BJR
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fanning, N F
Right arrow Articles by Redmond, H P
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fanning, N F
Right arrow Articles by Redmond, H P
British Journal of Radiology 75 (2002),861-873 © 2002 The British Institute of Radiology

Full paper

Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway

N F Fanning, MD, FRCSI1, B J Manning, MB, AFRCSI2, J Buckley, FRCR1 and H P Redmond, MCh, FRCSI2

Departments of 1 Radiology and 2 Surgery, Cork University Hospital, Wilton, Cork, Ireland


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml-1) of ionic (diatrizoate 69.6±2.9%; ioxaglate 58.9±2.0%) and non-ionic (iohexol 57.3±2.9%; iotrolan 57.1±2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7±1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml-1) treated neutrophils to control levels (23.0±3.5% to 61.2±5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and resolution of an inflammatory response.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Iodinated contrast media (ICM) are extensively used in diagnostic imaging to change the X-ray absorption of tissues. ICM should be biologically inert. However, there is an increasing recognition that these agents can alter cell function and viability. Recent reports suggest that ICM can induce apoptosis (programmed cell death) in a variety of non-immune cells including cardiac myocytes, renal tubular and glomerular cells, vascular endothelial cells and smooth muscle cells [1–5]. There have been no reports suggesting that ICM can trigger apoptosis in immune cells.

Neutrophils are the most abundant leukocyte in the blood circulation. These cells are exposed to high concentrations of ICM following intravascular injection. Neutrophils constitutively undergo apoptosis and regulation of this process is critical in controlling the duration of host response to injury and infection. Mature neutrophils have a short lifespan of 8–20 h, but this can be increased several fold if recruited into inflamed tissues in vivo, or exposed to pro-inflammatory mediators, such as lipopolysaccharide (LPS), in vitro [6]. Delayed expression of neutrophil apoptosis is associated with systemic pro-inflammatory syndromes, such as the acute respiratory distress syndrome and the systemic inflammatory response syndrome [7]. Accelerated expression of neutrophil apoptosis is associated with an increased susceptibility to sepsis [8]. A recent report suggests that ICM increase the incidence of both local and systemic septic complications in patients with mild acute pancreatitis [9]. Successful resolution of inflammation requires a balance between both pro- and anti-apoptotic intracellular signalling pathways in neutrophils. Any agent that alters the rate of constitutive and inflammatory neutrophil cell death could therefore have a significant deleterious effect on host immune defence and resolution of an inflammatory response.

The present report investigates and contrasts the effects on neutrophil survival of the four main types of ICM used clinically; high osmolar ionic monomer, low osmolar ionic dimer, low osmolar non-ionic monomer, and iso-osmolar non-ionic dimer (Table 1Go). We hypothesized that ICM induce neutrophil cell death in both unstimulated and LPS stimulated neutrophils. In non-immune cells the pro-apoptotic effects of ICM appear, in part, to be related to their colligative properties. However, direct alteration in expression of specific apoptotic proteins (decreased bcl-2, increased bax and p53) has been described [2]. Given that p38 mitogen-activated protein kinase (MAPK) activation in response to cellular stresses has been shown to accelerate apoptosis in neutrophils, we investigated the effect of ICM on p38 MAPK phosphorylation, an essential step in the activation of this signalling pathway. We also determined the effect of ICM on mitochondrial depolarization and the role of caspase activation in ICM-induced apoptosis.


View this table:
[in this window]
[in a new window]
  		Table 1. The physicochemical properties of iodinated contrast media (ICM)

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Preparation of purified neutrophil populations
Heparinized venous blood (lithium heparin 143 USP, Greiner Labortechnik, Kremsmuenster, Austria), was obtained from healthy adult volunteers. Neutrophils were isolated by sequential sedimentation in 6% Dextran (Sigma, Dorset, UK) (molecular weight 520 000) in 0.9% NaCl for 45 min at 22°C, centrifugation in Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, NJ) at 300 g for 30 min to pellet granulocytes and remaining erythrocytes, and centrifugation of the resuspended pellet over an 81% isotonic Percoll (Sigma, Dorset, UK) gradient at 350 g for 15 min to pellet erythrocytes. The diffuse layer at the interface containing neutrophils was harvested, washed, resuspended in medium and counted. Cell viability was greater than 98%, as determined by trypan blue exclusion. The preparation contained greater than 95% neutrophils, with eosinophils being the principle contaminant, as determined by Rapi-diff II (Diagnostic Developments, Lancashire, UK) staining on cytocentrifuged samples.

Culture conditions for neutrophils
Preparations of isolated neutrophils were maintained in Rosweli Park Memorial Institute 1640 medium (RPMI) supplemented with 10% platelet poor plasma (PPP), 2 mM L-glutamine, 100 U ml-1 penicillin, and 100 µg ml-1 streptomycin, at a concentration of 1 x 106 ml-1 in polypropylene round bottom tubes (Becton Dickinson, Franldin Lakes, NJ) at 37°C in a humidified CO2 incubator (5% CO2, 95% air). PPP was prepared by centrifugation of the plasma rich layer at 600 g for 30 min to pellet platelets leaving PPP above the pellet. PPP was then aliquoted into 1 ml vials and frozen at -80°C for subsequent experiments. All culture reagents were from GIBCO-BRL, Paisley, UK.

Experimental studies and solutions
Assessment of the apoptotic effect of ICM: concentration dependent study
Neutrophils were incubated with isoiodine concentrations of the four main types of ICM used in clinical practice; the high osmolar ionic monomer diatrizoate (Urografin; Schering AG, Berlin, Germany), the low osmolar ionic dimer ioxaglate 320 (Hexabrix; May & Baker Ltd, Dagenham, UK), the low osmolar non-ionic monomer iohexol 300 (Omnipaque; Nycomed Ireland Ltd, Cork, Ireland), and iso-osmolar non-ionic dimer iotrolan 300 (Isovist; Schering AG, Berlin, Germany). As the ICM studied contain differing concentrations of iodine per millilitre of contrast, an isoiodine concentration of contrast in milligrammes of iodine per millilitre was used as a measure of the total amount of contrast to which cells were exposed. A concentration dependent effect was investigated by exposing cells to increasing isoiodine concentrations (10 mg I ml-1, 20 mg I ml-1, 40 mg I ml-1 and 80 mg I ml-1) of the four ICM studied, with apoptosis assessed at 12 h culture by both annexin V flow cytometry and morphology. Neutrophils incubated with RPMI medium alone were used as a control. From knowing the total concentration of ICM in the commercially available solutions, stated in the manufacturers information sheet, the different concentrations of ICM being investigated were prepared from the commercially available solutions by calculating the volume of parent product to be added, so that the required concentration of ICM in mg I ml-1 was achieved in a total final volume of 1 ml. The final volume of all experimental solutions in this study was 1 ml. Accordingly, a 1 ml test solution contained 100 µl of 1 x 107 ml-1 neutrophils (1 x 106 ml-1 final neutrophil concentration), 100 µl of PPP (10% solution in final volume of 1 ml), the volume of the commercially available ICM required to achieve the concentration of ICM being investigated, and RPMI to make up the final volume of 1 ml. The effect of growth factor dilution by ICM on neutrophil apoptosis was specifically investigated (see below).

Assessment of the apoptotic effect of ICM: time dependent study
A time dependent effect of ICM on neutrophil apoptosis was determined by incubating neutrophils with isoiodine concentrations (20 mg I ml-1) of the four different ICM for 1–24 h and apoptosis assessed by dual staining with propidium iodide and annexin v-fluorescein isothiocyanate (V-FITC) at 1 h, 12 h and 24 h in culture. As a confirmatory study, apoptosis was also assessed at 12 h and 24 h by morphology. To investigate the effects of short duration of exposure on neutrophil apoptosis, additional experiments were performed in which neutrophils were incubated with iohexol 40 mg I ml-1 for 45 min and then washed in phosphate buffered saline to remove the contrast media. These neutrophils were then aged in vitro. Apoptosis was assessed at 12 h and 24 h by flow cytometry and compared with RPMI treated controls.

Assessment of the colligative properties of ICM on neutrophil apoptosis
Since the ICM differ with respect to osmolality and ionic strength (Table 1Go), we investigated the effects on neutrophil apoptosis of control solutions of similar hypertonicity, hyperosmolality and ionic strength as diatrizoate 20 mg I ml-1. Hyperosmolality refers to a solution that has a greater number of solute particles per kilogram of solvent than that of plasma (290 mOsmol kg-1); hypertonicity refers to the capacity of a solution to exert a greater osmotic force across a cell membrane than plasma and takes into account cell permeability to a solute. Hyperosmolar mannitol was used as a non-ionic, membrane-impermeable (non-ionic hypertonicity) control. Hyperosmolar NaCl was used as a functionally membrane-impermeable (ionic hypertonicity) control solute. As mannitol and NaCl solutions cause cell shrinkage by their hypertonicity, and the induction of apoptosis has been related to changes in cell volume in other experimental systems [10], hyperosmolar urea was used as an additional control. Urea is a cell membrane permeable solute. The urea solutions used are hyperosmolar but not hypertonic and therefore do not induce significant changes in cell volume (normotonic hyperosmolality control). Neutrophil cell suspensions were treated with experimental solutions for 12 h and apoptosis quantified by flow cytometry. Neutrophils incubated with RPMI medium alone were used as control.

Effect of growth factor dilution by ICM on neutrophil apoptosis
Given that growth factor deprivation may induce apoptosis [11], we analysed the effect of medium dilution by the experimental media using isotonic saline. RPMI was diluted 1:2 with 0.9% NaCl. Control media was full strength RPMI. Apoptosis was assessed at 12 h culture.

Assessment of the effects of specific constituent components of ICM on neutrophil cell death
All the ICM studied contain 0.1 mg ml-1 of disodium calcium edetate (EDTA). As EDTA is known to inhibit neutrophil adherance [12], neutrophils were incubated with EDTA at this concentration to exclude an effect on cell death. EDTA was purchased from Sigma (Dorset, UK). Apoptosis was quantified at 12 h culture by flow cytometry and compared with untreated controls.

Iodine is an essential component of ICM. The effect of elemental iodine on neutrophil cell death was determined by complexing iodine with 25% povidone (soluble polyvinylpyrrolidone; Geistlich Pharma AG, Wolhusen, Switzerland) to form a soluble povidone–iodine complex and co-culturing neutrophils with povidone–iodine at a concentration of 0.1 mg I ml-1, 1 mg I ml-1, 10 mg I ml-1, 20 mg I ml-1, 40 mg I ml-1 and 80 mg I ml-1. Apoptosis was quantified at 12 h culture by flow cytometry. Neutrophils incubated with povidone alone were used as an additional control.

Assessment of ICM effect on activated neutrophils
Neutrophils from healthy volunteers were activated with LPS (1000 ng ml-1). LPS treated neutrophils were then incubated with iohexol 40 mg I ml-1 for 24 h to ascertain the effects of ICM on activated neutrophil apoptosis. Apoptosis was assessed by annexin V-FITC assay at 1 h, 12 h and 24 h culture.

Assessment of the effect of p38 MAPK inhibition on LPS and iohexol treated cells
For experiments using the p38 MAPK inhibitor SB202190 (Biomol, Plymouth Meeting, PA), neutrophil cell suspensions were pre-incubated with 50 µM of inhibitor or saline vehicle control for 60 min before treatment with LPS (1000 ng ml-1) and iohexol (40 mg I ml-1) alone and in combination. Apoptosis was assessed at 12 h culture by flow cytometry.

Effect of caspase-cascade inhibition on ICM-induced apotposis
Neutrophil cell suspensions were treated with iohexol (40 mg I ml-1) or RPMI control, in the presence or absence of 100 µM of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) (Bachem Ltd, Essex, UK). Apoptosis was quantified at 12 h culture by flow cytometry to determine if inhibition of the caspase-cascade could block the proapoptotic effect of ICM.

Assessment of apoptosis
Immunofluorescence flow cytometry of annexin V-FITC binding
Translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an early event in apoptosis, occurring before any nuclear changes have occurred [13]. The binding of annexin V-FITC to phosphatidylserine in a Ca2+-dependent manner was used as a sensitive measure of neutrophil apoptosis. Dual staining with propidium iodide was employed to enable membrane-disrupted cells to be readily distinguished, as cells that have lost their membrane integrity may also stain positive with annexin V. Thus apoptotic cells stain positive for annexin V-FITC only, while necrotic cells stain positive for both annexin V-FITC and propidium iodide. Briefly, neutrophils (0.3 x 106 ml-1 cells) were dual stained with propidium iodide (Sigma, Dorset, UK) (final concentration 10 µg ml-1) and annexin V-FITC diluted in binding buffer (10 mM 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES)/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2) according to the manufacturer's instructions (Bender MedSystems, Heidelberg, Germany) for 5 min at room temperature. Neutrophils were analysed on a Becton Dickinson FACScan flow cytometer equipped with CellQuest software with excitation at 488 nm and emission collected through a 530/30 band pass filter for FITC (FL1-H) and a 585/42 band pass filter for propidium iodide (FL2-H). 20 000 events were collected while gating on physical parameters to exclude cell debris. Annexin V flow cytometry was used as the primary method of quantifying apoptosis in the experimental work.

Morphological assessment of apoptosis
As a supplementary method to annexin V-FITC assay, we quantified apoptosis by counting cytocentrifuged preparations of cells for morphological changes characteristic of apoptosis. Preparations of 105 cells were centrifuged at 100 g for 2 min onto glass slides using a Shandon cytocentrifuge (Shandon Instruments, Runcorn, UK). Apoptotic cells were identified in Rapi-diff II stained cytospins by their morphological features (cell shrinkage, nuclear pyknosis, chromatin condensation). A minimum of 500 cells per slide were counted using x 100 objective, and the percentage apoptosis calculated by (number of apoptotic cells/total number of cells) x 100. This method was used to confirm the results of annexin V-FITC assay in the concentration and time dependent experiments. Having verified the annexin V-FITC results with morphology, apoptosis in the remaining experiments was quantified by flow cytometry alone, with 20 000 cells per experiment analysed.

Western blot analysis
Isolated neutrophils were stimulated with ICM, or medium alone as control. Cells were washed twice with ice cold PBS and whole cell lysates were prepared with a 3% sodium dodecylsulphate (SDS) lysis buffer (10 mmol l-1 HEPES, pH 8.0; 1.5 mmol l-1 MgCl2; 10 mmol l-1 KCl; 1 mmol l-1 dithiothreitol; 0.5 mmol l-1 phenylmethanesulphonylfluoride; 0.1% NP-40; 3% SDS; and Complete (Boehringer Mannheim, Indianapolis, Inc) protease inhibitors), and protein was quantitated with the bicinchoninic acid protein assay (Pierce, Rockford, IL). Proteins were separated by SDS-polyacrylamide gel electrophoresis with 12% polyacrylamide gels. After electrophoresis proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 25 mM Tris, 192 mM glycine and 20% methanol. Membranes were blocked by incubating in 5% non-fat dried milk for 2 h. Rabbit polyclonal IgG specific for human p38 protein, as well as phospho-specific immunoglobin (IgG) antibodies to p38 MAPK, were used to assess p38 MAPK activation in conjunction with goat anti-rabbit IgG secondary antibody. Bands of immunoreactivity were detected using the chemiluminescence detection method.

Analysis of mitochondrial membrane potential
To detect variations in mitochondrial membrane potential at the single cell level, we utilized the cytofluorimetric technique developed by Cossarizza et al using the lipophilic dual emission styryl cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) (Molecular Probes, Leiden, The Netherlands) [14, 15]. JC-1 incorporates into the mitochondria and forms aggregates (red fluorescence at 590 nm (FL2-H)) at high mitochondrial membrane potential, and monomers (green fluorescence at 527 nm (FL1-H)) at low mitochondrial membrane potential [16, 17]. JC-1 was used at 5 µg ml-1 from a 5 mg ml-1 stock prepared in dimethylsulphoxide. Control cells and cells treated with iohexol 40 mg I ml-1 were incubated with JC-1 for 15 min at 37°C and fluoresence measured on a Becton Dickinson FACScan.

Statistics
Results are expressed as mean±standard error of the mean of the number (n) of independent experiments each using cells from separate, healthy donors. As the data were not normally distributed, mean values were compared using Mann-Whitney U-test. Differences were regarded as significant if p<0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The numerical values of apoptosis represent data from the annexin V flow cytometry assay for quantification of apoptosis.

ICM-induced neutrophil apoptosis is time dependent
All ICM studied induced time dependent apoptosis in neutrophils. Figure 1Go shows the time dependence of ICM-induced apoptosis using phosphadidylserine (PS) externalization as a marker of apoptosis. Compared with control neutrophils, the percentage of PS externalization was significantly increased at 12 h after exposure to all ICM studied and continued to rise progressively up to the maximum studied period of 24 h. Similar results were obtained when apoptosis was assessed by morphological criteria (data not shown). Ionic (diatrizoate and ioxaglate) ICM induced a more significant pro-apoptotic effect (p<0.01) than non-ionic (iohexol and iotrolan) ICM (p<0.05) at both 12 h and 24 h. Interestingly, diatrizoate had significantly greater apoptotic rates at 12 h and 24 h compared with ioxaglate; 69.6±2.6% vs 56.3±2.0% at 12 h (p<0.01) (Figure 1Go). Furthermore, we observed that even a short (45 min) exposure of neutrophils to iohexol caused a significant induction in apoptotic rates when neutrophils were aged for 12 h and 24 h in culture (p<0.05) (Table 2Go).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 1. Time course of apoptosis induced by iodinated contrast media (ICM). (a) Representative flow cytometry profiles taken at 12 h culture of annexin v-fluorescein isothiocyanate (V-FITC) and propidium iodide staining of: (A), control; (B) iotrolan 20 mg I ml-1; (C) iohexol 20 mg I ml-1; (D) ioxaglate 20 mg I ml-1; (E), diatrizoate 20 mg I ml-1. The percentage given in the top left quadrant indicate the percentage of annexin V-FITC positive apoptotic neutrophils. (b) Results expressed as mean percentage of apoptotic cells±standard error of the mean. *, p<0.05 and {ddagger}, p<0.01 for ICM treated vs control neutrophils.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Effect on neutrophil apoptosis of 45 min incubation of iohexol 40 mg I ml-1. Results expressed as mean percentage of apoptotic cells±standard error of the mean; n=4

 
ICM-induced neutrophil apoptosis is concentration dependent
The concentration dependence of ICM-induced apoptosis during a 12 h incubation period is illustrated in Figure 2Go. Results represent flow cytometry data, with similar results obtained by morphology. At concentrations of 20 mg I ml-1, ioxaglate, iohexol and iotrolan induced a significant increase in apoptotic rate compared with control medium, and with increasing concentrations of these ICM (40 mg I ml-1 and 80 mg I ml-1), further concentration dependent apoptosis (p<0.01) was observed. Unlike the other ICM studied, diatrizoate caused a significant (p<0.01) increase in apoptosis, even at a concentration of 10 mg I ml-1. Further increasing diatrizoate concentrations (20 mg I ml-1 and 40 mg I ml-1) resulted in a concentration dependent effect. At diatrizoate concentrations of 80 mg I ml-1 high neutrophil necrotic rates (49.6±5.1% vs 4.2±0.7% for controls; p<0.01) were observed, resulting in an apparent fall off in apoptotic rates. Diatrizoate at 80 mg I ml-1 resulted in a significant increase in total cell death (apoptosis and necrosis) compared with control medium (83.9±2.8% vs 51.8±1.6%; p<0.01). Figure 3Go shows induction of neutrophil apoptosis following ICM treatment on cytospin preparations.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 2. Concentration dependence of iodinated contrast media (ICM) induced apoptosis. Results expressed as mean percentage of apoptotic cells±standard error of the mean. *, p<0.05 and {ddagger}, p<0.01 for ICM treated vs control neutrophils; §, high necrotic rate.

 


View larger version (60K):
[in this window]
[in a new window]
  		Figure 3. Cell morphology of control and iodinated contrast media treated neutrophils. Cytospins of (a) control and (b) diatrizoate (20 mg I ml-1) treated neutrophils at 12 h culture. In control neutrophils, normal neutrophil morphology with a multi-lobulated nucleus is seen (arrow). Following diatrizoate treatment, characteristic morphological signs of neutrophil apoptosis, such as cell shrinkage, nuclear pyknosis and chromatin condensation are demonstrated (arrow). Magnification of figures x 1 000.

 
Physicochemical factors contribute to the toxicity of ICM
Since the ICM studied differ with respect to osmolality and ionic strength (Table 1Go), we investigated the effects of control solutions with similar hypertonicity, hyperosmolality and ionic strength to diatrizoate 20 mg I ml-1 on neutrophil apoptosis following incubation for 12 h (Figure 4Go). The more significant pro-apoptotic effects of the ionic contrast media (diatrizoate and ioxaglate) over non-ionic contrast media (iohexol and iotrolan) appear to be related to their ionicity, as hyperosmolar NaCl (ionic hypertonicity control) significantly (p<0.01) induced neutrophil apoptosis compared with the non-ionic hypertonic control (mannitol) and the normotonic hyperosmolality control (urea), which did not induce apoptosis at the concentrations studied.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 4. Effects of physicochemical properties of iodinated contrast media and growth factor dilution on neutrophil apoptosis. (a) Neutrophils were treated with experimental solutions for 12 h and apoptosis quantified by flow cytometry. Control, Rosweli Park Memorial Institute 1640 medium (RPMI)+10% platelet poor plasma; diatrizoate, 20 mg I ml-1; NaCl hypertonic, mannitol, and urea at same hyperosmolality as 20 mg I ml-1 diatrizoate; disodium calcium edetate (EDTA) 0.1 mg ml-1, 0.9% NaCl, RPMI diluted 1:2 with isotonic NaCl solution. Results expressed as mean percentage of apoptotic cells±standard error of the mean; {ddagger}p<0.01 for treated vs control neutrophils. (b) Neutrophils were cultured in RPMI containing 10% platelet poor plasma alone and treated with increasing concentrations (0.1–80 mg I ml-1) of povidone-iodine. Results expressed as mean percentage of necrotic cells±standard error of the mean; n=3–4; {ddagger}p<0.01 for treated vs control neutrophils.

 
Dilution of growth factor medium has no effect on neutrophil apoptosis
There was no significant difference between RPMI diluted with isotonic saline and control RPMI, excluding an effect of dilution of growth factor media by ICM solutions as a cause for their induction of apoptosis.

Povidone-iodine, but not EDTA, induces neutrophil cell death
An effect of ETDA (0.1 mg ml-1), a component of ICM, on neutrophil apoptosis was also excluded (Figure 4Go). The povidone–iodine complex induced a concentration dependent necrosis of neutrophils without an effect on apoptosis (Figure 4Go), with povidone alone having no effect on neutrophil cell death (data not shown).

ICM abrogates the survival effect of LPS treated neutrophils
Inflammatory mediators, such as LPS, are known to prolong neutrophil survival in vitro by delaying apoptosis [18]. We confirmed in our study that LPS induced a significant (p<0.01) delay in neutrophil apoptosis compared with controls at 12 h and 24 h (Figure 5Go). Interestingly, the strong pro-apoptotic effects of iohexol abrogated the survival effect of LPS, indicating that ICM have a pro-apoptotic effect on inflammatory neutrophils in vitro.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 5 Iodinated contrast media accelerates apoptosis in lipopolysaccharide (LPS) stimulated neutrophils. Neutrophils were cultured in Rosweli Park Memorial Institute 1640 medium containing 10% platelet poor plasma alone and treated with iohexol 40 mg I ml-1, with or without LPS (1000 ng ml-1). Apoptosis was assessed at 1 h, 12 h and 24 h by flow cytometry. Results expressed as mean percentage of apoptotic cells±standard error of the mean; n=4; *p<0.01 for LPS treated vs control neutrophils; {ddagger}p<0.01 for iohexol treated vs control; §p<0.01 for LPS+iohexol vs LPS alone. –{rectangle}–, control; –{triangleup}–, iohexol; –{diamond}–, LPS; –{circ}–, LPS+iohexol.

 
Iohexol induces neutrophil apoptosis independent of p38 MAPK signalling pathway
p38 MAPK activation in LPS treated neutrophils appears to mediate a survival pathway as LPS delayed apoptosis is partially reversed when p38 MAPK activity is blocked using the p38 MAPK inhibitor SB202190. However, SB202190 had no effect on spontaneous neutrophil apoptosis or on neutrophils treated with ICM (Table 3Go). Western blot analysis confirmed that p38 MAPK phosphorylation and activation was no different in ICM stimulated cells when compared with controls (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of p38 mitogen-activated protein kinase inhibition on lipopolysaccharide (LPS) and iohexol treated neutrophils. Results expressed as mean percentage of apoptotic cells±standard error of the mean as quantified by flow cytometry; n=4

 
Iohexol promotes loss of mitochondrial transmembrane potential
Mitochondria are increasingly recognized as important in the execution of the apoptotic programme in neutrophils [19]. We found that iohexol promotes the reduction in mitochondrial transmembrane potential up to a maximum studied period of 18 h (Figure 6Go).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 6. Iohexol promotes mitochondrial depolarization. Neutrophils were cultured in Rosweli Park Memorial Institute 1640 medium containing (a) 10% platelet poor plasma alone and (b) treated with iohexol 40 mg I ml-1. Mitochondrial transmembrane potential was measured using the styryl dye, JC-1, at 4 h, 10 h and 18 h in culture. Iohexol promotes a greater reduction in mitochondrial transmembrane potential compared with the control as assessed by increased formation of monomers (488nm-FL1-H). This effect was evident within 4 h of culture, with progressive loss of mitochondrial transmembrane potential at 10 h and almost complete mitochondrial depolarization at 18 h. Results representative of three experiments.

 
Induction of apoptosis by iohexol is dependent on activation of the caspase-cascade pathway
As reported previously [20], zVAD-fmk reduced the rate of spontaneous apoptosis, however, zVAD-fmk also inhibited the increase in apoptosis induced by iohexol (Figure 7Go). This demonstates that apoptosis induced by iohexol is dependent on caspase activation.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 7. Induction of apoptosis by iohexol is dependent on activation of the caspase-cascade pathway. Neutrophils were cultured in Rosweli Park Memorial Institute 1640 medium containing 10% platelet poor plasma alone and treated with iohexol 40 mg I ml-1, with or without zVAD-fmk (100 µM). Apoptosis was assessed at 12 h by flow cytometry. Results expressed as mean percentage of apoptotic cells±standard error of the mean; n=3; *p<0.05 for zVAD-fmk vs control neutrophils; {ddagger}p<0.05 for iohexol treated vs control; §p<0.05 for zVAD-fmk+iohexol vs iohexol alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study demonstrates that the four classes of ICM used clinically significantly induce neutrophil apoptosis in a time and concentration dependent manner in vitro (Figures 1–3GoGoGo). We observed an induction in neutrophil apoptosis at concentrations of 20 mg I ml-1, with a more significant pro-apoptotic effect with ionic media compared with non-ionic ICM. Ionic strength appears to mediate part of the toxic effects of diatrizoate and ioxaglate since the ionic control solution (hypertonic NaCl) significantly enhanced neutrophil apoptosis. However, ionicity alone does not explain why diatrizoate was the most toxic agent studied. Diatrizoate, unlike the other agents, induced apoptosis at concentrations as low as 10 mg I ml-1, and had significantly greater apoptotic rates at 12 h and 24 h compared with ioxaglate (Figure 1Go). We detected no effect of hypertonicity and hyperosmolality on neutrophil apoptosis at the concentrations studied (equivalent to 20 mg I ml-1 diatrizoate), as both the hypertonic and hyperosmolar mannitol solution and the isotonic and hyperosmolar urea media failed to induce neutrophil apoptosis (Figure 4Go). Previous reports indicate that highly hypertonic solutions may induce apoptosis in renal tubular cells. However, lower concentrations, as used in this study, failed to induce apoptosis [1, 21]. The reason for the greater toxicity of diatrizoate is unknown. A significant role of growth factor dilution by contrast media was excluded by incubating cells with isotonically diluted media without observing a toxic effect (Figure 4Go).

The incubation times and iodine concentrations used in the current experiments may be pathophysiologically relevant, since they may occur in the clinical setting in special circumstances. We observed a significant induction of apoptosis in neutrophils aged in vitro following a short (45 min) exposure of cells to iohexol (40 mg I ml-1) (Table 2Go). It has been reported that brief (1–15 min) exposure to ICM is sufficient to induce apoptosis in other cell systems [4]. This short incubation time may have relevance in vivo as the elimination half-life of contrast media in healthy individuals is approximately 120 min [22]. Exposure of neutrophils to ICM for longer incubation times (Figures 1–3GoGoGo) may have more relevance in patients with advanced renal failure, where the elimination half-life of contrast media can be up to 15 times that of normal. The iodine concentrations of ICM used in the present study were 10–80 mg I ml-1. We observed a pro-apoptotic effect on neutrophils at concentrations of 20 mg I ml-1 for ioxaglate, iohexol and iotrolan, and 10 mg I ml-1 for diatrizoate. Plasma concentrations of ICM of 10–20 mg I ml-1 are reached during routine intravascular injection [23, 24], but only for short times owing to the rapid distribution of ICM to the interstitial fluid (distribution half-life of contrast media of approximately 20 min [22]). The relevance of our findings on circulating neutrophils is therefore uncertain, and further study on the clinical effects of ICM on neutrophil apoptosis in vivo is warranted.

We also investigated the effect of specific components of ICM on neutrophil apoptosis to see if we could identify the toxic agent in contrast media. We excluded a significant role of EDTA on apoptosis by incubating cells with concentrations of EDTA comparable to that found in ICM and found no effect on the apoptotic effect (Figure 4Go). Excess iodine has been shown to induce thyroid cell apoptosis in goitrogen treated rats and has been reported to cause neutropenia in humans [25–27]. Iodine, due to its sufficiently high atomic number to maximize photoelectric absorption, is an essential component of ICM. All current ICM are based on a tri-iodinated benzene derivative. The iodine in contrast media is covalently bound in the benzene ring so that the concentration of free iodine in contrast media is minute and unlikely to be a factor in the chemotoxicity of contrast media. We attempted to investigate the effects of elemental iodine on neutrophil cell death using a soluble povidone–iodine complex. Povidone, in addition to solubilizing iodine, decreases the toxicity of iodine owing to its ability to form stable complexes. The amount of free iodine in the povidone–iodine complex is small (a few parts per million) as the iodine is ionically bound to the polyvinylpyrrolidone polymer [28]. We observed that the iodine complex induced neutrophil necrosis, not apoptosis, and at concentrations as low as 5 mg I ml-1 (Figure 4Go). There was no effect of povidone alone on neutrophil survival (data not shown). We have clearly demonstrated that iodine is toxic to neutrophils. We have also shown that diatrizoate at concentrations of 80 mg I ml-1 significantly increases necrotic cell death in neutrophils. This data would suggest that the chemical structure of ICM decreases, but does not eliminate, the toxicity of iodine with diatrizoate being the most toxic agent studied.

The effect of ICM on human neutrophils appears to differ from their reported effects in other cell systems. Hizóh et al [1] demonstrated that the ionic contrast agent diatrizoate (37–111 mg I ml-1) induced apoptosis in a canine renal epithelial cell line, however, the non-ionic low-osmolar contrast medium iopamidol (74 mg I ml-1) failed to induce apoptosis. A further in vitro study showed increased apoptosis in human vascular endothelial cells exposed to high concentrations (250 mg I ml-1) of diatrizoate, ioxaglate and iopromide, but not iotrolan [4]. Other studies have looked at the effect of ICM on cell viability in vitro using vital dye exclusion tests in vascular smooth muscle cells and renal tubular cells, and showed that hyperosmolar ionic ICM (diatrizoate) are most cytotoxic [21, 29, 30]. In an in vivo animal study, Zhang et al [2] illustrated that diatrizoate and iohexol induced apoptosis in cardiac myocytes, renal tubular and glomerular cells, and in vascular endothelial and smooth muscle cells of the heart and kidney. Heyman et al [31] demonstrated that iothalmate, an ionic ICM, increased renal medullary cell apoptosis in hydronephrotic kidneys. These reports indicate that ICM can induce apoptosis in a variety of cells, and that different cell types have different susceptibilities to ICM-induced apoptosis. Neutrophils appear to be more sensitive to the pro-apoptotic effects of ICM than other cell types, as all classes of ICM induce apoptosis, and at relatively low doses (20 mg I ml-1). The differences observed may be explained by the differences in cell biology. Neutrophils are terminally differentiated cells with a uniquely high rate of constitutive apoptosis. This makes them an ideal cell system to study the inherent effects of ICM on cell survival. Neutrophils, unlike other cells, do not express the anti-apoptotic protein bcl-2 but constitutively express a range of relatively long lived pro-apoptotic proteins of the bcl-2 family (bax, bid, bak and bad). Neutrophils do express relatively short lived survival proteins of the bcl-2 family, mcl-1 and A1. However, in the absence of survival signals e.g. LPS, the activity of the longer lived pro-apoptotic proteins will predominate and apoptosis will ensue [32]. Zhang et al [2] showed, in renal cells, that diatrizoate and iohexol decreased expression of bcl-2 and increased expression of bax and p53. Neutrophils do not express p53 or bcl-2 [33, 34], and differences in protein expression may help explain differences in observed apoptotic rates between various cell types.

The bacterial wall product LPS can rescue neutrophils from constitutive apoptosis. We demonstrated that iohexol abrogates the pro-survival effect of LPS on neutrophils, causing them to rapidly progress though the apoptotic pathway (Figure 5Go). This result indicates that iohexol can overcome the LPS-generated pro-survival signalling pathways in neutrophils. There has been much interest in the role of p38 MAPK in the regulation of neutrophil apoptosis and survival. The function of p38 MAPK activation in neutrophil apoptosis and survival is unclear. Certain stress stimuli, such as hyperosmolality or ultraviolet irradiation, appear to activate p38 MAPK and accelerate apoptosis [35], whereas hypoxia-mediated delay of apoptosis requires p38 MAPK activity. In contrast to Nolan et al [36], we observed that LPS-induced delayed apoptosis is reduced when p38 MAPK activity is inhibited (Table 3Go) suggesting that LPS, like hypoxia, may result in a p38 MAPK-generated survival signal. We therefore investigated if ICM-induced apoptosis is mediated by inhibition of the p38 MAPK survival signalling pathway. We found that p38 MAPK was not activated following treatment of neutrophils with ICM by Western blotting, and that the p38 MAPK inhibitor SB202190 had no effect on ICM treated neutrophils. These results indicate that ICM induce neutrophil apoptosis through a pathway independent p38 MAPK.

The signals transduced by many pro-apoptotic pathways converge to regulate activation of procaspase-3 [37–40]. Procaspase-3 can be directly activated by caspase-8 [41], or indirectly through the release of apoptosis-inducing factors, such as cytochrome c, from the mitochondria [42–49]. While it has been reported that mature neutrophils have a reduced number of phenotypically atypical neutrophils [32], Pryde et al [19] demonstrated that mitochondria play an important role in triggering neutrophil apoptosis. In addition, subcellular localization of ICM to mitochondria has been shown [50]. We therefore investigated the effect of ICM on mitochondrial membrane potential and the role of caspase activation in mediating the pro-apoptotic effects of ICM. We demonstrated that iohexol promotes loss of mitochondrial membrane potential (Figure 6Go), and that induction of apoptosis by iohexol is dependent on activation of the caspase-cascade pathway (Figure 7Go).

In conclusion, this report presents evidence demonstrating that apoptosis can be induced by ICM in immune cells in vitro. Specifically, ICM increase constitutive and inflammatory neutrophil apoptosis in a time and concentration dependent manner in vitro, with ionic ICM appearing to have a greater toxic effect. Neutrophils play a central role in host response to infection and injury. Agents that compromise neutrophil viability may affect host response to infection and resolution of an inflammatory response. Induction of apoptosis occurs through alteration of the neutrophil signal transduction pathways following exposure to ICM. We have demonstrated that iohexol acts to induce neutrophil apoptosis through a p38 MAPK independent pathway, promoting mitochondrial depolarization and caspase activation. This report adds to our understanding of the mechanism of ICM-induced apoptosis in neutrophils, and may provide insights into the pro-apoptotic mechanisms of ICM in other cells types.

Received for publication November 20, 2001. Revision received June 6, 2002. Accepted for publication June 13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Hizoh I, Strater J, Schick CS, Kubler W, Haller C. Radiocontrast-induced DNA fragmentation of renal tubular cells in vitro: role of hypertonicity. Nephrol Dial Transplant 1998;13:911–8.[Abstract/Free Full Text]
  2. Zhang J, Duarte CG, Ellis S. Contrast medium- and mannitol-induced apoptosis in heart and kidney of SHR rats. Toxicol Pathol 1999;27:427–35.[Medline]
  3. Beeri R, Symon Z, Brezis M, Ben-Sasson SA, Baehr PH, Rosen S, et al. Rapid DNA fragmentation from hypoxia along the thick ascending limb of rat kidneys. Kidney Int 1995;47:1806–10.[Medline]
  4. Zhang H, Holt CM, Malik N, Shepherd L, Morcos SK. Effects of radiographic contrast media on proliferation and apoptosis of human vascular endothelial cells. Br J Radiol 2000;73:1034–41.[Abstract]
  5. Heyman SN, Fuchs S, Jaffe R, Shina A, Ellezian L, Brezis M, et al. Renal microcirculation and tissue damage during acute ureteral obstruction in the rat: effect of saline infusion, indomethacin and radiocontrast. Kidney Int 1997;51:653–63.[Medline]
  6. Savill J. Apoptosis in resolution of inflammation. J Leukoc Biol 1997;61:375–80.[Abstract]
  7. Fanning NF, Kell MR, Shorten GD, Kirwan WO, Bouchier-Hayes D, Cotter TG, et al. Circulating granulocyte macrophage colony-stimulating factor in plasma of patients with the systemic inflammatory response syndrome delays neutrophil apoptosis through inhibition of spontaneous reactive oxygen species generation. Shock 1999;11:167–74.[Medline]
  8. Kusaba N, Kumashiro R, Ogata H, Sata M, Tanikawa K. In vitro study of neutrophil apoptosis in liver cirrhosis. Intern Med 1998;37:11–7.[Medline]
  9. Carmona-Sanchez R, Uscanga L, Bezaury-Rivas P, Robles-Diaz G, Suazo-Barahona J, Vargas-Vorackova F. Potential harmful effect of iodinated intravenous contrast medium on the clinical course of mild acute pancreatitis. Arch Surg 2000;135:1280–4.[Abstract/Free Full Text]
  10. Orlov SN, Dam TV, Tremblay J, Hamet P. Apoptosis in vascular smooth muscle cells: role of cell shrinkage. Biochem Biophys Res Commun 1996;221:708–15.[Medline]
  11. Duke RC, Cohen JJ. IL-2 addiction: withdrawal of growth factor activates a suicide program in dependent T cells. Lymphokine Res 1986;5:289–99.[Medline]
  12. MacGregor RR, Spagnuolo PJ, Lentnek AL. Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. N Engl J Med 1974;291:642–6.
  13. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995;182:1545–56.[Abstract/Free Full Text]
  14. Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993;197:40–5.[Medline]
  15. Cossarizza A, Ceccarelli D, Masini A. Functional heterogeneity of an isolated mitochondrial population revealed by cytofluorometric analysis at the single organelle level. Exp Cell Res 1996;222:84–94.[Medline]
  16. Reers M, Smith TW, Chen LB. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 1991;30:4480–6.[Medline]
  17. Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith TW, et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci U S A 1991;88:3671–5.[Abstract/Free Full Text]
  18. Lee A, Whyte MK, Haslett C. Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J Leukoc Biol 1993;54:283–8.[Abstract]
  19. Pryde JG, Walker A, Rossi AG, Hannah S, Haslett C. Temperature-dependent arrest of neutrophil apoptosis. Failure of Bax insertion into mitochondria at 15 degrees C prevents the release of cytochrome c. J Biol Chem 2000;275:33574–84.[Abstract/Free Full Text]
  20. Khwaja A, Tatton L. Caspase-mediated proteolysis and activation of protein kinase Cdelta plays a central role in neutrophil apoptosis. Blood 1999;94:291–301.[Abstract/Free Full Text]
  21. Schick CS, Haller C. Comparative cytotoxicity of ionic and non-ionic radiocontrast agents on MDCK cell monolayers in vitro. Nephrol Dial Transplant 1999;14:342–7.[Abstract/Free Full Text]
  22. Bourin M, Jolliet P, Ballereau F. An overview of the clinical pharmacokinetics of X-ray contrast media. Clin Pharmacokinet 1997;32:180–93.[Medline]
  23. Gmelin E, Rinast E, Ollrogge C. Arterial bolus curve after intravenous injection of ionic and nonionic contrast media. In: Taenzer V, Wende S, editors. Recent developments in nonionic contrast media. New York, NY: Thieme Medical Publishers, Inc.; 1989:67–9.
  24. Roobottom CA, Farrow R, Wells IP, Hurlock N. The effects of radiographic contrast media on leucocyte orientation. Br J Radiol 1993;66:778–80.[Abstract]
  25. Vitale M, Di Matola T, D'Ascoli F, Salzano S, Bogazzi F, Fenzi G, et al. Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress. Endocrinology 2000;141:598–605.[Abstract/Free Full Text]
  26. Burikhanov RB, Matsuzaki S. Excess iodine induces apoptosis in the thyroid of goitrogen-pretreated rats in vivo. Thyroid 2000;10:123–9.[Medline]
  27. Alvarez E. Neutropenia in a burned patient being treated topically with povidone-iodine foam. Plast Reconstr Surg 1979;63:839–40.[Medline]
  28. Schenck HU, Simak P, Haedicke E. Structure of polyvinylpyrrolidone-iodine (povidone-iodine). J Pharm Sci 1979;68:1505–9.[Medline]
  29. Haller C, Schick CS, Zorn M, Kubler W. Cytotoxicity of radiocontrast agents on polarized renal epithelial cell monolayers. Cardiovasc Res 1997;33:655–65.[Abstract/Free Full Text]
  30. Wang YX, Chan P, Morcos SK. The effect of radiographic contrast media on human vascular smooth muscle cells. Br J Radiol 1998;71:376–80.[Abstract]
  31. Heyman SN, Fuchs S, Jaffe R, Shina A, Ellezian L, Brezis M, et al. Renal microcirculation and tissue damage during acute ureteral obstruction in the rat: effect of saline infusion, indomethacin and radiocontrast. Kidney Int 1997;51:653–63.
  32. Moulding DA, Quayle JA, Hart CA, Edwards SW. Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival. Blood 1998;92:2495–502.[Abstract/Free Full Text]
  33. Hsieh SC, Huang MH, Tsai CY, Tsai YY, Tsai ST, Sun KH, et al. The expression of genes modulating programmed cell death in normal human polymorphonuclear neutrophils. Biochem Biophys Res Commun 1997;233:700–6.[Medline]
  34. Wei S, Liu JH, Epling-Burnette PK, Gamero AM, Ussery D, Pearson EW, et al. Critical role of Lyn kinase in inhibition of neutrophil apoptosis by granulocyte-macrophage colony-stimulating factor. J Immunol 1996;157:5155–62.[Abstract]
  35. Frasch SC, Nick JA, Fadok VA, Bratton DL, Worthen GS, Henson PM. p38 mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils. J Biol Chem 1998;273:8389–97.[Abstract/Free Full Text]
  36. Nolan B, Duffy A, Paquin L, De M, Collette H, Graziano CM, et al. Mitogen-activated protein kinases signal inhibition of apoptosis in lipopolysaccharide-stimulated neutrophils. Surgery 1999;126:406–12.[Medline]
  37. Sanghavi DM, Thelen M, Thornberry NA, Casciola-Rosen L, Rosen A. Caspase-mediated proteolysis during apoptosis: insights from apoptotic neutrophils. FEBS Lett 1998;422:179–84.[Medline]
  38. Korsmeyer SJ. Regulators of cell death. Trends Genet 1995;11:101–5.[Medline]
  39. Yamashita K, Takahashi A, Kobayashi S, Hirata H, Mesner PW Jr, Kaufmann SH, et al. Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production. Blood 1999;93:674–85.[Abstract/Free Full Text]
  40. Yuan JY, Horvitz HR. The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dev Biol 1990;138:33–41.[Medline]
  41. Stennicke HR, Jurgensmeier JM, Shin H, Deveraux Q, Wolf BB, Yang X, et al. Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem 1998;273:27084–90.[Abstract/Free Full Text]
  42. Kuwana T, Smith JJ, Muzio M, Dixit V, Newmeyer DD, Kornbluth S. Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c. J Biol Chem 1998;273:16589–94.[Abstract/Free Full Text]
  43. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997;91:479–89.[Medline]
  44. Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 1996;184:1331–41.[Abstract/Free Full Text]
  45. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis [see comments]. Science 1997;275:1132–6.[Abstract/Free Full Text]
  46. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 [see comments]. Cell 1997;90:405–13.[Medline]
  47. Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 1998;17:37–49.[Medline]
  48. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2000;2:156–62.[Medline]
  49. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996;86:147–57.[Medline]
  50. Guidolet J, Barbe R, Borsson F, Gateau O, Amiel M, Louisot P. Cardiovascular radiology. Subcellular localization of uro-angiographic contrast by 125I-labeled media. Invest Radiol 1980;15:S215–19.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF)
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fanning, N F
Right arrow Articles by Redmond, H P
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fanning, N F
Right arrow Articles by Redmond, H P

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
BJR DMFR IMAGING  ALL BIR JOURNALS