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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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Effects of A. actinomycetemcomitans leukotoxin (Ltx) on human macrophage IL-1β production and secretion. (a) ELISA quantification of the cell-associated and secreted IL-1β from macrophages exposed to 10 ng/ml A. actinomycetemcomitans Ltx or 100 ng/ml E. coli LPS (LPS) for 180 min. Mean values±S.D. of seven experiments with macrophages obtained from different donors. (b) Real-time RT-PCR quantification of mRNA of IL-1β in macrophages exposed to Ltx (10 ng/ml) or LPS (100 ng/ml) for 60 min in relation to the endogenous control GAPDH. Mean values±S.D. of three experiments from one macrophage donor. (c) Levels of mRNA for IL-1β in relation to the endogenous control GAPDH quantified by real time RT-PCR in macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (d) ELISA quantification of IL-1β secreted from macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (e) ELISA quantification of the cell-associated and secreted amounts of pro-IL-1β and IL-1β of macrophages exposed for 60 min to 10 ng/ml Ltx or 10 ng/ml heat-inactivated (HI) Ltx (pretreated at 70 °C for 30 min). Mean values±S.D. from three experiments from one macrophage donor. (f) Western blot analyses of cell lysates (Pellet) and supernatants (Sup) of macrophages exposed to 10 ng/ml Ltx or 100 ng/ml LPS for 60 min. GAPDH was used as endogenous control. Recombinant human IL-1β (rh IL1β, left lower band) was used for verification of the position of the 17 kDa IL-1β (active form). Representative results from two experiments with different macrophage donors are shown
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fig2: Effects of A. actinomycetemcomitans leukotoxin (Ltx) on human macrophage IL-1β production and secretion. (a) ELISA quantification of the cell-associated and secreted IL-1β from macrophages exposed to 10 ng/ml A. actinomycetemcomitans Ltx or 100 ng/ml E. coli LPS (LPS) for 180 min. Mean values±S.D. of seven experiments with macrophages obtained from different donors. (b) Real-time RT-PCR quantification of mRNA of IL-1β in macrophages exposed to Ltx (10 ng/ml) or LPS (100 ng/ml) for 60 min in relation to the endogenous control GAPDH. Mean values±S.D. of three experiments from one macrophage donor. (c) Levels of mRNA for IL-1β in relation to the endogenous control GAPDH quantified by real time RT-PCR in macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (d) ELISA quantification of IL-1β secreted from macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (e) ELISA quantification of the cell-associated and secreted amounts of pro-IL-1β and IL-1β of macrophages exposed for 60 min to 10 ng/ml Ltx or 10 ng/ml heat-inactivated (HI) Ltx (pretreated at 70 °C for 30 min). Mean values±S.D. from three experiments from one macrophage donor. (f) Western blot analyses of cell lysates (Pellet) and supernatants (Sup) of macrophages exposed to 10 ng/ml Ltx or 100 ng/ml LPS for 60 min. GAPDH was used as endogenous control. Recombinant human IL-1β (rh IL1β, left lower band) was used for verification of the position of the 17 kDa IL-1β (active form). Representative results from two experiments with different macrophage donors are shown

Mentions: The lactate dehydrogenase (LDH) leakage from human macrophages exposed to various concentrations of A. actinomycetemcomitans leukotoxin for 60 min indicated that a dose-dependent disruption of the membrane integrity occurred when leukotoxin concentrations were ≥1 ng/ml (Figure 1a). The uptake of early apoptotic (Yo-PRO-1) and necrotic/late apoptotic (propidium iodide, PI) markers in macrophages exposed to 1 or 10 ng/ml of leukotoxin (60 min) showed a similar pattern for both markers (Figure 1b). The morphology of the macrophages exposed to these concentrations of leukotoxin for 60 min was further analyzed by transmission electron microscopy (TEM) (Figure 1c). The proportion of normal cells (Figure 1c-i) decreased, while the proportions of necrotic cells (Figures 1c-II and c-IV) and apoptotic cells (Figures 1c-III and c-V) increased. Leukotoxin caused an activation and secretion of IL-18 and IL-1β from the affected macrophages (Figures 2d and e). Taken together, these results indicated that there existed a threshold level for leukotoxin sensitivity in each cell and that each of the affected cells activated signaling pathways that eventually led to cell death.


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)

Effects of A. actinomycetemcomitans leukotoxin (Ltx) on human macrophage IL-1β production and secretion. (a) ELISA quantification of the cell-associated and secreted IL-1β from macrophages exposed to 10 ng/ml A. actinomycetemcomitans Ltx or 100 ng/ml E. coli LPS (LPS) for 180 min. Mean values±S.D. of seven experiments with macrophages obtained from different donors. (b) Real-time RT-PCR quantification of mRNA of IL-1β in macrophages exposed to Ltx (10 ng/ml) or LPS (100 ng/ml) for 60 min in relation to the endogenous control GAPDH. Mean values±S.D. of three experiments from one macrophage donor. (c) Levels of mRNA for IL-1β in relation to the endogenous control GAPDH quantified by real time RT-PCR in macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (d) ELISA quantification of IL-1β secreted from macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (e) ELISA quantification of the cell-associated and secreted amounts of pro-IL-1β and IL-1β of macrophages exposed for 60 min to 10 ng/ml Ltx or 10 ng/ml heat-inactivated (HI) Ltx (pretreated at 70 °C for 30 min). Mean values±S.D. from three experiments from one macrophage donor. (f) Western blot analyses of cell lysates (Pellet) and supernatants (Sup) of macrophages exposed to 10 ng/ml Ltx or 100 ng/ml LPS for 60 min. GAPDH was used as endogenous control. Recombinant human IL-1β (rh IL1β, left lower band) was used for verification of the position of the 17 kDa IL-1β (active form). Representative results from two experiments with different macrophage donors are shown
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig2: Effects of A. actinomycetemcomitans leukotoxin (Ltx) on human macrophage IL-1β production and secretion. (a) ELISA quantification of the cell-associated and secreted IL-1β from macrophages exposed to 10 ng/ml A. actinomycetemcomitans Ltx or 100 ng/ml E. coli LPS (LPS) for 180 min. Mean values±S.D. of seven experiments with macrophages obtained from different donors. (b) Real-time RT-PCR quantification of mRNA of IL-1β in macrophages exposed to Ltx (10 ng/ml) or LPS (100 ng/ml) for 60 min in relation to the endogenous control GAPDH. Mean values±S.D. of three experiments from one macrophage donor. (c) Levels of mRNA for IL-1β in relation to the endogenous control GAPDH quantified by real time RT-PCR in macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (d) ELISA quantification of IL-1β secreted from macrophages exposed to Ltx (0–60 min) or LPS (60 min). Mean values±S.D. from three experiments from one macrophage donor. (e) ELISA quantification of the cell-associated and secreted amounts of pro-IL-1β and IL-1β of macrophages exposed for 60 min to 10 ng/ml Ltx or 10 ng/ml heat-inactivated (HI) Ltx (pretreated at 70 °C for 30 min). Mean values±S.D. from three experiments from one macrophage donor. (f) Western blot analyses of cell lysates (Pellet) and supernatants (Sup) of macrophages exposed to 10 ng/ml Ltx or 100 ng/ml LPS for 60 min. GAPDH was used as endogenous control. Recombinant human IL-1β (rh IL1β, left lower band) was used for verification of the position of the 17 kDa IL-1β (active form). Representative results from two experiments with different macrophage donors are shown
Mentions: The lactate dehydrogenase (LDH) leakage from human macrophages exposed to various concentrations of A. actinomycetemcomitans leukotoxin for 60 min indicated that a dose-dependent disruption of the membrane integrity occurred when leukotoxin concentrations were ≥1 ng/ml (Figure 1a). The uptake of early apoptotic (Yo-PRO-1) and necrotic/late apoptotic (propidium iodide, PI) markers in macrophages exposed to 1 or 10 ng/ml of leukotoxin (60 min) showed a similar pattern for both markers (Figure 1b). The morphology of the macrophages exposed to these concentrations of leukotoxin for 60 min was further analyzed by transmission electron microscopy (TEM) (Figure 1c). The proportion of normal cells (Figure 1c-i) decreased, while the proportions of necrotic cells (Figures 1c-II and c-IV) and apoptotic cells (Figures 1c-III and c-V) increased. Leukotoxin caused an activation and secretion of IL-18 and IL-1β from the affected macrophages (Figures 2d and e). Taken together, these results indicated that there existed a threshold level for leukotoxin sensitivity in each cell and that each of the affected cells activated signaling pathways that eventually led to cell death.

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