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The Journal of Neuroscience, July 1, 2002, 22(13):5403-5411
Activation of Group III Metabotropic Glutamate Receptors Inhibits
the Production of RANTES in Glial Cell Cultures
Gilbert
Besong1, *,
Giuseppe
Battaglia1, *,
Mara
D'Onofrio1, *,
Roberto
Di
Marco2,
Richard Teke
Ngomba1,
Marianna
Storto1,
Marzia
Castiglione4,
Katia
Mangano2,
Carla L.
Busceti1,
Ferdinando R.
Nicoletti2, 3,
Kevin
Bacon5,
Michael
Tusche5,
Ornella
Valenti6,
Peter Jeffrey
Conn6,
Valeria
Bruno1, 4, and
Ferdinando
Nicoletti1, 4
1 Istituto Neurologico Mediterraneo Neuromed,
86077 Pozzilli, Italy, Departments of 2 Microbiology and
Gynecology and 3 Biology/Section of General Pathology,
University of Catania, 95125 Catania, Italy,
4 Department of Human Physiology and Pharmacology,
University "La Sapienza", 00185 Rome, Italy,
5 Department of Biology, Bayer Yakuhin, Ltd. 6-5-1-3, Kunimidai, Kyoto, Japan, and 6 Department of Pharmacology,
School of Medicine, Emory University, Atlanta, Georgia
30322
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ABSTRACT |
The chemokine RANTES is critically involved in
neuroinflammation and has been implicated in the pathophysiology of
multiple sclerosis. We examined the possibility that activation of
G-protein-coupled metabotropic glutamate (mGlu) receptors regulates the
formation of RANTES in glial cells. A 15 hr exposure of cultured
astrocytes to tumor necrosis factor- and interferon-
induced a substantial increase in both RANTES mRNA and extracellular
RANTES levels. These increases were markedly reduced when astrocytes
were coincubated with L-2-amino-4-phosphonobutanoate
(L-AP-4), 4-phosphonophenylglycine, or
L-serine-O-phosphate, which selectively
activate group III mGlu receptor subtypes (i.e., mGlu4, -6, -7, and -8 receptors). Agonists of mGlu1/5 or mGlu2/3 receptors were virtually
inactive. Inhibition of RANTES release produced by L-AP-4
was attenuated by the selective group III mGlu receptor antagonist
(R,S)--methylserine-O-phosphate or by pretreatment of the cultures with pertussis toxin. Cultured astrocytes expressed mGlu4 receptors, and the ability of
L-AP-4 to inhibit RANTES release was markedly reduced in
cultures prepared from mGlu4 knock-out mice. This suggests that
activation of mGlu4 receptors negatively modulates the production of
RANTES in glial cells. We also examined the effect of
L-AP-4 on the development of experimental allergic
encephalomyelitis (EAE) in Lewis rats. L-AP-4 was
subcutaneously infused for 28 d by an osmotic minipump that
released 250 nl/hr of a solution of 250 mM of the drug.
Detectable levels of L-AP-4 (~100 nM) were
found in the brain dialysate of EAE rats. Infusion of
L-AP-4 did not affect the time at onset and the severity of
neurological symptoms but significantly increased the rate of recovery
from EAE. In addition, lower levels of RANTES mRNA were found in the
cerebellum and spinal cord of EAE rats infused with L-AP-4.
These results suggest that pharmacological activation of group III mGlu
receptors may be useful in the experimental treatment of
neuroinflammatory CNS disorders.
Key words:
chemokines; glial cultures; experimental allergic
encephalomyelitis; multiple sclerosis; mGlu4 receptor; leukocytes
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INTRODUCTION |
Chemokines constitute a growing
family of low molecular weight cytokines (8-14 kDa) that play a
pivotal role in many biological processes, from routine
immunosurveillance to the control of inflammation and cell infection by
human immunodeficiency virus. They are divided into four
distinct subfamilies (designated as or CXC,  or CC, or CX3C,
and  or C chemokines) and activate G-protein-coupled receptors,
which are named according to the classification of their specific
ligands (Bazan et al., 1997 ; Pan et al., 1997; Wells et al., 1998).
Chemokines have been implicated in the modulation of numerous
biological functions in both the developing and mature CNS, including
neuropoiesis, oligodendrocyte proliferation, regulation of synaptic
plasticity, and, particularly, leukocyte recruitment in response to
traumatic injury, stroke, autoimmunity, and inflammation (Mennicken et
al., 1999). Chemokines are the only group of inflammatory mediators
endowed with cell type-selective chemotactic activity, and hence,
they play a vital role in defining the cellular composition of
inflammatory infiltrates at the sites of tissue damage. Because of
this, chemokines are becoming potential targets for therapeutic intervention in inflammatory disorders of the CNS, including multiple sclerosis (MS) (Godiska et al., 1995; Miyagishi et al., 1997; Ransohoff
and Bacon, 2000) (see Discussion and references therein). Chemokines
are constitutively expressed at low-to-negligible levels in neurons,
astrocytes, and microglia, but are markedly upregulated in response to
proinflammatory cytokines, such as tumor necrosis factor- (TNF-)
and interferon- (IFN-) (Barnes et al., 1996; Mennicken et al.,
1999). The identification of membrane receptors that control the
induction of chemokines is an obligatory step in the search for drugs
acting on leukocyte recruitment in neuroinflammation. We now report
that pharmacological activation of group III mGlu receptors reduces the
synthesis and release of the -chemokine RANTES (from regulated upon
activation of normal T cell expressed and secreted) induced by TNF-
and IFN- in cultured astrocytes and increases the rate of functional
recovery in an in vivo model of neuroinflammation.
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MATERIALS AND METHODS |
Materials.
(S)-4-carboxy-3-hydroxyphenylglicine (4C3HPG),
L-2-amino-4-phosphonobutanoate
(L-AP-4),
(R,S)--methylserine-O-phosphate (MSOP), L-serine-O-phosphate
(L-SOP),
(RS)-3,5-dihydroxyphenylglycine (DHPG), and
(2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate
(2R,4R-APDC) were purchased from
Tocris Cookson Ltd. (Bristol, UK). 4-phosphonophenylglycine (PPG) was a
generous gift from Dr. Peter J. Flor and Dr. Fabrizio Gasparini
(Novartis Pharma, Basel, Switzerland). All other drugs or chemicals
were purchased from Sigma (Milan, Italy).
Preparation of primary cultures of mouse or rats cortical
astrocytes. Primary cultures of cortical astrocytes were prepared from neonate CD1 mice (Charles River, Calco, Italy), mGlu4 knock-out mice (Jackson Laboratories, Bar Harbor, MN), or Lewis rats (Charles River), as described by Rose et al. (1992). Dissociated cortical cells
were grown in MEM-Eagle's salts, supplemented with 10% horse serum,
10% fetal calf serum, 2 mM glutamine, 25 mM sodium bicarbonate, and 21 mM glucose. Cultures were kept at 37°C in a
humidified 5% CO2 atmosphere until they reached
confluence (7-14 d in vitro). The number of
microglial cells contaminating the cultures was assessed in cultures
fixed with 2% paraformaldehyde by using the lectin, isolectin
B4 Banderiera simplicifolia I, coupled
to biotin. After 30 min of incubation at room temperature with lectin
at 1:50 dilution, lectin binding was identified by using the ABC Vectastain kit (Vector Laboratories, Burlingame, CA). The efficacy of
the method for the detection of microglial cells was proven by staining
spinal cord sections of Lewis rats 11 d after immunization with
myelin basic protein (MBP) (data not shown).
Measurement of extracellular RANTES in the glial medium.
Glial cultures were washed with serum-free medium and, 1 hr later, were
incubated for 15 hr with IFN- (10 U/ml) and TNF- (0.1 ng/ml), in
the absence or presence of specific mGlu receptor ligands. At the end
of the incubation, the medium was removed and used for the
determination of RANTES using the mouse RANTES QUANTIKINE M immunoassay
ELISA kit (R & D Systems, Minneapolis, MN).
RNA extraction and Northern blot analysis of RANTES mRNA.
Total RNA was prepared from cultured astrocytes and from cerebellum and
spinal cord of Lewis rats (Chomczynski and Sacchi, 1987). Thirty
micrograms of total RNA were denatured, subjected to electrophoresis on
1% formaldehyde agarose gel, and transferred to a nylon membrane Hybond-N (Amersham Pharmacia Biotech, Milan, Italy). Membranes were
fixed by UV irradiation using an XL-1500 UV cross-linker (Spectrolinker, Spectronics Corporation, Westbury, NY) and stained with
0.04% methylene blue and 0.5 M sodium acetate.
Membranes were hybridized with a random primed
[-32P]-dCTP-labeled probe consisting
of RANTES 0.7 kb cDNA insert in a pCR II vector (Invitrogen, Groningen,
The Netherlands) cloned at the BstXI/NotI site.
Hybridizations were performed overnight at 42°C. Blots were washed
twice using 2× SSC-0.1% SDS for 15 min at 42°C and then twice with
0.1× SSC-0.1% SDS for 15 min at 42°C. The filters were then
exposed to Hyperfilm-MP (Amersham Pharmacia Biotech) and exposed at
 80°C for 18 hr. Filters were reprobed with -actin cDNA, and
autoradiograms were quantified by densitometry using a computerized
image-processing system (NIH Imaging, Bethesda, MD).
RT-PCR analysis of mGlu4 receptors. Two micrograms of total
RNA extracted from cultured astrocytes and 100 ng of random hexamers dissolved in 10 µl of RNase-free water were heated to 65°C for 10 min and then cooled on ice. Reverse transcriptase (RT) buffer (10 mM DTT, 500 µM dNTP,
and 200 U of Moloney murine leukemia virus RT enzyme) was added
to a final volume of 25 µl. The incubation was continued at 42°C
for 1 hr, and the reaction was terminated by a 10 min incubation at
99°C. PCR was performed for 35 cycles in final volume of 50 µl with
appropriate quantity of buffer and MgCl2, 200 µM of dNTP, 50 pmol of either forward or
reverse primers, and 2.5 U of AmpliTaq Gold (Perkin-Elmer Cetus Corp.,
Norwalk, CT). RT-PCR negative control was performed loading
dH2O instead of cDNA. Primers and PCR conditions
were as follows: mGlu4 receptor: GenBank accession #M90518; 1.5 mM MgCl2; annealing at
60°C; amplimer 567 bp; forward: 5'-TGAGCTACGTGCTGCTGGCG-3'; reverse: 5'-TGTCGGCTGACTGTGAGGTG-3'.
Western blot analysis of RANTES and mGlu4 receptors. Western
blot analysis in protein extracts from lysates of cultured astrocytes prepared from mice or rats or from mouse cerebral cortex (used as a
positive control) was performed as described by Ciccarelli et al.
(2000). RANTES expression was detected with 4 µg/ml of a monoclonal
antibody (R & D Systems). mGlu4a receptors were detected with 0.5 µg/ml of a polyclonal antibody raised against synthetic peptides
corresponding to the following amino acid sequences
CGGLETPALATKQTYVTNHAI corresponding to the putative intracellular
C-terminal domain of rat mGlu4a receptor (Upstate Biotechnology, Inc.
Lake Placid, NY; residues 893-912) (Bradley et al., 1996, 1999).
Induction of experimental allergic encephalomyelitis in Lewis
rats. For experimental allergic encephalomyelitis (EAE)
induction, Lewis rats (225-250 gm, body weight) were immunized in the
proximal portion of the tail with 50 µg of guinea pig MBP, 2 mg
Mycobacterium tuberculosis in 100 µl saline, and 100 µl
Freund's incomplete adjuvant. Animals were implanted with subcutaneous
osmotic minipumps (Alzet; Alza, Palo Alto, CA) containing 200 µl of
250 mM of L-AP-4 dissolved
in PBS, which release 250 nl/d for 28 d. Control animals were
implanted with minipumps containing PBS alone. Immunization was
performed 48 hr after implanting the osmotic minipumps. In standard
experiments, immunized animals developed clinical signs of EAE 10 d after immunization. Symptoms of EAE were scored using the disability
scale described by Godiska et al. (1995) in which 0 = absence of
clinical signs; 1 = loss of motor control in the tail; 2 = hindquarter weakness or the inability to turn over when placed on the
back; 3 = total hindquarter paralysis; 4 = total hindquarter
paralysis with incontinence and/or forearm involvement; and 5 = death caused by EAE. In addition, body weight was monitored every day
during the development of EAE.
Some animals were killed 12 d after immunization (i.e., at the
time of the peak of clinical symptoms) for the detection of RANTES in
the cerebellum and spinal cord. RANTES protein and mRNA levels were
assessed by Western and Northern analysis, as described above.
Immunohistochemical analysis of spinal cords. Two
established parameters of neuroinflammation, i.e., the expression of
major histocompatibility complex (MHC) class II antigens and the
presence of CD4+ cells were examined by
immunohistochemistry in Lewis rats immunized with MBP and treated with
L-AP-4, as described above. Animals were killed
by CO2 inhalation at 11 or 27 d after
immunization, and spinal cords were collected and snap-frozen in liquid
nitrogen. For immunohistochemical analysis, spinal cord cryostat
sections (10 µm) were exposed to appropriate dilutions of the mouse
FITC-conjugated monoclonal antibodies (Seralab, Crawley-Down, UK)
directed against the rat homologs of human CD4, (W3/25), and MHC class
II antigens (OX6). Coded slides were examined by fluorescence
microscopy at 40× magnification. Inflammation grade was assessed
blindly and scored from grade 1-4 in relation to the intensity of
immunostaining, density of immunopositive cells, and distribution of
clusters of immunopositive cells (total score: 0-12) (Di Marco et al., 2001).
Measurements of L-AP-4 levels by in vivo
microdialysis. To assess whether subcutaneously infused
L-AP-4 can penetrate the brain, we measured the
amount of the drug present in the striatal dialysate of freely moving
animals. Lewis rats, 250-300 gm, were implanted with microdialysis
intracerebral guides in the striatum using the following coordinates:
2.0 mm anterior to bregma, 2.6 mm lateral to the midline, and 4-6 mm
ventral, according to the atlas of Pellegrino et al. (1992), under
pentobarbital anesthesia (50 mg/kg, i.p.). After surgery, rats were
housed in separate cages in a temperature-controlled environment on a
12 hr light/dark cycle, with ad libitum access to water and
food, and allowed to recover. Forty-eight hours later rats were
implanted with subcutaneous osmotic minipumps (Alzet), containing
L-AP-4 (200 µl, 250 mM) that release 250 nl/d for 28 d of L-AP-4,
and were immunized 48 hr later as described above. Microdialysis was
performed 9 d after immunization. Twelve hours before the
experiment, a concentric vertical probe (2-mm-long and 0.5 mm in outer
diameter having a polycarbonate membrane; molecular cutoff: 20,000 Da;
CMA/12; CMA Microdialysis, Stockholm, Sweden) was inserted into the
intracerebral guide cannula, and rats were transferred to a plastic
bowl cage with a moving arm. The animals had ad libitum
access to water and food. The probe was perfused continuously with
artificial CSF (ACSF), at a flow rate of 0.1 µl/min, using a
microinjection pump. ACSF contained in mM: 150
NaCl, 3 KCl, 1.7 CaCl2, and 0.9 MgCl2. On the next day, perfusate sample
fractions were continuously collected by a fraction collector. Analysis
of L-AP-4 present in the dialysate was performed
by HPLC with fluorescence detection.
Measurement of extracellular RANTES in leukocytes.
Peripheral blood mononuclear cells (PBMCs) were prepared using Ficoll
HyPaque according to the manufacturer's recommended protocol. Enriched monocytes were obtained from whole PBMCs by incubation with RPMI 1640 + 10% FCS at 4°C, for 30 min at constant rotation. Nonaggregated cells
were removed, and monocyte-enriched pellet was resuspended in RPMI and
plated 2 × 10 6 cells per well in
6-well plates. After a 2 hr incubation at 37°C, nonadherent cells
were removed, and adherent cells were washed. Adherent cells were then
removed from the plate using trypsin. This enriched monocyte fraction
was 85-90% CD4+ by flow cytometric
analysis. CD8+ cells were obtained from
whole PBMCs by negative selection. Briefly, cells were incubated with
an antibody cocktail containing microbeads against CD4, 11b, 14, 16, 19, 36, 56, and IgE (Miltenyi Biotec) for 30 min at 4°C. Cells were
washed and passed over a permanent magnet. The negative fraction was
>95% CD8+ as determined by flow
cytometric analysis. Cells were preincubated with appropriate
concentration of L-AP-4 for 30 min at 37°C and seeded at 2 × 105 cells per well in
96-well plates. Phorbol-12-myristate-13-acetate (PMA) was added, and
cultures were incubated for 48 hr at 37°C. Supernatants were
collected and stored at 80°C. For the determination of
extracellular RANTES, human anti-RANTES antibody (Genzyme Techne) was
coated on a 96-well plate (10 µg/ml) at 4°C for 18 hr. Plate was
washed three times with PBS. Nonspecific sites were blocked by an
incubation with PBS supplemented with 5% BSA. After washing plate with
PBS + 0.05% Tween 20, 50 µl/well supernatant was added and incubated
at room temperature for 2 hr. A Europium-labeled anti-RANTES antibody
was added (a kind gift from H. Inbe, Department of Biological
Chemistry, Yamaguchi University, Yamaguchi, Japan) and incubated
for a further 2 hr at room temperature. Plate was washed using PBS + 0.05% Tween 20 at 300 µl/well. Enhancement solution (LKB-Wallac,
Gaithersburg, MD) was added at 100 µl/well and incubated at
room temperature for 10 min. The plate was then measured for Europium
counts using a Wallac Arvo SX Multi-Label counter (Wallac).
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RESULTS |
Activation of group III mGlu receptors reduces the production of
RANTES in primary cultures of glial cells
We used confluent cultures of glial cells, which were primarily
constituted of flat and polygonal type-1 astrocytes. Cultures were
virtually devoid of microglial cells (only 6-10 cells per well stained
with the lectin, Banderiera simplicifolia I). A 15 hr
exposure of mouse cultured glial cells to 0.1 ng/ml of TNF- plus 10 U/ml of IFN- led to a substantial increase in the amount of RANTES
released into the medium. L-AP-4 applied in
combination with TNF- and IFN- reduced extracellular RANTES
levels in a concentration-dependent manner, with an apparent
EC50 value of 6 µM (Fig.
1A). Among other mGlu
receptor ligands, L-SOP (300 µM) and PPG (100 µM)
were also able to reduce extracellular RANTES. 4C3HPG,
2R,4R-APDC, and DHPG (all at 100 µM) had negligible, if any, effect on the
cytokine-stimulated increase in extracellular RANTES (Fig.
1B). The group III mGlu receptor antagonist MSOP (100 µM) produced a slight increase in extracellular
RANTES on it own, but markedly reduced the inhibitory action of
L-AP-4 (30 µM) (Table
1). None of the mGlu receptor ligands
induced changes in extracellular RANTES in the absence of TNF- and
IFN- (data not shown). We also assessed intracellular RANTES mRNA
and protein levels in mouse cultured glial cells. Northern blot
analysis showed that RANTES mRNA levels were nearly undetectable in
untreated cultures but increased substantially after a 15 hr treatment
with TNF- and IFN-. This increase was markedly reduced by
L-AP-4 (100 µM) or PPG
(100 µM), but was minimally affected by
2R,4R-APDC (100 µM) or DHPG (100 µM)
(Fig. 2A,B). Similarly,
L-AP-4 reduced the cytokine-induced increase in
RANTES protein levels, and its action was attenuated in cultures
pretreated with pertussis toxin (PTX) (Fig.
3).
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Figure 1.
Extracellular RANTES levels in mouse cultured
astrocytes exposed to TNF- and IFN- in the absence or presence of
mGlu receptor ligands. A, Concentration-dependent effect
of L-AP-4 on the increase in extracellular RANTES induced
by TNF- and IFN- in cultured cortical astrocytes prepared from
wild-type or knock-out mouse cortical astrocytes. Addition of TNF-
and IFN- increased extracellular RANTES levels from 0.15 ± 0.02 to 6.7 ± 0.81 ng/mg protein in wild-type cultures and from
0.08 ± 0.04 to 5.9 ± 0.63 ng/mg protein in mGlu4(/)
cultures. Values were calculated as a percentage of cytokine-stimulated
RANTES release and represent the means ± SEM of six
determinations. *p < 0.05 (one-way ANOVA + Fisher's PLSD) as compared with TNF- and IFN- alone.
B, Effect of different mGlu receptor ligands on the
increase in extracellular RANTES induced by TNF- and IFN- in
mouse cultured astrocytes. Values were calculated from 6-12
determinations from at least three individual multiplates. All mGlu
receptor ligands were added at concentrations of 100 µM
with the exception of L-SOP (300 µM).
*p < 0.05 (one-way ANOVA and Fisher's PLSD) as
compared with values obtained in the absence of mGlu receptor ligands
().
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Figure 2.
RANTES mRNA levels in mouse cultured astrocytes
incubated for 15 hr with TNF- and IFN- in the absence or presence
of different mGlu receptor agonists. A, Representative
Northern Blot of RANTES mRNA in mouse cultures of astrocytes. In the
first lane, the spleen is shown as a positive control. Note that RANTES
mRNA levels were nearly undetectable in untreated cultures (), but
increased substantially after a 15 hr treatment with TNF- and
IFN-. This increase was markedly reduced by L-AP-4 or
PPG (both at 100 µM). B, Densitometric
analysis of RANTES mRNA levels in cultured astrocytes incubated for 15 hr with TNF- and IFN- in the absence or presence of 4C3HPG,
2R,4R-APDC
(APDC), L-AP-4, or PPG (all at 100 µM). Values were normalized by the amount of -actin
mRNA and represent the means ± SEM of three determinations.
*p < 0.05 (one-way ANOVA plus Fisher's
PLSD) versus cultures treated with TNF- and IFN- alone
().
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Figure 3.
Western blot analysis of RANTES in mouse cultured
astrocytes incubated for 15 hr with TNF- and IFN- in the absence
or presence of L-AP-4 (100 µM) and/or PTX
(0.5 µg/ml, preincubated for 16 hr before the incubation with the
cytokines). Note that PTX abolished the reduction in RANTES levels
induced by L-AP-4.
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Finally, we measured the ability of L-AP-4 to reduce
cytokine-stimulated RANTES production in cultured glial cells prepared from knock-out mice lacking mGlu4 receptors. In mGlu4(/) cultures a
15-hr treatment with TNF- + IFN- increases the extracellular levels of RANTES to the same extent as in cultures from wild-type mice.
L-AP-4 could still reduce cytokine-stimulated RANTES
release in mGlu4(/) cultures, but only at concentrations of 300 µM (Fig. 1A).
Detection of mGlu4 receptors in cultured glial cells
In immunoblots, mGlu4 antibodies labeled a major band at ~100
kDa, which corresponds to the receptor monomer. This band was detected
in cultured glial cells from wild-type mice or Lewis rats, but not in
cultures from mGlu4(/) mice (Fig.
4A). The presence of
mGlu4 receptors in cultured glial cells was confirmed by RT-PCR
analysis (Fig. 4B).
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Figure 4.
Expression of mGlu4a receptor in cultured
astrocytes. A, Western blot analysis of mGlu4a receptor
in cultured astrocytes from wild-type mice, mGlu4(/) mice, or Lewis
rats. Expression in the cerebral cortex of wild-type mice is shown as a
positive control. The antibody recognized a specific band corresponding
to the monomeric form of receptor at 100 kDa (arrow).
B, RT-PCR analysis of mGlu4a receptor mRNA in cultured
astrocytes from wild-type mice or Lewis rats. () and (+) refer to the
absence or presence of reverse transcriptase. Mouse cerebral cortex is
shown as reference tissue. NC represents a negative control in which
dH2O has been loaded instead of cDNA. Size markers
(M) are on the last lane on the
right.
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Effect of L-AP-4 on the development of EAE in
Lewis rats
To assess whether the modulation of RANTES formation in glial
cells has any functional relevance, we examined the effect of systemically injected L-AP-4 in the EAE model of
neuroinflammation in Lewis rats. Immunization of Lewis rats with guinea
pig MBP induced the classical symptoms of EAE after a latency of
10 d, as assessed by the disability scale and by the loss of body
weight. In standard experiments, the course of EAE was biphasic,
showing a peak at 11-13 d after immunization, followed by a drop in
the score during days 14-18 and by a plateau phase with scores of 1-1.5 lasting until day 23. No relapses were observed after full recovery of EAE. Figure 5 shows the
temporal profile of EAE in control rats and in rats implanted with a
subcutaneous osmotic minipump releasing 250 nl/d of a PBS solution
containing 250 mM of L-AP-4. The pump was
implanted 2 d before immunization. Detectable amounts of
L-AP-4 (105 ± 37 nM; n = 3) were detected in the striatal dialysate of freely moving rats,
suggesting that some amounts of subcutaneously infused
L-AP-4 can penetrate the brain. No gross abnormalities in motor behavior were observed in nonimmunized rats
treated with L-AP-4 or in immunized rats infused
with L-AP-4 before the clinical onset of EAE, as
detected by measuring locomotor activity in an open field apparatus
(data not shown). In rats treated with L-AP-4
there was no difference in the time at onset and in the peak of the
disability score of EAE, as compared with control rats implanted with a
minipump containing PBS alone, although L-AP-4-treated rats reached the peak of the
disability score 1 d later than control rats (i.e., 12 instead of
11 d after immunization). However, rats treated with
L-AP-4 showed a faster recovery rate from EAE, as
indicated by the absence of the plateau phase, and showed a full
recovery after 19 d (vs 24 or 25 d in control rats) (Fig.
5A,B). Independent groups of animals were used for
histological examination. Neuroinflammation was assessed by
immunohistochemical analysis of MHC-II+
and CD4+ cells in the spinal cord
(MHC-II+ cells in the spinal cord of
immunized animals are shown in the lower panel of Fig.
6A). We adopted a
semiquantitative scale with scores from 0 to 4 referred to the
intensity of staining, number of immunopositive cells within identified
clusters of cells, and distribution of clusters of immunopositive cells
(maximal score = 12; modified from Di Marco et al., 2001). Animals
were examined at day 11 or 27 after immunization. A substantial
increase in the neuroinflammation score for both MHC-II and CD4 was
observed in the spinal cord of immunized animals after 11 d, i.e.,
at the time of the peak of motor symptoms. The score was only slightly reduced at 27 d, when animals were apparently asymptomatic (Fig. 6B-D). Immunized rats that received
L-AP-4 subcutaneously had a lower disability
score at day 11 (Fig. 6B) and a lower number of
MCH-II+ cells in the spinal cord (Fig.
6C). The MCH-II score showed a trend to a reduction also at
27 d in immunized animals treated with
L-AP-4 (Fig. 6C). This trend with
L-AP-4 was also observed by scoring CD4
immunoreactivity at 11 and 27 d after immunization (Fig.
6D). In other animals we measured RANTES mRNA levels
in the cerebellum and spinal cord at the time of the peak of the disability score. Northern blot analysis showed that treatment with
L-AP-4 reduced RANTES mRNA levels in the
cerebellum and spinal cord of EAE rats (Fig.
7). Because systemically injected
L-AP-4 gave only a partial protection against
EAE, we decided to examine whether the drug had any effect on the
production of RANTES by other cells that contribute to the
pathophysiology of EAE, such as leukocytes. Experiments were performed
on human purified protein derivative-derived Th1 clones,
CD8+ leukocytes, and monocytes, in which
the production and release of RANTES was stimulated by PMA (0.1-10
ng/ml). Addition of L-AP-4 (0.1-1000
µM) had no effect on extracellular RANTES in
Th1 clones and showed a trend to reduction of extracellular RANTES in
CD8+ cells and monocytes. However, this
trend was observed only with high concentrations of
L-AP-4 (100 or 1000 µM)
and was not statistically significant (Fig.
8A-C).
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Figure 5.
Effect of L-AP-4 on the development of
EAE in Lewis rats. A, Temporal profile of body weight in
animals immunized with MBP implanted with an osmotic minipump, which
released 250 nl/d of PBS or a PBS solution containing 250 mM of L-AP-4. B, Temporal
profile of the disability score of EAE. Note that rats treated with
L-AP-4 showed a faster recovery rate from EAE than control
rats. Values express the means ± SEM of 10 animals per group.
*p < 0.05 (one-way ANOVA and Fisher's PLSD)
versus PBS-treated animals.
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Figure 6.
Immunohistochemistry of MHC-II+
and CD4+ cells in the spinal cord of nonimmunized or
immunized Lewis rats subcutaneously infused with L-AP-4. An
example of MHC-II immunostaining in the spinal cord of nonimmunized
(top panel) and immunized (bottom
panel) rats is shown in A. The disability
score of immunized animals killed at 11 or 27 d after MBP
injection and used for immunohistochemical analysis is shown in
B (mean + SEM of three to five determinations;
*p < 0.05; Student's t test vs the
respective group of rats subcutaneously infused with saline).
Quantification of MHC-II and CD4 immunostaining in nonimmunized
(untreated) or immunized (MBP) rats
subcutaneously infused with PBS or L-AP-4 is shown in
C and D, respectively. Immunized rats
were examined at day 11 or 27 after injection of MBP. Untreated rats
were examined 13 d after implantation of osmotic minipumps
releasing either saline or L-AP-4. Values are means + SEM
of three to five determinations; *p < 0.05 (one-way ANOVA and Fisher's PLSD, as compared with the respective
group implanted with minipumps releasing saline).
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Figure 7.
Expression of RANTES mRNA levels in the
cerebellum and spinal cord of EAE rats subcutaneously infused with PBS
or L-AP-4. A, Densitometric analysis of
RANTES mRNA levels in the cerebellum and spinal cord is shown in
A, and B, respectively. Values were
normalized by the amount of -actin mRNA and express the means ± SEM of three determinations. *p < 0.05 (one-way
ANOVA and Fisher's PLSD) versus treated animals. Subcutaneous infusion
of L-AP-4 in nonimmunized rats did not induce changes in
RANTES mRNA levels in the cerebellum and spinal cord (93 + 7.8% of
controls in the cerebellum and 137 + 28% of controls in the spinal
cord, 13 d after implantations of osmotic minipumps;
n = 4).
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Figure 8.
Effect of L-AP-4 on RANTES release in
Th1 clones (A), CD8+ leukocytes
(B), or monocytes (C), stimulated for 48 hr with 0.1 ng/ml of PMA. Values are means ± SEM.
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DISCUSSION |
RANTES is a chemokine that is gaining more and more interest
for its role in neuroinflammation. Similar to monocyte chemoattractant protein-1 (MCP-1) and interferon--inducible protein-10 (IP-10), RANTES is chemoattractant for monocytes, and contributes to the pathophysiology of immune disorders, including MS (Ransohoff, 1999).
RANTES can also attract memory T and NK cells, which are involved in
MS, and act as an antigen-independent activator of T cells in
vitro (Schall et al., 1990; Bacon et al., 1995; Taub et al.,
1995). In EAE, RANTES amplifies the inflammatory process, and its
expression correlates with the intensity of neuroinflammation (Glabinski et al., 1998; Ransohoff, 1999). In addition, RANTES has been
associated with the early formation of plaques (Simpson et al., 1998),
and the RANTES receptor CCR5 has been found in lymphocytes and
phagocytes of actively demyelinating lesions in MS (Bacon and
Oppenheim, 1998; Sorensen et al., 1999). Interestingly, the production
of RANTES by peripheral mononuclear cells is reduced after treating MS
patients with IFN--1b (Iarlori et al., 2000; Zang et al., 2001),
suggesting that the modulation of RANTES production may be a valuable
target in the pharmacological treatment of MS patients. We examined the
possibility that the production of RANTES could be modulated by the
activation of glial membrane receptors. In particular, we focused on
mGlu receptors, which are G-protein-coupled receptors activated by
glutamate and other excitatory amino acids. mGlu receptors form a
family of eight subtypes, which are subdivided into three groups on the
basis of their sequence homology, pharmacological profile, and
transduction pathways. Group I includes mGlu1 and -5 receptors, which
are coupled to inositol phospholipid hydrolysis and activated by DHPG.
Group II includes mGlu2 and -3 receptors, which are coupled to
Gi-proteins and activated by
2R,4R-APDC. 4C3HPG behaves as a
mixed mGlu2/3 agonist/mGlu1 antagonist. Group III includes mGlu4, -6, -7, and -8 receptors, which are also coupled to
Gi-proteins and selectively activated by
L-AP-4, PPG, and L-SOP (Pin
and Duvoisin, 1995; Schoepp et al., 1999). Whereas mGlu3 and -5 receptors are expressed by astrocytes (Miller et al., 1995; Petralia et
al., 1996; Ciccarelli et al., 1999), evidence for the expression of
group III mGlu receptors in glial cells is still lacking. Hence, we
used primary cultures of glial cells, expecting that mGlu3 or -5 receptor agonists could modulate the production of RANTES.
Unexpectedly, however, only group III mGlu receptor agonists (i.e.,
L-AP-4, PPG, or L-SOP)
reduced RANTES levels in cultures stimulated with TNF- and IFN-.
L-AP-4, the prototypic agonist of group III mGlu
receptors, produced the more substantial and reproducible effect on
RANTES levels, and its action was sensitive to the group III mGlu
receptor antagonist MSOP (Schoepp et al., 1999) and to PTX, which
inhibits the activity of Gi-proteins. The
reduction of RANTES mRNA levels by L-AP-4 or PPG
suggested that these drugs act at transcriptional level or decrease the stability of RANTES mRNA. The calculated EC50
value for L-AP-4 is consistent with the
activation of mGlu4 or -8 rather than mGlu7 receptors, which can be
recruited only by concentrations of L-AP-4 >100
µM (Schoepp et al., 1999). At least mGlu4
receptors appeared to be expressed by cultured glial cells. It is
unlikely that expression of mGlu4 receptors derived from contaminating
microglia, because only 6-10 microglial cells per well were detected
with a method that was highly efficacious in detecting microglia in the
inflammatory infiltrate of EAE rats (data not shown). In cultures from
mGlu4 knock-out mice, only concentrations of
L-AP-4 > 100 µM
were still able to reduce the production of RANTES. This indicates that
glial mGlu4 receptors primarily contribute to the regulation of RANTES formation in astrocytes. Whether the effect produced by 300 µM L-AP-4 reflects the
recruitment of mGlu7 receptors or rather represents a nonspecific
effect of the drug remains to be determined. Moving from the in
vitro data, we decided to examine the effect of
L-AP-4 on EAE in Lewis rats. Because EAE develops
gradually after a latency period of ~10 d, we continuously infused
L-AP-4 by means of a subcutaneous osmotic
minipump. We used this strategy because the increased permeability of
the blood-brain barrier during the development of EAE (Perry et al.,
1997) could allow a sufficient penetration of
L-AP-4 into the brain. To assess whether this
assumption was correct, we measured extracellular brain levels of
L-AP-4 by in vivo microdialysis. The
amount of L-AP-4 found in the dialysate (~100
nM) suggests that concentrations of
L-AP-4 in the range of 0.1-1
µM should be present in the extracellular
space. These concentrations may be sufficient to activate mGlu4
receptors (Schoepp et al., 1999). Infusion of
L-AP-4 neither delayed the time at onset nor
reduced the severity of EAE symptoms, although treated animals reached
the peak of the disability score 1 d later. Interestingly, however, treatment with L-AP-4 increased the rate
of recovery from EAE. The second phase of motor disability, which was
present in animals infused with PBS from day 18 to day 23 after
immunization, was not observed in animals that received
L-AP-4. Because EAE in Lewis rats represents a
model of neuroinflammation, but not of demyelination, we suggest that
L-AP-4 infusion reduces the inflammatory
infiltrate in the brain parenchyma to an extent that does not prevent
the development of EAE but allows a faster recovery from the disease
(Miyagishi et al., 1997). Accordingly, immunized animals treated with
L-AP-4 showed a reduced immunostaining for MHC
class II in the spinal cord and a trend to a reduction in CD4
immunostaining in the spinal cord at day 11 after immunization. The
partial reduction of RANTES mRNA levels produced by
L-AP-4 infusion in the cerebellum and spinal cord
of EAE rats might account for the 1 d delay in reaching the peak
of the disability score, the reduced extent of neuroinflammation, and
the lack of the delayed plateau phase of the disability score. The
possibility that the delayed plateau phase is sustained by the release
of RANTES from glial cells is interesting and deserves further
investigation. The lack of a more robust effect of
L-AP-4 on EAE may reflect the inability of the
drug to reduce the production of RANTES from other cells that directly
contribute to the inflammatory infiltrate, such as leukocytes.
Accordingly, L-AP-4 did not reduce the release of
RANTES stimulated by phorbol esters in lymphocytes or monocytes, although a trend to a reduction with high concentrations of
L-AP-4 was seen in
CD8+ lymphocytes and in monocytes.
In conclusion, present results offer the first demonstration that group
III mGlu receptors are expressed and functional in glial cells. The
unexpected finding that activation of these receptors reduces the
production of RANTES in astrocytes suggests that brain-permeable agonists may be useful in the experimental treatment of
neuroinflammatory disorders of the CNS. The potential usefulness of
group III mGlu receptor agonists in human pathology awaits the
demonstration that these drugs show good safety and tolerability when
systemically injected.
| |
FOOTNOTES |
Received Nov. 12, 2002; revised April 4, 2002; accepted April 11, 2002.
*
G.B., G.B., and. M.D. contributed equally to this work.
Correspondence should be addressed to Dr. Ferdinando Nicoletti,
Department of Human Physiology and Pharmacology, University of Rome
"La Sapienza", Piazzale Aldo Moro, 5, 00185 Rome, Italy. E-mail:
nicoletti{at}neuromed.it.
| |
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ABSTRACT
INTRODUCTION
