Characterization of the Phosphodiesterase (PDE) Pattern of in Vitro-Generated Human Dendritic Cells (DC) and the Influence of PDE Inhibitors on DC Function

doi:10.1006/pupt.1999.0220

Pulmonary Pharmacology & Therapeutics

Volume 12, Issue 6, 1 December 1999, Pages 377-386

https://doi.org/10.1006/pupt.1999.0220 Get rights and content

Abstract

During differentiation of human monocytes (CD14⁺/CD1a⁻) to CD14⁻/CD1a⁺dendritic cells (DC), a drastic decrease in PDE4 activity was observed, while activities of PDE1 and PDE3 substantially increased. DC released tumour necrosis factor-α (TNF) in response to lipopolysaccharide (LPS) challenge, which was abolished both by dexamethasone and the cyclic AMP-elevating drugs db-cAMP and PGE₂. In addition, rolipram, at PDE4-selective concentrations, blocked TNF release by 37 ± 5% (P<0.05 vs. control). The PDE3 inhibitor motapizone only marginally influenced TNF synthesis, but a synergistic inhibitory effect was noted in combination with rolipram. Qualitatively, similar inhibitory effects were observed in DC-stimulated T cell responses. Motapizone, lacking efficacy when used alone, increased the effect of rolipram in blocking CD4⁺T lymphocyte proliferation in response to antigen (Ag) (tetanus toxoid, TT; keyhole limpet hemocyanin, KLH) presented by DC and in allogeneic mixed leukocyte reactions (MLR). However, in these coculture systems the T cells rather than the DC seem to be the major target cells of PDE-inhibitor action. In summary, PDE inhibitors can affect DC function directly as demonstrated by blocking TNF release and their efficacy reflects the changes in the PDE activity profile during differentiation from their monocyte precursors. These results together with the known efficacy of PDE3/4 inhibitors in T cells support the concept of combined PDE3/4 inhibitors for asthma therapy.

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PDE3A is abundant in the cardiovascular system, including the myocardium, arterial and venous smooth muscle, bronchial, genitourinary and gastrointestinal smooth muscle as well as the epithelium, megakaryocytes, and oocytes, while PDE3B is highly expressed in adipose tissue (Reinhardt et al., 1995). In the lung, PDE3 was detected in alveolar macrophages, lymphocytes, monocytes, platelets, endothelial cells, as well as in epithelial cells and ASM cells (Beute et al., 2018; Chung, 2006; Gantner, Schudt, Wendel, & Hatzelmann, 1999; Wright, Seybold, Robichaud, Adcock, & Barnes, 1998; Zuo et al., 2018). A substantial body of evidence suggests that PDE3 inhibitors including siguazodan, SK&F94120 and org9935 are potent relaxants in ASM (Bernareggi, Belvisi, Patel, Barnes, & Giembycz, 1999; Nicholson et al., 1995; Torphy et al., 1993).
Chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and asthma, affect millions of people all over the world. Cyclic adenosine monophosphate (cAMP) which is one of the most important second messengers, plays a vital role in relaxing airway smooth muscles and suppressing inflammation. Given its vast role in regulating intracellular responses, cAMP provides an attractive pharmaceutical target in the treatment of chronic respiratory diseases. Phosphodiesterases (PDEs) are enzymes that hydrolyze cyclic nucleotides and help control cyclic nucleotide signals in a compartmentalized manner. Currently, the selective PDE4 inhibitor, roflumilast, is used as an add-on treatment for patients with severe COPD associated with bronchitis and a history of frequent exacerbations. In addition, other novel PDE inhibitors are in different phases of clinical trials. The current review provides an overview of the regulation of various PDEs and the potential application of selective PDE inhibitors in the treatment of COPD and asthma. The possibility to combine various PDE inhibitors as a way to increase their therapeutic effectiveness is also emphasized.
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Due to this dual role of cAMP signaling in APCs, here we assessed the activities of clinically relevant PDE inhibitors on DCs. Leukocytes including DCs predominantly express PDE3 and PDE4 [12,31]. Therefore, we applied the PDE3 inhibitors enoximone and milrinone as used for treatment of heart failure [32] and the pan PDE inhibitor ibudilast [33] used for treatment of asthma in Japan.
Phosphodiesterase 4 (PDE4) inhibitors serve to prevent degradation of the intracellular second messenger cAMP, resulting in broad anti-inflammatory effects on different cell types including immune cells. Agents that elevate cAMP levels via activation of adenylate cyclase have been shown to imprint a Th17-promoting capacity in dendritic cells (DCs). Therefore, we studied the potential of therapeutically relevant PDE inhibitors to induce a pronounced Th17-skewing capacity in DCs. Here we show that mouse bone marrow-derived (BM-) DCs when treated with the PDE4 inhibitor roflumilast (ROF, trade name: Daxas) in the course of stimulation with LPS (ROF-DCs) evoked elevated IL-17 levels in cocultured allogeneic T cells. In addition, as compared with control settings, levels of IFN-γ remained unaltered, while contents of Th2 cytokines (IL-5, IL-10) were diminished. ROF enhanced expression of the Th17-promoting factor IL-23 in BM-DCs. In line, neutralizing antibodies specific for IL-23 or IL-6 when applied to DC/T cell cocultures partially inhibited the IL17-promoting effect of ROF-DCs. Furthermore, ROF-DCs displayed a markedly diminished allogeneic T cell stimulatory capacity due to enhanced production of IL-10, which was restored upon application of IL-10 specific neutralizing antibody to DC/T cell cocultures. Both the IL-17-inducing and impaired T cell stimulatory capacity of BM-DCs were mimicked by a specific activator of protein kinase A, while stimulation of EPACs (exchange proteins of activated cAMP) did not yield such effects. Taken together, our findings suggest that PDE4 inhibitors aside from their broad overall anti-inflammatory effects may enhance the Th17-polarizing capacity in DCs as an unwanted side effect.
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Accordingly, hybrid inhibitors are predicted to have superior clinical efficacy over compounds that selectively block PDE4. Moreover, a PDE3 inhibitor may also have clinically relevant actions on certain pro-inflammatory and immune cells especially during concurrent PDE4 inhibition [108,112–119]. Despite the discontinuation of many hybrid PDE3/4 inhibitors, development of this drug class continues with compounds from Verona Pharma attracting interest, including two long-acting, pyrimido[6,1-a]isoquinolin-4-ones, which are derivatives of trequinsin [120,121].
Chronic obstructive pulmonary disease (COPD) defines a group of chronic inflammatory disorders of the airways that are characterised by a progressive and largely irreversible decline in expiratory airflow. Drugs used to treat COPD through actions mediated by cyclic AMP (cAMP) are restricted to long-acting and short-acting β₂-adrenoceptor agonists and, in a subset of patients with chronic bronchitis, a phosphodiesterase 4 inhibitor, roflumilast. These agents relax airway smooth muscle and suppress inflammation. At the molecular level, these effects in the airways are mediated by two cAMP effectors, cAMP-dependent protein kinase and exchange proteins activated by cAMP. The pharmacology of newer agents, acting through these systems, is discussed here with an emphasis on their potential to interact and increase therapeutic effectiveness.
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The level of cyclic AMP is regulated by many pathways, and phosphodiesterase (PDE) is responsible for the hydrolysis of cyclic nucleotides, leading to the reduction of intracellular levels of cyclic nucleotides. Among 11 distinct families of PDE that are known, PDE-4, a cyclic AMP specific PDE, has received a great deal of attention as a therapeutic target for the development of anti-asthmatic agents, since PDE-4 is a primary enzyme expressed in inflammatory and immunomodulatory cells (Dent et al., 1991; Essayan et al., 1997; Gantner et al., 1999; Tenor et al., 1996), and its inhibition improves many characteristics associated with the pathogenesis of asthma (Barnette, 1999; Torphy, 1998). Interestingly, PDE-4 expression in inflammatory cells is increased in asthmatic patients (Crocker et al., 2000; Mue et al., 1976), indicating its role in the development of asthma.
Phosphodiesterase-4 (PDE-4) is responsible for metabolizing adenosine 3′,5′-cyclic monophosphate that reduces the activation of a wide range of inflammatory cells including eosinophils. PDE-4 inhibitors are under development for the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Herein, we report a novel PDE-4 inhibitor, PDE-423 (3-[1-(3-cyclopropylmethoxy-4-difluoromethoxybenzyl)-1H-pyrazol-3-yl]-benzoic acid), which shows good in vitro and in vivo oral activities. PDE-423 exhibited in vitro IC₅₀s of 140 nM and 550 nM in enzyme assay and cell-based assay, respectively. In vivo study using ovalbumin-induced asthmatic mice revealed that PDE-423 reduced methacholine-stimulated airway hyperreactivity in a dose-dependent manner by once daily oral administration (ED₅₀ = 18.3 mg/kg), in parallel with decreased eosinophil peroxidase activity and improved lung histology. In addition, PDE-423 was effective in diminishing lipopolysaccharide-induced neutrophilia in vivo as well as in vitro. Oral administration of PDE-423 (100 mg/kg) had no effect on the duration of xylazine/ketamine-induced anesthesia and did not induce vomiting incidence in ferrets up to the dose of 1000 mg/kg. The present study indicates that a novel PDE-4 inhibitor, PDE-423, has good pharmacological profiles implicating this as a potential candidate for the development of a new anti-asthmatic drug.
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The most prominently expressed PDE subtypes in immune cells are PDE3 and PDE4 (Lugnier, 2006). In some cell types a combined inhibition of both enzyme activities is required to obtain maximal increases of cAMP and attenuation of inflammation (Gantner et al., 1997, 1999). In other cells PDE inhibitors have no effect unless they are combined with a cAMP-increasing stimulus (Selige et al., 2010a).
Inflammation is an important hallmark of all neurodegenerative diseases and activation of different glial populations may be involved in the progression of some of these disorders. Especially, the activation of astroglia can lead to long-term detrimental morphological changes, such as scar formation. Therefore, improved strategies to modulate inflammation in these cells are currently being investigated. We investigated the interaction of phosphodiesterase (PDE) 4 inhibitors, such as rolipram, with other agents raising cellular cAMP levels. When used alone, none of the PDE4 inhibitors increased cAMP levels. The adenylate cyclase activator forskolin, the β₂-adrenergic agonist clenbuterol and the mixed β₁/β₂-adrenergic agonist isoproterenol increased intracellular cAMP levels of cortical murine astrocytes. This increase was synergistically elevated by rolipram or the PDE4 inhibitor RO-201724, but not by inhibition of PDE3. Inflammatory stimulation of the cells with the cytokines TNF-α, IL-1β and IFN-γ strongly induced PDE4B and augmented overall PDE4 activity, while PDE3 activity was low. Clenbuterol and forskolin caused downregulation of cytokines and chemokines such as IL-6 and MCP-1. This effect was further enhanced by rolipram, but not by the PDE3 inhibitor milrinone. The cAMP-raising drug combinations attenuated the upregulation of TNF-α and IL-6 mRNA and the secretion of IL-6, but did not affect initial NF-κB signalling triggered by the stimulating cytokines. These results indicate that PDE4 may be a valuable anti-inflammatory target in brain diseases, especially under conditions associated with stimulation of cAMP-augmenting astrocyte receptors as is observed by clenbuterol treatment.
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^f1: Author for correspondence: Dr Armin Hatzelmann, Byk Gulden, Department of Biochemistry, POB 100310, D-78403 Konstanz, Germany. Fax: 49-7531-84-9-2690.

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Regular ArticleCharacterization of the Phosphodiesterase (PDE) Pattern of in Vitro-Generated Human Dendritic Cells (DC) and the Influence of PDE Inhibitors on DC Function

Abstract

Mol Med Today

Immunol Today

Immunol Today

TIBS

J Allergy Clin Immunol

J Allergy Clin Immunol

Biochem Pharmacol

J Allergy Clin Immunol

Immunol Today

Dendritic cells: from ontogenetic orphans to myelomonocytic descendants

Immunol Today

New insights into the mobilization and phagocytic activity of dendritic cells

J Exp Med

The endocytic activity of dendritic cells

J Exp Med

Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium

J Exp Med

Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature

Immunology

Isolation and characterization of human peripheral blood dendritic cells

J Immunol

Dendritic cells are required for the development of chronic eosiniphilic airway inflammation in response to inhaled antigen in sensitzed mice

J Immunol

Interaction of PDE4 inhibitors with enzymes and cell functions

In: Schudt C, Dent G, Rabe K F eds. Phosphodiesterase Inhibitors London: Academic Press

Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor- α

J Exp Med

Regular Article
Characterization of the Phosphodiesterase (PDE) Pattern of in Vitro-Generated Human Dendritic Cells (DC) and the Influence of PDE Inhibitors on DC Function