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(Journal of Leukocyte Biology. 2002;71:231-237.)
© 2002 by Society for Leukocyte Biology

Reversible phosphatidylserine expression on blood granulocytes related to membrane perturbation but not DNA strand breaks

Ming-Yu Yang, Hau Chuang, Rong-Fu Chen and Kuender D. Yang

Chang Gung Children’s Hospital at Kaohsiung, Chang Gung University, Taiwan

Correspondence: Dr. Kuender D. Yang, Office of Vice Superintendents, Chang Gung Children’s Hospital at Kaohsiung, 123 Ta-Pei Road, Niao-Sung, Kaohsiung 833, Taiwan. E-mail: yangkd{at}adm.cgmh.org.tw


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ABSTRACT
 
Exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane is recognized as an early indicator of programmed cell death (apoptosis) in plant and mammalian cells. Currently, there is no literature describing that PS expression on the surface of white blood cells is reversible. We found that a hypotonic 0.2% NaCl or NH4Cl lysing solution used to separate white blood cells from red blood cells induced a reversible PS expression on the cell surface of granulocytes and monocytes but not lymphocytes. This reversible PS expression was associated with change of plasma membrane potential but not degranulation-associated membrane mobilization or DNA fragmentation. In contrast, TNF-{alpha} induced an irreversible PS expression, associated with apoptotic DNA fragmentation shown on gel electrophoresis. The fact that hypotonic shock induced a reversible PS expression on granulocytes, and TNF-{alpha} induced an irreversible PS expression associated with apoptotic DNA fragmentation indicate the new insight that expression of PS on the outer cell surface does not always represent cell apoptosis. Also, the reversible PS expression was associated with altered plasma-membrane potential but not DNA strand breaks, indicating that early PS expression may be related to the membrane perturbation but not directly related to DNA fragmentation in certain types of cells.

Key Words: hypotonic shock • apoptosis • fragmentation • white blood cells • red blood cells • NaCl • NH4Cl


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INTRODUCTION
 
Apoptosis of mammalian cells is accompanied by several morphological changes in cell membrane, cytosol, and nucleus. Before cells undergo nuclear apoptotic DNA fragmentation, they have manifested with early cell-membrane asymmetry, including exposure of phosphatidylserine (PS) onto the outer leaflet of cell-plasma membrane and mitochondrial membrane permeability disruption [1 ]. Evidence suggests that death ligands such as tumor necrosis factor {alpha} (TNF-{alpha}) or Fas-ligands (Fas-L) bind to death receptors, TNF-{alpha}R or Fas, committing the cells to apoptosis [2 ]. The death signal is transmitted from the death receptor-associated death domains to the disruption of mitochondrial-permeability transition, release of cytochrome c, exposure of PS to the outer leaflet of cell-surface membrane, activation of caspases, chromatin condensation, and DNA fragmentation [2 3 4 ]. Flow cytometric analysis of the PS exposure onto the outer cell surface is recognized as an early indicator of cell apoptosis in plants [5 ] and mammalian cells [6 , 7 ] including granulocytes [8 ].

Granulocytes tend to have a short half-life in the blood circulation [9 ]. The mechanism that mediates spontaneous granulocyte death is believed to come about through programmed cell death activated by the interaction with coexpression of cell-surface Fas and Fas-L as well as TNF-{alpha} and TNF-{alpha}R [10 ]. It is not well-known how far along the progress of the programmed cell death can go and still be reversible. Lin and colleagues [11 ] demonstrated that antioxidants could rescue the Sindbis virus-induced apoptosis of neuroblastoma and prostate carcinoma cells. Overexpression of certain oncogenes such as Bcl-2 and Sek-1 could prevent cell apoptosis [11 , 12 ]. Studies in dexamethasone-induced lymphocyte apoptosis have shown that a fundamental feature of cell apoptosis is cell shrinkage, probably as a result of proteolysis rather than loss of water or increase of osmolarity [13 ]. Further studies by Bortner and Cidlowski [14 ] have demonstrated that several other cell types resist cell shrinkage-induced apoptosis, although thymocytes develop rapid apoptosis in response to the physical shrinkage induced by dexamethasone or hypertonic solutions. However, to date, there have been no studies indicating that hypotonic solutions might induce cell apoptosis, nor have there been any studies indicating that expression of PS on the outer leaflet of cell surface does not mean there is undergoing cell apoptosis. In a study to isolate white blood cells (WBC) from red blood cells (RBC) with hypotonic 0.2% NaCl or a hemolytic NH4Cl lysing buffer, we have found that hemolytic shock with NH4Cl or 0.2% NaCl as a lysing solution rapidly induced PS expression on the cell surface of granulocytes and monocytes but not lymphocytes. Although cell shrinkage has been shown to induce programmed cell death [13 ], we have demonstrated that hypotonic solution, which perturbs plasma membrane potential, does induce reversible PS expression but not degranulation-associated membrane mobilization or apoptotic DNA fragmentation.


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MATERIALS AND METHODS
 
Reagents and buffers
Ammonium chloride lysing buffer containing NH4Cl (150 mM) at pH = 7.2 was used to isolate WBC from hemolytic depletion of RBC [15 ]. NaCl (0.2%) was used as another hypotonic solution, whereas 1.6% NaCl was used as a hypertonic solution for the isotonic restoration of 0.2% NaCl hypotonic shock [15 , 16 ]. Phosphate-buffered saline (PBS; pH 7.3, 150 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.1 mM KH2PO4) and RPMI 1640 medium from Gibco-BRL (Grand Island, NY) were used as isotonic buffers. Normal saline-containing calcium chloride (2.5 mM) and HEPES (10 mM) at pH 7.3 were run as a binding buffer for the detection of PS expression with fluorescein isothiocyanate (FITC)-labeled Annexin-V (PharMingen, San Diego, CA). Plasma-membrane potential tracer, DiOC6 (3,3'-dipentyloxacarbocyanine), was purchased from Molecular Probes (Eugene, OR) and dissolved in dimethylsulfoxide (DMSO; Sigma Chemical Co., St. Louis, MO) and cell-stimulating agents: fMLP (formyl-methionyl-leucyl-phenylalanine) and PMA (phorbol myristate acetate) were purchased from Sigma Chemical Co. and dissolved in DMSO. The DMSO concentrations in the reactions were kept below 0.1% in all experiments.

Flow cytometric analysis of leukocyte PS expression and TdT-mediated biotin-BrdU nicked end-labeling (TUNEL) staining
Peripheral blood samples from healthy adult volunteers were drawn into heparin-rinsed syringes (10 U/ml) and aliquoted into 1.5-ml Eppendorf tubes for studies after informed consent was obtained. Human leukocytes were isolated by depletion of RBC with NH4Cl hemolytic solution for 20 min or hypotonic NaCl (0.2%) for 30 s followed by restoration of osmolarity with 1.6% NaCl before staining by FITC-labeled Annexin-V. Blood samples were also treated with and without TNF-{alpha} (50 ng/ml) to study whether the TNF-{alpha}-induced PS expression was irreversible. The blood leukocytes were stained with 5 µl FITC-labeled Annexin-V (50 µg/ml) in a binding buffer with 2.5 mM calcium and 10 mM HEPES at room temperature for 20 min, according to the manufacturer’s directions (PharMingen). The blood cells were washed in PBS twice before being fixed in 1% paraformaldehyde and suspended to 0.5 ml for flow cytometric analysis of PS expression [17 ]. Similarly, flow cytometric analysis of DNA fragmentation was detected by APO-BRDU (PharMingen) with flow cytometric analysis of streptavidin-FITC detection of biotin-BrdU incorporation. In brief, heparinized whole blood (1.0 ml) was treated with and without TNF-{alpha} (50 ng/ml) for 6 h. The blood without TNF-{alpha} treatment was separated into two parts: One ran as a negative control, and the other was subjected to hypotonic shock. To demonstrate a positive control for TUNEL staining, permealized leukocytes were treated with TACS-nuclease (R&D Systems, Minneapolis, MN) to create DNA strand breaks for end-labeling. Leukocytes harvested from the blood with and without TNF-{alpha} treatment or hypotonic shock were fixed in 3.7% formaldehyde for 10 min, followed by the cell permeabilization with cytonin (10 µg/ml) for 30 min at room temperature. After washing, the intracellular DNA strand breaks were determined by TUNEL at 37°C for 60 min. The DNA strand breaks end-labeled with biotin-BrdU were recognized by streptavidin-FITC and analyzed by flow cytometry.

Assessment of leukocyte viability, necrosis, and DNA fragmentation
Leukocyte viability was assessed by the trypan blue exclusion test [18 ]. Leukocyte suspension in 0.25 ml PBS was added into 0.25 ml trypan blue (0.25%) solution for 5 min before observing trypan blue exclusion cells under a light microscope (10x10 magnification field). Cell viability was defined as % viable cells = (total cells-blue-stained cells)/(total cells) x 100%. Cell necrosis was assessed by flow cytometric analysis of propidium iodide (PI; 2.5 µg/ml final concentration) fluorescence at 632 nm emission under 488 nm excitation [19 ]. DNA extracted from blood granulocytes, which were treated with and without hemolytic solutions, were subjected to assessment of apoptotic DNA fragmentation in 1.5% agarose gel electrophoresis in TBE buffer [90 mM Tris, 90 mM borate, and 2 mM ethylenediaminetetraacetate (EDTA), pH 8.3] as in one of our previously described studies [17 ].

Detection of membrane perturbation by hemolytic shock and TNF-{alpha}
The DiOC6 (50 nM) is used to probe plasma membrane potential [20 , 21 ]. Blood samples (1.0 ml) were preincubated with 50 nM of DiOC6 at room temperature in the dark for 10 min [20 ] before being treated with and without TNF-{alpha} (50 ng/ml) or hemolytic shock. At the end of the incubation period, cells were washed with PBS twice, resuspended in a total volume of 500 µl PBS, and analyzed by a flow cytometric gating of leukocytes. For each sample, 30,000 cells were acquired and analyzed with CellQuest software (Becton Dickinson, San Jose, CA). A higher fluorescence signal at 488-nm excitation and 520-nm emission indicated cells with hyperpolarization of membrane potential, whereas a lower fluorescence signal indicated cells with depolarization of membrane potential [20 ].

Determination of leukocyte degranulation
To explore whether PS exposure onto outer cell surface originated in cell degranulation, we measured extracellular myeloperoxidase (MPO) release to reflect degranulation of leukocyte primary granules [22 ] and assessed mobilization of adhesion molecules (CD11b) onto granulocyte cell surface to determine degranulation of leukocyte secondary granules [23 ]. The degree of primary degranulation shown with MPO release was positively controlled by the stimulation with PMA (32 nM) in 10 min. The CD11b mobilization onto outer cell surface was positively controlled by the stimulation with fMLP (10-7 M) for 10 min. MPO levels were determined by a commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems), and CD11b expression was assessed by flow cytometric analysis with a phycoerythrin (PE)-labeled anti-CD11b antibody as previously described [24 ].


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RESULTS
 
Spontaneous PS expression of blood leukocytes as determined by Annexin-V staining
Exposure of PS onto the outer cell-surface membrane has been recognized as an early indicator of granulocyte apoptosis [8 ]. Using flow cytometric analysis of PS expression with FITC-labeled Annexin-V, we investigated in vitro spontaneous apoptosis of leukocytes in a whole-blood model. As shown by PS expression, granulocytes and monocytes in the heparinized peripheral blood obtained from healthy adult volunteers underwent spontaneous apoptosis at a rate of 6.6 ± 1.6% and 3.6 ± 0.6%, respectively. In contrast, blood lymphocytes did not significantly undergo spontaneous apoptosis (0.6±0.2%), as shown in Figure 1.



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  		Figure 1. Spontaneous apoptosis of blood leukocytes. Localization of blood granulocytes, monocytes, and lymphocytes was established by flow cytometry (A). Spontaneous apoptosis of blood granulocytes (B) was determined by PS-positive staining based on a reference without specific Annexin-V staining. Spontaneous apoptosis of blood monocytes (C) was higher than those of blood lymphocytes (D), and spontaneous apoptosis of blood lymphocytes was significantly lower than those of blood granulocytes. Data presented are derived from one set of the representative experiment in six reproducible experiments.

Induction of PS expression on the blood leukocytes by RBC-depleting solutions
Numbers of RBC in the blood are usually 500 times higher than WBC. To characterize WBC in the blood, they are usually isolated by hemolytic depletion of RBC with NH4Cl lysing buffer or 0.2% NaCl hypotonic solution, followed by restoration of osmolarity with 1.6% NaCl [16 , 18 ]. Treatment of NH4Cl lysing buffer for 20 min was found to be able to induce hemolysis effectively but not leukolysis of WBC. The WBC viability, as determined by the trypan blue exclusion test, was always higher than 98% (99±0.5%), similar to the rate in one of our previous studies in which granulocytes isolated with 0.2% NaCl hypotonic depletion of RBC followed by 1.6% NaCl restoration of osmolarity had viability over 98% [16 , 18 ]. However, in this study, we found that hypotonic depletion of RBC with 0.2% NaCl significantly increased the PS expression on blood granulocytes and monocytes but not lymphocytes. The hypotonic shock-induced PS expression was not associated with an increase in the necrotic cells as determined by PI staining (Fig. 2 ). Similarly, NH4Cl depletion of RBC also significantly induced PS expression on granulocytes and monocytes but not lymphocytes (Fig. 2D) . It is not known whether the PS expression induced by hypotonic shock could progress to final DNA fragmentation of cell apoptosis. Studies were then performed to determine whether the hypotonic shock-induced PS exposure on the granulocyte cell surface was reversible or truly represented cell apoptosis as determined by DNA fragmentation.



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  		Figure 2. Induction of PS expression on blood leukocytes by RBC-depleting solutions. In a representative study, depletion of RBC with 0.2% NaCl for 30 s followed with restoration of the osmolarity by 1.6% NaCl caused PS expression on granulocytes (A) and monocytes (B) but not lymphocytes (C). The PS expression was not associated with cell necrosis as determined by PI staining (y-axis). Similarly, blood leukocytes exposed to NH4Cl RBC-depleting solution (pH=7.2) for 20 min also induced PS expression on granulocytes and monocytes but not lymphocytes (D). Data are represented as mean ± SE calculated from studies with six experiments.

Reversion of hemolytic shock-induced PS expression by isotonic buffers
There has been no literature indicating that hemolytic or hypotonic conditions could induce WBC apoptosis and no studies that indicate exposure of PS onto the outer cell surface does not represent cell apoptosis. In this study therefore we tried to clarify whether the hemolytic shock-induced PS expression on granulocytes was reversible after incubation of the granulocytes in RPMI 1640 medium and isotonic buffers. As shown in Figure 3 , the PS expression on blood granulocytes induced by the NH4Cl lysing buffer could be reversed in isotonic RPMI 1640 medium in a time-dependent way. The optimal time for the reversibility was 30–60 min after restoration of normal osmolarity.



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  		Figure 3. Reversible PS expression on the blood granulocytes in the NH4Cl depletion of RBC. Seven replicate, heparinized blood samples (0.2 ml) were treated with NH4Cl solution for 20 min before resuspended into isotonic RPMI 1640 medium. Each replicate sample was harvested for analysis of the PS expression on granulocytes at 0, 15, 30, 45, 60, 90, and 120 min. A time-dependent reversal of PS expression on granulocytes was gated and analyzed by a flow cytometry. Data are presented as mean ± SE calculated from eight duplicate studies.

TNF-{alpha} but not hypotonic solutions induced irreversible PS expression and DNA fragmentation of granulocytes. It is not known whether irreversible PS expression on granulocytes represents cell apoptosis or whether reversible PS expression represents cell apoptosis. We treated the blood samples with and without TNF-{alpha} (50 ng/ml) for 6 h, followed by hypotonic shock with restoration of osmolarity for 60 min in an isotonic medium (RPMI 1640) to test the postulate. It was found that RBC-depleting solutions induced PS expression of blood granulocytes, which was reversible by the incubation of granulocytes in isotonic RPMI 1640 for 60 min (Fig. 4 A ), whereas TNF-{alpha}-induced PS expression was not reversible by the incubation of granulocytes in isotonic RPMI 1640 for 60 min (Fig. 4B) . Further studies demonstrated that TNF-{alpha} but not RBC-depleting solution induced DNA fragmentation of the nuclei obtained from blood granulocytes (Fig. 4C) . Results from these studies indicate that RBC-depleting solutions can induce reversible granulocyte-PS expression but not granulocyte apoptosis and that TNF-{alpha} induces granulocyte apoptosis associated with irreversible PS exposure and DNA fragmentation.



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  		Figure 4. TNF-{alpha} but not RBC-depleting solutions induced irreversible PS expression and DNA fragmentation. (A) NH4Cl and 0.2% NaCl solutions induced reversible PS expression on granulocytes. Blood granulocytes in the heparinized blood treated with NH4Cl for 20 min or 0.2% NaCl for 30 s followed by normalized osmolarity with 1.6% NaCl revealed a higher initial PS expression followed by a significant reversal in the isotonic RPMI 1640 medium for 60 min. (B) TNF-{alpha} flipped PS expression on the blood granulocytes without reversal of the PS expression in the isotonic RPMI 1640 medium for 60 min. (C) TNF-{alpha} (lane 1) but not 0.2% NaCl (lane 2) or NH4Cl (lane 3) with restoration in isotonic solution for 60 min induced blood granulocyte DNA fragmentations, as demonstrated by the 1.5% agarose gel electrophoresis.

Hypotonic shock induced a transient decrease of plasma membrane potential but not DNA strand breaks or degranulation-associated membrane mobilization
As shown in Figure 5 , hypotonic shock was found to induce a decrease in total plasma membrane potential in granulocytes as determined by the DiOC6 fluorescent probe. The decrease of total membrane potential in granulocytes induced by hypotonic shock was restored to the level close to the controls without hypotonic treatment in 30 min after the restoration of osmolarity (Fig. 5A) . Similarly, the treatment with hypotonic shock did not induce DNA strand breaks, but TNF-{alpha} induced detectable DNA strand breaks as determined by flow cytometric analysis of TUNEL staining (Fig. 5B) .



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  		Figure 5. Hypotonic shock induced reversible plasma membrane potential changes but not DNA strand breaks as determined by TUNEL staining. (A) Hypotonic depletion of RBC induced a transient decrease in the total plasma membrane potential in granulocytes, which was reversible after incubating with isotonic RPMI 1640 for 30 min. (B) Determination of DNA strand breaks by TUNEL staining. The treatment with TNF-{alpha} for 6 h but not that without TNF-{alpha} treatment for 6 h followed by hemolytic depletion of RBC induced an increase in granulocytes with TUNEL staining. Blood granulocytes incubated with PBS and TACS-nuclease were run as negative and positive controls, respectively. The figure presented is a representative plot from four reproducible experiments.

Because membrane perturbation of leukocytes is usually associated with degranulation, to rule out the possibility that the outer cell surface-PS expression on granulocytes was a result of cell membrane mobilization after leukocyte degranulation, we assessed extracellular MPO release and membrane-integrin (CD11b) mobilization onto cell surface to reflect degranulation of primary [22 ] and secondary [23 ] granules, respectively. Results showed that hypotonic shock of blood leukocytes did not significantly increase extracellular MPO release, although there was prominent MPO release induced by PMA stimulation (Fig. 6 A ). Hypotonic shock also did not elicit degranulation of secondary granules, as demonstrated by CD11b mobilization onto the cell surface. On the contrary, fMLP induced a prominent mobilization of CD11b onto cell surface of granulocytes in 10 min (Fig. 6B) . These results suggest that plasma-membrane perturbation but not degranulation-associated membrane mobilization is involved in reversible PS expression onto the outer leaflet of the phagocyte cell membrane.



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  		Figure 6. Detection of leukocyte degranulation induced by hypotonic shock. (A) Degranulation of primary granules as determined by extracellular MPO release. Hypotonic shock for 30 s followed by normalized osmolarity for 10 min did not significantly induce extracellular MPO release, but a potent leukocyte stimulator, PMA (32 nM), dramatically augmented MPO release. (B) Degranulation of secondary granules as determined by mobilization of CD11b expression onto the granulocyte cell surface. Granulocytes from heparinized blood treated with hypotonic shock for 30 s followed by normalized osmolarity for 10 min did not increase the CD11b expression, whereas granulocytes from those treated with fMLP (10-7 M) revealed a significant increase in CD11b expression on the cell surface in 10 min.


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DISCUSSION
 
It is clear that programmed cell death demonstrates membrane asymmetry such as disruption of mitochondrial permeability and exposure of PS from the inner surface to the outer cell surface. The mechanism that mediates the PS exposure onto the outer cell surface is not clear. Although cell shrinkage has been found to induce cell apoptosis in certain lymphoid cells, enlarged cell volume has never been recognized as a signal to trigger cell apoptosis or PS expression onto cell-plasma membrane [13 , 14 ]. Using flow cytometric analysis of PS exposure, we have identified that hypotonic shock can induce a reversible PS exposure on the outer cell surface of blood granulocytes but did not result in apoptotic DNA fragmentation of blood granulocytes. In support of the idea that PS expression can be reversible and cannot be related to cell apoptosis, we showed that the reversible PS expression was associated with a reversible restoration of plasma-membrane potential but not with mitochondrial-membrane potential change or DNA fragmentation. Results from this study suggest that outer cell-surface exposure of PS is related to plasma-membrane perturbation but not to mitochondrial-membrane potential or apoptotic DNA fragmentation.

Many studies have demonstrated that PS exposure onto the outer leaflet of cell surface is an early signal committing the cells to apoptosis [1 , 5 6 7 8 ]. Although previous literature does not indicate that the outer cell-surface PS exposure is reversible, we found that PS expression and plasma-membrane potential were affected immediately after hypotonic shock but recovered in the isotonic buffer gradually in 1 h. The restoration of plasma-membrane potential occurred in 30 min (Fig. 5A) , earlier than the disappearance of PS expression (Fig. 3) , suggesting that PS exposure onto the outer surface may be mediated by disruption of plasma-membrane potential.

Studies with lymphoid and nonlymphoid cell lines have all demonstrated that hypotonic osmolarity does not induce cell apoptosis [13 , 14 ]. In this study, we found that hypo-osmolarity did induce a reversible PS expression on granulocytes and monocytes but not lymphoyctes. Therefore, this suggests that lymphocytes may possess a better intrinsic ability to maintain cell volume or membrane integrity and that phagocytes may be more susceptible to the membrane asymmetry affected by hypotonic treatment.

Many in vivo and in vitro conditions might influence the lifespan of phagocytes. For instance, some cytokines such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF do protect granulocytes from apoptosis, and certain cytokines such as TNF-{alpha} can promote granulocyte apoptosis [10 ]. Transendothelial migration and enhancement of integrin expression can delay granulocyte apoptosis, whereas enhancement of selectin expression promotes its apoptosis [25 ]. To clarify whether other pathophysiological conditions could also induce reversible PS expression, we found that TNF-{alpha} induced an irreversible PS expression on blood granulocytes (Fig. 4B) , but changes of pH values from 7.0 to 8.0 or doubled osmolarity (1.8% NaCl) for 30 min did not flip PS expression on granulocytes or lymphocytes (unpublished results). It remains to be determined whether certain membrane perturbations such as decrease of osmolarity that enlarges cell volume may attribute to redistribution of plasma membrane phospholipids resulting in regulation of reversible and irreversible exposure of PS on the outer leaflet of granulocyte cell membrane.


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ACKNOWLEDGEMENTS
 
This work was partly supported by grants NSC89-2320-B-182-007-M53 from National Science Council, Taiwan, R.O.C., and NMRP 097 from Chang Gung Memorial Hospital, Kaohsiung, Taiwan.

Received August 14, 2000; revised September 24, 2001; accepted September 28, 2001.


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REFERENCES
 
    1
  1. Castedo, M., Hirsch, T., Susin, S. A., Zamzami, N., Marchetti, P., Macho, A., Kroemer, G. (1996) Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis J. Immunol. 157,512-521[Abstract]
    2. 2
  2. Susin, S. A., Zamzami, N., Castedo, M., Douglas, E., Wang, H. G., Geley, S., Fassy, F., Reed, J. C., Kroemer, G. (1997) The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis J. Immunol. 186,25-37
    3. 3
  3. Marchetti, P., Castedo, M., Susin, S. A., Zamzami, N., Hirsch, T., Macho, A., Haeffner, A., Hirch, F., Geuskens, M., Kroemer, G. (1996) Mitochondrial permeability transition is a central coordinating event of apoptosis J. Exp. Med. 184,1155-1160[Abstract/Free Full Text]
    4. 4
  4. Schuler, M., Bossy-Wetzel, E., Goldstein, J. C., Fitzgerald, P., Green, D. R. (2000) p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release J. Biol. Chem. 275,7337-7342[Abstract/Free Full Text]
    5. 5
  5. O’Brien, I. E., Reutelingsperger, C. P., Holdaway, K. M. (1997) Annexin-V and TUNEL use in monitoring the progression of apoptosis in plants Cytometry 29,28-33[Medline]
    6. 6
  6. Naito, M., Nagashima, K., Mashima, T., Tsuruo, T. (1997) Phosphatidylserine externalization is a downstream event of interleukin-1 beta-converting enzyme family protease activation during apoptosis Blood 89,2060-2066[Abstract/Free Full Text]
    7. 7
  7. Rimon, G., Bazenet, C. E., Philpott, K. L., Rubin, L. L. (1997) Increased surface phosphatidylserine is an early marker of neuronal apoptosis J. Neurosci. Res. 48,563-570[Medline]
    8. 8
  8. Homburg, C. H., de Haas, M., von dem Borne, A. E., Verhoeven, A. J., Reutelingsperger, C. P., Roos, D. (1995) Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro Blood 85,532-540[Abstract/Free Full Text]
    9. 9
  9. Yang, K. D., Hill, H. R. (1997) Disorders of leukocyte functions Rimoin, D. L. Connor, J. M. Pyeitz, R. E. Emery, A. E. H. eds. Principles and Practice of Medical Genetics Edinburgh UK.
    10. 10
  10. Liles, W. C., Kiener, P. A., Ledbetter, J. A., Aruffo, A., Klebanoff, S. J. (1996) Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils J. Exp. Med. 184,429-440[Abstract/Free Full Text]
    11. 11
  11. Lin, K. I., Lee, S. H., Narayana, R., Baraban, J. M., Hardwick, J. M., Tatan, R. R. (1995) Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-kappa B J. Cell Biol. 131,1149-1161[Abstract/Free Full Text]
    12. 12
  12. Nishina, H., Fischer, K. D., Radvanyi, L., Shahinian, A., Hakem, R., Rubie, E. A., Berstein, A., Mak, T. W., Woodgett, J. R., Penninger, J. M. (1997) Stress-signaling kinase Sek-1 protects thymocytes from apoptosis mediated by CD95 and CD3 Nature 385,350-353[Medline]
    13. 13
  13. Benson, R. S., Heer, S., Dive, C., Watson, A. J. (1996) Characterization of cell volume loss in CEM-C7A cells during dexamethasone-induced apoptosis Am. J. Physiol. 270,C1190-C1203[Abstract/Free Full Text]
    14. 14
  14. Bortner, C. D., Cidlowski, J. A. (1996) Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes Am. J. Physiol. 271,C950-C961[Abstract/Free Full Text]
    15. 15
  15. Jackson, A. L., Warner, N. L. (1986) Preparation, staining, and analysis by flow cytometry of peripheral blood leukocytes Rose, N. R. Friedman, H. Fahey, J. L. eds. Manual of Clinical Laboratory Immunology ,226-235 American Society for Microbiology Washington, DC.
    16. 16
  16. Yang, K. D., Augustine, N. H., Gonzalez, L. A., Bohnsack, J. F., Hill, H. R. (1998) Effects of fibronectin on the interaction of polymorphonuclear leukocytes with unopsonized and antibody-opsonized bacteria J. Infect. Dis. 158,823-830
    17. 17
  17. Yang, K. D., Chen, M-Z., Teng, R-J., Yang, M-Y., Liu, H-C., Chen, R-F., Hsu, T-Y., Shaio, M-F. (2000) A model to study antioxidant regulation of endotoxemia-modulated neonatal granulopoiesis and granulocyte apoptosis Pediatr. Res. 48,829-834[Medline]
    18. 18
  18. Yang, K. D., Shaio, M. F., Augustine, N. H., Hill, H. R. (1994) Effects of fibronectin on phagocyte activation: implications in respiratory burst and actin polymerization J. Cell. Physiol. 158,347-353[Medline]
    19. 19
  19. Matteucci, C., Grelli, S., De Smaele, E., Fontana, C., Mastino, A. (1999) Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry Cytometry 35,145-153[Medline]
    20. 20
  20. Salvioli, S., Ardizzoni, A., Franceschi, C., Cossarizza, A. (1997) JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis FEBS Lett 411,77-82[Medline]
    21. 21
  21. Rottenberg, H., Wu, S. (1998) Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells Biochim. Biophys. Acta 1404,393-404[Medline]
    22. 22
  22. Koller, D. Y., Urbanek, R., Gotz, M. (1995) Increased degranulation of eosinophil and neutrophil granulocytes in cystic fibrosis Am. J. Respir. Crit. Care Med. 152,629-633[Abstract]
    23. 23
  23. Mukherjee, G., Rasmusson, B., Linner, J. G., Quinn, M. T., Parkos, C. A. (1998) Organization and mobility of CD11b/CD18 and targeting of superoxide on the surface of degranulated human neutrophils Arch. Biochem. Biophys. 357,164-172[Medline]
    24. 24
  24. Yang, K. D., Chao, C. Y., Shaio, M. F. (1998) Pentoxifylline synergizes with all-trans retinoic acid to induce differentiation of HL-60 myelocytic cells, but suppresses tRA-augmented clonal growth of normal CFU-GM Acta Haematol 99,191-199[Medline]
    25. 25
  25. Watson, R. W., Rotstein, O. D., Nathens, A. B., Patodo, J., Marshall, J. C. (1997) Neutrophil apoptosis is modulated by endothelial transmigration and adhesion molecule engagement J. Immunol. 158,945-953[Abstract]



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A model to study neurotropism and persistency of Japanese encephalitis virus infection in human neuroblastoma cells and leukocytes
J. Gen. Virol., March 1, 2004; 85(3): 635 - 642.
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