Induction of Indoleamine 2,3-Dioxygenase by Interferon-γ in Human Islets

  1. Suparna A. Sarkar,
  2. Randall Wong,
  3. Seija I. Hackl,
  4. Ong Moua,
  5. Ronald G. Gill,
  6. Alexander Wiseman,
  7. Howard W. Davidson and
  8. John C. Hutton
  1. From the Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver and Heath Sciences Center, Aurora, Colorado
  1. Address correspondence and reprint requests to John C. Hutton, Director of Research, Professor of Pediatrics, Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver and Health Sciences Center, 1775 Ursula St., Mail Stop B-140, Aurora, CO 80045-6511. E-mail: john.hutton{at}uchsc.edu

Abstract

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial, rate-limiting step of tryptophan (Trp) catabolism along the kynurenine (KYN) pathway, and its induction in cells of the immune system in response to cytokines has been implicated in the regulation of antigen presentation and responses to cell-mediated immune attack. Microarray and quantitative PCR analyses of isolated human islets incubated with interferon (IFN)-γ for 24 h revealed increased expression of IDO mRNA (>139-fold) and Trp-tRNA synthase (WARS) (>17-fold) along with 975 other transcripts more than threefold, notably the downstream effectors janus kinase (JAK)2, signal transducer and activator of transcription (STAT)1, IFN-γ regulatory factor-1, and several chemokines (CXCL9/MIG, CXCL10/IP10, CXCL11/1-TAC, CCL2, and CCL5/RANTES) and their receptors. IDO protein expression was upregulated in IFN-γ–treated islets and accompanied by increased intracellular IDO enzyme activity and the release of KYN into the media. The response to IFN-γ was countered by interleukin-4 and 1α-methyl Trp. Immunohistochemical localization showed IDO to be induced in cells of both endocrine, including pancreatic duodenal homeobox 1–positive β-cells, and nonendocrine origin. We postulate that in the short term, IDO activation may protect islets from cytotoxic damage, although chronic exposure to various Trp metabolites could equally lead to β-cell attrition.

Type 1 diabetes is a polygenic T-cell–dependent autoimmune disease characterized by the selective destruction of the β-cells of the islets of Langerhans (13). It appears to result from a breakdown in tolerance to β-cell antigens (4) in individuals who have inherent defects in critical immunomodulatory mechanisms (5) that increase the risk of a pathogenic rather than protective immune response to self (6). In female NOD mice, the progression to clinical type 1 diabetes occurs via an ordered series of events that begin at ∼3–4 weeks of age with infiltration (initially by macrophages and dendritic cells and subsequently by B and T lymphocytes) of the perivascular ducts and peri-islet regions of the pancreas (7). This is followed by invasive lymphocytic insulitis and subsequently by progressive β-cell destruction, with overt disease typically occurring by 4–6 months of age. The natural history of human type 1 diabetes is temporally more variable, does not show the same pronounced sex bias observed in most NOD colonies, and is accompanied by less insulitis. Nevertheless, it likely involves passage through similar checkpoints that mark key changes in the autoimmune process in NOD mice (8).

l-Tryptophan (Trp) is an essential amino acid in humans typically making up 1–1.5% of the total amino acid content of the body. It is also the precursor of a series of bioactive compounds, including serotonin, kynurenine (KYN), picolinic, and quinolinic acids (911). It is catabolized by a constiutively expressed high Km Trp 2,3-dioxygenase (TDO) in liver and by a low Km inducible enzyme, indoleamine 2,3-dioxygenase (IDO; EC 1.13.11.42) in other tissues. IDO is the rate-limiting enzyme in the catabolism of Trp to N-formylkynurenine and is normally limited to the small intestine, spleen, thymus, lung, and epididymis (12,13). In pregnancy, it is expressed by the syncitotrophoblast cells of the placenta and is believed to play a key role here in preventing fetal rejection (14). IDO is strongly induced by interferon (IFN)-γ (15), and intracellular bacterial, viral, and protozoan infections in many tissues and notably in synoviocytes, endothelial cells, activated macrophages, and dendritic cells during inflammation (16). The low Km for Trp (20 μmol/l) enables IDO to reduce intracellular free Trp to submicromolar levels, thereby suppressing the growth of intracellular parasites such as Toxoplasma gondii in infected cells (17,18). In a poorly vascularized site, extracellular Trp depletion may also contribute to IFN-γ–mediated killing of tumor cells and allogeneic transplants by a process of starvation (18,19).

The present study was aimed at investigating the response of human islets to cytokine-mediated inflammation that may typify autoimmune attack in preclinical diabetes or islet allograft rejection. We report a remarkable induction of transcripts for IDO in human islets along with other enzymes of Trp metabolism and chemotactic factors in response to IFN-γ. Increased IDO transcription was accompanied by changes in protein expression and enzyme activity and occurred in a variety of islet cells types, including endocrine cells. A concomitant induction of several genes from the janus kinase–signal transducer and activator of transcription (JAK-STAT) pathway and downstream targets such as chemokine and chemokine receptors may amplify the response.

RESEARCH DESIGN AND METHODS

Procurement and culture of human islets.

Human islets were prepared by collagenase digestion by the Islet Cell Resource Center at the University of Colorado at Denver and Health Sciences Center using the Edmonton protocol (cold ischemia time between 4 and 9 h). All donors were brain dead, heart-beating individuals from the state of Colorado who died in motor vehicle accidents. None had a previous history of diabetes or inflammatory diseases. Islet purity (75–80%) and viability (76–96%) were determined by dithizone and Syto13/ethidium bromide staining according to the Standard Operation Procedure Manual, Clinical Islet Laboratory, SMRI, (Edmonton Alberta, Canada). The islets were precultured for 12 to 24 h in Miami medium (CMRL 1066 supplemented medium 99-603-CV; Mediatech, Herndon, VA) containing 0.5% human serum albumin and then further cultured in the presence and absence of cytokines, namely 10 μg/ml interleukin (IL)-1β (500 units/ml), 25 μg/ml IFN-γ (500 units/ml), 25 μg/ml tumor necrosis factor-α (TNF-α) (2,500 units/ml), and a cocktail mixture of 2 μg/ml IL-1β (100 units/ml), 10 μg/ml IFN-γ (200 units/ml), and 10 μg/ml TNF-α (1,000 units/ml) (Roche Applied Science, Indianapolis, IN) in 5% CO2 and 37°C for 24 h. Islet preparations (2,000 islet equivalents) from two of the donors when transplanted under the kidney capsule of streptozotocin-induced diabetic Rag2−/− B6 mice normalized hyperglycemia, indicating long-term survival and functionality.

RNA isolation and microarray.

Total RNA was extracted from groups of 8,000–10,000 human islets from four unrelated donors (two men and two women, age 26–46.6 years) by TRIzol reagent (Invitrogen, Carlsbad, CA) and purified by RNeasy columns (Qiagen, Valencia, CA), and RNA quality was verified by capillary electrrophoresis (Agilent-2100 Bioanalyzer; Agilent, Palo Alto, CA). Biotin-labeled cRNA was synthesized from total RNA (6 μg) according to standard Affymetrix protocol (Affymetrix, Santa Clara, CA), and 15 μg was hybridized to human genome HG U133 Plus 2.0 chip. The basic experiment included untreated islet tissue (n = 4) and exposure to IL-1β (n = 3), TNF-α (n = 4), IFN-γ (n = 3), and cytokine cocktail (n = 3). Data were analyzed using the GeneSpring Software (Silicon Genetics, Palo Alto, CA). The dataset was normalized as follows: 1) Scanner intensity values below 0.01 were set to 0.01. 2) Each measurement was divided by the 50th percentile of all measurements in that particular sample. 3) Specific samples (cytokine treated; n = 13) were normalized to the median of the corresponding control sample (n = 4). Cross gene error model was based on replicates, and the average of base/proportion ration was calculated as 68.15. For statistical analysis, the data were categorized on the basis of the treatment parameters, and a parametric test was performed that assumes equal variances. A one-way ANOVA for multiple groups was chosen in Gene Spring software to initially filter the data. A multiple correction test in the form of Bonferroni Step Down (Holm) was chosen because, during testing a set of genes for statistical significance across various groups, some of the genes may be falsely considered as statistically significant. The purpose of a multiple testing correction was to keep the overall error rate/false positives to less than the P value cutoff (0.01) (20). This restriction tested 54,675 genes and estimated that ∼0.01 genes could be expected to pass the restriction by chance. Tukey’s test was used for post hoc analysis.

Quantitative real-time PCR with Taqman probes.

cDNA was prepared from total RNA (1 μg) using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). Samples equivalent to 0.1 μg of the original RNA sample were used as templates for amplification in the linear phase in a 5′ nuclease assay–based system using FAM dye–labeled Taqman MGB probes (Applied Biosystems, Foster City, CA) on a 96-well ABI 7000 PCR system instrument. Hypoxanthine phosphoribosyl transferase (HPRT) gene was selected for sample normalization based on preliminary experiments with the ABI control plate (part no. 430 9199). The cycle threshold values (CT) were measured in triplicate, and the samples were re-normalized to the CT values from islet samples without cytokine exposure (control group/calibrator), and data were quantified using 2−ΔΔCT method (ABI User Bulletin #2).

IDO enzyme assay and cellular production of KYN.

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. Groups of 1,000 human islets were cultured for 24 h in 1 ml medium with cytokines, recovered by centrifugation for 5 min at 800 × g and sonicated in 150 μl PBS containing a protease inhibitor cocktail (Set 2; Calbiochem, EMD Biosciences, San Diego, CA). The sonicate was centrifuged for 10 min at 10,000 × g, and the supernatant was assayed in triplicate by incubating a 40-μl sample with an equal volume of 100 mmol/l potassium phosphate buffer, pH 6.5, containing 40 mmol/l ascorbic acid (neutralized to pH 7.0), 100 μmol/l methylene blue, 200 μg/ml catalase, and 400 μmol/l l-Trp for 30 min at 37°C. The assay was terminated by the addition of 16 μl 30% (w/v) trichloroacetic acid (TCA) and further incubated at 60°C for 15 min to hydrolyze N-formylkynurenine to KYN. The mixture was then centrifuged at 12,000 rpm for 15 min, and KYN was quantified by mixing equal volume of supernatant with 2% (w/v) Ehrlich’s reagent in glacial acetic acid in 96-well micro-titer plate and reading the absorbance at 480 nm using l-KYN as standard. Protein in the islet samples was quantified by Bio-Rad Protein assay at 595 nm. For the detection of l-KYN in the islet culture supernatants, proteins were precipitated with 5% (w/v) TCA and centrifuged at 12,000 rpm for 15 min, and determination of KYN in the supernatant with Ehrlich’s reagent was as described above. IL-4 (10 μg/ml; 500–2,000 units/ml) and 1-α-methyl Trp (1-MT; 40 μmol/l) were added to the incubation media as indicated.

Western blot analyses.

Groups of 1,000–1,200 islets incubated for 24 h in Miami medium in the presence of cytokines were harvested and sonicated in PBS as above, and 50-μg protein samples were electrophoresed on 10% SDS–PAGE gels. COS7 cells (0.6 × 106 cells/60-mm3 petri dish) transfected with human-IDO plasmid (3 μg) or empty vector cells were used as positive and negative controls, respectively. Proteins were transferred electrophoretically onto polyvinylidine fluoride membranes by semidry method and blocked for 1 h with 5% (w/v) nonfat dry milk in Tris-buffered saline and 0.1% Tween and then incubated overnight with anti-human mouse IDO antibody (1:500; Chemicon, Temecula, CA), phospho-STAT p91, and STAT p91 (1:500; Zymed, San Francisco, CA). Immunoreactive proteins were visualized with ECL Plus Western blotting detection reagent (Amersham BioSciences, Buckinghamshire, U.K.) after incubation for 1 h with anti-mouse horseradish peroxidase–conjugated secondary antibody (Jackson Immunolabs, West Grove, PA).

Immunohistochemical detection of IDO.

Islets were fixed in 4% paraformaldehyde in PBS (Invitrogen) for 1 h, immobilized in molten 10% porcine skin gelatin blocks (37°C), and embedded in optimal cutting temperature compound. Immunofluorescent staining on islet tissue was performed on 7-μm sections that were stained with antibodies raised against pancreatic duodenal homeobox 1 (PDX1) and IDO. Antigen retrieval was performed in a water bath for 30 min in a buffer containing 10 mmol/l Tris and 1 mmol/l EDTA (pH 9.0) at 97°C. The sections blocked for 1 h with 5% normal goat serum in PBS. The tissues were then reacted with mouse monoclonal anti-human IDO antibody (1:20; Chemicon) and goat polyclonal anti-human PDX1 antibody (1:2,000; gift from Chris Wright, School of Medicine, Vanderbilt, TN) overnight at room temperature in a humid chamber. Secondary antibodies anti-goat (labeled with Cy3) and anti-mouse (labeled with Cy2) were purchased from Jackson Immunolabs and were used at a concentration of 1:200. The nuclei were stained with Hoechst 33258 (Molecular Probes, Eugene, OR). Images were acquired by Intelligent Imaging System software from an Olympus 1X81 inverted motorized microscope equipped with Olympus DSU (spinning disk confocal) and Hamamatsu ORCA IIER monochromatic CCD camera.

RESULTS

IDO mRNA induction by IFN-γ.

Human islets exposed for 24 h to IFN-γ, either alone or in combination with other cytokines, exhibited profound changes in a number of genes, many of which have been previously documented in either rodent islets or pancreatic β-cells purified by flow cytometry (21,22). Affymetrics HG U133 Plus 2.0 gene chips that enable concurrent analysis of 54,675 genes were used to analyze global gene expression profile of human islet preparations exposed to cytokines. In all, 975 transcripts appeared to be upregulated and 523 downregulated (more than threefold) by IFN-γ alone, and 1,197 transcripts appeared to be upregulated and 660 downregulated (more than threefold) by IFN-γ + IL-1β and TNF-α. The effect on IDO mRNA was the most remarkable (>139-fold, P = 7.99E-06) (Fig. 1) along with another enzyme, tryptophanyl t-RNA synthase (WARS) (>17-fold) (P = 4.89E-06) (Table 1). The effect of IFN-γ on these mRNAs was further enhanced by combination with TNF-α and IL-1β (>235-fold, P = 3.23E-06) and WARS (>19-fold, P = 2.57E-05), although TNF-α and IL-1β alone had modest effects. Two related enzymes of Trp metabolism (KYN monooxygenase), the mitochondrial WARS2 mRNAs were unaffected, as were major secretory products in islets such as insulin and glucagons (Fig. 1; data not shown). Several genes from the JAK-STAT pathway, which is thought to mediate the effects of IFN receptor binding, were also upregulated with IFN-γ pretreatment, including JAK2 (4.5-fold, P = 3.75E-04), STAT1α (4.2-fold, P = 2.07E-04), and IFN-γ regulatory factor-1 (IRF-1) (8.3-fold, P = 1.73E-04). Other transcripts showing dramatic responses to IFN-γ included the chemokines (C-X-C motif) ligand 9 (CXCL9) (106.4-fold, P = 1.43E-06), ligand 10 (CXCL10) (84.7-fold, P = 7.65E-07), ligand 11 (CXCL11) (80.6-fold, P = 5.07E-07), and ligand CCL5 or RANTES (31-fold, P = 2.95E-04) (Table 1). The genes most upregulated by IFN-γ are shown in Table 1. The remaining genes modified by IFN-γ and other cytokines will be described in a separate publication (S.A.S., B. Kutlu, R.W., A. Valentine, H.W.D., A.W., R.G.G., J.C.H., unpublished data) and will also be accessible at http://genespeed.uchsc.edu. The induction of IDO and WARS by IFN-γ and the further potentiation by the addition of TNF-α and IL-1β was confirmed by quantitative real-time PCR (Q-RTPCR) using the 5′ nuclease Taqman assay (Fig. 2). The induction of IDO and WARS transcription occurred within 6 h, peaked between 24 and 48 h, and was sustained for up to 86 h (Fig. 3). Consistent with the microarray data, no significant changes were seen in the expression of KYN under these conditions (Figs. 2 and 3). No difference in β-cell apoptosis between control and cytokine-treated islet preparations was noted by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling at the end of 24 h (data not shown), although previous studies have suggested that chronic exposure to these cytokine combinations may induce functional changes in human islets after 2–6 days (23) and apoptotic death of the β-cells after 6–9 days (24).

Detection of IDO protein and enzyme activity.

Western blot analysis of human islets after exposure to IFN-γ alone or in combination with TNF-α and IL-1β showed the induction of a single 45-kDa IDO immunoreactive component, a size consistent with that of the primary translation product (45,331 Da). IL-1β and TNF-α alone had little effect and did not appear to potentiate the response to IFN-γ, although synergism with IL-1β and TNF-α has been reported previously (25). Basal IDO protein expression in COS7 was undetectable but reached a level of expression that was 10- to 20-fold higher than treated islets after transfection with a cDNA construct under a cytomegalovirus promoter (Fig. 4).

IFN-γ signaling occurs through the JAK-STAT pathway, leading to the phosphorylation of STAT1α and its translocation to the nucleus, where it initiates transcription (26,27). Western blot analysis detected STAT1α protein (91 kDa) in the control sample and induction of expression with cytokine treatment, most noticeably with IFN-γ–treated tissue (Fig. 4). Blotting with an antibody specific to the transcriptionally active phospho-form of STAT indicated marginal induction by TNF-α and marked increase in response to IFN-γ alone or in combination with IL-1β and TNF-α (Fig. 4).

IDO enzyme activity in intact islet cells was quantified by the determination of the appearance of KYN in the islet culture media after 24 h of exposure to cytokines (Figs. 5 and 6). Treatment with IFN-γ but not IL-1β or TNF-α increased KYN production by the islets (Fig. 5), an effect that was partly suppressed by co-incubation with IL-4. The IDO competitive substrate inhibitor 1-MT blocked the KYN production induced by IFN-γ, without having any effect on the basal level of KYN in the medium. Determination of IDO catalytic activity in islet homogenates after treatment for 24 h with cytokines demonstrated the induction of IDO by IFN-γ but not with other cytokines. IL-4 pretreatment did not significantly induce IDO enzyme activity but countered the effect of IFN-γ. 1-MT inhibited the activity induced by IFN-γ (Fig. 6), as expected. Homogenates of nontransfected COS7 cells showed negligible basal IDO enzyme activity but marked increase upon transfection with a plasmid construct encoding human IDO under the cytomegalovirus promoter. The observed changes in IDO protein expression and enzyme activity suggested that IDO mRNA was translated into enzymatically active protein, although perhaps with a lesser efficiency than may have been indicated by the fold changes in IDO transcripts after cytokine incubation.

Immunohistochemical analysis performed on human islets under basal conditions demonstrated the presence of strong IDO immunoreactivity in a few scattered islet cells (Fig. 7). These included β-cells, as demonstrated by their immunoreactivity for transcription factor PDX1, but also other endocrine cells that showed a similar nuclear morphology and chromogranin immunostaining but lacked PDX-1 immunoreactivity. An increase in the number of IDO immunopositive cells was observed in response to IFN-γ treatment that included both PDX1 immunoreactive β-cells and other cells (Fig. 7). The cytokines IL-1β and TNF-α did not produce an obvious change in the number or intensity of staining, and in combination with IFN-γ, they appeared to decrease the number of immunopositive cells. In this respect, the response appeared to parallel the effects of these agents on the protein expression level (Fig. 4) more than the mRNA response (Fig. 1). As in the case of the basal immunostaining, it was not possible to ascribe the increased staining with IFN-γ treatment to a specific cell type, although it was clear from the distribution of immunoreactivity that it included PDX1-positive β-cells and other endocrine and nonendocrine cells.

DISCUSSION

Changes in Trp metabolism are likely to play a role in the ability of IFN-γ to induce a tolerizing phenotype in splenic CD8 dendritic cells and of CTLA4-Ig to correct the defective tolerogenesis young type 1A diabetic NOD mice (28). Further support for a tolerogenic role for IDO in islets in vivo has come from the observation that adenovirus-mediated IDO expression in transplanted NOD islets prolonged their survival in NOD/SCID recipients that received diabetogenic splenocytes (29). These effects are in part attributed to local depletion and perturbation of the function of activated B and T lymphocytes by local depletion of free Trp (19,30). It is not clear whether such in vitro observations have a counterpart in the interaction of antigen-presenting cells and effector and regulatory populations of T-cells in the insulitic lesion in the pancreas and whether the endocrine cells can participate in the process. IDO has not previously been described as a significant gene transcript in the pancreatic islet, as evidenced by the low frequency of appearance in pancreas cDNA libraries and its representation in Unigene EST gene profiling analyses (www.ncbi.nlm.nih.gov/Unigene/ESTProfileViewer) (1 clone from 14,299 expressed sequence tags in human islets). Nevertheless, IDO transcripts have been documented in recent microarray profiling experiments on islets incubated in vitro with cytokine, especially in response to IFN-γ (31).

In this study, we demonstrated that IFN-γ alone or in combination with IL-β and TNF-α induces >100-fold the level of IDO mRNA in human islets obtained from heart-beating cadaveric donors. These changes were accompanied by a concomitant increase in IDO protein in human islets and IDO enzymatic activity measured both in intact cells and in islet homogenates. These responses were largely attributable to an IFN-γ effect and not to a nonspecific effect on islet viability. IFN-γ under these conditions does not induce apoptosis, although this can be achieved with cocktail of IL-1β, TNF-α, and IFN-γ after several days of exposure in culture (21,24). Such a cocktail further increased IDO mRNA relative to IFN-γ alone but paradoxically appeared to reduce the level of active enzyme. This may in part be attributable to the production of reactive nitrogen species via IL-1β signaling through nuclear factor-κB to upregulate inducible nitric oxide synthase (32), which in concert with SOD produces peroxynitrite-mediated nitrosylation of tyrosine residues and inactivation of STAT1 and its phosphorylation (33). The inverse correlation between IDO induction and the apoptotic reaction to cytokines suggests that IDO is not deleterious and may even play a protective role.

Although Trp depletion is the favored mechanistic explanation for the tolerogenic effect of IDO, the Trp downstream catabolites KYN and picolinate can directly inhibit T-cell proliferation (34,35), suggesting that other elements of the KYN pathway can also significantly influence cellular function. The effects of chronic β-cell exposure to KYN are unknown, although its metabolites 3-OH-KYN and 3-hydroxyanthranilate can acutely block leucine-stimulated insulin secretion by rat islets (36). Another metabolite of Trp, quinolinate, is a potent agonist of NMDA (N-methyl-d-aspartate) receptors, which during central nervous system infection and AIDS-dementia complex has been implicated in neurotoxicity induced by activated macrophages (37). Both pancreatic β-cells (38) and T-cells (39) express glutamate receptors that may be relevant in this regard. Compared with TDO, IDO has a high Km for molecular oxygen with little activity expressed <100 μmol/l (40). It has been suggested that endogenous electron donors for IDO are superoxides generated from tetrahydrobiopterin, flavoenzymes, xanthine oxidase, gluthathione reductase, or reduced flavin or pyridine nucleotides (10) and that the enzyme may play a protective biological role as an antioxidant (41) and by suppression of the cell surface expression of major histocompatibility complex (MHC) class I molecules (42).

IDO is only one of many gene transcripts that are regulated by IFN-γ in islets. Several genes from the JAK-STAT signaling pathway showed increased expression, including JAK2, IRF-1, and STAT1α (Table 1). Downstream genes, notably the chemokines CCL5/RANTES, CXCL9, CXCL10, and CXCL11, were upregulated >25-fold (Table 1). The extravasation and recruitment of lymphocytes to the islet in response to these signals would likely exacerbate insulitis and result in further exposure of the tissue to inflammatory cytokines (43). Increased expression of Trp-tRNA synthase (WARS) in response to IFN-γ might compensate for a reduced availability of Trp for protein synthesis resulting from IDO activation. An anti-inflammatory role for WARS is also conceivable because the vascular endothelial WARS is proteolytically processed to an anti-angiogenic factor (44,45)

Elevated circulating levels of CXCL9, CXCL10, and CXCL11 are reported in type 1 diabetes in human serum (40) and suggest that the response to IFN-γ seen here in vitro may model a physiologically relevant response. Further information is needed concerning the spatio-temporal pattern of expression of IFN-γ–responsive genes and their modifiers such as IL-4 and IL-4 receptors. The available immunohistochemical data suggest a complex pattern of IDO expression in human islets (Fig. 7) that encompass a variety of cell types, including a heterogeneous pattern of expression in the major endocrine cell types, including PDX1-positive β-cells. Vascular endothelium, macrophages, and dendritic cells are other potential sites of IDO induction by cytokines based on studies in the other tissues, although these cell types could not account for the overall pattern of islet expression that we observe. Such complexity is perhaps not unexpected given that within the pancreas, islets that are heavily infiltrated with mononuclear cells can occur immediately adjacent to the others that are morphologically normal, both in long-standing human type 1 diabetic subjects and at any stage of disease development in NOD mouse. It attests to the importance of local factors and the dynamic instability of the inflammatory reaction. Within this scenario, we postulate that in the short-term, IDO activation may protect islets from cytotoxic damage through depletion of superoxides and maintenance of redox potential and perhaps a reduction in MHC class 1 surface expression. The release of Trp metabolites such as 3-OH-KYN, 3-hydroxyanthranilate, quinolinate, and picolinic acid could also provide bystander inhibition of immune cell function. However, in the longer term, the metabolites of Trp may adversely affect the endocrine cells, particularly the β-cell that has a low antioxidant capacity but generates reactive oxygen species through mitochondrial electron transfer as part of the sensing of metabolizable secretagogues. In addition, further downstream metabolites derived from NAD such as ADP ribose, NAADH, and cyclic ADP ribose could lead to intracellular Ca2+ build up to toxic levels and ultimately lead to β-cell attrition.

FIG. 1.

Expression of IDO, WARS, and KYN in human islets after 24 h of incubation with cytokines in Miami medium supplemented with 0.5% human serum albumin. The normalized intensity (log scale) from data obtained on HG U133 Plus 2.0 Affymetrix chip is shown. Each data point is the mean ± SE of three to four observations.

FIG. 2.

Expression of IDO, WARS, and KYN mRNA by quantitative PCR. Islets were cultured for 24 h in the presence of indicated cytokines, and Q-RTPCR was performed using a 5′ nuclease assay and FAM dye–labeled Taqman MGB probes with two PCR primers. Endogenous HPRT was used for normalization. Data (means ± SD, four donors) were quantified using 2−ΔΔCT method in log scale and expressed relative to an islet sample incubated in medium alone. *Significant differences between control and IFN-γ (P = 9.65E-05) or cytokine mixture (P = 0.000429).

FIG. 3.

Temporal expression of IDO, WARS, and KYN gene transcripts. Q-RTPCR in response to 500 units/ml IFN-γ (see legend to Fig. 2). Data (means ± SD, three donors) were quantified using 2−ΔΔCT method in log scale and expressed relative to an islet sample incubated for time zero.

FIG. 4.

Western blot analysis of IDO, STAT1α, and pSTAT1α expression. Islets were cultured for 24 h with indicated cytokines, and samples (50 μg protein) were electrophoresed on 10% SDS-PAGE before blotting with antibodies specific for IDO (45 kDa) and STAT1α (91 kDa). The blots were re-probed for phosphoSTAT1α (91 kDa). COS7 cells transfected with human IDO were used as controls. Similar results were obtained from three different donors.

FIG. 5.

KYN production by human islets in response to cytokines. Groups of 1,000 human islets were cultured for 24 h in 1 ml Miami medium under the indicated conditions, and l-KYN in the medium was assayed colorimetrically with Ehrlich’s reagent. COS7 cells transfected with human IDO plasmid served as controls. Results are the means ± SE (n = 4). *Significant differences between control and IFN-γ (P = 9.43E-05) and the inhibitory effect of IL-4 (P = 0.0006) and MT (P = 2.07E-06) on IFN-γ–induced KYN production (three donors).

FIG. 6.

IDO enzyme activity in cytokine-treated human islets. Islets were cultured as indicated for 24 h with cytokines then homogenized and assayed for IDO activity. COS7 cells transfected with human IDO plasmid were used as controls. IL-4 was added to the culture medium and 1-MT to tissue homogenate before assay. *Significant differences between control and IFN-γ (P = 0.009), cytokine mixture (P = 0.009), and the inhibitory effect of IL-4 on IFN-γ–induced IDO induction (P = 0.032) (three donors).

FIG. 7.

Cytokine-induced IDO immunoreactivity in human islets. Human islet preparations were exposed to IFN-γ for 24 h before double immunohistochemical localization of PDX1 (red) and IDO (green). A and B: Basal expression of IDO is seen in islet preparations incubated with media alone. C and D: IDO is upregulated in PDX1-positive β-cells and other non–β-cells. The nuclei were stained with Hoechst 33258 (blue). The scale bar represents 20 μm. The figure is representative of n = 4 experiments.

TABLE 1

Microarray analysis of most responsive IFN-γ–regulated transcripts in human islets

Acknowledgments

S.A.S. was a Juvenile Diabetes Research Foundation postdoctoral fellow and has received Juvenile Diabetes Research Foundation Grant 02-05-60294) and the provision of pancreatic islets and core resources from the Islet Cell Resource Center at the University of Colorado at Denver and Health Sciences Center (National Institutes of Health Grant 5-U42-RR-016599) and the Diabetes and Endocrinology Research Center cores (National Institutes of Health Grant P30-DK-57516).

We thank Dr. Chris Wright (School of Medicine, Vanderbilt, TN) for the gift of PDX1 antibody. We thank Travis Still and Iyabo Osifeso for their assistance with histology and Tony Valentine and Joshua Beilke for the isolation of islets.

Footnotes

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

    • Accepted September 19, 2006.
    • Received May 4, 2006.

REFERENCES

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