Regulation of mast cell survival and apoptosis
Author: Karlberg, Mats
Date: 2008-12-05
Location: Salen Medicin, huvudbyggnaden, huvudingången, plan 4, Hus A6:04, Karolinska universitetssjukhuset, Solna
Time: 09.00
Department: Institutionen för medicin / Department of Medicine
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Thesis (1.289Mb)
Abstract
Mature mast cells reside in the tissue as heavily granulated cells possessing a regulatory function in innate and adaptive immunity against pathogens, but also detrimentally in atopic, as well as, chronic inflammation-associated diseases such as allergic reactions and autoimmunity. Upon stimulation, mast cells release a vast variety of mediators that recruit and stimulate other immune cells, induce vasodilation and angiogenesis, and process and degrade proteins. Mast cells are long-lived cells and can survive the harsh process of degranulation followed upon IgE receptor activation. In human mast cells, IgE receptor activation induces increased expression of the anti-apoptotic protein Bfl-1/A1, which has been demonstrated crucial for activation-induced survival in mouse. Upon stimulation with the TH1 cytokine, IFNgamma mast cells upregulate the normally not expressed high affinity receptor for IgG. Here we show that IFNgamma stimulated human mast cells, activated via IgG receptor crosslinking, are rescued from cytokine deprivation-induced apoptosis and express increased levels of Bfl-1.
In mouse, the activation-induced mast cell survival has been correlated to the upregulation of A1. This anti-apoptotic protein is described to be under transcriptional control of the nuclear factor-kappaB (NF-kappaB), and being expressed upon activation in several cell types such as T- and B-lymphocytes. However, by using mast cells from NF-kappaB deficient mice, stable transfections of IkappaB-alpha super repressor, and promoter gene analysis with deleted NF-kappaB binding sites, we here demonstrate that NF-kappaB is not involved in activation-induced upregulation of A1 in mast cells. Instead, our data from using the calcineurin inhibitor, cyclosporin A, electrophoretic mobility shift assay and chromatin immunoprecipitation suggest NFAT to be the important regulator of A1 transcription. Bfl-1/A1 is a member of the Bcl-2 protein family, which control the intrinsic pathway of apoptosis. Interactions between pro- and anti-apoptotic members determine whether the cell will stay alive or enter apoptosis. Two pro-apoptotic proteins, Bax and Bak are effector proteins and essential for induction of apoptosis. We show that Bax has a prominent and Bak a minor role in mast cell apoptosis induced by cytokine deprivation. Only double deficient mast cells were totally rescued from cytokine deprivation-induced apoptosis, indicating that both proteins are contributing.
Activation of Bax and Bak is initialized by pro-apoptotic BH3-only members, which liberate them through interactions with the anti-apoptotic proteins. By using small molecular inhibitors of anti-apoptotic proteins, mimicking the BH3 binding domain, apoptosis can be induced. We demonstrate that mast cells enter apoptosis in presence of low concentrations of the BH3-only mimetic ABT-737. In vivo injections of the compound reveal a decrease in viability of both mast cells and lymphocytes. However, ex vivo treatment of peritoneal cells show a stronger response in mature mast cells than other cell types. Our data suggest ABT-737, or more bioavailable derivates of it, as a promising compound to be used in therapy to reduce mast cell number locally in tissue. Due to their potent orchestrating function of the immune system, mast cell activation can also have negative effects, for instance in autoimmune diseases and tumourigenesis. It is therefore important to understanding the regulation of survival and apoptosis in mast cells in order to control their effect. This thesis contributes by describing cell specific regulation of the survival protein A1, and by suggesting drug-induced apoptosis specifically in mast cells.
In mouse, the activation-induced mast cell survival has been correlated to the upregulation of A1. This anti-apoptotic protein is described to be under transcriptional control of the nuclear factor-kappaB (NF-kappaB), and being expressed upon activation in several cell types such as T- and B-lymphocytes. However, by using mast cells from NF-kappaB deficient mice, stable transfections of IkappaB-alpha super repressor, and promoter gene analysis with deleted NF-kappaB binding sites, we here demonstrate that NF-kappaB is not involved in activation-induced upregulation of A1 in mast cells. Instead, our data from using the calcineurin inhibitor, cyclosporin A, electrophoretic mobility shift assay and chromatin immunoprecipitation suggest NFAT to be the important regulator of A1 transcription. Bfl-1/A1 is a member of the Bcl-2 protein family, which control the intrinsic pathway of apoptosis. Interactions between pro- and anti-apoptotic members determine whether the cell will stay alive or enter apoptosis. Two pro-apoptotic proteins, Bax and Bak are effector proteins and essential for induction of apoptosis. We show that Bax has a prominent and Bak a minor role in mast cell apoptosis induced by cytokine deprivation. Only double deficient mast cells were totally rescued from cytokine deprivation-induced apoptosis, indicating that both proteins are contributing.
Activation of Bax and Bak is initialized by pro-apoptotic BH3-only members, which liberate them through interactions with the anti-apoptotic proteins. By using small molecular inhibitors of anti-apoptotic proteins, mimicking the BH3 binding domain, apoptosis can be induced. We demonstrate that mast cells enter apoptosis in presence of low concentrations of the BH3-only mimetic ABT-737. In vivo injections of the compound reveal a decrease in viability of both mast cells and lymphocytes. However, ex vivo treatment of peritoneal cells show a stronger response in mature mast cells than other cell types. Our data suggest ABT-737, or more bioavailable derivates of it, as a promising compound to be used in therapy to reduce mast cell number locally in tissue. Due to their potent orchestrating function of the immune system, mast cell activation can also have negative effects, for instance in autoimmune diseases and tumourigenesis. It is therefore important to understanding the regulation of survival and apoptosis in mast cells in order to control their effect. This thesis contributes by describing cell specific regulation of the survival protein A1, and by suggesting drug-induced apoptosis specifically in mast cells.
List of papers:
I. Karlberg M, Xiang Z, Nilsson G (2008). Fc gamma RI-mediated activation of human mast cells promotes survival and induction of the pro-survival gene Bfl-1. J Clin Immunol. 28(3): 250-5
Pubmed
II. Ullerås E, Karlberg M, Möller Westerberg C, Alfredsson J, Gerondakis S, Strasser A, Nilsson G (2008). NFAT but not NF-kappaB is critical for transcriptional induction of the prosurvival gene A1 after IgE receptor activation in mast cells. Blood. 111(6): 3081-9. Epub 2008 Jan 8
Pubmed
III. Karlberg M, Ekoff M, Labi V, Strasser A, Huang DCS, Nilsson G (2008). Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis. [Submitted]
IV. Karlberg M, Nilsson G (2008). The BH3-mimetic ABT-737 induces mast cell apoptosis in vitro and in vivo. [Manuscript]
I. Karlberg M, Xiang Z, Nilsson G (2008). Fc gamma RI-mediated activation of human mast cells promotes survival and induction of the pro-survival gene Bfl-1. J Clin Immunol. 28(3): 250-5
Pubmed
II. Ullerås E, Karlberg M, Möller Westerberg C, Alfredsson J, Gerondakis S, Strasser A, Nilsson G (2008). NFAT but not NF-kappaB is critical for transcriptional induction of the prosurvival gene A1 after IgE receptor activation in mast cells. Blood. 111(6): 3081-9. Epub 2008 Jan 8
Pubmed
III. Karlberg M, Ekoff M, Labi V, Strasser A, Huang DCS, Nilsson G (2008). Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis. [Submitted]
IV. Karlberg M, Nilsson G (2008). The BH3-mimetic ABT-737 induces mast cell apoptosis in vitro and in vivo. [Manuscript]
Issue date: 2008-11-14
Rights:
Publication year: 2008
ISBN: 978-91-7409-234-9
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