bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice

We have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl-xL, a product of bcl-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xL but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xL within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-xL are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-xL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation.     

Continued differentiation during B lymphopoiesis requires signals in addition to cell survival

During B lymphopoiesis, cells undergo successive rounds of division and growth arrest coupled to intermittent selection on the basis of Ig expression. It is unresolved whether differentiation requires specific signaling or is merely the consequence of sustained cell survival. Transgenic expression of the cell death antagonist, Bcl-2, promoted accumulation of B lymphoid cells in mice deficient in antigen receptor rearrangement (scid or rag-1-/-) and in mice lacking the IgM transmembrane domain (microMT). Continued differentiation occurred, however, only in the bcl-2/scid and bcl-2/microMT mice. The appearance of B lineage cells expressing CD21, CD22 and CD23 was associated with DHJH rearrangements which encode a truncated C mu-containing protein called D mu in bcl-2/scid mice and with expression of Ig heavy chain classes other than IgM in the bcl-2/ microMT mice. In neither case, however, were proliferating cells observed in the more mature B lineage compartments in the bone marrow. Thus, continued B cell development requires signaling via Ig heavy chain-containing receptors and is not simply a consequence of blocking apoptosis.     

Transgenic expression of VpreB-3 under the control of the immunoglobulin heavy chain enhancer and SV40 promoter

Recently we isolated a novel gene, VpreB-3 gene from the cDNA library of a pre-B cell clone. This gene is selectively expressed in pre-B and bone marrow-derived B cell lines. Its products are associated with the immunoglobulin heavy (IgH) chain in pre-B cell line. In the present study, to address the role of VpreB-3 on B cell development, transgenic mice carrying the VpreB-3 gene under the control of the IgH enhancer and SV40 promoter were produced. The transgenic mice expressed the VpreB-3 gene in bone marrow and spleen at a high level compared with control mice. In the thymus, the expression of the transgene was also detected, although its level was low. Flow cytometry analysis revealed that the frequency of CD45R+ and mu+ B cells were reduced in the bone marrow of 2 of the 11 transgenic mice and also reduced in the spleen of 1 of the transgenic mice. Furthermore, the results of a stromal cell-dependent B cell culture assay suggested that early B cell development, the differentiation from CD45R- B progenitor cells to CD45R+ pro-B cells, was delayed in the bone marrow cultures of 3 of the 5 transgenic mice compared with the control mice. These results suggested that VpreB-3 products may play some role in early B cell development at the stage of CD45R- B progenitor cells before the expression of surface mu chains.     

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During B cell development, the surface expression of CD79 alpha/CD79 beta heterodimers had been thought to begin in the pre-B cell stage where the heterodimers constitute pre-B cell receptors together with mu heavy and surrogate light chains. Thereafter, in mature B cells, CD79 alpha/CD79 beta associates with surface Ig to form B cell antigen receptors. In this study, we revealed by using newly established mAb that CD79 beta was expressed on the surface of pro-B cells which had not undergone the productive Ig gene rearrangement. Biochemical analysis showed that CD79 beta on pro-B cells existed either as monomers or as disulfide-linked heterodimers with CD79 alpha, non-covalently associated with four unidentified membrane molecules. Our finding that CD79 beta is expressed on earlier B-lineage cells than previously expected coincides with the recent study in which CD79 beta-deficient mice exhibit a blockade of B cell differentiation at the pro-B cell stage. Thus, it is speculated that the CD79 beta-containing complexes on pro-B cell surfaces may function to induce early B cell differentiation.     

Early expression of Ig mu chain from a transgene significantly reduces the duration of the pro-B stage but does not affect the small pre-B stage

During B cell development, V-J rearrangements at the Ig heavy mu chain (IgH mu chain) locus occur in early cycling precursors (pro-B stage). Subsequently, rearrangements at the Ig light (IgL) chain locus occur in late resting precursors (small pre-B stage). To study the effects of mu chain expression on the rate of B cell development, purified hematopoietic stem cells (HSC) bearing a mu chain transgene or wild-type HSC were transferred into immunodeficient RAG-2-/- mice and B cell development was followed over time. In addition, cycling B cell precursors were pulse-labeled by the injection of BrdU into transgenic and wild-type mice, and the production of BrdU-labeled kappa + and lambda + B cells was followed over time. These experiments suggested that early expression of the mu chain from the transgene significantly shortened the duration of the pro-B stage and immediately drove the precursors to differentiate into small pre-B cells. By contrast, the presence of the transgene did not affect the small pre-B stage, where IgL rearrangements occur. Thus, kappa and lambda rearrangements occurred only after the arrest of cell cycling as previously shown in wild-type mice, even when the mu chain is artificially expressed earlier in B cell development.     

bcl-2 alters the antigen-driven selection of B cells in mukappa but not in mu-only Xid transgenic mice

A point mutation in the pleckstrin homology domain of the mouse Bruton's tyrosine kinase (btk) gene results in an X-linked immune defect, Xid, characterized by immunologic unresponsiveness to polymeric carbohydrate Ags. In Xid mice, B cells specific for phosphocholine (PC) do not develop in peripheral lymphoid tissues because they either fail to be positively selected from the marrow or they are clonally deleted via an Ag-driven, receptor-mediated process. Overexpression of the bcl-2 gene allows PC-specific B cells to survive and mature in Xid mukappa anti-PC transgenic mice, but PC-specific B cells are not rescued by bcl-2 in Xid mu-only transgenic mice. The failure of bcl-2 to rescue PC-specific B cells, in mu-only transgenic mice suggests that either it does not correct the btk defect in the Ag-driven selection process that occurs in pre-B cells and/or in very immature B cells or that a btk-dependent proliferative phase is required for the selection and amplification of the PC-specific B cells in mu-only transgenic mice. The rescue of PC-specific B cells in mukappa transgenic mice indicates that bcl-2 can alter receptor-mediated B cell selection at late stages in B cell development. The rescued PC-specific B cells in Xid male mice do not exhibit an altered proliferation profile in response to B cell-stimulating agents compared with B cells from unmanipulated Xid mice; thus, they fail to respond to soluble anti-mu, or PC-dextran, but they proliferate in response to PC, anti-mu, or anti-id conjugated to Sepharose.     

Bcl-2 is expressed in human natural killer cells and is regulated by interleukin-2

Bcl-2 is a major anti-apoptotic protein expressed in many normal and malignant cells. Recently, low to absent expression was reported in human natural killer (NK) cells cultured in serum-free media which could be induced with stem cell factor. We investigated the expression of bcl-2 protein of NK cells in normal blood donors and compared the bcl-2 expression in CD56+ NK cells with CD3+ T cells. To determine bcl-2 reactivity, a three-color flow-cytometric technique was used. CD56+ CD3- NK cells had an average bcl-2 expression of 83% compared with CD3+ T cells. CD56 and CD3 double positive T cells had an average content of 111% compared with all peripheral CD3+ T lymphocytes. When peripheral mononuclear cells were cultured with interleukin-2 (IL-2), bcl-2 could be upregulated by IL-2 in all cell populations studied. The induction of bcl-2 in these cell populations paralleled the induction in CD56- T lymphocytes cultured under identical conditions. The induction of bcl-2 by IL-2 was confirmed by Western blotting. The maximum induction of bcl-2 by IL-2 was observed at an IL-2 dose of 100-1,000 U/ml. Our data confirm the anti-apoptotic protein bcl-2 as an activation- or proliferation-associated marker of normal NK cells which can be induced by IL-2.     

CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and independent forms

We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and beta 2m-/- mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23- CD43-) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in beta 2m-/- mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both beta 2m-/- and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the beta 2m-dependent form of CD1. They further suggest that a beta 2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.     

Apoptotic regulation in primitive hematopoietic precursors

Bcl-2 and bcl-xL function as suppressors of programmed cell death. The expression of bcl-2 protein in vivo is associated with long-lived hematopoietic cells such as mature lymphocytes and early myeloid progenitors. Bcl-xL, a homologue of bcl-2, is also expressed in lymphocytes and thymocytes. In contrast, the bcl-2-related proteins (bax, bad, and bak) act by promoting apoptotic cell death as shown from their expression in hematopoietic cell lines. We analyzed the expression of bcl-2 and bcl-x proteins in hematopoietic precursors obtained from various cell sources in adult mobilized peripheral blood collected from 13 patients with solid tumors, 8 adult bone marrow, and 12 umbilical cord blood. The analysis was based on the expression of the proliferation and activation specific antigens, CD38 and class II (HLA-DR). Similarly, we analyzed the expression of bcl-2-related proteins bcl-xL, bax, bad, and bak before and during ex-vivo expansion. Hematopoietic precursors expressing strongly the CD34 antigen (CD34(s+)) and lacking CD38 or HLA-DR expression were analyzed by using three-color immunofluorescence staining. The majority of CD34(+) cells expressed bcl-2 and unexpectedly showed a bimodal distribution of low and high expression. More cells that lacked or expressed low density CD38 expressed low bcl-2 than the more differentiated counterparts (those with high density CD38). Immaturity (ie, little or no HLA-DR) is associated with the expression of low bcl-2 compared with HLA-DR+. However, HLA-DR-/low population contained a lower number of cells expressing low bcl-2 (30% to 40%) than CD38(-/low) in comparable samples. The hematopoietic precursors with bcl-2(low) and bcl-2(high) formed a homogeneous population of undifferentiated lymphoid-like cells having a similar forward scatter. These cells expressed strongly the bcl-xL protein (>95%) but were bax low (4% to 12%), bad low (0% to 0.8%), and bak low (0% to 3%). The expression of apoptosis specific protein (ASP) was also low (3.4% +/- 3.1%) as was Annexin V. In addition, the CD34(+)/CD38(-) showed low cell cycle activity (<2.2%). Induction of apoptosis by overnight incubation of CD34 cells in serum-deprived medium resulted in the upregulation of bcl-2 as a single population histogram. Thus, these results suggest that in quiescent hematopoietic precursors, the bcl-2 protein plays a less prominent role as a survival promoter than bcl-xL and that the low bcl-2 expression did not promote apoptosis. During day 10 of ex vivo expansion of CD34(+) cells in liquid culture containing stem cell factor, interleukin-3 (IL-3), IL-6, IL-1beta, and erythropoietin, the CD34(+)/CD38(-) cells expressed high bcl-2 as a single population histogram, and greater than 90% were bcl-xL high. However, the expression of pro- and apoptotic antigens increased: bax (10% to 15%), bad (5% to 8%), bak (6% to 14%), and ASP (6% to 10%). These results show the importance of monitoring the expression of these proteins when defining the culture conditions for ex vivo expansion.     

Limited importance of CD40/CD40L interaction in the B7-dependent generation of anti-MOPC-315 cytotoxic T lymphocyte activity by tumor bearer splenic cells stimulated in vitro in the presence of tumor necrosis factor

We have previously illustrated the importance of B7-2 expression for the enhanced generation of cytotoxic T lymphocyte (CTL) activity by stimulation cultures of tumor bearer splenic cells to which tumor necrosis factor alpha (TNFalpha) has been added. Here we show that the B7-1 molecule is also important for CTL generation by such stimulation cultures, although to a much lesser extent than the B7-2 molecule. In addition, we show the importance of CD40/CD40L interaction for the expression of the B7-2 molecule, but not the B7-1 molecule, by tumor bearer splenic cells stimulated in vitro in the presence of TNF. The CD40/CD40L interaction is also shown to be important for the generation of CTL activity by tumor bearer splenic cells stimulated in vitro in the presence of exogenous TNF. However, the CD40/CD40L interaction is less important for the generation of enhanced CTL activity than for the expression of an elevated level of B7-2. Specifically, blockade of CD40/CD40L interaction, which reduced the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the presence of TNF to the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the absence of exogenous TNF, failed to reduce the level of CTL generated to the level generated by tumor bearer splenic cells stimulated in the absence of exogenous TNF. Finally, blockade of CD40/CD40L interaction was inferior to blockade of B7-2/CD28 interaction in inhibiting the generation of CTL activity by tumor bearer splenic cells stimulated in the presence of exogenous TNF. Thus, although CD40/CD40L interaction is important for the generation of enhanced CTL activity by stimulation cultures of tumor bearer splenic cells to which TNF has been added, TNF also mediates its potentiating effect for CTL generation by such stimulation cultures via other mechanisms that are independent of CD40/CD40L interaction but dependent on B7-2 expression.     

The expression of murine B cell CD23, in vivo, is regulated by its ligand, IgE

CD23, the low-affinity IgE receptor, is believed to participate in immune responses by mediating antigen capture for presentation by B cells and by shedding fragments with immunomodulatory properties. The number of CD23 molecules on B cells is increased during allergic responses and infection with helminths. This can be attributed in part to regulation of CD23 expression by cytokines, including IL-4. In addition, there is evidence that CD23 can be induced on cultured B cells by its ligand, IgE. In the current study we use IgE-deficient (IgE-/-) mice to establish the effects of IgE on CD23 expression by B cells in vivo, in the absence of allergic or parasitic stimuli. The spleens of IgE-/- and wild-type mice contained similar proportions of CD23+ B lymphocytes. However, cells from IgE-/- mice were found to have nearly 3-fold less CD23 on their surface. The mutant B cells had a corresponding defect in their ability to bind IgE. CD23 could be normally induced on IgE-/- B cells after culture with IL-4 or CD40 ligand, indicating that these cells had no inherent defect in CD23 biosynthesis. CD23 expression and IgE-binding capacity were both restored when splenocytes from IgE-/- mice were cultured in the presence of IgE. IgE-induced up-regulation of CD23 could be elicited in vivo as well. In IgE-/- mice, i.v. infusion of IgE corrected CD23 expression to wild-type levels. Our results demonstrate that IgE directly participates in CD23 regulation in vivo. This positive feedback loop may constitute a mechanism for the amplification of ongoing allergic responses.     

A combination of IL-10 and direct contact with plasma cell tumors decreases CD23 expression on splenic B cells

We and others have previously found that splenic B cells from plasma cell tumor-bearing mice exhibit decreased CD23 expression. In the present study we further examined the mechanism of CD23 down-regulation by plasma cell tumors. We show here that although direct contact is required between the tumor cells and B cells, it is not sufficient, since fixed tumor cells do not induce the same reduction in CD23 expression. We have identified IL-10, a cytokine produce by the tumors, as the sole soluble factor that contributes to decreased CD23 expression on B cells induced by plasma cell tumors because 1) Abs to IL-10 prevent the loss of CD23 induced by plasma cell tumors both in vitro and in vivo; 2) engineered IL-10 negative variants of these tumors are reduced in their ability to down-regulate CD23 expression; 3) rIL-10 alone induces partial, but significant, decreases in CD23 expression on normal splenic B cells; and 4) the addition of IL-10 and fixed tumor cells to cultures of normal splenocytes decreases CD23 expression to levels similar to those in cocultures with live tumor cells. Collectively, these results demonstrate that plasma cell tumors down-regulate CD23 expression on B cells by a coordinate mechanism of IL-10 plus contact-mediated events and reveal a novel role for IL-10 in the regulation of CD23 expression on B cells that is suggestive of host B cell activation in the presence of the tumor.     

CD40 ligand expressed on B cells in the BXSB mouse model of systemic lupus erythematosus

Male BXSB mice, unlike female BXSB, develop a severe early onset lupus-like disease that has been linked to an intrinsic B cell defect. In investigating this B cell defect the present study showed that male, but not female, BXSB contained a higher percentage of large, activated splenic B cells that were more responsive to anti-CD40 mAb-induced proliferation. The hyperactivity of the large B cells from the male mice was also observed in the absence of anti-CD40 mAb or any other stimuli. In examining the mechanism of the B cell hyperactivity, it was found that 20% of unstimulated large B cells from male mice, unlike large B cells from female mice, expressed CD40 ligand (CD40L), a molecule normally expressed on activated CD4+ cells. The percentage of large B cells from the male BXSB that expressed CD40L was increased to 43% by stimulation with LPS. A functional role for CD40L expression on B cells was confirmed by showing that CD40-Ig blocked the spontaneous proliferation of the large B cells from male mice. In addition, the stimulatory capacity of the large B cells from the male mice was demonstrated by their ability to induce DNA synthesis in small B cells in a CD40L-dependent manner. These results demonstrated that large B cells from male BXSB expressed functionally active CD40L. It is likely that the B cell CD40L expression and increased susceptibility to CD40 signaling due to an intrinsic B cell hyperactivity promotes autoimmune disease in BXSB mice.     

Cutaneous CD30+ lymphoproliferative disorders: expression of bcl-2 and proteins of the tumor necrosis factor receptor superfamily

The spectrum of CD30+ cutaneous lymphoproliferative disorders is characterized by the histology of a high-grade lymphoma but frequent clinical regression of skin lesions in lymphomatoid papulosis (LyP) and occasional regression in CD30+ large cell lymphomas (LCLs). A recent study shows that apoptosis may be a significant mechanism of regression of LyP (Arch Dermatol 133:828-833, 1997). Therefore, we studied expression of proteins that induce apoptosis, including CD27, CD40, CD95, and nerve growth factor receptor (NGF-R), as well as anti-apoptotic protein bcl-2 in skin lesions from 25 patients within the spectrum of CD30+ cutaneous lymphoma. Our results show consistent expression of CD95 (APO-1/Fas), but rare or absent expression of CD27, CD40, and NGF-R on tumor cells from both regressing LyP lesions and nonregressing CD30+ lymphomas. Bcl-2 was expressed at low levels in LyP and at high levels in pleomorphic CD30+ lymphomas. These results indicate that, in addition to CD30, CD95 expression is preferentially expressed at high levels in all cutaneous CD30+ lymphomas and suggest that CD95 may play a role in the regression of CD30+ skin lesions. Expression of bcl-2 appears to protect tumor cells from apoptosis in CD30+ lymphoproliferative disorders.     

IL-2 receptor alpha-chain expression is independently regulated in primary and secondary lymphoid organs

The IL-2R is composed of three chains: IL-2R alpha, IL-2R beta, and IL-2R gamma. In mice, IL-2Ra is critical and determines IL-2 binding to the tripartite IL-2R complex. To extend our previous studies, which demonstrated that IL-2 regulates IL-2R alpha expression in vitro, we have analyzed expression in IL-2-deficient mice in vivo. As in control animals, CD4- CD8- thymocytes and bone marrow-derived B220+ pre-B cells were IL-2R alpha positive. In contrast, activated lymph node and splenic CD4 T cells (CD4+ CD69+) were found to be IL-2R alpha negative, whereas approximately 20% of the same cell populations from the MLR/lpr strain, which also accumulate large numbers of CD4-activated T cells in the presence of intact IL-2, retained expression. A similar pattern of IL-2R alpha expression was found among splenic CD8 cells from IL-2(-/-) and IL-2(+/-) animals. These findings demonstrate that in primary lymphoid organs, IL-2 is not directly involved in IL-2R alpha expression. However, at the level of mature lymphocytes, and more specifically CD4 T cells, IL-2 remains in vivo, as in vitro, the most critical cytokine controlling both IL-2R alpha expression and sensitivity to IL-2.     

Resistance to apoptosis and elevated expression of Bcl-2 in clonally expanded CD4+CD28- T cells from rheumatoid arthritis patients

Patients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28- T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28- T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28- T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28-CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28- T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28- T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28- T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.     

In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death

Gene targeting experiments have demonstrated that the expression of immunoglobulin heavy chain in the pre-B cell receptor (pBCR) and of heavy and light chains in the B cell antigen receptor (BCR) marks checkpoints in early B cell development that the cells have to pass to survive. To investigate whether the persistence of mature B cells in the peripheral immune system also depends on BCR expression, we have generated a transgenic mouse in which the BCR can be inducibly ablated through V region gene deletion. Ablation leads to rapid death of mature B lymphocytes, which is preceded by down-regulation of MHC antigens and up-regulation of CD95 (Fas) and can be delayed by constitutive bcl-2 expression.     

Differential expression of CD22 (Lyb8) on murine B cells

Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging IgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculating B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, in combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets.     

Ligation of CD40 potentiates Fas-mediated activation of the cysteine protease CPP32, cleavage of its death substrate PARP, and apoptosis in Ramos-Burkitt lymphoma B cells

The proliferation and survival of a B cell population is necessarily tightly controlled to prevent the arisal of malignancy or autoimmunity. The expansion or elimination of a B cell clone is determined through a complex interaction of the tumour necrosis factor receptor/nerve growth factor receptor family members CD40 and Fas, which are expressed on the B cell surface, with their respective physiological ligands (CD40L and FasL) expressed on the surface of CD4+ T cells. The regulation of B cell growth by signals transduced through CD40 and Fas contributes to the maintenance of peripheral tolerance and likely takes place and in the germinal centres (GC) of secondary lymphoid tissues. In this study, we investigate the relationship between the expression of Fas and B cell survival following engagement of CD40 and Fas in the Epstein-Barr virus-genome-negative Ramos-Burkitt lymphoma (Ramos-BL) B cell line model of GC B lymphocyte selection during maturation of the humoral immune response. We now present evidence that Ramos-BL B cells constitutively express both Fas and FasL on their surface and that expression of Fas, but not FasL, is enhanced following ligation of CD40. Coligation of CD40 and Fas, triggers for growth inhibition, activation of the interleukin-1 beta-converting enzyme, now caspase, family member CPP32 (caspase-3) but not Ich-1L (caspase-2), cleavage of its death substrate poly(ADP-ribose) polymerase, and apoptosis from the G1 phase of cell cycle; engagement of Fas alone fails to trigger for growth inhibition and apoptosis but enhances AgR-mediated cellular death. This CD40-potentiated Fas-triggered growth inhibition and apoptosis occurs in the presence of CD40-induced expression of the anti-apoptotic proteins Bcl-xL and Bcl-2. Taken together, these data indicate that ligation of CD40 facilitates efficient coupling of Fas to the caspase-mediated pathway of apoptosis.