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International Immunology Advance Access originally published online on December 15, 2005
International Immunology 2006 18(1):139-150; doi:10.1093/intimm/dxh356
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© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells

Nadia Berkova1,*, Sybille Lair-Fulleringer1,*, Françoise Féménia1, Dominique Huet1, Marie-Christine Wagner2, Kamila Gorna1, Frédéric Tournier3, Oumaïma Ibrahim-Granet4, Jacques Guillot1,5, René Chermette1,5, Pascal Boireau1 and Jean-Paul Latgé4

1 INRA, AFSSA, ENVA, UPVM, UMR 956; 22 rue Curie, Maisons Alfort Cedex F-94700, France
2 Plate-Forme de Cytométrie, Institut Pasteur, Paris, France
3 Laboratoire de Cytophysiologie et Toxicologie Cellulaire, Université Paris 7, Paris, France
4 Unité des Aspergillus, Institut Pasteur, Paris, France
5 Service de Parasitologie-Mycologie, Ecole Nationale Vétérinaire d'Alfort, 7 av de Général de Gaulle, Maisons Alfort Cedex 94704, France

Correspondence to: N. Berkova; E-mail: nberkova{at}vet-alfort.fr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
A major innate immune response to inhaled conidia of the opportunistic pathogen Aspergillus fumigatus (Af) is the synthesis of pro-inflammatory cytokines, which include tumour necrosis factor (TNF)-{alpha}, a known inducer of apoptosis. Modulation of host cell apoptosis has been reported to be one of the mechanisms whereby pathogens overcome host cell defences. Our study was designed to investigate whether or not Af conidia could modulate apoptosis induced by TNF-{alpha} or staurosporine (STS). Exposure of epithelial cells treated by these inducers and exposed to Af conidia decreased the number of apoptotic cells detected by Annexin V staining, analysis of nuclear morphology, terminal deoxynucleotidyl transferase-mediated fluorescein–dUTP nick end-labelling reaction and immunoblotting. Inhibition of apoptosis by Af conidia was seen in cells of the A549 pneumocyte II line, human tracheal epithelial 16HBE and primary human respiratory cells. Inhibition of apoptosis by Af conidia was also observed when apoptosis was induced by co-cultivating A549 cells with activated human alveolar macrophages. Unlike Af conidia, conidia of Cladosporium cladosporioides as well as latex beads or killed Af conidia have no inhibitory effect on TNF-{alpha} or STS-induced apoptosis. For TNF-induced apoptosis, the observed anti-apoptotic effect of Af conidia was found to be associated with a significant reduction of caspase-3.

Keywords: apoptosis, epithelial cells, fungi, programmed cell death


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Aspergillus fumigatus (Af) is a saprophytic mould, which is responsible for the majority of invasive mycosis in patients undergoing chemotherapy or organ transplantation (1). It propagates through airborne conidia (spores), which are inhaled into the small airways where they may germinate and initiate an infection. The invasion of airway epithelial cells is therefore a key step in the etiology of aspergillosis, especially since it has been shown that these cells are able to phagocytose Af conidia which can survive at least for some time in these cells (2–4).

Pathogenic micro-organisms have evolved different mechanisms to survive in the host environment: modulation of host cell apoptosis is one of these mechanisms. Escherichia coli (5) and Candida albicans (6) have been found to induce apoptosis in neutrophils, and Cryptococcus neoformans has been shown to induce apoptosis in inflammatory cells in granulomas of rats with cryptococcal meningitis (7, 8). In contrast, inhibition of host cell apoptosis by the yeast form of Histoplasma capsulatum and the protozoa Theileria parva and Toxoplasma may provide an intracellular niche for the pathogens by extending the life span of infected host cells (9–11). Previous studies have shown that gliotoxin, the most abundant mycotoxin produced by Af, induces apoptosis in many types of cells (12). However, this secondary metabolite is only produced by hyphae and nothing is known about the influence of conidia of Af on host cell apoptosis.

Apoptotic cells show characteristic morphological changes, including cell shrinkage, nuclear/chromatin condensation, inter-nucleosomal cleavage of DNA, membrane blebbing and formation of apoptotic bodies (13). Caspases, which are responsible for many biochemical and morphological changes associated with apoptosis, are synthesized as inactive pro-enzymes, most of which have to be activated by proteolytic cleavage (14). There are various alternative inducers of apoptosis such as the proteins of the tumour necrosis factor receptor (TNFR) family and stimuli such as cytotoxic agents, cell metabolites and other pathological insults (15, 16). The receptor-mediated (extrinsic) signalling pathway involves the formation of a death-inducing signalling complex (DISC), with subsequent activation of the initiator caspase, which either directly activates the effector caspases or cleaves cytosolic factors, leading to the release of cytochrome-c and activation of another initiator caspase, and subsequently of effector caspases (17–19). The chemically mediated (intrinsic) pathway, which is classically linked to mitochondrial disturbance, is characterized by the release of cytochrome-c from mitochondria, following the activation of the initiator caspases, which in turn activate effector caspases (14, 16). Caspase-3 is one of the effector caspases which is situated at nodal points in both apoptotic pathways. When activated by proteolytic cleavage, caspase-3 cleaves vital cellular proteins involved in the apoptotic process (19, 20). However, a caspase-independent apoptotic pathway has also been identified recently (21, 22).

The studies described in this report were designed to investigate whether Af conidia could induce or modify apoptosis and hence affect host cell survival. Exposure of alveolar macrophages to Af conidia results in the synthesis of TNF-{alpha} (23), which in turn may induce apoptosis in epithelial cells. Here, we describe in vitro experiments which have been mainly carried out with a transformed human type II pneumocyte cell line (A549) as a model for alveolar epithelial cells, using TNF-{alpha} and staurosporine (STS) as inducers of apoptosis. Cycloheximide (CXH), a translational inhibitor, has been used together with TNF, to favour DISC-dependent activation of initiator caspase (24–26). STS was selected as an alternative inducer of apoptosis for the investigation of the chemical-mediated pathway. The results of the morphological analysis of stained nuclei, flow cytometry, terminal deoxynucleotidyl transferase-mediated fluorescein–dUTP nick end-labelling (TUNEL) assay and western blot analysis demonstrated that Af conidia only slightly induce apoptosis in untreated A549 cells. In contrast, Af conidia drastically inhibit apoptosis initiated by either TNF-{alpha} or STS. The mechanism of inhibition of apoptosis following STS treatment remains unknown, while conidia-mediated inhibition of cytokine-induced death appears to be caspase-3 dependent. The inhibition of apoptosis by Af conidia was also observed in the human tracheal epithelial 16HBE cell line, in airway epithelial primary culture cells as well as in A549 cells co-cultivated with activated human alveolar macrophages (HAM) cells in the absence of the addition of TNF or STS: this points to the biological significance of our findings.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Fungal strains and growth conditions
Af CBS 144.89 and Cladosporium cladosporioides IP1232 (Institut Pasteur, Paris, France) were used throughout this study. Conidia of Af and C. cladosporioides were obtained from cultures grown on YM agar (0.3% yeast extract, 2% malt extract, 0.5% peptone and 0.5% agar) for 3 days at 37°C and 25°C, respectively. Conidia were harvested by flooding the plates with sterile distilled water and then suspending the conidia in 0.15 M PBS. For some experiments, Af conidia were killed by incubation in a 3% PFA solution overnight followed by washings with 0.2 M glycine and PBS.

Human cells and growth conditions
Type II pneumocyte cell line A549 derived from a human lung carcinoma was obtained from American Type Culture Collection [ATCC CCL 185 (27)] and maintained in Kaighn's modification of HAM's F12 medium supplemented with 10% of FCS (Invitrogen), pen/strep (16 mg ml–1 penicillin and 100 mg l–1 streptomycin), 2 mM L-glutamine and 1.5 g l–1 sodium bicarbonate. The cells were grown until confluent. Unless specified otherwise, all incubations were at 37°C in an incubator with a humidified atmosphere of 5% CO2. Trypsin/EDTA (Invitrogen) was used to release adherent cells for subculturing when this was required.

Human tracheal epithelial SV40-transformed cells (16HBE) were provided by Boisvieux-Ulrich (Cytophysiology and Toxicology laboratory, University of Paris 7, France). 16HBE cells were maintained in Eagle MEM medium (Invitrogen) with 2% UltroserG (Invitrogen), pen/strep, 2 mM L-glutamine (Sigma) and 1.5 g l–1 sodium bicarbonate (Sigma) and were grown until confluent (28).

Primary epithelial cells were obtained from human nasal turbinates (HNT) of patients undergoing turbinectomy (P. Herman, CHU Lariboisière, Paris, France) as described previously (29). Briefly, HNT were washed in Dulbecco's modified Eagle medium DMEM/F12 (Invitrogen) and incubated with 2 mg ml–1 pronase (Protease XIV; Sigma,) in DMEM/F12 supplemented with pen/strep, at 4°C for 16–20 h under slow rotary agitation (80 r.p.m.). After washing, aggregates were discarded and dissociated cells were filtered using a 30-µm pore filter. The cell suspension was then plated for 2 h at 37°C on plastic dishes (Falcon) to eliminate contaminating fibroblasts. After centrifugation, the supernatant containing the epithelial cells was plated on collagen type I-coated wells and cultivated in DMEM/F12 supplemented with 10% FCS and pen/strep.

HAM were obtained from a bronchoalveolar lavage of a patient who had received a lung transplant (B. Philippe, Foch Hospital, Suresnes, France). Cells were harvested by centrifugation (400 x g for 10 min at 4°C), re-suspended in RPMI complete medium containing 10% FCS and seeded on cell culture plates at 106 per well. Non-adherent cells were removed by washing after 2 h incubation.

Induction of apoptosis
Either A549 or 16HBE cells were seeded at 5 x 105 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips (Marienfeld, Germany) in 12-well plates (Nunc, NuclonTM Surface) in triplicate and grown for 16 h at 37°C. Primary epithelial cells were seeded at 5 x 106 cells per well and grown for 48 h. After washing the cover slips with PBS–BSA (PBS-5% BSA, Fraction V, Sigma), the inducers of apoptosis were added to the wells in 1 ml of medium. To induce apoptosis, two different types of inducers were used: (i) 1 µM STS (Sigma) and (ii) 20 ng ml–1 of TNF-{alpha} (Sigma) with 2 µg of CHX (Sigma). After 7 h of incubation conidia germinated, however, the apoptotic features of cells, incubated with TNF-{alpha} in the absence of CHX, appeared much later (data not shown). To efficiently induce apoptosis and decrease the time of incubation, TNF-{alpha} was used together with CHX, since CHX has been shown to suppress survival signals induced by an engagement of TNFR (23). The presence of CHX is therefore only required to allow us to study the role of conidia on induced apoptosis in the absence of conidial germination.

To investigate early stages of apoptosis by flow cytometry, the induction of apoptosis was performed for 1 h, a period when maximal numbers of apoptotic cells and minimal number of necrotic cells were detected (data not shown). The control cells were incubated either with CHX or medium alone. We did not detect any difference between CHX-treated cells (data not shown) and cells incubated with medium alone. None of the treatments inducing apoptosis affected the viability of conidia (data not shown).

To study the late stages of apoptosis, A549 cells were incubated with STS or TNF-{alpha} for 6 h: this duration of treatment was defined by results reported by others (25, 30) and on our own unpublished observation showing the absence of the 17-kDa active form of caspase-3 after 4 h of incubation.

HAM cells have also been used as a source of TNF for apoptosis induction. In order to estimate the optimal incubation time, preliminary assay of apoptosis induction in A549 with 20 ng ml–1 of TNF-{alpha} during different periods (6 h, 24 h and 48 h) was performed. The highest number of apoptotic cells was found after 24 h incubation (data not shown). Therefore, for a 24-h incubation, different doses of TNF-{alpha} were tested by morphological criteria for apoptosis induction in A549 cells. Apoptotic features of cells were observed at a concentration of TNF-{alpha} as low as 5 ng ml–1 of TNF-{alpha} (data not shown). A549 cells were seeded at 5 x 105 cells per well on 18-mm diameter cover slips in 12-well plates and grown for 16 h. After washing with PBS-5% BSA, the cover slips bearing A549 cells were transferred onto the surface of an 18-h-old culture of alveolar macrophages stimulated with 1 µg ml–1 of LPS for the same time of culture. Under these culture conditions, HAM produced 10 ng ml–1 of TNF-{alpha}, as measured in culture supernatant using a commercial ELISA kit (BD Biosciences).

Exposure to conidia
Following washing of A549, 16HBE or primary culture cells with PBS, Af conidia ranging from 5 x 106 to 108 conidia per millilitre of medium were added to the cells simultaneously with the inducer of apoptosis. In experiments when activated HAM have been used as a source of TNF, 107 conidia per millilitre have been added to the cells. The lower concentration of conidia was chosen on the basis of the results of Wasylnka and Moore (31); the higher concentrations were chosen to investigate dose dependence. After incubation, unbound conidia were removed by washing wells with PBS-5% BSA. The resulting number of apoptotic cells was compared with cultures incubated either with the inducers or the medium (control). To test for the specificity of the effect of live Af conidia, some wells received instead 5 x 106 to 108 C. cladosporioides conidia, latex beads or PFA-killed Af conidia.

Flow cytometry analysis
A549 cells were labelled with an FITC–Annexin V and stained with propidium iodide (PI) using an apoptosis detection kit (Sigma). The procedure consists of (i) the binding of FITC–Annexin V to phosphatidylserine which translocates from the interior to the exterior of the cell membranes at the start of the apoptotic process and (b) the binding of PI to DNA in cells where the membrane has been totally compromised (32). A549 cells were prepared as described above. Induction of apoptosis was performed for 1 h. Harvested cells were centrifuged for 5 min at 250 x g and after washing with PBS were re-suspended in binding buffer (10 mM HEPES/NaOH, pH 7.5 containing 140 mM NaCl and 2.5 mM CaCl2) at a concentration of 1 x 106 cells per millilitre. Five microlitres of 1:10 dilution of FITC–Annexin V conjugate (50 µg ml–1 in 50 mM Tris–HCl, pH 7.5, containing 100 mM NaCl) and 10 µl of PI solution (100 µg ml–1 in 10 mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl) were added to 500 µl of cell suspension and incubated in the dark at room temperature for 15 min. A suitable dilution of FITC–Annexin V conjugate was determined in preliminary experiments. The relative level of apoptotic cells was detected using a FACScan system (BD Biosciences) using CellQuest 3.3 software (33).

Detection of apoptotic body formation and chromatin condensation
Nuclei of the control cells, of cells treated with the inducers of apoptosis for 6 h and of cells exposed to Af conidia in the presence or absence of inducers, together with untreated control cells, were stained with DNA binding dye Hoechst 33342 and then analysed using a fluorescence microscope. The cover slips were washed with PBS-5% BSA and the cells fixed with PBS-4% (wt/vol) PFA (pH 7.4) for 1 h. Chromatin condensation and apoptotic body formation was assessed by staining with Hoechst 33342 (1.5 µM; Sigma) for 30 min at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield (Vector Laboratory). Samples were viewed with a Zeiss fluorescence microscope using x400 magnifications. For each sample, cells from five random fields were counted and the percentage of apoptotic cells was calculated as the number of apoptotic cells divided by the total number counted multiplied by 100.

TUNEL assay of DNA fragmentation
TUNEL technology, based on labelling of DNA strand breaks, was performed using an In Situ Cell Death Detection kit (Roche Molecular Biologicals) (34). After induction of apoptosis for 6 h at 37°C, cover slips were washed with PBS-5% BSA, the cells were fixed with PBS-4% (wt/vol) PFA (pH 7.4) for 1 h and then permeabilized in a solution containing 0.1% Triton X-100/0.5% sodium citrate for 30 min at room temperature. Cover slips were washed again and 50 µl of TUNEL reaction mixture was added for 1 h and incubated in a humidified dark chamber at 37°C. After detection of incorporated FITC by anti-FITC antibody, conjugated with alkaline phosphatase, substrate was added and stained cells were analysed by light microscopy.

Caspase analysis
In order to monitor the presence of both pro-caspase-3 and the active form of caspase-3, western blot analysis of the cell lysates was performed. Following the induction of apoptosis, cells exposed (or not) to Af conidia and untreated controls without conidia were simultaneously harvested by trypsinisation. After centrifugation at 200 x g, the cell pellets were re-suspended in 30 µl of buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.1% triton, 0.1% SDS and a cocktail of protease inhibitors: 2.5 mM orthovanadate, 10 mM paranitrophenylphosphate, 10 µg ml–1 leupeptine and 10 µg ml–1 aprotinin. The resulting lysates were centrifuged for 10 min at 15 000 x g at 4°C. Protein concentration was determined using a bicinchoninic acid-based protein assay (Interchim) and equal amounts of protein were mixed with 2x Laemmli sample buffer (62.5 mM Tris–HCL pH 6.8; 8.4% SDS, 5% mercaptoethanol, 8.5% glycerol, 0.25% bromophenol blue) and maintained at 100°C for 7 min before loading 20 µg of cell extract per lane onto 15% SDS-polyacrylamide gels (35). After electrophoresis, the separated proteins and molecular weight standards were electrophoretically transferred onto 0.2 µm Immuno-Blot PVDF membranes. The membranes were blocked with 10% skimmed milk in 0.1 M Tris buffer, 0.9% NaCl, 0.05% Tween 20, pH 7.2, and 3% normal goat serum (Sigma), incubated for 3 h with polyclonal rabbit antibody specific for human caspase-3 (Abcam, UK) diluted 1:250 in the blocking solution and after washing were incubated with goat anti-rabbit antibody (1:1000) coupled with peroxidase (Sigma) for 1 h. The membranes were then washed in PBS and incubated with enhanced chemoluminescence reagents (Amersham). The optical density of the bands, with the relative molecular weights 32 and 17 kDa corresponding to pro-caspase-3 and the active form of caspase-3, was estimated using the ImageQuant system. To compare the intensities of the bands in the different lines, the values of the bands were divided by the value of the band corresponding to pro-caspase-3 in the control untreated cells without conidia.

To confirm the involvement of caspase, different concentrations of the caspase inhibitor Z-VAD.FMK or FMK negative control ranging from 0.125 to 0.5 mM were added simultaneously with the inducer of apoptosis to the cells exposed to conidia. The percentage of apoptosis was assessed according to the nuclear morphology of the cells stained with 1.5 µM Hoechst 33342.

Statistical analysis
At least three different assays were performed per experiment. The differences in the number of apoptotic cells for TNF- and STS-induced apoptosis exposed or not to conidia were assessed by analysis of variance. P-values <0.05 were considered to be significant. Tukey's honestly significant difference test was applied for comparison of means between groups. Means followed by the same letter are not significantly different. The values are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Inhibition of apoptosis in A549 cells exposed to Af conidia
Flow cytometry analysis.
Flow cytometry of FITC–Annexin V-labelled cells stained with PI allowed an analysis of the early stages of apoptosis of the host cells. At the highest concentration, Af conidia inhibited TNF- and STS-induced apoptosis of A549 cells. The level of apoptotic cells, after 1 h incubation with TNF or STS was 14 ± 3% and 20 ± 1.5%, respectively: an example of one replicate is shown in Fig. 1(A). TNF treatment of cells and simultaneous exposure to Af conidia leads to a statistically significant (P < 0.05) dose-dependent decrease of the number of apoptotic cells from 14 ± 3% in the controls to 1.5 ± 2% with 108, to 6.7 ± 2% with 107 and to 9.8 ± 1% with 5 x 106 conidia (Fig. 1B). The equivalent effect in STS-treated cultures was from 20 ± 1.5% in controls to 7 ± 1.5%, 13 ± 1% and 17 ± 1.2%, respectively (P < 0.05). In an untreated control, 2.5 ± 0.5% apoptotic cells were found. No apoptosis was induced in the cultures following addition of 107 or 5 x 106 Af conidia. Spontaneous apoptosis was weakly increased to 4.5 ± 1% when cells were exposed to 108 conidia (P < 0.05) (Fig. 1B).



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  		Fig. 1. (A) An example of contour diagram of FITC–Annexin V/PI obtained by flow cytometry of A549 cells exposed to Af conidia and treated with the inducers of apoptosis is shown. FITC–Annexin V fluorescence is presented on the horizontal axis and PI fluorescence on the vertical axis. The lower left quadrant of the cytograms represents the viable cells (FITC–/PI–) and the upper right quadrant represents the necrotic cells (FITC+/PI+), while apoptotic cells were recorded in the lower right quadrant (FITC+/PI–) and dead cells in the upper left quadrant (FITC–/PI+). In each case, the fluorescence of 10 000 cells was assessed. Percentage of the early-stage apoptotic cells is marked in the right lower quadrant. Abbreviations: (A549), control untreated A549 cells; (A549 + STS), A549 cells treated with 1 µm of STS; (A549 + STS + 108 conidia), A549 cells treated with 1 µm of STS and exposed to 108 of conidia; (A549 + 108 conidia), A549 cells exposed to 108 of conidia; (A549 + TNF), A549 cells treated with 20 ng ml–1 of TNF-{alpha} and 2 µg of CXH; (A549 + TNF + 108 conidia), A549 cells treated with 20 ng ml–1 of TNF-{alpha}, 2 µg of CXH and exposed to 108 of conidia. (B) Inhibition of apoptosis is depending on the dose of the conidia. The percentage of apoptotic cells was calculated with data from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; –, absence of TNF, STS or conidia.

 
Detection of apoptotic body formation and chromatin condensation
The anti-apoptotic role of Af conidia was verified on advanced stages of apoptosis, when TNF- or STS-treated cells were simultaneously exposed to conidia for 6 h (see Fig. 2(A) as a representative example of the inhibition of late stages of apoptosis by conidia). Quantification of the differences in the number of apoptotic cells for TNF- and STS-induced apoptosis showed that 45 ± 5% of TNF-treated and 23 ± 4% of STS-treated cells displayed features of apoptosis, while CHX-treated cells (data not shown) or untreated control cells were unaffected by apoptosis (Fig. 2B). Exposure of A549 cells to Af conidia inhibited the apoptotic process: the number of apoptotic cells induced by both inducers was significantly decreased (P < 0.05) in the samples of cells treated with the inducers and exposed to Af conidia (Fig. 2B). Figure 2 shows that the anti-apoptotic effect of Af conidia was dose dependent. Exposure to 5 x 106, 107 and 108 conidia decreased the number of apoptotic cells from 45 ± 5% in control to 33 ± 3%, 25 ± 2% and 20 ± 2% for TNF-induced apoptosis and from 23 ± 4% in control to 16 ± 2%, 10 ± 2% and 7 ± 1.5% (P < 0.05) for STS-induced apoptosis, respectively. In cell cultures containing 5 x 108 conidia without inducers of apoptosis, 1.5% of the cells were apoptotic.



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  		Fig. 2. (A) Conidial inhibition of apoptotic body formation/chromatin condensation induced by 6 h of STS or TNF treatment. After 6 h of treatment with 5 x 106 conidia (the late stage of apoptosis), in the presence or absence of the inducers of apoptosis (1 µm STS or 20 ng ml–1 of TNF and 2 µg of CXH per millilitre), cells were fixed with PBS-4% PFA for 1 h. Chromatin condensation and apoptotic bodies' formation were assessed by staining with Hoechst 33342. Solid arrows indicate apoptotic bodies. The broken line arrows indicate Af conidia. One representative experiment is shown. (B) Inhibition of late stage of apoptosis also depends on the dose of the conidia. The percentage of cells with apoptotic bodies was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; –, absence of TNF, STS or conidia. Abbreviations are as in Fig. 1.

 
Detection of nuclear DNA nicks using TUNEL technology
The inhibitory effect of Af conidia on advanced stage of apoptosis was confirmed by TUNEL. After 6 h incubation, the cells treated either with TNF-{alpha} or STS have shown intense positive TUNEL staining in comparison with control cells or cells exposed only to Af conidia (Fig. 3). The number of apoptotic cells after TNF and STS treatment reached 53 ± 6% and 20 ± 4%, respectively. In contrast, control cells remained TUNEL negative, while only 1% of cells exposed to 107 conidia per millilitre were TUNEL positive. The TUNEL assay has also confirmed that exposure to conidia inhibited TNF- and STS-induced apoptosis in A549 cells. Exposure to 107 conidia per millilitre decreased the number of TUNEL-stained cells in TNF-treated culture from 53 ± 6% to 22 ± 4% (P < 0.05) and in STS-treated culture from 20 ± 4% to 5 ± 2% (P < 0.05).



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  		Fig. 3. Example of the inhibition of apoptotic cell death by Af conidia using TUNEL. The arrows indicate positive TUNEL staining. One representative experiment is shown. Same treatment as cells of Fig. 2; legend and abbreviations as in Fig. 1 caption.

 
Implication of caspase-3 in the inhibition of TNF-induced apoptosis by Af conidia
Since caspase-3 has been implicated as a general effector of apoptosis (36), we used western blot analysis to test whether or not caspase-3 activation took place in cells treated with the inducers of apoptosis and Af conidia. Anti-caspase-3 antibody detected a 32-kDa band corresponding to pro-caspase-3 in the control cell lysate (Fig. 4). After 6 h of TNF treatment, the 32-kDa band disappeared and a 17-kDa band corresponding to the active form of caspase-3 appeared. In TNF-treated cells exposed to Af conidia, the intensity of the 17-kDa band decreased, while the 32-kDa pro-caspase-3 was not detected. The effect was dose dependent: the ratio of the intensity of the 17-kDa band in control to that in TNF-treated cells was 0.80, which dropped to 0.75 and 0.30 for cultures treated with 107 and 108 conidia, respectively. The intensity of the 17-kDa band in lysate of the cells exposed to 5 x 106 conidia was equal to those in TNF-treated cells without conidia (data not shown). These results suggest that conidial inhibition of apoptosis in TNF-treated A549 cells was due to the degradation of either caspase-3 or pro-caspase-3. In contrast, in the lysates of cells treated with STS or cells treated with STS and 107 or 108 conidia, the 32-kDa band only was observed. The results showed that under our experimental conditions, STS-triggered apoptosis of A549 cells was caspase-3 independent.



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  		Fig. 4. Western blot analysis of pro-caspase-3 processing in TNF- and STS-treated A549 cells. Control A549 cells, cells exposed to Af conidia in the presence or absence of inducers of apoptosis (1 µm STS or 20 ng ml–1 of TNF and 2 µg of CXH per millilitre) and cells treated only with the inducers of apoptosis for 6 h (the late stage of apoptosis) were harvested and cell extracts were used for western blot analysis. Pro-caspase-3 and caspase-3 were detected by anti-caspase-3 antibody. At the left side, the molecular weight standards are shown. The arrows indicate active 17-kDa caspase-3. The values of the band intensities were divided by the value of the band corresponding to pro-caspase-3 in the control cells. The ratio is shown at the bottom of the figure. Abbreviations are as in Fig. 1.

 
Involvement of caspase during TNF-induced apoptosis was confirmed by the use of the caspase inhibitor Z-VAD.FMK. Exposure to Z-VAD.FMK did not affect STS-induced apoptosis (Fig. 5). Supplementation of conidia with 0.125 to 0.5 mM Z-VAD. FMK did not increase the inhibition of apoptosis induced by the conidia (Fig. 5). In contrast, Z-VAD. FMK inhibited TNF-induced apoptosis in a dose-dependent manner, while control cultures without the reagent showed no change in the number of apoptotic cells (Fig. 5). Furthermore, it was shown that the inhibitory effect of Z-VAD.FMK was additive to the effect induced by conidia, suggesting that the inhibitions by Z-VAD.FMK and conidia might both target caspase (Fig. 5).



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  		Fig. 5. Caspase inhibitor Z-VAD.FMK does not modulate STS-induced apoptosis (A) but increases conidial inhibition of TNF-induced apoptosis (B). A549 cells were seeded at 5 x 105 cells per well on cover slips and grown for 16 h at 37°C. Caspase inhibitor Z-VAD.FMK or FMK negative control ranging from 0.125 to 0.5 mM was added simultaneously with the inducer of apoptosis to the cells exposed or not to conidia. Nuclear morphology was assessed by staining with the nuclear dye Hoechst 33342. The percentage of the late-stage apoptotic cells was calculated from four experiments. Results are presented as means ± SEM. Means followed by the same letter are not significantly different. +, presence; –, absence of TNF, STS, conidia or inhibitor.

 
Inhibition of apoptosis in TNF- and STS-treated cells is induced specifically by Af conidia
As shown in Fig. 6, 5 x 106 Af conidia strongly inhibited TNF-induced apoptosis from 45 ± 5% to 27 ± 2% for STS-induced apoptosis and from 19 ± 3% to 11 ± 2% for STS-induced apoptosis. Cladosporium cladosporioides conidia, even at doses as high as 108, had no inhibitory effect. Percentage of apoptotic cells exposed to C. cladosporioides was 44 ± 2% versus 45 ± 5% in control cells for TNF-induced apoptosis and 18 ± 2% versus 19 ± 3% in control cells for STS-induced apoptosis, respectively (P > 0.05). Moreover, exposure to 2-µm latex beads or PFA-fixed conidia did not induce a significant reduction of apoptosis induced by STS or TNF (Fig. 6). These results support the conclusion that the inhibition of apoptosis is specific and depends on live conidia of Af.



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  		Fig. 6. Af conidia specifically inhibit TNF- and STS-induced apoptosis of A549 cells. A549 cells were treated with the inducers of apoptosis (1 µm STS or 20 ng ml–1 of TNF and 2 µg of CXH per millilitre) for 6 h in the presence or absence of 5 x 106 conidia of Af, Cladosporium cladosporioides, PFA-fixed conidia or latex beads. Apoptotic cells were assessed by staining with nuclear dye Hoechst 33342. The percentage of apoptotic cells was calculated from triplicates of four experiments. Results are presented as means ± SEM. Means followed by the same letter are not significantly different. +, presence; –, absence of TNF, STS or conidia.

 
Af conidia inhibit apoptosis in cells other than A549
Inhibition of apoptosis following exposure to (107) conidia also occurred in human tracheal epithelial 16HBE cells treated with TNF and STS, (Fig. 7) although the 16HBE cells were less sensitive to the apoptotic inducers than A549 cells: for example, for TNF and STS, 16HBE cells showed that 16% and 18% of cells, respectively, were apoptotic, the relative numbers were 4% and 6% after exposure to conidia. There were no signs of apoptosis in cultures exposed only to 107 conidia or in control cultures receiving no conidia.



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  		Fig. 7. Af conidia decreased the number of apoptotic cells in 16HBE cells. HBE cells were treated for 6 h with 5 x 106 conidia, in the presence or absence of the inducers of apoptosis. Chromatin condensation and apoptotic body formation were assessed by staining with Hoechst 33342. The number of apoptotic cells was compared with control and with cultures incubated either with the inducers or with conidia. The percentage of apoptotic cells was calculated on the base of the data from four experiments. Means followed by the same letter are not significantly different. +, presence; –, absence of TNF, STS or conidia.

 
After 6 h of incubation with TNF or STS, epithelial primary cells obtained from HNT displayed apoptotic features in 15 ± 3% of TNF-treated cells and 14 ± 4% of STS-treated cells while untreated control cells were unaffected by apoptosis (Fig. 8). The exposure of HNT cells to Af conidia inhibited TNF- or STS-induced apoptosis. Exposure to 107 conidia decreased the number of apoptotic cells from 15 ± 3% to 4 ± 3% (P < 0.05) for TNF-induced apoptosis and from 14 ± 4% to 3 ± 1.5% (P < 0.05) for STS-induced apoptosis (Fig. 8). There were no signs of apoptosis in cell cultures containing 107 conidia without inducers of apoptosis or in control cultures receiving no conidia. Exposure to PFA-fixed conidia did not reduce the number of apoptotic cells significantly.



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  		Fig. 8. Inhibition of apoptosis in airway primary culture cells exposed to Af conidia. Primary epithelial cells obtained from the HNT were seeded at 5 x 106 cells per well on cover slips and grown for 48 h at 37°C. Induction of apoptosis with STS and TNF and exposure to 5 x 106 conidia have been performed as described above. (A) Analysis of nuclear morphology. The arrows indicated apoptotic bodies. (B) Quantification of the apoptotic inhibition. Means followed by the same letter are not significantly different. +, presence; –, absence of either TNF, STS or conidia.

 
Conidia also inhibited the apoptosis of A549 cells induced by their co-culture with LPS-activated HAM (as the source of TNF for apoptosis induction). In the co-culture growth conditions, the amount of apoptotic cells was low (6 ± 1%). After exposure to Af conidia, the number of apoptotic cells was significantly reduced (to 2 ± 1.5%) (P < 0.05) (Fig. 9).



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  		Fig. 9. Af conidia inhibit apoptosis induced by co-culture of HAM with A549 cells. HAM were stimulated with 1 µg ml–1 of LPS and grown for 18 h at 37°C. Subsequently the cover slips containing A549 cells were transferred into the wells with HAM and exposed or not to 5 x 106 Af conidia. The number of A549 apoptotic cells assessed by staining with dye Hoechst 33342 was compared in co-culture containing either control HAM or non-stimulated HAM incubated with conidia. Means followed by the same letter are not significantly different. +: presence; –: absence of either LPS or conidia.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The influence of Af conidia on host cell apoptosis has been investigated in vitro using epithelial cells since they are the likely primary target of inhaled Af conidia. We examined the effect of Af conidia on apoptosis of A549 cells treated with TNF-{alpha}. TNF-{alpha} was selected as an inducer of apoptosis since its role during Af infection has been well established: the release of TNF-{alpha} has been demonstrated ex vivo, using whole blood from healthy volunteers, stimulated by non-viable conidia and hyphal fragments from Af (23). Furthermore, it has been shown that TNF-{alpha} is produced in the lungs of normal and immunocompromised mice (2 and 6 ng ml–1, respectively) in animals responding to the intra-tracheal administration of Af conidia at concentrations that are compatible with those used in this study (37, 38). Resident alveolar macrophages are responsible for the early release of TNF-{alpha}, whereas the later release and higher levels of this cytokine are produced by recruited neutrophils (37). Our study has shown for the first time that Af conidia do not promote apoptosis of epithelial cells but in contrast are able to inhibit early, as well as late stages of drug-induced apoptosis. The biological significance of this finding is emphasized by the inhibition of apoptosis of A549 cells co-incubated with human-activated macrophages or human respiratory epithelial primary culture cells. Alteration of apoptosis could play a pivotal role in the first step of the interaction between Af and the host since it would allow intracellular conidium survival with subsequent germination and host invasion.

It has been reported that many pathogenic agents interact at check(nodal)-points in the cascade of events leading to apoptosis (39). For example, baculovirus contains an anti-apoptotic factor that inhibits caspases (40); EBV (41) and herpes virus (42) encode proteins that are homologues of the cellular anti-apoptotic protein Bcl-2. Thus, in the case of Af conidia a strong inhibition of induced apoptosis yet also show a weak augmentation of spontaneous apoptosis.

The complexity of the apoptotic process is illustrated in our study where Af conidia are shown to have an inhibitory effect and the response to Af is morphotype specific. Such findings were repeatedly observed in immunological reactions to conidia and hyphae of Af (43, 44). In the case of apoptosis, the various fungal forms elicit a different response: conidia inhibit apoptosis whereas hyphae secrete gliotoxin, a well known inducer of apoptosis in many cell types, including macrophages and monocytes (12, 45–47).

Processing of pro-caspase-3 was observed in TNF-induced apoptotic cells, resulting in the complete loss of an inactive 32-kDa pro-caspase-3 and the appearance of an active 17-kDa form. We have shown that the level of active caspase-3 as well as pro-caspase-3 was inhibited by the exposure of TNF-treated cells to Af conidia. We do not know whether the inhibition of TNF-induced apoptosis by Af conidia takes place upstream or downstream of caspase-3 activation. A variety of pathogens can inhibit caspase-3, for example, the anti-apoptotic effect of cytomegalovirus infection was shown to be associated with a significant reduction of caspase-3 activity (48), and the intracellular pathogen Leishmania major was found to inhibit the spontaneous apoptosis of neutrophils via a mechanism involving the inhibition of caspase-3 activation (49). Another intracellular pathogen, Chlamydophila pneumoniae, inhibits apoptosis induced by treatment with drugs or by death receptor ligation. The observed inhibition was associated with a lack of caspase-3 activation in infected cells: the protective effect was confined to the cells inhabited by Chlamydophila (25). It has been reported that herpes simplex virus (HSV-1) interferes with apoptotic processes in infected cells: HSV-1 was able to prevent apoptosis which was induced by various stimuli, including treatment with TNF-{alpha} and STS. A further parallel with the situation seen after Af conidia treatment is that wild-type HSV-1 infection itself could induce low-level apoptosis in cells (50, 29). It was concluded that HSV-1 acted at multiple metabolic checkpoints.

The other apoptosis inducer used was STS. If conidia similarly inhibit STS- and TNF-induced apoptosis, the mechanisms of induction and inhibition are clearly different for these two drugs. TNF-dependent apoptosis is induced through the caspase pathway which is blocked by Z-VAD.FMK which in contrast has no effect on STS-induced apoptosis and pro-caspase-3 remains uncleaved. These observations indicate that STS-induced apoptosis in A549 cells does not require caspase activity. The lack of processing of pro-caspase-3 in cells exposed to STS was also reported for HeLa H21 cells (30). While HeLa H21 cells were efficiently killed after TNF or STS treatment, extensive processing of caspase-3 was only observed after TNF treatment. Recently, it was shown that STS-induced apoptosis of some cancer cells, including A549 cells, is caspase independent (51). These results support the view that various apoptotic stimuli may activate different steps of apoptosis. At this stage a role for other proteolytic caspases cannot be excluded.

Using three different convergent methods, we have shown that Af conidia inhibited apoptosis induced by two divergent pro-apoptotic stimuli, TNF and STS. The nature and mode of action of the conidial factors that block apoptosis are unknown: conidial molecules could be present in the airborne conidia and released upon contact with the cell or synthesized de novo in the presence of the epithelial cells; two or several molecules may inhibit the caspase-dependent and caspase-independent apoptotic pathways or both pathways could be inhibited by the same molecule: at this stage, we are unable to exclude dependence on the presence of conidia in early stages of germination, not distinguishable by standard light microscopy, releasing active products. Further experiments are required to fully understand the role of conidia in the inhibition of apoptosis.


   Acknowledgements
 
This work was supported in part by a grant from The Institut National de la Recherche Agronomique (Transversalité INRA 2000 ‘Mycotoxines’ AIP INRA A-263) and by a Fellowship (S.L-F.) from The University Paris XII—Val de Marne, Paris, France. We thank David Dresser (School of Biological Sciences, University of Edinburgh, UK) and Svirshchevskaya Elena (Institute of Bioorganic Chemistry, Russia) for significant discussions and critical reading of the manuscript, Jacqueline Sarfati (Unité des Aspergillus, Institut Pasteur, Paris, France) for technical assistance and Emmanuelle Boisieux-Ulrich (Université Paris 7) for the helpful suggestions in some experiments. We are also thankful to the reviewers for their suggestions.


   Abbreviations
 
Af   Aspergillus fumigatus
CHX   cycloheximide
DISC   death-inducing signalling complex
HAM   human alveolar macrophages
HNT   human nasal turbinates
HSV-1   herpes simplex virus
PI   propidium iodide
STS   staurosporine
TNF   tumour necrosis factor
TUNEL   terminal deoxynucleotidyl transferase-mediated fluorescein–dUTP nick end labelling

   Notes
 
* These authors made an equal contribution to this work. Back

Transmitting editor: A. Cooke

Received 7 December 2004, accepted 17 October 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781.[Web of Science][Medline]
  2. Botterel, F., Cordonnier, C., Barbier, V. et al. 2002. Aspergillus fumigatus causes in vitro electrophysiological and morphological modifications in human nasal epithelial cells. Histopathology 17:1095.
  3. Paris, S., Boisvieux-Ulrich, E., Crestani, B. et al. 1997. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells. Infect. Immun. 65:1510.[Abstract]
  4. Wasylnka, J. A. and Moore, M. M. 2003. Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells. J. Cell Sci. 15:579.
  5. Watson, R. W., Redmond. H. P., Wang, J. H., Condron, C. and Bouchier-Hayes, D. 1996. Neutrophils undergo apoptosis following ingestion of Escherichia coli. J. Immunol. 156:3986.
  6. Rotstein, D., Parodo, J., Taneja, R. and Marshall, J. C. 2000. Phagocytosis of Candida albicans induces apoptosis of human neutrophils. Shock 14:278.[Web of Science][Medline]
  7. Goldman, D. L., Casadevall, A., Cho, Y. and Lee, S. C. 1996. Cryptococcus neoformans meningitis in the rat. Lab. Invesigt. 75:759.
  8. Chiapello, L. S., Aoki, M. P., Rubinstein, H. R. and Masih, D. T. 2003. Apoptosis induction by glucuronoxylomannan of Cryptococcus neoformans. Med. Mycol. 41:347.[CrossRef][Web of Science][Medline]
  9. Heussler, V. T., Machado, J., Jr, Fernandez, P. C. et al. 1999. The intracellular parasite Theileria parva protects infected T cells from apoptosis. Proc. Natl Acad. Sci. USA 96:7312.[Abstract/Free Full Text]
  10. Nash, P. B., Purner, M. B., Leon, R. P., Clarke, P., Duke, R. C. and Curiel, T. J. 1998. Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis. J. Immunol. 160:1824.[Abstract/Free Full Text]
  11. Medeiros, A. I., Bonato, V. L., Malheiro, A., Dias, A. R., Faccioli, L. H. and Silva, C. L. 2002. Histoplasma capsulatum inhibits apoptosis and Mac-1 expression in leucocytes. Scand. J. Immunol. 56:392.[CrossRef][Web of Science][Medline]
  12. Stanzani, M., Orciuolo, E., Lewis, R. et al. 2004. Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes. Blood 16
  13. Wyllie, A. H., Kerr, J. F. and Currie, A. R. 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68:251.[Medline]
  14. Thornberry, N. A. and Lazebnik, Y. 1998. Caspases: enemies within. Science 281:1312.[Abstract/Free Full Text]
  15. Ashkenazi, A. and Dixit, V. 1998. Death receptors: signaling and modulation. Science 281:1305.[Abstract/Free Full Text]
  16. Kroemer, G., Zamzami, N. and Susin, S. A. 1997. Mitochondrial control of apoptosis. Immunol. Today 18:44.[CrossRef][Web of Science][Medline]
  17. Kischkel, F. C, Hellbardt, S., Behrmann, I. et al. 1995. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14:5579.[Web of Science][Medline]
  18. Scaffidi, C., Fulda, S., Srinivasan, A. et al. 1998. Two CD95 (Apo-1/Fas) signaling pathways. EMBO J. 17:1675.[CrossRef][Web of Science][Medline]
  19. Sun, X. M., MacFarlane, M., Zhuang, J., Wolf, B. B., Green, D. R. and Cohen, G.M. 1999. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem. 274:5053.[Abstract/Free Full Text]
  20. Stennicke, H. R., Jurgensmeier, J. M., Shin, H. et al. 1998. Pro-caspase-3 is a major physiologic target of caspase-8. J. Biol. Chem. 273:27084.[Abstract/Free Full Text]
  21. Susin, S. A., Daugas, E., Ravagnan, L. et al. 2000. Two distinct pathways leading to nuclear apoptosis. J. Exp. Med. 192:571.[Abstract/Free Full Text]
  22. Chose, O., Sarde, C. O., Gerbod, D., Viscogliosi, E. and Roseto, A. 2003. Programmed cell death in parasitic protozoans that lack mitochondria. Trends Parasitol. 19:559.[CrossRef][Web of Science][Medline]
  23. Warris, A., Netea, M. G., Wang, J. E. et al. 2003. Cytokine release in healthy donors and patients with chronic granulomatous disease upon stimulation with Aspergillus fumigatus. Scand. J. Infect. Dis. 35:482.[CrossRef][Web of Science][Medline]
  24. Higuchi, M., Singh, S. and Aggarwal, B. 1995. Characterization of the apoptotic effect of human tumor necrosis factor: development of highly rapid and specific bioassay for human tumor necrosis factor and lymphotoxin using human target cells. J. Immunol. Methods 178:173.[CrossRef][Web of Science][Medline]
  25. Rajalingam, K., Al-Younes, H., Muller, A., Meyer, T. F., Szczepek, A. J. and Rudel, T. 2001. Epithelial cells infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) are resistant to apoptosis. Infect. Immun. 69:7880.[Abstract/Free Full Text]
  26. Galvan, V. and Roizman, B. 1998. Herpes simplex virus 1 induces and blocks apoptosis at multiple steps during infection and protects cells from exogenous inducers in a cell-type-dependent manner. Proc. Natl Acad. Sci. USA 95:3931.[Abstract/Free Full Text]
  27. Lieber, M., Smith, B., Szakal, A., Nelson-Rees, W. and Todaro, G. 1976. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17:62.[Web of Science][Medline]
  28. Gruenert, D. C., Basbaum, C. B., Welsh, M. J., Li, M., Finkbeiner, W. E. and Nadel, J. A. 1988. Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40. Proc. Natl Acad. Sci.USA 85:5951.[Abstract/Free Full Text]
  29. Booth, B. W., Adler, K. B., Bonner, J. C., Tournier, F. and Martin, L. D. 2001. Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha. Am. J. Respir. Cell Mol. Biol. 25:739[Abstract/Free Full Text]
  30. Janicke, R. U., Sprengart, M. L., Wati, M. R. and Porter, A. G. 1998. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem. 273:9357.[Abstract/Free Full Text]
  31. Wasylnka Julie, A. and Moore Margo, M. 2002. Uptake of Aspergillus fumigatus conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein. Infect. Immun. 70:3156.[Abstract/Free Full Text]
  32. Vermes, I., Haanen, C., Steffens-Nakken, H. and Reutelingsperger, C. 1995. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184:39.[CrossRef][Web of Science][Medline]
  33. Koopman, G., Reutelingsperger, C. P., Kuijten, G. A., Keehnen, R. M., Pals, S. T. and van Oers, M. H. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415.[Abstract/Free Full Text]
  34. Gorczyca, W., Gong, J. and Darzynkiewicz, Z. 1993. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 53:1945.[Abstract/Free Full Text]
  35. Laemmli, U. K. 1970. Cleavage of structural proteins of the head of the bacteriophage T4. Nature 277:680.
  36. Jacobson, M. D., Weil, M. and Raff, M. C. 1996. Role of Ced-3/ICE-family proteases in STS-induced programmed cell death. J. Cell Biol. 133:1041.[Abstract]
  37. Mehrad, B., Strieter, R. M. and Standiford, T. J. 1999. Role of TNF-{alpha} in pulmonary host defense in murine invasive aspergillosis. J. Immunol. 162:1633.[Abstract/Free Full Text]
  38. Taramelli, D., Malabarba, M. G., Sala, G., Basilico, N. and Cocuzza, G. 1996. Production of cytokines by alveolar and peritoneal macrophages stimulated by Aspergillus fumigatus conidia or hyphae. J. Med. Vet. Mycol. 34:49.[Web of Science][Medline]
  39. Muller, A. and Rudel, T. 2001. Modification of host cell apoptosis by viral and bacterial pathogens. Int. J. Med. Microbiol. 291:197.[CrossRef][Web of Science][Medline]
  40. Crook, N. E., Clem, R. J. and Miller, L. K. 1993. An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J. Virol. 67:2168.[Abstract/Free Full Text]
  41. Henderson, S., Huen, D., Rowe, M., Dawson, C., Johnson, G. and Rickinson, A. 1993. Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc. Natl Acad. Sci. USA 90:8479.[Abstract/Free Full Text]
  42. Derfuss, T., Fickenscher, H., Kraft, M. S. et al. 1998. Antiapoptotic activity of the herpesvirus saimiri-encoded Bcl-2 homolog: stabilization of mitochondria and inhibition of caspase-3 like activity. J. Virol. 72:5897.[Abstract/Free Full Text]
  43. Bellocchio, S., Moretti, S., Perruccio, K. et al. 2004. TLRs govern neutrophil activity in aspergillosis. J. Immunol. 173:7406.[Abstract/Free Full Text]
  44. Bozza, S., Perruccio, K., Montagnoli, C. et al. 2003. A dendritic cell vaccine against invasive aspergillosis in allogeneic hematopoietic transplantation. Blood 102:3807.[Abstract/Free Full Text]
  45. Tomee, J. F. and Kauffman, H. F. 2000. Putative virulence factors of Aspergillus fumigatus. Clin. Exp. Allergy 30:476.[CrossRef][Web of Science][Medline]
  46. Waring, P., Eichner, R. D., Mullbacher, A. and Sjaarda, A. 1988. Gliotoxin induced apoptosis in macrophages unrelated to its antiphagocytic properties. J. Biol. Chem. 263:18493.[Abstract/Free Full Text]
  47. Kweon, Y. O., Paik, Y. H., Schnabl, B., Qian, T., Lamasters, J. J. and Brenner, D. A. 2003 Gliotoxin-mediated apoptosis of activated human hepatic stellate cells. J. Hepatol. 39:38.[Medline]
  48. Schroeder, B., Christensen, M. R. and Worthen, G. S. 2002. Human cytomegalovirus protects endothelial cells from apoptosis induced by growth factor withdrawal. J. Clin. Virol. 25(Suppl. 2):149.[CrossRef]
  49. Aga, E., Katschinski, D. M., van Zandbergen, G. et al. 2002. Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major. J. Immunol. 169:898.[Abstract/Free Full Text]
  50. Aubert, M., O'Toole, J. and Blaho, J. A. 1999. Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1. J. Virol. 73:10359.[Abstract/Free Full Text]
  51. Cummings, B. S., Kinsey, G. R., Bolchoz, L. J. and Schnellmann, R. G. 2004. Identification of caspase-independent apoptosis in epithelial and cancer cells. J. Pharmacol. Exp. Ther. 310:126.[Abstract/Free Full Text]

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