Rescue from anti‐IgM‐induced programmed cell death by the B cell surface proteins CD20 and CD40

Abstract

Programmed cell death (PCD), or apoptosis, is characterized by several morphologic alterations and eventual cleavage of nuclear DNA into oligonucleo-some-length fragments. We defined a human B cell line, Ramos, that responds with PCD following ligation of surface IgM. Of the DNA in Ramos cells 3%-10% was fragmented as early as 4 h after IgM ligation. Propidium iodide staining demonstrated that 20%-40% of Ramos cells became apoptotic by 18 h and further established that cells transiting into the S phase of the cell cycle were susceptible to PCD. Addition of several agents to the Ramos cells abrogated anti-IgM-induced PCD, including the phorbol 12-myristate 13-acetate (PMA). In contrast to the effect of PMA, the 4 alpha PMA isomer of PMA neither activated protein kinase C (PKC) nor rescued the cells from anti-IgM-induced PCD, confirming a role for PKC in negating apoptosis. To explore the effect of physiologic signals on anti-IgM-induced PCD, antibodies against the CD20 or CD40 molecules were added in concert with anti-IgM. Both CD20 and CD40 synergize with anti-IgM to augment proliferation but neither molecule activates PKC in Ramos cells. Both anti-CD20 and anti-CD40 reduced the number of cells undergoing anti-IgM-induced PCD. Unlike the effect of anti-CD40, addition of anti-CD20 to anti-IgM-stimulated cells negated PCD only in a subset of cells. Maximal rescue occurred following the addition of anti-CD40 and occurred by 4 h and at least up to 20 h of culture. These data show that (a) PCD can be initiated in B cells entering the S phase of the cell cycle, (b) PCD can be triggered by engagement of surface IgM in the absence of ancillary signals or PKC activation, and (c) rescue from PCD can occur by several mechanisms, either PKC dependent or PKC independent.

Autocrine proliferating B cells are susceptible to terminal differentiation and apoptosis

“…The medium background was below detection limit of the assay 1989). This is in agreement with the observation that PKC activation rescued several lymphoma cells from anti-Iginduced cell death: Peyer's patch B-cells (Motyka et al, 1993), Burkitt's lymphoma cells (Knox et al, 1992), Ramos cells (Valentine and Licciardi, 1992), and the immature BKS-2 lymphoma cells (Muthukkumar et al, 1993). Moreover, PKC activation prevents germinal center B cells (Knox et al, 1992), ilial Peyer's patch B cells (Motyka et al, 1993), and resting splenic B cells (Illera et al, 1993) from spontaneous apoptosis.…”

Translocation of Full-length Bid to Mitochondria during Anoikis

“…Most Bcl-2 proteins associate with other mitochondrial components and form stable complexes that can be isolated by gel filtration. Bax translocates to mitochondria as a monomer before forming stable oligomeric complexes (35)(36)(37). We therefore asked whether Bid associated with other mitochondrial proteins following translocation to mitochondria and whether we could detect it in fractions that contained Bax.…”

Stimulation-induced gene expression in Ramos B-cells

“…[37][38][39] Very little is known about the progression of apoptosis after the crosslinking of IgM. Apoptosis in Ramos cells begins only 4 h after the stimulation, 25,40 and reaches a maximum around 24 h. 38 Under certain conditions, non apoptotic cells that survive BCR stimulation show diminished expression of the surface molecules integrin VLA-4 and BCR after 24 h. 41 In our study, the a4 subunit of VLA-4, Iga, Igb and the C region of IgM are indeed downregulated 24 h after stimulation, indicating that only a fraction of Ramos B-cells become apoptotic during the experiment. The amount of apoptotic cells was small since no RNA degradation was detected.…”