Regulation of TCR-induced IFN-{gamma} release from islet-reactive non-obese diabetic CD8+ T cells by prostaglandin E2 receptor signaling

Vidya Ganapathy, Tatyana Gurlo, Hilde O. Jarstadmarken and Hermann von Grafenstein

School of Pharmacy, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA

Correspondence to: H. von Grafenstein


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Prostaglandins (PG) are released during tissue injury and inflammation, and inhibit immune responses at many points. PG may be one of several factors that protect not only against injury-induced, but also spontaneous, organ-specific autoimmune disease. Here we show that the production of PGE2, normally produced at a very low rate in islets of Langerhans, is significantly increased in inflamed islets of non-obese diabetic (NOD) mice. We investigated a possible role of PGE2 in controlling TCR-dependent release of IFN-{gamma} from islet-reactive NOD CD8+ T cells. PGE2 inhibited anti-TCR antibody-triggered release of IFN-{gamma} from CD8+ T cell clone 8D8 and from polyclonal cytotoxic T lymphocytes (CTL). Using receptor subtype selective agonists, we present evidence that the effect of PGE2 is mediated by EP2 and EP4 receptors, both of which are coupled to an increase in intracellular cAMP production. The cAMP analogs 8-Br-cAMP and Sp-cAMPS mimic the effect of EP2/EP4 receptor agonists, inhibiting TCR-triggered IFN-{gamma} release from NOD CD8+ T cells in a dose-dependent manner. The inhibitory effect of PGE2 was largely reversed by IL-2 added at the time of culture initiation and decreased with increasing strength of stimulation through the TCR. Resting CTL were more sensitive to PGE2 than recently expanded CTL and NOD CD8+ T cells remained insensitive to PGE2 for a longer time than BALB/c cells. Our study suggests that PGE2 may be part of a regulatory network that controls local activation of T cells and may play a role in the balance between the development of islet autoimmunity or maintenance of tolerance.

Keywords: cell cell interactions, cytokine, cytotoxic T lymphocyte, diabetes, rodent


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The non-obese diabetic (NOD) mouse is an excellent animal model for the study of the pathogenesis of insulin-dependent diabetes mellitus (IDDM) because it shares many features with the human disease (1,2). IDDM is an organ-specific autoimmune disorder characterized by chronic inflammation of the islets of Langerhans and by T cell-mediated destruction of islet ß cells (1,3). In NOD mice and in humans, the etiology of IDDM is considered to be multifactorial with disease development requiring the interactions of a multitude of cells, which coordinate their function by means of contact-dependent and soluble factors. Some of these factors and the cells that produce them are thought to be disease promoting while others are considered to be protective. IFN-{gamma}, produced by Th1 CD4+ T cells and CD8+ T cells, has been implicated as a key disease-enhancing cytokine (4), while IL-4 produced by Th2 CD4+ T cells as well as systemic IL-10 and transforming growth factor-ß are considered to be protective factors (5). Although the Th1–Th2 dichotomy may be an oversimplification, an imbalance of positive and negative regulatory signals may influence the development of autoimmune disease (3). It is therefore essential to identify and characterize not only factors and mechanisms that promote disease, but also those that limit or delay anti-islet immunity and have the potential to protect against disease development.

Prostaglandin (PG) E2 is a candidate protective factor because it leads to the down-regulation of T cell-dependent immune responses. It has been proposed that one of the functions of PGE2 is to protect against immunity to self antigens when released during tissue injury and inflammation (6), and PGE2 is thought to mediate some of the immunosuppressive effects of tumors (7,8). The same reasons may argue for a protective role of PGE2 against spontaneous autoimmune disease, although this possibility has not been addressed in detail. In the experimental autoimmune encephalomyelitis model, local synthesis of PGE2 was found to be associated with the resolution of brain lesions after the induction of oral tolerance (9). Recent studies have provided evidence for cytokine-induced synthesis of PGE2 in islets (10). Although in that study PGE2 was proposed to mediate ß cell cytotoxic effects of pro-inflammatory cytokines, it is equally plausible that PGE2 has a protective effect against IDDM given its well-known immunosuppressive function. An increase in the production of PGE2 in islets was found to be associated with the protective effect of oral insulin (11). A more detailed understanding of PGE2's multiple effects on T cell subsets and their function is clearly necessary before an understanding of its role can be reached.

Most of the general information about the effect of PGE2 on T cell development and function that is available supports a potential protective role of PGE2 in IDDM. PGE2 has been implicated in the differentiation of CD4+ T cells and the activation of subsets of CD4+ T cells. Thus, it has been shown that PGE2, when present at the time of T cell priming, facilitates the in vitro differentiation of naive CD4+ T cells into Th2 rather than Th1 cells (12). Recently, it has been suggested that PGE2 may influence the differentiation of Th subsets indirectly, through effects on antigen-presenting cells, and that this mechanism is defective in autoimmune prone animals, thereby favoring Th1 cell development (13). Subtype-specific effects of PGE2 were also observed for differentiated Th1 and Th2 cells. PGE2 was shown to inhibit IL-2 and IFN-{gamma} production in both short- and long-term clonal Th1 T cells, while IL-4 production by Th2 T cells was not inhibited and IL-5 was slightly enhanced (14,15). Although this differential effect was complicated by the fact that it was dependent on the presence of exogenously added IL-2 (15), it implies that Th1 cells are more sensitive to the inhibitory effect of PGE2 than Th2 cells. Since Th1 cells and their cytokines are thought to promote development of IDDM, whereas Th2 cells are thought to be protective (5) or neutral (4), these data suggest that PGE2 may play a role in disease development that has until now not been recognized. A similar dichotomy of T cell phenotypes is thought to exist in CD8+ T cells (16). We and others have proposed that CD8+ T cells are an important source of IFN-{gamma} during the development of IDDM (17,18, see also 4). In contrast to the considerable amount of information that is available on the influence of PGE2 on CD4+ T cell function, little is known as to how PGE2 affects CD8+ T cells. In previous studies we have shown that IL-2-driven proliferation of CTLL-2 cells, a cell line derived from CD8+ T cells, could be inhibited by PGE2 (19). However, the role of PGE2 in the regulation of TCR-mediated CD8+ T cell responses such as release of IFN-{gamma} is not known.

PGE2 mediates a broad range of physiological effects in different tissues. The diversity of PGE2 effects can be attributed to the molecular diversity of receptors (20). Four subtypes of pharmacologically active PGE2 receptors differing in their mode of signal transduction have been characterized (2124). All known PGE2 receptor subtypes, termed EP1–EP4, are coupled to intracellular signaling via heterotrimeric GTP-binding proteins. EP1 receptors activate phosphatidylinositol turnover and intracellular Ca2+ release via a poorly characterized G-protein-mediated mechanism (25,26). EP2 and EP4 receptors are coupled via Gs protein to an increase in intracellular cAMP production (20,21,27,28). EP3 receptors exist in multiple forms that differ in their cytoplasmic tail that couple the receptor to intracellular different signaling pathways, but have the same extracellular ligand binding characteristics (22,23).

Most studies on intracellular signaling events following PGE2 receptor activation have focused on EP2/EP4 receptors expressed by CD4+ T cells and events downstream of receptor-induced cAMP production such as inhibition of IL-2 gene expression (24,29). Although some evidence is available suggesting that regulation of IFN-{gamma} synthesis has elements in common with IL-2 gene expression (30,31), IL-2 and IFN-{gamma} are not regulated concordantly in all situations (32). Anergic cells, for example, are characterized by an inability to produce IL-2 and to proliferate (33) while maintaining effector function such as production of IFN-{gamma} (34,35). Little is known about a role of PGE2 in regulating IFN-{gamma} production in CD8+ T cells and any signaling mechanisms that might mediate such PGE2-induced effects.

In the studies reported here we demonstrated that PGE2 is produced by inflamed but not healthy NOD islets, and determined the effect of PGE2 on TCR-dependent release of IFN-{gamma} from cloned and polyclonal islet-reactive NOD CD8+ T cells. Using receptor subtype-specific agonists we identified PGE2 receptor subtypes and intracellular signals that mediate the effects PGE2 on IFN-{gamma} release. We also investigated how PGE2 signaling can be overcome and how it is integrated with other signals that act on T cells. Significant differences between NOD and BALB/c mice in the time-dependent TCR–PGE2 signal integration may point to a disease-promoting abnormality in NOD mice.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and reagents
PGE2, 8-bromo-cAMP, avidin–horseradish peroxidase, o-phenylenediamine (OPD), murine rIL-2 and murine rIFN-{gamma} were obtained from Sigma (St Louis, MD). Misoprostol, sulprostone and a PGE2 enzyme immunoassay kit were obtained from Cayman Chemicals (Ann Arbor, MI). Sp-cAMPS and 19(R)OH-PGE2 were obtained from Biomol (Plymouth Meeting, PA). Murine rIL-7 was purchased from Life Technologies (Gaithersburg, MD). C18 columns were obtained from Waters (Milford, MA). NS-398 (36) was purchased from Calbiochem (La Jolla, CA). Indo-1-AM was obtained from Molecular Probes (Eugene, OR).

TCX6310 cells were kindly provided by Dr F. Melchers (Basel Institute for Immunology). Hybridomas H57-597 (anti-{alpha}ßTCR), GK1.5 (anti-CD4) and 3.155 (anti-CD8) were purchased from ATCC (Rockville, MD), and hybridoma YCD3-1 (anti-CD3{varepsilon}) was a kind gift from Dr C. A. Janeway, Jr (Yale University). Anti-{alpha}ßTCR mAb was purified from hybridoma culture supernatant using GammaBind Plus Sepharose (Pharmacia Biotech, Piscataway, NJ) columns. Anti-CD3, anti-CD4 and anti-CD8 mAb were used in the form of diluted hybridoma supernatant. FITC–goat F(ab')2 anti-rat IgG antibody was obtained from Caltag (South San Francisco, CA).

The tissue culture medium (TCM) used for cell culture and for all experiments was based on Click's medium (Irvine Scientific, Santa Ana, CA) which was supplemented with 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), 40 µM ß-mercaptoethanol (Sigma) and 10% FBS (Hyclone, Logan, UT). For routine T cell culture we used the supernatant of TCX6310 cells (37) as a source of IL-2.

Mice
NOD, B10.BR and BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME), and were bred and maintained in the USC animal facility under pathogen-free conditions. The spontaneous incidence of diabetes in our NOD colony reaches 65–70% in female mice by 20 weeks of age and diabetes usually commences by 13 weeks of age. For experiments, 8- to 12-week-old mice were used.

PGE2 production in islets
Islets were prepared by collagenase digestion as described elsewhere (38). Islets were cultured for 16 h and the number of mononuclear cells emanating from them was monitored. Islets were then divided into two groups: those that did not show any sign of infiltration and those that were severely infiltrated as indicated by having none or >10 cells around them respectively. Between 25 and 50 islets were cultured in 250 µl of TCM for 40 h and PGE2 release was measured by competitive ELISA (Cayman PGE2 enzyme immunoassay kit). PGE2 was extracted from the culture supernatant as described by Powell (39). Briefly, two parts (typically 400 µl) of ethanol/water (10/90, v/v) were added to one part (typically 200 µl) of the supernatant to be assayed. The resulting mixture was adjusted to pH 3.0 (0.1 N HCl) and applied to a pre-activated 3 ml C18 column. Column pre-activation and extraction of PGE2 were performed following the Cayman enzyme immunoassay kit protocol.

Islet-reactive polyclonal CTL and clones
The generation of islet-reactive CTL and cloning of CD8+ T cells is described in detail elsewhere (18). Briefly, spleen cells from newly diabetic female NOD mice were cultured with NOD islets of Langerhans in the presence of IFN-{gamma} (10 U/ml), IL-2 (10 U/ml) and IL-7 (10 ng/ml) for 5 days. T cell clusters surrounding disintegrating islets were picked using a pipette and pooled. Cells were re-stimulated with islets or the ß cell line NIT-1 (40) 4 times at 7–10 day intervals. Polyclonal CTL obtained this way potently destroyed islets and were used for cloning. For cloning by limiting dilution, T cells were stimulated using anti-CD3 mAb and mitomycin C-treated NOD spleen cells as described (18). In this study we used CD8+ T cell clone 8D8, which was one of 18 clones obtained.

For routine maintenance, CTL and 8D8 cells were stimulated with anti-CD3 mAb every 3–4 weeks. T cells (1–5x105) were cultured with mitomycin C-treated spleen cells (5x106) in 5 ml of TCM in the presence of YCD3-1 (anti-CD3{varepsilon} mAb) cell culture supernatant diluted 1:20. After 48 h, T cells were washed and treated with 40 U/ml IL-2 and 10 ng/ml IL-7. Two days later cells were fed once more with the same medium, and after that every 3–4 days with TCM alone or, during every other feeding cycle, with TCM supplemented with IL-2 and IL-7 (10 U/ml and 10 ng/ml respectively). In most experiments T cell clones were used 15 days post-stimulation. Dead cells were removed using Lymphocyte Separation Medium (Organon Teknika, Durham, NC). In some experiments T cells were stimulated with anti-CD3 mAb and splenocytes to study the influence of full T cell activation versus return to a resting state on the sensitivity to PGE2. In these experiments the anti-CD3 mAb was removed after 48 h by washing the cells as above. The stimulatory effect of any remaining anti-TCR antibody bound to Fc receptors of splenocytes does not last beyond 48 h, because most antigen-presenting cells in splenocytes die during this time. After this 48 h stimulation period, T cells were maintained in culture without stimulation for various time intervals and then used for experiments.

Polyclonal CD8+ T cells
Spleen cells (1–2x106) were stimulated with anti-CD3 mAb and splenocytes, and cultured as above. To purify CD8+ T cells, cultured spleen cells were incubated with anti-CD4 mAb (supernatant from hybridoma GK1.5, diluted 1 in 3) for 30 min on ice, washed and treated with diluted (1:10) pooled rabbit complement (ICN, Irvine, CA) for 45 min at 37°C. The resulting cell preparation was analyzed by flow cytometry and was found to contain >95% of CD8+ T cells.

Flow cytometry
For the detection of CD8-expressing cells, polyclonal cells were stained with anti-CD8 mAb 3.155 and with FITC–F(ab')2 goat anti-rat IgG as second-step antibody. Cells were analyzed using a FACStar flow cytometer (Becton Dickinson, San Jose, CA).

Stimulation of IFN- {gamma} release from T cells
Unless otherwise indicated, IFN-{gamma} release from T cells was stimulated using anti-{alpha}ßTCR mAb H57-597 immobilized in tissue culture plates (Falcon; 96-well, flat-bottom plate coated with 1 µg/ml mAb in PBS at 4°C overnight). Clonal T cells were seeded in antibody-coated plates at a density of 1x104 cells/well and polyclonal CD8+ T cells at 3x104 cells/well. Immediately after seeding the cells, PGE2, PGE2 agonists or cAMP analogs were added to the culture at the concentrations indicated in the figure legends. After 24 h, supernatant was collected and stored at –80°C. PGE2, misoprostol and sulprostone were dissolved in DMSO and dilutions were made in TCM. The final concentration of DMSO in culture was less then 0.18% and at that concentration did not have any influence on IFN-{gamma} release from T cells (data not shown).

IFN- {gamma} assay
The concentration of IFN-{gamma} in the culture supernatant was measured by sandwich ELISA using paired anti-cytokine mAb (PharMingen, San Diego, CA), following protocols recommended by the manufacturer. Primary mAb was immobilized in Immulon-4 plates (Dynatech, Boston, MA). The sensitivity of the assay was 1 U/ml (67 pg/ml).

Measurement of intracellular free calcium in P815 cells
P815 cells, known to express EP3 receptors coupled to an increase of intracellular free calcium (41), were used as a control for the efficacy of the EP3 receptor agonist sulprostone. To load P815 cells with Ca2+-indicator, they were incubated with 3 mM of Indo-1-AM for 30 min at 37°C. The cells were then washed and kept in the form of a pellet at 4°C until assayed. The cell pellet was resuspended in serum-free tissue culture medium prior to the assay. Indo-1-AM-loaded cells (5x106) were stimulated with PGE2, sulprostone (10–6 M) or incubated with digitonin to determine maximal intracellular free calcium. The fluorescence intensity was recorded online with a fluorescence spectrophotometer.

Measurement of uterine contractions
Uteri were excised from NOD mice, cut in half and mounted in a tension transducer. The bath solution was HBSS (Life Technologies), kept at 37°C and bubbled with 95% O2/5% CO2. Various concentrations of PGE2 or sulprostone were added and tension development was monitored.

Curve fitting and statistical analysis
Theoretical dose–effect curves were fitted to the data as described earlier (19). Briefly, the following expression was used for the concentration-dependent effect of PGE2:


ln(L) ranges from 1 (full inhibition) to 0 (no inhibition). L (ligand) refers to the concentration of PGE2, n is the number of theoretical binding sites and ki are individual binding constants of these sites. {alpha}i is the relative contribution of site i to the total inhibitory effect when it is fully occupied by ligand. The sum of all these contributions is 1 (i.e. 100%). For fitting of ln(L) to the experimental data, the sum of square deviations of all data points from the theoretical curve was set to a minimum by varying the binding constants ki, as well as {alpha}i. For each experiment a scaling factor was used that allowed the data to be fitted to a curve ranging from 1 to 0. Statistical significance of a second or third binding constant was assessed by calculating the F ratio and corresponding P values due to the introduction of the additional constant as compared to a simpler model without it (42). P < 0.001 was considered to be significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Endogenous production of PGE2 in islets
During development of diabetes in the NOD mouse, the islets of Langerhans become infiltrated with mononuclear cells. This phenomenon, also known as islet inflammation or insulitis, exhibits a large degree of heterogeneity at any one time. Heavily inflamed islets and islets undergoing destruction co-exist with seemingly healthy islets in the same pancreas. Because PGE2 is produced at sites of inflammation, it is likely that inflamed NOD islets produce this mediator. To address this question, islets were isolated from pancreata of NOD mice and inflamed islets were separated from non-inflamed islets based on the number of mononuclear cells emanating from them during an overnight culture (see Methods and 18). Figure 1Go shows that inflamed islets produce significant amounts of PGE2, in contrast to islets that do not show signs of inflammation. The amount of PGE2 produced by non-inflamed islets was comparable to that produced by B10.BR islets and could be increased somewhat by stimulation with lipopolysaccharide, but never reached the high levels produced by inflamed NOD islets. PGE2 production by these islets was catalyzed by the inducible form of cyclooxygenase as it was inhibited by NS-398, a specific inhibitor of this enzyme.



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Fig. 1. PGE2 release from islets. Islets from B10.BR mice (B10.BR), NOD islets without any sign of infiltration (NOD) or severely infiltrated NOD islets (NODinf) (see Methods) were cultured for 40 h in the presence or absence of lipopolysaccharide (10 µg/ml) as indicated. The specific COX-2 inhibitor NS-398 (10 µM) (36) was added as indicated. Data are the mean ± SEM of three measurements.

 
PGE2 inhibits TCR-dependent IFN-{gamma} release
PGE2 produced by inflamed NOD islets is likely to affect the function of cells in the inflammatory infiltrate, including CD8+ T cells. The following experiments were conducted to investigate the effects of PGE2 on islet-reactive CD8+ T cells. Throughout this study, the NOD CD8+ T cell clone 8D8 was used which had been described earlier (18). This clone destroys islets in vitro and releases IFN-{gamma} in response to islets. Before studying PGE2 effects on IFN-{gamma} release, it was of interest to determine the time course and dose range of anti-TCR antibody for triggering IFN-{gamma} secretion from 8D8 cells. Two weeks after expansion with soluble anti-TCR antibody and splenocytes, 8D8 cells were stimulated with immobilized anti-TCR antibody in the absence of splenocytes. To do this, 8D8 cells were added to tissue culture plates coated with varying concentrations of anti-TCR antibody. In contrast to stimulation with anti-TCR antibody and splenocytes, stimulation with anti-TCR antibody alone did not cause proliferation of 8D8 cells (data not shown) but effectively triggered IFN-{gamma} release. A concentration of 1 µg/ml of anti-{alpha}ßTCR antibody in the coating buffer generated a half-maximal response and we chose this concentration for studies of PGE2 effects. We next performed time course studies of IFN-{gamma} release from TCR-activated clone 8D8. After 24 h, IFN-{gamma} release began to level off and had reached a level one half of that released by 96 h (data not shown). We chose the 24 h time interval to study the PGE2-dependent modulation of TCR triggered IFN-{gamma} release.

To determine the effect of PGE2 on IFN-{gamma} release from cloned islet-specific CD8+ T cells, 8D8 cells were activated with a half-maximal concentration of immobilized anti-{alpha}ßTCR antibody. Various concentrations of PGE2 were added at the same time. As shown in Fig. 2Go(A), PGE2 inhibited TCR-dependent IFN-{gamma} release in a dose-dependent manner. At a concentration of 10–6 M, PGE2 abolished the release of IFN-{gamma} from 8D8 cells. PGE2 inhibited polyclonal CTL (Fig. 2BGo) with similar potency as clone 8D8, indicating that this effect was a general characteristic of islet-reactive NOD CD8+ T cells and not restricted to the clonal cell line used in this study. The inhibitory effect of PGE2 was not due to toxicity, because the number of Trypan blue excluding cells did not decline, but rather increased slightly, after culture with PGE2 (data not shown).



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Fig. 2. PGE2 inhibits IFN-{gamma} release from clone 8D8 (A) and polyclonal CTL (B). T cells were stimulated with 1 µg/ml of immobilized anti-{alpha}ßTCR mAb in the presence of the indicated concentrations of PGE2. Data are the mean ± SEM of three experiments. Error bars are omitted in (B) because their size is comparable to that of the symbols.

 
EP2 receptors mediate the inhibitory effect of PGE2 on IFN-{gamma} release
There are several receptors for PGE2 that have different extracellular binding sites and affinities for PGE2, and its analogs and inhibitors. On the intracellular side, PGE2 receptor subtypes are coupled to different signaling pathways and consequently will interface differently with other signals acting on T cells. It is therefore important to know which of the known PGE2 receptor subtypes might be involved in the inhibition of IFN-{gamma} release. To address this question, we stimulated IFN-{gamma} release from clone 8D8 in the presence of various receptor subtype-specific agonists. As shown in Fig. 3Go(A), sulprostone, an EP1/EP3 receptor agonist, was ineffective up to a concentration of 10–5 M. Sulprostone was active because it increased the intracellular free calcium concentration in the mastocytoma cell line P815 and caused contractions of murine uterus tissue (data not shown). To further confirm that sulprostone was active under the conditions of our experiments, the agonist was cultured at 37°C in TCM for 16 h. When used after this culture period, sulprostone still potently induced contraction of uterine smooth muscle (data not shown). The data indicate that EP1/EP3 receptor subtypes are not involved in the PGE2-mediated inhibition of IFN-{gamma} release from NOD CD8+ T cells.



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Fig. 3. Effect of PGE2 agonists on TCR-dependent IFN-{gamma} release from clone 8D8. Cloned T cells were stimulated with immobilized anti-{alpha}ßTCR mAb (1 µg/ml) in the presence of the indicated concentrations of the EP1/EP3 agonist sulprostone (A), the EP2/EP3/EP4 agonist misoprostol (B) and the EP2 agonist 19(R)OH-PGE2 (C). Data are the mean ± SEM of four experiments.

 
In contrast, the EP2/EP3/EP4 receptor agonist misoprostol inhibited IFN-{gamma} release (Fig. 3BGo). Having ruled out EP1 and EP3 receptors, activation of EP2 and/or EP4 receptors are the remaining candidate mediators of PGE2 effects. The inhibitory effect of misoprostol occurred within the concentration range that would be expected from published constants of this agonist for binding to EP2 (2.5x10–7 M) and EP4 (6.7x10–8 M) receptors (43). To distinguish between EP2 and EP4 receptors we used 19(R)OH-PGE2, an agonist that is selective for EP2 receptors. The dose–effect curve for 19(R)OH-PGE2 showed that it was an effective inhibitor (Fig. 3CGo) and the Kd value was comparable to a published value (1.5x10–8 M) for this agonist (44). These data provide definitive support for EP2 receptors being involved in the effect of PGE2.

EP4 receptors contribute to suppression of IFN-{gamma} release by PGE2
To our knowledge, an EP4 selective agonist is not available at the present time that could be used to define the precise role of EP4 receptor subtype in the inhibition of IFN-{gamma} release. In order to evaluate whether the effect of PGE2 binding was due to more than one receptor subtype, we pooled the data from three experiments and performed curve-fitting analysis of the dose–effect curves (Fig. 4Go). Because the above findings have left only EP2 and EP4 receptors as candidate mediators of PGE2 effects, any evidence for two active sites would implicate EP4 receptors in addition to EP2 receptors. Our analysis showed that a model with two distinct PGE2 receptors fitted the data better than a model with one receptor (Fig. 4Go). The introduction of the second active binding site was highly statistically significant as judged by the F ratio (F = 19.97) and P value (P = 1.4x10–7) of the two- versus one-receptor model. The introduction of a third active site did not significantly improve the goodness of fit. The calculated binding constants for the two sites are 3.4x10–8 M (range 1.5–6.8x10–8 M) and 1.0x10–9 M (range 0.6–2.8x10–9 M) which are close to 1.2x10–8 M (range 0.9–1.5x10–8 M) and 1.9x10–9 M (range 1.5–2.5x10–9 M), the reported binding constants of PGE2 for EP2 and EP4 receptors respectively (43). This analysis provides evidence in support for the involvement of EP4 in addition to EP2 receptors. It also allows an estimate of the contribution of both receptor subtypes to the total inhibitory effect of PGE2. The EP4 receptor accounts for ~57 ± 6% (SEM) of the effect with EP2 receptors contributing the remaining 43%.



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Fig. 4. Dose–effect analysis of the PGE2 effect on IFN-{gamma} release from clone 8D8. Data from three experiments were pooled and theoretical curves corresponding to a one-receptor model (interrupted line) or a two-receptor model (solid line) were fitted to the data. Data from individual experiments were multiplied with a constant to fit on a scale ranging from 1 (no inhibition) to 0 (maximal inhibition). Residual IFN-{gamma} release at 10–5 M PGE2 was considered non-inhibitable and was subtracted from the data before curve fitting. Non-inhibitable IFN-{gamma} release was consistently <10% of maximal IFN-{gamma} release.

 
Analogs of cAMP mimic the inhibitory effect of PGE2 on IFN-{gamma} release
The above experiments with PGE2 agonists implicate EP2 and EP4 receptor subtypes in the effect of PGE2 on IFN-{gamma} release, both of which are coupled to adenylate cyclase. These results predict that the effect of PGE2 is caused by an increase of the concentration of intracellular cAMP. Therefore, cAMP analogs should inhibit IFN-{gamma} release from cloned CD8+ T cells. The data in Fig. 5Go(A) show that this is indeed the case. Both 8-Bromo-cAMP and Sp-cAMPS completely inhibited IFN-{gamma} release at a concentration of 10–3 M. Identical results were obtained when experiments were performed with polyclonal CTL (Fig. 5BGo). These data, together with those on PGE2 receptor subtype-specific agonists, strongly suggest that cAMP-coupled EP2/EP4 receptor subtypes are involved in PGE2-mediated inhibition of IFN-{gamma} release.



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Fig. 5 . Effect of cAMP analogs on TCR-dependent IFN-{gamma} release from clone 8D8 (A) and from polyclonal CTL (B). T cells were stimulated with 1 µg/ml of immobilized anti-{alpha}ßTCR in the presence of indicated concentrations of 8-Bromo-cAMP (•) and Sp-cAMPS ({blacktriangleup}). Data are the mean ± SEM of three experiments.

 
The sensitivity to PGE2 depends on the T cell activation state
Given the inhibitory effect of PGE2 on IFN-{gamma} release from CD8+ T cells at 2 weeks after expansion, it was important to know if these effects are similar in resting and activated CD8+ T cells. To address this question, we compared the sensitivity of NOD CD8+ T cells to PGE2 at different time points after full activation with anti-TCR antibody and splenocytes. When stimulated with anti-TCR antibody in the presence of splenocytes, the CD8+ T cells used in this study become fully activated and begin to proliferate. The role of splenocytes in this protocol is 2-fold: to cross-link the TCR by binding anti-TCR antibody via Fc receptors and to provide co-stimulatory signals. The contribution of splenocytes is essential for full activation since stimulation with anti-TCR antibody alone, soluble or immobilized, does not trigger proliferation, although it effectively elicits IFN-{gamma} release. After 48 h, stimulation was arrested by washing out the anti-TCR antibody in solution. Figure 6Go(A) shows that at 5–7 days after initiation of expansion, i.e. 3–5 days after arrest of stimulation, PGE2 has very little effect on IFN-{gamma} release from CD8+ T cells. At 8–9 days after expansion, CD8+ T cells still remain rather insensitive to PGE2. The concentration of PGE2 that is required for ~70% inhibition was close to 10–5 M and half-maximal inhibition occurred at ~10–7 M. In contrast, at 12–15 days after expansion, the sensitivity to PGE2 was restored. The dose for half-maximal inhibition had shifted to ~10–8 M. Therefore, the sensitivity to PGE2 is critically dependent on the time after proliferation-inducing signaling. Resting cells are more sensitive to PGE2 than recently expanded CD8+ T cells.



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Fig. 6. (A) Resting CD8+ T cells are more sensitive to the inhibitory effect of PGE2 than activated CD8+ T cells. At 5–7 (•), 8–9 ({blacksquare}) or 12–15 ({blacktriangleup}) days after expansion with anti-CD3{varepsilon} mAb and splenocytes, polyclonal CD8+ T cells were re-stimulated with immobilized anti-{alpha}ßTCR antibody (1 µg/ml) in the presence of the indicated concentrations of PGE2. The data are the mean ± SEM of three experiments. (B) IL-2 largely reverses the inhibitory effect of PGE2 on TCR-dependent IFN-{gamma} release from clone 8D8 and polyclonal CD8+ T cells. Cells were stimulated with immobilized 1 µg/ml anti-{alpha}ßTCR mAb in the presence or absence of 1x10–7 M PGE2. IL-2 (10 U/ml) was added, as indicated, at the time of stimulation. The data are the mean ± SEM of three experiments. (C) The inhibitory effect of PGE2 on IFN-{gamma} release from CD8+ T cells depends on the strength of stimulation through the TCR. At 8–9 days after expansion with anti-CD3{varepsilon} mAb and splenocytes, polyclonal CD8+ T cells were stimulated with 0.1 (closed bars) or 1 (open bars) µg/ml of immobilized anti-{alpha}ßTCR mAb in the presence of the indicated concentrations of PGE2. The data are the mean ± SEM of three experiments.

 
The inhibitory effect of PGE2 is blunted by exogenous IL-2
T cells integrate multiple signals and modify their response to antigenic stimulation, some of which may counteract the inhibitory effect of PGE2. IL-2 is among the signals that are known to modulate IFN-{gamma} production by CD8+ T cells (45). Although IFN-{gamma}-producing CD8+ T cells, such as those used in this study, frequently are poor producers of IL-2 (46), they may receive this cytokine from other cells particularly Th1 CD4+ T cells in the islet inflammatory infiltrate or in lymphoid tissue. It was therefore of interest to determine the role of exogenous IL-2 in PGE2-mediated inhibition of IFN-{gamma} release from CD8+ T cells. To address this question, we added IL-2 to our culture at the time of stimulation through the TCR together with a concentration of PGE2 that caused half-maximal inhibition of IFN-{gamma} release. For this experiment, 8D8 cells and polyclonal CD8+ T cells were used 2 weeks after expansion. In controls, either PGE2 or IL-2 was omitted. As shown in Fig. 6Go(B), we found that IL-2 at a concentration of 10 U/ml blunted the inhibitory effect of PGE2.

The TCR signal strength determines the sensitivity to PGE2
Signal integration by T cells can occur by tuning of signaling thresholds for antigenic stimulation. For example, co-stimulatory signals reduce the signal strength that is required for proliferation (32,47), whereas inhibitory signals may increase the TCR signaling threshold. The data above show that the responsiveness to PGE2 is diminished for some time after proliferation-inducing signals. This effect disappears with time. To investigate whether the diminished sensitivity to PGE2 after expansion of CD8+ T cells was due to such tuning of the TCR signaling threshold, we reduced the TCR signal strength at a time when the cells were still insensitive to PGE2. Figure 6Go(C) shows that reducing the anti-TCR concentration to 0.1 µg/ml restored the sensitivity to PGE2 even at 8–9 days after expansion. Therefore, signals that stimulate proliferation do not abolish sensitivity to PGE2, but alter the threshold at which TCR signals become sensitive to PGE2.

After activation NOD CD8+ T cells remain insensitive to PGE2 longer than BALB/c CD8+ T cells
The NOD mouse is prone to autoimmune disease, and this could in part be due to abnormalities in the integration of positive and negative regulatory signals. To address this question we compared the influence of PGE2 on CD8+ T cells derived from NOD and BALB/c mice. As shown in Fig. 7Go, we found that 5–7 days after expansion, CD8+ T cells from BALB/c mice were more sensitive to PGE2 than those from NOD mice. However, the sensitivity to PGE2 of CD8+ T cells derived from either strain was similar at 12–15 days after expansion. It therefore appears that NOD CD8+ T cells remain insensitive to PGE2 longer than CD8+ T cells from BALB/c mice.



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Fig. 7. Activated NOD CD8+ T cells are less sensitive to the inhibitory effect of PGE2 than activated BALB/c CD8+ T cells. NOD (squares) or BALB/c (triangles) polyclonal CD8+ T cells were re-stimulated, in the presence of indicated concentrations of PGE2, with immobilized anti-{alpha}ßTCR antibody (1 µg/ml) after 5–7 (closed symbols) or 12–15 (open symbols) days after expansion with anti-CD3{varepsilon} mAb and splenocytes. The data are the mean ± SEM of three experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our studies have shown that PGE2 inhibits IFN-{gamma} release from cloned NOD CD8+ T cells and polyclonal CTL. We have identified EP2 and EP4 receptor subtypes as mediators of this inhibition, and were able to conclusively exclude the involvement of EP1 and EP3 receptors. Both EP2 and EP4 receptors signal through the heterotrimeric GTP binding protein Gs by elevating the intracellular concentration of cAMP (20,21,27,28,48). Our finding that two different membrane permeable cAMP analogs mimic the effect of PGE2 receptor signaling confirmed a link between a rise in intracellular cAMP and inhibition of TCR-triggered IFN-{gamma} release in NOD CD8+ T cells.

In our experimental system T cell activation and IFN-{gamma} release occur in the absence of a second signal. It seems therefore likely that cAMP interferes at some point with downstream events of TCR signaling. What might be a possible point at which both signaling pathways converge? The transcriptional activator NF-AT is dephosphorylated by TCR-dependent activation of calcineurin but is phosphorylated by cAMP-activated protein kinase A (4951). NF-AT may therefore be one of several possible downstream points where TCR and cAMP signaling intersect. Regulation of IFN-{gamma} transcription is thought to be mediated by NF-AT binding sites within introns of the IFN-{gamma} gene (31). Signal convergence at NF-AT phosphorylation and dephosphorylation may also explain the effect of IL-2 since this cytokine also signals, among other pathways, through NF-AT (52).

The results of our study were obtained with islet-reactive CD8+ T cell clones isolated from diabetic NOD mice, a widely used animal model for IDDM. In NOD mice and humans, the development of IDDM is characterized by a lymphocytic infiltrate in and around the islets of Langerhans. It has been proposed that two distinct phases can be distinguished, early benign insulitis and a later malignant phase associated with islet ß cell destruction (53,54), but it is not known what triggers the transition. It has been suggested that the early benign infiltrate is dominated by IL-4-producing Th2 T cells, whereas the destructive phase is characterized by a dominance of Th1 T cells producing IL-2 and IFN-{gamma} (5). PGE2, synthesized in islets, may be one of the factors that influences the development and function of T cell subsets in the islet infiltrate because PGE2 inhibits IFN-{gamma} release from Th1 cells and drives polarization of T cell differentiation toward the Th2 phenotype. PGE2 may be another, as yet little recognized, but important protective factor preventing islet destruction during the early phase of inflammation. Recently, a similar dichotomy of T cell subsets has been described for CD8+ T cells and the subsets corresponding to Th1 and Th2 cells have been termed Tc1 and Tc2 cells (55). The islet destructive clones and polyclonal cells used in this study are potent producers of IFN-{gamma} and in that regard would be analogous to the Th1 subset of CD4+ T cells. We and others have postulated that CD8+ T cells play not only a role in killing ß cells but are also potent producers of IFN-{gamma} (18,56). The inhibition of IFN-{gamma} release by PGE2 described in this study suggests that PGE2 may be protective not only by inhibiting Th1 CD4+ T cells but also by way of its effects on CD8+ T cells. PGE2 may act as a negative regulator that limits activation of IFN-{gamma}-dependent events in the islet environment during the development of IDDM. PGE2 may act not only by inhibiting CD8+ T cell function, but also by promoting, together with IFN-{gamma}, the induction of negative regulatory CD8+ T cells (57,58), which may produce PGE2 themselves. Some of the suppressive effects of CD8+ T cells in tumor bearing hosts were attributed to PGE2-induced CD8+ T cells (7) and oral tolerance was thought to be mediated in part by PGE2-producing CD8+ T cells (9).

The data in this study show that large amounts of PGE2 are produced in inflamed islets. Given the role of PGE2 as a negative regulator of both CD8+ T cells (and of Th1 cells), how is it possible that disease still develops? In this study we have shown that the inhibitory effect of PGE2 on CD8+ T cells can be overcome by signals that lead to the expansion of CD8+ T cells. This change in sensitivity could be due to activation-dependent alterations of intracellular signal integration or to alterations of PGE2 receptor expression (59,60). In our system, the effect of PGE2 can be largely abrogated even in resting cells by IL-2, a Th1-derived pro-inflammatory factor. It is likely that PGE2 acts in concert with other protective factors such as IL-4 and transforming growth factor-ß, and it will be important to uncover the time-dependent interplay between several protective and disease-promoting factors for an understanding of the transition from benign to malignant insulitis and progression to IDDM. In this study, we report significant differences between the time-dependent sensitivity of NOD and BALB/c CD8+ T cells. After full activation, NOD cells remain insensitive to PGE2 for a longer time than BALB/c CD8+ T cells. Whether this difference is significant in regard to disease development remains to be determined. These data do suggest, however, that PGE2 may be a less effective negative regulator of CD8+ T cells in NOD mice than in BALB/c mice.

The putative role of PGE2 has implications with regard to the use of non-steroidal anti-inflammatory drugs in persons at risk of developing type I diabetes. Many of the currently used non-steroidal anti-inflammatory drugs block the increase of PGE2 synthesis during inflammation and may prevent important immunomodulating actions of PGE2 (61). Since PGE2 is inhibitory for CD4+ and CD8+ T cell effector function, reducing PGE2 levels over a long period of time could lead to an imbalance of positive and negative control of T cell function and adverse effects associated with this lack of control.


    Acknowledgments
 
This work was supported by National Institutes of Health grant DK49717. We thank Drs. Gunther Dennert and Sarah Hamm-Alvarez for critically reading this manuscript, and Donald MacDougall for making the tension transducer available to us.


    Abbreviations
 
IDDM insulin-dependent diabetes mellitus
NOD non-obese diabetic
OPD o-phenylenediamine
PG prostaglandin
TCM tissue culture medium

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: J.-F. Bach

Received 8 June 1999, accepted 18 February 2000.


    References
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 Introduction
 Methods
 Results
 Discussion
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