Cytoprotective response of A1, a Bcl-2 homologue expressed in mature human neutrophils and promyelocytic HL-60 cells, to oxidant stress-induced cell death

The ability to generate reactive oxidative intermediates is one of the quintessential properties of mature human neutrophils. Endogenously generated oxidants have been shown to be an important mechanism underlying neutrophil cell death. In acute lung inflammation, newly recruited neutrophils further encounter external oxidants, including reactive oxygen and nitrogen intermediates. In our present study, we showed that A1, a constitutive and inducible Bcl-2 homologue expressed in mature circulating human neutrophils, might confer the protection from hydrogen peroxide (H₂O₂)- and peroxynitrite (ONOO)-induced cell death. Utilizing the myeloid precursor cell line, HL-60, we further examined the hypothesis that A1 was capable of conferring cytoprotective activity against these oxidative stresses. Whereas the control-transfected HL-60 cells expressed small amounts of A1 and were sensitive to the biologically relevant, cell death-inducing oxidants, H₂O₂ and ONOO, the stable transfectants that overexpressed A1 were significantly more tolerant. Furthermore, there was a correlation between the level of A1 expression and the anti-apoptotic activity. Thus, our results suggest a cytoprotective role of A1 in mature human neutrophils under oxidant stresses in host defense and inflammation.     

Regulation of A1/Bfl-1 expression in peripheral splenic B cells

We analyzed here the expression of the prosurvival Bcl-2 homologue A1 in peripheral B cell compartment. We observed that A1 mRNA are highly expressed in peripheral B cells as compared with other anti-apoptotic genes of the Bcl-2 family such as bcl-xl and bcl-2 itself. The expression of A1 is up-regulated in immature B cells at the transition between transitional type 1 (T1) and type 2 (T2) cells, and remained highly expressed in mature (M) B cells. We, therefore, analyzed the effect of B cell antigen receptor (BCR) and BAFF receptor (BAFF-R) engagement on the regulation of A1 in total B220(+) cells but also FACS-sorted immature T1, T2 and M B cells. We demonstrated that only BCR engagement up-regulated the expression of A1 mRNA and protein. These results suggest that A1 may play a key role in antigen-dependent signals that are required for survival and/or proliferation of peripheral B cells.     

Bcl-2-related protein family gene expression during oligodendroglial differentiation

Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.     

Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa

Neutrophils enter the peripheral blood from the bone marrow and die after a short time. Molecular analysis of spontaneous neutrophil apoptosis is difficult as these cells die rapidly and cannot be easily manipulated. We use conditional Hoxb8 expression to generate mouse neutrophils and test the regulation of apoptosis by extensive manipulation of B-cell lymphoma protein 2 (Bcl-2)-family proteins. Spontaneous apoptosis was preceded by downregulation of anti-apoptotic Bcl-2 proteins. Loss of the pro-apoptotic Bcl-2 homology domain (BH3)-only protein Bcl-2-interacting mediator of cell death (Bim) gave some protection, but only neutrophils deficient in both BH3-only proteins, Bim and Noxa, were strongly protected against apoptosis. Function of Noxa was at least in part neutralization of induced myeloid leukemia cell differentiation protein (Mcl-1) in neutrophils and progenitors. Loss of Bim and Noxa preserved neutrophil function in culture, and apoptosis-resistant cells remained in circulation in mice. Apoptosis regulated by Bim- and Noxa-driven loss of Mcl-1 is thus the final step in neutrophil differentiation, required for the termination of neutrophil function and neutrophil-dependent inflammation.     

The anti-apoptotic effect of leukotriene B4 in neutrophils: a role for phosphatidylinositol 3-kinase, extracellular signal-regulated kinase and Mcl-1

The constitutive commitment of neutrophils to apoptosis is a key process for the control and resolution of inflammation and it can be delayed by various inflammatory mediators including leukotriene B4 (LTB4). The mechanisms by which LTB4 contributes to neutrophil survival are still unclear and the present work aims at identifying intracellular pathways underlying this effect. Inhibition of human neutrophil apoptosis by LTB4 was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and by the specific MEK inhibitor PD98059. In contrast, inhibitors of p38 MAPK, Jak2/3 and Src did not hinder the anti-apoptotic effect of LTB4. We also investigated the effects of members of the Bcl-2 family as they play a crucial role in the regulation of programmed cell death. When neutrophils were incubated with LTB4 for 1 to 6 h, the mRNA levels of the anti-apoptotic protein Mcl-1 were upregulated approximately 2-fold, while those of the pro-apoptotic protein Bax were downregulated 3- to 4-fold, as determined by real-time PCR. Accordingly, Western blot analysis revealed that the expression of Mcl-1 was upregulated in presence of LTB4, while flow cytometric analysis revealed that Bax protein was downregulated. Furthermore, the modulatory effects of LTB4 on Mcl-1 and Bax proteins were abolished in the presence of either wortmannin or PD98059. Taken together, these results demonstrate the participation of PI3-K and MEK/ERK kinases, as well as regulatory apoptotic proteins such as Mcl-1 and Bax, in the anti-apoptotic effects of LTB4 in human neutrophils.     

Cyclic AMP delays neutrophil apoptosis via stabilization of Mcl-1

Human neutrophils underwent spontaneous apoptosis, which was accompanied by degradation of Mcl-1, but not other anti-apoptotic molecules (cIAP1, cIAP2, A1, survivin and Bcl-2). Spontaneous neutrophil apoptosis and Mcl-1 degradation were prevented by cyclic AMP (cAMP) agonists (dibutyryl cAMP and prostaglandin E(1)), and the effects of cAMP agonists on neutrophils were highly resistant to cycloheximide, a protein synthesis inhibitor, although slight increase in Mcl-1 mRNA expression was induced by cAMP agonists. Proteasome inhibitors (epoxomicin and lactacystin) also prevented spontaneous neutrophil apoptosis and Mcl-1 degradation to the same extent as cAMP agonists, and no additive effect was obtained by combination of cAMP agonists and proteasome inhibitors. These findings suggest that cAMP agonists, like proteasome inhibitors, delay neutrophil apoptosis primarily via stabilization of Mcl-1.     

Delayed apoptosis by neutrophils from COPD patients is associated with altered bak, bcl-xl, and mcl-1 mRNA expression

ABSTRACT: BACKGROUND: Delayed neutrophil apoptosis may be an important factor in the persistent inflammation associated with chronic obstructive pulmonary disease (COPD). Bcl-2 family proteins are important regulators of neutrophil apoptosis. We determined the mRNA levels of proapoptotic Bak and anti-aptototic Bcl-xl and Mcl-1 members of the Bcl-2 family in unstimulated peripheral blood neutrophils from patients with mild to moderate COPD and compared these to neutrophils from healthy controls. METHODS: Neutrophils were isolated from peripheral blood samples of 47 COPD patients (smokers: N = 24) and 47 healthy controls (smokers: N = 24). Percentages of apoptotic cells were determined at 4, 24, and 36 h for unstimulated neutrophils cultured in vitro. Neutrophil mRNA expression of Bak, Bcl-xl, and Mcl-1 was determined by real-time polymerase chain reaction (PCR). FEV1 (% predicted) and FVC were determined by spirometry and correlations between mRNA levels and lung function parameters were determined. RESULTS: The percentages of apoptotic cells among unstimulated neutrophils from COPD patients were significantly lower compared to cells from controls after 4, 24, and 36 h in culture; smoking history had only a minimal effect on these differences. Unstimulated neutrophils from COPD patients had significantly lower Bak mRNA expression and higher expressions of Bcl-xl and Mcl-1 mRNA than cells from healthy controls. Again, smoking history had only a minimal effect on these trends. Bak mRNA expression was significantly positively correlated with both %predicted FEV1 and the FEV1/FVC ratio, while Bcl-xl and Mcl-1 mRNA expressions were significantly negatively correlated with %predicted FEV1 and the FEV1/FVC ratio. CONCLUSIONS: The genes for pro-apoptotic Bak, and anti-apoptotic Bcl-xl and Mcl-1 may be important in regulating the delayed neutrophil apoptosis observed in COPD, which may contribute to COPD pathogenesis. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1605269445677066.     

Distinct roles of Bcl-2 and Bcl-Xl in the apoptosis of human bone marrow mesenchymal stem cells during differentiation

Background

Adult mesenchymal stem cells (MSCs) can be maintained over extended periods of time before activation and differentiation. Little is known about the programs that sustain the survival of these cells.

Principal Findings

Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in response to different cell death inducers. Conversely, the same inducers can readily induce apoptosis when hMSCs are engaged in the early stages of differentiation. The survival of undifferentiated cells is linked to the expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is expressed at similar levels in undifferentiated and differentiated hMSCs while Bcl-2 is expressed only in differentiated cells. In undifferentiated hMSCs, the down-regulation of Bcl-Xl is associated with an increased sensitivity to apoptosis while the ectopic expression of Bcl-2 induced apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in undifferentiated hMSCs.

Significance

In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an anti-apoptotic activity under all conditions.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

SAHA treatment overcomes the anti-apoptotic effects of Bcl-2 and is associated with the formation of mature PML nuclear bodies in human leukemic U937 cells

Bcl-2 protects tumor cells from the apoptotic effects of various antineoplastic agents. Increased expression of Bcl-2 has been associated with poor response to chemotherapy in various malignancies, including leukemia. Therefore, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. We undertook this study to examine whether SAHA (suberoylanilide hydroxamic acid) overcomes the resistance by Bcl-2 in human leukemic cells, with a specific focus on the involvement of PML-NBs. Experiments were conducted with Bcl-2-overexpressing human leukemic U937 cells. Since we previously demonstrated that overexpression of Bcl-2 attenuates resveratrol-induced apoptosis in human leukemic U937 cells, resveratrol-treated U937 cells were used as a negative control. The present study indicates that SAHA at 1-7 μM, the dose range known to induce apoptosis in various cancer cells, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2-overexpressing human leukemic U937 cells. Notably, we observed that SAHA-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in human leukemic U937 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that could help bypass the resistance to apoptosis conferred by Bcl-2. Elucidating exactly how PML regulates Bcl-2 will require further work.     

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb–BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.     

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.     

Triggering receptor expressed on myeloid cells-1 in neutrophil inflammatory responses: differential regulation of activation and survival

Polymorphonuclear neutrophils (PMN) are crucial in the innate host defense by their ability to rapidly accumulate in inflamed tissues and clear a site of infection from microbial pathogens by their potent effector mechanisms. The triggering receptor expressed on myeloid cells (TREM)-1 is a recently described activating receptor on PMN with an important role in inflammation. However, the effects of TREM-1 stimulation on a cellular level remain to be further defined. To characterize TREM-1-mediated activation of human PMN, we evaluated the effect of receptor ligation on PMN effector functions. Activation via TREM-1 induces immediate degranulation of neutrophilic granules resulting in the release of IL-8, respiratory burst, and phagocytosis. TREM-1 ligation synergizes with the activation by the Toll-like receptors (TLR) ligands LPS, Pam(3)Cys, and R-848. In contrast, no synergy between TREM-1- and TLR-mediated stimulation was observed concerning PMN survival, whereas TLR-mediated stimuli protect PMN from apoptosis, concurrent TREM-1 activation neutralizes these anti-apoptotic effects. These results give a new perspective for the regulation of neutrophil inflammatory responses emphasizing the importance of TREM-1 in innate immunity.     

PRAM-1 is required for optimal integrin-dependent neutrophil function

PML-retinoic acid receptor alpha (RARalpha) regulated adaptor molecule 1 (PRAM-1) is an intracellular adaptor molecule that is upregulated during the induced granulocytic differentiation of promyelocytic leukemic cells and during normal human myelopoiesis. This report describes the generation of PRAM-1-deficient mice and an analysis of the function of this adaptor in neutrophil differentiation and mature neutrophil function. We demonstrate here that neutrophil differentiation is not impaired in PRAM-1-deficient mice and that PRAM-1-deficient neutrophils function normally following engagement of Fcgamma receptors. In contrast, mature PRAM-1-null neutrophils exhibit significant defects in adhesion-dependent reactive oxygen intermediate production and degranulation. Surprisingly, other integrin-dependent responses, such as cell spreading and activation of several signaling pathways, are normal. Together, these findings demonstrate the uncoupling of key integrin-dependent responses in the absence of PRAM-1 and show this adaptor to be critical for select integrin functions in neutrophils.     

DMSO及RA对GPI-80分子表达的不同影响

以往的研究表明GPI-80的表达可能与髓系细胞的分化相关。DMSO及RA是两种不同的中性粒细胞的诱导分化剂,均可刺激HL-60白血病细胞向中性粒细胞分化。GPI-80是人糖基化磷脂酰肌醇锚糖蛋白,被认为是潜在的β2-黏合素分子依赖的白细胞黏附的调节剂,主要在人中性粒细胞上表达。本研究通过RT—PCR、流式细胞仪及Western—blot分析,检测分化细胞的GPI-80表达,并分析GPI-80的表达与CD11b及CD71表达之间的关系。结果表明GPI-80在RA诱导的类中性粒细胞上只有mRNA水平上的微弱表达,用流式细胞仪和Western—blot分析均检测不到,且RA可抑制GPI-80的表达;相反GPI-80在DMSO诱导的类中性粒细胞上有明显的表达,且随DMSO的浓度增加及诱导时间的延长而增强。GPI-80的表达出现在CD11b上调表达及CD71下调表达之后,提示GPI-80表达与DMSO诱导分化的类中性粒细胞的成熟密切相关。RA不能明确诱导GPI-80的表达,反而抑制GPI-80的表达,提示可能两者诱导HL-60细胞分化时所激活的信号传递通路不同。     

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3^−/− mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3^−/− progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-X_L rescued survival; however, Bcl-X_L-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3^−/− cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-X_L permitted differentiation of G6PC3^−/− cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation and survival thus underlies neutropenia in G6PC3^−/− deficiency, both originating from a reduced ability to utilize glucose. Hoxb8-dependent cells are a model to study differentiation and survival of the neutrophil lineage.     

The differential effect of dexamethasone on granulocyte apoptosis involves stabilization of Mcl-1L in neutrophils but not in eosinophils

In the absence of activation signals, circulating human neutrophils and eosinophils undergo spontaneous apoptosis. The glucocorticoid dexamethasone (Dex) accelerates apoptosis in inflammatory cells such as eosinophils, but uniquely delays neutrophil apoptosis. Corresponding to the opposite effects of Dex on granulocyte apoptosis, we demonstrate that in neutrophils and eosinophils Dex oppositely affects expression of the anti-apoptotic Bcl-2 family protein Mcl-1L. Mcl-1L expression declines over time in vitro; however, Dex maintains Mcl-1L expression in neutrophils. In contrast, Dex accelerates Mcl-1L protein loss in eosinophils. Neither Mcl-1S, a pro-apoptotic splice variant, nor Bax were affected. Dex treatment in the presence of a translation inhibitor stabilized existing Mcl-1L protein in neutrophils, while Mcl-1L stability in eosinophils was unaffected. Accordingly, delay of neutrophil apoptosis by Dex was prevented by antisense Mcl-1L siRNA. Our findings suggest that regulation of Mcl-1L degradation plays an important role in the opposite effects of Dex on granulocyte apoptosis.