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Published as doi: 10.1096/fj.07-9002com.
 (The FASEB Journal. 2008;22:786-796.)
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(The FASEB Journal. 2008;22:786-796.)
© 2008 FASEB

Glucocorticoids regulate innate immunity in a model of multiple sclerosis: reciprocal interactions between the A1 adenosine receptor and β-arrestin-1 in monocytoid cells

Shigeki Tsutsui*,1, David Vergote*,{dagger},1, Neda Shariat*, Kenneth Warren{dagger}, Stephen S. G. Ferguson{ddagger} and Christopher Power*,{dagger},2

* Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada;

{dagger} Department of Medicine, University of Alberta, Edmonton, Alberta, Canada; and

{ddagger} Cell Biology Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada

2Correspondence: Department of Medicine, University of Alberta, Heritage Medical Research Centre, Rm. 611, Edmonton, AB T6G 2S2, Canada. E-mail: chris.power{at}ualberta.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Desensitization of seven transmembrane receptors (7TMRs), which are modulated by the β-arrestins, leads to altered G protein activation. The A1 adenosine receptor (A1AR) is an antiinflammatory 7TMR exhibiting reduced expression and activity in both multiple sclerosis (MS) and the murine MS model, experimental autoimmune encephalomyelitis (EAE) in monocytoid cells. Herein, we report that β-arrestin-1 expression was increased in brains of MS patients relative to non-MS brains, whereas A1AR expression was concomitantly reduced. This inverse relationship between β-arrestin-1 and A1AR was confirmed in cultured monocytoid cells as β-arrestin-1 overexpression resulted in a down-regulation of A1AR together with the internalization of the surface receptor. Moreover, a physical interaction between β-arrestin-1 and A1AR was demonstrated in monocytoid cells. Proinflammatory cytokines regulated the A1AR/β-arrestin-1 interactions, while A1AR activation also modulated proinflammatory cytokines expression. During EAE, β-arrestin-1 and A1AR expression in the spinal cord displayed a similar pattern compared to that observed in MS brains. EAE-induced neuroinflammation and neurobehavioral deficits were suppressed by glucocorticoid treatments, accompanied by concurrent reduced β-arrestin-1 and enhanced A1AR expression. Thus, the interplay between β-arrestin-1 and A1AR in the central nervous system during neuroinflammation represents a reciprocal regulatory mechanism through which neuroprotective therapeutic strategies for neuroinflammatory diseases might be further developed.—Tsutsui, S., Vergote, D., Shariat, N., Warren, K., Ferguson, S. S. G., Power, C. Glucocorticoids regulate innate immunity in a model of multiple sclerosis: reciprocal interactions between the A1 adenosine receptor and β-arrestin-1 in monocytoid cells.


Key Words: central nervous system • cytokines • experimental autoimmune encephalomyelitis • G protein-coupled receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACTIVATION OF INNATE IMMUNE responses occurs in the central nervous system (CNS) during trauma, stroke, spinal cord injury, and chronic neurological diseases including multiple sclerosis (MS) (1) . Microglia and macrophages play pivotal roles in the innate immune response in terms of initiating and sustaining immune responses to microbial, neoplastic, and neural antigens in the CNS, together with their antigen presenting and phagocytic properties (2 , 3) . The activation of monocytoid cells in the CNS and secretion of proinflammatory molecules contribute to the pathogenesis of neurological disorders including MS (3 , 4) , Alzheimer’s disease (5) , HIV-associated dementia (6) , and intracerebral hemorrhage (7) . The A1 adenosine receptor (A1AR) is a seven-transmembrane receptor (7TMR) that activates G proteins and is highly expressed on microglia/macrophages and neurons but not on infiltrating lymphocytes (8) . The A1AR has been implicated in the control of systemic immune activation through its regulation of cytokine expression and release in monocytoid cells (9) . Engagement of the A1AR by adenosine exerts protective effects in the CNS and maintains tissue integrity by modulating immune activation (10) .

MS is the prototypic neuroimmune disorder of the CNS, which classically exhibits a relapsing-remitting course accompanied by ongoing inflammatory demyelination with ensuing axonal and neuronal injury (11) . One of its neuropathological hallmarks is multifocal perivascular infiltration of mononuclear cells in the CNS. The most widely held hypothesis is that MS is a T cell-driven autoimmune reaction against myelin-related proteins, although the etiology of MS remains unknown (11 12 13) . Nonetheless, innate immunity also participates in MS initiation and progression coupled with the selective expression and release of inflammatory mediators by activated immune cells, such as cytokines (14) , chemokines (15 , 16) , and matrix metalloproteinases (MMPs) (17) . Recent studies indicate that very early events in MS pathogenesis are characterized by microglial activation, together with apoptosis of oligodendrocytes (18) . A1AR function is diminished in peripheral blood mononuclear cells from MS patients (19) , and reduced A1AR expression is also evident in brain monocytoid cells among MS patients, reflecting a selective down-regulation of the β transcript that encodes the A1AR (8) . Moreover, A1AR engagement and activation can regulate the extent of neuroinflammation and associated demyelination in experimental autoimmune encephalomyelitis (EAE) (20) . Modulation of most 7TMRs by the β-arrestins has been described, although the outcome of the interaction, internalization or signaling, depends on the individual circumstances and molecular profile. However, the interface between A1AR and the β-arrestins in monocytoid cells remains unclear. Of interest, autoantibodies to β-arrestin-1 have been detected in sera from MS patients, suggesting aberrant or overexpression of β-arrestin-1 (21 , 22) . This finding was confirmed by a report showing that β-arrestin-1 protein expression increased significantly in splenocytes during EAE (23) . Moreover, arrestins have been used to induce experimental autoimmune uveoretinitis, an animal model for CNS autoimmune diseases that affects the retina (24) . Earlier studies indicated that the A1AR displayed an affinity for arrestins in smooth muscle cells (25) , but no evidence indicates that the β-arrestins interact with the A1AR as they do for many other 7TMRs in the CNS.

High-dose glucocorticoid (pulse) treatment is common therapeutic strategy for treating relapses in MS (26) . The goal of MS therapy is prevention of ongoing tissue destruction, including demyelination and axonal loss, which results in subsequent permanent disabilities. Glucocorticoid treatment also modulates cytokine expression (27) , inhibits leukocyte migration (28) , and induces T cell apoptosis (29) , which controls CNS inflammation and may prevent the tissue damage. Importantly, neuronal A1AR expression is up-regulated by glucocorticoids (30) , and this effect may in part explain the neuroprotective effects of glucocorticoids. The findings of the A1AR’s diminished expression on monocytoid cells in MS and EAE (8 , 19 , 20) , together with these related effects mediated by glucocorticoids, led us to hypothesize that glucocorticoids modulated neuroinflammation by up-regulating the A1AR pathway, possibly through influencing β-arrestin expression. Herein, we report that β-arrestin-1 expression is increased in conjunction with reduced A1AR abundance in the CNS of MS patients and mice with EAE. Indeed, aberrant expression of β-arrestin-1 and A1AR was reversed by glucocorticoid treatment. Hence, up-regulation of A1AR pathway by glucocorticoids with a concomitant reduction in β-arrestin-1 expression may be a pivotal mechanism that can be exploited further in the development of therapies for multiple sclerosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures
U937 human monocytoid cells (American Type Culture Collection, Manassas, VA, USA) were cultured as previously reported (31) . Culture media was replaced with AIM-V serum-free medium (Life Technologies, Inc., Langley, OK, USA) for all experimental procedures. U937 cells were pretreated with adenosine amine congener (ADAC; 0.1, 1 or 10 µM) for 1 h and treated with 5 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma, Oakville, ON, Canada) for 4 h. For chronic ADAC treatment study, U937 cells were pretreated with the A1AR agonists ADAC (1 µM) and N6-cyclohexyladenosine (CHA; 0.1 µM) or the A2AR agonist CGS21680 (1 µM) for 3 days and treated with 1 or 10 µM dexamethasone (DEX) for 24 h. U937 cells were pretreated with 1 or 10 µM DEX for 1 h and treated with 50 ng/ml PMA for 4 h. Total cellular RNA was isolated from the above cells, and A1AR, tumor necrosis factor-{alpha} (TNF-{alpha}), IL-1β, IL-10, MMP-9, MMP-12, and β-arrestin-1 RNA expression was quantified using real-time RT-PCR analysis as described below. Previously described plasmid encoding the wild-type β-arrestin-1 was a generous gift from Dr. Vsevolod V. Gurevich (Vanderbilt University, Nashville, TN, USA) (32) . U937 cells were transfected with this plasmid using the DMRIE-C transfection reagent (Invitrogen, Gaithersburg, MD, USA) according to the manufacturer’s recommandations.

Human brain tissue samples
Brain tissue (frontal white matter) was collected at autopsy from each experimental group (MS, n=10 and non-MS, n=10) and stored at –80°C, as described previously (8 , 20 , 33 , 34) .

Induction and treatment of EAE
To induce myelin-oligodendrocyte glycoprotein (MOG)-EAE, female C57BL/6 mice (Charles River, Montréal, QC, Canada) were used, as previously reported (20 , 35) . Treatments, including clinically accepted doses of prednisone (PDE)- and DEX-related experiments, were initiated at the onset of neurobehavioral abnormalities. EAE animals were treated initially with 10 mg/kg PDE for 5 days from the onset and subsequently at 1 mg/kg PDE until day 30 by oral gavage, recapitulating clinical protocols (36) . Intraperitoneal injection of DEX (200 µg/kg) or saline was initiated at disease onset and continued until day 30 post-EAE induction. Animals were assessed daily for EAE severity, as reported previously (20) . Animals were killed by cardiac puncture under methoxyflurane anesthesia at day 30. Spinal cords were removed from euthanized animals, immersed in 10% neutral buffered formalin, and embedded in paraffin as previously reported (37) . Spinal cord tissue was also homogenized and then lysed in TRIzol (Invitrogen) according to the manufacturer’s guidelines, or fixed in 10% neutralized buffered formalin for immunohistochemistry and immunofluorescent staining. Animals were housed and handled according to Canadian Council on Animal Care guidelines, and this study was approved by the University of Calgary Animal Care Committee.

Immunohistochemistry
Paraffin sections were prepared from human and mouse CNS tissues samples, as previously reported (34 , 38) . To detect human β-arrestin-1, an anti-β-arrestin-1 (1:250 dilution; BD Transduction Laboratories, Mississauga, ON, Canada) polyclonal antibody was used together with anti-human CD45 monoclonal antibody (Zymed Laboratories Inc., San Francisco, CA, USA). Previously reported immunostaining protocols were performed for single- and double-labeling (33) .

Immunofluorescence and confocal laser scanning microscopy
Sections (4 µm) taken from the lumbar spinal cord region were immunostained. Deparaffinized sections were preincubated with 10% normal goat serum/2% BSA/0.2% Triton X-100 overnight at 4°C to prevent nonspecific binding (20) . Antigen retrieval was achieved as described previously (8) . Double-staining was performed using Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500 dilution; Invitrogen) to detect the Iba-1 antibody (1:500; Wako, Osaka, Japan) and Cy3-conjugated goat anti-mouse secondary antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA) to detect the mouse anti-MBP (1:1000 dilution; Sternberger Monoclonals, Lutherville, MD, USA) and anti-CD3 (CD3-{xi}, 6B10.2,1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) monoclonal antibodies. Specificity of staining was confirmed by omitting the primary antibody. The images from each spinal cord section were scanned by an Olympus Fluoview (FV300, Tokyo, Japan) confocal laser scanning microscope (20) .

Real-time RT-PCR
Total cellular RNA was isolated from human frontal subcortical white matter, mouse lumbar-sacral spinal cords, or cultured cells. The synthesis of cDNA and PCR reactions were performed as described previously (39) . All mouse and human primer sequences were established previously (20 , 33) except those as follows: mouse β-arrestin-1: sense, 5'-GGACACGAATCTGGCTTCCA-3'; antisense, 5'-ACGATGATGCCCAGGATTTC-3'; human β-arrestin-1: 5'-TCATGTCGGACAAGCCCTTGC-3'; antisense, 5'-CTCTTTGGGCTTGGGGTGCAT-3'; human GRK-2: sense, 5'-AGCGATAAGTTCACACGGTT-3'; antisense, TCGCACCGCTCCGAGATGGTGA-3'. Semiquantitative analysis was performed by using the SYBR-green dye on a BIO-RAD i-Cycler. Real-time fluorescence measurements were performed, and data were normalized to the GAPDH RNA level and expressed as RNA relative fold change (RFC).

Coimmunoprecipitation
U937 cells (6x106) were seeded in 6 ml of complete medium before being lysed on ice with lysis buffer (10 mM Tris-HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.5% Nonidet P-40; protease inhibitor cocktail 1:800 (Calbiochem, San Diego, CA, USA). The protein extract was centrifuged, and half of the resulting supernatant was used for A1AR immunoprecipitation. The A1AR and interacting proteins were coimmunoprecipitated with 15 µl A1AR rabbit antiserum (Alpha Diagnostic Inc, San Antonio, TX, USA) using protein A/G-agarose (Santa Cruz Biotechnology). After extensive washes in RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% Na-deoxycholate; 150 mM NaCl; 1 mM EDTA; protease inhibitor cocktail 1:800) to ensure specific interactions, immunoprecipitated complexes were released from the antibody-agarose complex by incubating 30 min at 4°C with 0.1 µg/µl of the A1AR antibodies epitope (Alpha Diagnostic Inc.). Proteins were then solubilized in Laemmli buffer and used for subsequent Western blot analysis.

Western blot analysis
Protein extracts were prepared from brain tissue samples with cell lysis buffer (20 mM Tris-HCl, 1% Triton X-100, 0.05% SDS, 5 mg sodium deoxycholate, 150 mM NaCl, and 1 mM PMSF), and concentrations were determined by BCA assay (Pierce, Rockford, IL, USA). Protein (50 µg) was separated by 10% SDS-polyacrylamide and transferred onto nitrocellulose membranes followed by blocking with 10% skimmed milk (20) . Membranes were then probed with polyclonal antisera to A1AR (1:1000 dilution; Alpha Diagnostic Inc.), monoclonal antibody to β-arrestin-1 (1:250 dilution; BD Transduction Laboratories), GRK-2 (1:1000 dilution, Millipore, Billerica, MA, USA), or HRP-conjugated β-actin (1:200, Millipore) overnight at 4°C followed by washing with TBST. Goat anti-rabbit or anti-mouse secondary antibody conjugated to HRP (1:5000 dilution; Millipore) was used to detect the primary antibody bound to the protein. After several washes, peroxidase activity on the membrane was detected by ECL (Roche Diagnostics, Laval, QC, Canada).

Flow cytometry
Aliquots of 106 U937 cells were either fixed with 2% paraformaldehyde or fixed and permeabilized using the Cytofix/CytopermTM kit (BD Biosciences) for 20 min at 4°C before incubation for 20 min at 4°C with 20 µg/ml of anti-A1AR polyclonal antisera (Alpha Diagnostic Inc.), followed by 50 µg/ml of Alexa Fluor 488-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). U937 cells were then resuspended in 500 µl of PBS before analysis. Data were collected from 15000 events for each experimental condition using a FACScan flow cytometer (Becton Dickinson, Oakville, ON, Canada). U937 incubated in the absence of antibody served as controls for each sample.

Statistical analysis
Statistical analyses were performed using GraphPad InStat version 3.0 (GraphPad Software, San Diego, CA, USA) for both parametric and nonparametric comparisons. When indicated, Dunnett’s Multiple Comparison Test was applied by comparing experimental treatments to the control. P values of less than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dysregulation of the A1AR/β-arrestin-1 system in MS brains
The expression of β-arrestins is closely coupled with the function of several 7TMRs in the CNS leading to modulatory effects on 7TMR sensitization and signaling (40) . To determine whether β-arrestin expression was dysregulated in the human CNS during inflammatory conditions, we examined corresponding transcript and protein levels in brain tissue from MS and non-MS patients. β-arrestin-1 RNA expression in normal appearing white matter was significantly increased in MS patients compared to non-MS (Fig. 1 A, left panel), while G protein-coupled receptor kinase (GRK)-2 transcript expression was significantly reduced in MS patients’ brains. A1AR transcript levels also exhibited a significant reduction in brains from MS patients relative to non-MS patients (Fig. 1A , right panel). β-arrestin-2 expression did not differ significantly in white matter between MS and non-MS patients (data not shown). Similar to RNA expression pattern, β-arrestin-1 protein immunoreactivity (55 kDa) was also increased markedly, although both GRK-2 (80 kDa) and A1AR (36 kDa) immunoreactivities were decreased in MS white matter compared to non-MS (Fig. 1B ). β-arrestin-1 immunoreactivity was increased in brain sections from MS patients (Fig. 1C , right panel) and was detected chiefly in CD45-positive microglia/macrophages in white matter (Fig. 1C , inset), while brain sections from non-MS patients showed minimal β-arrestin-1 immunoreactivity (Fig. 1C , left panel). Thus, β-arrestin-1 and the A1AR transcript and protein levels were inversely correlated in MS brains, suggesting that β-arrestin-1 up-regulation might contribute to the regulation of A1AR availability in MS as shown for other 7TMRs.


Figure 1
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  		Figure 1. β-arrestin-1 and A1AR expression in vivo. A) Analysis of mean relative fold change (RFC) (mean±SE) in β-arrestin-1, GRK-2, and A1AR transcript levels in MS and non-MS brains showed increased β-arrestin-1 levels in MS patients, but mean GRK-2 and A1AR RNA levels were reduced in MS brains. B) Western blots confirmed that β-arrestin-1 protein was induced in MS patient (n=3) brains (MS) while GRK-2 and A1AR protein levels were lower in MS brains compared with non-MS (n=3) brains (non-MS). C, D) Immunohistochemistry showed that β-arrestin-1 is highly expressed on microglia in MS brains (D), while non-MS sections (C) showed minimal β-arrestin-1 immunoreactivity. Inset: Double-staining of β-arrestin-1 and CD45 showed that β-arrestin-1 immunoreactivity was detected chiefly in CD45-positive microglia/macrophages in white matter. (Original magnification, x400; Student’s t test; **P<0.01).

β-arrestin-1 directly regulates A1AR availability
To investigate the role of β-arrestin-1 in the regulation of A1AR, U937 monocytoid cells were transfected with a plasmid expressing β-arrestin-1. Eight hours after transfection, β-arrestin-1 expression was significantly up-regulated both at the RNA and at the protein levels (Fig. 2 A, B). Of interest, GRK-2 and A1AR levels were concomittantly decreased, suggesting that β-arrestin-1 is directly involved in the regulation of the A1AR system (Fig. 2A, B ). Of interest, β-arrestin-1 overexpression did not have any effect on the cytokines TNF-{alpha} and IL-1β RNA expression (data not shown). Because one of the well-described roles of β-arrestins is the desensitization of receptors by internalization, the A1AR abundance at the cell surface was evaluated by flow cytometry. Overexpression of β-arrestin-1 was associated with a decreased detection of A1AR at the membrane level of monocytoid cells, while the overall abundance of A1AR did not change (Fig. 2C ). Moreover, coimmunoprecipitation experiments demonstrated β-arrestin-1 immunoreactivity (55 kDa) in samples previously precipitated with an A1AR antibody, which was preserved despite repeated washing steps, indicating that β-arrestin-1 was physically associated with the A1AR (Fig. 2D , lane 5). Thus, A1AR availability was closely related to β-arrestin-1 level and its regulation was dependent on a physical interaction with β-arrestin-1. These findings suggested that the dysregulation of β-arrestin-1 in MS might be responsible for the perturbation in A1AR pathway occurring in MS patients.


Figure 2
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  		Figure 2. β-arrestin-1 regulates A1AR expression and its availability at the cell membrane. Human monocytoid (U937) cells transfected with a β-arrestin-1 expressing plasmid showed a significant increase of β-arrestin-1 expression 8 h after transfection. This was associated with a decreased expression of GRK-2 and A1AR both at the RNA (A) and the protein level (B) compared with the transfection with the empty vector. C) Flow cytometry showed a decrease in A1AR level in nonpermeabilized cells (left panel) in β-arrestin-1 transfected U937 (full line) compared to U937 transfected with an empty vector (dotted line) but not in permeabilized cells (right panel). Black histograms corresponds to the background signal detected in absence of A1AR antibody. D) Coimmunoprecipitation studies demonstrated β-arrestin-1 directly interacts with the A1AR: Western blot displayed β-arrestin-1 immunoreactivity (lane 1, protein sample extracted from U937 cells; lanes 2–4 represent 3 serial washings of the lysate-antibody complex; lane 5 reflects protein released from the lysate-antibody complex by addition of a soluble A1AR epitope subsequent to the 3 washing steps. (Student’s t test; *P<0.05; **P<0.01)

Regulation of the A1AR/β-arrestin-1 system by inflammation
Glucocorticoids are well-described antiinflammatory molecules (41 , 42) . The ability of the prototypic synthetic glucocorticoid, DEX, to inhibit inflammation was confirmed in our in vitro model in which expression of the PMA-induced proinflammatory cytokines IL-1β and TNF-{alpha} were reversed by DEX in a concentration-dependent manner (Fig. 3 A, B). Because glucocorticoids act through intracellular receptors to regulate gene expression, including A1AR, we hypothesized that beneficial effects of glucocorticoid treatment in MS therapy may be mediated by glucocorticoid-induced A1AR up-regulation. DEX treatment of monocytoid cells resulted in the up-regulation of A1AR expression, which was inversely correlated with β-arrestin-1 expression (Fig. 3C ). However, DEX did not affect GRK-2 expression significantly ex vivo (data not shown). Earlier studies indicate that inflammation diminishes A1AR expression (8 , 19 , 20) and, hence, we treated monocytoid cells with TNF-{alpha} (Fig. 3D ) or IL-1β (data not shown), disclosing that these two inflammatory cytokines down-regulated A1AR expression. Moreover, DEX treatment of TNF-{alpha}- or IL-1β-treated U937 cells reversed the reduction of A1AR RNA levels. Indeed, DEX also unregulated A1AR transcript abundance on resting (undifferentiated) monocytoid cells (data not shown). To simulate the elevated extracellular levels of adenosine and their effects observed during chronic inflammation, we next investigated whether the A1AR-specific agonist ADAC could modulate A1AR expression in monocytoid cells, together with influencing pro- and antiinflammatory gene expression. ADAC treatment significantly suppressed the PMA-induced proinflammatory molecules, TNF-{alpha} (Fig. 3E ) and MMP-9 (data not shown) RNA expression in a concentration-dependent manner while having no effects on cytokine and MMP expression in unstimulated cells. In contrast, the antiinflammatory cytokine, IL-10 RNA, abundance was enhanced by ADAC treatment but again with no effects in resting cells (data not shown). To examine the effects of protracted A1AR activation on its expression, we treated resting monocytoid cells with the A1AR agonists ADAC and CHA for 72 h, which did not influence A1AR RNA expression (Fig. 3F –G). However, chronic treatment with both A1AR agonists significantly inhibited the up-regulation of A1AR expression induced by DEX (Fig. 3F-G ). Of interest, ADAC and CHA enhanced β-arrestin-1 transcript abundance in resting monocytoid cells (Fig. 3F-G ), which was reduced by concomitant DEX treatment, although DEX treatment alone had no effect on β-arrestin-1 expression in untreated cells. This effect on both elements of the A1AR/β-arrestin-1 system was specific to A1AR stimulation, as the treatment with the A2AR agonist CGS21680, while also reversing DEX-mediated A1AR up-regulation, did not alter the β-arrestin-1 expression (Fig. 3H ). Thus, activation of the A1AR by ADAC regulated cytokine and MMP expression in activated monocytoid cells while chronic A1AR activation also resulted in a concurrent induction of β-arrestin-1.


Figure 3
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  		Figure 3. Inflammation regulates the A1AR/β-arrestin-1 system. PMA-induced IL-1β (A) and TNF-{alpha} (B) RNA expression was significantly suppressed by 1 and 10 µM of the glucocorticoid DEX. DEX treatment significantly increased A1AR expression and decreased β-arrestin-1 expression in PMA-stimulated U937 (C). D) TNF-{alpha} treatment (40 µg/ml) up-regulated β-arrestin-1 and down-regulated A1AR expression in U937. E) ADAC treatment (0.1 to 10 µM) of U937 monocytoid cells significantly suppressed PMA-induced proinflammatory molecules, TNF-{alpha} RNA expression. F–H) Although chronic ADAC (1 µM), CHA (0.1 µM), or CGS21680 (1 µM) treatment did not down-regulate A1AR RNA expression, DEX-induced A1AR up-regulation was significantly inhibited by chronic ADAC, CHA, or CGS21680 treatment. Concomitant DEX treatment significantly reduced ADAC- or CHA-induced β-arrestin-1. CGS21680 did not affect β-arrestin-1 expression level (A–E, unpaired t test; F–H, Dunnett’s multiple comparison test. *P<0.05; **P<0.01; ***P<0.001).

Glucocorticoid treatment reduces neurobehavioral and neuropathological outcomes of EAE
To determine the in vivo effects of glucocorticoid treatment on the MS course, we examined neurobehavioral deficits in prednisone- (PDE), DEX-, and saline-treated mice following MOG-induction of EAE. The disease course in this MS model was a chronic progressive-relapsing phenotype (Fig. 4 A). Both PDE and DEX treatments significantly reduced the severity of neurobehavioral deficits in EAE animals compared to the saline-treated EAE group (Fig. 4A ). In addition, the cumulative (Fig. 4B ) and maximum neurological disability (Fig. 4C ) was significantly reduced in both PDE- and DEX-treated animals compared to saline-treated EAE animals.


Figure 4
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  		Figure 4. Glucocorticoid treatment reduces neurological deficits in EAE. A) Neurobehavioral analyses during EAE showed that the disease course in this model was a chronic progressive-relapsing phenotype. Both PDE and DEX treatments significantly reduced the severity of neurobehavioral deficits in EAE animals compared to the saline-treated EAE group. The cumulative (B) and maximum (C) neurological disability was significantly reduced in both PDE- and DEX-treated animals compared to saline-treated EAE animals. (Mann-Whitney U test; *P<0.05; **P<0.01).

Immune activation with demyelination and axonal injury are the principal neuropathological features of both MS and EAE (43) . To determine the in vivo neuropathological effects of glucocorticoid treatment, we examined the spinal cords from EAE mice treated with PDE, DEX, or saline (control) treatments. Oral PDE, parenteral DEX, or saline treatments of animals were initiated at the time of onset of EAE neurobehavioral abnormalities and continued until day 30 after MOG injection. Immunoreactivity of the microglial/macrophage marker, Iba-1, was markedly enhanced in white matter of lumbar spinal cord with microglial hypertrophy and macrophage infiltration in saline-treated EAE animals (Fig. 5 E) compared to healthy control animals (Fig. 5A ). However, both PDE (Fig. 5I ) and DEX (Fig. 5M ) treatments reduced Iba-1 immunoreactivity in EAE mice. CD3-immunopositive cell infiltrates in white matter were also apparent in EAE animals (Fig. 5F ) compared to healthy controls (Fig. 5B ). As expected, CD3 immunoreactivity was significantly reduced by both PDE (Fig. 5J ) and DEX (Fig. 5N ) treatments of animals with EAE. The integrity of myelin, evaluated by MBP immunoreactivity in saline-treated EAE animals, was also significantly reduced (Fig. 5G ) relative to healthy controls (Fig. 5C ). Indeed, the loss of MBP immunoreactivity in saline-treated EAE animals showed contemporaneous enhancement of Iba-1 immunopositivity (Fig. 5E ). Likewise, the reduced MBP immunoreactivity evident during EAE was reversed by both PDE- and DEX-treatment of EAE animals compared to the saline-treated EAE group (Fig. 5K, O , respectively). Of particular interest was the finding that a loss of silver-positive axons occurred in EAE-induced mice (Fig. 5H ), relative to healthy controls (Fig. 5D ), which was prevented by glucocorticoid treatments including both PDE (Fig. 5L ) and DEX (Fig. 5P ). The above findings were quantified, revealing that both glucocorticoids significantly reduced Iba-1 (Fig. 5Q ) and CD3 (Fig. 5R ) immunoreactivity in EAE mice while concurrently significantly enhancing MBP expression (Fig. 5S ). Likewise, PDE- and DEX-treated animals exhibited an improvement in axonal counts compared to saline-treated EAE-induced mice (Fig. 5T ). Thus, these findings demonstrate that both oral PDE and parenteral DEX reduced neuroinflammation with concomitant improved preservation of myelin and axonal integrity, resulting in the reduction of neurobehavioral deficits during EAE.


Figure 5
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  		Figure 5. Neuropathological changes in lumbar spinal cord during EAE. Iba-1 immunoreactivity on microglia/macrophage was markedly enhanced in white matter with microglial hypertrophy and macrophage infiltration in saline-treated EAE animals (E), compared to healthy control animals (A). Both PDE (I) and DEX (M) treatments reduced Iba-1 immunoreactivity in EAE mice. CD3-immunopostive cell infiltrates in white matter were also apparent in EAE animals (F) compared to healthy controls (B), but CD3 immunoreactivity was significantly reduced by PDE (J) or DEX (N) treatments of animals with EAE. MBP immunoreactivity in saline-treated EAE animals was also significantly reduced (G) relative to healthy controls (C). Indeed, the loss of MBP immunoreactivity in saline-treated EAE animals showed concurrent enhancement of Iba-1 immunopositivity (E, G). MBP immunoreactivity was increased in both PDE- and DEX-treated EAE animals compared the saline-treated EAE group (K and O, respectively). Of particular interest, there was a loss of silver-positive axons in EAE-induced mice (H), relative to healthy controls (D), which was prevented by glucocorticoid treatments including both PDE (L) and DEX (P). Quantitation of Iba-1, CD3, MBP and silver detection revealed that both glucocorticoids significantly reduced Iba-1 (Q) and CD3 (R) immunoreactivity in EAE mice while concurrently significantly enhancing MBP expression (S). Likewise, PDE- and DEX-treated animals exhibited a significant improvement in axonal counts compared to untreated EAE-induced mice (T). (n=4 per group). (Dunnett’s multiple comparison test; *P<0.05; **P<0.01).

A1AR down-regulation in EAE is prevented by glucocorticoid treatment
Previous studies have reported that A1AR RNA and protein levels in monocytoid cells were reduced in the blood and brain of patients with MS and also in EAE spinal cords with associated dysregulation of cytokine expression (8 , 19 , 20) . To determine if A1AR down-regulation in EAE is regulated by glucocorticoid treatment, including DEX and PDE treatment, we examined A1AR and β-arrestin-1 RNA expression in PDE-, DEX-, and saline-treated mice following EAE induction. A1AR RNA expression was significantly down-regulated at the onset and day 3 post-EAE induction in EAE animals compared to control animals. Both PDE and DEX treatments significantly up-regulated A1AR expression at day 3, and their effects persisted until day 30 (Fig. 6 A). In contrast, β-arrestin-1 RNA expression was significantly up-regulated at the onset and on day 3, compared to controls, while both PDE and DEX treatment inhibited the up-regulation of β-arrestin-1 in EAE animals, again lasting until day 30 (Fig. 6B ). Of note, both PDE and DEX had minimal effects at all times on A1AR and β-arrestin-1 expression in healthy animals except for β-arrestin-1 transcript abundance, which was down-regulated at day 30. We next examined proinflammatory cytokine expression at the onset, day 3 and day 30 in MOG-induced EAE mice. TNF-{alpha} (Fig. 6C ) and IFN-{gamma} (Fig. 6D ) showed highest transcript levels at disease onset, but IFN-{gamma} exhibited spontaneously reduced levels at day 3. However, DEX treatment reduced TNF-{alpha} (Fig. 6C ) and IFN-{gamma} (Fig. 6D ) expression at day 3, while PDE treatment was effective in reducing only TNF-{alpha} expression at day 3 (Fig. 6C ). However, all of these latter cytokines’ expression was significantly reduced by both PDE and DEX treatments at day 30 after EAE induction (Fig. 6C, D ). In addition, both MMP-12 and IL-1β, which was also induced in EAE animals, showed significant suppression by both PDE and DEX at both day 3 and day 30 post-EAE induction (data not shown). Conversely, the antiinflammatory cytokine, IL-4, was significantly down-regulated in EAE animals, while both PDE and DEX treatments enhanced its expression at day 3 and day 30 post-EAE induction (data not shown). Hence, the improved neurobehavioral performance observed with glucocorticoid treatments reflected diminished neuropathology, together with enhanced A1AR and reduced β-arrestin-1 expression during EAE.


Figure 6
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  		Figure 6. A1AR, β-arrestin-1 and proinflammatory cytokine expression in the CNS following induction of EAE. A) PDE and DEX treatments significantly up-regulated A1AR expression at day 3 and 30. B) β-arrestin-1 RNA expression was significantly up-regulated in saline-treated EAE animals at the onset and day 3 compared to healthy controls, while both PDE and DEX treatment inhibited the up-regulation of β-arrestin-1 in EAE animals. C, D) TNF-{alpha} (C) and IFN-{gamma} (D) showed highest transcript levels at disease onset, and glucocorticoid treatment significantly reduced all proinflammatory cytokines’ expression both at day 3 and 30. (A, B, Dunnett’s multiple comparison test; C, D, unpaired t test; *P<0.05; **P<0.01; ***P<0.001).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we report that β-arrestin-1 protein and transcript abundance was markedly increased in conjunction with reduced A1AR expression in the CNS of MS patients and animals with EAE, compared to controls. Indeed, we found a direct, albeit reciprocal relationship in monocytoid cells, between the comparative expression of β-arrestin-1 and the A1AR in that β-arrestin-1 expression was increased while the adenosine A1AR was diminished following immune activation both in vitro and in vivo. Moreover, increased β-arrestin-1 expression was sufficient to down-regulate A1AR expression and availability at the cell membrane through a physical interaction. Cell treatment with the A1AR agonist, ADAC, diminished proinflammatory cytokine expression, but chronic A1AR activation up-regulated β-arrestin-1 and prevented A1AR induction by glucocorticoids. Glucocorticoid treatments exerted marked neuroprotective effects in EAE, as evidenced by reduced demyelination and axonal injury. The neuroprotective effects of glucocorticoids were also associated with a reversal of the reciprocal aberrant interaction between A1AR and β-arrestin-1 expression in the CNS during EAE with a concurrent down-regulation of proinflammatory molecules. Hence, these studies demonstrate that β-arrestin-1 is a key regulator for A1AR availability in MS (Fig. 7 ).


Figure 7
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  		Figure 7. Reciprocal interactions between glucocorticoid-regulated inflammation and A1AR/β-arrestin-1 system. Neuroinflammation induces β-arrestin-1 expression in monocytoid cells, resulting in suppression of the A1AR, which is reversed during glucocorticoid (GC) treatment. Chronic A1AR activation by ADAC inhibits the induction of A1AR expression by GC.

The 7TMR superfamily represents cell surface receptors for many neuromodulators and more than 90% of nonsensory 7TMRs are expressed in the CNS, where they play important roles in many neural cell functions (44) . 7TMRs function as mediators of (slow) neuromodulators rather than (fast) neurotransmitters in CNS, and under- or overactivity of several 7TMRs expressed in the brain contribute to pathological conditions (45) . A unique feature of 7TMR signaling systems is their ability to retain a memory of prior activation or signaling tone (46) . Thus, protracted receptor activation leads to desensitization of a receptor and reduced expression in same cases, while diminished activation leads to sensitization as previously shown for the A1AR (25) . Activation of 7TMRs by specific agonists desensitizes the receptor through phosphorylation mediated by members of G protein-coupled receptor kinases (GRKs). The phosphorylated receptor is subsequently bound by individual β-arrestins, which promotes 7TMR endocytosis (47) . GRK-2 is suppressed in EAE (47) , and likewise here it was reduced in the brains of MS patients, making it unlikely that GRK-2 contributes to aberrant A1AR expression in MS and EAE. However, it is plausible that the GRKs might have other roles in MS pathogenesis such as modulating chemokine receptor expression. β-arrestins are multifunctional endocytic adaptor proteins and are ubiquitously expressed, including within the brain (48) . Many studies have demonstrated that GRKs and β-arrestins play critical roles in the internalization of 7TMRs, such as β-adrenergic receptors, chemokine receptors, proteinase-activated receptors, and adenosine A2B receptor (40 , 49) . Some of these 7TMRs exert anti- or proinflammatory properties initially through G protein phosphorylation preceding the activation of signaling molecules regulating the expression of inflammatory molecules. The roles of β-arrestins in these actions remain obscure (50) . However, it is increasingly recognized β-arrestins are directly involved in signaling cascades, including transactivation of receptor tyrosine kinases, induction of MAP kinases, chemotaxis, and prevention of apoptosis (47) . We have previously reported that the A1AR is down-regulated on blood-derived monocytic cells and microglia in MS brains (8 , 19) and spinal cords in MOG-EAE (20) , while up-regulation of A1AR reduces the severity of EAE-related neurobehavioral and neuropathological outcomes (20) . Nonetheless, our present findings suggest that the A1AR and its antiinflammatory effects may be modulated by β-arrestin-1. Indeed, no previous reports have shown direct interaction between the β-arrestins and the A1AR internalization, although our coimmunoprecipitation studies revealed that β-arrestin-1 physically interacted with A1AR in monocytoid cells. β-arrestins usually require activation and conformational changes of the cognate 7TMR for binding, but in the present monocytoid cell culture system the A1AR is likely tonically activated through the release and presence of adenosine in the culture medium. These findings suggest that, like other 7TMRs, A1AR expression is regulated by β-arrestin-1 and that the immune activation occurring in MS/EAE brains influences the β-arrestin-1/A1AR system on a reciprocal basis. These interactions thus have a direct impact on neuropathologies of autoimmune origin, which could be modulated by glucocorticoids (Fig. 7) .

While β-arrestins are largely recognized for their roles in 7TMR desensitization, they also mediate numerous other effects, especially in the area of cellular signaling (40) . β-arrestins regulate cAMP levels, MAP kinases activity, and genes that regulate chemotaxis. Likewise, the A1AR has been shown to down-regulate cAMP levels and modulate Akt and PI3 kinase activity (51) . The present ex vivo and in vivo observations reflect changes in closely coupled transcript and protein levels for both β-arrestin-1 and A1AR, although the interface between these two genes might be a protein:protein interaction. Hence, the complementary changes in transcript and protein levels likely reflect activation of parallel processes. The transcriptional control of the A1AR and β-arrestin-1 remains unclear, but several reports highlight potential candidate mechanisms. The expression of human A1AR gene is controlled by promoters A and B (52) . Promoter A contains binding sites for GATA-4 and Nkx2.5 (53) , and promoter B has a glucocorticoid response element (GRE) binding site and an AP-1 site (52) . β-arrestin-1 is strongly induced by intracellular cAMP pathway (54) , and the expression of β-arrestin-1 protein is increased by GM-CSF (55) , a soluble factor required for EAE onset (56 , 57) . Our data, together with these previous reports, suggest that both A1AR and β-arrestins can be regulated by immune responses and that A1AR/β-arrestin-1 system modulates inflammation in MS/EAE. A previous study showed that the mitogen PHA diminished β-arrestin-1 expression in lymphocytes, whereas GRK-2 and -3 were up-regulated (58) . Although the A1AR is not expressed on lymphocytes (8) , this latter study underscores the cell-dependent and reciprocal effects of mitogenic stimulation on β-arrestin-1 expression.

The effects of glucocorticoids are far-reaching with altered calcium metabolism, cell viability, and suppression of adaptive immune mechanisms, including lymphocyte activation (59) . High-dose glucocorticoids remain a cornerstone of MS treatment with presumed effects on immune events within the CNS and the peripheral immune system. Indeed, the glucocorticoid family affects transcriptional expression of many genes through interactions with its intracellular glucocorticoid receptor (GR). After binding to the glucocorticoid receptor, the ligand/receptor complex enters the nucleus and binds to GRE or other domains, which regulate gene expression (60) . Computer analysis of the A1AR B promoter sequence revealed a GRE monomer-binding site and an AP1 site that has been shown to be a target of GR interaction (61 62 63) . Supporting these computer analyses, previous reports have shown that treatment with the synthetic glucocorticoid DEX increases expression of the A1AR, whereas the adenosine A2 receptor was down-regulated in smooth muscle cells (30) . DEX treatment of adrenalectomized rats also showed a marked increase in A1AR in brain tissue but not A2aAR (64) . Our findings extend the studies of glucocorticoid properties in the CNS by showing that DEX exerts inhibitory effects on β-arrestin-1 expression ex vivo and in vivo with concomitant inverse consequences on A1AR abundance. As previous studies showed that arrestins are key molecules for the onset of autoimmune disease affecting the CNS and that autoantibodies targeting arrestins were found in MS patients (21 , 22 , 24) , glucocorticoid effects on β-arrestin-1 expression might mediate, at least partially, the beneficial outcome of glucocorticoid treatment in MS/EAE. Not surprisingly, these actions were accompanied by diminished neuroinflammation and demyelination. However, glucocorticoids also suppressed axonal injury in EAE, an effect recapitulated by recent clinical studies indicating that "black holes" on MRI scans of MS patients were reduced with glucocorticoid therapy (65) . Although we found that both DEX and prednisone demonstrated protective effects in terms of reduced neuroinflammation and demyelination, oral prednisone appeared to have less beneficial effects on neurobehavioral abnormalities than parenteral DEX. However, we did not measure plasma levels of glucocorticoids to ensure equivalent tissue concentrations for the two drugs. Nonetheless, the neuroprotective effects mediated by glucocorticoids in terms of axonal preservation is an important observation that warrants further investigation in terms of clinical benefits and underlying mechanisms.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Arthur Clark for providing tissues and Stephanie Skinner for manuscript preparation. S. T. is a Multiple Sclerosis Society of Canada (MSSC) Fellow, S.S.G.F. holds Canada Research Chair (Tier 2) in Molecular Neuroscience. C. P. holds Canada Research Chair (Tier 1) in Neurological Infection and Immunity. These studies were supported by MSSC and a CIHR-Interdiciplinary Health Research Team. The authors have no financial conflict of interest.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication May 14, 2007. Accepted for publication September 13, 2007.


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