The impact of Bcl-2 family members on mast cell survival and apoptosis

Sammanfattning: Mast cells are long-lived effector cells of the immune system perhaps best known for their involvement in allergic diseases. There are several acute and chronic inflammations where mast cell accumulation, activation and release of mediators are important for the initiation and perpetuation of the inflammation. Better knowledge of mechanisms regulating the number of mast cells and their activity is desirable. Mast cells do not represent a homogenous population as the surrounding microenvironment will affect their effector profile and numbers. It is known that, in contrast to many inflammatory cells, activated mast cells have the capacity to recover and thereby be activated again (Fc epsilon Receptor I (FcepsilonRI) activation-induced survival). In this thesis we investigated the effect of B-cell lymphoma-2 (Bcl-2) family members on mast cell survival and apoptosis in murine and human mast cells. Murine bone marrow cells were cultured in two different ways, generating mucosal-like mast cells (MLMCs) and connective tissue-like mast cells (CTLMCs). Our in vitro-derived MLMCs and CTLMCs were found to display similar differences in chymase expression, proliferation rate and histamine content as mucosal mast cells (MMCs) and connective tissue mast cells (CTMCs) in vivo. This suggests that MLMCs and CTLMCs represent a useful in vitro model for committed mast cell lineages. Moreover, we found that CTLMCs, but not MLMCs, exhibit upregulation of the anti-apoptotic Bcl-2 family member A1 and activation-induced survival upon Fc?RI crosslinking. Similarly to murine mast cells, Fc?RI crosslinking of in vitro-derived human mast cells lead to upregulation of the human homologue of A1, bfl-1, and by the use of bfl-1 siRNA we demonstrate bfl-1 to be crucial for activation-induced human mast cell survival. Furthermore, the activation-induced survival of human mast cells is sustained in response to the inhibitors ABT-737 and roscovitine which indicate a minor role for the targeted anti-apoptotic Bcl-2 family members Bcl-XL, Bcl-2, Bcl-w and Myeloid cell leukemia-1 (Mcl-1). Taken together, we provide evidence that mast cell populations differ in their ability to survive allergic reactions and identify the Bcl-2 family member A1/Bfl-1 as a potential target for treatment of allergic diseases. The Bcl-2 homlogy 3 (BH3)-only protein Bcl-2-interacting modulator of cell death (Bim) has been found to play a role in cytokine deprivation-induced apoptosis of murine mast cells although overexpression of anti-apoptotic Bcl-2 protects mast cells more potently than loss of Bim. This indicates that other proteins, besides Bim, might be involved in this process. We describe the BH3-only protein p53 upregulated modulator of apoptosis (Puma) to be critical for the induction of mast cell apoptosis following cytokine deprivation and treatment with the DNA-damaging agent etoposide. Our data also suggest the involvement of the transcription factor Forkhead box O3A (FOXO3a) in the regulation of cytokine deprivation-induced apoptosis and the expression of Puma. Mast cells deficient for FOXO3a were markedly resistant to cytokine deprivation and overexpression of constitutively active FOXO3a caused an upregulation of Puma. We further examined the role of the two pro-apoptotic effector proteins Bcl-2-associated X protein (Bax) and Bcl-2 homologue antagonist/killer (Bak) in cytokine deprivation-induced apoptosis. Although both proteins were expressed we found a major role for Bax but not Bak in mediating mast cell apoptosis. Taken together, this identifies the pro-apoptotic Bcl-2 family members Puma and Bax to be critical for induction of apoptosis which suggest a plausible role for these pro-apoptotic proteins in the regulation of mast cell numbers in vivo.