Activation of human B cells. Comparison of the signal transduced by IL-4 to four different competence signals

The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.     

Induction and regulation of casein kinase II during B lymphocyte activation.

This study evaluates the regulation of casein kinase II (CK II) activity in resting B cells induced to enter the cell cycle. The induction of B cell cycle progression PMA and ionomycin results in an oscillatory expression of CK II. This kinase activity is also elicited after direct physical interaction between B cells and activated, fixed Th cells, indicating that the increase seen in CK II activity is probably associated with the delivery of the competence-inducing signal to resting B cells. The selective inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, during PMA and ionomycin-induction of B cell cycle progression, inhibits the expression of CK II activity. The addition of polyamines to cytosolic preparations recovered from cells in which ODC is inhibited results in the appearance of CK II activity, showing that the ODC inhibitor does not directly inhibit the kinase. The treatment of B cells with cycloheximide results in the appearance of CK II activity within 15 min, and this induction is partially explainable by a cycloheximide-elicited increase in cellular levels of polyamines. The artificial elevation of cellular levels of cAMP simultaneous with the addition of PMA and ionomycin results in a 150 to 200% increase in detectable CK II levels, suggesting that the cAMP-dependent signaling cascade may participate during the early regulation of CK II. In contrast, the inhibition of protein kinase C does not adversely influence the early expression of CK II, while actually enhancing kinase activity by 18 h poststimulation.     

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.     

The role of class II molecules in human B cell activation. Association with phosphatidyl inositol turnover, protein tyrosine phosphorylation, and proliferation.

Cross-linking class II molecules on resting human B cells can initiate phosphatidyl inositol turnover and an increase in intracellular calcium concentration levels comparable with that seen with the cross-linking of surface Ig receptors. The calcium response is most evident on dense B cell fractions: buoyant cells are less responsive, even though the levels of class II expression are similar on dense and buoyant tonsillar B cells. Human B cell lines exhibit the same absence of correlation between intensity of the calcium signal and levels of surface class II expression, indicating that responsiveness is related to the state of differentiation of the cell rather than the amount of class II expressed. Cross-linking class II on normal B cells or B cell lines caused accumulation of inositol phosphates, suggesting class II induces calcium release from intracellular stores, rather than through direct regulation of calcium channels. The calcium response mediated through class II was completely abolished by bringing the protein tyrosine phosphatase, CD45, into close proximity with surface class II. This result indicated that protein tyrosine phosphorylation might regulate the signal transduced through this molecule. In support of this notion we found that tyrosine phosphorylation is induced when small dense tonsillar B cells are stimulated with either anti-Ig or with antibodies to class II. Finally, in B cell proliferation assays we show that cross-linking class II molecules on dense tonsillar B cells synergize strongly with suboptimal concentrations of PMA or IL-4. The significance of these results is discussed with regard to the cognate signal between B and T lymphocytes.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.     

Molecular signals in B cell activation. I. Differential refractory effects of incomplete signaling by ionomycin or PMA relate to autocrine IL-2 production and IL-2R expression

The molecular signals required by resting (G0) B cells for the induction of cell cycle entry, IL-2 production, and high-affinity IL-2 receptor (IL-2R) expression were defined and the effects of incomplete activation signals on the subsequent response to complete signals were examined. Highly enriched rabbit peripheral blood B cells were activated with a calcium ionophore, ionomycin, and a protein kinase C (PKC) activating phorbol ester, phorbol myristate acetate (PMA). It was observed that cell cycle entry to early G1 was induced by either reagent acting alone, but both reagents were required to stimulate IL-2 production, IL-2R expression, and DNA synthesis. These effects of ionomycin and PMA were shown to be mediated by increased intracellular calcium ion concentration [Ca2+]i and PKC activation, respectively. Although, increased [Ca2+]i or PKC activation each led to cell cycle entry, the subsequent response of these preactivated cells to complete activation with both signals was different: Cells pretreated with PMA alone for up to 24 hr could progress further to DNA synthesis after the addition of ionomycin. In contrast, cells activated with ionomycin alone, or those cultured without any stimulus, progressively lost the ability to show DNA synthesis after complete activation. The failure to progress to DNA synthesis in these two cases was, however, differentially regulated by the ability of these cells to produce IL-2 and to express IL-2R. Ionomycin-pretreated cells retained the ability to produce IL-2 but showed about 70% reduction in the numbers of IL-2R; whereas cells cultured without any stimulus lost the ability to produce IL-2 after subsequent complete activation, but showed lesser reduction in IL-2R expression.     

Studies of the in vitro activation and differentiation of human B lymphocytes. II. Optimization of activation by anti-immunoglobulin antibody bound to beads: analysis of the role of autocrine effects on B-cell proliferation and of T-cell help in B-cell differentiation

We report experiments attempting to optimize the proliferative response of human B cells to rabbit anti-immunoglobulin antibody (RAHIg)-linked beads (anti-Ig beads). By choosing polyacrylamide beads of small size (3 micron) and coupling anti-Ig to them at high concentrations, beads were obtained which were both B-cell specific and more highly mitogenic than other than anti-Ig reagents and B-cell mitogens (SAC, protein A). Using these beads to activate B cells, the augmentation of the anti-Ig-induced proliferative response by added T-cell-derived growth factors was largely eliminated at high cell densities although the effect of these factors was still evident at low cell densities. However, when cultures were performed in round-bottom vessels which crowded the B cells together, the response to anti-Ig beads was independent of T-cell factors even at low B-cell densities, suggesting that normal B cells triggered by anti-Ig beads are able to maintain their own proliferation. In contrast to the proliferative response, even with the most potent anti-Ig bead preparations, no differentiation (Ig production or expression of terminal differentiation markers) was evident unless T-cell help was provided.     

Lymphoma models for B cell activation and tolerance. VI. Reversal of anti-Ig-mediated negative signaling by T cell-derived lymphokines

We have recently described three "immature" B cell lymphomas which are exquisitely sensitive to growth inhibition by anti-Ig reagents and may serve as models for tolerance induction in normal B cells. These cells are inhibited from cell cycle progression into S after receiving a negative signal in early G1. In this paper, we demonstrate that the growth inhibition by anti-Ig can be prevented and reversed by the addition of supernatants from T cell lines. One such line, called Tova, produces factors which restore normal levels of DNA synthesis in the presence of concentrations of anti-Fab or anti-kappa immunoglobulins which cause up to a 90% inhibition of thymidine incorporation in a 2- to 3-day culture period. This factor is at least partially effective when added up to 24 hr after anti-Ig to unsynchronized lymphoma cells and it does not alter the growth of control cultures. Studies using synchronized lymphoma cells indicated that the T cell factor permitted cycle progression into S when added during the early G1 exposure to anti-kappa and was less effective when added late in G1. Preliminary characterization suggests that both B cell growth factor II (interleukin 5) and B cell stimulatory factor 1 (interleukin 4) have additive activity in this system, although another unidentified lymphokine may also be involved. The relevance of T cell reversal of Ig receptor-mediated negative signaling to neonatal B cell tolerance is emphasized.     

Differential responsiveness of immature- and mature-stage murine B cells to anti-IgM reflects both FcR-dependent and -independent mechanisms.

Mature and immature B cells differ in their responses to antigen receptor crosslinking. Whereas mature B cells enter cell cycle in response to such stimulation, immature B cells exhibit proliferative unresponsiveness and undergo induced tolerance following surface immunoglobulin (sIg) engagement. Previous studies evaluating antigen receptor-mediated negative signaling have utilized intact goat anti-immunoglobulin (anti-Ig) antibodies as polyclonal ligands based upon observations that the Fc portion of these reagents does not interact with and mediate negative signaling through the FcR on mature B cells. Thus, the negative effects of goat anti-Ig on immature B cells have been attributed solely to signals mediated via their antigen receptors. In the studies reported here we show that the activation unresponsiveness inherent to immature B cells is FcR independent. However, we also show that immature B cells are sensitive to FcR-mediated inhibition and that these effects can be mediated by intact goat antibodies at concentrations that promote positive activation signals in mature B cells. Our results demonstrate that inhibition of immature B cell LPS responses by anti-Ig antibodies, used in previous studies as an in vitro model for B cell tolerance induction, is an FcR-mediated phenomenon. We show that developmentally associated anti-Ig-mediated inhibition of LPS requires the use of intact antibodies, and that this inhibition can be blocked by the anti-FcR monoclonal antibody 2.4G2. Flow cytometric analysis of FcR-positive B cells indicates that both mature and immature B cells express equivalent levels of FcR gamma. Therefore, the sensitivity of immature, but not mature, cells to intact goat anti-mu antibodies suggests that either FcRs or their associated inhibitory pathways change during B cell development.     

Pre-exposure of human B cells to recombinant IL-1 enhances subsequent proliferation

The reported effects of the monocyte-derived cytokine IL-1 on human B lymphocytes are both varied and controversial. IL-1 has been reported to augment both proliferation and Ig secretion of previously activated human B cells. In the present study highly purified splenic B cells were cultured with rIL-1 before, simultaneously with, and after the addition of the polyclonal B cell mitogen, anti-Ig. rIL-1 had no significant effect on B cell proliferation when added simultaneously with or after B cell activation with anti-Ig. However, incubation of splenic B cells with rIL-1 for 24 h before stimulation with anti-Ig appeared to enhance mitogenesis. With the observation that rIL-1 exerted effects on resting B cells, the effect of rIL-1 on several events which accompany B cell activation was examined. rIL-1 failed to stimulate RNA synthesis, effect increases in cell size or intracellular Ca2+ levels, or lead to the hyperexpression of MHC class II or B cell activation Ag. These studies suggest that rIL-1 does not activate B cells but primes them to respond to subsequent activation.     

Inhibitory effect of anti-class II antibodies on human B-cell activation

The role of class II antigens for B-cell activation was analyzed using purified human B cells and anti-class II monoclonal antibodies. The stimulation of purified B cells with Staphylococcus aureus Cowan I induced proliferation and differentiation into immunoglobulin-producing cells in the presence of interleukin-1 and T-cell-derived factors (B-cell growth factor and B-cell differentiation factor). The addition of anti-class II monoclonal antibodies inhibited B-cell responses. However, anti-class I monoclonal antibody did not inhibit B-cell responses. When mitomycin C and cycloheximide-treated B cells were added to the induction culture of B cells as the stimulator, B-cell responses were enhanced in a dose-dependent manner. Furthermore, the stimulator B cells also partially restored the suppressed B-cell responses which were induced by the pretreatment of B cells with anti-class II antibody. This enhancing effect of stimulator B cells on B-cell responses was inhibited by the pretreatment of stimulator B cells with anti-class II antibody. The treatment of B cells with anti-class II antibody and complement depleted the activity of both responder B cells and stimulator B cells. These results suggest that cellular interaction among B cells exists in the B-cell activation induced with Staphylococcus aureus, Cowan I and anti-class II antibody inhibits B-cell activation by interfering in this cellular interaction.     

A polyclonal model for B cell tolerance. I. Fc-dependent and Fc-independent induction of nonresponsiveness by pretreatment of normal splenic B cells with anti-Ig.

We have developed a simple and adaptable, polyclonal model for B cell nonresponsiveness that is based on the inhibitory activity of anti-Ig as a surrogate for Ag. In our system the induction phase (treatment with anti-Ig) is separated from the challenge phase (Ag or mitogen), so that the critical events in each phase can be evaluated. Our results show that T cell-depleted B cells precultured for 18 to 24 h with rabbit anti-Ig reagents are rendered unresponsive to challenge with either Ag, fluorescein coupled to Brucella abortus (FL-BA), or mitogen (LPS). This state of nonresponsiveness (anergy) is reflected by an inhibition of a prototype response to the fluorescein hapten, as well as total Ig and IgG synthesis, but no reduction in proliferation to LPS. Interestingly, mitogen-induced polyclonal antibody formation was consistently reduced by 90% by treatment with either F(ab')2 or intact IgG anti-Ig. In contrast, the Ag-driven (FL-BA) response of pretreated B cells was inhibited by only 50 to 70%. Moreover, the latter effect usually required pretreatment with intact IgG anti-Ig, a result that suggests the importance of an Fc-dependent negative signal affecting the B cell's response to FL-BA. Furthermore, pretreatment and coculture of B cells with IL-4 blocked the Fc-dependent inhibition of the FL-BA responsiveness. These results, as well as kinetics experiments establishing a 4-h latent period, suggest that simple blocking of surface Ig receptor on target B cells is not responsible for the induction of anergy. Pretreated B cells displayed unique phenotypic changes after treatment with anti-Ig, including a diminution of Thy-1 expression in response to LPS + IL-4, as well as a reduction in membrane IgM and J11d expression (i.e., they were IgMlo, IgDmed, and J11dlo, as recently reported for anergic B cells in transgenic mice). These results suggest that B cell anergy can be induced in mature B cells by both Fc-dependent and Fc-independent processes that lead to unique phenotypic changes and may reflect egress from G0 in the absence of T cell help. The significance of these changes to tolerance mechanisms is discussed.     

B cells activated by lipopolysaccharide,but not by anti-Ig and anti-CD40 antibody,induce anergy in CD8+ T cells: role of TGF-beta 1

B cells recognize Ag through their surface IgRs and present it in the context of MHC class II molecules to CD4(+) T cells. Recent evidence indicates that B cells also present exogenous Ags in the context of MHC class I to CD8(+) T cells and thus may play an important role in the modulation of CTL responses. However, in this regard, conflicting reports are available. One group of studies suggests that the interaction between B cells and CD8(+) T cells leads to the activation of the T cells, whereas other studies propose that it induces T cell tolerance. For discerning this dichotomy, we used B cells that were activated with either LPS or anti-Ig plus anti-CD40 Ab, which mimic the T-independent and T-dependent modes of B cell activation, respectively, to provide accessory signals to resting CD8(+) T cells. Our results show that, in comparison with anti-Ig plus anti-CD40 Ab-activated B cells, the LPS-activated B cells (LPS-B) failed to induce significant levels of proliferation, cytokine secretion, and cytotoxic ability of CD8(+) T cells. This hyporesponsiveness of CD8(+) T cells activated with LPS-B was significantly rescued by anti-TGF-beta1 Ab. Moreover, it was found that such hyporesponsive CD8(+) T cells activated with LPS-B had entered a state of anergy. Furthermore, LPS-B expresses a significantly higher level of TGF-beta1 on the surface, which caused the observed hyporesponsiveness of CD8(+) T cells. Therefore, this study, for the first time, provides a novel mechanism of B cell surface TGF-beta1-mediated hyporesponsiveness leading to anergy of CD8(+) T cells.     

Lymphoma models for B-cell activation and tolerance. IX. Efficient reversal of anti-Ig-mediated growth inhibition by an activated TH2 clone.

We have utilized several B-cell lymphomas that are growth inhibited by anti-Ig reagents as models for tolerance induction. In a previous communication, we demonstrated that the growth inhibition by anti-Ig can be partially prevented by the recombinant lymphokine, IL-4. In this paper, we report that complete protection of B lymphomas from anti-Ig was provided by a type 2 helper cell clone, D10.G4, when these T cells were activated by monoclonal anti-CD3. Conditioned medium from anti-CD3-stimulated D10.G4 cells also provided protection from anti-Ig. In contrast, little protection was observed with activated cells from a type 1 T-cell clone, A.E7. Furthermore, we show that combinations of IL-4 and tumor necrosis factors (both TNF alpha and TNF beta), as well as IL-4, effected partial protection by themselves and enhanced the activity of the other lymphokine if used in a pretreatment protocol. However, anti-cytokine antibodies were ineffective at reversing the T-cell-mediated protection. The possibility that direct T:B-cell contact mediates part of the protective signal is discussed.     

CD3 pathway of T-cell activation. II. Role of HLA-class I molecules in early events

The role of distinct regions of HLA class I molecules in regulating T-cell activation via the CD3-antigen receptor complex was investigated. Monoclonal antibodies (MoAbs) which recognize monomorphic and polymorphic epitopes on HLA Class I molecules were shown to inhibit T-cell proliferation to OKT3. These MoAbs have differential effects on the synthesis of interleukin-2 (IL-2) and IL-2 receptor expression. Cell cycle analysis demonstrated that these MoAbs function both in inhibiting cell cycle entry (G0-G1 shift) and in blocking cell cycle progression (G1-S shift) of activated T cells. Furthermore, these MoAbs have regulatory effects on the alternate pathway of T-cell activation via the CD2 molecule, T-cell activation induced by PHA, and activation induced by the phorbol ester PMA in conjunction with the calcium ionophore Ionomycin. Thus these MoAbs have different effects depending upon the pathway of T-cell activation. The results indicate that HLA class I molecules are selectively involved in the sequence of intracellular events leading to T-cell activation and proliferation.     

Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide

The expression of two membrane glycoproteins, RL388 antigen and transferrin receptor (TfR), was examined on murine B cells stimulated with lipopolysaccharide (LPS) in vitro. Immunofluorescent staining with monoclonal antibodies and flow cytofluorometric analysis were used to monitor the expression of these markers as a function of the time in culture, the state of membrane Ia antigen expression, the position in cell cycle, and the degree of B-cell differentiation. Freshly explanted splenic B cells expressed low levels of RL388 antigen and TfR. Following LPS stimulation, increased expression of RL388 antigen was detectable by 8 to 12 hr of culture, a time span characterized by increased Ia antigen expression, blast transformation, and G0 to G1 phase transition. The increased expression of TfR was apparent later and correlated with entry into late G1 phase and the onset of S phase. LPS-stimulated cell cultures treated with actinomycin D (G0/G1 block) exhibited increased expression of Ia antigen, but neither RL388 antigen nor TfR, whereas hydroxyurea treatment (G1/S block) allowed expression of all three markers. These results indicate that hyperexpression of RL388 antigen and TfR occurs during G1 phase and that these events are subsequent to Ia antigen hyperexpression. Finally, B cells in late G1 through M phase of the cell cycle simultaneously express high levels of RL388 antigen and TfR. These findings suggest that the expression patterns of RL388 antigen and TfR might be useful parameters for defining compartments of the murine B-cell cycle.     

Effect of priming with a thymus-independent antigen on susceptibility to B-cell tolerance.

The effects of priming on the susceptibility of B-cell subsets to tolerance induction have been tested in a model system in which anti-immunoglobulin (anti-Ig) has been employed as a surrogate for tolerogen. T-cell-depleted B cells were primed in vitro with fluorescein or trinitrophenylated Ficoll (a thymus-independent (TI) antigen) and then exposed overnight to anti-Ig to attempt to induce B-cell anergy. Primed cells were relatively resistant to this tolerance protocol and resistance was hapten specific. The dose response and kinetics suggested that this process was not due to receptor blockade or modulation, but was an active process. Moreover, this priming for resistance to tolerance was reproduced in vivo upon intraperitoneal treatment with haptenated Ficoll. Such in vivo priming for tolerance resistance was long-lasting and did not occur with a thymus-dependent priming protocol with fluoresceinated hemocyanin. These results are discussed in terms of TI priming to drive B cells into cycle and express novel functional and phenotypic properties.     

Withdrawal of 2-mercaptoethanol induces apoptosis in a B-cell line via fas upregulation

Mouse lymphoid cell cultures are dependent on reducing agents in their culture medium to allow proliferation and survival of the cells. In the case of the mouse CD5⁺-pre-B cell line SPGM-1, withdrawal of 2-mercaptoethanol (2-ME) resulted in rapid inhibition of proliferation and subsequent cell death by apoptosis. The pathways leading to cell death by withdrawal of 2-ME or by incubation with ionomycin, a known inducer of apoptosis, were compared. Both kinds of stimulation resulted in apoptosis of the whole population, but cell death occurred with different kinetics. Only apoptosis induced by ionomycin was inhibited by coincubation with the phorbol ester PMA, while apoptosis induced by withdrawal of 2-ME was not. Overexpression of the human bcl-2 proto-oncogene in these cells delayed the death process induced by either method. SPGM-1xbcl-2 cells accumulated in the G₀/G₁ and G₂/M cell cycle phases after removal of 2-ME from the medium, whereas treatment with ionomycin resulted in an arrest only in the G₀/G₁ transition. Interestingly, both stimuli induced the expression of the Fas receptor, but with different kinetics, while the Fas ligand (FasL) was expressed constitutively in SPGM-1 cells. These data demonstrate that withdrawal of 2-ME and incubation with ionomycin both induce rapid cell death by apoptosis, possibly mediated by an autocrine Fas/FasL loop. Although the initial pathways activated by the two forms of treatment must be different, they converge on a common level controlled by the anti-apoptotic gene product Bcl-2. J. Cell. Physiol. 177:68–75, 1998. © 1998 Wiley-Liss, Inc.     

Effect of gamma radiation on resting B lymphocytes. II. Functional characterization of the antigen-presentation defect

The effect of radiation on three discrete Ag-presentation functions in resting B cells was examined: 1) Ag uptake and processing, 2) expression of processed Ag in the context of functional class II molecules, and 3) provision of necessary co-stimulatory, or "second," signals. Analysis of radiation's effect on B cell presentation of intact vs fragmented Ag or its effect on presentation by Ag-pulsed B cells indicated that damage to Ag uptake and processing could not account for the bulk of the radiation-induced Ag-presentation defect. Experiments with phosphatidylinositol hydrolysis as an indirect measure of TCR occupancy suggested that irradiation caused a fairly rapid (within 1 to 2 h) decrease in the ability of the B cell APC to display a stimulatory combination of Ag and class II molecule. Ag dose-response analyses demonstrated that when presenting a fragment of the Ag pigeon cytochrome c to a T cell clone, 3000 rad-treated B cell APC were able to stimulate approximately 50% as much phosphatidylinositol turnover as unirradiated B cells. It was also found that, in contrast to their inability to initiate T cell proliferation, and similarly to chemically cross-linked splenocytes, heavily irradiated resting B cells plus Ag induced a state of Ag hyporesponsiveness in T cell clones. This effect on T cells had the same Ag- and MHC-specificity as did receptor occupancy required for proliferation, indicating that heavily irradiated resting B cells bear functional class II molecules. Co-culture of T cells with allogeneic B cells and syngeneic heavily irradiated B cells or chemically cross-linked splenic APC plus Ag resulted in T cell proliferation and interfered with the induction of the hyporesponsive state. This co-stimulatory function was radiosensitive in resting allogeneic B cells. Together, these data support the hypothesis that the major functional consequences of radiation to resting B cell APC are a reduction in the effective display of Ag plus class II molecules and, probably what is more important, a loss in the ability to provide APC-derived co-stimulatory signals.