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Originally published online as doi:10.1189/jlb.0606375 on November 29, 2006

Published online before print November 29, 2006
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(Journal of Leukocyte Biology. 2007;81:809-817.)
© 2007 by Society for Leukocyte Biology

Prostaglandin I2 analogs inhibit Th1 and Th2 effector cytokine production by CD4 T cells

Weisong Zhou*,1, Timothy S. Blackwell*, Kasia Goleniewska*, Jamye F. O’Neal*, Garret A. FitzGerald{dagger}, Margaret Lucitt{dagger}, Richard M. Breyer{ddagger} and R. Stokes Peebles, Jr.*

Departments of
* Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, and
{ddagger} Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA; and
{dagger} Department of Medicine and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

1 Correspondence: Center for Lung Research, T-1218 MCN, Vanderbilt University Medical Center, Nashville, TN 37232-2650, USA. E-mail: weisong.zhou{at}vanderbilt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An anti-inflammatory effect of PGI2 has been suggested by increased inflammation in mice that are deficient in the PGI2 receptor (IP) or in respiratory syncytial viral- or OVA-induced CD4 T cell-associated responses. To determine the mechanism of the anti-inflammatory effect, we hypothesized that PGI2 analogs inhibit CD4 T cell effector cytokine production. To test this hypothesis, we activated purified CD4 T cells with anti-CD3 and anti-CD28 antibodies under Th1 and Th2 polarizing conditions for 4 days and restimulated the T cells with anti-CD3 in the presence of PGI2 analogs for 2 days. We found that PGI2 analogs (cicaprost and iloprost) inhibited the production of Th1 cytokines (IFN-{gamma}) and Th2 cytokines (IL-4, IL-10, and IL-13) in a dose-dependent pattern. The inhibitory effect was partially dependent on the IP receptor signaling and was correlated with elevated intracellular cAMP and down-regulated NF-{kappa}B activity. Pretreatment of the CD4 T cells with 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, to inhibit a key signaling molecule in the cAMP pathway, protein kinase A (PKA), attenuated the suppressive effect of PGI2 analogs significantly, suggesting that PKA, in part, mediates the inhibition of the cytokine production. These data indicate that PGI2 analogs have an immune-suppressive effect on previously activated and differentiated CD4 T cells in vitro and suggest that PGI2 may have a similar function in vivo.

Key Words: prostanoids • cAMP • lymphocytes • mouse


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PGI2, also known as prostacyclin, is an arachidonic acid metabolite in the cyclooxygenase (COX) pathway and plays modulatory roles in inflammatory and immune responses. In a previous study, we demonstrated that PGI2 had an anti-inflammatory effect using a respiratory syncytial viral (RSV) infection model with a Th1-dominant immune response [1 ]. Mice, which were deficient in the PGI2 receptor (IP) exhibited more severe illness with increased weight loss and greater production of the Th1 cytokine IFN-{gamma} in the lung after RSV infection [1 ]. In contrast, selective overexpression of PGI2 synthase in mouse lung epithelium inhibited RSV-induced weight loss and IFN-{gamma} production in the lung [1 ]. The IP receptor deficiency also augmented the Th2 immune responses and inflammation in OVA-induced allergic responses. Compared with the wild-type (WT) control mice, IP knockout (KO) mice exhibited elevated levels of serum IgE and Th2 cytokines (IL-4 and IL-5) in bronchoalveolar lavage (BAL) fluid [2 , 3 ]. An anti-inflammatory function of PGI2 was also suggested by a study using an adoptive T cell transfer model, in that an increased allergic lung inflammation was associated with lower levels of stable PGI2 metabolite 6-keto-PGF1a in the BAL fluid of COX-2-inhibited mice [4 ]. These studies suggest that PGI2 suppresses Th1 and Th2 responses. However, the mechanism by which PGI2 and signaling, through its receptor, exert an anti-inflammatory effect is still unclear.

Activation of CD4 T cells results in differentiation of Th1 and Th2 cell subsets as defined by profiles of effector cytokine production [5 , 6 ]. Th1 cells produce IFN-{gamma} and play a critical role in the clearance of intracellular pathogens such as viruses and tumors [7 , 8 ]. Th2 cells express IL-4, IL-5, and IL-13 and are crucial for the protection against extracellular pathogens and the induction of allergic responses [9 ]. To drive CD4 T cell differentiation, polarizing cytokines, specifically, IL-12 for Th1 and IL-4 for Th2, are required. In addition to the cytokine milieu, PGs have been shown to affect immune and inflammatory responses [10 , 11 ]. PGs are produced in the COX pathway by the action of two enzyme isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most types of cells, and COX-2 expression is induced by inflammatory stimuli such as microbial infections [12 ]. The COX-2 products, mainly PGI2 and PGE2 [13 ], which are present at the sites of inflammation, have been proposed to limit inflammatory processes [11 ]. In physiological conditions, PGI2 is unstable and has a half-life in the order of seconds [14 ]. As a result of this property, it is likely that PGI2 functions locally at the sites of inflammation. Consequently, the cells such as effector T lymphocytes, which are recruited to the inflammatory sites, may be affected by the local action of PGI2. It has been reported that in vitro treatment of DO11.10 T cells with PGI2 and its analog carba-PGI2, during T cell activation and differentiation, resulted in increased IL-10 production [4 ]. However, the effect of PGI2 on previously activated and differentiated effector T cells remains to be determined. Such an effect is relevant to the in vivo function of PGI2 on T cells.

PGI2 exerts its functions by selective binding to and activating the G-protein-coupled IP receptor [15 ]. In humans, the IP receptor is expressed in multiple tissues including lung, liver, kidney, platelets, heart, and aorta, suggesting a broad range of biological activities for PGI2 [15 , 16 ]. Stimulation of the IP receptor leads to an increase in intracellular cAMP and activation of downstream protein kinase A (PKA). It has been shown that the cAMP-signaling pathway is responsible for the vasodilatory and antiplatelet aggregatory effects of PGI2 [17 ].

PGI2 and its analog iloprost are used clinically to treat pulmonary hypertension. The suppressive effects of PGI2 and its analogs on the immune system may have important implications in their clinical applications. In this study, we hypothesized that PGI2 analogs would inhibit CD4 T cell effector cytokine production. We used purified CD4 T cells from mouse spleens and in vitro activated and differentiated the cells under Th1- and Th2-polarizing conditions. We determined the effect of PGI2 analogs on the effector cytokine production by the differentiated Th1 and Th2 cells and examined the role of cAMP, PKA, and NF-{kappa}B in the PGI2 analog-mediated effect. We also investigated whether the effect of PGI2 analogs was dependent on the PGI2 receptor signaling by using CD4 T cells from IP KO mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
WT C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). IP KO mice were generated by homologous recombination in embryonic stem cells and were backcrossed to the C57BL/6 background for more than 10 generations [18 ]. NF-{kappa}B reporter transgenic mice, referred to as HIV-long-terminal repeat (LTR)/luciferase (HLL) mice on a C57BL/6/DBA background, were generated as described [19 ]. HLL mice carry the Photinus luciferase gene cDNA driven by the proximal 5' HIV-1-LTR promoter [20 , 21 ]. The proximal HIV-LTR contains two NF-{kappa}B motifs and is a well-characterized NF-{kappa}B-responsive promoter [21 ]. In primary culture, NF-{kappa}B activation is absolutely required for transcriptional activity of the proximal HIV-LTR [22 , 23 ]. Therefore, luciferase expression in HLL cells is used as a surrogate marker for NF-{kappa}B activity. The NF-{kappa}B-GFP-luciferase (NGL)-transgenic mouse line, on a C57BL/6/DBA background, was generated as described previously [24 ]. In NGL mice, luciferase gene expression was driven by the human herpes virus minimal thymidine kinase promoter containing four tandem copies of a 36-base enhancer (each containing two NF-{kappa}B-binding sites) from the 5' HIV-LTR. Expression of the luciferase gene in NGL cells has been shown to reflect NF-{kappa}B activation [24 ]. In caring for animals, the investigators adhered to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (revised 1996).

Reagents
Cicaprost was a gift from Dr. Manuela Huebner (Schering AG, Berlin, Germany). Iloprost was obtained from Cayman Chemicals (Ann Arbor, MI). Indomethacin, forskolin, and 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cAMPS), were purchased from Sigma-Aldrich (St. Louis, MO). Anti-CD3 (2C11), anti-IL-4 (11B11), and anti-IFN-{gamma} (R4-6A2) mAb were purified from tissue-culture supernatant of hybridoma cells (American Type Culture Collection, Manassas, VA).

CD4 T cell culture
CD4 T cells were purified from the splenic cells of WT, IP KO, HLL, and NGL mice with antimurine CD4 magnetic microbeads (Miltenyi Biotec, Auburn, CA). The purified CD4 T cells (purity >94%, as assessed by flow cytometry) were resuspended at 1 x 106 cells/ml in RPMI-1640 medium (Mediatech, Inc., Herndon, VA) supplemented with 10% FBS (HyClone, Logan, UT), 4 mM L-glutamine, 1 mM sodium pyruvate, 55 µM ß-ME, 10 mM HEPES, 100 units/ml penicillin, and 100 µg/ml streptomycin. Primary T cell stimulation was performed by incubating CD4 T cells with plate-bound anti-CD3 and anti-CD28 (PharMingen, San Diego, CA) in 96-well plates for 4 days [25 ]. To differentiate T cells to Th1 and Th2 lineages, IL-12 (5 ng/ml) and anti-IL-4 (10 µg/ml) were added to the culture for Th1 differentiation and IL-4 (5 ng/ml) and anti-IFN-{gamma} (10 µg/ml) for Th2 differentiation during the primary stimulation. To stimulate effector cytokine production, the cells, which had been activated and differentiated for 4 days, were restimulated with plate-bound anti-CD3 for 2 days.

Cell treatment
PGI2 analogs (cicaprost and iloprost), indomethacin, and a cAMP-elevating agent (forskolin) were added once directly to the culture medium at the time of restimulation. Culture supernatant was collected at 2 days after restimulation for cytokine measurements. The concentrations of these agents were indicated in Results. As these agents were dissolved in different aqueous solutions or organic solvents, the vehicle solutions were diluted in the same way and used to treat cells with the same protocols as control treatments. For combined treatment with two agents, cells were pretreated with Rp-8-Br-cAMPS (100 µM) for 30 min at 37°C before incubation with PGI2 analogs.

PCR
Quantitative PCR was used to measure the mRNA levels of T-bet, Gata-3, the IP receptor, and ß-actin. Total RNA was extracted from polarized T cells at Day 4 after the primary stimulation by using TRIzol reagent (Invitrogen, Carlsbad, CA), treated with DNase I, and reverse-transcribed using Superscript III RT and oligo dT(16) (Invitrogen), according to the manufacturer’s protocols. The cDNA products were amplified by SYBR Green real-time PCR with gene-specific primers. The primer sequences are: T-bet: 5'-GGCGG TACCA GAGCG GCAAG TG-3' (forward) and 5'-CGGGG CTGGT ACTTG TGGAG AGACT-3' (reverse); Gata-3: 5'-GGCGA GATGG TACCG GGCAC TA-3' (forward) and 5'-CCCCA TTAGC GTTCC TCCTC CAGA-3' (reverse); IP: 5'-CCGCC AACAG AGACG CCACC AT-3' (forward) and 5'-CGGGC ACACA GGCAA CACAA CCA-3' (reverse); ß-actin: 5'-GACGA TGCTC CCCGG GCTGT A-3' (forward) and 5'-CGACC AGAGG CATAC AGGGA CAGC-3' (reverse). Real-time PCR reactions were performed in duplicate for each sample by using iTAQ Real-Time SYBR Green Supermix with 5-carboxy-X-rhodamine, succinimidyl ester (ROX) (BioRad Laboratories, Hercules, CA) and iCycler (BioRad Laboratories). The thermocycling condition was: Stage 1, 95°C for 3 min for one cycle, and Stage 2, 95°C for 15 s and 60°C for 45 s for 45 cycles. The PCR amplification products were analyzed on an agarose gel (Fisher Scientific, Pittsburg, PA) to confirm the absence of nonspecific amplification. Standard curves were generated for each gene using increasing amounts of RNA input. Relative levels of gene expression in the samples were extrapolated from the standard curves. ß-Actin gene expression was used to normalize for the amount of RNA input.

For semiquantitative PCR, PCR reactions were terminated in the linear phase of amplification (at 24 cycles for the IP receptor gene and 18 cycles for ß-actin). PCR products were analyzed by agarose gel electrophoresis with ethidium bromide.

Assays
Cytokines (IL-4, IL-10, IL-13, and IFN-{gamma}), in cell culture supernatant, were measured by ELISA Quantikine® or Duoset® kits from R&D Systems (Minneapolis, MN). Cells were counted by using Guava ViaCount Flex reagent and Guava EasyCyte flow cytometer (Guava Technologies, Hayward, CA). For cAMP measurement, T cells that had been activated for 4 days were seeded at 1 x 106 cells/ml in 96-well plates and treated with cicaprost (2000 nM), iloprost (2000 nM), and forskolin (50 nM) for 10 min at 37°C. The levels of intracellular cAMP were measured with a low pH cAMP assay kit (R&D Systems). Luciferase activity in HLL and NGL CD4 T cells after PGI2 analog treatments for 6 h was measured with a luciferase reporter assay system from Promega (Madison, WI).

Statistics
The P values were calculated by using unpaired Student’s t-test. Values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PGI2 analogs inhibit effector cytokine production by Th1 and Th2 cells
Purified splenic CD4 T cells from WT mice were first stimulated with anti-CD3 and anti-CD28 antibodies under Th1 (IL-12 plus anti-IL-4) and Th2 (IL-4 plus anti-IFN-{gamma}) conditions for 4 days. These cells were then restimulated with anti-CD3 for 2 days. This protocol was intended to mimic in vivo antigen stimulation of naïve CD4 T cells for activation and differentiation, followed by antigen re-encounter of activated Th1 and Th2 cells for effector cytokine induction. During the primary stimulation, the CD4 T cells were activated and proliferated. The proliferated cells formed colonies, which were readily visible when examined with a dissecting microscope at days 3 and 4. To examine the efficiency of the Th1 and Th2 conditions on T cell differentiation and polarization, we measured cytokine production by the cells after restimulation. Cells under Th1 condition produced 5.7-fold more IFN-{gamma} (436±14 ng vs. 77±9.1 ng/ml) and 654-fold less IL-4 (0.08±0.02 ng/ml vs. 52±1.7 ng) than those under Th2 condition (Fig. 1 A), indicating an effective Th1/Th2 differentiation. We also examined the mRNA levels of two transcription factors, T-bet and Gata-3, in the cells by real-time PCR after 4 days of primary stimulation. We chose these two transcription factors, as T-bet expression is up-regulated in Th1 but not in Th2 cells, whereas Gata-3 expression is increased in Th2 but not Th1 cells [26 ]. T cells stimulated under Th1 conditions produced 12-fold more T-bet mRNA and sevenfold less Gata-3 mRNA than those stimulated under Th2 conditions (Fig. 1B) , confirming that the polarization of T cells under Th1 and Th2 conditions was effective. The CD4 T cells differentiated along the Th1 and Th2 pathways also produced detectable levels of the IP receptor mRNA (Fig. 1C) , and there was no significant difference in the mRNA levels between Th1 and Th2 conditions as shown by real-time PCR (Fig. 1B) .


Figure 1
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  		Figure 1. CD4 T cell differentiation and expression of the PGI2 IP receptor mRNA. (A) CD4 T cells were stimulated under Th1 and Th2 conditions for 4 days followed by TCR restimulation for 2 days. Cytokines (IFN-{gamma} and IL-4) were assayed by ELISA. (B and C) Total RNA was isolated from CD4 T cells, which were stimulated under Th1 and Th2 conditions for 4 days and used for (B) real-time PCR or (C) semiquantitative PCR with primers specific for T-bet, Gata-3, the IP receptor, and ß-actin genes. (C) PCR reactions were terminated in the linear phase of amplification (at 24 cycles for IP and 18 cycles for ß-actin), and PCR products were analyzed by agarose gel electrophoresis with ethidium bromide. (A and C) Data are representative of three experiments, or (B) data are the mean ± SEM of n = 3 experiments.

 
To test the hypothesis that PGI2 analogs inhibit CD4 T cell effector cytokine production, we treated the cells at the time of restimulation with PGI2 analogs including cicaprost and iloprost at concentrations of 2, 20, 200, and 2000 nM. To examine the effect of endogenously produced PGs on the T cell response, the cells were treated at the time of restimulation with a nonselective COX inhibitor, indomethacin, to suppress endogenous PG production. Treatment of the activated and differentiated cells with cicaprost or iloprost decreased the production of IFN-{gamma} by Th1 cells and IL-4, IL-10, and IL-13, by Th2 cells in a dose-dependent pattern compared with vehicle treatments (Table 1 and Fig. 2 A). Specifically, cicaprost and iloprost at 200 and 2000 nM inhibited IFN-{gamma} production significantly by Th1 cells, and both analogs at 20, 200, and 2000 nM suppressed IL-4, IL-10, and IL-13 production significantly by Th2 cells (Fig. 2A) . Treatment of the cells with vehicle reagents did not change the levels of effector cytokine production significantly (Table 1) . Similarly, indomethacin did not change the production of these four cytokines (Table 1 and Fig. 2A ). The magnitude of the inhibitory effect of each PGI2 analog on Th2 cytokine production appeared to be greater than that on Th1 cytokine production. Treatment with PGI2 analogs did not have a significant effect on the total number of viable cells (Fig. 2B) , suggesting that the inhibitory effect of PGI2 analogs on effector cytokine production was not a result of cell killing. These data indicated that PGI2 analogs did not affect the survival of previously activated and differentiated CD4 cells but nonetheless, inhibited the effector cytokine production by Th1 and Th2 cells.


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  		Table 1. PGI2 Analogs Inhibit Effector Cytokine Production by Th1 and Th2 Cells

 

Figure 2
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  		Figure 2. PGI2 analogs inhibit effector cytokine production by differentiated Th1 and Th2 cells. CD4 T cells were stimulated under Th1 and Th2 conditions for 4 days followed by TCR restimulation for 2 days. PGI2 analogs [cicaprost (cica) and iloprost (Ilop)] and indomethacin were added at the time of restimulation. (A) Cytokines were measured by ELISA. (B) Numbers of viable cells were analyzed with Guava ViaCount Flex reagent. Data shown are the mean ± SEM of (A) n = 4 or (B) n = 3 experiments. *, P < 0.05, versus vehicles.

 
The inhibitory effect of PGI2 analogs is partially dependent on the IP receptor
To test whether the PGI2 analogs suppressed cytokine production in an IP-dependent manner, experiments were performed using WT and IP KO mice in side-by-side experiments. Although treatment of WT and IP KO T cells with cicaprost and iloprost at 2000 nM resulted in significant inhibition of Th1 (IFN-{gamma}) and Th2 (IL-4, IL-10, and IL-13) cytokines compared with vehicle-treated cells, the percentages of the cytokine inhibition on IP KO T cells were significantly lower than those on WT T cells (Fig. 3 ). These results indicate that the deficiency of the IP receptor signaling attenuated the effect of the analogs and suggest that the cicaprost- and iloprost-mediated inhibitory effect on the effector cytokine production was partially dependent on the signaling through the IP receptor.


Figure 3
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  		Figure 3. PGI2 analog-mediated effect on the effector cytokine production is partially dependent on the IP receptor. WT and IP KO CD4 T cells were stimulated under Th1 and Th2 conditions for 4 days followed by TCR restimulation for 2 days. PGI2 analogs (cicaprost and iloprost at 2000 nM) were added at the time of restimulation. Cytokines were measured by ELISA. Data shown are the mean ± SD of n = 3 wells and are representative of three experiments. *, P < 0.05, versus vehicles; {dagger}, P < 0.05, IP KO mice versus WT controls.

 
PGI2 analogs increase intracellular cAMP in Th1 and Th2 cells
As activation of the IP receptor signaling leads to increased levels of cAMP in smooth muscle cells and monocytes [27 , 28 ], we sought to examine whether the PGI2 analogs also increased cAMP levels in the T cells. As shown in Figure 4 A, treatment of CD4 T cells, which had been activated under Th1 and Th2 conditions for 4 days with cicaprost or iloprost at 2000 nM, resulted in a two- to threefold increase of intracellular cAMP. As a positive control, forskolin induced elevated levels of cAMP in Th1 and Th2 cells (Fig. 4A) . Therefore, the inhibitory effect of PGI2 analogs on effector cytokine production was associated with increased intracellular cAMP levels.


Figure 4
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  		Figure 4. The role of the cAMP-signaling pathway in the PGI2 analog-mediated effect on T cells. CD4 T cells were stimulated under Th1 and Th2 conditions for 4 days. (A) The differentiated cells were treated with PGI2 analogs (cicaprost and iloprost at 2000 nM) or forskolin at 50 nM for 10 min at 37°C. Intracellular cAMP was measured by a R&D Systems cAMP assay kit. (B) The differentiated cells were pretreated without (Mock) or with Rp-8-Br-cAMPS at 100 µM for 30 min, followed by treatment with cicaprost and iloprost at 2000 nM at the time of restimulation. Cytokines in culture supernatant were assayed by ELISA. (A) Data are the mean ± SD of n = 3 wells and are representative of three experiments. (B) Data are the mean ± SEM of two experiments. *, P < 0.05.

 
The role of the cAMP-signaling pathway in the PGI2 analog-mediated effect on the effector cytokine production was investigated further by using a cAMP antagonist, Rp-8-Br-cAMPS, which selectively blocks the activation of PKA. Pretreatment of the differentiated Th1 and Th2 cells with Rp-8-Br-cAMPS, followed by exposure to cicaprost and iloprost, increased the production of IFN-{gamma} significantly by Th1 cells and IL-4 by Th2 cells by 50–100%, indicating an attenuation of PGI2 analog-mediated effect (Fig. 4B) . The significant attenuation, although not a full abrogation, of PGI2 analog-mediated effect by the PKA inhibitor, suggests an involvement of PKA signaling pathway in PGI2 analog-induced suppression of Th1 and Th2 cytokine production.

PGI2 analogs down-regulate NF-{kappa}B activity in Th1 and Th2 cells
It has been demonstrated that activation of NF-{kappa}B enhances the expression of IFN-{gamma} and IL-4 [29 , 30 ]. Therefore, we investigated whether the PGI2 analog-mediated inhibitory effect on T cells was associated with a down-regulation of NF-{kappa}B activity. We used CD4 T cells from NF-{kappa}B reporter mice (HLL mice), in which luciferase gene expression was used as a surrogate marker of NF-{kappa}B activity. Treatment of the cells with cicaprost or iloprost at the time of restimulation inhibited NF-{kappa}B activity in a dose-dependent pattern, compared with vehicle-treatment controls (Table 2 and Fig. 5 A). In contrast, treatment with indomethacin at 2000 nM did not affect NF-{kappa}B activity (Table 2) .


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  		Table 2. PGI2 Analogs Suppress NF-{kappa}B Activity in Th1 and Th2 Cells

 

Figure 5
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  		Figure 5. PGI2 analogs suppress NF-{kappa}B activity in Th1 and Th2 cells. CD4 T cells of (A) HLL and (B) NGL NF-{kappa}B reporter mice were stimulated for 4 days under Th1 and Th2 conditions followed by restimulation with anti-CD3 for 6 h in the presence of increasing concentrations of cicaprost, iloprost, or indomethacin. Luciferase activity was measured by a luciferase reporter assay system. Data shown are the mean ± SEM of n = 2 experiments. *, P < 0.05, versus vehicle controls.

 
To further examine the inhibitory effect of PGI2 analogs on NF-{kappa}B activity, we used another transgenic NF-{kappa}B reporter mouse line (NGL mice). As shown in Figure 5B , treatment of NGL T cells with iloprost and cicaprost at the time of restimulation decreased NF-{kappa}B activity dose-dependently in Th1 and Th2 cells. This result confirms that iloprost and cicaprost inhibit NF-{kappa}B activity in polarized T cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An anti-inflammatory effect of PGI2 has been suggested by in vivo studies using murine models of RSV infection and OVA-induced allergic responses [1 2 3 4 ]. In the present study, we have shown that PGI2 analogs have a direct, suppressive effect on effector cytokine production by differentiated Th1 and Th2 cells. The inhibition of effector cytokine production is associated with elevated levels of intracellular cAMP and is partially dependent on cAMP-dependent activation of PKA. These findings suggest that the direct immune suppressive effects on CD4 T cells may be a mechanism for the anti-inflammatory effect of PGI2 shown in RSV- and OVA-induced inflammations [1 2 3 4 ].

PGI2 analogs inhibited effector cytokine production, specifically IFN-{gamma} production by Th1 cells and IL-4, IL-10, and IL-13, by Th2 cells. To our best knowledge, this report is the first showing the effect of PGI2 analogs on previously activated and differentiated effector T cells. Iloprost and cicaprost did not change IL-5 production significantly by the polarized Th2 cells (data not shown), suggesting that these analogs differentially inhibit IFN-{gamma}, IL-4, IL-10, and IL-13 expression. Our results are different from the report by Jaffar and colleagues [4 ], in that they found that in vitro treatment of T cells increased the production of IL-10 by Th2 cells but not IL-4 and IL-5 by Th2 cells and IFN-{gamma} by Th1 cells. In Jaffar’s study, they used T cells of DO11.10 mice on a BALB/c background, treated the cells with PGI2 or carba-PGI2 during the primary stimulation under Th1 and Th2 conditions, and then restimulated the cells in the absence of PGI2 and its analog. In our study, we used CD4 T cells on a C57BL/6 background, polarized the cells under Th1 or Th2 conditions in the absence of PGI2 analogs, and treated the differentiated T cells with PGI2 analogs during the period of restimulation. Experiments directly comparing different mouse strains, experimental protocols, and reagents should help to explain the difference. In Jaffar’s study, PGI2-induced up-regulation of IL-10 production by Th2 cells was associated with the suppressive effect of COX products on allergic inflammation. Our data suggest that PGI2 may exert its antiallergic function by inhibiting the production of Th2 effector cytokines directly, such as IL-4 and IL-13 as well.

The inhibitory effect of PGI2 analogs on the production of IFN-{gamma}, IL-4, and IL-13 by CD4 T cells strongly suggests an immune-suppressive function of PGI2 analogs. However, the overall effect of PGI2 analogs on the immune system may be complicated by their ability to inhibit IL-10 expression. IL-10 is considered to be an anti-inflammatory cytokine, and decreased IL-10 production by T cells may attenuate PGI2-induced immune suppression. It should be noted that PGI2 analogs increased IL-10 production but decreased IL-12 and TNF-{alpha} production by macrophages [31 ] and dendritic cells (DC) [32 ] and W. Zhou and R. S. Peebles, Jr. [unpublished data], indicating that PGI2 may affect multiple types of cells to exert its immune-modulatory function. These results also suggest that PGI2 analogs may affect Th1 and Th2 differentiation by altering cytokine profiles produced by DC.

Our data indicate that the effect of PGI2 analogs on Th1 and Th2 cytokine production is partially mediated by the cAMP-signaling pathway. This is consistent with the inhibitory effect of other cAMP-elevating agents such as forskolin, cholera toxin, and rolipram on T cell cytokine production [33 34 35 36 37 38 ]. cAMP-elevating agents have been shown to inhibit the production of Th1 cytokines (IFN-{gamma} and IL-2) in most of the published studies (e.g., refs. 33 , 36 37 39 40 ). However, depending on experimental systems in different studies, the effects of cAMP-elevating agents on Th2 cytokine production appear to vary. cAMP-elevating agents inhibited [33 37 41 42 43 ] or increased Th2 cytokine (IL-4) production [35 36 44 45 ]. For instance, forskolin and PGE2 decreased IL-4 production by a human T cell line [33 ] but increased IL-4 production by primary, cultured T cells [35 ]. Similarly, PGE2 inhibited IL-5 production by human peripheral mononuclear cells [46 ] but increased IL-5 production by DO11.10 T cells or lymphoma cells [36 ], suggesting cell type-dependent effects. In our study, we did not find significant changes in IL-5 production by PGI2 analog-treated splenic CD4 T cells, which were polarized under Th1 and Th2 conditions. Differential cytokine responses to cAMP-elevating agents reflect the complexity of the cAMP-signaling pathway and its impact on the downstream gene expression.

The mechanism of cAMP-induced modulation of cytokine expression is not well understood. It has been reported that PKA-dependent and independent signaling pathways may play a role. PKA is a signaling molecule immediately downstream of cAMP and is activated by elevated levels of intracellular cAMP. In the present study, the important role of PKA on PGI2 analog-mediated effect is suggested in that iloprost- and cicaprost-mediated inhibition of Th1 (IFN-{gamma}) and Th2 (IL-4) cytokine production is attenuated by a PKA inhibitor, Rp-8-Br-cAMPS. The partial, but not complete, reversal of PGI2 analog-mediated effect on cytokine production by treatment of cells with Rp-8-Br-cAMPS also suggests an existence of the PKA-independent pathway for the immune-suppressive function of PGI2 analogs.

It has been reported that PGE2 also inhibited IFN-{gamma} and IL-4 production by T cells [33 37 39 ]. The similar inhibitory effect of PGI2 analogs and PGE2 on the production of these cytokines suggests a T cell inhibitory function in common between PGI2 and PGE2. The PGI2-mediated inhibition on effector cytokine production is only partially dependent on the IP receptor signaling, suggesting that other types of PG receptors may play a role in PGI2 analog-mediated effects. This is consistent with previous findings that iloprost may bind to EP1 and EP3 receptors and cicaprost to the EP4 receptor at high concentrations, although both analogs bind to the IP receptor with the highest affinity [47 ]. As a result of the instability of PGI2 in aqueous solutions, it is difficult to determine the physiological concentration of PGI2, which is present in inflammatory conditions. However, given that iloprost exhibited an equilibrium dissociation constant for the IP receptor with a Kd value of 9.8 nM [15 ], and PGI2 stimulated cAMP activity at 6.6 nM in NCB-20 cells that express IP [48 ], it may be reasonable to estimate that PGI2 concentrations, which are physiologically functional, may be between 10 and 100 nM. Therefore, our data, showing significant inhibition of Th1 and Th2 cytokines by cicaprost and iloprost at concentrations of 20–200 nM, appear to have a physiological relevance.

In the present study, PGI2 analogs inhibited the NF-{kappa}B activity in the differentiated Th1 and Th2 cells, as shown by using T cells from two lines of transgenic NF-{kappa}B reporter mice. Therefore, PGI2 analog-mediated inhibition of effector cytokine production is associated with not only the elevated cAMP but also down-regulation of NF-{kappa}B activity. Consistent with our finding, other studies have demonstrated that NF-{kappa}B activation enhances the expression of IFN-{gamma} [29 ] and IL-4 in vitro [30 ]. In addition, Aronica and co-workers [49 ] reported that inhibition of the NF-{kappa}B/Rel-signaling pathway resulted in impaired Th1 responses in a footpad model of delayed type hypersensitivity but increased Type 2 responses in a model of allergic pulmonary inflammation. In that study, inhibition of NF-{kappa}B function led to decreased IFN-{gamma} production but increased IL-4 production by lymph node cells from the immune-sensitized and challenged mice [49 ]. The opposite effects of NF-{kappa}B on IL-4 expression shown in these studies may be because of intrinsic differences between in vitro and in vivo studies. Although the inhibitory effect of a PGI2 analog, treprostinil, on NF-{kappa}B has been demonstrated in human alveolar macrophages [50 ], our data are the first to show the inhibition of NF-{kappa}B activity by PGI2 analogs in CD4 T cells. Previous reports suggested that PKA activation negatively regulates multiple components in the T cell signaling pathways, including NF-{kappa}B and the nuclear accumulation of NF-AT [51 52 53 ]. It remains to be determined whether iloprost- and cicaprost-mediated inhibition of NF-{kappa}B activity is dependent on the elevation of cAMP and activation of PKA.

PGs were originally considered to be proinflammatory mediators, as they are often up-regulated by inflammatory stimuli, and inhibition of PG production by nonsteroid, anti-inflammatory drugs, aspirin and indomethacin, down-regulated inflammation in diseases such as rheumatoid arthritis. However, more recent studies indicate that individual PGs, including PGI2 and PGE2, may also have anti-inflammatory function in vivo and in vitro [54 ]. Those PGs, in the setting of inflammation, may contribute to the limitation, instead of stimulation, of inflammation. In this report, we demonstrated that PGI2 analogs suppressed the effector cytokine production by differentiated Th1 and Th2 cells, suggesting that PGI2 may have a direct effect on CD4 T cells in vivo.


   ACKNOWLEDGEMENTS
 
This work was supported by the American Academy of Allergy, Asthma and Immunology Interest Section Award (R01-AI-054660 and R01-HL-069949).

Received June 6, 2006; revised October 23, 2006; accepted October 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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