Mitochondrial regulation of apoptosis during B cell selection

Katz, Elad (2000) Mitochondrial regulation of apoptosis during B cell selection. PhD thesis, University of Glasgow.

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Abstract

This thesis has focused on identifying key signalling events during antigen-driven B cell development, particularly those concerned with the regulation and commitment to apoptosis. The research comprised both biochemical and physiological aspects of cell fate decisions, mainly focusing on the immature-mature B cell transition, as well as other later selection events in the spleen, lymph nodes and the peritoneal cavity. With respect to the molecular aspects, the work has concentrated mainly on identifying mitochondria-related changes and the role of cell cycle proteins in the events downstream of B cell receptor (BCR) ligation which lead to apoptosis. A novel phospholipase A2-dependent mechanism of mitochondrial disruption was identified as being responsible for the transduction of antigen receptor-mediated apoptosis of an immature B cell line, WEHI-231. The product of phospholipase A2, arachidonic acid, rather than one of its metabolites, directly causes a collapse in the mitochondrial potential which results in apoptosis. Regulation of this mitochondrial dysfunction also involves the Bcl-2 family. Pro-apoptotic and anti-apoptotic members of this family are both elevated in response to BCR stimulation. Nonetheless, a rescue signal via CD40 elevates BCl-XL expression and prevents Bid and Bad from translocating to the mitochondria. Indeed, BCl-XL overexpression is sufficient to reverse both BCR and arachidonic acid- induced apoptosis in WEHI-231 cells. Intriguingly, this novel cPLA2- dependent pathway of BCR-induced apoptosis in WEHI-231 is executed in a Caspase-independent manner and hence differs from the classical description of apoptosis. This novel form of apoptosis appears to be excuted by Cathepsin B which appears to act as an alternative killer protease to exert the destruction of cell components. In order to address the physiological relevance of these findings, an ex vivo model has also been devised to study the development of immature B cells in the bone marrow. This model has been used to identify the major cellular intermediates in this process and to observe some of the signals required for immature and transitional B cell selection. The detection of different B cell populations and determination of their cell cycle profiles, after BCR stimulation, has allowed the determination of sequence of events during B cell selection. For example, immature B cell apoptosis bears some of the hallmarks of the mechanisms identified in WEHI-231 B cells. In addition, this model has made it possible to examine the key factors regulating transitional B cell deletion. Interestingly, the use of Xid mice, which are defient in the PTK Btk, showed that Btk was essential for the maturation of transitional B cells in response to BCR stimulation. Moreover, it was shown that transitional B cells can undergo apoptosis in response to anti-Fas treatment and this may involve also a collapse in the mitochondrial potential. Further work with cells from mice immunocompromised by a parasite-derived molecule, ES-62 which is capable of desensitising BCR stimulation in vivo, showed that such proteins can also compromise the maturation of B cells in the bone marrow. This results in the breaking of tolerance and greater rates of B cell maturation, presumably as a consequence of ES-62 raising the threshold of apoptosis. Taken together, these observations challenge the instructive model for B cell selection, which relies on BCR-specificity being the sole determinant of cell fate decisions. This new evidence suggests that B cell selection may also be dependent on stochastic parameters such as Fas expression, cell density or other environmental factors. Similar investigations were performed on B cells from the periphery, which are involved in cell fate determination, in particular, B cells from Germinal Centres and the peritoneal cavity. It was established that apoptosis and proliferation of B cells differed depending on their maturation and activation status. These novel observations tracked the formation of Germinal Centres, at both sites, ex vivo. The molecular mechanisms regulating the responses of Germinal Centre B cells were also explored. Two main observations were made. Firstly, Btk-/- Germinal Centre B cells were found to be hyperproliferative in response to BCR stimulation, whilst non- Germinal Centre B cells were found to be hyposensitive. Secondly, BCR-induced apoptosis of Germinal Centre B cells appeared to be Caspase-dependent, similarly to that observed for transitional B cells. Furthermore, using HEL-BCR transgenic mice, it was shown that the primary and secondary responses to antigen are different in the spleen and in the lymph nodes. Finally, B-1 cells from the peritoneal cavity were shown to exhibit restricted proliferative responses and did not respond strongly to a secondary stimulation via the BCR. In addition, and in contrast to the situation in immature B cells, but similar to Germinal Centre B cells, BCR-induced apoptosis of B-1 cells appears Caspase-dependent.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Maggie Harnett
Keywords: Biochemistry, Cellular biology, Developmental biology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-72772
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72772

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