Plasma membrane phospholipid asymmetry precedes DNA fragmentation in different apoptotic cell models

Chan, Andrew; Reiter, Rudolf; Wiese, Stefan; Fertig, Georg; Gold, Ralf

doi:10.1007/s004180050317

Cited by 49 publications

(37 citation statements)

References 27 publications

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“…2 D and E). The higher proportion of Annexin V-positive cells compared with TUNEL-positive cells is consistent with most cells still being in the early stages of apoptosis at 7 h postinfection (36). The robust ability of serotype 4 to induce apoptosis is also observed at lower MOI (Fig.…”

Section: S Pneumoniae Induces Dna Damage Responses In Alveolar Epithsupporting

confidence: 75%

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

Rai

Parrish

Tay

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

131

102

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Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H 2 O 2 , plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H 2 O 2 , greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H 2 O 2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniaeinduced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.DNA damage | Streptococcus pneumoniae | hydrogen peroxide | γH2AX | Ku80

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Section: S Pneumoniae Induces Dna Damage Responses In Alveolar Epithsupporting

confidence: 75%

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

Rai

Parrish

Tay

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

131

102

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“…Furthermore, the TUNEL assay is prone to false positive and negative staining (15,24). ISEL and PI detect specific DNA fragments and loss of cell membrane integrity, respectively, which are features of inevitable PCD as they occur after each other during the execution phase (1,2,16,(25)(26)(27). From our background of research with stem cells, we questioned if detection of AnV (within CD34ϩ cells) means that the point-of-no-return of PCD has been passed.…”

Section: Discussionmentioning

confidence: 99%

The dynamic process of apoptosis analyzed by flow cytometry using Annexin‐V/propidium iodide and a modified in situ end labeling technique

Span¹,

Pennings

Vierwinden

et al. 2001

Cytometry

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BackgroundTo study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin‐V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death.MethodsApoptosis was induced in Jurkat cells by γ‐irradiation, incubation with camptothecin (CPT), or cytosine β‐D‐arabinofuranoside (Ara‐C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze‐thaw procedure. Various AnV/PI− CD34+ fractions were cultured in a single‐cell single‐well (SCSW) assay.ResultsJurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI− cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80–90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI− fractions (overall range 23–62%). Within total AnV+ and AnV+/PI− populations, only a minority of CD34+ cells showed ISEL positivity (range 4–8% and 0.8–6%, respectively). Different fractions of AnV+/PI− CD34+ cells did have clonogenic capacity.ConclusionsPCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo‐nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV‐fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI− CD34+ cells in the SCSW assay. Cytometry 47:24–31, 2002. © 2001 Wiley‐Liss, Inc.

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“…This method has been described in detail (12,19). In brief, apoptosis in freshly prepared autologous thymocytes was induced by adding 0.1 g/ml methylprednisolone (MP; Hoechst, Frankfurt am Main, Germany).…”

Section: Preparation Of Autologous Apoptotic Thymocytesmentioning

confidence: 99%

“…The assay that we used for our experiments is a modified version of a readout system that has been used on many different cell types (12,18,19,(23)(24)(25)(26)(27) and was adjusted to microglia cells (12). In brief, 3 ϫ 10 5 microglia cells per well were cultured overnight.…”

Section: In Vitro Phagocytosis Assaymentioning

confidence: 99%

Microglial Phagocytosis of Apoptotic Inflammatory T Cells Leads to Down-Regulation of Microglial Immune Activation

Magnus

Chan

Grauer

et al. 2001

The Journal of Immunology

Self Cite

126

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Apoptotic cell death is an established mechanism to terminate an inflammatory response in rodent or human brains. Microglia, as the resident phagocyte, is a strong candidate for the clearance of apoptotic lymphocytes. Apoptosis was induced in cultured autologous thymocytes and in myelin basic protein (MBP)-specific, encephalitogenic T cells from Lewis rats by the addition of 0.1 μg/ml methylprednisolone. The amount of phagocytosis of apoptotic cells was assessed using an in vitro phagocytosis assay. Supernatants were collected to measure microglial cytokine secretion. The state of immune activation in microglia was investigated by a T cell proliferation assay and by flow cytometric analysis of microglial surface expression of immune molecules. Microglia ingested specifically apoptotic cells (apoptotic thymocytes as well as MBP-specific T cells) in contrast to nonapoptotic control cells (p < 0.0001). Subsequent secretion of the proinflammatory cytokines TNF-α and IL-12 was significantly decreased, while the secretion of IL-10 and TGF-β was not affected. Furthermore, ingestion of apoptotic cells led to increased microglial MHC class II expression without concomitant increase in MHC class I, costimulatory molecules, and ICAM expression. The Ag-specific activation of MBP-specific T cells in cocultures with microglia that had ingested apoptotic cells was significantly less than that of identical T cells that interacted with nonphagocytosing microglia. Together with negative results obtained in a trans-well system, this is in support of a cell contact-mediated effect. Microglia might play an important role in the clearance of apoptotic cells. The uptake of apoptotic cells by microglia is tolerogenic and results in a reduced proinflammatory cytokine production and a reduced activation of encephalitogenic T cells. This might help to restrict an autoimmune inflammation and minimize damage in the inflamed brain.

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Plasma membrane phospholipid asymmetry precedes DNA fragmentation in different apoptotic cell models

Cited by 49 publications

References 27 publications

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

Streptococcus pneumoniaesecretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

The dynamic process of apoptosis analyzed by flow cytometry using Annexin‐V/propidium iodide and a modified in situ end labeling technique

Microglial Phagocytosis of Apoptotic Inflammatory T Cells Leads to Down-Regulation of Microglial Immune Activation

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