Change in the frequency of apoptosis after low- and high-dose X-irradiation of human lymphocytes.

The purpose of the present study was to correlate the type and frequency of cell death in human lymphocytes receiving variable doses of X-irradiation. Monocyte-depleted lymphocyte fractions were exposed in vitro to variable doses of X-rays of 0-20 Gy (0-2000 rads) and incubated for 4 and 16 h. An assessment of the mode of cell death (apoptosis vs. classical necrosis) was carefully evaluated using a multidisciplinary approach using light, fluorescence and electron microscopy (EM), and dye exclusion assays. Eosin Y exclusion assays indicated the absence of classical necrosis occurring in short-term cultures (4 h postirradiation). An assessment of cell counts, however, revealed a mean decrease of 4% at 0 Gy and 13% at 10-20 Gy (1000-2000 rads). The predominant mode of cell death was apoptosis, but the percent apoptotic cells (determined by EM) did not parallel this increase in cell loss with increasing radiation and actually decreased at doses above 5 Gy (500 rads). The discrepancy between percent cell loss and percent apoptosis was explained by a proposed change in overall duration of the apoptotic process. In long-term cultures (16 h postirradiation), a combination of classical necrosis, classical apoptosis, and combined apoptosis and necrosis (secondary necrosis of apoptotic cells) was apparent and was associated with a marked decrease in viability. Irradiation effects on lymphocytes showing none of the morphologic features of apoptosis or classical necrosis in short-term culture were evidenced by an increase in nuclear lobation. The results of this study indicate that the vast majority of peripheral blood lymphocytes are radioresistant. The use of irradiation in an in vitro model to study the biochemical events of the apoptotic process is also evaluated.     

Apoptosis of cytotoxic T-cells in histiocytic necrotizing lymphadenitis

 Cell death is necrotic or apoptotic. In histiocytic necrotizing lymphadenitis (HNL), apoptosis is the main form of cell death, resulting in the creation of nuclear debris that is one of the characteristic features of HNL. To investigate the cell type of apoptotic cells, 12 cases of HNL were analyzed using the immunohistochemical staining for TIA-1, a cytotoxic granule of either cytotoxic T or NK cells. One quarter to over half of all apoptotic cells were positive for TIA-1, and some of the nuclear debris was also positive. The necrotic lesions of HNL were found to consist of nuclear debris, apoptotic cells, histiocytes and lymphocytes. The lymphocytes were mainly CD8-positive T-cells or CD4-positive cells, while B- and NK cells were only rarely observed. The number of TIA-1-positive lymphocytes was more closely related to the number of CD8-positive cells than to the number of CD4 cells. In double staining, the TIA-1 positive lymphocytes were mainly CD8 positive, but rarely CD4 positive. In HNL, then, CD8-positive cytotoxic T-cells are likely to undergo apoptosis. Received: 4 November 1997 / Accepted: 9 March 1998     

B cell apoptosis accelerates the onset of murine lupus

To investigate whether the increased rate of lymphocyte apoptosis in systemic lupus erythematosus is involved in the onset of the disease, apoptotic or necrotic T or B lymphocytes from various cell lines were injected intraperitoneally into pre-autoimmune (NZBxNZW)F1 mice (BW) and non-autoimmune BALB/c mice. The intraperitoneal production of cytokines and chemokines, the specific T cell response in the spleen, and the production of anti-histone and anti-dsDNA Ab were investigated. The onset of the disease was characterized by creatinine levels and evaluation of glomerular IgG deposits. In BW, but not in BALB/c mice, injection of apoptotic and not necrotic cells up-regulated IL-6 and IL-10 in resident macrophages. Administration of apoptotic cells augmented the number of Th2 and B lymphocytes recruited in the peritoneal cavity. Only the treatment with apoptotic B cells promoted a systemic Th2 autoimmune response to H2 histones, associated with earlier occurrence of high levels of anti-dsDNA autoantibodies, higher creatinine levels and more numerous glomerular IgG deposits than in BW controls not injected with apoptotic B cells. In genetically susceptible mice exposure to apoptotic of B, but not T, lymphocytes can elicit a Th2 response to H2 histones that helps B cell production of anti-dsDNA Ab and finally triggers the onset of lupus.     

Role of Nr13 in regulation of programmed cell death in the bursa of Fabricius

Apoptotic cell death is developmentally regulated in the chicken bursa of Fabricius. Although apoptosis is low in the embryonic bursa, cell death increases markedly after hatching. The expression of Bcl2 family cell death antagonists was examined to identify the genes that regulate bursal cell apoptosis. The expression of Bcl-xL, A1, and Mcl1 was detected in both embryos and hatched birds, whereas Nr13 was expressed at high levels in embryonic bursa, and decreased significantly after hatching, correlating inversely with apoptosis. The oncogene v-reland phorbol myristate acetate, two known inhibitors of bursal cell apoptosis, induced Nr13 expression. Overexpression of Nr13 in DT40 bursal lymphoma cells protected them from low serum-induced apoptosis. The mechanism of inhibition of apoptosis by Nr13 is likely to involve a critical BH4 domain and interaction with death agonist Bax. Deletion of the BH4 domain converted Nr13 into a death agonist. Bax coimmunoprecipitated with Nr13 and Bax was induced, whereas Nr13 levels diminished when bursal lymphoblasts were induced to apoptosis by dispersion. Bursal transplantation studies demonstrated that Nr13 could prevent the in vivo programmed elimination of bursal stem cells after hatching, suggesting that Nr13 plays a role in maintaining bursal stem cells.     

Immune deficiency following thermal trauma is associated with apoptotic cell death

Thermal injury-associated specific immune deficiency occurs despite indicators of systemic activation of the lymphoid compartment. We investigated the possibility that postburn immune failure and T cell activation are causally related through activation-induced (apoptotic) cell death. The relationship between the cellular immune response and cell mortality was examined in cultures of peripheral blood mononuclear cells (PBMC) from 14 immunosuppressed patients with extensive burns (35–90% total body surface area). Impaired cellular immunity coincided with significantly reduced cell viability as ascertained by propidium iodide staining and dye reduction assays. Following stimulation with the mitogenic lectin, phytohemagglutinin (PHA), the majority of DNA in patient cultures was fragmented, suggesting the occurrence of apoptotic cell death. Even without stimulation a portion of patient cells was apoptotic as indicated by oligonucleosomal bands on agarose gel electrophoresis. Exogenous interleukin-2 or phorbol ester markedly reduced constitutive as well as PHAinduced DNA fragmentation.In situ demonstration of DNA strand breaks in freshly isolated patient PBMC, by a TdT-based labeling technique, confirmed that a larger fraction (up to 60%) of circulating lymphocytes was undergoing apoptosis on the periphery. These novel observations suggest that apoptosis may play a major role in thermal injury-related cellular immunodeficiency.     

Versatility of merocyanine 540 for the flow cytometric detection of apoptosis in human and murine cells

Since apoptosis plays many roles in development, immune function, and disease, there is an ongoing need to identify inexpensive and reliable fluorochromes for the quantitation of apoptosis. Merocyanine 540 (MC540) binds to the outer membrane of cells and readily fluoresces in the highly disordered membranes of apoptotic cells making them readily detectable by flow cytometry. Protocols for the effective labeling and gating of MC540br apoptotic cells are provided. For example, MC540br cells from dexamethasone (Dex) treated thymocytes were found to be equivalent in proportion to apoptotic cells noted in the propidium iodide (PI) stained and annexin-V stained populations. Sorting of the MC540br cells followed by counterstaining with PI demonstrated that these cells resided in the low DNA fluorescent or sub-G1 region and were small in size based on light scatter. Dexamethasone, etoposide, irradiation, and a calcium ionophore were used to induce cell death with equivalent numbers of apoptotic cells obtained with MC540 and PI. Moreover, apoptotic human bone marrow (BM) B cells, neutrophils, Jurkat T cells, and testicular cells could readily be identified with MC540. The latter is particularly noteworthy since some of the standard methods for identifying cell death have not worked well with human cells. The versatility of this dye is such that it was also possible to phenotypically label cells stained with MC540 to analyze apoptosis in heterogenous populations of cells. Finally, the rate of detection of apoptotic cells after treatment of thymocytes with dexamethasone at 2, 4, 6, and 8 h with MC540 was shown to be equivalent to PI and annexin-V. Taken together, the data demonstrate that when proper precautions are taken, MC540 is a reliable, versatile, and inexpensive fluorochrome that can be used to identify apoptotic cells of human or murine origin even in heterogenous populations that require multicolor labeling.     

Lymphocyte apoptosis as an immune subversion strategy of microbial pathogens

Apoptosis is a component of cellular death in several immunological reactions. Lymphocyte apoptosis is a feature of negative selection of thymic lymphocytes. Target cells die by apoptosis during their interaction with cytotoxic T cells. Antigens derived from apoptotic cells can be cross-presented by antigen presenting cells (APCs). In these examples, apoptotic death is a beneficial feature for the individual. The apoptosis of cells also occurs during infection with a variety of microorganisms, but this process can be detrimental to the handling of the infection by the host. Here, we aim to highlight some of the recent advances in understanding why apoptosis can be a detrimental event during infection. We will focus on recent research with the intracellular bacterial pathogen Listeria monocytogenes, which demonstrates how apoptosis is induced, some of the host pathways that are exploited and the immunological consequences of cell death. We propose that L. monocytogenes causes lymphocyte death by enhancing the cell-death programs of the host. The presence of apoptotic lymphocytes downregulates early innate immunity, creating a permissive environment for bacterial growth.     

Monocyte viability on titanium and copper coated titanium

The role of apoptosis/cell death in the inflammatory response at the implanted materials is unexplored. Two surfaces with different cytotoxic potential and in vivo outcomes, titanium (Ti) and copper (Cu) were incubated in vitro with human monocytes and studied using a method to discriminate apoptotic and necrotic cells (Annexin V/PI staining). Further, staurosporine, a potent inducer of apoptosis, was added to the surface adherent monocytes. Lactate dehydrogenase (a marker of cell membrane injury) and TNF-alpha and IL-10, cytokines, previously suggested to play a major role in the monocyte apoptosis, were assayed in the culture medium. The results demonstrated that Ti surfaces displayed enhanced monocyte survival and production of IL-10 and TNF-alpha. Cu adherent cells exhibited apoptotic signs as early as 1h after incubation. In contrast to Ti, after 48 h the predominance of apoptotic cells switched to apoptotic/necrotic cells on Cu surfaces. Staurosporine treatment of Ti adherent cells mediated similar type of cell death. LDH and cytokine contents were low around Cu surfaces, partly explained by interference between Cu ions and LDH and cytokines. This study suggests that material properties rapidly influence the onset of human monocyte apoptosis and progression to late apoptosis/necrosis. Early detection of apoptosis and cell death may be important for the understanding of the biological response to implanted materials.     

Bovine herpesvirus 1-induced apoptosis: phenotypic characterization of susceptible peripheral blood mononuclear cells

Summary.  Bovine herpesvirus 1 (BHV-1), a member of the it Alphaherpesvirinae, induces apoptotic cell death in peripheral blood mononuclear cells (PBMC). To investigate the process by which BHV-1 induces apoptosis, we determined the susceptibility of the three main PBMC subpopulations to BHV-1-induced apoptosis. This study shows that BHV-1 can induce apoptosis individually in T lymphocytes, B lymphocytes and monocytes. This conclusion is based on the following findings: (i) BHV-1 substantially reduces the percentages of viable T and B lymphocytes in PBMCs. (ii) Concomitant detection of cell phenotype and apoptosis indeed showed higher percentages of apoptotic T lymphocytes and B lymphocytes in BHV-1-infected PBMCs than in mock-infected cells. (iii) Each individual PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes) undergo apoptosis when incubated with BHV-1. These data also suggest that BHV-1 does not require the recruitment of one or more individual PBMC subpopulations (e.g. cytotoxic cells) to induce apoptosis. Finally, we observed that BL-3 cells which have been characterized as bovine tumoral Blymphocytes also undergo apoptosis when incubated with BHV-1. Therefore, the use of the BL-3 cell line provides a new experimental model to investigate the apoptotic process induced by BHV-1 in vitro. Accepted November 17, 1997 Received September 2, 1997     

Prostaglandin E2 induces apoptosis in immature normal and malignant B lymphocytes.

The purpose of this research was to determine whether prostaglandin E2 (PGE2), a major product of macrophages which can kill certain murine B cell lymphomas, induces death by a necrotic mechanism or by an alternate pathway called apoptosis. CH31 is a phenotypically "immature" B cell lymphoma which resembles immature neonatal B cells in its susceptibility to killing by reagents which cross-link surface immunoglobulin (sIg). In the present study we first show that PGE2, but not the closely related prostanoid, PGF2 alpha, kills CH31 lymphoma cells. In contrast, CH12, a phenotypically "mature" lymphoma which is not negatively affected by sIg cross-linking, is not induced to die after exposure to PGE2. Agarose gel electrophoresis demonstrated that the DNA of PGE2-treated CH31, but not CH12 cells, is cleaved into characteristic 200 base pair oligonucleosomal fragments indicative of an apoptotic mechanism of death. However, a necrotic form of death, indicated by random DNA cleavage which produces a smear following electrophoresis, could be induced by treatment of CH12 or CH31 with anti-class II MHC antibodies and complement. The apoptotic mechanism of CH31 cell killing by PGE2 was confirmed using scanning electron microscopy which demonstrated the unique membrane blebbing and bubbling pathognomonic of this form of death. Finally, using a recently devised flow cytometric method to study apoptosis in heterogeneous cell populations, we compared the ability of anti-IgM, PGE2, or PGF2 alpha to induce apoptosis in B lymphocytes from neonatal or adult mice. Anti-IgM, and to a lesser extent PGE2, but not PGF2 alpha, induces apoptosis in a fraction of neonatal B cells. None of these treatments induced cell death in B lymphocytes from mature mice. Overall, these observations suggest that PGE-secreting cells such as macrophages, which inhabit the B cell microenvironments of lymphoid organs, may eliminate a subset of immature B lymphocytes and may be important in controlling the spread of PGE-sensitive malignant B lymphoma cells.     

Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry.

Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a heterogeneous sample.     

Cell death mechanisms following traumatic brain injury

Neuronal and glial cell death and traumatic axonal injury contribute to the overall pathology of traumatic brain injury (TBI) in both humans and animals. In both head-injured humans and following experimental brain injury, dying neural cells exhibit either an apoptotic or a necrotic morphology. Apoptotic and necrotic neurons have been identified within contusions in the acute post-traumatic period, and in regions remote from the site of impact in the days and weeks after trauma, while degenerating oligodendrocytes and astrocytes have been observed within injured white matter tracts. We review and compare the regional and temporal patterns of apoptotic and necrotic cell death following TBI and the possible mechanisms underlying trauma-induced cell death. While excitatory amino acids, increases in intracellular calcium and free radicals can all cause cells to undergo apoptosis, in vitro studies have determined that neural cells can undergo apoptosis via many other pathways. It is generally accepted that a shift in the balance between pro- and anti-apoptotic protein factors towards the expression of proteins that promote death may be one mechanism underlying apoptotic cell death. The effect of TBI on cellular expression of survival promoting-proteins such as Bcl-2, Bcl-xL, and extracellular signal-regulated kinases, and death-inducing proteins such as Bax, c-Jun N-terminal kinase, tumor-suppressor gene, p53, and the calpain and caspase families of proteases are reviewed. In light of pharmacologic strategies that have been devised to reduce the extent of apoptotic cell death in animal models of TBI, our review also considers whether apoptosis may serve a protective role in the injured brain. Together, these observations suggest that cell death mechanisms may be representative of a continuum between apoptotic and necrotic pathways.     

Survival of activated human T lymphocytes is promoted by retinoic acid via induction of IL-2

At the end of an immune response, most activated T cells spontaneously undergo programmed cell death (apoptosis). In the present study we show that all-trans retinoic acid (atRA), a major vitamin A metabolite, can inhibit the spontaneous apoptosis of activated human T lymphocytes in vitro. Isolated peripheral blood T lymphocytes were activated by 12-O-tetradecanoyl phorbol 13-acetate and cultured for up to 11 days without any further stimuli. With time, a gradual increase in cell death was observed. This spontaneous death of activated T cells was apoptotic, as demonstrated by cell shrinkage, DNA fragmentation and depolarization of the mitochondrial membrane. In the presence of physiological concentrations of atRA, the percentage of T cells exhibiting these apoptotic features was significantly reduced. After 5 days of stimulation, the percentage of TUNEL+ T cells decreased from 28 to 12% in the presence of atRA. The anti-apoptotic effect of atRA was mimicked by the retinoic acid receptor (RAR)-selective agonists 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and AM-580, and totally abrogated by the RAR-selective antagonist Ro 41-5253. Cytokines of the IL-2 family have been shown to improve the survival of activated T cells. Strikingly, we found that the ability of atRA to inhibit apoptosis was significantly correlated with its ability to increase the production of IL-2. Furthermore, a blocking anti-IL-2 receptor antibody completely abrogated the anti-apoptotic effect of atRA. Together, these results suggest that retinoic acid inhibits spontaneous apoptosis of activated T lymphocytes through a RAR-dependent increase in IL-2 production.     

Fas、Caspase-3和抗核小体抗体在参与SLE患者淋巴细胞凋亡紊乱中的作用

目的探讨SLE患者免疫功能紊乱与淋巴细胞凋亡信号传导途径异常之间的关系。方法应用流式细胞术测定SLE患者淋巴细胞表面Fas、FasL及细胞质中活化caspase-3的表达率,并测定凋亡细胞百分率（AnnexinV^＋PI^-）和坏死细胞百分率（AnnexinV^＋PI^＋）。应用ELISA方法测定血清中抗核小体抗体浓度。结果与健康对照组相比,稳定期和活动期SLE患者组淋巴细胞中凋亡细胞和坏死细胞百分率均显著增加（P〈0．05）,淋巴细胞表面Fas、FasL及细胞质中活化caspase-3的表达率也显著增加（P〈0．05）。与稳定期SLE患者组相比,活动期SLE患者组淋巴细胞中坏死细胞百分率显著增加（P〈0．05）,凋亡细胞百分率略有增加但无统计学意义（P〉0．05）。活动期患者组淋巴细胞表面Fas、FasL以及细胞质中活化caspase-3的表达率略有增加但无统计学意义（P〉0．05）。活动期SLE患者组抗核小体抗体浓度显著高于健康对照组和稳定期患者组（P〈0．05）。SLE患者凋亡细胞百分率和活化caspase-3的表达率与补体C3浓度水平呈负相关关系（P〈0．05）。结论Fas信号传导通路在SLE患者淋巴细胞凋亡紊乱中发挥了重要作用。caspase-3的活化是早期提示淋巴细胞凋亡的重要信号。SLE患者淋巴细胞凋亡活化程度与疾病活动程度和免疫效应功能紊乱密切相关,而淋巴细胞凋亡异常程度与抗核小体抗体水平的高低密切相关。淋巴细胞凋亡加速在SLE患者免疫病理损伤加重和免疫细胞调控紊乱中扮演了重要角色。     

Differential roles of JNK in ConA/GalN and ConA-induced liver injury in mice

Tumor necrosis factor-α-mediated liver injury can be induced by several different means; however, the signaling events and mechanisms of cell death are likely different. We investigated the mechanism of both apoptotic and necrotic hepatocyte cell death as well as the role of c-Jun NH₂-terminal kinase (JNK) in the ConA and ConA/D-galactosamine (GalN) models of murine liver injury. ConA alone induced primarily necrotic cell death with no caspase activation, whereas ConA/GalN induced apoptosis in addition to necrotic cell death. The bi-modal death pattern in the ConA/GalN model was confirmed by the use of transgenic mice expressing a dominant-negative form of Fas-associated death domain in which the mice were resistant to apoptotic but not necrotic cell death. JNK1 and, more significantly, JNK2 participated in the induction of hepatocyte apoptosis in response to ConA/GalN. Deletion of JNK led to the stabilization of FLIP_L, reduced caspase-8 activation, decreased Bid cleavage, and inhibition of the mitochondrial apoptosis pathway. In contrast, JNK did not participate in necrotic death induced by ConA either alone or in combination with GalN. As such, JNK-deficient mice remained susceptible to necrotic liver injury in both model systems. Thus, ConA and ConA/GalN mouse models induce liver injury with different mechanisms of cell death, and JNK contributes to apoptotic but not necrotic cell death. These findings further elucidate the specific pathways involved in tumor necrosis factor-α-mediated liver injury.     

Caspase-independent cell death in T lymphocytes

T lymphocyte death is essential for proper function of the immune system. During the decline of an immune response, most of the activated T cells die. Cell death is also responsible for eliminating autoreactive lymphocytes. Although recent studies have focused on caspase-dependent apoptotic signals, much evidence now shows that caspase- independent, necrotic cell death pathways are as important. An understanding of the molecular control of these alternative pathways is beginning to emerge. Damage of organelles including mitochondria, endoplasmic reticulum or lysozymes, leading to an increase in calcium and reactive oxygen species and the release of effector proteins, is frequently involved in caspase-independent cell death.     

Propagation and titration of infectious bursal disease virus,including non-cell-culture-adapted strains,using ex vivo-stimulated chicken bursal cells

Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.     

Role of Mtd/Bok in normal and neoplastic B-cell development in the bursa of Fabricius

B-cell development in the bursa of Fabricius is accompanied by extensive apoptotic cell death. Apoptosis, however, is suppressed during c-myc-induced neoplasia. The experiments described here suggest that Mtd/Bok may drive apoptosis during normal development, and that this activity is blocked during myc-induced tumorigenesis. Bursal Mtd/Bok expression increases during development, correlating with the onset of intense, spontaneous apoptosis after hatching. Two isoforms of Mtd/Bok were characterized: WT-chMtd/Bok, found predominantly in the mitochondria and a less abundant form, lacking the presumptive transmembrane domain, Mtd/Bok deltaTM, found predominantly in the cytosol. Over-expression of Mtd/Bok deltaTM in a bursal lymphoma-derived cell line, DT40, reduced mitochondrial function and sensitized DT40 cells to apoptotic stimuli, while WT-chMtd/Bok had a diminished phenotype in these cells. In contrast, retroviral transduction of bursal stem cells with WT-chMtd/Bok ablated normal stem cell function in transplantation experiments, and produced extensive apoptosis in myc-induced pre-neoplastic bursal populations, but not in tumor cells.     

Loss of lineage antigens is a common feature of apoptotic lymphocytes

The analysis of apoptosis in cell populations involves the detection of their specific lineage antigen (LAg) expression. This experimental approach relies on their assumed constant expression, but it is unclear whether such expression is actually maintained during cell death. We examined whether the loss of LAgs is a common feature of apoptotic lymphocytes and whether some might completely lose their LAgs. The changes in the expression of CD3, CD5, CD8, CD4, CD28, CD56, and CD19 were monitored in highly purified lymphocyte populations obtained by negative selection in a fluorescence-activated cell sorter. These were cultured for 24 h with or without phytohemagglutinin or staurosporin. For each LAg-positive subset studied, apoptosis was consistently more common among cells showing partial or total loss of LAg expression compared with cells maintaining their initial LAg levels. The kinetics of expression loss was rapid for CD8, CD56, and CD28, and more than 80% of initial expression was lost in the early stages of apoptosis but was slower for CD3, CD5, and CD4. For CD3 and CD5, expression was dependent on the apoptotic stimulus used. It is interesting that loss of antigen expression was independent of cell size. This phenomenon was also found in nonmanipulated, highly pure CD19 B lymphocytes of peripheral blood mononuclear cells from B chronic lymphocytic leukemia patients. Loss of LAg expression appeared to be a common feature of apoptotic lymphocytes under all the conditions assayed. The different kinetic patterns of LAg loss suggest apoptotic cells might actively regulate this process.     

The role of bcl-xL in CD40-mediated rescue from anti-μ-induced apoptosis in WEHI-231 B lymphoma cells

The phenotypically immature B cell lymphoma WEHI-231 undergoes apoptotic cell death when cultured with anti-immunoglobulin (Ig) antibodies, via a bcl-2-independent mechanism. We have therefore studied the role of the bcl-2-related protein bcl-x in controlling cell death in WEHI-231. We find that overexpression of the long form of bcl-x (bcl-x_L) renders these cells refractory to anti-Ig-induced cell death. Stimulation of WEHI-231 via CD40 has similar protective effects. We show here that ligation of CD40 rapidly induces the appearance of the bcl-x_L protein in WEHI-231, while stimulation via sIgM, sIgD, CD5 or CD45 receptors, or with phorbol esters plus ionomycin does not. WEHI-231 cells also rapidly undergo massive apoptosis following culture with thapsigargin, a specific inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum: this is also reversed by anti-CD40, or by overexpression of bcl-x_L. We, therefore, conclude that bcl-x_L plays a key role in the regulation of antigen receptor-mediated apoptosis via CD40 in WEHI-231. In addition, the fact that this protein is not induced in WEHI-231 in response to phorbol dibutyrate plus ionomycin points to a fundamental signaling defect in these cells, which could conceivably be a reflection of their immature, apoptosis-susceptible phenotype.