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* Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232;
Department of Microbiology, Fukushima Medical University, Fukushima, Japan;
LINCO Research, St. Charles, MO 63304;
Departments of Medicine and Pharmacology, University of Pennsylvania, Philadelphia, PA 19104; and
¶ Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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, IL-1, IL-6) and chemokines (MIP-1, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-
B activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI2 signaling through the IP may exert anti-inflammatory effects by acting on DC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Dendritic cells (DCs) play important roles in inflammation and in innate and adaptive immune responses to pathogens and nonself substances. They present Ag in the context of MHC molecules to T cells leading to the development of immune responses. In addition, DCs produce a variety of mediators including cytokines and chemokines that may either up- or down-regulate immune and inflammatory responses. Because excessive inflammatory responses may cause tissue damage and be harmful to the host, resolution of inflammation is required for the recovery of normal tissue function and tissue homeostasis. In vivo studies in RSV infection and in OVA-induced allergic responses (1, 2, 3) suggest that PGI2 is one of the mediators that might contribute to the limitation of inflammation. The anti-inflammatory function of PGI2 may be mediated by direct effects of PGI2 on DCs that respond to RSV infection and allergens.
PGI2 exerts its function through the Gs protein-coupled IP and signaling through the IP leads to increased levels of intracellular cAMP (4, 5). Because PGI2 is very unstable in aqueous solution, PGI2 analogs with more chemical stability have commonly been used in laboratory research and clinical applications. PGI2 analogs, such as iloprost and cicaprost, have been shown to inhibit production of proinflammatory cytokine TNF- and GM-CSF, while increasing the production of an anti-inflammatory cytokine IL-10 by human peripheral mononuclear cells in vitro (6, 7, 8, 9). In another study, the PGI2 analog treprostinil inhibited production of multiple cytokines including IL-6, TNF-, GM-CSF, and IL-1 by human alveolar macrophages and blocked NF-B nuclear translocation (10). However, the effect of PGI2 analogs on DCs has not been well-studied. We hypothesized that PGI2 analogs exert anti-inflammatory activity by suppressing DC activation, maturation, and T cell stimulatory function. To test this hypothesis, we examined the in vitro effect of three PGI2 analogs, iloprost, cicaprost, and treprostinil, on LPS-induced cytokine and chemokine production, maturation, and NF-B activity of bone marrow (BM)-derived DCs (BMDCs). We used BMDCs of IP–/– mice to study the receptor specificity of the effects of PGI2 analogs. We also examined the effect of PGI2 analogs on DC ability to stimulate Ag-specific T cell proliferation by using BMDCs and CD4 T cells of DO11.10 mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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IP–/– mice were generated by homologous recombination in embryonic stem cells and were backcrossed to the C57BL/6 background for >10 generations (11). Age- and sex-matched C57BL/6J mice (from The Jackson Laboratory) were used as wild-type control mice (IP+/+). NF-B reporter transgenic mice, referred to as HLL (HIV-long terminal repeat (LTR)/luciferase) mice on a C57BL/6/DBA background, were generated as described (12). HLL mice carry the Photinus luciferase gene cDNA driven by the proximal 5' HIV (HIV-1) LTR promoter (12, 13). The proximal HIV-LTR contains two NF-B motifs and is a well-characterized NF-B-responsive promoter (14). In primary culture, NF-B activation is required for transcriptional activity of the proximal HIV-LTR (15, 16). Therefore, luciferase expression in HLL cells is used as a surrogate marker for NF-B activity. DO11.10 mice that express OVA-specific transgenic TCR specific for OVA peptide 323-339 were obtained from The Jackson Laboratory. 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. M. Huebner (Schering, Berlin, Germany). Iloprost was obtained from Cayman Chemicals. Treprostinil was provided by United Therapeutics. Indomethacin, ibuprofen, forskolin, N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate sodium (db-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cAMPS) were purchased from Sigma-Aldrich. Anti-IL-10 mAb and control rat IgG1 were purchased from BD Pharmingen.
Dendritic cells
BMDCs and macrophages were generated using a previously described method with modifications (17). Briefly, the BM in femurs and tibias of IP–/–, IP+/+, HLL, and DO11.10 mice was flushed out with RPMI 1640 medium (Mediatech) and a single-cell suspension was prepared by passing the BM solution through a 19-gauge needle five times. After lysis of RBC, the cells were passed through a nylon cell strainer with a mesh size at 70 µm. The cells were then washed with RPMI 1640 medium and resuspended at 5 x 105 cells/ml in complete RPMI 1640 medium containing 5% heat-inactivated FBS (HyClone), 50 µg/ml gentamicin, and 55 µM 2-ME. GM-CSF (R&D Systems) was added to the cell solution at 20 ng/ml. The cells were cultured at day 0 in 6-well plates (Corning) and maintained at 37°C in humidified air containing 5% CO2. On days 3 and 6, half of the culture medium was replaced with fresh complete medium containing 20 ng/ml GM-CSF. At day 8, non- and loosely adherent cells were harvested. Greater than 60% of the harvested cells were CD11c+ and >95% were CD11b+. These cells were either simultaneously activated with LPS (1 µg/ml) and treated with PGI2 analogs or further purified with anti-murine CD11c magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. The purified DCs (purity >94% CD11c+ as assessed by flow cytometry) were treated with PGI2 analogs in the presence of LPS (1 µg/ml). The levels of PGE2 and stable metabolite of PGI2, 6-keto-PGF1, in the culture supernatant were measured by a modified stable isotope dilution assay that used gas chromatography-negative ion chemical ionization-mass spectrometry as previously described (18).
Cell treatment with PGI2 analogs and cAMP-elevating agents
BMDCs were treated by adding diluted PGI2 analogs (cicaprost, iloprost, treprostinil), indomethacin, ibuprofen, and vehicle solutions once directly to the culture medium in the presence of LPS (1 µg/ml). The cells were further cultured for 24 h before the culture supernatant was harvested for cytokine and chemokine measurements and the cells were stained for flow cytometric analyses. Specific concentrations of these agents were used as indicated in Results. BMDCs were also treated with cAMP-elevating agents including db-cAMP (500 µM), forskolin (5 µM), and forskolin plus IBMX (100 µM) in the presence of LPS (1 µm/ml). In some experiments, IL-10 neutralizing Ab (20 µg/ml) or control rat IgG1 (20 µg/ml) were added together with PGI2 analogs. Because the PGI2 analogs and cAMP-elevating agents were dissolved in different aqueous solutions or organic solvents, vehicle solutions were diluted in the identical fashion as the respective analogs and agents and used to treat cells with the same protocols as control treatments. The vehicle solutions were: methyl acetate for iloprost, distilled water for cicaprost, db-cAMP, and Rp-8-Br-cAMPS, specifically formulated citrate-buffered solution from United Therapeutics for treprostinil, ethanol for indomethacin and ibuprofen, and DMSO for forskolin and IBMX. For combined treatment with two reagents, cells were pretreated with Rp-8-Br-cAMPS (200 µM) and IBMX (100 µM) for 30 min at 37°C followed by addition of PGI2 analogs and forskolin (5 µM), respectively.
Cytokine measurement
Multiple cytokines and chemokines (see list in Table I) in the cell culture supernatant were measured with a commercially available LINCOplex Mouse Cytokine/Chemokine kit (LINCO Research) using fluorescently labeled microsphere beads and a Luminex reader. In some experiments, Quantikine and Duoset ELISA kits (R&D Systems) were used to measure the secretion of cytokines and chemokines (IL-12 p70, TNF-, GM-CSF, MIP-1, and MCP-1) according to the manufacturer’s instructions.
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IP–/– and IP+/+ BMDCs were seeded in 5-ml round-bottom tubes (BD Biosciences) at 1 x 106 cells/tube in 1 ml of complete medium and treated with iloprost, cicaprost, and forskolin in the presence of LPS (1 µg/ml) for 10 min at 37°C. Intracellular cAMP was assayed by using a low pH cAMP assay kit (R&D Systems) according to the manufacturer’s instructions.
Transduction with adenoviral vectors
A replication-deficient adenoviral vector that expressed constitutively active IB kinase 2 (cIKK2) and a control adenoviral vector that expressed GFP (AdeGFP) were constructed as previously described (19). For adenoviral transduction, purified HLL BMDCs at a concentration of 1 x 107 cells/ml were incubated with cIKK2 or AdeGFP (at a multiplicity of infection of 200) at 37°C for 4 h. The cells were washed and cultured in 96-well plates for 12 h before being treated with PGI2 analogs and cAMP-elevating agents. The efficiency of transduction was indicated by >60% of BMDCs being visibly green under observation through a dissecting microscope 24 h after AdeGFP transduction (data not shown).
Luciferase assay
Purified BMDCs of HLL mice were seeded at 1 x 106 cells/ml in complete medium in 96-well plates (200 µl/well) and treated with PGI2 analogs, cAMP-elevating agents, or vehicle solutions in the presence of LPS (1 µg/ml) for 4 h. HLL BMDCs that had been transduced with either cIKK2 or AdeGFP were seeded at 1 x 106 cells/ml for treatments in the absence of LPS for 4 h. Luciferase activity was measured by using the luciferase reporter assay system (Promega) and Luminometer Monolight 3010 (Analytical Luminescence Laboratories) according to manufacturer’s instructions. LPS stimulation of the BMDCs increased luciferase activity 3-fold compared with the control cells without LPS stimulation; cIKK2-tranduction increased luciferase activity 2.3-fold compared with the cells transduced with AdeGFP.
Flow cytometric analysis
IP–/– and IP+/+ BMDCs were treated with iloprost and vehicle solutions for 24 h. Non- and loosely adherent cells were harvested for staining with fluorochrome-labeled Abs against CD11b, CD11c, CD86, CD40, and MHC class II (I-Ab; BD Pharmingen) and analyzed using a LSR II flow cytometer (BD Biosciences). A total of 10,000 live cell events as gated on forward and side scatter characteristics was acquired. CD86, CD40, and MHC class II expression were analyzed on a live DC population gated on CD11b and CD11c double-positive cells.
Stimulation of CD4 T cells by DCs
CD4 T cells were purified from the spleens of DO11.10 mice with anti-murine CD4 magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. The cells were labeled with 5 µM CFSE (Molecular Probes) in PBS for 10 min at room temperature. The reaction was quenched with PBS containing 25% serum. The cells were washed twice with complete medium before being cocultured with DCs.
BMDCs of DO11.10 mice were treated with iloprost (400 nM), cicaprost (400 nM), or vehicle solutions in the presence of LPS (1 µg/ml) and 100 µg/ml OVA (Sigma-Aldrich) for 24 h. The DCs were washed with medium three times and specific numbers of DCs (5,000 and 20,000 cells) in each PGI2 analog- or vehicle-treated group were used for coculture with CFSE-labeled CD4 T cells (2 x 105 cells) for 4 days. The cells were analyzed for CFSE fluorescence intensity by flow cytometry. A reduction of CSFE intensity by half is an indication of a cell division. The cells were also subjected to viable cell counting using a Guava PCA 96 system and Guava ViaCount Flex Reagent (Guava Technologies). The culture supernatant was used for IL-5 and IL-13 measurement.
Statistics
The p values were calculated by using the unpaired Student t test. Values of p < 0.05 were considered significant.
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Results |
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To test the hypothesis that PGI2 analogs down-regulate proinflammatory cytokine production by inhibiting activation of DCs, we differentiated BM precursor cells of C57BL/6J IP+/+ mice to BMDCs and macrophages in GM-CSF for 8 days, then activated the mixed cell population with LPS and treated them with exogenous PGI2 analogs including iloprost, cicaprost, and treprostinil for 24 h. The cell culture supernatant was assayed for cytokine production. To examine the effect of endogenous PGs on the function of BMDCs, we treated the cells with a nonselective cyclooxygenase (COX) inhibitor, indomethacin, to inhibit endogenous prostanoid production. The cells differentiated in GM-CSF for 8 days expressed the IP as analyzed by RT-PCR (data not shown, primer sequences were: forward, 5'-CCGCC AACAG AGACG CCACC AT-3'; reverse, 5'-CGGGC ACACAG GCAAC ACAAC CA-3'). Treatment of these cells with the three PGI2 analogs significantly inhibited the production of LPS-induced proinflammatory cytokine IL-12 p70 in a dose-dependent pattern (Fig. 1). In contrast, the three analogs dose-dependently increased the production of anti-inflammatory IL-10 (Fig. 1). In the culture supernatant of the cells stimulated with LPS, PGE2 was measured to be <1 nM and the stable metabolite of PGI2, 6-keto-PGF1, was below the limit of detection. Inhibition of endogenous PGs with indomethacin at 40 and 400 nM did not change the production of these cytokines (Fig. 1, only showing the data for 400 nM indomethacin). The lack of effect by the COX inhibitor indomethacin on BMDC cytokine production was confirmed by the use of an alternative COX inhibitor, ibuprofen, at the same doses (data not shown). These data indicate that the endogenously produced PGs did not have a measurable overall impact on the cytokine production.
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were under the limit of detection. PGI2 analogs, iloprost, cicaprost, and treprostinil, significantly decreased LPS-induced production of IL-12, TNF-, IL-1, IL-6, MIP-1, and MCP-1, but increased the production of IL-10 (Fig. 2A). Because IL-10 has been shown to be an inhibitor of proinflammatory cytokine expression and exogenously added IL-10-suppressed IL-12 and TNF- production by BMDCs (20, 21, 22, 23), the inhibitory effect of PGI2 analogs on the proinflammatory cytokine and chemokine production might have been mediated by the concurrent elevation of IL-10 expression. To examine this possibility, an IL-10-specific neutralizing Ab was added to the culture during the period of treatment with PGI2 analogs. Neutralization of IL-10 did not abrogate the inhibitory effect of PGI2 analogs (Fig. 2B), although addition of anti-IL-10 to cicaprost-treated BMDC culture resulted in an undetectable level of IL-10 in the culture supernatant (Fig. 2B). These data indicate that the PGI2 analog-mediated inhibition is not a result of concurrent stimulation of IL-10 expression.
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We next investigated the role of IP signaling in PGI2-induced changes of cytokine and chemokine production by using IP–/– and IP+/+ DCs. As demonstrated in Fig. 3, treatment of purified IP+/+ BMDCs with the PGI2 analogs iloprost (Fig. 3A) and cicaprost (Fig. 3B) dose-dependently inhibited LPS-induced production of IL-12, TNF-, IL-1, IL-6, MIP-1, and MCP-1; the PGI2 analogs up-regulated the production of IL-10 as compared with the appropriate vehicles. In contrast, treatment of IP–/– DCs with iloprost and cicaprost at the same concentrations did not significantly affect LPS-induced production of any of these cytokines and chemokines compared with vehicle treatments, indicating an essential role of IP-dependent signaling in PGI2 analog-mediated modulation of the cytokine and chemokine production.
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B activity in BMDCsStimulation of the IP leads to increased intracellular cAMP levels (24, 25). To examine the effect of IP signaling on cAMP production in DCs, purified BMDCs were treated with cicaprost and iloprost in the presence of LPS for 10 min and analyzed for intracellular cAMP production. As shown in Fig. 4A, iloprost and cicaprost significantly elevated intracellular cAMP levels up to 10- and 6-fold, respectively, in IP+/+ BMDCs, but not in IP–/– BMDCs. These data suggest that the modulatory effect of PGI2 analogs on cAMP production was dependent on IP signaling. Forskolin induced similar levels of cAMP in both IP+/+ and IP–/– BMDCs, indicating that IP–/– BMDCs were able to generate a cAMP response to non-IP-dependent stimulation.
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production, and 4- and 6-fold increases in MIP-1 production by the cells treated with cicaprost and iloprost, respectively. This suggests the PKA inhibitor significantly attenuated the effect of iloprost and cicaprost on the cytokine production. However, under the PKA-inhibited condition, the PGI2 analog-treated cells still produced significantly less TNF- and MIP-1 compared with appropriate vehicle-treated cells. Cells treated with iloprost in the presence of Rp-8-Br-cAMPS produced 3.7-fold less TNF- than vehicle-treated cells (1.1 ± 0.175 ng/ml vs 4.1 ± 0.38 ng/ml, p < 0.05) and 7-fold less MIP-1 (9.4 ± 2.0 ng/ml vs 66.4 ± 6.0 ng/ml, p < 0.05). Cells treated with cicaprost in the presence of Rp-8-Br-cAMPS produced 6.4-fold less TNF- than vehicle-treated cells (0.74 ± 0.065 ng/ml vs 4.7 ± 0.175 ng/ml, p < 0.05) and 11.7-fold less MIP-1 (6.3 ± 1.1 ng/ml vs 73.6 ± 2.0 ng/ml, p < 0.05). These data indicate that the PKA inhibition did not fully abrogate the effect of the PGI2 analogs and suggest that the effect of iloprost and cicaprost were partially dependent on PKA-mediated signaling. Alternatively, because Rp-8-Br-cAMPS discriminates between the two isozymes of PKA and prefers type I PKA (26), this PKA inhibitor may not have sufficient potency to inhibit type II PKA which may be functional in the cAMP-signaling pathway.
It has been shown that NF-B plays a critical role in DC activation and expression of proinflammatory cytokines and chemokines (27). To test the hypothesis that the anti-inflammatory effect of PGI2 was associated with inhibition of NF-B activation, we examined the effect of PGI2 analogs on NF-B transcriptional activity in BMDCs that were derived from NF-B reporter HLL mice. In cells from these mice, luciferase expression can be assayed as a measure of NF-B activity (12, 13). Treatment of purified HLL BMDCs with PGI2 analogs in the presence of LPS significantly decreased LPS-induced luciferase activity (Fig. 4C), suggesting an inhibitory effect of the analogs on NF-B activity. Treatment of the DCs with indomethacin did not change NF-B activity. Treatment of cells with a cAMP analog, db-cAMP, and cAMP-elevating agents, IBMX plus forskolin, also resulted in similar reduction of luciferase expression (Fig. 4C), suggesting that up-regulation of intracellular cAMP inhibits NF-B activity. Cicaprost, db-cAMP, and IBMX plus forskolin also inhibited cIKK2-induced luciferase activity (Fig. 4C), suggesting that factors downstream of IKK2 in the NF-B signaling pathway are affected. Preincubation of HLL BMDCs with the PKA inhibitor, Rp-8-Br-cAMPS, significantly increased luciferase activity by 25–42% (Fig. 4D), suggesting a partial role of cAMP signaling in PGI2 analog-mediated inhibition of NF-B function.
Inhibitory effect of PGI2 analogs on CD86, CD40, and MHC class II expression on DCs
To examine the effect of PGI2 on LPS-induced DC maturation, we measured cell surface expression of CD86, CD40, and MHC class II by flow cytometry after treatment of IP–/– and IP+/+ BMDCs with iloprost, cicaprost, or vehicle solutions in the presence of LPS. Both analogs markedly decreased the expression of CD86, CD40 and MHC class II on DCs (Fig. 5A). The percentage of cells staining for high levels of CD86, CD40, or MHC class II was reduced >60% after the treatment of PGI2 analogs compared with vehicle-treated cells (Fig. 5B). In contrast, iloprost and cicaprost did not change the levels of CD86, CD40, and MHC class II expression on IP–/– DCs, although IP–/– BMDCs had similar levels of expression of the three molecules as IP+/+ BMDCs in response to LPS (data not shown). These results suggested that iloprost and cicaprost inhibited LPS-induced activation and maturation of the DCs and the inhibitory effect on the expression of CD86, CD40, and MHC class II is mainly mediated by the IP-mediated signaling pathway.
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Based on the inhibition of BMDC activation and maturation by PGI2 analogs, we hypothesized that BMDCs treated with iloprost and cicaprost would have decreased ability to activate naive CD4 T cells in an Ag-specific fashion. To test this hypothesis, we treated DO11.10 BMDCs with iloprost (400 nM), cicaprost (400 nM), or vehicle solutions in the presence of LPS (1 µg/ml) and OVA (100 µg/ml) for 24 h. The cells were washed three times and cocultured with freshly purified and CFSE-labeled DO11.10 CD4 T cells for 4 days. We found that treatment of BMDCs with OVA was necessary to stimulate DO11.10 CD4 T cell proliferation and the production of IL-5 and IL-13, indicating that the T cell response was OVA specific. Treatment of the BMDCs with iloprost or cicaprost in the presence of OVA and LPS markedly decreased their ability to stimulate DO11.10 CD4 T cell proliferation as indicated by a significant decrease in the number of divided CFSE-labeled T cells (Fig. 6A) and significantly reduced numbers of total T cells in the culture (Fig. 6B), compared with vehicle-treated DCs. In addition, treatment of the BMDCs with iloprost and cicaprost led to decreased IL-5 and IL-13 production by the CD4 T cells (Fig. 6C). Therefore, iloprost and cicaprost not only inhibited the expression of CD86, CD40, and MHC class II, but also suppressed the ability of BMDCs to stimulate T cells in an Ag-specific manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B activity. Furthermore, these analogs also suppressed DC maturation and function by decreasing CD86, CD40, and MHC class II expression and by inhibiting DC-induced T cell proliferation and cytokine production. These findings suggest that the previously described anti-inflammatory activity of PGI2 and IP signaling (1, 2, 3) may involve inhibition of DC activation, maturation, and function.
Treatment of BMDCs with PGI2 analogs resulted in differential effects on cytokine and chemokine expression. All three PGI2 analogs, iloprost, cicaprost, and treprostinil, dose-dependently inhibited the production of IL-12, TNF-, IL-1, IL-6, MIP-1, and MCP-1, but increased IL-10 production by the BMDCs, consistent with the finding of Jozefowski et al. (29) that iloprost decreased IL-12, TNF-, and IL-6 production by BM-derived cells. The cytokines and chemokines that were suppressed by the PGI2 analogs have a variety of functions in inflammatory and immune responses. They may induce fever and acute phase response proteins (IL-1, IL-6, and TNF-) (30, 31, 32), drive Th1 cell differentiation and cell-mediated immune response (IL-12) (33), and recruit macrophages and monocytes to inflamed areas (MIP-1 and MCP-1) (34). In contrast, IL-10 plays a major role in suppressing inflammatory responses by inhibiting the expression of multiple proinflammatory cytokines (35) and by limiting Th1 immune responses (36). Therefore, PGI2 analogs inhibited the production of proinflammatory cytokines and chemokines while increasing IL-10 production, suggesting an overall anti-inflammatory effect on BMDCs.
What are the molecular mechanisms of PGI2 analog-mediated modulation of the production of inflammatory mediators by DCs? Activation of the IP has been shown to elevate intracellular cAMP, an important second messenger, in smooth muscle cells (24) and macrophages (25). Increased cAMP leads to activation of PKA and phosphorylation of the cAMP response element binding protein (CREB), which binds to the CREB element in the target genes for transcription activation (37). It has been shown that induction of cAMP has been associated with the modulatory effects of cAMP-elevating agents including db-cAMP, forskolin, and the phosphodiesterase inhibitor IBMX. These reagents inhibited IL-12, TNF-, and MIP-1, while increasing IL-10 production by BMDCs (22, 38, 39). A similar effect of cAMP-elevating agents has been reported on BM-derived macrophages (40). We found that iloprost and cicaprost IP-dependently increased cAMP production in BMDCs in a dose-dependent pattern. The elevated cAMP was associated with the IP-dependent inhibition of the proinflammatory cytokine and chemokine production, and CD86, CD40 and MHC class II expression. The effect on cytokine production was partially attenuated by Rp-8-Br-cAMPS, an inhibitor of PKA in the cAMP-signaling pathway. These data suggest an involvement of the cAMP-signaling pathway in the PGI2 analog-mediated effect on DCs. The IP-dependent effect of PGI2 analogs on the production of cAMP and secretion of cytokines and chemokines suggests an important role of IP signaling in the anti-inflammatory function of PGI2.
Another important finding of this study was that PGI2 analogs suppressed NF-B activity in BMDCs. NF-B is a critical transcription factor in the molecular regulation of inflammatory cytokine and chemokine expression (27). Known NF-B-dependent genes include IL-12, TNF-, IL-6, MCP-1, KC, and IL-1
(12, 27, 41, 42). Elevation of intracellular cAMP by cAMP-elevating agents decreased LPS-induced NF-B function as shown in this study and in several other studies (37, 43, 44, 45) and this effect was partially attenuated by Rp-8-Br-cAMPS, suggesting that PGI2 analogs may suppress NF-B function by activating the cAMP-signaling pathway. Although the mechanism of NF-B inhibition by cAMP remains to be investigated, it is possible that elevated levels of cAMP result in more activated CREB. Because both transcription factors, CREB and NF-B, use CREB-binding protein (CBP) as coactivator (46), more activated CREB may compete with NF-B for a limited amount of CBP, leading to decreased NF-B activity. This possibility is strongly supported by a recent study showing that activation of CREB resulted in up-regulation of CREB-dependent IL-10 production and suppression of NF-B-dependent expression of inflammatory cytokines (IL-12, TNF-, IL-6) by human monocytes (47). PGI2 analogs were capable of inhibiting NF-B activity in cells expressing the constitutively active form of IKK2, suggesting that a process downstream of IKK2 activity in the NF-B signaling pathway, possibly involving CBP, was affected. Other possible mechanisms for PGI2 analog/cAMP-mediated NF-B inhibition include blocking nuclear translocation of NF-B (10) and decreasing degradation or up-regulation of IB (48). The action of PGI2 analogs makes them effective inhibitors of proinflammatory cytokines and chemokines and our data indicate that this inhibitory effect of PGI2 analogs is associated with the decrease of NF-B activity.
PGE2 is another PG that has anti-inflammatory properties (22, 38, 49). Similar to the effect of PGI2 analogs, PGE2 has been shown to inhibit IL-12, TNF-, and IL-6 production by BMDCs, suppress MHC class II expression, and enhance IL-10 production (22, 29, 38, 39). Studies showed that DCs may be activated to produce high levels of proinflammatory cytokines and remain at sites of inflammation or develop into CCR7-expressing cells that allow them to migrate to the draining lymph nodes and induce Ag-specific immune responses (50, 51). These migratory DCs produce low levels of inflammatory cytokines (50). PGE2 promotes the generation of the migratory DCs, inhibits proinflammatory cytokine production (50), and up-regulates CCR7 expression (52, 53). For generation of migrating DCs, PGE2 is required at early time points of DC maturation (53). The stimulatory effect of PGE2 on DC migration is dependent on the signaling through EP2 and/or the EP4 receptor (53, 54). The effect of PGE2 is believed to be mediated by cAMP pathway because activation of EP2 and EP4 receptor signaling results in elevated levels of intracellular cAMP (55, 56). Although it remains to be determined whether PGI2 analogs also affect the commitment of DCs to migratory DCs, the similar anti-inflammatory function of PGI2 and PGE2 suggest that these PGs produced in inflammatory sites may act together to modulate the inflammatory and immune processes through the cAMP-signaling pathway.
Our in vitro data indicate that endogenous PGs seem not to contribute to the modulation of cytokine and chemokine production by DCs in an autocrine manner. Our result is consistent with the finding of Jozefowski et al. (29). DCs may be affected in vivo by PGs produced by neighboring cells at the sites of inflammation, leading to decreased DC activation, maturation, and function. In the model of OVA-induced allergic inflammation in the lung, multiple PGs including PGI2, PGE2, PGF2, and PGD2 were detected in the bronchoalveolar lavage fluid (57, 58, 59). The source of PGI2 may be endothelial cells that constitutively express PGI2 synthase (60). Macrophages in the lung may be another source of PGI2 (61). Because of the instability of PGI2, the physiologic concentration of PGI2 in the site of inflammation is difficult to determine. However, considering that iloprost exhibited an equilibrium dissociation constant for the IP with a Kd value of 9.8 nM (62), and PGI2 stimulated adenylyl cyclase activity with an EC50 (the concentration that leads to 50% maximal response) at 6.6 nM in NCB-20 cells (63), it may be reasonable to estimate physiologically functional levels of PGI2 in a range of 10–100 nM. Therefore, our in vitro data showing the effect of iloprost and cicaprost at a range of 4–400 nM on cytokine expression, cAMP production, and T cell stimulatory function of DCs appear to have a physiological relevance.
In summary, we have demonstrated that PGI2 analogs suppress the production of multiple proinflammatory cytokines and chemokines, and increase the anti-inflammatory cytokine IL-10 production by BMDCs. The analogs also inhibit DC maturation and their function to activate naive CD4 T cells in an Ag-specific manner. The modulatory effects were associated with elevated intracellular cAMP production and decreased NF-B activity, suggesting an involvement of cAMP and NF-B-signaling pathways in mediating the PGI2 analog-induced effects. The IP-dependent modulatory effect of PGI2 analogs on DC activation and maturation suggest that PGI2 may exert its anti-inflammatory function by acting on DCs via an IP-mediated signaling pathway.
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Footnotes |
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1 This work was supported by the American Academy of Allergy, Asthma, and Immunology Interest Section Award, R01-AI-054660, R01-HL-069949, and P30 CA68485. 
2 Address correspondence and reprint requests to Dr. Weisong Zhou, Center for Lung Research, Vanderbilt University Medical Center, T1218, Medical Center North, Nashville, TN 37232-2650. E-mail address: weisong.zhou{at}vanderbilt.edu
3 Abbreviations used in this paper: RSV, respiratory syncytial virus; IP, the PGI2 receptor; DC, dendritic cell; BM, bone marrow; BMDC, BM-derived DC; LTR, long terminal repeat; IBMX, 3-isobutyl-1-methylxanthine; IKK, IB kinase; cIKK2, constitutively active IKK 2; COX, cyclooxygenase; PKA, protein kinase A; CBP, CREB-binding protein.
Received for publication February 17, 2006. Accepted for publication October 31, 2006.
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in LPS-stimulated human peripheral blood mononuclear cells. Immunopharmacology 26: 259-264. [Medline] generation. Br. J. Pharmacol. 122: 149-157. [Medline]B nuclear translocation in human alveolar macrophages. J. Biol. Chem. 277: 33344-33348. B activation in a transgenic mouse model of systemic inflammation. Am. J. Respir. Crit Care Med. 162: 1095-1101. B activation in endotoxin-treated mice. Am. J. Respir. Crit. Care Med. 164: 873-878. B in vitro. Genes Dev. 6: 761-774. B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO J. 14: 1552-1560. [Medline]B kinase expression in airway epithelium generates neutrophilic lung inflammation. J. Immunol. 170: 1091-1098. and in alloreactions induced by human lung dendritic cells and macrophages. Am. J. Respir. Cell Mol. Biol. 13: 83-90. [Abstract], and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells. Cell Immunol. 228: 99-109. [Medline]-induced nitric oxide synthase in human monocytes. Nephron 91: 391-398. [Medline]B and IB proteins: an overview. Semin. Cancer Biol. 8: 63-73. [Medline]B-mediated transcription. J. Immunol. 159: 5450-5456. [Abstract]B activation after intratracheal endotoxin. Am. J. Physiol. 277: L823-L830. [Medline]B pathway genes in dendritic cells (DCs) or macrophages assessed by gene expression profiling. J. Cell Biochem. 83: 281-290. [Medline]B-mediated transcription in human monocytic cells and endothelial cells. J. Biol. Chem. 271: 20828-20835. B activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2. FEBS Lett. 400: 315-318. [Medline]-Adrenergic agonists exert their "anti-inflammatory" effects in monocytic cells through the IB/NF-B pathway. Am. J. Physiol. 279: L675-L682. B-mediated transcription. J. Immunol. 159: 5450-5456. [Abstract]This article has been cited by other articles:
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