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Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival.

Ekoff M, Lyberg K, Krajewska M, Arvidsson M, Rak S, Reed JC, Harvima I, Nilsson G - PLoS ONE (2012)

Bottom Line: FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1.ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L), Bcl-2, Bcl-w and Mcl-1.Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden. maria.ekoff@ki.se

ABSTRACT
Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine). Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L), Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

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siRNA targeting BFL-1 but not MCL-1 diminishes activation-induced survival of human mast cells.(A) The upregulation of BFL-1 and MCL-1 following IgECL in CBMCs is abolished following targeted siRNA treatment as verified 30 hours post-transfection by quantative PCR. Cells were transfected with 100 nM siRNA, sensitized with 1 μg/mL of IgE and challenged with 2 μg/mL anti-IgE before expression was determined. Data correspond to one representative experiment using one donor. Similar result was obtained for another donor. (B) BFL-1 but not MCL-1 siRNA treated CBMCs show decreased survival upon IgECL compared to control siRNA treated cells. Cells were transfected, sensitized with 1 μg/mL of IgE and 24 hours post-transfection cytokine-deprived and challenged with 2 μg/mL anti-IgE before being enumerated 24 hours later using the vital dye trypan blue. N=6. (C) The upregulation of BFL-1 and MCL-1 following IgECL in LAD-2 cells is abolished following targeted siRNA treatment as described above. (D) BFL-1 but not MCL-1 siRNA treated LAD-2 cells show decreased survival upon IgECL compared to control siRNA treated cells. N=4. Change in viability is percentage viable cytokine-deprived cells deducted from viable cytokine-deprived cells following IgECL. (E) Bfl-1 is down-regulated in LAD-2 cells following siRNA treatment targeting BFL-1 as compared to control siRNA in immunohistochemical stainings for Bfl-1 expression.
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pone-0039117-g003: siRNA targeting BFL-1 but not MCL-1 diminishes activation-induced survival of human mast cells.(A) The upregulation of BFL-1 and MCL-1 following IgECL in CBMCs is abolished following targeted siRNA treatment as verified 30 hours post-transfection by quantative PCR. Cells were transfected with 100 nM siRNA, sensitized with 1 μg/mL of IgE and challenged with 2 μg/mL anti-IgE before expression was determined. Data correspond to one representative experiment using one donor. Similar result was obtained for another donor. (B) BFL-1 but not MCL-1 siRNA treated CBMCs show decreased survival upon IgECL compared to control siRNA treated cells. Cells were transfected, sensitized with 1 μg/mL of IgE and 24 hours post-transfection cytokine-deprived and challenged with 2 μg/mL anti-IgE before being enumerated 24 hours later using the vital dye trypan blue. N=6. (C) The upregulation of BFL-1 and MCL-1 following IgECL in LAD-2 cells is abolished following targeted siRNA treatment as described above. (D) BFL-1 but not MCL-1 siRNA treated LAD-2 cells show decreased survival upon IgECL compared to control siRNA treated cells. N=4. Change in viability is percentage viable cytokine-deprived cells deducted from viable cytokine-deprived cells following IgECL. (E) Bfl-1 is down-regulated in LAD-2 cells following siRNA treatment targeting BFL-1 as compared to control siRNA in immunohistochemical stainings for Bfl-1 expression.

Mentions: Having demonstrated a minor role of Bcl-XL, Bcl-2, Bcl-W and possibly also Mcl-1 we next examined how inhibition of BFL-1 affects activation-induced survival of CBMC and LAD-2 cells. To inhibit gene expression, cells were electroporated using siRNA oligonucleotides targeting BFL-1, MCL-1 or non-targeting siRNA. Activation-induced upregulation of both BFL-1 and MCL-1 at the mRNA level following IgECL were abolished by siRNA treatment, as demonstrated by quantative PCR (Fig. 3A and C). The expression of BCL-2, BCL-XL, or C-KIT was not affected (data not shown). In contrast, activation-induced survival was diminished by BFL-1 targeting siRNA but not by MCL-1 targeting siRNA (Fig. 3B and D). We could also demonstrate, by immunohistochemical staining, Bfl-1 to be down-regulated on the protein level in CBMC and LAD-2 cells by BFL-1 siRNA treatment as compared to non-targeting siRNA (fig. 3E). Taken together our results demonstrate the critical role of Bfl-1 in prolonging the survival of cytokine-deprived human mast cells after IgECL and support our hypothesis that Bfl-1 is a major survival factor promoting activation-induced survival. Mcl-1 by comparison, seems to play a minor role in activation-induced mast cell survival, even though IgECL induces Mcl-1 expression.


Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival.

Ekoff M, Lyberg K, Krajewska M, Arvidsson M, Rak S, Reed JC, Harvima I, Nilsson G - PLoS ONE (2012)

siRNA targeting BFL-1 but not MCL-1 diminishes activation-induced survival of human mast cells.(A) The upregulation of BFL-1 and MCL-1 following IgECL in CBMCs is abolished following targeted siRNA treatment as verified 30 hours post-transfection by quantative PCR. Cells were transfected with 100 nM siRNA, sensitized with 1 μg/mL of IgE and challenged with 2 μg/mL anti-IgE before expression was determined. Data correspond to one representative experiment using one donor. Similar result was obtained for another donor. (B) BFL-1 but not MCL-1 siRNA treated CBMCs show decreased survival upon IgECL compared to control siRNA treated cells. Cells were transfected, sensitized with 1 μg/mL of IgE and 24 hours post-transfection cytokine-deprived and challenged with 2 μg/mL anti-IgE before being enumerated 24 hours later using the vital dye trypan blue. N=6. (C) The upregulation of BFL-1 and MCL-1 following IgECL in LAD-2 cells is abolished following targeted siRNA treatment as described above. (D) BFL-1 but not MCL-1 siRNA treated LAD-2 cells show decreased survival upon IgECL compared to control siRNA treated cells. N=4. Change in viability is percentage viable cytokine-deprived cells deducted from viable cytokine-deprived cells following IgECL. (E) Bfl-1 is down-regulated in LAD-2 cells following siRNA treatment targeting BFL-1 as compared to control siRNA in immunohistochemical stainings for Bfl-1 expression.
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getmorefigures.php?uid=PMC3376125&req=5

pone-0039117-g003: siRNA targeting BFL-1 but not MCL-1 diminishes activation-induced survival of human mast cells.(A) The upregulation of BFL-1 and MCL-1 following IgECL in CBMCs is abolished following targeted siRNA treatment as verified 30 hours post-transfection by quantative PCR. Cells were transfected with 100 nM siRNA, sensitized with 1 μg/mL of IgE and challenged with 2 μg/mL anti-IgE before expression was determined. Data correspond to one representative experiment using one donor. Similar result was obtained for another donor. (B) BFL-1 but not MCL-1 siRNA treated CBMCs show decreased survival upon IgECL compared to control siRNA treated cells. Cells were transfected, sensitized with 1 μg/mL of IgE and 24 hours post-transfection cytokine-deprived and challenged with 2 μg/mL anti-IgE before being enumerated 24 hours later using the vital dye trypan blue. N=6. (C) The upregulation of BFL-1 and MCL-1 following IgECL in LAD-2 cells is abolished following targeted siRNA treatment as described above. (D) BFL-1 but not MCL-1 siRNA treated LAD-2 cells show decreased survival upon IgECL compared to control siRNA treated cells. N=4. Change in viability is percentage viable cytokine-deprived cells deducted from viable cytokine-deprived cells following IgECL. (E) Bfl-1 is down-regulated in LAD-2 cells following siRNA treatment targeting BFL-1 as compared to control siRNA in immunohistochemical stainings for Bfl-1 expression.
Mentions: Having demonstrated a minor role of Bcl-XL, Bcl-2, Bcl-W and possibly also Mcl-1 we next examined how inhibition of BFL-1 affects activation-induced survival of CBMC and LAD-2 cells. To inhibit gene expression, cells were electroporated using siRNA oligonucleotides targeting BFL-1, MCL-1 or non-targeting siRNA. Activation-induced upregulation of both BFL-1 and MCL-1 at the mRNA level following IgECL were abolished by siRNA treatment, as demonstrated by quantative PCR (Fig. 3A and C). The expression of BCL-2, BCL-XL, or C-KIT was not affected (data not shown). In contrast, activation-induced survival was diminished by BFL-1 targeting siRNA but not by MCL-1 targeting siRNA (Fig. 3B and D). We could also demonstrate, by immunohistochemical staining, Bfl-1 to be down-regulated on the protein level in CBMC and LAD-2 cells by BFL-1 siRNA treatment as compared to non-targeting siRNA (fig. 3E). Taken together our results demonstrate the critical role of Bfl-1 in prolonging the survival of cytokine-deprived human mast cells after IgECL and support our hypothesis that Bfl-1 is a major survival factor promoting activation-induced survival. Mcl-1 by comparison, seems to play a minor role in activation-induced mast cell survival, even though IgECL induces Mcl-1 expression.

Bottom Line: FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1.ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L), Bcl-2, Bcl-w and Mcl-1.Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden. maria.ekoff@ki.se

ABSTRACT
Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine). Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L), Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

Show MeSH
Related in: MedlinePlus