TNF-{alpha} renders human peritoneal mesothelial cells sensitive to anti-Fas antibody-induced apoptosis

Jinn-Yang Chen1,4, Chin-Wen Chi2, Hui-Ling Chen5, Chiung-Pei Wan1, Wu-Chang Yang1 and An-Hang Yang3,4

1 Division of Nephrology, Department of Medicine, 2 Department of Medical Research and Education, 3 Division of Ultrastructural and Molecular Pathology, Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, 4 Institute of Clinical Medicine, School of Medicine, National Yang-Ming University and 5 Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan

Correspondence and offprint requests to: Dr An-Hang Yang, Division of Ultrastructural and Molecular Pathology, Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei, Taiwan 112. Email: jychen{at}vghtpe.gov.tw



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Fas-mediated apoptosis is important in the regulation of immune response. Human peritoneal mesothelial cells (HPMCs) are able to regulate peritoneal inflammation, but the role of Fas in HPMCs is not clear. This study addresses the mechanisms of Fas-mediated apoptosis in HPMCs.

Methods. Tumour necrosis factor-{alpha} (TNF-{alpha}) primed HPMCs were stimulated with agonistic anti-Fas antibody. The expression of Fas was evaluated by real-time reverse transcription polymerase chain reaction (TaqMan quantitative polymerase chain reaction) and flow cytometry. Apoptosis was assessed by nuclear morphology, TUNEL assay, fractional DNA content and cytokeratin 18 cleavage. Caspase activation and bcl-2 expression were analysed by western blotting. The phagocytosis of apoptotic HPMCs was demonstrated by immunofluorescence and transmission electron microscopy.

Results. Cultured HPMCs constitutively expressed Fas, and the Fas expression was upregulated by TNF-{alpha}. TNF-{alpha} primed HPMCs underwent apoptosis after anti-Fas antibody treatment, and the apoptotic HPMCs could be phagocytosed by macrophages. TNF-{alpha} was able to downregulate bcl-2 expression. Activation of caspase-3 and caspase-8 was noted during the apoptotic process. The inhibitors of either caspase-3 or caspase-8 could prevent the Fas-induced apoptosis in HPMCs. We also detected increased HPMC apoptosis in dialysate effluent during the recovery phase of peritonitis in peritoneal dialysis patients.

Conclusions. TNF-{alpha} directs HPMCs to commit apoptosis via the Fas/Fas ligand pathway through a modulation of Fas and bcl-2. Our study shows that HPMCs undergo apoptosis during peritonitis and suggests that the apoptosis of HPMCs may be related to the resolution of peritoneal inflammation.

Keywords: apoptosis; caspase; Fas; mesothelial cells; tumour necrosis factor-{alpha}



   Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apoptosis is a critical process important for tissue remodelling, the maintenance of immune homeostasis and the resolution of inflammation [1]. Recent data showed that the removal of apoptotic cells by phagocytes might suppress inflammation, modulate macrophage function and regulate immune responses [2,3].

The Fas/APO-1 (CD95) antigen belongs to the tumour necrosis factor/nerve growth factor receptor family [4]. Upon activation by natural Fas ligand (FasL) or agonistic anti-Fas antibody, Fas antigens are trimerized, transducing the signal to Fas-associating protein with death domain (FADD) [5]. FADD then activates the caspase cascade: first caspase-8 followed by caspase-3, caspase-6 and caspase-7 [5]. Fas receptor is constitutively expressed in cells of many human tissues [6], and the Fas/FasL system is implicated in the control of immune response [6].

In addition to their function as a barrier, human peritoneal mesothelial cells (HPMCs) play an important role in peritoneal inflammatory response. In response to bacterial products and macrophage-derived cytokines, HPMCs produce interleukin-1 (IL-1), IL-6, IL-8, monocyte chemoattractant protein-1 and RANTES, thus amplifying the inflammatory signals and recruiting leukocytes into the peritoneal cavity [7,8].

Although peritoneal dialysate was reported to induce apoptosis of HPMCs in vitro [9], whether apoptosis plays a role in regulating HPMC mass in vivo is not clear, and information pertaining to the function of the Fas/FasL system in the peritoneal cavity is limited. It remains unknown whether HPMCs express Fas and undergo apoptosis through the Fas/FasL pathway. It is our hypothesis that cytokines upregulate Fas expression in HPMCs and that anti-Fas antibody induces apoptosis in HPMCs.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mesothelial cell culture
HPMCs were obtained from human omentum as described previously with modification [10]. Omentum collected from consenting non-uraemic patients undergoing elective abdominal surgery was incubated in 0.05% (w/v) trypsin and 0.01% (w/v) EDTA for 20 min at 37°C. The harvested mesothelial cells were centrifuged at 150 g for 5 min and then resuspended in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 (DMEM/F-12, Sigma, St Louis, MO, USA) containing 10% fetal calf serum (FCS, Life Technologies, Grand Island, NY, USA). Media were supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM -glutamine. Cells were seeded into 25 cm2 plastic flasks (Nalge Nunc International, Naperville, IL, USA) and cultured in 5% CO2 at 37°C. HPMCs were characterized by positive immunofluorescence stain for cytokeratin and vimentin. These cells did not express factor VIII, desmin and UEA-I. Transmission electromicroscopy also showed the presence of numerous surface microvilli, pinocytotic vesicles and abundant cytoplasmic microfilaments. All experiments were conducted with cells from the second or third passage, and each experiment was repeated at least three times.

After reaching near-confluence, HPMCs were incubated with cytokines [tumour necrosis factor-{alpha} (TNF-{alpha}) and/or interferon-{gamma} (IFN-{gamma}), R & D Systems, Minneapolis, MN, USA] or control medium (DMEM/F-12 containing 0.1% FCS) for 48 h and then treated with antibody.

Assessment of Fas expression
Flow cytometric analysis. Near-confluent HPMCs were cultured for 72 h in the presence of cytokines or control medium. HPMCs were detached with 0.5 mM EDTA, washed with phosphate-buffered saline (PBS) and pelleted by centrifugation at 300 g for 5 min. Single cell suspensions were obtained by filtering through a nylon mesh. HPMCs were incubated for 30 min with a fluorescein isothiocyanate (FITC)-conjugated anti-Fas antibody (clone LOB 3/17, Serotec, Kidlington, UK) or FITC-conjugated mouse IgG1 isotype control (clone W3/25, Serotec) at room temperature and analysed by flow cytometry (FACSCalibur, Beckton Dickinson, San Jose, CA, USA). Cell debris was excluded from analysis by selective gating based on forward and right-angled scatter. A total of 10 000 events were collected for each sample, and the data were displayed on a logarithmic scale of increasing green-fluorescence intensity. Mean cell fluorescence was calculated by using CELLQuest software (Beckton Dickinson), and the mean cell fluorescence acquired with the control IgG1 was subtracted from that obtained with the anti-Fas antibody.

Real-time RT–PCR analysis. Total RNA was extracted using a commercial kit RNAzol B (Tel-Test, Friendswood, TX, USA). First-strand cDNA was synthesized from 5 µg of total RNA with Superscript II RNase H Reverse Transcriptase (Life Technologies) using Oligo(dT) as primers.

The mRNA level of Fas was quantified by real-time reverse transcription polymerase chain reaction (RT–PCR) on the ABI Prism 7000 Sequence Detection System software version 1.0 (PE Applied Biosystems, Foster City, CA, USA). PCR was performed using TaqMan Pre-Developed Assay Reagents (TaqMan PDARs, PE Applied Biosystems) according to the manufacturer’s instruction. The principle of real-time RT–PCR has been described in detail elsewhere [11]. Briefly, a sequence specific probe was labelled with a 5'-reporter dye (6-carboxy-fluorescin, FAM) and a 3'-non-fluorescent quencher (Minor Groove Binder, MGB). When the probe is intact, reporter dye emission is quenched. However, during the extension phase of the PCR, the nuclease activity of the Taq DNA polymerase cleaves the hybridized probe, and, due to the separation of reporter and quencher dye, a fluorescence signal is released and monitored by the sequence detector. A computer algorithm normalizes the signal to an internal reference dye and calculates the threshold cycle number (Ct). The amount of Fas expression was normalized by endogenous control (ß-actin) and calculated by {triangleup}{triangleup}Ct Method according to the manufacturer’s instruction (PE Applied Biosystems). Quantitative PCR was performed using the TaqMan Universal PCR Master Mix (PE Applied Biosystems), cDNAs corresponding to 100 ng of total RNA, and 200 nM probe and 900 nM primers were mixed in a 25 µl final reaction mix (one PCR cycle 50°C for 2 min and 95°C for 10 min; 50 PCR cycles 60°C for 1 min and 95°C for 15 s).

Western blot analysis of bcl-2 and caspases
After treatment, HPMCs were washed three times with PBS and lysed by two 10 s sonications with lysis buffer (25 mM Tris, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM phenylmethylsulfonylfluoride, 4 mM EDTA, 4 mM EGTA and 1% Triton X-100). The lysates were spun at 12 500 g for 15 min, and the protein concentration was measured with Bradford’s method. Twenty-five micrograms of total protein was separated on 13% SDS–polyacrylamide gel and transferred to polyvinylidene difluoride membrane. Membranes were blocked overnight at 4°C with 5% non-fat dry milk in PBS containing 0.1% Tween-20 and probed with monoclonal mouse antibodies against bcl-2 (Transduction Laboratories, Lexington, KY, USA), caspase-3 (Imgenex, San Diego, CA, USA) or caspase-8 (Upstate Biotechnology, Lake Placid, NY, USA). The membranes were subsequently incubated for 1 h with peroxidase-conjugated goat anti-mouse IgG (Dako A/S, Glostrup, Denmark), visualized by enhanced chemiluminescence (Supersignal, Pierce, Rockford, IL, USA) and recorded on Kodak BioMAX film. Membranes were then stripped and re-probed with monoclonal anti-{alpha}-tubulin antibody as an internal control (Amersham Pharmacia Biotech, Little Chalfont, UK). Quantitation of bcl-2, caspase-3 and caspase-8 protein bands was performed by densitometry using an AlphaImager 2000 system (Alpha Innotech, San Leandro, CA, USA).

Assessment of cell death
Histologic study. Near-confluent HPMCs grown on chamber slide (lab-Tek II, Nalge Nunc International) were first primed with cytokine for 48 h and then treated with agonistic anti-Fas IgM antibody (clone CH11, Upstate Biotechnology) or control IgM (Dako A/S) for 24 h. For nucleus morphology observation, cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min and incubated in propidium iodide (PI, 1 µg/ml) and RNase A (100 µg/ml) for 30 min at 37°C.

To detect DNA fragmentation in HPMCs, cells were washed with PBS containing 0.9% bovine serum albumin (BSA) and fixed with 4% paraformaldehyde for 30 min at 4°C. Subsequently, cells were washed and subjected to incubation in a TUNEL reaction mixture containing fluorescein-dUTP and TdT (In situ Cell Death Detection kit, fluorescein, Roche Molecular Biochemicals, Mannheim, Germany). Control cells were incubated in a TUNEL reaction mixture containing fluorescein-dUTP but not TdT.

To assess cytoskeletal change, HPMCs were fixed in methanol at –20°C for 30 min and washed twice with PBS containing 1% BSA and 0.1% Tween-20. Cells then underwent a 60 min incubation at room temperature with a fluorescein-conjugated M30 CytoDeath antibody that recognizes a neo-epitope on cleaved CK 18 (Roche Molecular Biochemicals).

Flow cytometric analysis. Near-confluent HPMCs were incubated with cytokines for 48 h before incubation with an agonistic anti-Fas IgM antibody (clone CH11) or a non-specific IgM antibody. After a 24 h exposure, attached cells were harvested by trypsin-EDTA treatment, mixed with the detached cells, fixed with 70% ethanol at –20°C for 30 min and extracted with 0.2 M phosphate-citrate buffer at pH 7.8 for 30 min. Cells were resuspended for 30 min in 10 µg/ml PI and 200 µg/ml RNase A and then analysed by flow cytometry. The proportion of apoptotic cell death was determined by counting cells with hypodiploid DNA content.

To evaluate DNA fragmentation and cytoskeletal changes, the adhered and the detached cells were combined and then treated as described above. Cellular DNA was counterstained using PI (5 µg/ml).

Phagocytosis assay. Human macrophages were isolated from peripheral blood monocytes of normal donors as described previously [12] and cultured on chamber slide and Transwell (Nalge Nunc International) for 7 days. Monolayers of HPMCs were treated with cytokines and anti-Fas antibody as described above and then detached with trypsinization. The harvested HPMCs were incubated with adhered macrophages for 1 h in medium containing 15% human serum. The interaction was terminated by ice-cold PBS washing. Macrophages were labelled with anti-CD68 (Dako A/S), and the apoptotic HPMCs were stained with M30. For electron microscopy, cells were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide and then dehydrated in increasing concentrations of ethanol. The resulting thin sections were viewed under an electron microscope.

Caspase inhibition. In some experiments, TNF-{alpha} primed HPMCs were incubated with Z-D(OMe)-E(OMe)-V-D(OMe)-FMK (Z-DEVD-FMK) and Z-I-E(OMe)-T-D(OMe)-FMK (Z-IETD-FMK) (R & D Systems) for 2 h before the addition of agonistic anti-Fas or control IgM antibody. Z-DEVD-FMK and Z-IETD-FMK are cell-permeable peptides and irreversibly inhibit caspase-3 and caspase-8, respectively.

Detection of apoptotic HPMCs in dialysate effluent. Cells were collected from overnight (8 h) peritoneal dialysate effluent (PDE, glucose 1.36%) on days 5, 15 and 28 after the peritonitis episode from 15 continuous ambulatory peritoneal dialysis (CAPD) patients. Non-infective PDE (glucose 1.36%) was collected from 20 CAPD patients. The time on CAPD, previous peritonitis incidence and total mesothelial cell mass measured by CA 125 of the peritonitis group were similar to those of the control group. Cells were fixed with ice-cold methanol, stained with M30 and then analysed by flow cytometry.

Statistical analyses
Data were analysed by two-way ANOVA followed by the Bonferroni multiple comparison procedure. Differences between histograms on flow cytometry analysis were assessed by Kolmogorov–Smirnov statistics. Differences were considered significant when P < 0.05.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fas was constitutively expressed in HPMCs and its expression was upregulated by TNF-{alpha}
Constitutive expression of Fas in cultured HPMCs was shown by flow cytometry (Figure 1) and real-time RT–PCR analysis. The mean cell fluorescence increased by 55.1% after a 72 h incubation with TNF-{alpha} (5 ng/ml) (P < 0.001). Quantitative RT–PCR showed that Fas mRNA increased by 4.6-fold after stimulating with TNF-{alpha} (5 ng/ml) for 48 h. IFN-{gamma} (0.5, 5 and 50 ng/ml) did not increase the expression of Fas in HPMCs. Both flow cytometry and quantitative RT–PCR showed that stimulation with a combination of TNF-{alpha} (5 ng/ml) and IFN-{gamma} (5 ng/ml) did not have a synergistic effect on Fas expression.



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Fig. 1. Flow cytometric analysis of surface Fas expression in HPMCs. HPMCs incubated in TNF-{alpha} or control medium were stained with anti-Fas (clone LOB 3/17 A) or isotype control. Dotted line, isotype control; empty histogram, un-primed HPMCs; solid histogram, HPMCs treated with TNF-{alpha} (5 ng/ml) for 48 h. Data are from a representative experiment.

 
Anti-Fas antibody-induced apoptosis in TNF-{alpha} primed HPMCs
Un-primed HPMCs were resistant to anti-Fas antibody (clone CH11)-induced cytotoxicity (Figure 2A). TNF-{alpha} primed HPMCs treated with anti-Fas (50 ng/ml) demonstrated bright, small and condensed nuclei (Figure 2B). Flow cytometric analysis showed a hypodiploid DNA peak when TNF-{alpha} primed HPMCs were treated with anti-Fas (see Figure 4B). IFN-{gamma} primed HPMCs did not show cytotoxicity after anti-Fas antibody treatment (data not shown).



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Fig. 2. Immunofluorescence photomicrographs of HPMCs. PI staining of HPMCs is illustrated after incubated in (A) control medium and (B) TNF-{alpha} (5 ng/ml) + anti-Fas (clone CH11, 50 ng/ml). (C) TNF-{alpha} primed HPMCs stimulated with anti-Fas showed positive TUNEL staining. (D) Staining of un-primed HPMCs with anti-pan-cytokeratin (clone C-11, Sigma) showed a normal distributed pattern of cytokeratin. Magnification: x270. (E) TNF-{alpha} primed HPMCs stimulated with anti-Fas were stained with M30 Cytodeath. (F) Apoptotic HPMCs stained with M30 CytoDeath antibody were engulfed by macrophages. Magnification: x540. The results shown are representative of three independent experiments using cells prepared from three different donors.

 
Apoptosis of HPMCs was confirmed by TUNEL staining (Figure 2C). Flow cytometry study showed that the log fluorescence intensity of HPMCs increased after TNF-{alpha} primed HPMCs were treated with anti-Fas antibody and there was an apoptotic peak (see Figure 4E).



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Fig. 4. Flow cytometric analysis of HPMCs. (AC) DNA histogram. The percentages of apoptotic HPMCs with hypodiploid DNA content are shown in the right upper corner. (DF) TUNEL stain. (GI) M30 reactivity. (A, D and G) Un-primed HPMCs treated with anti-Fas (clone CH11, 50 ng/ml); (B, E and H) TNF-{alpha} primed HPMCs stimulated with anti-Fas (50 ng/ml); (C, F and I) TNF-{alpha} primed HPMCs preincubated with Z-IETD-FMK (50 µM) for 2 h before the addition of anti-Fas antibody.

 
M30 reactivity was observed in TNF-{alpha} primed HPMCs stimulated with anti-Fas antibody, and an aggregated granular appearance of cytokeratin was demonstrated (Figure 2E). Flow cytometry analysis showed that the log fluorescence intensity increased after TNF-{alpha} primed HPMCs were treated with anti-Fas antibody, and M30 reactivity was observed in a significant proportion of cells (see Figure 4H).

Both immunofluorescence and electron microscopy showed that the apoptotic HPMCs could be phagocytosed by macrophages (Figures 2F and 3).



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Fig. 3. Electronmicrograph of a macrophage that engulfed apoptotic HPMCs. Transmission electron microscopy showed that an apoptotic body was engulfed by macrophages.

 
Apoptosis was completely blocked by preincubating TNF-{alpha} primed HPMCs with 50 µM Z-IETD-FMK (Figure 4C, F and I) or 100 µM Z-DEVD-FMK (data not shown) for 2 h before adding anti-Fas.

Western blot analysis of bcl-2, caspase-3 and caspase-8
Cultured HPMCs constitutively expressed bcl-2. TNF-{alpha} treatment downregulated the expression of bcl-2 in HPMCs by 46.6% (Figure 5).



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Fig. 5. Western blot analysis of bcl-2 expression after TNF-{alpha} treatment in HPMCs. Lane 1, lysate prepared from Jurkat cell line was used as positive control; lane 2, HPMCs incubated in control medium; lane 3, HPMCs treated with TNF-{alpha} (5 ng/ml) for 48 h. The result shown is representative of three independent experiments using cells prepared from three different donors.

 
Anti-Fas antibody induced proteolytic processing of caspase-3 and caspase-8 in TNF-{alpha} primed HPMCs (Figure 6). The levels of procaspase-3 decreased and active caspase-3 increased from 12 to 24 h. Similarly, the levels of procaspase-8 decreased with time from 12 to 24 h.



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Fig. 6. Western blot analysis of caspase-3 and caspase-8 activation in HPMCs. TNF-{alpha} primed HPMCs treated with anti-Fas antibody had proteolytically processed caspase-3 (A) and caspase-8 (B) proteins. Lane 1, HPMCs incubated in control medium; lane 2, un-primed HPMCs treated with anti-Fas (50 ng/ml); lanes 3 and 4, TNF-{alpha} primed HPMCs stimulated with anti-Fas for 12 and 24 h, respectively. The results shown are representative of three independent experiments using cells prepared from three different donors.

 
Increased apoptosis of HPMCs in peritonitis PDE
A significant increase of apoptotic HPMCs was observed on days 5, 15 and 28 after the peritonitis episode in the PDE of peritonitis patients (Table 1).


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Table 1. Percentage of HPMC apoptosis in PDE from CAPD patients

 


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we have shown that TNF-{alpha} primed HPMCs underwent apoptosis after anti-Fas antibody treatment and that these apoptotic HPMCs could be phagocytosed by macrophages. Growing evidence suggests that, after engulfment of apoptotic cells, macrophages become actively anti-inflammatory and release suppressive mediators such as TGF-ß and IL-10 [2]. There is also evidence demonstrating that, during the resolution of inflammation, excess resident cells are cleared by apoptosis [13]. HPMCs have a strong regenerative capacity during the reparative phase of peritonitis. However, peritoneal biopsy and PDE analysis have disclosed a prolonged reparative phase after peritonitis and marked morphologic changes of peritoneum in long-term PD patients [14,15]. Lindic et al. [14] found that, after peritonitis, the peritoneum surface was denuded and covered with fibrin and fibrous tissue in most CAPD patients during catheter removal or re-insertion. Williams et al. [15] reported that peritoneum morphology changes and mesothelial cells denudation occurred in long-term PD patients. Recently, Lai et al. [16] reported a progressive increase in the percentage of mesothelial cells or dead cells in the total cell population in PDE up to 6 weeks after the peritonitis episode. Our analysis of PDE revealed that, in comparison with non-infective control, there was increased mesothelial cell apoptosis during the recovery phase of peritonitis and that this phenomenon lasted for at least 4 weeks. Usually, tissue inflammation would be induced if cells died by necrosis. Therefore, our results imply that HPMC apoptosis might be related to the resolution of peritoneal inflammation.

The reason for the prolonged repair after peritonitis in CAPD patients was not very clear. Our previous report demonstrated that peritoneal dialysate induced apoptosis of HPMCs in culture condition [9]. Generally, cells in active proliferation are more susceptible to damage and will then undergo apoptosis. Probably the bio-incompatible PD fluids aggravate the apoptosis and even impair the phagocytic ability of peritoneal macrophages and the subsequent removal of apoptotic cells. Failed clearance of apoptotic cells might result in failure to promote a reprogramming of macrophages from ‘pro-inflammatory’ to ‘reparative’ [3]. Therefore, future studies should examine the influence of blockade of local apoptotic process in the peritoneal cavity or the interference of engulfment of apoptosis by macrophage on the peritoneal reparative process during peritonitis in an animal model. The difference between conventional lactate-based PD fluids and newer bicarbonate-based PD fluids in terms of PDE mesothelial cell apoptosis during peritonitis should also be investigated.

Fas-mediated apoptosis was regulated at multiple steps. Increased surface Fas density by cytokine stimulation enhanced the susceptibility to FasL or agonistic anti-Fas antibody- induced apoptosis [6]. However, the collaboration of several survival factors, such as bcl-xL, bcl-2 and Fas-associated phosphatase-1 (FAP-1) was also important in regulating Fas-mediated apoptosis [17,18]. The present study revealed upregulation of Fas expression and downregulation of bcl-2 in HPMCs after TNF-{alpha} treatment. Therefore, our results suggest that TNF-{alpha} creates a pro-apoptotic environment favourable for Fas-mediated apoptosis in HPMCs and that there is an important synergism between intracellular apoptotic molecules induced by TNF-{alpha} and Fas. Our dosage of TNF-{alpha} probably reflected that, under in vitro conditions, the concentrations of cytokines necessary to stimulate HPMCs are a bit higher than those detected in PDE during peritonitis.

IFN-{gamma} plays a role in controlling the phenotype of infiltrating leukocytes during the course of peritoneal inflammation [19] and has been reported to upregulate Fas expression in several cell lines [6]. However, HPMCs did not respond to IFN-{gamma} in terms of modulation of Fas at the concentration ranges we used, and our results suggested that IFN-{gamma} is not involved in the regulation of Fas-mediated apoptosis in HPMCs.

An important question arising from our findings is what would be the source of FasL in vivo? In vitro evidence indicates that biologically active soluble FasL is released by normal human monocytes/macrophages during phagocytosis and following activation with phytohaemagglutinin or superantigen [20,21] and that the soluble Fas can function as a cytokine to induce apoptosis in susceptible cells [20]. However, considerable attention has also been directed to FasL expression in activated lymphocytes [6]. In vitro studies have shown that T-lymphocytes use the Fas/FasL pathway and the perforin system to kill target cells [22]. Therefore, macrophages and/or lymphocytes are possible sources of FasL in the peritoneal cavity.

In conclusion, our results reveal that HPMCs undergo apoptosis during peritonitis. The apoptosis of HPMCs might be related to the resolution of peritoneal inflammation and subsequent peritoneum repair.



   Acknowledgments
 
We gratefully thank Ms Ming-Ling Hsu, Ms Yuh-Fang Chang and Ms Pui-Ching Lee for their technical assistance. This work was supported by research grant # 216 (2000) from Taipei Veterans General Hospital and research grant NSC-90-2314-B-075-075 from the National Science Council, Republic of China. Part of this manuscript was previously submitted in abstract form to the 2001 ASN/ISN World Congress of Nephrology, San Francisco.

Conflict of interest statement. None declared.



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 Introduction
 Materials and methods
 Results
 Discussion
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Received for publication: 15. 2.02
Accepted in revised form: 4. 4.03





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