Selective Induction of cAMP Phosphodiesterase PDE4B2 Expression in Experimental Autoimmune Encephalomyelitis

By Reyes-Irisarri, Elisabet Sanchez, Antonio J; Garcia-Merino, Juan Antonio; Mengod, Guadalupe

Abstract Experimental autoimmune encephalomyelitis (EAE) in Lewis rats is the most widely used animal model for multiple sclerosis. Cyclic adenosine monophosphate (cAMP) has been associated with neuroinflammation. The aim of this study was to investigate the possible involvement of different cAMP-specific phosphodiesterase (PDE) isoenzymes by analyzing their expression in the brain of EAE rats. We found in the brain of EAE animals that there was a dramatic increase in the mRNA expression levels of the PDE4B isozyme detected around blood vessels from the spinal cord to the upper midbrain. There was a single splicing form of the 4 splice variants that are known for PDE4B: PDE4B2, which showed increased expression levels. This overexpression is localized around the blood vessels and parenchyma in infiltrating T cells and macrophages/microglia. These results support the role played by the activation of the PDE4B2 gene in the neuroinflammatory process in EAE rats.

Key Words: Lymphocytes, Microglia, Multiple sclerosis, Neuroinflammation, PDE4, PDE7.

INTRODUCTION

Multiple sclerosis (MS) is a chronic inflammatory, neurodegenerative disease of the CNS, characterized by the destruction of myelin sheaths leading to lesions (1). The composition of the inflammatory infiltrates together with the local expression of cytokines, chemokines, and other immune response- related molecules suggest that the basis for the inflammatory reaction is an immunologic process mediated by T cells. This process disrupts the blood-brain barrier, which leads to the recruitment of other inflammatory cells. In addition, cytokines produced by activated T cells in the lesions induce the activation of effector cells (i.e. macrophages or microglia), which are ultimately responsible for demyelination and tissue damage (2). Experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, is an inflammatory disease induced in genetically susceptible animals by active immunization against myelin antigens of the CNS (3, 4).

The inflammatory process in MS and EAE lesions is dominated by T lymphocytes and activated macrophages/ microglia and is associated with the expression of major histocompatibility complex antigens, adhesion molecules and chemokines, which appear to be pivotal in the initiation and propagation of this process (5). In addition to T cells, macrophages and activated microglia have been found in areas with active tissue injury, attached to degenerating myelin sheaths and axons (6, 7).

An increase in intracellular cAMP levels is usually accompanied by an inhibition of certain functions of different types of cells involved in the immune response (8, 9). Intracellular levels of cAMP are regulated by adenylyl cyclases and cyclic nucleotide 3′,5′- phosphodiesterases (PDEs). Four different PDE4 genes (PDE4A, PDE4B, PDE4C, and PDE4D) and 2 PDE7 genes (PDE7A and PDE7B) have been identified in rats and humans (10). The mRNAs coding for the PDE4 isoenzymes (11, 12) and for PDE7 and PDE8 (11, 13-15) have been found in the brains of rats, monkeys, and humans. PDE4 isozymes are highly expressed in different immune and inflammatory cells, where they may act as important regulators of inflammatory processes (16, 17); moreover, their expression is differentially modulated in lipopolysaccharide (LPS)-treated rats and mice (18-21).

Treatment with rolipram or other PDE4 inhibitors prevents or reduces the pathologic process in EAE rats (22, 23) and mice (24). Several therapeutic strategies involving PDE4 inhibition have been proposed for MS treatment (25-27).

The possible involvement of cAMP through PDEs in inflammation prompted us to analyze the expression of PDE4 and PDE7 isoenzymes as well as the 4 mRNA splice variants of PDE4B in the brain of EAE rats.

MATERIALS AND METHODS

Induction of EAE and Clinical Evaluation

EAE was induced in Lewis rats (Charles River, Les Oncins, France) as described elsewhere (28). Briefly, an inoculum containing 50 [mu]g of guinea pig myelin basic protein (Sigma-Aldrich Chemie, Steinheim, Germany) and 500 [mu]g of Mycobacterium tuberculosis (strain H37Ra; Difco, Detroit, MI) in incomplete Freund’s adjuvant (Difco) was injected subcutaneously into the hind footpads. Control rats were injected with M. tuberculosis and incomplete Freund’s adjuvant in the same way. Rats were examined daily for the presence of neurologic signs using the following scale: 0 = no EAE; 1 = partial loss of tail tonicity; 2 = loss of tail tonicity; 3 = unsteady gait and mild paraparesis; 4 = hindlimb paralysis; and 5 = death. Grades 3 and 4 were often accompanied by urinary and fecal incontinence. Animal procedures were performed according to European Union guidelines (86/609/EU) for the use of laboratory animals.

Tissue Preparation

The rats were killed by decapitation 10 and 13 days after the inoculum. The brains were frozen on dry ice and stored at – 20[degrees]C. Tissue sections, 14-[mu]m thick, were cut on a microtome-cryostat (HM500 OM; Microm, Walldorf, Germany), thaw- mounted onto 3-aminopropyltriethoxysilane (Sigma-Aldrich Chemie)- coated slides, and stored at -20[degrees]C until use.

Hybridization Probes

The oligonucleotides used to detect the mRNAs coding for different PDEs, TCRbeta (T-cell receptor active beta-chain C- region, T cell marker) and PAFR (platelet-activated factor receptor, microglia marker) are shown in the Table They were all synthesized and high-performance liquid chromatography-purified by Isogen Bioscience BV (Maarsden, The Netherlands). The hybridization conditions to detect all mRNAs have been described elsewhere (11, 12, 15).

TABLE. List of the Oligonucleotides Used

The mRNA regions for each mRNA analyzed were chosen because they share no similarity with each other. Evaluation of the oligonucleotide sequences with the basic local alignment search tools of EMBL and GenBank databases indicated that the probes do not present any significant similarity with mRNAs other than their corresponding targets in the rat. The specificity of the autoradiographic signal obtained in the in situ hybridization histochemistry experiments was confirmed by performing a series of routine controls (29), such as the use of different oligonucleotides for the same mRNA to obtain identical hybridization patterns, competition with the same unlabeled oligonucleotide for the nonspecific hybridization signal, determination of the T^sub m^ of the hybrids, and others. The probes used to visualize the 4 PDE4B mRNA splicing variants have been described elsewhere (E. Reyes- Irisarri and G. Mengod, unpublished observations, 2007).

Oligonucleotides for each PDE were labeled at their 3′-end using [alpha-^sup 33^P]dATP (3000 Ci/mmol, New England Nuclear, Boston, MA) and terminal deoxynucleotidyltransferase (Calbiochem, San Diego, CA), obtaining a final specific activity of 0.9 to 2.7 x 10^sup 4^ Ci/mmol. They were purified using ProbeQuant G-50 Micro Columns (GE Healthcare, Buckinghamshire, UK) (15). PAFR and TCRbeta oligonucleotides were nonradioactively labeled with recombinant terminal deoxynucleotidyltransferase (Roche Diagnostics, Penzberg, Germany) and digoxigenin (DIG)-11-dUTP (Boehringer Mannheim, Mannheim, Germany) according to a procedure described previously (30).

In Situ Hybridization Histochemistry Procedure

The protocols for single- and double-labeled in situ hybridization histochemistry were based on procedures described previously (31, 32) and are published elsewhere (15, 33). Frozen tissue sections were brought to room temperature, fixed for 20 minutes at 4[degrees]C in 4% paraformaldehyde in PBS (1 x PBS: 8 mM Na^sub 2^HPO^sub 4^), 1.4 mM KH^sub 2^PO^sub 4^, 136 mM NaCl, 2.6 mM KCl), washed for 5 minutes in 3 x PBS at room temperature and twice for 5 minutes each in 1 x PBS, and incubated for 2 minutes at 20[degrees]C in a solution of predigested pronase (Calbiochem) at a final concentration of 24 U/mL in 50 mM Tris-HCl, pH 7.5 and 5 mM EDTA. The enzymatic activity was stopped by immersion for 30 seconds in 2 mg/mL glycine in 1 x PBS. Tissues were finally rinsed in 1 x PBS and dehydrated through a graded ethanol series. For hybridization, radioactively labeled and nonradioactively labeled probes were diluted in a solution containing 50% formamide, 4 x standard saline citrate (SSC) (1 x SSC: 150 mM NaCl and 15 mM sodium citrate), 1 x Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin), 10% dextran sulfate, 1% sarkosyl, 20 mM phosphate buffer, pH 7.0, 250 [mu]g/mL yeast tRNA, and 500 [mu]g/mL salmon sperm DNA. The final concentrations of radioactive and DIG-labeled probes in the hybridization buffer were in the same range (approximately 1.5 nM). Tissue sections were covered with hybridization solution containing the labeled probe(s), overlaid with Nescofilm coverslips (Bando Chemical, Kobe, Japan) and incubated overnight at 42[degrees]C in humid boxes. Sections were washed 4 times (45 minutes each) in 0.6 M NaCl, 10 mM Tris-HCl, pH 7.5, at 60[degrees]C, and once in the same buffer at room temperature for 10 minutes.

Development of Radioactive and Nonradioactive Hybridization Signals

Hybridized sections were treated as described in (32). After washing, the slides were immersed for 30 minutes in a buffer containing 0.1 M Tris-HCl pH 7.5, 1 M NaCl, 2 mM MgCl^sub 2^ and 0.5% bovine serum albumin (Sigma-Aldrich Chemie) and incubated overnight at 4[degrees]C in the same solution with alkaline phosphate-conjugated antidigoxigenin-Fab fragments (1:5000; Boehringer Mannheim). They were then washed 3 times (10 minutes each) in the same buffer (without antibody) and twice in an alkaline buffer containing 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, and 5 mM MgCl^sub 2^. Alkaline phosphatase activity was developed by incubating the sections with 3.3 mg of nitroblue tetrazolium and 1.65 mg of bromochloroindolyl phosphate (Gibco BRL, Gaithersburg, MD) diluted in 10 mL of alkaline buffer. The enzymatic reaction was blocked by extensive rinsing in the alkaline buffer containing 1 mM EDTA. The sections were then briefly dipped in 70% and 100% ethanol, air-dried, and dipped into Ilford K5 nuclear emulsion (Ilford, Mobberly, Cheshire, UK) diluted 1:1 with distilled water. They were exposed in the dark at 4[degrees]C for 4 to 6 weeks and finally developed in Kodak D19 (Kodak, Rochester, NY) for 5 minutes and fixed in Ilford Hypam fixer (Ilford). For film autoradiography, some hybridized sections were exposed to Biomax-MR (Kodak) films for 2 to 4 weeks at -70[degrees]C with intensifying screens.

Immunohistochemistry

Sections adjacent to those used for in situ hybridization histochemistry were also examined by immunohistochemistry. To detect the different cell types, the following primary antibodies were used: mouse anti-rat CD11b (1:100; Serotec, Oxford, UK) for microglia; mouse anti-neuronal-specific nuclear protein (1:1000; Chemicon International, Temecula, CA) for the neuronal cell population; and rabbit anti-human CD3 (1:100; Dako Cytomation, Glostrup, Denmark) for T cells. The secondary antibodies used were biotinylated anti-mouse IgG (H+L) (1:300; Vector Laboratories, Burlingame, CA) and biotinylated anti-rabbit IgG (H+L) (1:100; Vector Laboratories).

The sections were fixed for 20 minutes at 4[degrees]C in 4% paraformaldehyde, rinsed in 1 x PBS for 5 minutes, and incubated for 15 minutes in a solution containing 0.3% H^sub 2^O^sub 2^ (Sigma- Aldrich) in 1 x PBS. After rinsing in 1 x PBS for 5 minutes, preincubation and incubation with primary and biotinylated antibodies were performed in a 1 x PBS solution containing 2% normal goat serum (Vector Laboratories) for those sections incubated with biotinylated antirabbit antibodies and with 2% normal goat serum and 2% normal rat serum for those incubated with biotinylated antimouse antibodies. Primary antibodies were incubated for 1 hour at 37[degrees]C and secondary antibodies at 37[degrees]C for 30 minutes. Control slides were incubated without primary antibodies. After rinsing in 1 x PBS at room temperature, the slides were incubated at 37[degrees]C for 30 minutes in ABC solution (Vectastain Elite ABC Kit; Vector Laboratories) according to the manufacturer’s procedures. The color reaction was performed with diaminobenzidine tetra hydrochloride (DAB) solution I (0.05 M Tris-HCl,7 0.3 mg/mL DAB [Sigma-Aldrich Chemie], 10 [mu]L/mL dimethyl sulfoxide [Sigma- Aldrich Chemie], and 0.64 mg/mL NaN^sub 3^ [Merck, Darmstadt, Germany]) and with DAB solution II (0.05 M Tris-HCl, 0.3 mg/mL DAB; 0.64 mg/mL NaN^sub 3^; and 0.06 [mu]L/mL H^sub 2^O^sub 2^ [Sigma- Aldrich Chemie]) at room temperature for 5 minutes each. After rinsing in 1 x PBS and in distilled water the sections were mounted in Mowiol (Calbiochem).

Preparation of Figures

Photographs of the film autoradiograms of the hybridized tissue sections were taken with a Wild 420 Leica microscope equipped with a digital camera (DXM1200 F; Nikon, Tokyo, Japan) and ACT-1 Nikon Software. Microphotography of the hybridized tissue slides was performed using a Zeiss Axioplan microscope equipped with a digital camera (DXM1200 F) and ACT-1 Nikon Software. Figures were prepared for publication using Adobe Photoshop software (Adobe Software, San Jose, CA). The contrast and brightness of images were the only variables adjusted digitally. For anatomical reference, sections close to those used were stained with cresyl violet.

RESULTS

Expression of cAMP-Specific Phosphodiestease Enzymes in EAE Rat Brain

We first analyzed the expression patterns of the PDE4 and PDE7 isozymes at the lower brainstem level of 13-day EAE rats and compared them with those for control rats. The results are shown in the photographs in Figure 1. PDE4C expression was completely absent (Fig. 1E, F). PDE4A (Fig. 1A, B) and PDE4D (Fig. 1G, H) showed a similar hybridization pattern in both EAE and control rat brain. In contrast, PDE4B (Fig. 1C, D) expression presented additional dark patches of autoradiographic grains in a dispersed distribution only in EAE rats. No differences were observed in the expression of PDE7A (Fig. 1I, J) and PDE7B (Fig. 1K, L) between control and EAE brains.

Expression of PDE4B in EAE Rat Brain at Different Disease Stages

Next, we analyzed the expression of PDE4B at different brain levels and at 2 different times of EAE induction, 10 and 13 days postinoculum. The results are shown in Figure 2. The overexpression of PDE4B in EAE rats killed 10 days after the inoculum (Fig. 2B, E, H, K, N) was limited to the lower parts of the brainstem. Hybridization levels were high from the spinal cord to the lower brainstem and the majority can be recognized in the areas close to the brain ventricles. At a higher, anterior, level, no abnormal PDE4B expression was seen. In contrast, when rats were killed 13 days postinoculum (Fig. 2C, F, I, L, O), the patchy distribution of PDE4B hybridization was seen from the spinal cord to more rostral brainstem levels. In this case this PDE4B overexpression was not concentrated close to the ventricles but rather extended throughout the microvasculature of the brainstem.

FIGURE 1. cAMP-phosphodiesterase (PDE) mRNA expression in the brain of experimental autoimmune encephalomyelitis (EAE) rats. Film autoradiogram images from coronal brain sections from control (A, C, E, C, I, K) and 13-day post-inoculation (B, D, F, H, I, L) rats obtained after in situ hybridization for mRNA coding for PDE4A (A, B), PDE4B (C, D), PDE4C (E, F), PDE4D (C, H), PDE7A (I, J), and PDE7B (K, L). Note the dotted autoradiographic specific hybridization signal obtained for PDE4B mRNA after EAE induction (B^sub 2^). Scale bar = 2.5 mm.

Expression of PDE4B mRNA Splicing Forms in EAE Rat Brain

Because PDE4B was the only PDE enzyme that showed an overexpression in EAE rat brain, our next step was to determine which of the 4 known PDE4B mRNA splicing forms was responsible. The results are shown in Figure 3, in which PDE4B2 (Fig. 3B) was the only splicing variant that showed a similar hybridization pattern observed with the oligonucleotide probe that recognized all isoforms for PDE4B, as shown in Figure 2L.

Identification of the Cells Containing the Upregulated PDE4B2 mRNA

To identify the type of cells that overexpressed PDE4B2 mRNA in EAE rat brain, we performed the following experiments. First, we stained a coronal rat brain section with hematoxylin and eosin close to the section hybridized with the PDE4B2 oligonucleotide probe and found that most of the positive hybridizing cells were located in and close to brain vessels (not shown). We then performed double in situ hybridization experiments using a digoxigenin-labeled oligonucleotide complementary to the corresponding cell marker mRNA together with the radioactively labeled PDE4B2 probe. It was seen that the type of cells responsible for the upregulation of PDE4B expression could not be assigned to a single population. Some brain T cells surrounding the brain vessels and some immediately adjacent to them, identified either by their hybridization with an oligonucleotide complementary to the TCRbeta mRNA (Fig. 4C) or by immunohistochemical reaction with an anti-CD3 antibody (Fig. 4D), expressed PDE4B2 mRNA. PDE4B2 mRNA was also found to be expressed in macrophages or microglia, identified by their hybridization with an oligonucleotide for the cell marker PAFR (Fig. 4E) or by their reaction with an anti-CD11b antibody (Fig. 4F). Some T cells and some macrophages/microglia could be seen 10 days postinoculum, close to the ventricular areas of the brainstem, whereas after 13 days cells were found in the parenchyma.

FIGURE 2. PDE4B mRNA expression in experimental autoimmune encephalomyelitis (EAE) rat brain. Film autoradiogram images from rat brain sections at 4 different coronal levels from control (A, D, G, J, M), 10-day (B, E, H, K, N), and 13-day (C, F, I, L, O) postinoculation obtained after in situ hybridization to detect PDE4B mRNA. The accumulation of PDE4B can be observed from the spinal cord (O) to the midbrain level (F) in 13-day inoculated rats. Scale bar = 2.5 mm.

DISCUSSION

Using an animal model of multiple sclerosis, the EAE rat, we present the first description of the upregulation of expression of a PDE4B mRNA splice variant (PDE4B2) in the brain, thus suggesting a possible involvement of this isoform in the development of the disease.

PDE7A has been found in both B cells (34) and T cells (in particular the alternative isoforms PDE7A1 and PDE7A3 [35]). A critical role of PDE7A in T cell proliferation and interleukin-2 production has been suggested by the use of specific antisense oligonucleotides, which blocked these functions (36). This suggestion was reinforced by Nakata et al (37), who found that T cell proliferation and cytokine synthesis were downregulated by selective inhibition of PDE7A rather than PDE4. However, the observation that PDE7A knockout mice (PDE7A^sup -/-^) had functional T cells challenged previous hypotheses concerning the crucial role of this enzyme in T cell regulation (38). According to all these reports we expected to find PDE7A mRNA expression in the infiltrating T cells. Our observation of the lack of PDE7A expression in these T cells contradicts this prediction on the possible role that this phosphodiesterase could play in a T cell- mediated disease such as EAE. The reason for this discrepancy remains unexplained and deserves further study. PDE4 isoenzymes have been considered a possible target for anti-inflammatory drugs because of their widespread expression pattern (in the CNS as well as in the periphery) and their involvement in the control of cell responses in inflammatory disease models, including EAE (21, 39). In humans, circulating monocyte PDE4B gene expression is selectively induced by LPS (40, 41). These results point to the possible role of PDE4B in inflammatory processes. Jin et al (18-20) have shown, using either PDE4B or PDE4D knockout mice, that LPS stimulation of mouse inflammatory cells (peripheral leukocytes and peritoneal and lung macrophages) induced PDE4B mRNA accumulation and increased PDE4 activity. LPS-induced tumor necrosis factor-alpha (TNF-alpha) secretion was lower in mice deficient in PDE4B but not in those lacking PDE4D, which indicates that the induction of PDE4B expression is essential for LPS-activated TNF-alpha responses. The authors suggested that because there is no change in the cAMP levels in the cells deficient in PDE4B, a minor pool of cAMP regulated by PDE4B is involved in Toll-like receptor signaling.

FIGURE 3. PDE4B splice variant expression in experimental autoimmune encephalomyelitis (EAE) rat brain. Film autoradiogram images from 13-day postinoculum rat brain sections visualizing the mRNAs coding for PDE4B1 (A), PDE4B2 (B), PDE4B3 (C), and PDE4B4 (D). Scale bar = 3 mm.

Our results show a selective increase in PDE4B2 mRN A levels in the brain as a consequence of the induction of EAE in rats. We also observed a selective increase in PDE4B2 mRNA expression in some peripheral organs of LPS-treated rats (E. Reyes Irisarri and G. Mengod, unpublished data, 2007). This PDE4B2 behavior is consistent with that reported by other authors and discussed above for PDE4 in the periphery of some animal inflammation models and bears out the conclusion of Jin et al (18) that PDE4B2 is the major PDE4 form inducible by LPS (in their case) and the only form induced by autoimmune neuroinflammation of EAE rats (present results). The activation of resident microglia and perivascular macrophages and the recruitment of T cells in the CNS are among the most consistent changes observed in EAE rats (42, 43). We observed that the increase in PDE4B occurs in T cells and macrophages or microglia, which suggests that this isoenzyme is directly involved in EAE disease and that its function in these cells is highly specialized.

The PDE4B2 mRNA induction observed mainly in infiltrated T cells and macrophages/microglia in EAE rat brain seems to coincide with the peak of the expression observed for several cytokines, TNF- alpha, interferon-gamma, leukotriene, interleukin-beta and TGF-beta (22, 44, 45) on day 13 in the same animal model. This upregulation of PDE4B2 expression observed is also probably located in activated macrophages or microglia cells because of the morphology of the cells and the fact that “resting” microglia in control brain do not express this isoform. Taken together, these findings suggest that the induction of PDE4B2 expression in the cellular infiltrates of EAE rat brain could be necessary for cytokine production.

Stimulation of T cells through their antigen receptors (TCRs) produces a marked increase in intracellular cAMP levels, most probably due to the activation of adenylate cyclase by a G protein coupled to the TCR (46). However, high levels of cAMP inhibit many of the biologic responses of the T cell (36), so antigen receptor- mediated signaling must be coordinated with the activation of PDEs. There is evidence of the involvement of a biochemical pathway in the coordination of cAMP-PDE activation to limit the magnitude and the duration of the increase in cAMP levels resulting from the TCR signaling (47). Arp et al have recently shown that de novo expression of PDE4B2, previously shown to be associated with the CD3epsilon chain of TCR (48), enhances T cell activation and increases interleukin-2 production (49). This effect correlates with the intracellular distribution of PDE4B2, found within lipid rafts after T cell stimulation. The authors proposed that a compartmentalization of PDE4B2 occurs close to the immunologic synapse and sites of maximal cAMP generation at early T cell activation times. Another compartmentalization has been proposed for PDE4s, but particularly for PDE4D5, which interact with beta- arrestin, thereby recruiting PDEs to ligand-activated receptors (50). Such an interaction provides a mechanism by which cAMP- degrading enzymes can be delivered close to the point of cAMP production in an agonist-dependent fashion. In this way, compartmentalized PDEs may be recruited to different areas of the cell where they can be involved in different signaling pathways (51). Consequently, the induction of PDE4B2 observed in our study suggests that this isoform could be located in precise compartments in the infiltrating cells, helping to maintain low cAMP levels in the micro-compartments and thus allowing full activation of the cytokine responses. Preliminary data obtained in our laboratory from EAE rats treated with rolipram according to the protocol established by Sanchez et al (52) show a decrease in the number of cellular infiltrates, which correlates well with the absence of the PDE4B2 overexpression (data not shown) described above and that is characteristic of this model.

FIGURE 4. PDE4B2 mRNA expression in T cells and macrophages/ microglia around blood vessels of experimental autoimmune encephalomyelitis (EAE) rat cerebellar white matter. (A) Panel shows a digital dark-field microphotograph of emulsion-dipped section, where the hybridization signal for PDE4B2 mRNA is seen as white silver grains. (B) Shows a higher magnification of (A). (C) and (E) are bright-field photomicrographs showing the simultaneous detection of PDE4B2 mRNA (dark silver grains) with a DIG-labeled oligonucleotide probe for T-cell receptor active beta-chain C- region, T cell marker (TCRp) mRNA in some T cells (C) or with a DIG- labeled oligonucleotide probe for platelet-activated factor receptor, microglia marker (PAFR) mRNA in some macrophages/ microglia (E). White arrowheads point to cells positive for either DIG-TCRagr; mRNA (C) or DIG-PAFR mRNA (E), black arrowheads mark cellular profiles positive for radioactively labeled PDE4B2 mRNA, and black and white arrowheads show double-labeled cells. Higher magnification displays double labeled cells (inset). Immunohistochemistry for CD3 to detect T cells (D) and CD-11b to detect macrophages/microglia (F) was performed on adjacent sections. Arrows in (F) point to some of the activated macrophages/microglia. Yellow lines in (B) mark the blood vessel limits. Scale bars = (A) 100 [mu]m; (B-F) 20 [mu]m; inset, 5 [mu]m.

In summary, in the present work we described upregulation in the expression of 1 of the PDE4B splicing variants (PDE4B2) in the T cells and in macrophages/microglia in perivascular cuffs and parenchyma in the spinal cord and brainstem of rats with EAE. These findings have important physiologic consequences and are indicative of a very precise and specialized role of PDE4B2 in some cellular subpopulations. These results support the role played by PDE4B2 in inflammation and its importance as a therapeutic target for the treatment of neuroinflammatory diseases using subtype-selective PDE4B inhibitors.

ACKNOWLEDGMENTS

We thank Rocio Martin for technical assistance and Robin Rycroft for English corrections.

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Elisabet Reyes-Irisarri, PhD, Antonio J. Sanchez, BSc, Juan Antonio Garcia-Merino, MD, PhD, and Guadalupe Mengod, PhD

From the Department of Neuropharmacology (ERI, GM), Instituto de Investigaciones Biomedicas de Barcelona, Consejo Superior de Investigaciones Cientificas (Institut d’Investigacions Biomediques August Pi i Sunyer), Barcelona, Spain; and Neuroimmunology Unit (AJS, JAGM), Universidad Autonoma de Madrid, Hospital Puerta de Hierro, Madrid, Spain.

Send correspondence and reprint requests to: Guadalupe Mengod, Department of Neuropharmacology Instituto de Investigaciones Biomedicas de Barcelona, CSIC (IDIBAPS). c/Rossello 161, 6a. E- 08036 Barcelona, Spain; E-mail: gmlnqr@iibb.csic.es

This work was supported by grants awarded by Ministerio de Educacion y Ciencia and Fondo Europeo de Desarrollo Regional Funds (SAF2003-02083 and SAF2006-10243).

Copyright Lippincott Williams & Wilkins Oct 2007

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