overcome the diminished stimulatory capacity of the “older” ATP preparation. As shown (Figure 2), cells stimulated with LPS and subsequently with ATP that was prepared 2 min (Figure 2A), 10 min (Figure 2B), or 20 min (Figure 2C) before addition, all secrete IL-1β. However, IL-1β secretion decreased with increasing time in between the preparation and the stimulation with ATP. Also, at each time point 3 mM ATP induces a greater IL-1β secretion than 1 mM ATP. However, the 5 and 10 mM concentrations do not increase the response further. Thus, the maximum response in this cell model is observed at 3 mM ATP. Nevertheless, even this response is not able to overcome the preparation effect; with a 20 min 3 mM ATP preparation IL-1β secretion is still lower than with a 2 min 1 mM ATP preparation.

Bottom Line: Subsequently, adenosine triphosphate (ATP) is added to the cells in order to trigger the P2X7 receptor resulting in processing and secretion of mature IL-1β.However, it is often reported that secretion is due to cytotoxic effects of ATP with P2X7 receptor-activation-related cell death.More importantly we show that these conclusions can only be drawn under stringent experimental conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Radboud University Medical Center, Nijmegen Institute for Infection, Inflammation and Immunity (N4i) , Nijmegen , Netherlands.

ABSTRACT
Interleukin-1β is a potent proinflammatory cytokine, of which processing and secretion are tightly regulated. After exposure to various stimuli, mononuclear phagocytes synthesize the inactive precursor (pro-IL-1β), which is then cleaved intracellularly by caspase-1 and secreted. A widely used method for in vitro secretion of IL-1β employs LPS-primed human peripheral blood monocytes. Subsequently, adenosine triphosphate (ATP) is added to the cells in order to trigger the P2X7 receptor resulting in processing and secretion of mature IL-1β. However, it is often reported that secretion is due to cytotoxic effects of ATP with P2X7 receptor-activation-related cell death. We have challenged this concept and demonstrate IL-1β specific secretion, since there is no increase in cell death and IL-1α and IL-18 are not released in the same cultures. More importantly we show that these conclusions can only be drawn under stringent experimental conditions.

No MeSH data available.


Related in: MedlinePlus