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Am J Physiol Renal Physiol 293: F801-F812, 2007. First published July 3, 2007; doi:10.1152/ajprenal.00044.2007
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Proapoptotic activity of indoleamine 2,3-dioxygenase expressed in renal tubular epithelial cells

Kanishka Mohib,1,2 Qiunong Guan,2 Hong Diao,2 Caigan Du,1,2,3,4 and Anthony M. Jevnikar1,2,3,4

1Departments of Medicine and Microbiology and Immunology, The University of Western Ontario, London, Ontario; 2The Robarts Research Institute, London, Ontario; 3The Lawson Health Research Institute, London, Ontario; and 4The Multi-Organ Transplant Program, London Health Sciences Center, London, Ontario, Canada

Submitted 27 January 2007 ; accepted in final form 28 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exposure of renal tubular epithelial cells (TEC) to IFN-{gamma}/TNF-{alpha} leads to Fas/FasL-mediated self-injury, which contributes to allograft rejection. Indoleamine 2,3-dioxygenase (IDO) converts tryptophan to N-formyl-kynurenine and contributes to immune privilege in tissues by increasing Fas-mediated T cell apoptosis. However, renal expression of IDO and its role in promoting Fas-mediated TEC death have not been examined. IDO expression was analyzed by RT-PCR and Western blot. Apoptosis was measured by fluorescence-activated cell sorting analysis and terminal deoxytransferase-mediated dUTP nick end labeling. We demonstrated that functional IDO is expressed in TEC and is increased by IFN-{gamma}/TNF-{alpha} exposure. Increased IDO activity promoted TEC apoptosis, whereas inhibition of IDO by its specific inhibitor 1-methyl-D-tryptophan attenuated IFN-{gamma}/TNF-{alpha}-mediated TEC apoptosis and augmented TEC survival. Transgenic expression of IDO resulted in increased TEC apoptosis in the absence of proinflammatory cytokine exposure, supporting a central role for IDO in TEC injury. Inhibition of IDO-mediated TEC death by a caspase-8-specific inhibitor (Z-IETD-FMK), as well as the absence of an IDO effect in Fas-deficient and FasL-deficient TEC, supports a Fas/FasL-dependent, caspase-8-mediated mechanism for IDO-enhanced TEC death. These data suggest that renal IDO expression may be deleterious during renal inflammation, because it enhances TEC self-injury through Fas/FasL interactions. Thus attenuation of IDO may represent a novel strategy to promote kidney function following ischemia and renal allograft rejection.

tubular epithelium; apoptosis

INDOLEAMINE 2,3-DIOXYGENASE (IDO; EC 1.13.11.17 [EC] ) is a rate-limiting enzyme in the kynurenine enzymatic pathway that converts tryptophan to N-formyl-kynurenine, required for the production of the cellular cofactor NAD+ (24). In mammals, IDO has been found in many types of cells and tissues, including antigen-presenting cells [e.g., dendritic cells (DC) and macrophages] and tumors (16, 21, 32). The expression of IDO is induced mainly in response to LPS, IFN-{gamma}, and weakly by TNF-{alpha} (39, 41–44). IDO was initially described for its ability to inhibit the growth of intracellular pathogens such as Chlamydia subspecies following engulfment by macrophages, through the depletion of the essential amino acid tryptophan (3, 9). More recently, IDO activity in DC and macrophages has been shown to participate in T cell activation-induced cell death related to growth arrest in the G0 stage of the cell cycle and sensitization to Fas-induced death (27, 31, 32). Moreover, IDO has been shown to enhance anti-Fas antibody (clone CH11)-mediated death of several human tumor cell lines (19). These observed enzymatic activities place IDO expression by parenchymal cells in a pivotal position to influence inflammatory events.

IDO expression has been previously observed in the kidney (8, 19, 30) and thus suggested to play an important role in acute rejection and chronic renal failure (6, 35, 38). However, expression by renal tubular epithelium and the precise physiological function of this enzyme, particularly in the pathogenesis of Fas-mediated renal cell apoptosis, has not been investigated. Tubular epithelial cells (TEC) are a major type of parenchymal cell in the kidney and are able to express important proinflammatory molecules such as transforming growth factor (TGF)-beta, TNF-{alpha}, Fas/Fas ligand (FasL), and adhesion proteins such as ICAM and VCAM (2, 22, 33, 40). We and others have previously demonstrated that TEC express basal levels of both Fas and FasL and that Fas expression increases in response LPS, oxidative stress, and inflammatory cytokines such as IFN-{gamma} and TNF-{alpha} (11, 25, 26, 33). Importantly, TEC can interact through Fas and FasL on adjacent cells during inflammation, resulting in "fratricide" that contributes to kidney injury associated with ischemia and immune injury during rejection (10, 34, 36). Therefore, we investigated a potential harmful role for renal IDO expression in promoting TEC death under inflammatory conditions. We have now demonstrated that IDO expression can promote Fas/FasL-mediated TEC death in response to IFN-{gamma} and TNF-{alpha} exposure. The in vitro harmful effects of IDO could translate into a similar role for the enzyme in diverse forms of in vivo inflammation, including renal allograft rejection.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cells. Primary renal proximal TEC were isolated from the kidney cortex of C3H Hej or C57BL/6 mice as previously described (10). Cloned TEC cell lines were immortalized by origin-deficient SV40 DNA as described previously (10) and were obtained from C57BL/6 (NG1.1), C3H Hej (CS3.7), MRL-lpr, and MRL-gld mice. The phenotype of TEC was confirmed by the expression of E-cadherin, CD26 (dipeptidyl peptidase), and CD13 (alanine aminopeptidase) using fluorescence-activated cell sorting (FACS) analysis. All TEC were grown in K1 medium, made up of DMEM and Ham's F12 (50:50; Invitrogen GIBCO, Burlington, ON, Canada), supplemented with 10% fetal bovine serum, hormone mix (5 µg/ml insulin, 34 pg/ml triiodothyronine, 5 µg/ml transferrin, 1.73 ng/ml sodium selenite, and 18 ng/ml hydrocortisone) and 25 ng/ml EGF (Sigma-Aldrich, Mississauga, ON, Canada). Cell death assays were carried out in serum-free K1 medium.

NG1.1 cell characterization. Cells (2.5 x 105) were seeded to each well of 24-well plates overnight (Sarstedt, Newton, NC). The next day, the cells were removed with 0.5% trypsin-EDTA (Invitrogen GIBCO) and washed with 1x PBS twice. Following the washes, the cells were incubated in 10% goat serum for 5 min on ice. One microliter of anti-E-cadherin, anti-CD13, anti-CD26, or isotype control antibody (BD Biosciences, Mississauga, ON, Canada) was added, and the cells were incubated for an additional 30 min on ice. Cells were washed two times with 1x PBS before FACS analysis.

RNA isolation and RT-PCR. Cells (2.5 x 106) were plated overnight in each 60-mm petri dish (Sarstedt) and induced the following day with IFN-{gamma}, TNF-{alpha}, or IFN-{gamma}/TNF-{alpha} in combination (BD PharMingen, Mississauga, ON, Canada). In all experiments, each cytokine was added to a final concentration of 10 ng/ml. One milliliter of Trizol (Invitrogen GIBCO) was added to each petri dish at the designated time, and the cells were removed using a rubber policeman (Sarstedt). RNA isolation was carried out using the manufacturer's protocol. RT-PCR was performed using total isolated RNA. Briefly, 5 µg of RNA from each sample were used for reverse transcription with SuperScript II (Invitrogen GIBCO). PCR amplification of IDO cDNA was carried out using primers 5'-CATAAGACAGAATAGGAGGC (sense) and 5'-GAAGATGTGGGCTTTGCTCTA (antisense) for 32 cycles (28 cycles for inducible transgenic system). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were used as an internal control for the amount of RNA used in each sample. The PCR products were visualized on a 1% agarose gel with 0.5 µg/ml ethidium bromide.

Western blot analysis. Cells (2.5 x 106) were plated overnight on each 60-mm petri dish (Sarstedt) and stimulated with 10 ng/ml IFN-{gamma}, TNF-{alpha}, or IFN-{gamma}/TNF-{alpha} in combination (BD PharMingen). The cells were collected at the indicated times by using a rubber policeman (Sarstedt), spun down, and treated with equal volumes of lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% Nonidet-P 40, 1 mM DTT, and a protease cocktail (Roche Diagnostics, Mannheim Germany)] and 5x SDS sample buffer [20 mM Tris·HCl (pH 6.8), 5% (wt/vol) SDS, 10% (vol/vol) beta-mercaptoethanol, 2 mM EDTA, and 0.02% bromphenol blue]. The samples were run on a 12% SDS-polyacrylamide gel, followed by transfer to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked using 5% milk in TBS-T (20 mM Tris·HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) for 1 h. The IDO protein was detected using a primary polyclonal rabbit antibody (1:3,000), kindly provided by Dr. Andrew Mellor (Medical College of Georgia, Augusta, GA), using 2.5% milk in TBS-T and a goat anti-rabbit peroxidase-conjugated secondary antibody (Sigma-Aldrich). The IDO-specific protein bands were visualized using an enhanced chemiluminescence assay (ECL; Amersham Pharmacia Biotech, Little Chalfont, UK). Blots were reprobed using anti-beta-actin antibody (Sigma-Aldrich) for total beta-actin protein as protein loading control.

Tryptophan depletion assay. Tryptophan depletion was measured according to the method of Denkla and Dewey as modified previously (5, 9). Specifically, 1.5 x 105 cells were grown overnight in a 24-well plate. The cells were treated with 200 µl of medium alone or medium containing 10 ng/ml IFN-{gamma}/TNF-{alpha} for 24 h, and the supernatant was collected for analysis. The supernatant (50 µl) was mixed with 1.5 ml of 10% ice-cold trichloroacetic acid (TCA; Sigma-Aldrich), spun for 20 min at 10,000 g, and then transferred to a glass tube with 0.2 ml of 2% formaldehyde and 0.1 ml of 6.0 x 10–3 M FeCl3 (Sigma-Aldrich) in 10% TCA. The tubes were capped and boiled for 1 h to convert the remaining tryptophan to norharman, which was then measured with a spectrofluorometer using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

Apoptosis determination. Two methods were used for apoptosis determination. For FACS analysis with 7-amino-actinomycin D or propidium iodide (7-AAD/PI) and annexin V staining, 2.5 x 105 cells were seeded to each well of 24-well plates (Sarstedt) and grown overnight. The cells were then treated with 10 ng/ml of IFN-{gamma} or IFN-{gamma}/TNF-{alpha} (BD PharMingen) in the absence or presence of the IDO inhibitor 1-methyl-D-tryptophan (1-MT; Sigma-Aldrich) dissolved in PBS using 0.1 N NaOH. The cells were harvested by a brief trypsinization with 0.5% trypsin-EDTA (Invitrogen GIBCO) and washed twice with 1x PBS. Each sample received 5 µl of annexin V-FITC and either 10 µl of Via Probe (7-AAD) or 5 µl of 100 µg/ml PI in annexin V binding buffer (BD Bioscience). The cells were left to incubate for 15 min in the dark. Flow cytometry was carried out using a Becton-Dickinson flow cytometer (BD Bioscience). For terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) assay, 1.0 x 106 cells were grown overnight in a six-well plate and treated with 10 ng/ml of IFN-{gamma}/TNF-{alpha} in combination along with various concentrations of 1-MT for 48 h. The cells were removed from the wells using 0.5% trypsin EDTA (Invitrogen GIBCO), washed twice with PBS, resuspended with 1% paraformaldehyde, and then incubated on ice for 15 min, followed by two washes with PBS. Cells were suspended in 1 ml of ice-cold 70% ethanol for 20 min on ice and washed twice with PBS. A Roche TUNEL kit was used from which a labeling reaction solution was prepared according to the instructions provided. The reaction mixture (50 µl), containing FITC-labeled dNTPs and terminal deoxytransferase, was added to each sample. The samples were incubated at 37°C for 1 h, during which time they were gently shaken every 15 min. Finally, the cells were washed two times with PBS and analyzed using a Becton-Dickinson flow cytometer. Picolinic acid and quinolinic acid (Sigma-Aldrich) were dissolved in 1x PBS and used at the indicated concentrations.

Stable shRNA vector construction. Stable shRNA expression and silencing was carried out using a modified pHEX vector. The pHEX vector kindly provided by Dr. C. Strathdee is based on a herpes simplex virus expression pHEX-6300 and contains a fused gene encoding Zeocin resistance and green fluorescent protein (GFP) (37). An H1 promoter was used to replace the hEF1{alpha} promoter for short hairpin RNA (shRNA) expression. NG1.1 cells were also transfected with pHEX-control vector containing a nonspecific shRNA sequence (5'-AAT CGC ATA GCG TAT GCC GTT-3') and pHEX-IDO (5'-GCA ATA TTG CTG TTC CCT ACT-3') (DNA Integrated Technologies, Coralville, IA). The cells were checked for GFP expression, and selection was carried out with 500 µg/ml Zeocin (Invitrogen GIBCO).

Caspase activation assay. Cells (1.0 x 106) were seeded to each well on a six-well plate (Sarstedt) overnight. The next day, the cells were pretreated with either PBS or PBS containing the indicated concentration of 1-MT for 24 h. Cells were treated with 10 ng/ml IFN-{gamma} for 3 h, and the cells were collected as described above. Caspase-8 activation was assessed using Western blot analysis and anti-caspase-8 antibodies (1:500; Santa Cruz Biotechnology, Santa Cruz, CA).

Inducible transgenic expression of IDO in TEC. The Drosophila-based ecdysone-inducible vector system consisting of the pIND and pVgRXR plasmids (Invitrogen GIBCO) was used to express IDO in TEC. IDO cDNA was cloned into the pIND multiple cloning site, and TEC were transfected using Lipofectamine 2000 (Invitrogen GIBCO). Stable expression of TEC was established using G418 (neomycin) selection (Sigma-Aldrich) (up to 0.5 mg/ml). Next, cells were transfected with a second vector (pVgRXR), to produce both ecdysone receptor (VgEcR) and retinoid receptor (RXR). TEC with expression of both vectors were subsequently selected using both Zeocin and G418 (Invitrogen GIBCO). Finally, the addition of 10 µM ponasterone A (Sigma-Aldrich) (the inducer) was used to dimerize the VgEcR and RXR to transactivate the 5xE/GRE response element on pIND, leading to the expression of IDO in TEC.

Statistical analysis. Two-way ANOVA and t-tests (two-tailed distribution) were used for comparisons between groups with StatView software (SAS Institute, Cary, NC). A P value ≤0.05 was considered significant. Comparison between treatment groups are expressed as mean viability (lower left quadrant) ± SE (%).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Upregulation of IDO expression and activity in TEC in response to inflammatory cytokines. To study the role of IDO in renal tubular epithelial cell injury, we used the NG1.1 in vitro cell line. NG1.1 cells have a typical proximal tubular cell morphology, exhibit contact inhibition in monolayers, and form tight junctions with zonula occludens (ZO)-1 (not shown). Phenotypically, NG1.1 cells express E-cadherin and CD13, as well as CD26 (Fig. 1), and the levels of these markers are comparable to those in a cloned cell line from C3H Hej mice (CS3.7 TEC). IDO expression was detected in TEC in response to proinflammatory cytokines. TEC treated with IFN-{gamma} upregulated IDO mRNA expression by 4 h and maintained high levels up to 8 h (Fig. 2A). At the protein level, the same trend was observed with IFN-{gamma} inducing upregulation of IDO within 4 h of treatment which has maintained up to 8 h (Fig. 2D). We found that TNF-{alpha} alone was able to weakly induce IDO expression. In combination with IFN-{gamma}, there was an additive increase in the level of IDO transcript and protein compared with IFN-{gamma} or TNF-{alpha} alone (Fig. 2, B, C, E, and F). Expression of IDO was confirmed in C3H Hej primary cells and CS3.7 TEC (data not shown), indicating that expression is not strain or clone cell dependent. These data demonstrate the ability of TEC to quickly upregulate IDO under inflammatory conditions.


Figure 1
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  		Fig. 1. NG1.1 cells express tubular epithelial cell (TEC) markers. NG1.1 TEC were incubated with 10% goat serum and stained with anti-E-cadherin (A), anti-CD13 (B), and anti-CD26 (C) (solid lines) or isotype control antibody (shaded lines).

 

Figure 2
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  		Fig. 2. Indoleamine 2,3-dioxygenase (IDO) is upregulated by NG 1.1 TEC in response to IFN-{gamma}, TNF-{alpha}, or IFN-{gamma}/TNF-{alpha} combined. A–C: TEC were treated with 10 ng/ml IFN-{gamma} (A), TNF-{alpha} (B), or IFN-{gamma}/TNF-{alpha} in combination (C) for 4 and 8 h. IDO mRNA levels were detected using RT-PCR. GAPDH was used to control for the amount of RNA used in each sample. D–F: Western blot of NG1.1 TEC treated with 10 ng/ml IFN-{gamma} (D), TNF-{alpha} (E), and IFN-{gamma}/TNF-{alpha} in combination (F) for 4 and 8 h. The IDO-specific band (~45 kDa) was detected using a rabbit-anti-IDO antibody in Western blot. beta-Actin was used as a protein loading control. Graphs display density ratios (means ± SD) of IDO/beta-actin for respective Western blots. Data are representative of 3 separate experiments.

 
IDO enzyme activity can be determined by catalysis of tryptophan to produce NAD+ (1). To confirm whether upregulation of IDO expression resulted in an increase of enzyme activity, we measured tryptophan depletion in cytokine-induced TEC cultures. NG1.1 TEC were treated with TNF-{alpha} and IFN-{gamma}, and residual tryptophan levels in supernatants were determined by conversion of tryptophan into norharman as described in METHODS (5, 9). The mean fluorescence emission level of norharman in the supernatants from cytokine-treated TEC cultures was 51,990 ± 3,692, which was lower than that from untreated cultures (83,100 ± 5,261) (P = 0.0005) and reflects functional IDO enzyme activity. To further confirm IDO activity, 1-MT, a specific IDO inhibitor, was added to cultures. As observed, the addition of 1-MT inhibited cytokine-induced tryptophan depletion (Fig. 3). These data indicate that cytokine-induced reduction of tryptophan was due to upregulation of IDO activity. Interestingly, basal IDO activity was present in TEC, since IDO protein was observed even in untreated TEC (Fig. 2, D and F), and tryptophan levels were increased over baseline controls in supernatants of cells exposed to 1-MT in the absence of cytokine. Tryptophan depletion observed in TNF-{alpha}- and IFN-{gamma}-treated cells correlates with IDO mRNA and protein expression in TEC and suggests that IDO activity may be upregulated in a similar fashion in vivo.


Figure 3
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  		Fig. 3. Tryptophan is depleted in supernatants of TEC grown in IFN-{gamma}/TNF-{alpha}. NG1.1 TEC were grown with medium alone or medium containing 10 ng/ml IFN-{gamma}/TNF-{alpha} in the absence or presence of 1-methyl-D-tryptophan (1-MT) for 24 h. The supernatant was collected, and residual tryptophan was converted to norharman. Norharman levels were measured by a spectrofluorometer using 360 and 460 nm as excitation and emission wavelengths, respectively. Results are means ± SE of 3 separate experiments. *P = 0.0005 compared with medium alone.

 
Proapoptotic function of IDO in cytokine-induced TEC apoptosis. IDO activity has previously been shown to augment activation-induced cell death of T cells through sensitization to Fas-mediated apoptosis (27). Moreover, a direct correlation between IDO silencing and Fas-mediated cell death has been observed in several tumor cell lines (19). Our previous work has demonstrated that stimulation of TEC with IFN-{gamma} and TNF-{alpha} results in increased Fas expression and a Fas/FasL-dependent form of self-injury or fratricide (10), and others have suggested that Fas/FasL interaction within the kidney may be harmful (4, 11, 26, 34, 35, 38). Given that both Fas and IDO are induced by IFN-{gamma} and TNF-{alpha}, we tested the potential proapoptotic capacity of IDO in this Fas-mediated TEC fratricide. Monolayers of NG1.1 TEC were treated with both IFN-{gamma} and TNF-{alpha} along with increasing concentrations of 1-MT. 1-MT had no effect on cell viability but was able to inhibit IFN-{gamma}/TNF-{alpha}-induced TEC apoptosis in a dose-dependent manner, as shown in a representative experiment (Fig. 4). Consistent with a death-inducing effect of IDO in TEC, 1-MT treatment increased cell viability on average in four experiments, from 39 ± 6% in cytokine-treated cultures to 60 ± 4% in cytokine cultures containing 750 µg/ml 1-MT (P = 0.02). A similar proapoptotic role for IDO was noted in primary TEC cultures, as shown in a representative experiment (Fig. 5). Addition of 1-MT (750 µg/ml) to IFN-{gamma}-treated C3H TEC cultures increased cell viability from 57 ± 8% in IFN-{gamma}-treated cells to 76 ± 4% (P = 0.03). These results were further confirmed in NG1.1 TEC by TUNEL assay (Fig. 6). In this assay, a decrease in the level of apoptotic cell death with DNA fragmentation was noticed in cultures treated with IFN-{gamma}/TNF-{alpha} along with 1-MT. Hence, our data demonstrate a proapoptotic role of IDO in IFN-{gamma}/TNF-{alpha}-mediated TEC apoptosis, which can be blocked by using the IDO-specific inhibitor 1-MT.


Figure 4
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  		Fig. 4. IDO enhances TEC death following exposure to proinflammatory cytokines. NG1.1 TEC were treated with 10 ng/ml TNF-{alpha}/IFN-{gamma} alone or in combination with the IDO inhibitor 1-MT (T/I + 1-MT) for 72 h in K1 serum-free medium. The cells were stained with annexin V-FITC/propidium iodide (PI), and fluorescence-activated cell sorting (FACS) analysis was used to detect cell death. Results are representative of 4 separate experiments.

 

Figure 5
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  		Fig. 5. IDO enhances primary TEC death following exposure to IFN-{gamma}. C3H primary cells were treated with IFN-{gamma} alone or IFN-{gamma} in combination with the IDO inhibitor 1-MT for 24 h in K1 serum-free medium. The cells were stained with annexin V-FITC/PI, and cell death was measured using FACS analysis. Results are representative of 5 separate experiments.

 

Figure 6
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  		Fig. 6. Cytokine-induced TEC apoptosis is blocked by IDO inhibition. NG1.1 TEC were treated with 10 ng/ml TNF-{alpha}/IFN-{gamma} or T/I + 1-MT for 48 h. DNA strand breaks were detected by terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) assay using FITC-conjugated dNTP. TUNEL-positive cells were detected using FACS analysis. Data are representative of 3 independent experiments.

 
To discount any cytoprotective properties of 1-MT, a stable shRNA vector containing an optimal sequence targeting the IDO transcript was constructed to directly block expression of the enzyme. NG1.1 cells were transfected with pHEX-IDO (containing an IDO-specific sequence) and a pHEX-control vector (containing a nonspecific sequence). The efficacy of pHEX-IDO silencing was determined by detecting IDO transcript levels following 24 and 48 h of IFN-{gamma} treatment. TEC transfected with pHEX-IDO had markedly lower levels of the IDO transcript compared with TEC transfected with the pHEX-control, which was most evident by 48 h (Fig. 7, A and B). Western blotting displayed the same trend with only minimal IDO protein detected in NG1.1-pHEX-IDO cells compared with NG1.1-pHEX-control cells at 48 h (Fig. 7C). Subsequently, NG1.1-pHEX-IDO-transfected cells were found to be more resistant to IFN-{gamma}-induced cell death than NG1.1-pHEX-control cells (Fig. 7D). Although there was an increase in cell death with IFN-{gamma} in pHEX-IDO-silenced TEC, overall levels were reduced compared with nonsilenced TEC, and survival was increased in pHEX-IDO cells (Fig. 7D). The small increase in death even with IDO silencing may be directly attributed to effects of IFN-{gamma} on these cells, exclusive of IDO participation. In contrast, improvement in survival of cytokine untreated pHEX-IDO cells may be due to the elimination of basal IDO protein expression and reduced Fas-mediated fratricide and is consistent with basal levels of apoptosis noted in 1-MT control cultures and in Fas-deficient TEC (10). Together, our data demonstrate that apoptosis is ameliorated by IDO inhibition with 1-MT or shRNA silencing and strongly suggest a proapoptotic role for IDO expression in the IFN-{gamma}/TNF-{alpha}-mediated TEC death.


Figure 7
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  		Fig. 7. IDO gene silencing blocks cytokine-induced TEC death. IDO gene silencing was carried out using a stable pHEX short hairpin RNA (shRNA) expression vector. NG1.1-pHEX-control and NG1.1-pHEX-IDO were treated with 10 ng/ml IFN-{gamma} for 24 (A) and 48 h (B). IDO expression levels were assessed using RT-PCR. GAPDH was used to control for the amount of RNA used in each sample. C: IDO protein levels were detected by Western blotting in pHEX-control and pHEX-IDO following 48 h of IFN-{gamma} treatment. D: pHEX-control and pHEX-IDO cells were treated for 48 h with 10 ng/ml IFN-{gamma}. Cells were stained with annexin V-phycoerythrin(PE)/7-AAD, and cell death was analyzed using FACS. Graphs display density ratios (means ± SD) of IDO/GAPDH or IDO/beta-actin for RT-PCR and Western blot, respectively. Data are representative of 3 separate experiments for A–D.

 
To further support the proapoptotic activity for IDO expression in TEC, we created an inducible transgenic expression system in which IDO expression could be increased in the absence of cytokine exposure. Observed effects of IDO expression on cell death could therefore be more directly attributed to the activity of the enzyme. Augmented expression of IDO was controlled in cloned NG1.1 TEC using an ecdysone-based inducible system by the addition of ponasterone A (Fig. 8A). In this system, induced IDO expression decreased TEC viability, as shown in a representative experiment (Fig. 8B). On average in three separate experiments, viability decreased from 81 ± 1 to 67 ± 3% (P = 0.02). Ponasterone/IDO-induced apoptosis was abrogated by the addition of 1-MT in a dose-dependent fashion (Fig. 8B), supporting the suggestion that IDO expression has a direct proapoptotic effect in TEC in the absence of cytokines. Ponasterone treatment of nontransfected NG1.1 cells did not have an effect on cell viability (data not shown). Together, our data strongly support the suggestion that IDO-induced TEC death involves a mechanism that is independent of a direct effect of IFN-{gamma} or TNF-{alpha}.


Figure 8
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  		Fig. 8. Transgenic expression of IDO promotes cell death. A: IDO cDNA-transfected TEC were grown in medium alone (control) or medium containing 10 µM ponasterone A (PonA) to induce IDO. IDO mRNA levels were detected using RT-PCR. GAPDH was used to control for the amount of RNA used in each sample. B: TEC were grown in K1 serum-free medium alone (uninduced) or medium containing either 10 µM PonA to induce IDO or 10 µM PonA in combination with 1-MT. Cells were stained with annexin V-FITC/7-AAD and analyzed using FACS. Graph displays density ratios (means ± SD) of IDO/GAPDH. Results are representative of 3 separate experiments.

 
Requirement of Fas and FasL interaction in proapoptotic function of IDO. Our previous studies have demonstrated that TEC fratricide is dependent on the interaction of Fas and FasL coexpressed by TEC upon exposure to the proinflammatory cytokines IFN-{gamma} and TNF-{alpha} (10). Thus the expression of IDO may augment TEC death by an effect on Fas-mediated death. To determine whether an association exists between IDO and Fas, we studied TEC lines obtained from MRL-lpr and MRL-gld mice deficient in Fas and FasL, respectively. IFN-{gamma} treatment of MRL-lpr and MRL-gld TEC had only a small effect on cell death, as we had previously reported (Fig. 9, A and B) (10). Addition of the IDO inhibitor along with IFN-{gamma} in either lpr or gld cells did not significantly alter cell death (P = 0.2 and P = 0.7, respectively). There was a small improvement in lpr TEC given 1-MT along with IFN-{gamma}, but this was not significant in analysis of repeated experiments (P = 0.2) (Fig. 9A). The results suggest that altering IDO activity in TEC lacking Fas or FasL has no additional benefit in preserving survival.


Figure 9
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  		Fig. 9. IDO-mediated TEC death is Fas/FasL dependent. Fas-deficient MRL-lpr cultured TEC (A) and FasL-deficient MRL-gld TEC (B) were treated with 10 ng/ml IFN-{gamma} or IFN-{gamma} in combination with 1-MT for 24 h. Cells were stained with annexin-V-FITC/PE and 7AAD/PI. Results are representative of at least 3 separate experiments.

 
To establish an association between enhanced IDO expression and Fas/FasL-mediated interaction, we tested the effect of IDO expression on caspase-8 activation, since this would be expected to increase with Fas engagement. NG1.1 cells were preincubated with 750 µg/ml 1-MT or PBS overnight and exposed to IFN-{gamma}, and cell lysates were tested for caspase-8 activation. Cells pretreated with 1-MT had no or minor activation of caspase-8 compared with TEC pretreated with PBS (Fig. 10A). The effect of IDO on caspase-8 activation was also examined by utilizing the transgenic inducible IDO system and Z-IETD-FMK, a caspase-8-specific inhibitor. Monolayers of transfected TEC were pretreated with Z-IETD-FMK for 6 h, and then TEC were induced for IDO expression with 10 µM ponasterone A for 96 h. Ponasterone treatment of cells in the absence of cytokine resulted in a significant decrease in viability compared with noninduced cells (Fig. 10B). Pretreatment with Z-IETD-FMK conferred resistance to this IDO-augmented death, and viability increased on average in three experiments from 66 ± 3 to 77 ± 4% (P = 0.048) (Fig. 10B). These data suggest that a primary effect of IDO in augmenting death may be related to Fas and FasL interaction and subsequent activation of caspase-8. However, metabolites produced in the kynurenine pathway may also contribute to TEC death by their ability to induce activation of caspase-8 directly (13, 17), which might account for the slight improvement in survival detected in lpr-TEC treated with IFN-{gamma} and 1-MT.


Figure 10
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  		Fig. 10. IDO expression enhances caspase-8 activation and TEC death. A: NG1.1 cells were preincubated with 750 µg/ml 1-MT or PBS for 24 h and then treated with IFN-{gamma} for 3 h. B: NG1.1 IDO-inducible transgenic cells were pretreated for 6 h with 100 µM caspase-8 inhibitor Z-IETD-FMK, and then IDO was induced using 10 µM Pon A for 96 h. Cell death was measured by staining the cells with annexin V-FITC and 7-AAD and subsequently analyzed using FACS. Graph displays density ratios (means ± SD) of IDO/beta actin for Western blot. Results are representative of 3 separate experiments.

 
Metabolites generated downstream of IDO activity enhance IFN-{gamma}/TNF-{alpha}-mediated TEC death. There are two possible pathways through which IDO could induce TEC cell death. IDO activity has been shown to mediate cell death by depleting tryptophan in the microenvironment, thus causing cellular starvation (27). Alternatively, cell death could occur through the accumulation of kynurenine pathway metabolites, some of which are associated with cytotoxicity (13, 17). As shown in Fig. 3, tryptophan levels in TEC cultures were not completely depleted following IFN-{gamma}/TNF-{alpha} treatment for 24 h, as well as after 72 h (data not shown). The addition of picolinic acid and quinolinic acid, downstream kynurenine pathway metabolites, in combination with IFN-{gamma}/TNF-{alpha} markedly enhanced TEC death (Fig. 11A). Importantly, picolinic acid and quinolinic acid added to TEC grown in medium alone did not have a significant effect on baseline cell death, and neither metabolite was capable of enhancing cytokine-mediated cell death of MRL-gld TEC (Fig. 11B). These data suggest that IDO activity enhances Fas/FasL-mediated TEC death through the generation and subsequent accumulation of kynurenine pathway metabolites.


Figure 11
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  		Fig. 11. Kynurenine metabolites generated downstream of IDO activity promote TEC death through a Fas/FasL-mediated pathway. NG1.1 TEC (A) and FasL-deficient MRL-gld TEC (B) were treated with 10 ng/ml IFN-{gamma}/TNF-{alpha} alone or in combination with picolinic acid and quinolinic acid. Cell death was measured by staining with annexin V-FITC/7-AAD and analyzed using FACS. Data are representative of at least 3 separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There is substantial data on the expression and potential role of renal tubular cell Fas/FasL interaction on kidney injury during inflammation (23, 26, 34). The absence of Fas and FasL in MRL-lpr and MRL-gld mice, respectively, has been demonstrated to protect TEC from cytokine-mediated cell death and ischemia-reperfusion injury (10, 25). The susceptibility of TEC to Fas/FasL- and caspase-8-dependent self-injury and/or fratricide presents a considerable challenge to strategies aimed at preventing renal injury during inflammation. Fas/FasL-mediated injury in TEC cannot be prevented by only limiting inflammatory cells during ischemia or allograft rejection, since local factors capable of influencing Fas/FasL-induced death include ATP depletion, nitric oxide generation, and IL-2 release (11, 15, 23). Therefore, greater understanding of the regulation of Fas/FasL injury in TEC is required. Although it has been previously reported that IDO expression and activity are important for activation-induced cell death of T cells through sensitization of Fas (27)- and Fas-mediated apoptosis of human tumor cell lines (19), the present report is the first to demonstrate that IDO expression by TEC in response to inflammatory cytokines is directly linked to Fas-induced TEC death. Given that TEC represent the majority of parenchymal cells within the renal cortex, the expression of IDO by these cells may have a major influence on acute and chronic injury during renal inflammation (6, 19, 35).

The capacity of 1-MT to inhibit cytokine-induced TEC death provides substantial support that IDO expression contributes to inflammatory injury of TEC and suggests that therapeutics that target IDO in vivo may be of benefit in various models of kidney injury. Therefore, pharmacological inhibition of IDO using 1-MT or analogs may represent a means of preventing renal injury induced by ischemia-reperfusion as well as acute rejection.

The mechanism by which IDO augments Fas-mediated TEC death in response to cytokines is not defined but appears to require caspase-8 activation. It has been shown that cytokines such as IFN-{gamma} and TNF-{alpha} can directly activate caspases, making conclusions regarding IDO-associated cell death in cytokine-treated cells difficult (7, 20). However, our data collectively suggest that IDO regulates caspase-8 activation following Fas/FasL interaction in TEC, leading to apoptosis. In addition, our data also indicate that IDO-mediated cell death may not be limited to apoptosis, because Fas/FasL or caspase-8 activation may influence TEC necrosis through a "necrapoptosis" pathway involving the destabilization of mitochondrial permeability transition (MPT) (28). This is most evident in Figs. 4 and 5, showing that 1-MT treatment decreased the annexin V+/7-AAD+ population the greatest, whereas the annexin V+/7-AAD– population remained unchanged.

There are several possibilities by which IDO might induce cell death. Lee et al. (27) demonstrated that deprivation of tryptophan is detrimental to activated and proliferating T cells, which require this essential amino acid. Since all the TEC used in our studies were taken from growth-arrested confluent monolayers cultured in serum-free medium, it is unlikely that IDO affected TEC susceptibility to cytokine-induced death based on a cell cycle effect. Furthermore, because high levels of tryptophan were present in the medium of even cytokine-stimulated TEC, an inhibitory effect through tryptophan depletion cannot explain our observations. Alternatively, it has been suggested that metabolites produced by the kynurenine enzymatic pathway can have an effect on cell death through the direct activation of caspase-8 and the release of cytochrome c (13, 17). Accordingly, the addition of picolinic acid and quinolinic acid to cytokine-stimulated cultures enhanced cell death, supporting an important role for kynurenine metabolites in our system. Indeed, although Fas- and FasL-deficient TEC are resistant to cytokine-induced self-injury and fratricide (10, 11), there was a modest but consistently detectable benefit of 1-MT on TEC survival even in the absence of Fas or FasL, which would be supportive of such a metabolite effect (13).

Recently, there have been several reports regarding the overexpression of IDO to provide immune privilege to the lung and skin, as well as islet cells (1, 18, 29). Although this approach may be of benefit in tissues in which Fas and FasL interactions are limited within the parenchymal cells, the present data would suggest some caution in cells which coexpress Fas and FasL. In cells such as kidney TEC, overexpression of IDO would be expected to enhance self-injury and fratricide in vivo. However, the observation that kynurenine metabolites may be important mediators of cell death presents the prospect that inhibition of a downstream enzyme in the pathway may be of benefit. By identifying a different target in the kynurenine pathway with a greater influence on TEC death, IDO activity could be left untouched or even enhanced to increase immune privilege of parenchymal cells. IDO over expression, while limiting Fas signaling or caspase-8 activation using shRNA, might also enhance protection from the immune system without precipitating self-injury (12).

Conclusion. We have shown for the first time that IDO expression by TEC in response to inflammatory cytokines can augment caspase-8 activation and enhance TEC death. Renal IDO expression may be deleterious during renal inflammation, because it can augment TEC self-injury through Fas/FasL interactions. Thus attenuation of IDO may represent an important strategy to promote kidney survival following ischemia and renal allograft rejection.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from the Canadian Institutes of Health Research (to A. M. Jevnikar and C. Du).


   ACKNOWLEDGMENTS
 
We thank Pamela Gardner for secretarial assistance and Drs. Joaquim Madrenas, Ewa Cairns, and Robert Zhong for invaluable advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Jevnikar, Division of Nephrology, Dept. of Medicine, Univ. of Western Ontario, Univ. Campus, 339 Windermere Road, London, ON, Canada N6A 5A5 (e-mail: jevnikar{at}uwo.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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