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Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin.

Kelk P, Abd H, Claesson R, Sandström G, Sjöstedt A, Johansson A - Cell Death Dis (2011)

Bottom Line: This toxin selectively kills human leukocytes by inducing apoptosis and lysis.A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18.In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Periodontology, Department of Odontology, Faculty of Medicine, Umeå University, Umeå, Sweden. peyman.kelk@odont.umu.se

ABSTRACT
Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.

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Related in: MedlinePlus

Caspase-1 and p38 inhibition, their effects on A. actinomycetemcomitans leukotoxin-induced lysis and IL-1β secretion from human macrophages. (a) Flow cytometric analysis of p38 and NF-κB (p65) phosphorylation in human macrophages exposed to 10 ng/ml Ltx for 5 min as compared with control cells. Mean values±S.D. of three experiments from macrophages obtained from different donors. (b) Extracellular release of LDH and (c) ELISA quantification of IL-1β secretion of human macrophages exposed to 0, 3 or 10 ng/ml leukotoxin for 60 min. The experiments were conducted without or with selective inhibitors: for p38 (SB 203580 or SKF-8600, 10 μM) or caspase-1 (Ac-YVAD-CMK or Z-VAD-FMK, 100 μM). Mean values±S.D. of four experiments with different macrophage donors
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fig3: Caspase-1 and p38 inhibition, their effects on A. actinomycetemcomitans leukotoxin-induced lysis and IL-1β secretion from human macrophages. (a) Flow cytometric analysis of p38 and NF-κB (p65) phosphorylation in human macrophages exposed to 10 ng/ml Ltx for 5 min as compared with control cells. Mean values±S.D. of three experiments from macrophages obtained from different donors. (b) Extracellular release of LDH and (c) ELISA quantification of IL-1β secretion of human macrophages exposed to 0, 3 or 10 ng/ml leukotoxin for 60 min. The experiments were conducted without or with selective inhibitors: for p38 (SB 203580 or SKF-8600, 10 μM) or caspase-1 (Ac-YVAD-CMK or Z-VAD-FMK, 100 μM). Mean values±S.D. of four experiments with different macrophage donors

Mentions: Western blot analyses of cell lysates from macrophages showed that exposure to leukotoxin (1 or 10 ng/ml) or LPS (100 ng/ml) for 15- or 30-min activated phosphorylation of mitogen-activated protein kinase (MAPK) p38 and the total levels of inhibitor of NF-κBα (IκBα) were not affected by the leukotoxin (data not shown). Time-course registration of p38 phosphorylation in macrophages by fluorescence-activated cell sorting (FACS) showed a more rapid effect by the leukotoxin than by the LPS (data not shown). Macrophages exposed to 10 ng/ml leukotoxin for 5 min showed a 10-fold increase in phosphorylated p38 as compared with the unchallenged control cells, while the levels of phosphorylated nuclear factor κB (p65) (NF-κB (p65)) in these cells were not affected (Figure 3a). To investigate the involvement of p38 and caspase-1 in the leukotoxin-induced cell lysis and IL-1β secretion from macrophages, the experiments were conducted in the presence of selective inhibitors. Neither of the two inhibitors used for p38 (SB 203580 or SKF-86002) affected the leukotoxin-induced cell lysis or IL-1β secretion (Figures 3b and c). In contrast, both selective inhibitors for caspase-1 (Ac-YVAD-CMK or Z-VAD-FMK) reduced the leukotoxin-induced lysis of macrophages to some extent (Figure 3b) and the leukotoxin-induced IL-1β secretion to levels similar to those found in unchallenged cells (Figure 3c). The combination of inhibitors for caspase-1 (Ac-YVAD-CMK) and p38 (SB 203580) had no additive inhibitory effect (Figures 3b and c). The presence of a p38 inhibitor (SB 203580) did not interfere with the secretion of any of the tested cytokines (IL-1β, tumor necrosis factor (TNF)-α and interleukin-6 (IL-6)) from leukotoxin-challenged macrophages, while this inhibitor (SB 203580) decreased the E. coli LPS-induced cytokine secretion (data not shown).


Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin.

Kelk P, Abd H, Claesson R, Sandström G, Sjöstedt A, Johansson A - Cell Death Dis (2011)

Caspase-1 and p38 inhibition, their effects on A. actinomycetemcomitans leukotoxin-induced lysis and IL-1β secretion from human macrophages. (a) Flow cytometric analysis of p38 and NF-κB (p65) phosphorylation in human macrophages exposed to 10 ng/ml Ltx for 5 min as compared with control cells. Mean values±S.D. of three experiments from macrophages obtained from different donors. (b) Extracellular release of LDH and (c) ELISA quantification of IL-1β secretion of human macrophages exposed to 0, 3 or 10 ng/ml leukotoxin for 60 min. The experiments were conducted without or with selective inhibitors: for p38 (SB 203580 or SKF-8600, 10 μM) or caspase-1 (Ac-YVAD-CMK or Z-VAD-FMK, 100 μM). Mean values±S.D. of four experiments with different macrophage donors
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101819&req=5

fig3: Caspase-1 and p38 inhibition, their effects on A. actinomycetemcomitans leukotoxin-induced lysis and IL-1β secretion from human macrophages. (a) Flow cytometric analysis of p38 and NF-κB (p65) phosphorylation in human macrophages exposed to 10 ng/ml Ltx for 5 min as compared with control cells. Mean values±S.D. of three experiments from macrophages obtained from different donors. (b) Extracellular release of LDH and (c) ELISA quantification of IL-1β secretion of human macrophages exposed to 0, 3 or 10 ng/ml leukotoxin for 60 min. The experiments were conducted without or with selective inhibitors: for p38 (SB 203580 or SKF-8600, 10 μM) or caspase-1 (Ac-YVAD-CMK or Z-VAD-FMK, 100 μM). Mean values±S.D. of four experiments with different macrophage donors
Mentions: Western blot analyses of cell lysates from macrophages showed that exposure to leukotoxin (1 or 10 ng/ml) or LPS (100 ng/ml) for 15- or 30-min activated phosphorylation of mitogen-activated protein kinase (MAPK) p38 and the total levels of inhibitor of NF-κBα (IκBα) were not affected by the leukotoxin (data not shown). Time-course registration of p38 phosphorylation in macrophages by fluorescence-activated cell sorting (FACS) showed a more rapid effect by the leukotoxin than by the LPS (data not shown). Macrophages exposed to 10 ng/ml leukotoxin for 5 min showed a 10-fold increase in phosphorylated p38 as compared with the unchallenged control cells, while the levels of phosphorylated nuclear factor κB (p65) (NF-κB (p65)) in these cells were not affected (Figure 3a). To investigate the involvement of p38 and caspase-1 in the leukotoxin-induced cell lysis and IL-1β secretion from macrophages, the experiments were conducted in the presence of selective inhibitors. Neither of the two inhibitors used for p38 (SB 203580 or SKF-86002) affected the leukotoxin-induced cell lysis or IL-1β secretion (Figures 3b and c). In contrast, both selective inhibitors for caspase-1 (Ac-YVAD-CMK or Z-VAD-FMK) reduced the leukotoxin-induced lysis of macrophages to some extent (Figure 3b) and the leukotoxin-induced IL-1β secretion to levels similar to those found in unchallenged cells (Figure 3c). The combination of inhibitors for caspase-1 (Ac-YVAD-CMK) and p38 (SB 203580) had no additive inhibitory effect (Figures 3b and c). The presence of a p38 inhibitor (SB 203580) did not interfere with the secretion of any of the tested cytokines (IL-1β, tumor necrosis factor (TNF)-α and interleukin-6 (IL-6)) from leukotoxin-challenged macrophages, while this inhibitor (SB 203580) decreased the E. coli LPS-induced cytokine secretion (data not shown).

Bottom Line: This toxin selectively kills human leukocytes by inducing apoptosis and lysis.A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18.In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Periodontology, Department of Odontology, Faculty of Medicine, Umeå University, Umeå, Sweden. peyman.kelk@odont.umu.se

ABSTRACT
Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1β and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.

Show MeSH
Related in: MedlinePlus