Bourke R. Maddox and David W. Scott

Immunology Department, American Red Cross Holland Laboratory, Rockville, MD 20855 USA

Received 03/29/96; Accepted 06/10/96; On-line 06/25/96

Adult murine spleen cells will undergo dose-, time- and temperature-dependent apoptosis in vitro if sufficient crosslinking of membrane IgM occurs via anti-IgM treatment (9-11). The data presented herein confirm that mature splenic B cells will be driven to PCD (apoptosis), but differences in the susceptibility of B-cell subsets to apoptosis can be seen in this system. These results are reminiscent of our recent observation of resistance to tolerance induction in murine B1 cells in the peritoneum (3) and the spleen (4). Indeed, these results suggest that the differential susceptibility to tolerance of murine B cells may reflect these signaling differences for apoptosis. Immunoglobulin receptor crosslinking also is important as the first step in B-cell activation for cell cycle entry (13, 14). Clearly, the maturity and lineage of the target B cells, as well as second signals like CD40 and cytokines, play a role in driving B-cell proliferation (15, 16). The data presented herein indicate that strain and subset differences also play a role in B-cell apoptosis.

Crosslinking of membrane IgM causes initial tyrosine phosphorylation of a number of substrates, as well as phosphatidyl inositol hydrolysis, calcium mobilization and downstream effector events to lead to exit from G0 and entry into the cell cycle (17). In our experience, the ability to drive B-cell proliferation and apoptosis generally do not correlate. For example, mitogenic monoclonal anti-m reagents do not induce apoptosis unless hyper-crosslinked (9, 10), yet such antibodies can drive resting B cells into cycle reproducibly. Moreover, CD45 knockout mice (18), which can not be driven into cycle with any form of anti-µ, are nonetheless susceptible to apoptosis induced by goat anti-µ.(9). Finally, xid mice show the same separation of proliferation and apoptosis: anti-induces PCD but can not drive xid B cells into S (19). Therefore, the ability to drive apoptosis and cell cycle entry, even abortively, does not correlate.

Interestingly, in CD45 knockout and xid spleen cells (9,19), anti-m causes a reduction in the p27 kinase inhibitor (20) known to regulate cell cycle entry in other cell types. It will be important to discern whether the anti-µ signaling defect is upstream or downstream from p27 modulation in 6.1 transgenic B cells. These studies are planned.

Our results are contrary to previously published data (21) suggesting normal proliferation in response to soluble anti-IgM in Sp6, but not M167 transgenic mice, which are predominantly B2-like in the spleen. Several differences between these studies should be noted. First, very high doses of different antibodies were used to induce proliferation in their system (21); indeed, in our hands, polyclonal anti-µ at >10µg/ml induces extensive B-cell PCD and minimal proliferation. However, a more likely explanation is that the background genes for these transgenic strains may differ (C57Bl vs. Balb/c). In both their studies and ours, transgenic B cells did proliferate normally in response to LPS and PWM. These data suggest that the differences noted may be downstream from initial tyrosine phosphorylation events (3, 4) since this event is relatively normal in Sp6 and 3.83 transgenics, and only modestly affected in 6.1 transgenic B cells (4).

Clearly, further studies are necessary to pinpoint the defect(s) leading to diminished B-cell apoptosis and proliferation in 6.1 transgenic B1-type cells compared to conventional B cells. Nonetheless, our results suggest that one reason for the resistance of B1 cells to tolerance induction is a failure to undergo apoptosis when their surface IgM is sufficiently crosslinked.