Secondary Necrotic Cells Research Articles

Oxidation of the alarmin high-mobility group box 1 protein (HMGB1) during apoptosis

The architectural chromosomal protein high-mobility group box 1 protein (HMGB1) acts as an alarmin when released from cells. It is involved in the pathogenesis of inflammatory and autoimmune diseases. HMGB1 can undergo post-translational modifications including oxidation. However, the mechanisms and functional relevance of HMGB1 oxidation are not yet understood. Increased concentrations of reactive oxygen species (ROS) have been reported during apoptosis and necrosis. Hence, we investigated the oxidative status of HMGB1 in dead cells. Immunoblot analyses under reducing and non-reducing conditions revealed that HMGB1 is oxidized in dead cells. Moreover, tagging of oxidized cysteine residues by a maleimide moiety linked to polyethylene glycol showed that HMGB1 passively released from primary and secondary necrotic cells was predominantly oxidized. Also HMGB1 in plasma of patients with systemic lupus was reversibly oxidized. In conclusion, HMGB1 undergoes reversible oxidative modifications at cysteine residues during cell death, which may modulate its biological properties.

Nucleosome‐releasing factor: a new role for factor VII‐activating protease (FSAP)

Plasma proteins such as early complement components and IgM are involved in the removal of late apoptotic or secondary necrotic (sn) cells. We have recently described how a plasma protease that could be inhibited by the protease inhibitor aprotinin was essential to remove nucleosomes from sn cells. An obvious candidate, plasmin, did indeed have nucleosome-releasing factor (NRF) activity. However, recalcified plasma (r-plasma) retained its NRF activity after plasminogen depletion, which suggests the existence of another protease responsible for NRF activity in plasma. In this study we have used size-exclusion and anion-exchange chromatography to purify the protease responsible for NRF activity in plasma. SDS-PAGE analysis of chromatography fractions containing NRF activity revealed a protein band corresponding with NRF activity. Sequence analysis showed this band to be factor VII-activating protease (FSAP). We developed monoclonal antibodies to FSAP and were able to completely inhibit NRF activity in plasma with monoclonal antibodies to FSAP. Using affinity chromatography we were able to purify single-chain (sc) FSAP from r-plasma. Purified scFSAP efficiently removes nucleosomes from sn cells. We report that factor VII-activating protease may function in cellular homeostasis by catalyzing the release of nucleosomes from secondary necrotic cells.

The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti‐inflammatory phenotype

Proteins of the annexin family bind to phospholipids in a Ca2+ dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.

Apoptosis and necrosis: Detection, discrimination and phagocytosis

Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.

Apoptosis-associated antigens recognized by autoantibodies in patients with the autoimmune liver disease primary biliary cirrhosis

There is growing evidence that the onset of autoimmune disorders can be linked to the inefficient removal of apoptotic cells. Since defects in the elimination of apoptotic cells lead to secondary necrosis and subsequent release of intracellular components, this might explain the generation of autoantibodies against intracellular antigens. Accordingly, we wanted to investigate, whether antibodies from patients with the autoimmune liver disease primary biliary cirrhosis (PBC) recognize self-proteins generated and released during apoptosis. Using Western blot analyses we could detect intracellular antigens with serum IgG from PBC patients but not with serum IgG from healthy donors in lysates of Jurkat T-leukemia, HepG2 hepatoma, and HT-29 colon-carcinoma cells. Interestingly, PBC serum IgG also recognized caspase substrates in cells undergoing apoptosis induced by staurosporine or TRAIL (TNF-related apoptosis inducing ligand). In addition to intracellular antigens, serum IgG from PBC patients detected caspase-dependent antigens in the supernatants of apoptotic (secondary necrotic) cells and antigens on the surface of apoptotic Jurkat cells. Among the caspase substrates recognized by PBC serum IgG we could identify the components PDC-E2 and -E1beta of the known autoantigen PDC (pyruvate dehydrogenase complex). Thus, caspase-mediated processing of intracellular proteins might generate de novo autoantigens that upon release contribute to the generation of autoantibodies and autoimmune diseases as PBC.

A plasma nucleosome releasing factor (NRF) with serine protease activity is instrumental in removal of nucleosomes from secondary necrotic cells

We observed that interaction of secondary necrotic (sn) cells with human serum or plasma leads to loss of DNA staining. The decrease turned out to be a result of nucleosome release and was specific for apoptotic cells as necrotic cells did not show this phenomenon. We named this activity in plasma nucleosome releasing factor (NRF). NRF activity was completely inhibited by trypsin inhibitors suggesting that a serine protease is involved. Upon testing a number of plasma candidate serine proteases we found that plasmin did have NRF activity. However, plasminogen-deficient plasma still had NRF activity indicating that NRF is not plasmin. We conclude that a yet unidentified plasma serine protease is involved in removal of nucleosomes from sn cells.

Toll-like Receptor 2 and Type 1 Diabetes

Clearance of apoptotic cells by phagocytosis helps prevent the leakage of cellular contents that may stimulate inflammation or autoimmunity. Nonobese diabetic (NOD) mice develop type I diabetes (T1D) because of the impaired phagocytosis of apoptotic pancreatic β cells by macrophages; however the mechanism involved is unclear. Kim et al . (see commentary by Filippi and von Herrath) examined the responses of mouse macrophages [which enter the pancreas at the onset of T1D and later act as antigen-presenting cells (APCs) for T cells] to exposure to MIN6N8 insulinoma cells in various stages of radiation-induced cell death. Whereas incubation of macrophages with apoptotic MIN6N8 cells did not stimulate production of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), as measured by enzyme-linked immunosorbant assay (ELISA), secondary necrotic (SN) cells resulted in substantial TNF-α production. SN cells are late-stage, intact apoptotic cells that can be distinguished from apoptotic cells by flow cytometry on the basis of cell size and staining for markers of cell death and are characteristic of apoptotic cells that escaped phagocytosis. Gel-shift experiments showed that incubation of macrophages with SN MIN6N8 cells resulted in activation of the transcription factor nuclear factor-κB (NF-κB). Exposure of macrophages from Tlr2 –/– mice to SN cells resulted in a lower amount of TNF-α production than did exposure of wild-type macrophages to the cells. Tlr2 –/– mice were more resistant to T1D induced by pharmacological killing of β cells than were wild-type mice (as assessed by histological analysis of islets). Coculture and adoptive transfer experiments in mice showed that TLR2 was necessary for the maturation of APCs and the subsequent activation and proliferation of T cells that mediate T1D. These results link the accumulation of apoptotic cells with TLR2-dependent initiation of autoimmunity, raising the possibility of targeting TLR2 signaling in apoptosis-related diseases and autoimmune disorders. H. S. Kim, M. S. Han, K. W. Chung, S. Kim, E. Kim, M. J. Kim, E. Jang, H. A. Lee, J. Youn, S. Akira, M.-S. Lee, Toll-like receptor 2 senses β-cell death and contributes to the initiation of autoimmune diabetes. Immunity 27 , 321-333 (2007). [PubMed] C. M. Filippi, M. G. von Herrath, Islet β-cell death: Fuel to sustain autoimmunity? Immunity 27 , 183-185 (2007). [Online Journal]

Immunological Consequences of Apoptotic Cell Phagocytosis

Cells undergo apoptosis in development, tissue homeostasis, and disease and are subsequently cleared by professional and nonprofessional phagocytes. There is now overwhelming evidence that phagocyte function is profoundly altered following apoptotic cell uptake, with consequences for the ensuing innate and adaptive immune response. Pathogens and tumors exploit the changes in macrophage function following apoptotic cell uptake. Here, we will outline the consequences of apoptotic cell phagocytosis and illustrate how apoptotic cells could be used to manipulate the immune response for therapeutic gain.

Mechanism for proliferation inhibition by various selenium compounds and selenium-enriched broccoli extract in rat glial cells

The objective of this study was to investigate the differential effects of various selenium (Se) compounds and Se-enriched broccoli extracts on cell proliferation and the possible mechanism responsible for the Se-induced growth inhibition. C6 rat glial cells were incubated with graded concentrations up to 1000 nM of selenite, selenate, selenomethionine (SeM), Se-methyl-selenocysteine (SeMCys), high-Se broccoli (H-SeB) extract or low-Se broccoli (L-SeB) extract for 24 and 48 h. MTT results indicated that all Se sources and levels examined inhibited C6 cell proliferation at 48 h. The results from cell cycle progression and apoptosis analysis indicated that SeM, SeMCys, H-SeB or L-SeB treatments at the concentration of 1000 nM reduced the cell population in G(0)/G(1) phase, but induced G(2)/M phase arrest and increased apoptosis and secondary necrosis in C6 cells at 24 h. The populations of apoptotic cells and secondary necrotic cells were increased by all Se sources examined. The COMET assay indicated that there was no significant DNA single-strand break found for all Se treatments in C6 cells for 48 h. In addition, the Se-induced proliferation inhibition may involve a hydrogen peroxide (H(2)O(2))-dependent mechanism with elevated cellular glutathione peroxidase (cGPX) activity. Both H-SeB and L-SeB inhibited C6 cell proliferation but H-SeB was less inhibitory than L-SeB. The proliferation inhibition by H-SeB in C6 cells is apparently related to the increased H(2)O(2) with the elevated cGPX activity, but the inhibition by L-SeB was H(2)O(2)-independent without change in cGPX activity.

In vitro measurement of cell death with the annexin A5 affinity assay

One of the hallmarks of cell death is the cell surface-expression of phosphatidylserine. Expression of phosphatidylserine at the cell surface can be measured in vitro with the phosphatidylserine-binding protein annexin A5 conjugated to fluorochromes. This measurement can be made by flow cytometry or by confocal scanning-laser microscopy. The annexin A5 affinity assay comprises the incubation of cells stimulated to execute cell death with fluorescence-labeled annexin A5 and propidium iodide. Living cells are annexin A5-negative and propidium iodide negative, cells in the early phases of cell death are annexin A5 positive-and propidium iodide-negative, and secondary necrotic cells are annexin A5-positive and propidium iodide-positive. The entire procedure takes about 30 minutes for flow cytometry and 45 minutes for confocal scanning-laser microscopy. Various precautions and considerations are discussed further in the protocol described here.

Lectins detect changes of the glycosylation status of plasma membrane constituents during late apoptosis

Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of apoptotic cell material and their potentially histotoxic contents is a prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the apoptotic cells that are associated with their recognition by macrophages are the subject of this report. Apoptosis and necrosis in primary cells and cell lines were induced by various stimuli. The binding profile of 23 different lectins for vital, apoptotic, and necrotic cells were analyzed by flow cytometry. We observed that lectins were able to attach to the cell surfaces of vital and dying cells. Some lectins exhibited membrane destructive properties and, consecutively, changed the morphology of the cells as detected by flow cytometry. Other lectins did not show differences in their binding to viable and apoptotic cells. Those lectins were, therefore, not used for analyses of surface changes. The lectins Griffonia simplificolia II (GSL II), Narcissus pseudonarcissus (NPn), and Ulex europaeus I (UEA I) showed no cytotoxic activity and bound preferentially to dying cells. Primary and secondary necrotic cells displayed an equal staining intensity, which was substantially higher than for apoptotic cells. The binding of GSL II, NPn, and UEA to dying cells increased in a time-dependent manner and was delayed to AxV positivity and the decrease in the mitochondrial membrane potential of apoptotic cells. The kinetic of the lectin staining correlated with the increase in subG1-DNA. GSL II, NPn, and UEA are specific for N-acetylglucosamine, mannose, and fucose, respectively. According to their binding specificity, we conclude that N-acetylglucosamine-, mannose-, and fucose-containing epitopes are increasingly exposed on cells undergoing apoptosis.

Betanodavirus induces phosphatidylserine exposure and loss of mitochondrial membrane potential in secondary necrotic cells, both of which are blocked by bongkrekic acid

In this study, we show how the red spotted grouper nervous necrosis virus (RGNNV) causes loss of mitochondrial membrane potential and promotes host secondary apoptotic necrosis. RGNNV viral proteins such as protein α (42 kDa) and protein A (110 kDa) were quickly expressed between 12 h and 24 h postinfection (p.i.) in GL-av cells. Annexin V staining revealed that the NNV infection of GL-av cells induced phosphatidylserine (PS) externalization and development of bulb-like vesicles (bleb formation) at 24 h p.i. NNV infection also induced DNA fragmentation detectable by TUNEL assay between 12 h (8%) and 72 h (32%) p.i. Bongkrekic acid (1.6 μM; BKA) blocked permeability of the mitochondrial permeability transition pore, but cyclosporine A (CsA) did not block secondary necrosis. Finally, secondary necrotic cells were not engulfed by neighboring cells. Our data suggest that RGNNV induces apoptotic death via opening the mitochondrial permeability transition pore thereby triggering secondary necrosis in the mid-apoptotic phase.

Complement Activation by Apoptotic Cells Occurs Predominantly via IgM and is Limited to Late Apoptotic (Secondary Necrotic) Cells

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation.Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM.We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy.

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.

Inhibitory effect of IGF-I on induced apoptosis in mouse preimplantation embryos cultured in vitro

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation. Camptothecin (0.01 μg/ml) and actimomycin D (0.005 μg/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed. Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P>0.001; actinomycin D, P>0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased ( P<0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development ( P>0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.

Serum amyloid P component binds to late apoptotic cells and mediates their uptake by monocyte-derived macrophages.

Some pentraxins, such as C-reactive protein, bind to apoptotic cells and are involved in the clearance of these cells. We undertook this study to determine whether serum amyloid P component (SAP; a pentraxin that, when deficient in mice, results in lupus-like disease) binds to apoptotic cells and to assess the functional consequences of SAP binding for their phagocytosis by macrophages. Human peripheral blood monocytes were isolated and cultured for 7 days to obtain monocyte-derived macrophages. Jurkat cells were irradiated with ultraviolet B to induce apoptosis. After 4 hours, a mean +/- SEM of 54.0 +/- 5.1% of these cells stained with annexin V and were propidium iodide negative (early apoptotic [EA] cells). After 24 hours, 77.3 +/- 2.7% of cells stained positive with both annexin V and propidium iodide (late apoptotic [LA] cells or secondary necrotic cells). EA and LA cells were incubated with fluorescein isothiocyanate-labeled SAP in the presence or absence of Ca(2+), and binding was measured by flow cytometry. Phagocytosis was tested by incubation of macrophages with EA or LA cells in the presence of normal human serum (NHS) and quantified as a phagocytosis index (PI; number of Jurkat cells internalized by 100 macrophages). Experiments were repeated with SAP-depleted serum and after reconstitution with increasing concentrations of SAP. The majority of LA cells did bind SAP in the presence of Ca(2+), whereas EA cells did not. SAP depletion of NHS resulted in a 50% decrease in the PI for LA cells, and complete restoration of the PI could be demonstrated with SAP reconstitution up to 100 microg/ml. SAP depletion had no effect on phagocytosis of EA cells. SAP binds to LA cells and is involved in the phagocytosis of these cells by human monocyte-derived macrophages. This may have consequences for diseases such as systemic lupus erythematosus, in which phagocytosis of apoptotic cells is decreased.

The dynamics of morphological changes during in vitro aging of bovine virgin mammary gland neutrophils

The present study was an in vitro analysis of the dynamics of bovine mammary gland neutrophil apop&amp;shy;tosis based on the detection of morphological changes. The neutrophils were isolated from mammary glands of five virgin heifers. The mammary glands were lavaged, the suspensions were then bacteriologically examined, and total and differential cell counts were made. The cells were cultivated in vitro for 4 hours. After 2, 3 and 4 hours of cultivation, they were panoptically stained, and the proportions of apoptotic neutrophils and trypan blue positive neutrophils were determined. Neutrophil apoptosis and impaired cytoplasmic membrane integrity of neutrophils were already observed in the mammary gland lavages (11.9% and 0.8%, respectively). During the cultivation, a progressive increase in the number of apoptotic neutrophils in various stages of apoptosis &amp;ndash; karyopyknosis, zeiosis and apoptotic bodies &amp;ndash; was observed. Karyopyknotic neutrophils represented a dominant part of the apoptotic neutrophil population in the course of the whole cultivation. The most intensive increase was observed in zeiosis, whereas the levels of apoptotic bodies remained the same. After 4 hours of cultivation, 31.7% apoptotic neutrophils and 9.8% trypan blue positive neutrophils (i.e. Secondary necrotic cells) were found. The results of this work show that spontaneous apoptosis and secondary neutrophil necrosis must be taken into account during in vitro cultivations of bovine mammary gland neutrophils.

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6–11 μm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 441–457, 2001.

Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.

Effect of Calcium Channel Blockers, Nifedipine and Benidipine, on Death of Cultured Mouse Mesangi al Cells

We examined the effects of the short–acting calcium channel blocker (CCB) nifedipine and the long–acting CCB benidipine on the death of mouse cultured mesangial cells induced by tumor necrosis factor alpha (TNF–α) and/or cycloheximide (CHX). Cell death was evaluated by a morphological study using semithin sections. The dead cells were divided into three types, i.e., apoptotic cells (type 1), necrotic cells (type 3) and other types of dead cells, the so–called ‘secondary necrotic cells‘ or ‘postapoptotic necrotic cells’ (type 2). In the morphological study with semithin sections, cells in the presence of TNF–α or CHX and nifedipine or benidipine showed low percentages of all dead cell types with 24 h incubation. Both nifedipine and benidipine have protective effects against TNF–α or CHX. It is postulated that CCB might inhibit the apoptotic or necrotic processes by TNF–α or CHX with 24 h incubation. With 36 h incubation, CCB increased the percentages of all types of dead cells except for treatment with 1×10^–5 M benidipine and CHX. It appears that these cell–protective effects might be decreased after treatment with TNF–α or CHX and CCB for 36 h. In conclusion, the short–acting CCB nifedipine and the long–acting CCB benidipine have protective effects on mouse cultured mesangial cells against TNF–α or CHX . However, nifedipine and benidipine did not inhibit specific types of cell death using semithin sections in this study.