Abstract
Background Dendritic cells (DCs) are professional antigen-presenting cells and have critical roles in regulating immune responses. Prostaglandin I2 (PGI2) analogs are considered to be potential treatments for asthma. However, the effect of PGI2 analogs on human monocyte-derived DCs (MDDCs) is still not clearly understood.
Methods Human MDDCs were pretreated with iloprost and treprostinil (2 PGI2 analogs) or forskolin (an adenyl cyclase activator) before lipopolysaccharide (LPS) stimulation. In some cases, I prostanoid (IP) receptor and E prostanoid receptor antagonists were added before the PGI2 analog treatment. tumor necrosis factor α (TNF-α) was measured by enzyme-linked immunosorbent assay. The expression of costimulatory molecules was assessed by flow cytometry. T-cell polarization function was investigated by measuring interferon γ, interleukin 13 (IL-13), and IL-17A production by T cells cocultured with iloprost-treated MDDCs.
Results Iloprost and treprostinil suppressed LPS-induced TNF-α expression in MDDCs. This effect could be reversed by an IP receptor antagonist, CAY10449, but not by E prostanoid receptor antagonists. Forskolin conferred a similar effect. Iloprost suppressed the LPS-induced expression of costimulatory molecules, including CD80, CD86, CD40, and HLA-DR. Iloprost-treated MDDCs increased IL-17A production by T cells.
Conclusions Prostaglandin I2 analogs may exert anti-inflammatory effects by suppressing TNF-α expression via the IP receptor-cyclic adenosine monophosphate pathways and by inhibiting the expression of costimulatory molecules in human MDDCs.
Asthma is a chronic inflammatory disorder of the airway. In the airways of patients with allergic asthma, the accumulation of inflammatory cells, including eosinophils, lymphocytes, neutrophils, and mast cells, can be observed.1Tumor necrosis factor α (TNF-α), a pleiotropic proinflammatory cytokine, is critically involved in the pathogenesis of asthma. Increased TNF-α messenger RNA and protein levels have been reported in the airways of patients with asthma.2Tumor necrosis factor α can be produced by macrophages, monocytes, dendritic cells (DCs), neutrophils, mast cells, eosinophils, and structural cells. Tumor necrosis factor α is also a chemoattractant for neutrophils and eosinophils and is involved in the activation of T cells.3Evidence has emerged to suggest that TNF-α might have a central role in severe refractory asthma because of its roles in neutrophil recruitment, in the induction of resistance to steroids, and in airway remodeling via the stimulation of fibroblast growth and maturation.4Recently, TNF-α has been proposed to be a useful biomarker in refractory asthma,4and targeting TNF-α may have therapeutic potential in treating refractory asthma.3
Dendritic cells are professional antigen-presenting cells that orchestrate the innate and adaptive immune responses. Dendritic cells initiate the immune response by upregulating MHC and costimulatory molecules and by producing a variety of inflammatory mediators, such as cytokines and chemokines. Dendritic cells are produced in the bone marrow and migrate into tissues via the bloodstream. However, under some conditions, monocytes can be recruited to the sites of inflammation and differentiate into macrophages or DCs, depending on the surrounding maturation and differentiation factors.5Therefore, DCs developed from CD14+ monocytes under granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) stimulation have been studied extensively for their medical implications. In asthma, DCs both initiate the polarization of and maintain adaptive T helper 2 (TH2) cells in response to inhaled allergens.6For example, monocyte-derived DCs (MDDCs) can produce TNF-α in response to common allergens, such as BG60 or Derp2, via DC-specific ICAM3-grabbing nonintegrin (DC-SIGN).7In addition, Derp1 can activate human MDDCs to promote TH2 cell responses through its effects on polarizing cytokines, costimulatory molecules, and cell surface receptors.8
Prostaglandins (PGs) are synthesized from arachidonic acid by cyclooxygenases and are considered to be proinflammatory molecules. However, prostaglandin I2 (PGI2) exerts anti-inflammatory effects via the I prostanoid (IP) receptor-cyclic adenosine monophosphate (cAMP) axis.9Iloprost, a stable PGI2 analog administered in an inhaled form, is widely accepted for the treatment of pulmonary arterial hypertension. In animal models, iloprost suppresses the cardinal features of asthma by inhibiting airway DC function, which induces TH2 differentiation,10and is thus considered to be a candidate for the treatment of asthma. We have previously shown that iloprost might induce tolerance by enhancing IL-10 production and by suppressing TNF-α in human plasmacytoid DCs, suggesting that PGI2 analogs have anti-inflammatory effects in humans.11Recently, we also showed that PGI2 analogs enhance growth-related oncogene (GRO) α, a neutrophil chemoattractant, in human MDDCs through the IP receptor-cAMP pathway.12However, whether PGI2 analogs modulate TNF-α expression, an important cytokine in severe and refractory asthma, or the maturation of human DCs has not been studied. In the present study, we investigated the in vitro effects of 2 PGI2 analogs, iloprost and treprostinil, on lipopolysaccharide (LPS)-stimulated TNF-α production in MDDCs. We also investigated the effects of iloprost on the expression of costimulatory molecules and the T-cell polarization activity of MDDCs.
MATERIALS AND METHODS
Isolation and Culture of MDDCs
The study protocol was approved by the Institutional Review Board of Kaohsiung Medical University Hospital. Peripheral blood samples were obtained from healthy individuals who had no personal or family history of allergic or systemic diseases (n = 6) after informed consent was obtained. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over Ficoll-Histopaque (Pharmacia Biotech, Uppsala, Sweden) then separated into a low-density fraction enriched in DCs by centrifuging for 30 minutes at 300g. Blood monocytes were magnetically sorted from PBMCs using CD14 cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions. To differentiate monocytes into immature MDDCs, isolated monocytes were cultured with 20 ng/mL of recombinant human GM-CSF (Biosource, Camarillo, CA) and 20 ng/mL of recombinant human IL-4 (R&D Systems, Abingdon, UK). Fresh medium was added every 2 days, and the cells were used in experiments after 7 days of cultivation.12Before each experiment, the morphology of the DCs was checked, and the DCs were analyzed for the expression of CD11c, CD209 (DC-SIGN), HLA-DR, and CD14. In all cases, the MDDCs used were CD14-negative (<0.7%) and highly expressed CD11c (>96%), CD209 (>88%), and HLA-DR (>97%) by flow cytometry (Fig. 1) and had a typical morphology. Monocyte-derived DCs were cultured in 24-well, round-bottom plates (105 cells/well) in 500 μL of RPMI 1640 medium (Sigma-Aldrich, St Louis, MO) buffered with NaHCO3 and containing 10% heat-inactivated, endotoxin-tested fetal calf serum, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin. Monocyte-derived DCs were treated with varying doses of the 2 PGI2 analogs, iloprost and treprostinil (10−10 to 10−7 M), or an adenyl cyclase activator, forskolin, for 2 hours and were stimulated with LPS (0.2 μg/mL; Escherichia coli-derived; Sigma-Aldrich). Supernatants were collected for the measurement of TNF-α 24 hours after LPS stimulation. In some cases, MDDCs were pretreated with an IP receptor antagonist (CAY10449), an E prostanoid 1 (EP1) receptor antagonist (SC19220), an EP2 receptor antagonist (AH 6809), or an EP4 receptor antagonist (GW627368X) 1 hour before the treatment of the cells with iloprost or treprostinil, and the cells were stimulated with LPS 2 hours after iloprost or treprostinil treatment. All IP and EP receptor antagonists were purchased from the Cayman Chemical Company (Cayman Chemical, Ann Arbor, MI).
Flow Cytometric Analysis
To analyze the surface markers of MDDCs generated from CD14+ monocytes after a 7-day cultivation with GM-CSF and IL-4, MDDCs were used for direct immunofluorescence staining with fluorescein isothiocyanate-labeled monoclonal antibodies to CD11c or CD209 and with phycoerythrin-labeled monoclonal antibodies to HLA-DR or CD14. To investigate the effect of PGI2 analogs on MDDC maturation, MDDCs were cultured in 12-well, round-bottom plates (106 cells/1 mL per well), treated with iloprost (10−7 M) for 2 hours, and stimulated with LPS for 48 hours. Monocyte-derived DCs were harvested and washed twice with PBS and were used for direct immunofluorescence staining with fluorescein isothiocyanate-labeled monoclonal antibodies to CD40 or CD80 and phycoerythrin-labeled monoclonal antibodies to CD86 or HLA-DR. All fluorescence-conjugated monoclonal antibodies were purchased from eBioscience (San Diego, CA). The surface markers of the MDDCs were analyzed using a FACScan flow cytometer and CellQuest software (BD Bioscience, Franklin Lakes, NJ).
MDDC and Autologous T-cell Coculture
CD4+ T cells were isolated from PBMCs using human CD4 MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. The MDDCs were pretreated with iloprost (10−7 M) for 2 hours and then stimulated with LPS (0.2 μg/mL) for 24 hours. The MDDCs were harvested and washed twice with PBS then cocultured with isolated autologous T cells in 24-well, round-bottom plates with 1 mL/well (105 MDDCs/106 T cells). Five days after the initiation of the coculture, the supernatants were collected to measure interferon γ (IFN-γ), IL-13, and IL-17A to evaluate the effects of iloprost-treated MDDCs on T-cell polarization.
Enzyme-Linked Immunosorbent Assay
The TNF-α, IFN-γ, IL-13, and IL-17A concentrations in the cell supernatants were determined using a commercially available enzyme-linked immunosorbent assay-based system (R&D Systems, Minneapolis, MN). The assays were performed using the protocols recommended by the manufacturer.
Statistical Analysis
For each experiment, 3 replicates were performed for each subject's MDDCs, and at least 3 subjects' MDDCs were used to confirm the results presented. All data are presented as the mean (SD). Differences between the experimental and control groups were analyzed using the Wilcoxon signed rank test. P < 0.05 was considered to be indicative of a significant difference between the groups.
RESULTS
PGI2 Analogs Suppress LPS-Induced TNF-α Expression in MDDCs
To examine the potential effect of PGI2 analogs on the expression of TNF-α in human DCs, MDDCs were treated with varying doses of iloprost or treprostinil, either alone or in combination with LPS (0.2 μg/mL). As shown in Figure 2A, LPS-induced TNF-α expression in MDDCs was significantly suppressed in the presence of iloprost in a dose-dependent manner (10−10 to 10−7 M after 24 hours of LPS stimulation). Lipopolysaccharide-induced TNF-α expression in MDDCs was also significantly suppressed in the presence of treprostinil in a dose-dependent manner (10−10 to 10−7 M after 24 hours of LPS stimulation; Fig. 2B). However, iloprost (10−10 to 10−7 M) or treprostinil (10−10 to 10−7 M) alone had no effect on TNF-α expression in MDDCs (data not shown).
PGI2 Analogs Suppress LPS-Induced TNF-α Expression in MDDCs via the IP Receptor-cAMP Pathway
Prostaglandin I2 analogs are reported to exert their anti-inflammatory effects via the IP receptor or EP receptor.13The signal transduction of the IP and EP receptors induces an increase in intracellular cAMP.14,15To examine whether the suppressive effect of PGI2 analogs on LPS-induced TNF-α expression in MDDCs was mediated by the IP or EP receptors, MDDCs were pretreated with an IP receptor antagonist (CAY10449) or EP receptor antagonists. As shown in Figure 3, A and B, CAY10449 partially reversed the suppression of LPS-induced TNF-α expression by iloprost and treprostinil in MDDCs in a dose-dependent manner. However, the EP1, EP2, or EP4 receptor antagonists did not reverse the iloprost- or treprostinil-mediated suppression of LPS-induced TNF-α expression (Fig. 3, C and D). The IP or EP receptor antagonists alone had no effect on TNF-α expression (data not shown). Next, we used an adenyl cyclase activator, forskolin, to examine whether increasing intracellular cAMP would have a similar effect to iloprost and treprostinil on TNF-α expression. As shown in Figure 3E, forskolin suppressed TNF-α expression in LPS-stimulated MDDCs. These data suggest that iloprost and treprostinil suppress TNF-α expression via the IP receptor-cAMP pathway in LPS-stimulated MDDCs.
Iloprost Suppresses the LPS-Induced Up-Regulation of CD40, HLA-DR, CD80, and CD86 in MDDCs
After exposure to antigen, DCs respond by producing immunostimulatory mediators and upregulating MHC and costimulatory molecules and become mature DCs with an increased capability for antigen presentation and immunostimulation.16The full activation of naive T cells requires 2 signals, the interaction of the T-cell receptor with the MHC and antigenic peptide complex on the antigen-presenting cells, and the expression of costimulatory molecules.17We next examined whether iloprost regulates the expression of the costimulatory molecules CD40, HLA-DR, CD80, and CD86. The expression of the cell surface markers was investigated using flow cytometric analysis after treatment with LPS for 48 hours with or without pretreatment with iloprost. As shown in Figure 4, iloprost suppressed LPS-induced CD40 and HLA-DR expression. Iloprost also suppressed LPS-induced CD80 and CD86 expression in MDDCs. These results suggest that iloprost inhibits the LPS-induced maturation of MDDCs.
Iloprost-Treated MDDCs Enhance IL-17A Production by T Cells
Because of the inhibitory effect of iloprost on MDDC maturation, we next investigated whether iloprost could regulate the polarization of T cells induced by LPS-stimulated MDDCs. Autologous T cells were cocultured with iloprost/LPS-treated or LPS-treated MDDCs, and cytokine production, including that of he TH1 cytokine IFN-γ, the TH2 cytokine IL-13, and the TH17 cytokine IL-17A, was measured. As shown in Figure 5, the iloprost-treated MDDCs had no effect on IFN-γ production by T cells. There was a trend that the iloprost-treated MDDCs suppressed IL-13 production. However, the effect was not statistically significant. Unexpectedly, the iloprost-treated MDDCs enhanced IL-17A production by T cells (Fig. 5).
DISCUSSION
Dendritic cells bridge the innate and adaptive immune responses and regulate inflammation by a variety of mediators, including cytokines and chemokines.18In atopic patients, MDDCs modulate T-cell differentiation toward a TH2 response,19and the function of MDDCs can be altered by antiasthmatic medications.20Therefore, understanding the regulatory pathways controlling the expression of cytokines and costimulatory molecules in MDDCs would greatly help in advancing our understanding of the modulatory effects of medications. Lipopolysaccharide is ubiquitously present in the environment and has been identified as an important trigger for the exacerbation of asthma.21,22In a mouse model of allergic sensitization, low levels of inhaled LPS were shown to induce TH2 responses to the inhaled antigen, and the process required the activation of antigen-containing DCs.23Lipopolysaccharide is also an important adjuvant for efficient sensitization and for the accumulation of neutrophils and eosinophils in the murine asthmatic airway.24In the present study, we demonstrated for the first time a role for PGI2 analogs in LPS-stimulated human MDDCs. Prostaglandin I2 analogs suppressed LPS-induced TNF-α production and inhibited the up-regulation of costimulatory molecules, including CD40, HLA-DR, CD80, and CD86. These data illustrate that the anti-inflammatory effects of the PGI2 analogs could be due to the alteration of the function of human MDDCs.
Tumor necrosis factor α is important in the pathophysiology of asthma and allergic diseases. Tumor necrosis factor α is released in response to LPS by MDDCs, leading to an inflammatory response. Recent studies have reported an association between increased levels of TNF-α and severe refractory asthma phenotypes.2-4,25Tumor necrosis factor α has a central role in the pathogenesis of severe refractory asthma by recruiting neutrophils, inducing resistance to steroids, and stimulating the myocyte and fibroblast proliferation that leads to airway remodeling.4An anti-TNF-α antibody is a recent and novel therapeutic option for the treatment of severe refractory asthma, but the efficacy is still controversial, and the treatment has some severe adverse effects.3In the present study, we found that both iloprost and treprostinil significantly suppressed LPS-induced TNF-α expression in human MDDCs, suggesting that these compounds have therapeutic potential for the treatment of patients with severe or refractory asthma.
Emerging evidence suggests that the anti-inflammatory effects of PGI2 are mediated through the activation of IP receptors.9,11In IP-deficient mice, allergen-induced airway remodeling can develop as a result of TH2 cytokines, IgE, or airway eosinophilic inflammation.26The immunomodulatory functions of the PGI2 analogs can also be mediated through the EP receptors.9Activation of the IP/EP receptors has been shown to elevate intracellular cAMP in several types of cells, including macrophages,9DCs,13smooth muscle cells,27and THP-1 cells.28,29Herein, we demonstrated that the suppressive effects of iloprost and treprostinil on TNF-α expression are IP-dependent, and an adenyl cyclase activator (forskolin) could confer similar effects. These data suggest that iloprost and treprostinil may suppress LPS-induced TNF-α expression at least partly via the IP receptor-cAMP pathway.
Before allergen challenge, immature DCs express low levels of MHC class II and costimulatory molecules, and they are not effective in antigen presentation or T-cell stimulation.30Allergic airway inflammation can be induced and enhanced by DC maturation,31which can be detected by the expression of costimulatory molecules, which activate T-cell proliferation and differentiation. MHC-II signaling is necessary to trigger TH2 effector cytokine production.31During allergen sensitization, CD40 signaling is fundamental for triggering isotype class switching to IgE.32CD40 also increases TH2 cytokine production and suppresses the number of regulatory T cells.33CD80/86 costimulation is necessary for the differentiation of TH2 cells from naive T cells.34In allergic patients, CD80 and CD86 are upregulated on the surface of alveolar macrophages.35,36In the present study, iloprost suppressed the expression of MHC-II (HLA-DR), CD40, CD80, and CD86 and therefore inhibited the maturation of MDDCs. These data provide the mechanism by which iloprost reduces TH2-mediated inflammation.
Another interesting finding of the present study was the effect of iloprost on the T-cell polarization function of MDDCs. Although we observed the trend that iloprost-treated MDDCs decreased IL-13 (a TH2 cytokine) production by T cells, the effect was not statistically significant. In addition to the well-characterized lineages of TH1 and TH2 cells, a third T helper cell subset that produces IL-17A, termed TH17, has been recently linked to autoimmune and allergic inflammation.37IL-17A can attenuate the TH2 response by downregulating TH2 cytokines and chemokines.38However, IL-17A can promote airway neutrophils and hyperreactivity by acting synergistically with the ongoing TH2 response.24Increasing evidence suggests that IL-17A, acting either directly or indirectly, significantly stimulates neutrophil maturation, migration, and function.39Surprisingly, in the present study, the results of the MDDC/T-cell coculture experiment revealed that iloprost-treated MDDCs could significantly enhance IL-17A production by T cells, although the level of IL-17A was low. Intriguingly, in our previous work, we reported that PGI2 analogs could significantly enhance the expression of the neutrophil chemoattractant GRO-α in human MDDCs and may thus recruit neutrophils.12Whether PGI2 analogs have significant effects on neutrophil recruitment in vivo needs further investigation.
The findings of our study increase our understanding of the anti-inflammatory effects of PGI2 analogs. Prostaglandin I2 analogs can suppress TNF-α expression and induce the anti-inflammatory cytokine IL-10 in murine bone marrow-derived DCs.10,13In the murine model, iloprost suppresses the cardinal features of asthma by inhibiting lung myeloid DCs10and blocks allergic pulmonary inflammation by preventing the recruitment of TH2 cells into the airway.40In human studies, PGI2 analogs have been shown to suppress TNF-α expression in human primary monocytes41and in plasmacytoid DCs.11Very recently, Muller et al.42reported that iloprost inhibits inflammatory cytokine production by human MDDCs and induces the generation of regulatory T cells. The present study additionally demonstrated the role of PGI2 analogs in regulating the expression of costimulatory molecules for the maturation of human MDDCs, supporting the current evidence for the candidacy of the PGI2 analog as a modulatory agent in treating asthma.
In conclusion, the present study has revealed that PGI2 analogs suppress LPS-induced TNF-α expression via the IP receptor-cAMP pathway and inhibit maturation by suppressing costimulatory molecule expression in human MDDCs. These data address the effectiveness of PGI2 analogs in humans and have implications for the therapeutic potential of PGI2 analogs in treating asthma.