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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

IL-1 concentrations. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or 1 mM ATP for 15 min. All cytokines were measured in the same preparations (n = 12). (A) Significant increased IL-1β levels after stimulating LPS-primed PBMCs with 1 mM ATP compared to LPS-priming only. (B) LPS-induced IL-1β mRNA expression. (C) Intracellular pro-IL-1β levels. (D) LPS-induced intracellular IL-1α concentrations. No IL-1α could be detected in supernatants. (E) PBMCs incubated for 24 h with 1 or 10 ng/mL LPS (n = 16) secrete significantly more IL-1α than unstimulated PBMCs, in a dose-dependent manner. (F) Comparison of IL-1β secretion after adding ATP 2 or 20 min after preparation (n = 6). IL-1β levels were determined in a separate experiment, using the same setup. All data are represented as mean + SEM. IL-1β levels detected in (A,F) represent both mature and pro-IL-1β, whereas in (C) specifically intact pro-IL-1β levels were detected.
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Figure 1: IL-1 concentrations. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or 1 mM ATP for 15 min. All cytokines were measured in the same preparations (n = 12). (A) Significant increased IL-1β levels after stimulating LPS-primed PBMCs with 1 mM ATP compared to LPS-priming only. (B) LPS-induced IL-1β mRNA expression. (C) Intracellular pro-IL-1β levels. (D) LPS-induced intracellular IL-1α concentrations. No IL-1α could be detected in supernatants. (E) PBMCs incubated for 24 h with 1 or 10 ng/mL LPS (n = 16) secrete significantly more IL-1α than unstimulated PBMCs, in a dose-dependent manner. (F) Comparison of IL-1β secretion after adding ATP 2 or 20 min after preparation (n = 6). IL-1β levels were determined in a separate experiment, using the same setup. All data are represented as mean + SEM. IL-1β levels detected in (A,F) represent both mature and pro-IL-1β, whereas in (C) specifically intact pro-IL-1β levels were detected.

Mentions: In human PBMCs priming with LPS alone for 3 h led to IL-1β secretion (mean 0.25 ng/mL ± 0.07 SEM, n = 12), which was significantly elevated when followed by ATP exposure (mean 1.53 ng/mL ± 0.46 SEM, p < 0.001, n = 12) (Figure 1A). Secretion of another IL-1 family member cleaved by caspase-1, IL-18, was not increased (IL-18 concentration in supernatant in all samples below level of detection). Upon LPS-priming, IL-1β mRNA expression increased 21-fold. However, ATP stimulation did not further influence the IL-1β mRNA expression (Figure 1B). Priming also induced the expression of intracellular pro-IL-1β protein (Figure 1C). The additional pulse with ATP had no effect on these concentrations. There was an increase in intracellular total IL-1β concentration after ATP (Figure 1A).


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)

IL-1 concentrations. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or 1 mM ATP for 15 min. All cytokines were measured in the same preparations (n = 12). (A) Significant increased IL-1β levels after stimulating LPS-primed PBMCs with 1 mM ATP compared to LPS-priming only. (B) LPS-induced IL-1β mRNA expression. (C) Intracellular pro-IL-1β levels. (D) LPS-induced intracellular IL-1α concentrations. No IL-1α could be detected in supernatants. (E) PBMCs incubated for 24 h with 1 or 10 ng/mL LPS (n = 16) secrete significantly more IL-1α than unstimulated PBMCs, in a dose-dependent manner. (F) Comparison of IL-1β secretion after adding ATP 2 or 20 min after preparation (n = 6). IL-1β levels were determined in a separate experiment, using the same setup. All data are represented as mean + SEM. IL-1β levels detected in (A,F) represent both mature and pro-IL-1β, whereas in (C) specifically intact pro-IL-1β levels were detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: IL-1 concentrations. PBMCs were incubated with either medium or 1 μg/ml LPS for 3 h, and additionally with medium or 1 mM ATP for 15 min. All cytokines were measured in the same preparations (n = 12). (A) Significant increased IL-1β levels after stimulating LPS-primed PBMCs with 1 mM ATP compared to LPS-priming only. (B) LPS-induced IL-1β mRNA expression. (C) Intracellular pro-IL-1β levels. (D) LPS-induced intracellular IL-1α concentrations. No IL-1α could be detected in supernatants. (E) PBMCs incubated for 24 h with 1 or 10 ng/mL LPS (n = 16) secrete significantly more IL-1α than unstimulated PBMCs, in a dose-dependent manner. (F) Comparison of IL-1β secretion after adding ATP 2 or 20 min after preparation (n = 6). IL-1β levels were determined in a separate experiment, using the same setup. All data are represented as mean + SEM. IL-1β levels detected in (A,F) represent both mature and pro-IL-1β, whereas in (C) specifically intact pro-IL-1β levels were detected.
Mentions: In human PBMCs priming with LPS alone for 3 h led to IL-1β secretion (mean 0.25 ng/mL ± 0.07 SEM, n = 12), which was significantly elevated when followed by ATP exposure (mean 1.53 ng/mL ± 0.46 SEM, p < 0.001, n = 12) (Figure 1A). Secretion of another IL-1 family member cleaved by caspase-1, IL-18, was not increased (IL-18 concentration in supernatant in all samples below level of detection). Upon LPS-priming, IL-1β mRNA expression increased 21-fold. However, ATP stimulation did not further influence the IL-1β mRNA expression (Figure 1B). Priming also induced the expression of intracellular pro-IL-1β protein (Figure 1C). The additional pulse with ATP had no effect on these concentrations. There was an increase in intracellular total IL-1β concentration after ATP (Figure 1A).

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