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B-cell receptor (BCR) signals are essential for B-cell differentiation, homeostasis and negative selection, which are regulated by the strength and quality of BCR signals. Recently, we identified a new adaptor protein, Swiprosin-1, in lipid rafts of B-cell lines that undergo apoptosis after BCR stimulation. During murine B-cell development, Swiprosin-1 exhibited highest expression in immature B cells of the bone marrow, but was also expressed in resting and activated splenic B cells and in non-lymphoid tissue, especially in the brain. Ectopic expression of Swiprosin-1 in the immature murine B-cell line WEHI231 enhanced spontaneous and BCR-induced apoptosis. In contrast, short hairpin RNA (shRNA)-mediated downregulation of Swiprosin-1 impaired specifically spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle arrest and upregulation of the cell cycle inhibitor p27(Kip1). In accordance, Swiprosin-1 abundance regulated net cell growth of WEHI231 cell populations through reciprocal regulation of Bcl-xL, but not Bim, thereby controlling spontaneous apoptosis. Swiprosin-1-enhanced apoptosis was blocked through nuclear factor kappaB-activating stimuli, namely B-cell-activating factor of the TNF family, anti-CD40 and lipopolysaccharide (LPS). This correlated with enhanced BCR-induced IkappaB-alpha phosphorylation and degradation in cells expressing a Swiprosin-1-specific shRNA. Finally, ectopic Swiprosin-1 expression enhanced BCR-induced cell death in primary, LPS-stimulated splenic B cells. Hence, Swiprosin-1 may regulate lifespan and BCR signaling thresholds in immature B cells.

Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.

The transmembrane receptor Notch1 plays a crucial role in differentiation and apoptosis of hematopoietic cells. To investigate the influence of Notch1 on apoptosis and cell growth of mature murine B cells, we transduced the murine B-lymphoma line NYC 31.1 with a constitutively active, intracellular form of human Notch1 (Notch1-ICT). NYC cells represent mature activated B cells that can be induced to undergo apoptosis by crosslinking of the B-cell receptor (BCR). In contrast to investigations in immature chicken B-cell lines, transduced Notch1-ICT did not affect cell cycle progression, cell growth or surface IgM levels in NYC cells and resulted only in a slight induction of apoptosis. However, BCR-crosslinking enhanced apoptosis, but did not influence cell cycle progression in Notch1-ICT-positive NYC cells. These data imply a distinct function of Notch1 in mature murine B-cells as compared to immature chicken B cells and provide further evidence for Notch1's involvement in B-cell differentiation and development.