Difference in apoptosis induction between surface IgD and IgM.

In the classic 'two-signal' model for B cell activation, signal 1 through the antigen receptor plus signal 2 through lymphokine receptors and CD40 leads to proliferation, but signal 1 alone leads to tolerance or anergy. In a protocol designed to deliver signal 1 in vitro with anti-delta without signal 2, purified small dense B cells from untreated mice exposed to any of three monoclonal anti-delta antibodies or to polyclonal anti-delta in vitro showed modest S phase entry at 50 microg/ml. In contrast, at low doses (0.1-0.5 microg/ml) of anti-delta, there was no cell cycle entry at 64 h, but apoptosis was accelerated at 16 h. Polyclonal anti-mu and three monoclonal anti-mus did not show this early apoptosis induction. Anti-CD40 and IL-4 inhibited apoptosis in B cells treated with 0.5 microg/ml anti-delta and increased S phase entry at 10 microg/ml anti-delta. Low-dose anti-delta (but not anti-mu) induced increased B7-2 and class II MHC expression on a subset of B cells, many of which were in apoptosis. Larger transient increases in c-Myc and Egr-1 expression were seen with low-dose anti-delta than with anti-mu, followed by an abrupt fall below baseline, a sequence previously linked to apoptosis. There was no change in Bcl-2, Bcl-x(L) or Bax. These data suggest a functional difference between delta and mu cross-linking on resting spleen B cells. A BCR stimulus sufficient for early activation events, but insufficient for full G1 entry, may lead to apoptosis.

Direct evidence that iron deprivation induces apoptosis in murine lymphoma 38C13.

We found that the mouse B cell lymphoma 38C13 underwent apoptosis in vitro when deprived of iron by three independent methods: (1) exposure to a synergistic pair of rat IgG monoclonal antibodies against the mouse transferrin receptor; (2) exposure to the iron chelator deferoxamine (DFO), and (3) exposure to a defined culture medium without any added iron (iron-poor medium). When each antibody was present at a concentration of 5 micrograms/ml, the number of living cells declined to approximately 25% after a 24-hour incubation. After 48 h, there were no surviving cells. When DFO was present at a concentration of 10 microM, the effects were similar, but delayed by 24 h. when iron-poor medium was used, the effects and kinetics were similar to those seen with antibody treatment. For each method of iron deprivation, the reduction in cell viability correlated with the development of apoptosis, as assessed by DNA fragmentation analysis and propidium iodide staining. Electron microscopy studies provided additional confirmation of apoptotic cell death. The addition of 500 microM ferric citrate completely prevented apoptosis for each of the three methods of iron deprivation. These studies provide new and compelling evidence to support the view that iron deprivation can specifically induce apoptosis and serve to strengthen the rationale for further studies of iron deprivation as a form of cancer treatment.

Fc receptor off-signal in the B cell involves apoptosis.

By linking surface Ig to the FcR Fc gamma RII on the mouse B lymphocyte surface, whole anti-Ig has been shown to block cell cycle entry and subsequent Ab production, a phenomenon called the "Fc receptor off-signal." IL-4 or blocking Ab to Fc gamma RII, present with whole anti-Ig, restores cell cycle progress to the levels observed with F(ab')2 anti-Ig. The current study demonstrates that under "off-signal" conditions with whole anti-Ig, the early entry of B cells into apoptosis was accelerated relative to medium alone or equimolar F(ab')2 anti-Ig. All reagents tested which opposed the whole-anti-Ig-induced blockade of B cell cycle entry also protected B cells from apoptosis (IL-4, PMA, dextran sulfate, and the monoclonal anti-Fc gamma RII 2.4G2). This protective effect was most evident at 4 to 12 h, waning at later times. Low dose cycloheximide partially protected B cells from whole anti-Ig-induced apoptosis, but acted as a survival factor, failing to advance B cells from G0 phase or stimulate thymidine incorporation. Additive early apoptosis-associated membrane changes were transiently seen when whole anti-Ig was combined with other apoptosis-accelerating agents (trifluoperazine, staurosporine, dexamethasone, ionomycin, high-dose cycloheximide), but hypodiploid nuclei did not show this effect. B cells from bcl-2 transgenic mice showed less apoptosis when cultured with whole anti-Ig, or with any of the other agents tested. At 4 h the bcl-2-associated reduction in hypodiploid nuclei was greater than the reduction in membrane unpacking, but by 16 h this difference was much less. These results suggest that acceleration of apoptosis contributes to the inhibition of proliferation induced by whole anti-Ig.

Membrane unpacking and the rapid disposal of apoptotic cells.

There is evidence that macrophages can recognize phosphatidylserine (PS) on the surface of apoptotic thymocytes, where PS exposure relates to looser packing ('unpacking') of the polar headgroups of the outer lamina, detectable by lipophilic dyes (Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L. and Henson, P.M. (1992) J. Immunol. 148, 2207). We have shown that membrane unpacking also occurs in B cells, where this event actually precedes DNA cleavage (Mower, D.A. Jr., Peckham, D.W., Illera, V.A., Fishbaugh, J.K., Stunz, L.L. and Ashman, R.F. (1994) J. Immunol. 152, 4832). This paper demonstrates that the time interval between membrane unpacking (detected as merocyanine 540 binding) and DNA cleavage (detected by flow cytometry of propidium iodide stained nuclei) also occurs in both T cells and thymocytes. The tight coupling of these two apparently distinct events is emphasized by their co-regulation by a variety of agents which accelerate or inhibit apoptosis. One hypothesis to explain the very low numbers of free apoptotic cells seen in vivo is that macrophages can recognize cells with unpacked membranes and destroy them before they cleave their DNA. In support of this hypothesis, we demonstrated that parenteral cycloheximide triggers a wave of apoptosis in the spleen detected by merocyanine 540 as well as by hypodiploid nuclei. Significantly, both parameters returned from peak values at 2 h virtually to normal by 4 h, testifying to the existence of a rapid disposal mechanism in vivo for cells with unpacked membranes as well as hypodiploid nuclei.

Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis.

Mature resting mouse spleen B cells progress stochastically into apoptosis at a uniform rate over the first 16 h in vitro in 3 stages. In stage 1, early apoptotic B cells decreased the normal phospholipid packing of their plasma membranes, detected as increased binding of the lipophilic dye merocyanine 540, and also decreased in volume, detected as decreased forward scatter. In stage 2 there was abrupt internucleosomal cleavage of DNA, quantitated as hypodiploid nuclei by flow cytometry. Some stage 2 cells entered stage 3, where the plasma membrane became permeable to propidium iodide. B cells in later stages of this sequence retained the characteristics of earlier stages, whereas nonapoptotic B cells remained in their original state. Cycloheximide increased the progression of B cells through these three stages, whereas dextran sulfate inhibited stage 1 more effectively than stages 2 or 3. Increased orthogonal scatter also occurred late in some of the cells that had passed through stage 1, but did not correlate well with propidium iodide permeability. Fresh small dense spleen B cells contained 5% to 7% stage 1 cells but only about 1% stage 2 cells. Macrophages have been reported to destroy preferentially apoptotic thymocytes by recognizing plasma membrane alterations deriving from loose packing of phospholipid head groups. The recognition of stage 1 rather than stage 2 B cells by macrophages may help to keep the proportion of apoptotic cells low in vivo.

Regulation of apoptosis in vitro in mature murine spleen T cells.

Previously it has been shown that thymocytes undergo apoptosis, a form of programmed cell death, in response to glucocorticoids. This classic form of apoptosis is prevented by inhibition of protein synthesis. The current paper demonstrates that mature T cells also undergo apoptosis, but that the regulation of apoptosis in spleen T cells differs from that of thymocytes. Mature mouse spleen T cells were shown to die by apoptosis, not necrosis, when cultured without an added stimulus. Assays for apoptosis included internucleosomal DNA cleavage by gel electrophoresis, percent fragmentation of DNA by the diphenylamine method, and percent of cells with hypodiploid DNA by flow cytometry. The percent of apoptotic cells was 2% in fresh spleen T cells, and increased at least until 16 h, when 21% were apoptotic. Dexamethasone caused apoptosis in both thymus and spleen T cells, but only thymocytes showed a requirement for protein synthesis in dexamethasone-induced death. Cycloheximide increased apoptosis in spleen T cells, indicating that apoptosis was controlled by newly synthesized protective proteins. Spontaneous apoptosis was decreased in spleen T cells by protein kinase C activation, and was increased by H7 and staurosporine, which inhibits protein kinases, in contrast with the behavior of thymocytes. The protein kinase A/G inhibitor HA1004 also decreased spleen T cell apoptosis. The contrasting effects of cycloheximide on thymocytes and spleen T cells occurred over the same concentration range, and the same was true for PMA. The dexamethasone dose-response curves were similar, except that a greater proportion of spleen T cells were dexamethasone-resistant. These data support the hypothesis that the apoptosis program in T cells undergoes a transition during their maturation, such that apoptosis in mature T cells is regulated more like that of mature B cells than that of thymocytes.

Apoptosis in splenic B lymphocytes. Regulation by protein kinase C and IL-4.

Small dense splenic B lymphocytes from adult specific pathogen-free mice were shown to undergo apoptosis in vitro as indicated by internucleosomal DNA fragmentation, hypodiploid DNA content of isolated nuclei, and morphologic features by electron microscopy. Unstimulated cultures showed spontaneous apoptosis increasing gradually and monotonically from < 2 to 32% of B cells by 16 h. The rate of accumulation of apoptotic cells was reduced by the addition of IL-4 or PMA, but not by the inactive phorbol ester, 4 alpha PDD. In contrast, inhibitors of protein kinase C (H7 and staurosporine) increased the percentage of cells undergoing apoptosis to > 70% by 12 h; HA 1004, genistein, and herbimycin A all had no effect on apoptosis. Thus, protein kinase C activity regulates apoptosis, but there is no evidence that protein kinases A and G and tyrosine kinases are involved. Cycloheximide increased apoptosis, indicating that apoptosis may be restrained in B cells by the presence of one or more labile protective proteins. The percentage of apoptotic cells measured by flow cytometry and the percentage of fragmented DNA measured by the diphenylamine method were nearly equal, regardless of the method of apoptotic regulation. Together with the absence of nuclei with flow cytometric properties intermediate between normal and apoptotic, these results suggest that in individual B cells apoptosis progresses rapidly to completion. These data suggest a fundamental change in our concept of the life-style of the "resting" B cell: instead of a dormant cell remaining unchanged until it receives activation signals, the mature spleen B cell appears programmed to die by apoptosis unless rescued by specific agents, such protein kinase C activators or IL-4.

Polyclonal activation of rat B cells. II. Dextran sulfate as a cofactor in mitogen-induced and antigen-induced differentiation of rat B lymphocytes.

Salmonella typhimurium mitogen (STM) is a polyclonal activator of rat B lymphocytes, triggering them to proliferate, but not differentiate, to antibody-secreting cells. When lymphokines in the form of a supernatant from Con A-stimulated splenocytes (CAS) are added to B cell cultures activated by STM, only a small number of cells are driven to differentiate. Only with the addition of a third signal provided by the polyanionic polysaccharide dextran sulfate (DXS) is significant rat B cell differentiation observed. In this study, we have shown that this requirement for DXS is not unique to the STM mitogen. LPS, Staphylococcus aureus Cowan I-fixed cells, and anti-Ig antibody all induced rat B cell proliferation with little differentiation, even in the presence of CAS. DXS was necessary to induce differentiation in all cultures costimulated with mitogen and CAS. The requirement for DXS for optimal B cell differentiation is also observed with other lymphokine preparations such as the supernatants from PMA-stimulated EL-4 cells and PHA-stimulated human T cells. Furthermore, this augmentative effect of DXS in rat B cell differentiation was not confined to polyclonal activation systems. Ag-specific IgG secretion was also increased when DXS was added to Ag and CAS costimulated cultures of B cells harvested from the draining lymph nodes of rats immunized with DNP-keyhole limpet hemocyanin. Within the polyclonal activation system, a method of staged additions of STM, DXS, and CAS to B cell cultures was used to investigate the role of DXS during B cell differentiation. Optimal differentiation occurred only when DXS was present in the B cell cultures in conjunction with CAS. The augmentation in differentiation seen with DXS did not appear to be due to the recruitment of an additional CAS-responsive B cell subset, because cycling, low density B cell blasts showed large increases in IgM secretion with subsequent exposure to DXS and CAS. These studies suggest tha DXS acts as a cofactor to various differentiation factors, augmenting polyclonal and Ag-specific rat B cell differentiation. The relevance of DXS to in vivo immune responses is discussed.

Two mechanisms of transferrin receptor induction by anti-Ig in B cells.

By cross-linking surface Ig to the Fc gamma R, whole (IgG)-rabbit anti-Ig antibodies have been shown to substantially inhibit proliferation induced by LPS and F(ab')2 anti-Ig, and polyphosphoinositide hydrolysis induced by F(ab')2 anti-Ig. Surprisingly, however, whole anti-Ig was unable to inhibit induction of transferrin receptor (TfR) expression by LPS or F(ab')2 anti-Ig. Indeed, whole anti-Ig on its own induced TfR as early as 4 h. TfR induction by F(ab')2 anti-Ig and by LPS was accompanied by an early increase in TfR mRNA, and was prevented by inhibitors of protein and RNA synthesis and therefore can be ascribed to a transcriptional mechanism. In contrast, whole anti-Ig induced TfR even in the presence of protein and RNA synthesis inhibitors. Little or no TfR mRNA was detectable after 4 or 16 h of exposure to whole anti-Ig, whereas increased TfR mRNA was evident after 4 h of F(ab')2 anti-Ig or LPS. Antibody to the Fc gamma R (2.4G2) restores the ability of whole anti-Ig to generate increased TfR expression via the transcriptional route. We conclude that whole anti-Ig induces TfR mostly by using preexisting TfR molecules through a mechanism different from the transcriptional mechanism triggered by F(ab')2 anti-Ig and LPS.

HLA-DRB1 and -DRB4 genes are differentially regulated at the transcriptional level.

The mechanism regulating the differential expression of DR beta 1 and DR beta 4 proteins on the surfaces of cultured EBV-transformed DR7 cell lines was investigated at the level of gene transcription. Steady-state levels of mRNA coding for DR7 beta 1 and DR beta 4 proteins were evaluated with the use of locus-specific oligonucleotide probes and relative transcriptional activity at the DR7B1 and DRB4 gene loci was measured with nuclear run-on transcription assays. Steady-state levels of DR7B1 mRNA were found to be three- to nine-fold higher than those of DRB4 in DR7,Dw7 cell lines, but no mature steady-state DRB4 mRNA was detectable in a DR7,Dw11 cell line which shows no detectable DR beta 4 protein. Run-on transcription assays performed with nuclei isolated from DR7,Dw7 cells showed a five-fold excess of DRB1 over DRB4 transcriptional activity, but, surprisingly, no difference in activity at the two DRB loci in nuclei isolated from DR7,Dw11 cells. Both DRB mRNA were quite stable as indicated by actinomycin D time course experiments. Thus the differential expression of the DR7 beta 1 and DR beta 4 proteins in the DR7,Dw7 cell lines arises at the level of mRNA production; the lack of detectable DR beta 4 protein in the DR7,Dw11 cell line, however, appears to be due to a different mechanism in which transcribed DRB4 sequences fail to be processed into mature mRNA molecules.

Polyclonal activation of rat B cells. I. A single mitogenic signal can stimulate proliferation, but three signals are required for differentiation.

A water-soluble, proteinaceous preparation derived from the cell walls of Salmonella typhimurium Re mutants has recently been tested in our laboratory for its ability to act as a mitogen for rat lymphocytes. We have found this preparation (STM) to be a potent stimulator of B lymphocyte proliferation, as measured both by 3H-TdR incorporation and by cell cycle analysis performed with flow cytofluorometry. STM stimulates approximately 50% of rat B cells to enter cycle. Previous investigations by others have shown that at least two sets of signals are required for B cell differentiation; a) proliferation signals that may consist of both a stimulator of B cell conversion from G0 to G1 and growth factors, and b) differentiation signals that probably include at least two B cell differentiation factors (BCDF). When STM was tested in a differentiation system it did not drive purified B cells to differentiate to PFC, either alone or when supplemented with a supernatant from concanavalin A-stimulated spleen cells (CAS). However, when both CAS and dextran sulfate (DXS) were supplied to the STM-stimulated cells, a large number of PFC resulted. DXS does not act by stimulating an additional, CAS-responsive B cell subset, since it has only a marginal effect upon 3H-TdR uptake and does not increase the number of B cells in cycle when used together with STM. We postulate that the two agents may be acting sequentially: STM stimulates the B cells to proliferate, and DXS drives the proliferating cells to become responsive to CAS. This suggests that the signals for B cell differentiation must consist of at least three activities: a trigger to stimulate the cells to proliferate, a factor to drive the cells to a BCDF-responsive state, and a BCDF that can drive the cells to secrete antibody.

A specific estrogen receptor in the mouse spleen. Characterization and evidence of physiological regulation.

We have used the synthetic estrogen [3H]-R2858 (11 beta-methoxy-17-ethnyl-1,3,5(10)-estratriene-3,17-diol) to characterize a cytoplasmic estrogen receptor in the mouse spleen. This receptor is specific for estrogens and binds to the steroids with high affinity. The binding is abolished by pronase or 37 C treatment, and the 8S peak is shifted to the 4S region on sucrose gradients under conditions of high salt. The concentration of spleen receptor increased significantly during pregnancy, and decreased after immunization with foreign protein. Estrogen receptors may be confined to certain subpopulations of spleen cells that change in numbers during pregnancy or after immunization.