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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

The effect of fresh ATP preparations on cell membrane integrity. (A) Cells that lost membrane integrity stain positive for AnnexinV and PI, and can be late apoptotic or necrotic. (B) Cells that stain only positive for AnnexinV are early apoptotic but have not lost membrane integrity. Higher concentrations of ATP cause more cell death (A) than a low concentration. ATP induces early apoptosis in all concentrations (B). Results from two independent experiments with a total of six donors were pooled and represented as mean + SEM.
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Figure 3: The effect of fresh ATP preparations on cell membrane integrity. (A) Cells that lost membrane integrity stain positive for AnnexinV and PI, and can be late apoptotic or necrotic. (B) Cells that stain only positive for AnnexinV are early apoptotic but have not lost membrane integrity. Higher concentrations of ATP cause more cell death (A) than a low concentration. ATP induces early apoptosis in all concentrations (B). Results from two independent experiments with a total of six donors were pooled and represented as mean + SEM.

Mentions: To investigate the effects of the different ATP concentrations on cell viability we further used AnnexinV-PI staining. At the early stages of apoptosis, cells start exposing phosphatidylserine (PS) to the external cellular environment, making them positive for AnnexinV-FITC staining. Loss of membrane integrity in late apoptotic or necrotic cells renders them permeable to PI. After 3 h LPS followed by 15 min ATP stimulation, only higher ATP concentrations (≥3 mM) induce cells to become late apoptotic or necrotic (AnnexinV+/PI+), although only at a low percentage (Figure 3A). Early apoptotic cells (AnnexinV+/PI−) were observed at all ATP concentrations (Figure 3B). In the absence of LPS cells were more sensitive to the ATP stimulus, than in the presence of LPS, as indicated by the black bars and white bars, respectively (Figure 3).


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)

The effect of fresh ATP preparations on cell membrane integrity. (A) Cells that lost membrane integrity stain positive for AnnexinV and PI, and can be late apoptotic or necrotic. (B) Cells that stain only positive for AnnexinV are early apoptotic but have not lost membrane integrity. Higher concentrations of ATP cause more cell death (A) than a low concentration. ATP induces early apoptosis in all concentrations (B). 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 3: The effect of fresh ATP preparations on cell membrane integrity. (A) Cells that lost membrane integrity stain positive for AnnexinV and PI, and can be late apoptotic or necrotic. (B) Cells that stain only positive for AnnexinV are early apoptotic but have not lost membrane integrity. Higher concentrations of ATP cause more cell death (A) than a low concentration. ATP induces early apoptosis in all concentrations (B). Results from two independent experiments with a total of six donors were pooled and represented as mean + SEM.
Mentions: To investigate the effects of the different ATP concentrations on cell viability we further used AnnexinV-PI staining. At the early stages of apoptosis, cells start exposing phosphatidylserine (PS) to the external cellular environment, making them positive for AnnexinV-FITC staining. Loss of membrane integrity in late apoptotic or necrotic cells renders them permeable to PI. After 3 h LPS followed by 15 min ATP stimulation, only higher ATP concentrations (≥3 mM) induce cells to become late apoptotic or necrotic (AnnexinV+/PI+), although only at a low percentage (Figure 3A). Early apoptotic cells (AnnexinV+/PI−) were observed at all ATP concentrations (Figure 3B). In the absence of LPS cells were more sensitive to the ATP stimulus, than in the presence of LPS, as indicated by the black bars and white bars, respectively (Figure 3).

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