Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2013 Sep 17;170(3):602–613. doi: 10.1111/bph.12308

Comparative assessment of PDE 4 and 7 inhibitors as therapeutic agents in experimental autoimmune encephalomyelitis

C González-García 1,†,*, B Bravo 1,*, A Ballester 1, R Gómez-Pérez 1,‡, C Eguiluz 2, M Redondo 3, A Martínez 3, C Gil 3, S Ballester 1
PMCID: PMC3791998  PMID: 23869659

Abstract

BACKGROUND AND PURPOSE

PDE4 inhibition suppresses experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, side effects hinder PDE4 inhibitors clinical use. PDE7 inhibition might constitute an alternative therapeutic strategy, but few data about the anti-inflammatory potential of PDE7 inhibitors are currently available. We have used the EAE model to perform a comparative evaluation of PDE4 and PDE7 inhibition as strategies for MS treatment.

EXPERIMENTAL APPROACH

Two PDE7 inhibitors, the sulfonamide derivative BRL50481 and the recently described quinazoline compound TC3.6, were assayed to modulate EAE in SJL mice, in comparison with the well-known PDE4 inhibitor Rolipram. We evaluated clinical signs, presence of inflammatory infiltrates in CNS and anti-inflammatory markers. We also analysed the effect of these inhibitors on the inflammatory profile of spleen cells in vitro.

KEY RESULTS

TC3.6 prevented EAE with efficacy similar to Rolipram, while BRL50481 had no effect on the disease. Differences between both PDE7 inhibitors are discussed. Data from Rolipram and TC3.6 showed that PDE4 and PDE7 inhibition work through both common and distinct pathways. Rolipram administration caused an increase in IL-10 and IL-27 expression which was not found after TC3.6 treatment. On the other hand, both inhibitors reduced IL-17 levels, prevented infiltration in CNS and increased the expression of the T regulator cell marker Foxp3.

CONCLUSIONS AND IMPLICATIONS

These results provide new information about the effects of Rolipram on EAE, underline PDE7 inhibition as a new therapeutic target for inflammatory diseases and show the value of TC3.6 to prevent EAE, with possible consequences for new therapeutic tools in MS.

Keywords: PDE, inhibitor, EAE, inflammation

Introduction

Experimental autoimmune encephalomyelitis (EAE) is considered as a good model of the human demyelinating disease multiple sclerosis (MS) (Constantinescu et al., 2011). EAE has proved to be useful to study mechanisms underlying MS pathogenesis and to carry preclinical testing of potential therapies for the human disease (Pierson et al., 2012). In fact, many of the drugs in current use have been previously assayed in this animal model (Teitelbaum et al., 1971; Blaszczyk et al., 1978; Yednock et al., 1992). This model has also shed light on mechanisms of action of therapeutic approaches for MS initially unrelated to EAE studies (Martin-Saavedra et al., 2007; 2008; Axtell et al., 2010; Inoue et al., 2012). Although different EAE models, depending on the antigen, species, strain or experimental condition used can determine different disease evolution (chronic or relapsing-remitting progression); some general features are shared by most EAE models. Administration of components of the CNS as antigen coupled to adjuvant to stimulate an innate immune response triggers an inflammatory reaction through activation of auto-responsive T-cells in lymph nodes and spleen able to migrate to the CNS and to cross the blood-brain barrier (BBB), where they recognize their specific antigen in the context of resident antigen presenting cells microglia or astrocytes, or immigrant macrophages or dendritic cells. These events favour an inflammatory environment with CNS injury and motor disability. Autoreactive T helper (Th) cells with Th1 (IFN-γ producers) or Th17 (IL-17 producers) phenotype are able to mediate EAE development (Chen et al., 2012). Nevertheless, current trends consider IL-17 pathway as a more suitable therapeutic target over IFN-γ due to the dual role of the latter on the course of the disease (Pierson et al., 2012).

Several immunomodulating cytokines like IL-4, IL-10 and IL-27 have the ability to restrain the disease (Bettelli et al., 1998; Falcone et al., 1998; Fitzgerald et al., 2007b; Wang et al., 2008). EAE development is also restricted by Foxp3+ T regulator (Treg) cells (TGF-β producers) (Kohm et al., 2003; McGeachy et al., 2005). Sustained increase of cAMP mediates a variety of immunoregulatory signals which lead to attenuation of immune reactions and inflammation (Brudvik and Tasken, 2012). Several compounds able to increase the intracellular levels of this second messenger have long shown anti-inflammatory properties and potential to control EAE (Reder et al., 1994; Genain et al., 1995; Correa et al., 2010). Inhibition of cAMP PDE leads to increased levels of intracellular cAMP, as these enzymes are responsible for cAMP catabolism (Mosenden and Tasken, 2011). Mainly PDE4 inhibition has been reported to improve the clinical signs of EAE (Sommer et al., 1995; Martinez et al., 1999; Sanchez et al., 2005; Paintlia et al., 2009). However, due to the difficulties to separate beneficial anti-inflammatory from adverse emetic effects of PDE4 inhibitors, efforts have been focused on finding inhibitors of other PDE that might provide therapeutic alternatives. Special interest has been directed to PDE7 inhibition as an anti-inflammatory strategy (Smith et al., 2004; Giembycz and Smith, 2006; Castano et al., 2009; Jerez et al., 2013), and some promising results using PDE7 inhibitors in EAE have been recently reported (Redondo et al., 2012a; 2012b).

In the present study, we have analysed in depth the anti-inflammatory capabilities of PDE7 inhibition, and performed a comparison between the mechanisms by which PDE4 and PDE7 inhibitors are able to ameliorate EAE. We assayed the PDE4 inhibitor Rolipram, as a well-known anti-inflammatory drug and assayed two chemically different PDE7 inhibitors drugs, the sulfonamide derivative BRL50481 (IC50 for PDE7A1 inhibition of 0.26 1 μM) (Smith et al., 2004) and the newer quinazoline type PDE7 inhibitor TC3.6 (IC50 for PDE7A1 inhibition of 1.04 μM), which has shown neuroprotective properties in an in vivo stroke model (Redondo et al., 2012c). Our results show that TC3.6, but not BRL50481, is as efficient as Rolipram in the control of EAE. In addition, we provide new information about the therapeutic action of Rolipram on EAE, and reveal that inhibition of PDE4 or PDE7 by TC3.6 act through both common and different anti-inflammatory mechanisms.

Methods

Animals, EAE induction and treatments

All experiments were conducted according to the institutional ethical and safety guidelines. SJL and C57BL/6 mice were used to perform EAE experiments and to obtain T-cells for in vitro assays, respectively. Murine strains were purchased from Charles River, Barcelona, Spain; SJL mice were acquired for each experiment, while C57BL-6 were breed and maintained at the animal facility of the Institution. Mice were housed in groups of 4–5 animals. Induction of EAE was performed in SJL mice by myelin basic protein (MBP) inoculation as previously described (Martin-Saavedra et al., 2007). A healthy control group was established for each experiment by dispensation of the same treatment without MBP. For treatments, PDE inhibitors were daily administrated since the day of MBP inoculation (D0) by i.p. injection at the indicated dose for each case in a solution of 14% DMSO and 5% Tocrisolve (R&D Systems, Oxon, UK). Mice inoculated with 14% DMSO and 5% Tocrisolve made up the control untreated EAE group for each experiment. PDE4 inhibitor Rolipram and PDE7 inhibitor BRL50481 were purchased from Sigma-Aldrich (Madrid, Spain) and Tocris Bioscience respectively. The 3-phenyl-2,4-dithioxo-1,2,3,4-tetrahydroquinazoline PDE7 inhibitor TC3.6 was synthesized as previously described (Castano et al., 2009). Clinical signs of EAE were scored according to a 0–5 scale as follows: no clinical signs, 0; loss of tail tonicity, 1; limp tail, 2; rear limbs weakness, 3; paralysis of two limbs, 4; full paralysis of four limbs, 5. Mice were killed at day 10–15 post-inoculation by CO2 inhalation. When any mouse reached score 4 before day 10–15 post-inoculation, it was killed in advanced. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).

Histopathology

Intact brains were removed from mice on day 15 post-inoculation, dissected and fixed by 10% buffered formalin. Paraffin-embedded blocks were cut into 4-μm thick sections and stained with haematoxylin and eosin and analysed by light microscopy to reveal inflammatory lesions. Quantification of perivascular infiltrates was carried out by examining four sections along the brain of each animal; sections were classified as positive or negative for infiltrates and numerical data were analysed by Fisher's test.

Cell isolation and culture

For analysis of in vivo PDE inhibitors effects, lymph nodes or spleens were removed from EAE animals on day 10–15 post-inoculation. Total spleen cells, lymph node cells (LNC), CD4+ cell purification and CNS cell isolation were performed as described (Martin-Saavedra et al., 2008). Cells from each group of EAE animals were pooled, washed and suspended in Click's medium (Click et al., 1972) before in vitro treatment.

In vitro T-cell phenotype polarization

For in vitro T helper phenotype polarization, spleen cells from C57BL/6 mice were used. Most of the in vitro results obtained for C57BL/6 strain were reproduced for C3H mice to exclude possible strain specificity results. In general, the experiments were performed at 10 μM of TC3.6 and Rolipram. When other doses were used, it is indicated at the corresponding figure. Freshly isolated spleen total population or purified CD4+ T-cells were in vitro stimulated by coated-plate anti-CD3 (YCD3-1, 50 μg mL−1) (Portolés et al., 1989) and soluble anti-CD28 clone 37.51 (0.5 ng mL−1, eBioscience, Hatfield, UK) in Click's medium. Specific phenotype culture conditions were carried out in the presence of 10 ng mL−1 of IL-12 (Prepotech, London, UK) and 25 μg mL−1 of anti-IL-4 (11B11; ATCC HB188) for Th1 phenotype induction; or 20 ng mL−1 of IL-6 (eBioscience), 5 ng mL−1 of TGFβ (eBioscience), 25 μg mL−1 of anti-IL-4 (11B11; ATCC HB188) and 25 μg mL−1 of anti-IFN-γ (R46A2; ATCC HB170) for Th17 polarization. For Th0 samples, only anti-CD3 and anti-CD28 were used to supplement the medium. Except for Th17 induction, after 24 h of culture, 50 u mL−1 of IL-2 (Prepotech) were added. After a total of 3 or 7 days of culture, cells were harvested, extensively washed and restimulated as indicated for each experiment. For CD11b+ cells, depletion anti-CD11b antibody (Mac-1α, Miltenyi Biotec, Madrid, Spain) was used following the manufacturer's instructions. For IL-27 neutralization, anti-IL-27p28 antibody (R&D Systems) (1 μg mL−1) was added.

T-cell proliferation and soluble cytokine detection

Proliferation was measured by the colorimetric assay based on thiazolyl blue tetrazolium bromide use (Mosman, 1983). A 2 × 105 cells were split on plate-bound anti-CD3 p-96 microtitre wells supplemented with soluble anti-CD28 during 72 h. Cytokines released to the medium were quantified by ELISA. Captured and biotin-conjugated antibodies, respectively were eBio17CK15A5 and eBio17B7 (e-Bioscience) for IL-17A; Jes5-2A5 and Jes5-16E3 (BD-Bioscience, Madrid, Spain) for IL-10; R4-6A.2 and XMG1.2 BD Bioscience) for IFN-γ; and 11B11 and BVD6-24G2 (Becton-Dickinson, Madrid, Spain) for IL-4. Each sample was assayed in quadruplicate.

Measurement of mRNA expression

Retrotranscription assay and quantitative real-time PCR conditions for Foxp3, IL-17, IL-4 and IFN-γ were as previously reported (Martin-Saavedra et al., 2008). Primers used for IL-27p28 were: 5′-CTCAGAGAGGGAGCAGCTGT−3′, as forward, and 5′-TCAGGTGTCATCCCAAGTGTCC-3′, as reverse. The PCR product quality was checked by a melting curve analysis for each sample and the reaction efficiencies were checked to be near 2. Each result was normalized to the housekeeping β-actin gene expression. Relative quantification of gene expression analysis was performed using the Pfaffl method (Pfaffl, 2001).

Surface and intracellular protein staining for flow cytometry analysis

After washing in staining buffer (PBS containing 0.5% BSA, 2 mM EDTA, pH 7.2), 5 × 105 cells were incubated for 15 min at 4°C with FITC-anti-CD4 purchased from BD-Bioscience. Cells were permeabilized and fixed with BD Cytofix/Cytoperm Plus (BD Biosciences) for intracellular staining with PE-anti-IL-17 (BD Biosciences), according to the manufacturer instructions.

Flow cytometry data management

Data were acquired on FACSCanto (Becton-Dickinson) flow cytometres and analysed by FACSDiva (Becton-Dickinson).

Statistics

Statistical analyses were performed with GraphPad Prism version 5.02 (GraphPad software, Inc, La Jolla, CA, USA). T-test with Welch's correction was used for unpaired data, and Fisher's test for contingency table to perivascular infiltrates quantification.

Results

PDE7 inhibition by TC3.6 prevents EAE

To perform a comparative analysis of PDE4 and PDE7 as therapeutic targets in EAE, we carried out treatments of this disease with the PDE4 inhibitor Rolipram, and the selective PDE7 inhibitors BRL50481 and TC3.6 (Figure 1A). As expected, PDE4 inhibition by Rolipram prevented the clinical signs of EAE after MBP inoculation (Figure 1B). No differences between Rolipram doses from 2.5 to 10 μg g−1 of body weight were found. The thioxoquinazoline derivative TC3.6, which targets PDE7, showed to be as efficient as Rolipram to control EAE at doses of 10 or 2.5 μg g−1. On the contrary, BRL50481, the other PDE7 inhibitor tested, had no effect on the course of the disease even at 10 μg g−1. Accordingly, histopathological analysis by haematoxylin and eosin staining of CNS sections revealed that drastically reduced perivascular inflammatory infiltrates in Rolipram-treated animals as compared to EAE control mice, while in BRL50481 treated-mice lesions were as extensive and frequent as in the EAE control group (Figure 1C–F). Treatment with TC3.6, in turn, significantly decreased the size and number of the perivascular infiltrates found in the analysed sections, in correlation with the differences found between both PDE7 inhibitors for effects on clinical signs.

Figure 1.

Figure 1

Open in a new tab

Effect of PDE inhibitors treatment on clinical score of MBP-EAE in SJL mice. (A): Chemical compounds used as PDE inhibitors. (B): Groups of seven mice were established by vehicle without antigen (healthy control), antigen MBP (EAE control), or MBP and the PDE inhibitor specified on the figure. Daily doses of inhibitors at μg g−1 of body weight are indicated in brackets. One representative experiment out of three independent assays is shown. Score values were calculated as the average of the evaluations assigned by four independent observers in blind inspection to each mouse (n = 28/group). SEM for daily values is shown. (C), (D): different magnifications of four-micrometre paraffin sections of cerebellum and hippocampus from animals killed on day 15 post-inoculation of MBP. Scale bars are indicated in the lower left micrograph for each panel. Sections were subjected to haematoxylin and eosin staining. Arrows show perivascular infiltrates. (E): Quantification of sections positive and negative for perivascular infiltrates to each group of mice. Contingency table by Fisher test was analysed by GraphPad software (***P < 0.0001, *P < 0.005). (F): Frequency of sections positive for infiltrates. Values are the average of three independent experiments and are shown as mean + SD.

Rolipram and TC3.6 restrain T-cell activity during EAE

T-cell activity from axillary and inguinal LNC was assayed by stimulation with anti-CD3 and anti-CD28, or with antigen (MBP). In EAE animals, LNC showed a pre-activated state and therefore higher proliferative response than cells from the healthy control group to both kind of stimulus. The BRL50481-treated group did not show differences with untreated mice. However, proliferation levels of cells from Rolipram and TC3.6 groups were reduced to nearly healthy control values. Such reduction was particularly noticeable for antigen stimulated cultures (Figure 2A), suggesting a peripheral tolerance to MBP. It is well known that Foxp3+ Treg cells are involved in tolerance and can help to control EAE (O'Connor and Anderton, 2008; Selvaraj and Geiger, 2008). To address if the therapeutic effect of Rolipram or TC3.6 correlates with higher Foxp3 expression, relative mRNA levels were determined by quantitative real-time PCR. LNC from animals treated with these PDE inhibitors expressed higher levels of Foxp3 mRNA than cells obtained from untreated or BRL50481-treated mice (Figure 2B). Foxp3+ Treg and Th17 cell subtypes are established through mutually exclusive pathways (Bettelli et al., 2006). Measurements of IL-17 expression in LNC showed reduced levels of IL-17 mRNA and soluble protein for Rolipram or TC3.6 groups as compared to the EAE group, in inverse correlation to Foxp3 mRNA levels (Figure 2C). Such result was also found for purified CD4+ and for total spleen cells (not shown).

Figure 2.

Figure 2

Open in a new tab

Influence of EAE treatment with PDE inhibitors on T-cell activity. LNC from each group of animals were pooled and stimulated by anti-CD3 and anti-CD28 (αCD3+αCD28) or by antigen (MBP) at 1x106 cells mL−1 for 72 h. Proliferation was quantified by thiazolyl blue tetrazolium bromide colorimetric method (A), Foxp3 mRNA was measured by qRT-PCR (B), and IL-17 expression was assayed by ELISA and by qRT-PCR (C). Proliferation data are given as optical density. Relative quantification of mRNA is expressed as percentages of control EAE values (considered as 100%). Soluble IL-17 results are expressed as ng mL−1 of protein secreted to the culture medium. Error bars are mean + SD.

Different pathways for IL-17 decrease by PDE4 and PDE7 inhibition

We next analysed whether the negative control of IL-17 expression by Rolipram and TC3.6 was due to a direct effect on Th17 cells. Purified CD4+ cells from naïve mice were in vitro committed to Th17 phenotype by TGFβ and IL-6 in the presence of each PDE inhibitor. After Rolipram or TC3.6 removal, cells were exposed to a second round of TCR stimulation by anti-CD3. Measurements of IL-17 production showed that, in agreement with our in vivo results, cultures defined to Th17 in the presence of TC3.6 secreted lower levels of this cytokine than control cultures, suggesting a direct effect of TC3.6 on IL-17 producer cells. Unexpectedly, Rolipram led to higher IL-17 production than control cultures. Results were similar after 3 or 7 days of culture in the presence of PDE inhibitors previous to restimulation (Figure 3A). As IL-6 is an extremely strong inductor of IL-17 producer cells, we analysed the effect of Rolipram on anti-CD3 stimulated CD4+ cultures in the absence of IL-6 and TGFβ to avoid excessive pressure towards Th17 phenotype. In these conditions, Rolipram also increased the IL-17 production in a dose-dependent way. Furthermore, the IL-17 mRNA levels were also increased by Rolipram in cells not subjected to IL-6 action (Figure 3B). Intracellular IL-17 staining also showed that Rolipram mediates an increase in the percentage of Th17 cells as an effect independent of the presence of IL-6, even if CD4+ cells are subjected to Th1 conditions by IL-12 (Figure 3C, D).

Figure 3.

Figure 3

Open in a new tab

IL-17 expression by CD4+ exposed to in vitro PDE4 and PDE7 inhibitors treatments. CD4+ cells purified from naïve mice were cultured at Th17 (A,C), Th0 (B,D) or Th1 (D) conditions. After three (D3) or seven (D7) days of culture, PDE inhibitors were removed and cells were subjected to a second round of anti-CD3 stimulation before IL-17 expression analysis. Where it is not indicated, primary cultures were 3 days long. Soluble protein was detected by ELISA (A,B); mRNA was measured by qRT-PCR (B); and IL-17 producer cells were quantified by FACS of cells double-stained with anti-CD4-FITC and anti-IL17-PE (C,D). Results for mRNA quantification and for IL-17+ cells in left panel of (C) are presented as percentages of control EAE values (100%). Three independent experiments in which every sample was assayed in quadruplicate were performed for each panel. Error bars are mean + SD. FACS histograms data correspond to representative experiments and numbers on them indicate percentages of IL-17+.

In order to explain the discrepancy between these in vitro results for purified CD4+ cells and the decrease of IL-17 production mediated by Rolipram in vivo, we determined the IL-17 levels produced by a total spleen cell population exposed to Rolipram in vitro. In this case, neither after the first cell stimulation (Figure 4A) nor after a second round of anti-CD3 challenge (Figure 4B) was Rolipram able to increase the secretion of IL-17 at any doses assayed. On the other hand, soluble IL-17 was reduced by 50% after 3 days in the presence of Rolipram. Because activity of CD11b+ cells, including macrophages, neutrophils and dendritic cell populations, has a great influence on T helper subsets of CD4+ cells, we next analysed the effect of Rolipram on a spleen cell population depleted of the CD11b+ fraction. Similarly to purified CD4+ cells, IL-17 production was increased by Rolipram in a dose-dependent way in cultures depleted of CD11b+ cells. In addition, this effect persisted even after removal of Rolipram during a second round of anti-CD3 stimulation. Interestingly, the supernatant of a total spleen population culture used as a conditioned medium to incubate purified CD4+ cells blocked the increase of IL-17 expression, suggesting a soluble factor as responsible of such blockade. (Figure 4B).

Figure 4.

Figure 4

Open in a new tab

Influence of CD11b+ cells on IL-17 expression in the presence of Rolipram. (A,B): Purified CD4+ (white), total spleen cell population (grey), or spleen population depleted of the CD11b+ fraction (black) were in vitro stimulated by anti-CD3 and anti-CD28 in the presence of Rolipram at the concentration (μM) indicated in brackets. Soluble IL-17 levels were measured by ELISA after 3 days of culture (A) or after a second round of anti-CD3 stimulation once Rolipram was removed (B). (C): Total spleen cell population was cultured during 3 days, the resulting supernatant was used as a conditioned medium to purified CD4+ cell stimulation in the presence of Rolipram. Cultures were carried out in fresh Click medium (white), in Click medium one-half diluted with conditioned medium supplemented with 50% fresh FBS (grey), or in whole conditioned medium supplemented with 10% fresh FBS (black). Values are expressed as percentage of IL-17 production in respect of each control culture without Rolipram (100%). Error bars are mean + SD.

IL-27 and IL-10 levels are enhanced by Rolipram but not by TC3.6

CD11b+ cells, such as macrophages or dendritic cells, produce IL-27 which has a negative regulatory activity on Th17 cells (Batten et al., 2006; Stumhofer et al., 2006; Fitzgerald et al., 2007a; Diveu et al., 2009). To study the possible involvement of IL-27 in the control of the IL-17 increase mediated by Rolipram, we used an anti-IL27 antibody to neutralize this cytokine in a total spleen population culture. In these conditions, anti-IL-27 had no effect on the IL-17 levels produced by cultures treated with TC3.6. However, Rolipram was able to increase the IL-17 production, resembling the effects on purified CD4+ cells (Figure 5A). This result attributes to IL-27 the prevention of the IL-17 increase mediated by Rolipram. Moreover, Rolipram led to a rise of IL-27 mRNA levels, while TC3.6 had no effect on the expression of this cytokine (Figure 5B). On the other hand, increased levels of IL-27 mRNA in spleen and spinal cord infiltrates were detected when mice were treated with Rolipram during EAE (Figure 5C).

Figure 5.

Figure 5

Open in a new tab

In vivo and in vitro IL-27 expression in response to Rolipram. (A): IL-17 expression under IL-27 blockade. A total spleen cell population was stimulated by anti-CD3 and anti-CD28. PDE inhibitor used and neutralizing anti-IL-27 antibody addition are indicated at the bottom. Values for samples of each PDE inhibitor are shown as percentage in respect of controls without anti-IL-27 (100%) (B): Quantification of IL-27 mRNA in cultures of spleen cells treated with Rolipram or TC3.6. Numbers in brackets indicate concentration in μM. Data are expressed as percentages in respect of control without PDE inhibitor (100%) (C): Quantification of IL-27 mRNA in spleen and spinal cord from mice subjected to EAE treatment with Rolipram or TC3.6. Values are expressed as percentages of expression in respect of untreated EAE (100%). Error bars are mean + SD for A and B, and mean + SEM with n = 10 for C.

In addition to IL-17, other cytokines produced by Th cells can be involved in the development and control of EAE. Thus, we analysed the effect of PDE4 or PDE7 inhibition during EAE on the levels of expression of IFN-γ, IL-4 and IL-10. No significant differences could be observed for IFN-γ or IL-4 expression between untreated controls and Rolipram or TC3.6 treated animals (Figure 6A, B). However, for IL-10 expression, mRNA and secreted protein levels were significantly increased in cells from animals which were treated with Rolipram, while TC3.6 treatment did not resulted in differences as compared with cells from untreated EAE animals. A similar result was obtained for lymph nodes, spleen, pure CD4+ cells and spinal cord mononuclear cells from animals killed at day 10 post-inoculation of MBP (Figure 6C).

Figure 6.

Figure 6

Open in a new tab

Expression of IFN-γ, IL-4 and IL-10 after treatment of EAE with Rolipram or TC3.6. Groups of healthy and EAE controls, and Rolipram or TC3.6 treated SJL mice were established. Mice were sacrificed at day 10 post-inoculation. Cells were obtained from spleen, lymph node and spinal cord; in vitro stimulated by anti-CD3 and anti-CD28 (αCD3+αCD28) or by antigen MBP (indicated on each panel). Expression of IFN-γ (A) and IL-4 (B) are expressed as absolute values of soluble protein obtained by ELISA. Expression of IL-10 (C) was measured by ELISA and by qRT-PCR and results are shown as percentages of expression in respect of EAE controls without treatment (100%). Error bars are mean + SEM with n = 8–15.

Discussion and conclusions

The data presented here show the new PDE7 inhibitor TC3.6 as a promising anti-inflammatory tool and points to PDE7 inhibition as a new therapeutic target for inflammatory diseases. This heterocyclic quinazoline compound is as efficient as Rolipram to prevent the clinical signs of EAE. However, the previously described PDE7 inhibitor BRL50481 could not avoid the development of the disease after antigen inoculation of mice. As both PDE7 inhibitors, BRL50481 and TC3.6, have similar in vitro inhibition potencies on the target, with IC50 values of 0.26 μM for BRL50481 (Smith et al., 2004) and 1 μM for TC3.6 (Castano et al., 2009), their different anti-inflammatory behaviour found in vivo might be due to differences in their physico-chemical properties. Since BRL50481 and TC3.6 have very different chemical structures, it is reasonable to think that they could have different pharmacokinetic properties, such as cell penetration ability and BBB permeation. In fact, we have strong data which support that TC3.6 is able to cross the BBB, since it has shown efficacy in vivo when a stroke model is used, and it is neuroprotective after i.p. administration in such model (Redondo et al., 2012c). On the other hand, as far as we know, no data regarding the BBB permeation capability of BRL50481 have been published. Nevertheless, differences in ability to cross BBB could not explain all the differences found between both PDE7 inhibitors since divergence between effects of each drug spans peripheral immune events as effects on T-cell activity. Taking into account that no previous in vivo studies have been reported for BRL50481, the lack of peripheral effect could be due to low cell penetrability in vivo of this pharmacological tool.

Inhibition of PDE4 and PDE7 by Rolipram and TC3.6, respectively seem to share some mechanisms to control EAE. Both treatments reduce frequency and extent of perivascular infiltration of immune cells in CNS. Our results show that Rolipram and TC3.6 administration are also followed by common effects at primary immune response. They similarly induce tolerance to MBP in LNC in correlation with increased levels of Foxp3 expression, suggesting involvement of induction of Treg development. Likewise, decreased production of the proinflammatory cytokine IL-17 is mediated by both PDE4 and PDE7 inhibitors. However, IL-17 decrease seems to be achieved through different pathways by each drug. As shown by our in vitro data, TC3.6 leads to reduction of IL-17 expression by a direct effect on purified CD4+ cells, while Rolipram-mediated IL-17 decrease is only reached in the presence of CD11b+ cells, indicating that this cell fraction acts as a necessary mediator for IL-17 reduction through PDE4 inhibition. In fact, Rolipram direct action on purified CD4+ cells caused an increase in IL-17 production. Such effect remains after removal of the drug, suggesting that it is mediated by deviation of Th phenotype in favour of Th17. This hypothesis is supported by cytokine intracellular staining of cytokines after phenotype definition assays in the presence of the PDE4 inhibitor. This tendency towards Th17 cell differentiation provided by Rolipram overcomes even the strong influence of IL-12 to direct naïve CD4+ cells to Th1 phenotype. Research in progress will try to elucidate the pathways through which Rolipram confers advantage to Th17 cell subtype in the absence of CD11b+ cells. Nevertheless, the EAE experiments show that the net outcome of the in vivo treatment with Rolipram is a reduction of the IL-17 production by CD4+ cells. Taking into account the aforementioned in vitro data, we can infer that such net anti-inflammatory effect of PDE4 inhibition might be mediated by the CD11b+ fraction. Cell-to-cell contact should not be required for such intermediation since cell-free supernatant from a spleen cell culture was enough to limit the levels of IL-17, which is indicative of the involvement of a soluble factor. There are several reasons to assign this function to IL-27. First, IL-27 is one of the main negative regulators of Th17 development. Definitive studies reported that the absence of IL-27 signalling exacerbates chronic inflammation in correlation with an increased number of Th17 cells (Batten et al., 2006; Stumhofer et al., 2006). Second, IL-27 is mainly produced by antigen-presenting cells such as activated monocytes, macrophages and monocyte-derived dendritic cells, all of them included in the CD11b+ population. Third, a role in down-modulation of Th17 cells has been previously proposed for CD11b+ cells (Ehirchiou et al., 2007; Valaperti et al., 2008). And finally, our results regarding IL-27 neutralization in cultures treated with Rolipram revealed that this cytokine participates in restraining the IL-17 production. On the other hand, spleen cells treated with Rolipram showed an increased IL-27 expression. Consistent with these in vitro results, spleen cells and mononuclear cells infiltrating the spinal cord obtained from animals treated with Rolipram expressed higher levels of IL-27 mRNA. In contrast, neither in vivo nor in vitro treatment with TC3.6 induced increased IL-27 expression.

Considering all the experimental evidence, it can be outlined that the decrease of IL-17 production detected in vivo by PDE4 inhibition during EAE could result from the conjunction of two events: expansion of the CD4+Foxp3+Treg cell population and increase of the IL-27 production by CD11b+ cells. According to the results related to IL-27, the anti-inflammatory decrease of IL-17 production in PDE7 inhibition by TC3.6 can only be ascribed to the direct effect on CD4+ cells. Foxp3+Treg cells are powerful suppressors of T-cell effector activity (Shevach, 2009). Besides, adaptive Foxp3+ cell establishment compete in particular with Th17 phenotype since these two cell types are directed through mutually exclusive pathways. Both of them are TGFβ dependent and the decision between one or the other's fate is made by IL-6 which blocks Treg cell development and promotes Th17 phenotype definition (Bettelli et al., 2006). The increase in Foxp3 expression mediated by Rolipram and TC3.6 does not correlate with decreased IFN-γ or IL-4 production. Thus, most likely, the decrease of IL-17 associated to the increase of Foxp3 expression could be attributed to a competition between Foxp3+ and IL-17 producer cells rather than to suppressor activity of Foxp3+ cells, which might not be enhanced enough to restrain cytokine production by other Th subtypes.

In addition to the differential effect on IL-27 mediated by Rolipram and TC3.6 treatments of EAE, the expression of the anti-inflammatory cytokine IL-10 is also only modified by Rolipram. IL-10 is an important suppressive cytokine with a pivotal role in inhibition of both antigen presentation and subsequent proinflammatory cytokine release. Its involvement in induction of T-cell tolerance with anti-inflammatory responses has been found in several organ specific autoimmune diseases, and in particular in EAE (Bettelli et al., 2003). Some authors have associated the negative control of IL-17 by IL-27 to a mechanism dependent on IL-10 (Fitzgerald et al., 2007b; Murugaiyan et al., 2009). The influence of Rolipram on IL-10 expression has been previously reported in in vitro studies and in collagen-induced arthritis (Ross et al., 1997; Kambayashi et al., 2001). We now show a correlation between increased IL-10 production and the therapeutic effect of Rolipram on EAE.

In summary, using the heterocyclic quinazoline derivative PDE7 inhibitor TC3.6, we show here for the first time that inhibition of PDE7 during EAE reduces perivascular infiltrates in CNS, T-cell proliferation and IL17 levels, and increases Foxp3 expression. These events correlate with effective prevention of the clinical signs of the disease. Besides, we describe here new effects of Rolipram on EAE which could contribute to mediate its anti-inflammatory activity, such as reduction in IL-17 production and increase in IL-27 and IL-10 expression. Although PDE4 and PDE7 inhibition act not only through shared but also through distinct mechanisms, TC3.6 confers as efficient protection against EAE as Rolipram, emerging as an alternative to PDE4 inhibitors as anti-inflammatory tool and, hopefully, as a new therapeutic approach for the control of Th17 cell-mediated diseases. Moreover, TC3.6 has recently shown its ability to promote oligodendrocyte precursor differentiation and survival (Medina-Rodríguez et al., 2013). Thus, we consider TC3.6 as a promising new drug candidate for MS pharmacological treatment, with both anti-inflammatory and neuroregenerative properties.

Acknowledgments

This work was sponsored by grants from Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Acción estratégica en Salud (PS09/00604 and SAF2009-13015-C02-01) and Instituto de Salud Carlos III (MPY 1402/0909 and RD07/0060/0015). R.G-P was supported by a grant from Instituto de Salud Carlos III. Authors are grateful to F. Javier Hernández and Marta Blanco for the valuable technical assistance. Authors also wish to thank Dr. R. Murillas for the critical reading of the manuscript.

Glossary

BBB

blood-brain barrier

EAE

encephalomyelitis autoimmune experimental

LNC

lymph node cells

MBP

myelin basic protein

MS

multiple sclerosis

Treg

T regulator

Conflict of interest

Authors declare no conflict of interest.

References

  1. Axtell RC, de Jong BA, Boniface K, van der Voort LF, Bhat R, De Sarno P, et al. T helper type 1 and 17 cells determine efficacy of interferon-beta in multiple sclerosis and experimental encephalomyelitis. Nat Med. 2010;16:406–412. doi: 10.1038/nm.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Batten M, Li J, Yi S, Kljavin NM, Danilenko DM, Lucas S, et al. Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells. Nat Immunol. 2006;7:929–936. doi: 10.1038/ni1375. [DOI] [PubMed] [Google Scholar]
  3. Bettelli E, Das MP, Howard ED, Weiner HL, Sobel RA, Kuchroo VK. IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice. J Immunol. 1998;161:3299–3306. [PubMed] [Google Scholar]
  4. Bettelli E, Nicholson LB, Kuchroo VK. IL-10, a key effector regulatory cytokine in experimental autoimmune encephalomyelitis. J Autoimmun. 2003;20:265–267. doi: 10.1016/s0896-8411(03)00048-9. [DOI] [PubMed] [Google Scholar]
  5. Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. doi: 10.1038/nature04753. [DOI] [PubMed] [Google Scholar]
  6. Blaszczyk B, Gieldanowski J, Karakoz I. Experimental allergic encephalomyelitis in chickens. Arch Immunol Ther Exp (Warsz) 1978;26:743–746. [PubMed] [Google Scholar]
  7. Brudvik KW, Tasken K. Modulation of T cell immune functions by the prostaglandin E(2) – cAMP pathway in chronic inflammatory states. Br J Pharmacol. 2012;166:411–419. doi: 10.1111/j.1476-5381.2011.01800.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Castano T, Wang H, Campillo NE, Ballester S, Gonzalez-Garcia C, Hernandez J, et al. Synthesis, structural analysis, and biological evaluation of thioxoquinazoline derivatives as phosphodiesterase 7 inhibitors. ChemMedChem. 2009;4:866–876. doi: 10.1002/cmdc.200900043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen SJ, Wang YL, Fan HC, Lo WT, Wang CC, Sytwu HK. Current status of the immunomodulation and immunomediated therapeutic strategies for multiple sclerosis. Clin Dev Immunol. 2012;2012:1–16. doi: 10.1155/2012/970789. 970789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Click RE, Benck L, Alter BJ. Enhancement of antibody synthesis in vitro by mercaptoethanol. Cell Immunol. 1972;3:156–160. doi: 10.1016/0008-8749(72)90237-7. [DOI] [PubMed] [Google Scholar]
  11. Constantinescu CS, Farooqi N, O'Brien K, Gran B. Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS) Br J Pharmacol. 2011;164:1079–1106. doi: 10.1111/j.1476-5381.2011.01302.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Correa JO, Aarestrup BJ, Aarestrup FM. Effect of thalidomide and pentoxifylline on experimental autoimmune encephalomyelitis (EAE) Exp Neurol. 2010;226:15–23. doi: 10.1016/j.expneurol.2010.04.007. [DOI] [PubMed] [Google Scholar]
  13. Diveu C, McGeachy MJ, Boniface K, Stumhofer JS, Sathe M, Joyce-Shaikh B, et al. IL-27 blocks RORc expression to inhibit lineage commitment of Th17 cells. J Immunol. 2009;182:5748–5756. doi: 10.4049/jimmunol.0801162. [DOI] [PubMed] [Google Scholar]
  14. Ehirchiou D, Xiong Y, Xu G, Chen W, Shi Y, Zhang L. CD11b facilitates the development of peripheral tolerance by suppressing Th17 differentiation. J Exp Med. 2007;204:1519–1524. doi: 10.1084/jem.20062292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Falcone M, Rajan AJ, Bloom BR, Brosnan CF. A critical role for IL-4 in regulating disease severity in experimental allergic encephalomyelitis as demonstrated in IL-4-deficient C57BL/6 mice and BALB/c mice. J Immunol. 1998;160:4822–4830. [PubMed] [Google Scholar]
  16. Fitzgerald DC, Ciric B, Touil T, Harle H, Grammatikopolou J, Das Sarma J, et al. Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis. J Immunol. 2007a;179:3268–3275. doi: 10.4049/jimmunol.179.5.3268. [DOI] [PubMed] [Google Scholar]
  17. Fitzgerald DC, Zhang GX, El-Behi M, Fonseca-Kelly Z, Li H, Yu S, et al. Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated T cells. Nat Immunol. 2007b;8:1372–1379. doi: 10.1038/ni1540. [DOI] [PubMed] [Google Scholar]
  18. Genain CP, Roberts T, Davis RL, Nguyen MH, Uccelli A, Faulds D, et al. Prevention of autoimmune demyelination in non-human primates by a cAMP-specific phosphodiesterase inhibitor. Proc Natl Acad Sci U S A. 1995;92:3601–3605. doi: 10.1073/pnas.92.8.3601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Giembycz MA, Smith SJ. Phosphodiesterase 7A: a new therapeutic target for alleviating chronic inflammation? Curr Pharm Des. 2006;12:3207–3220. doi: 10.2174/138161206778194123. [DOI] [PubMed] [Google Scholar]
  20. Inoue M, Williams KL, Oliver T, Vandenabeele P, Rajan JV, Miao EA, et al. Interferon-beta therapy against EAE is effective only when development of the disease depends on the NLRP3 inflammasome. Sci Signal. 2012;5:ra38. doi: 10.1126/scisignal.2002767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jerez MJ, Jerez M, González-García C, Ballester S, Castro A. Combined use of pharmacophoric models together with drug metabolism and genotoxicity ‘ ‘in silico' ’ studies in the hit finding process. J Comput Aided Mol Des. 2013;27:79–90. doi: 10.1007/s10822-012-9627-1. [DOI] [PubMed] [Google Scholar]
  22. Kambayashi T, Wallin RP, Ljunggren HG. cAMP-elevating agents suppress dendritic cell function. J Leukoc Biol. 2001;70:903–910. [PubMed] [Google Scholar]
  23. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol. 2010;160:1577–1579. doi: 10.1111/j.1476-5381.2010.00872.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kohm AP, Carpentier PA, Miller SD. Regulation of experimental autoimmune encephalomyelitis (EAE) by CD4+CD25+ regulatory T cells. Novartis Found Symp. 2003;252:45–52. discussion 52-44, 106-114. [PubMed] [Google Scholar]
  25. McGeachy MJ, Stephens LA, Anderton SM. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous system. J Immunol. 2005;175:3025–3032. doi: 10.4049/jimmunol.175.5.3025. [DOI] [PubMed] [Google Scholar]
  26. McGrath J, Drummond G, McLachlan E, Kilkenny C, Wainwright C. Guidelines for reporting experiments involving animals: the ARRIVE guidelines. Br J Pharmacol. 2010;160:1573–1576. doi: 10.1111/j.1476-5381.2010.00873.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martinez I, Puerta C, Redondo C, Garcia-Merino A. Type IV phosphodiesterase inhibition in experimental allergic encephalomyelitis of Lewis rats: sequential gene expression analysis of cytokines, adhesion molecules and the inducible nitric oxide synthase. J Neurol Sci. 1999;164:13–23. doi: 10.1016/s0022-510x(99)00050-7. [DOI] [PubMed] [Google Scholar]
  28. Martin-Saavedra FM, Flores N, Dorado B, Eguiluz C, Bravo B, Garcia-Merino A, et al. Beta-interferon unbalances the peripheral T cell proinflammatory response in experimental autoimmune encephalomyelitis. Mol Immunol. 2007;44:3597–3607. doi: 10.1016/j.molimm.2007.03.002. [DOI] [PubMed] [Google Scholar]
  29. Martin-Saavedra FM, Gonzalez-Garcia C, Bravo B, Ballester S. Beta interferon restricts the inflammatory potential of CD4+ cells through the boost of the Th2 phenotype, the inhibition of Th17 response and the prevalence of naturally occurring T regulatory cells. Mol Immunol. 2008;45:4008–4019. doi: 10.1016/j.molimm.2008.06.006. [DOI] [PubMed] [Google Scholar]
  30. Medina-Rodríguez EM, Arenzana EJ, Pastor J, Redondo M, Palomo V, García de Sola R, et al. Inhibition of endegenous Phosphodiesterase 7 promotes oligodendrocyte precursor differentiation and survival. Cell Mol Life Sci. 2013 doi: 10.1007/s00018-013-1340-2. (in press) [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mosenden R, Tasken K. Cyclic AMP-mediated immune regulation–overview of mechanisms of action in T cells. Cell Signal. 2011;23:1009–1016. doi: 10.1016/j.cellsig.2010.11.018. [DOI] [PubMed] [Google Scholar]
  32. Mosman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  33. Murugaiyan G, Mittal A, Lopez-Diego R, Maier LM, Anderson DE, Weiner HL. IL-27 is a key regulator of IL-10 and IL-17 production by human CD4+ T cells. J Immunol. 2009;183:2435–2443. doi: 10.4049/jimmunol.0900568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. O'Connor RA, Anderton SM. Foxp3+ regulatory T cells in the control of experimental CNS autoimmune disease. J Neuroimmunol. 2008;193:1–11. doi: 10.1016/j.jneuroim.2007.11.016. [DOI] [PubMed] [Google Scholar]
  35. Paintlia AS, Paintlia MK, Singh I, Skoff RB, Singh AK. Combination therapy of lovastatin and rolipram provides neuroprotection and promotes neurorepair in inflammatory demyelination model of multiple sclerosis. Glia. 2009;57:182–193. doi: 10.1002/glia.20745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pierson E, Simmons SB, Castelli L, Goverman JM. Mechanisms regulating regional localization of inflammation during CNS autoimmunity. Immunol Rev. 2012;248:205–215. doi: 10.1111/j.1600-065X.2012.01126.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Portolés P, Rojo J, Golby A, Bonneville M, Gromkowski S, Greenbaum L, et al. Monoclonal antibodies to murine CD3ε define distinct epitopes, one of which may interact with CD4 during T cell activation. J Immunol. 1989;142:4169–4175. [PubMed] [Google Scholar]
  39. Reder AT, Thapar M, Sapugay AM, Jensen MA. Prostaglandins and inhibitors of arachidonate metabolism suppress experimental allergic encephalomyelitis. J Neuroimmunol. 1994;54:117–127. doi: 10.1016/0165-5728(94)90238-0. [DOI] [PubMed] [Google Scholar]
  40. Redondo M, Brea J, Perez DI, Soteras I, Val C, Perez C, et al. Effect of phosphodiesterase 7 (PDE7) inhibitors in experimental autoimmune encephalomyelitis mice. Discovery of a new chemically diverse family of compounds. J Med Chem. 2012a;55:3274–3284. doi: 10.1021/jm201720d. [DOI] [PubMed] [Google Scholar]
  41. Redondo M, Palomo V, Brea J, Perez DI, Martin-Alvarez R, Perez C, et al. Identification in silico and experimental validation of novel phosphodiesterase 7 inhibitors with efficacy in experimental autoimmune encephalomyelitis mice. ACS Chem Neurosci. 2012b;3:793–803. doi: 10.1021/cn300105c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Redondo M, Zarruk JG, Ceballos P, Perez DI, Perez C, Perez-Castillo A, et al. Neuroprotective efficacy of quinazoline type phosphodiesterase 7 inhibitors in cellular cultures and experimental stroke model. Eur J Med Chem. 2012c;47:175–185. doi: 10.1016/j.ejmech.2011.10.040. [DOI] [PubMed] [Google Scholar]
  43. Ross SE, Williams RO, Mason LJ, Mauri C, Marinova-Mutafchieva L, Malfait AM, et al. Suppression of TNF-alpha expression, inhibition of Th1 activity, and amelioration of collagen-induced arthritis by rolipram. J Immunol. 1997;159:6253–6259. [PubMed] [Google Scholar]
  44. Sanchez AJ, Puerta C, Ballester S, Gonzalez P, Arriaga A, Garcia-Merino A. Rolipram impairs NF-kappaB activity and MMP-9 expression in experimental autoimmune encephalomyelitis. J Neuroimmunol. 2005;168:13–20. doi: 10.1016/j.jneuroim.2005.03.024. [DOI] [PubMed] [Google Scholar]
  45. Selvaraj RK, Geiger TL. Mitigation of experimental allergic encephalomyelitis by TGF-beta induced Foxp3+ regulatory T lymphocytes through the induction of anergy and infectious tolerance. J Immunol. 2008;180:2830–2838. doi: 10.4049/jimmunol.180.5.2830. [DOI] [PubMed] [Google Scholar]
  46. Shevach EM. Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity. 2009;30:636–645. doi: 10.1016/j.immuni.2009.04.010. [DOI] [PubMed] [Google Scholar]
  47. Smith SJ, Cieslinski LB, Newton R, Donnelly LE, Fenwick PS, Nicholson AG, et al. Discovery of BRL 50481 [3-(N,N-dimethylsulfonamido)-4-methyl-nitrobenzene], a selective inhibitor of phosphodiesterase 7: in vitro studies in human monocytes, lung macrophages, and CD8+ T-lymphocytes. Mol Pharmacol. 2004;66:1679–1689. doi: 10.1124/mol.104.002246. [DOI] [PubMed] [Google Scholar]
  48. Sommer N, Loschmann PA, Northoff GH, Weller M, Steinbrecher A, Steinbach JP, et al. The antidepressant rolipram suppresses cytokine production and prevents autoimmune encephalomyelitis. Nat Med. 1995;1:244–248. doi: 10.1038/nm0395-244. [DOI] [PubMed] [Google Scholar]
  49. Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, Johnson LM, et al. Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat Immunol. 2006;7:937–945. doi: 10.1038/ni1376. [DOI] [PubMed] [Google Scholar]
  50. Teitelbaum D, Meshorer A, Hirshfeld T, Arnon R, Sela M. Suppression of experimental allergic encephalomyelitis by a synthetic polypeptide. Eur J Immunol. 1971;1:242–248. doi: 10.1002/eji.1830010406. [DOI] [PubMed] [Google Scholar]
  51. Valaperti A, Marty RR, Kania G, Germano D, Mauermann N, Dirnhofer S, et al. CD11b+ monocytes abrogate Th17 CD4+ T cell-mediated experimental autoimmune myocarditis. J Immunol. 2008;180:2686–2695. doi: 10.4049/jimmunol.180.4.2686. [DOI] [PubMed] [Google Scholar]
  52. Wang J, Wang G, Sun B, Li H, Mu L, Wang Q, et al. Interleukin-27 suppresses experimental autoimmune encephalomyelitis during bone marrow stromal cell treatment. J Autoimmun. 2008;30:222–229. doi: 10.1016/j.jaut.2007.10.001. [DOI] [PubMed] [Google Scholar]
  53. Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L, Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature. 1992;356:63–66. doi: 10.1038/356063a0. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES