title: Dissection of the differential mechanisms of apoptosis used during b cell maturation
Creators: name: Carter, Natalie Annis
Publisher: name: University of Glasgow
Year: 2005
Subjects: subject: Cellular biology.

description: Ligation of the B cell receptor (BCR) on B lymphocytes results in differential biological outcomes depending on the maturation state of the cell. Thus, mature B lymphocytes become activated and proliferate (clonal expansion) in response to BCR crosslinking, whilst immature B cells either become unresponsive, alter the specificity of their BCR, or undergo apoptosis (clonal deletion). Furthermore, mature B cells can be induced to undergo growth arrest and apoptosis following coligation of the BCR and Fc?Rllb by immune complexes during negative feedback of B cell responses. The precise molecular events downstream of BCR signalling that are responsible for these distinct outcomes remain to be established. This study has focused upon identifying the important signalling mechanisms linking BCR ligation with regulation of the cell cycle and induction of apoptosis in immature and mature B lymphocytes. The murine B cell line WEHI 231 is widely used as a model for clonal deletion of immature B lymphocytes. This is because it has the cell surface phenotype of an immature B lymphocyte, and as such responds to BCR ligation by undergoing growth arrest and apoptosis. Moreover, WEHI 231 cells can be rescued from BCR-mediated apoptosis by co-engagement of CD40, mimicking T cell help. Such BCR-mediated apoptosis has previously been associated with both mitochondrial translocation and activation of PLA2 and a loss of mitochondrial membrane integrity. This study extended these findings by identifying that the product of PLA2, arachidonic acid, acts as the causal metabolite in the initiation of such BCR-mediated apoptosis. Furthermore, it demonstrated that the metabolism of arachidonic acid can provide a dynamic switch from apoptotic to proliferative signalling. For example, ligation of the BCR leads to the build up of arachidonic acid which leads to disruption of the mitochondrial membrane potential (MMP) and hence apoptosis. Whereas, in contrast to this, co-engagement of CD40 leads to the induction of C0X2 and hence the metabolism of arachidonic acid to prostaglandin E2 (PGE2) which promotes proliferative signalling. Therefore arachidonic acid metabolism acts as a dynamic, molecular switch from apoptotic to proliferative signalling. This study also demonstrated that upregulation of BclXL(as induced by CD40) acts to protect from arachidonic acid-mediated loss of mitochondrial membrane integrity and apoptosis. Furthermore, co-engagement of CD40 was found to increase the association of Bak with BclXL indicating that BclXL may act to sequester the pro-apoptotic Bak and hence inhibit the opening of the mitochondrial permeability pore. These data suggest that modulation of Bcl-2 family member induction and function is integral to such BCR-mediated apoptosis and T cell-derived rescue. In addition, it has been demonstrated that upregulation of BclXL can additionally function to downmodulate BCR-induced calcium signals providing an additional mechanism for the downregulation of mPLA2 activation and hence consequent apoptotic signalling. Previous work in this laboratory had highlighted the importance of dynamic ERK signalling in both proliferation and apoptosis of immature B cells. Thus, during BCR-mediated growth arrest and apoptosis the sustained ERK signalling, found in proliferating WEHI 231 cells. Is abrogated. Therefore a causal role for such sustained ERK signalling in immature B cell proliferation was investigated by manipulating endogenous ERK activation by expressing constitutively active RasV12 constructs in WEHI 231 cells. Such RasV12 expressing WEHI 231 cells were refractory to both BCR-mediated growth arrest and apoptosis for up to 24 h emphasising the importance of the uncoupling of ERK signalling in BCR- mediated cell cycle arrest and apoptosis. In an attempt to identify key regulatory elements involved, the role of SHIP and Dok in targeting the ERKMAPK pathway was explored by expressing dominant negative constructs. However, these constructs did not provide release from BCR-mediated apoptosis. (Abstract shortened by ProQuest.).