Transient down-regulation of PKC-zeta RNA following crosslinking of membrane IgM on WEHI-231 B lymphoma cells.

Regulation of protein kinase C (PKC) isoform mRNAs has been studied in the immature, murine B lymphoma WEHI-231 by the MAPPing protocol and by slot blot analysis of unamplified mRNA. This membrane IgM (mIgM)-positive cell line has been previously used as a model to study signal transduction by mIgM in immature B lymphocytes and the role of those signals in the induction of immune tolerance in the B cell compartment. Stimulation of the cells by anti-mu antibodies, phorbol ester, or Ca2+ ionophore caused growth arrest and death of the cells. IL 4 and IL 5 slowed the growth of the cells. Of these stimuli, only anti-mu stimulation affected PKC mRNA levels. Anti-mu treatment caused a transient decrease in the amount of PKC-zeta isoform mRNA within 3 hr. Within 24 hr levels returned toward normal. Anti-mu had little or no effect on the expression of mRNA for the alpha, beta, delta, or epsilon isoforms of PKC. WEHI-231 cells do not express PKC-gamma. Although anti-mu treatment blocked progression of the cells from the G0/G1 stage into the S phase of cell cycle, viable sort selected cells in either the G0/G1 or the S/G2/M phases showed no clear difference in the expression of PKC-zeta message. Thus, there is not preferential regulation of expression of PKC-zeta during stages of the cell cycle. The results show that mIgM on WEHI-231 cells can transduce a signal that is not mediated by PKC or Ca2+ mobilization alone. The signal causes transient, selective down-regulation of mRNA encoding the zeta PKC isoform.     

Antisense macrophage migration inhibitory factor (MIF) prevents anti-IgM mediated growth arrest and apoptosis of a murine B cell line by regulating cell cycle progression

Macrophage migration inhibitory factor (MIF) is involved in the generation of cell-mediated immune responses. Recently it has been reported that MIF also plays a role in cell proliferation and differentiation. In the present study, using a B-cell line, WEHI-231, and its stable MIF-antisense transfectant, WaM2, as a representative transfectant, we investigated the mechanism underlying regulation of the cell growth by MIF. WaM2 cells produced less MIF than vector control or parental WEHI-231 cells. Reduced and increased proportions were seen in G1 and S-phase cells, respectively, in WaM2 as compared with WEHI-231. Growth arrest and apoptosis after stimulation via surface Ig (sIg) were less prominent in WaM2 cells than those in WEHI-231. However, the addition of recombinant rat MIF did not reverse the inhibition of the growth arrest and apoptosis induced in WaM2 by cross-linking sIg. Almost the same amount of p27kip1 expression was detected in WaM2 cells as those in WEHI-231 and vector control cells. Cross-linking of sIg elevated the p27kip1 level equally in these cells irrespective of the MIF-antisense expression. Taken together, it seems that MIF plays a role in inducing apoptosis in B cells upon IgM cross-linking by regulating the cell cycle via a novel intracellular pathway.     

Programmed cell death by bcl-2-dependent and independent mechanisms in B lymphoma cells.

Programmed cell death (PCD) or apoptosis is a common form of cellular demise during embryogenesis, tumorigenesis and clonal selection in the immune system. The bcl-2 proto-oncogene has been recently implicated as a potential physiological regulator of the PCD pathway. Gene transfer studies have shown that overexpression of bcl-2 blocks apoptosis mediated by several stimuli in cultured cell lines and promotes the survival of B and T lymphocytes in transgenic mice. However, it remains unclear whether under normal conditions bcl-2 is responsible for controlling cell death. We have investigated the role of bcl-2 in the antimembrane IgM (mIgM)-induced apoptotic death of WEHI-231 B cell lymphoma, a model that mimics clonal deletion of immature B cells by antigen. Signalling of mIgM receptors triggered downregulation of both bcl-2 RNA and protein, and induced apoptosis in WEHI-231 B cells. This effect appeared to be specific since (i) the levels of beta 2-microglobulin and beta-actin RNA remain unchanged and (ii) signalling of the apoptosis-resistant B cell lymphoma line BAL-17 with anti-mu was not associated with downregulation of bcl-2 RNA. However, stable expression of bcl-2 by transfection did not rescue WEHI-231 B cells from apoptosis, yet WEHI-231 cells overexpressing bcl-2 were more resistant to programmed cell death induced by heat-shock.(ABSTRACT TRUNCATED AT 250 WORDS)     

The CD40-inducible Bcl-2 family member A1 protects B cells from antigen receptor-mediated apoptosis

CD40 activation is necessary for thymus-dependent humoral immune responses and rescuing both phenotypically immature WEHI-231 B lymphoma cells from B cell antigen receptor-induced cell death and germinal center B cells from spontaneous apoptosis. As some effects of CD40 are probably mediated by differences in gene expression, cDNA expression arrays and RNase protection assays were used to identify the anti-apoptotic Bcl-2 homolog A1 as a CD40-inducible gene in B cell lines and purified germinal center B cells. Sustained CD40-induced A1 upregulation correlated with CD40-mediated rescue of WEHI-231 cells from anti-IgM-induced apoptosis. Moreover, overexpression of A1 specifically protected WEHI-231 cells from anti-IgM-induced apoptosis but not cell death triggered by certain other stimuli.     

Thy28 partially prevents apoptosis induction following engagement of membrane immunoglobulin in WEHI-231 B lymphoma cells

Thy28 protein is conserved among plants, bacteria, and mammalian cells. Nuclear Thy28 protein is substantially expressed in testis, liver, and immune cells such as lymphocytes. Lymphocyte apoptosis plays a crucial role in homeostasis and formation of a diverse lymphocyte repertoire. In this study, we examined whether Thy28 affects induction of apoptosis in WEHI-231 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Once they were established, the Thy28-overexpressing WEHI-231 cells showed similar expression levels of IgM and class I major histocompatibility complex (MHC) molecule compared with controls. The Thy28-overexpressing cells were considerably resistant to loss of mitochondrial membrane potential (ΔΨm), caspase-3 activation, and increase in annexin-positive cells upon mIg engagement. These changes were concomitant with an increase in G1 phase associated with upregulation of p27^Kip1. The anti-IgM-induced sustained activation of c-Jun N-terminal kinase (JNK), which was associated with late-phase hydrogen peroxide (H₂O₂) production, was partially reduced in the Thy28-expressing cells relative to controls. Taken together, the data suggest that in WEHI-231 B lymphoma cells, Thy28 regulates mIg-mediated apoptotic events through the JNK-H₂O₂ activation pathway, concomitant with an accumulation of cells in G1 phase associated with upregulation of p27^Kip1 in WEHI-231 B lymphoma cells.     

Up-regulation of c-fos expression is a component of the mIg signal transduction mechanism but is not indicative of competence for proliferation

Control of entry into and progression through the early phases of cell cycle in B lymphocytes is poorly understood at the molecular level. Products of the c-fos proto-oncogene have been implicated in regulation of G0 to G1 cell cycle phase transition and cell proliferation in other systems. In view of these observations, the relationship between signals generated through receptor Ig which alter the B cells position in cell cycle and relative level of c-fos expression was investigated. Not unexpectantly, anti-Ig under conditions which promote G0-G1 and G1-S phase transition was observed to selectively up-regulate expression of c-fos. More interestingly, however, anti-Ig-induced cross-linking of surface Ig on the WEHI-231 B lymphoma also caused rapid and transient up-regulation of c-fos mRNA levels although it was associated with inhibition of proliferation of these cells. These results are important because they show that 1) c-fos expression is inducible in both normal and transformed B lymphocytes as a consequence of signals generated through receptor Ig, and 2) up-regulation of c-fos expression is not positively linked to B cell proliferation but rather appears to be a component of the surface Ig signal transduction mechanism. Finally, studies utilizing phorbol diesters suggest that pathways leading through protein kinase C are involved in both the growth inhibition and c-fos expression WEHI-231 following membrane-associated Ig cross-linking.     

Physicochemical and functional properties of murine B cell-derived B cell growth factor II (WEHI-231-BCGF-II)

A murine B lymphoma cell line, WEHI-231, constitutively secreted a kind of B cell stimulatory factor (BSF) that induced proliferation and IgM secretion in splenic B cells as well as BCL1 cells. Growth- and differentiation-promoting activities were not separated by various kinds of chromatographies on the basis of the m.w., isoelectric point, or hydrophobicity, and the degree of both activities in crude supernatants, DEAE-Toyopearl, TSK-3,000SWG, Mono P, and Phenyl-5PW fractions increased in a dose-dependent manner with complete correlation. The partially purified factor (WEHI-231-BGDF) did not show any other activities, such as IL 1, IL 2, interferon, or colony stimulating factor. WEHI-231-BGDF induced proliferation and IgM secretion in activated B cells with low density, but not in small resting B cells. WEHI-231-BGDF showed synergistic effect with dextran sulfate but not with anti-Ig in the induction of proliferation or IgM secretion in small resting B cells. WEHI-231-BGDF did not show any effect on Xid B cells. The relationship with several T cell-derived BSF and the significance of B cell-derived BSF in the B cell responses are discussed.     

Involvement of cell cycle progression in survival signaling through CD40 in the B-lymphocyte line WEHI-231

The CD40 molecule transmits a signal that abrogates apoptosis induced by ligation of the antigen receptor (BCR) in both primary B cells and B-cell lines such as WEHI-231. Expression of Bcl-xL and A1, antiapoptotic members of the Bcl-2 family, is enhanced by CD40 ligation, and is suggested to mediate CD40-induced B-cell survival. CD40 ligation also promotes cell cycle progression by increasing the levels of cyclin-dependent kinases (CDKs) required for cell cycle progression, and reducing expression of the CDK inhibitor p27(kip1). Here we demonstrate that cell cycle inhibition by retrovirus-mediated p27(kip1) expression does not modulate the levels of Bcl-xL or A1, but significantly reduces the survival of BCR-ligated WEHI-231 cells by CD40 ligation. This indicates that cell cycle progression is crucial for CD40-mediated survival of B cells.     

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca(2+)](c) in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca(2+) entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca(2+)](c) by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn(2+) quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca(2+) influx pathway of WEHI-231 was hardly permeable to Mn(2+). The intractable increase of [Ca(2+)](c) may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.     

Quantitative determination of the induction of apoptosis in a murine B cell line using flow cytometric bivariate cell cycle analysis.

WEHI-231 cells have been used extensively as a model of tolerance induction in B cells. Recent evidence has shown that anti-IgM treatment of WEHI-231 cells resulted in the induction of apoptosis. In this study, using acridine orange staining and flow cytometric analysis, we demonstrated that apoptotic cells are detected as a distinct population of cells separate from the cells in normal cell cycle progression. The validity of analysis gates was confirmed by cell sorting of the apoptotic population versus normal cells and subsequent gel analysis. Using this technique, we have demonstrated that F(ab')2 anti-mu, A23187, or PMA induced apoptosis in the WEHI-231 cells. The addition of LPS reversed apoptotic induction as seen previously with the WEHI-231 cell line. In contrast, however, PMA did not prevent the induction of apoptosis in anti-mu-treated cells. Additionally, we were interested in determining if the induction of apoptosis was restricted to a specific phase of cell cycle. Since growth inhibition results in most cells arresting in the G1 phase of cell cycle, we wanted to demonstrate apoptosis as a G1-dependent event. This was examined with WEHI-231 cells treated with known cell cycle inhibitors. Interestingly, inhibition of cells in each phase of cycle resulted in the induction of apoptosis. LPS was able to inhibit the induction of apoptosis with each of the cell cycle inhibitors except actinomycin D. Furthermore, we have demonstrated that the WEHI-231 cells contain a Ca(2+)-Mg(2+)-dependent preexisting endonuclease.     

Antigen receptor-induced cell cycle arrest in WEHI-231 B lymphoma cells depends on the duration of signaling before the G1 phase restriction point.

Stimulation of antigen receptors on WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-immunoglobulin M [IgM]) causes irreversible growth arrest. This may be a model for antigen-induced tolerance to self components in the immune system. Antigen receptor stimulation also causes inositol phospholipid hydrolysis, producing diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which causes release of calcium from intracellular stores. To better understand the nature of the antigen receptor-induced growth arrest of WEHI-231 cells, we have examined the basis for it. WEHI-231 cells in various phases of the cell cycle were isolated by centrifugal elutriation, and their response was evaluated following treatment with either anti-IgM or pharmacologic agents that raise intracellular free calcium levels and activate protein kinase C. Treatment with anti-IgM or the pharmacologic agents did not lengthen the cell cycle. Instead, growth inhibition was solely the result of arrest in the G1 phase. The efficiency of G1 arrest increased with the length of time during which the cells received signaling before reaching the G1 phase arrest point. Maximum efficiency of arrest was achieved after approximately one cell cycle of receptor signaling. These results imply that anti-IgM causes G1 arrest of WEHI-231 cells by slowly affecting components required for S phase progression, rather than by rapidly inhibiting such components or by rapidly activating a suicide mechanism. Antigen receptor stimulation was twice as effective as stimulation via the mimicking reagents phorbol dibutyrate and ionomycin. Thus, although the phosphoinositide second messengers diacylglycerol and calcium probably play roles in mediating the effects of anti-IgM on WEHI-231 cells, other second messengers may also be involved.     

Role of phosphoinositide-derived second messengers in mediating anti-IgM-induced growth arrest of WEHI-231 B lymphoma cells

Anti-IgM irreversibly inhibits the growth of WEHI-231 B lymphoma cells and induces phosphoinositide hydrolysis--producing diacylglycerol, which activates protein kinase C, inositol 1,4,5-trisphosphate, which induces the release of calcium from intracellular storage sites into the cytoplasm, and other inositol polyphosphates. The roles of two of the possible second messengers, cytoplasmic free calcium and diacylglycerol, in mediating the action of anti-IgM on WEHI-231 cells were assessed by elevating [Ca2+]i with ionomycin and by activating protein kinase C with phorbol 12,13-dibutyrate (PdBu). The combination of 250 nM ionomycin and 4 to 7 nM PdBu was found to cause growth arrest and cell volume decrease responses in WEHI-231 cells which were similar to those caused by anti-IgM, although clearly slower. Both anti-IgM and the combination of mimicking reagents induced growth arrest of WEHI-231 cells in the G1 phase of the cell cycle. In both cases, this growth arrest was mitigated by addition of bacterial LPS. Moreover, 250 nM ionomycin plus 4 to 7 nM PdBu did not inhibit the growth of two other murine B lymphoma cell lines, each of which did exhibit increased phosphoinositide hydrolysis but not growth arrest in response to anti-Ig. Taken together, these results suggest that ionomycin and PdBu, at the concentrations used, did not inhibit WEHI-231 growth by general toxicity, but rather by mimicking the effects of the natural second messengers generated from Ag receptor cross-linking. Thus, the phosphoinositide-derived second messengers Ca2+i and diacylglycerol are capable of playing important roles in mediating the action of anti-IgM on WEHI-231 B lymphoma cells. However, the response of WEHI-231 cells to anti-IgM could not be fully reproduced with ionomycin and phorbol diester. These results suggest that another second messenger induced by anti-IgM may also play an important role in mediating the growth arrest of these cells.     

CD40 ligand rescues inhibitor of differentiation 3-mediated G1 arrest induced by anti-IgM in WEHI-231 B lymphoma cells

The engagement of membrane-bound Igs (mIgs) results in growth arrest, accompanied by apoptosis, in the WEHI-231 murine B lymphoma cells, a cell line model representative of primary immature B cells. Inhibitor of differentiation (Id) proteins, members of the helix-loop-helix protein family, functions in proliferation, differentiation, and apoptosis in a variety of cell types. In this study, we analyzed the involvement of Id protein in mIg-induced growth arrest and apoptosis in WEHI-231 cells. Following stimulation with anti-IgM, expression of Id3 was up-regulated at both the mRNA and protein levels; this up-regulation could be reversed by CD40L treatment. Retrovirus-mediated transduction of the Id3 gene into WEHI-231 cells resulted in an accumulation of the cells in G(1) phase, but did not induce apoptosis. E box-binding activity decreased in response to anti-IgM administration, but increased after stimulation with either CD40L alone or anti-IgM plus CD40L, suggesting that E box-binding activity correlates with cell cycle progression. WEHI-231 cells overexpressing Id3 accumulated in G(1) phase, which was accompanied by reduced levels of cyclin D2, cyclin E, and cyclin A, and a reciprocal up-regulation of p27(Kip1). Both the helix-loop-helix and the C-terminal regions of Id3 were required for growth-suppressive activity. These data suggest that Id3 mimics mIg-mediated G(1) arrest in WEHI-231 cells.     

Peroxisome proliferator-activated receptor gamma-mediated NF-kappa B activation and apoptosis in pre-B cells

The role of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte physiology has been exploited for the treatment of diabetes. The expression of PPARgamma in lymphoid organs and its modulation of macrophage inflammatory responses, T cell proliferation and cytokine production, and B cell proliferation also implicate it in immune regulation. Despite significant human exposure to PPARgamma agonists, little is known about the consequences of PPARgamma activation in the developing immune system. Here, well-characterized models of B lymphopoiesis were used to investigate the effects of PPARgamma ligands on nontransformed pro/pre-B (BU-11) and transformed immature B (WEHI-231) cell development. Treatment of BU-11, WEHI-231, or primary bone marrow B cells with PPARgamma agonists (ciglitazone and GW347845X) resulted in rapid apoptosis. A role for PPARgamma and its dimerization partner, retinoid X receptor (RXR)alpha, in death signaling was supported by 1) the expression of RXRalpha mRNA and cytosolic PPARgamma protein, 2) agonist-induced binding of PPARgamma to a PPRE, and 3) synergistic increases in apoptosis following cotreatment with PPARgamma agonists and 9-cis-retinoic acid, an RXRalpha agonist. PPARgamma agonists activated NF-kappaB (p50, Rel A, c-Rel) binding to the upstream kappaB regulatory element site of c-myc. Only doses of agonists that induced apoptosis stimulated NF-kappaB-DNA binding. Cotreatment with 9-cis-retinoic acid and PPARgamma agonists decreased the dose required to activate NF-kappaB. These data suggest that activation of PPARgamma-RXR initiates a potent apoptotic signaling cascade in B cells, potentially through NF-kappaB activation. These results have implications for the nominal role of the PPARgamma in B cell development and for the use of PPARgamma agonists as immunomodulatory therapeutics.     

Identification of a prominent nuclear protein associated with proliferation of normal and malignant B cells

Experiments were undertaken to identify nuclear proteins that might be involved in regulation of the mitogenic process in B lymphocytes. Murine splenic B lymphocytes were purified and cultured with anti-Ig insolubilized onto Sepharose (anti-Ig/Sepharose) for 16 hr and labeled with [35S]methionine. Nuclei were isolated and the nuclear proteins were analyzed by two-dimensional gel electrophoresis. Anti-Ig/Sepharose induced a prominent increase in the synthesis and abundance of a 40 kDa/pI 5 nuclear protein (p40/pI-5). Inhibition of anti-Ig/Sepharose-induced mitogenesis by pretreatment of the cells with phorbol-12-myristate-13-acetate was associated with a specific inhibition (63%) of p40/pI-5. Subcellular fractionation experiments showed that p40/pI-5 is not detected in the soluble fraction of resting or activated B cells, indicating that this protein is located exclusively in the nucleus. Analysis of the expression of p40/pI-5 relative the cell cycle showed that the synthesis of this protein was increased during G1 phase and gradually reduced during S phase of the cell cycle. Abundant amounts of p40/pI-5 were also found in the rapidly proliferating B lymphoma cells, WEHI-231, and growth arrest of these cells by anti-mu was found to be associated with a marked inhibition (68%) of this protein. Taken collectively these results suggest that the nuclear protein p40/pI-5 may have an important role in regulation of the proliferation of normal and malignant B lymphocytes.