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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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P2X7 receptor expression and ATP release of MNL. (a–c) show unchallenged MNL examined by FACS and (d and e) show leukotoxin-exposed MNL. (a) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. (b) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. (c) Illustration of P2X7R expression in R1 and R2 by representative histograms. (d) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μM). (e) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)
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fig6: P2X7 receptor expression and ATP release of MNL. (a–c) show unchallenged MNL examined by FACS and (d and e) show leukotoxin-exposed MNL. (a) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. (b) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. (c) Illustration of P2X7R expression in R1 and R2 by representative histograms. (d) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μM). (e) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)

Mentions: The two different populations, R1 and R2, in the MNL-suspension were gated (Figure 6a) and the composition of leukocytes subsets (CD14+, CD3+ and CD19+) and P2X7R expression was documented by specific antibodies analyzed by FACS (Figure 6b). The majority of the P2X7R+ cells were found in the R1-population, which also consisted of mainly CD14+ cells, while the R2-population primarily was CD3+ cells without P2X7R-expression (Figures 6b and c). The high leukotoxin-sensitivity of the R1-population was further indicated by PI-staining examined by FACS. The leukotoxin-exposed (10 ng/ml) MNL showed a time-dependent decrease in living cells for the R1-population, but less pronounced for the R2-population (Figure 6d). Presence of oATP inhibited the leukotoxin-induced decrease in viable MNL (Figure 6d). Further, luminescence analysis of ATP-release from the macrophages showed a rapid extracellular ATP release induced by the leukotoxin (1 and 10 ng/ml) (Figure 6e).


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)

P2X7 receptor expression and ATP release of MNL. (a–c) show unchallenged MNL examined by FACS and (d and e) show leukotoxin-exposed MNL. (a) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. (b) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. (c) Illustration of P2X7R expression in R1 and R2 by representative histograms. (d) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μM). (e) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: P2X7 receptor expression and ATP release of MNL. (a–c) show unchallenged MNL examined by FACS and (d and e) show leukotoxin-exposed MNL. (a) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. (b) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. (c) Illustration of P2X7R expression in R1 and R2 by representative histograms. (d) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μM). (e) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)
Mentions: The two different populations, R1 and R2, in the MNL-suspension were gated (Figure 6a) and the composition of leukocytes subsets (CD14+, CD3+ and CD19+) and P2X7R expression was documented by specific antibodies analyzed by FACS (Figure 6b). The majority of the P2X7R+ cells were found in the R1-population, which also consisted of mainly CD14+ cells, while the R2-population primarily was CD3+ cells without P2X7R-expression (Figures 6b and c). The high leukotoxin-sensitivity of the R1-population was further indicated by PI-staining examined by FACS. The leukotoxin-exposed (10 ng/ml) MNL showed a time-dependent decrease in living cells for the R1-population, but less pronounced for the R2-population (Figure 6d). Presence of oATP inhibited the leukotoxin-induced decrease in viable MNL (Figure 6d). Further, luminescence analysis of ATP-release from the macrophages showed a rapid extracellular ATP release induced by the leukotoxin (1 and 10 ng/ml) (Figure 6e).

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