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The Journal of Neuroscience, May 1, 1999, 19(9):3440-3447
Fc RII/CD23 Is Expressed in Parkinson's Disease and
Induces, In Vitro, Production of Nitric Oxide and Tumor
Necrosis Factor- in Glial Cells
Stéphane
Hunot1,
Nathalie
Dugas2,
Baptiste
Faucheux1,
Andreas
Hartmann1,
Marc
Tardieu2,
Patrice
Debré3,
Yves
Agid1,
Bernard
Dugas3, 4, and
Etienne C.
Hirsch1
1 Institut National de la Santé et de la
Recherche Médicale, Unité 289, Mécanismes et
Conséquences de la Mort Neuronale, Hôpital de la
Salpêtrière, F-75013 Paris, France,
2 Laboratoire "Virus, Neurone et Immunité,"
Unité de Formation et de Recherche Kremlin Bicêtre, F-94276
Le Kremlin-Bicêtre Cedex, France, 3 Centre National
de la Recherche Scientifique, Unité de Recherche Associée
625, Laboratoire d'Immunologie Cellulaire et Tissulaire,
Hôpital de la Salpêtrière, F-75013 Paris, France,
and 4 Oxykine Therapeutics, F-75116 Paris, France
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ABSTRACT |
Oxidative stress is thought to be involved in the mechanism of
nerve cell death in Parkinson's disease (PD). Among several toxic
oxidative species, nitric oxide (NO) has been proposed as a key element
on the basis of the increased density of glial cells expressing
inducible nitric oxide synthase (iNOS) in the substantia nigra (SN) of
patients with PD. However, the mechanism of iNOS induction in the CNS
is poorly understood, especially under pathological conditions. Because
cytokines and Fc RII/CD23 antigen have been implicated in the
induction of iNOS in the immune system, we investigated their role in
glial cells in vitro and in the SN of patients with PD
and matched control subjects. We show that, in vitro,
interferon- (IFN-) together with interleukin-1 (Il-1) and
tumor necrosis factor- (TNF-) can induce the expression of CD23
in glial cells. Ligation of CD23 with specific antibodies resulted in
the induction of iNOS and the subsequent release of NO. The activation
of CD23 also led to an upregulation of TNF- production, which was
dependent on NO release. In the SN of PD patients, a significant
increase in the density of glial cells expressing TNF-, Il-1, and
IFN- was observed. Furthermore, although CD23 was not detectable in the SN of control subjects, it was found in both astroglial and microglial cells in parkinsonian patients. Altogether, these data demonstrate the existence of a cytokine/CD23-dependent activation pathway of iNOS and of proinflammatory mediators in glial cells and
their involvement in the pathophysiology of PD.
Key words:
inflammation; iNOS; astrocytes; microglial cells; neurodegenerative disease; postmortem
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INTRODUCTION |
The cardinal neuropathological
characteristic of Parkinson's disease (PD) is a massive loss of
dopaminergic neurons in the substantia nigra (SN). Although the
mechanism by which these neurons degenerate is still unknown, extensive
postmortem studies have provided evidence to suggest that oxidative
stress is a key component of the pathogenesis of PD (Hirsch, 1993 ;
Jenner, 1998). However, the nature and origin of the free radicals
involved in this oxidative stress are not fully known. In line with
this, several reports have suggested a deleterious role of the nitric
oxide (NO) radical in animal models of the disease in which
degeneration of nigral dopaminergic neurons was induced by
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Hantraye et al., 1996;
Przedborski et al., 1996; Matthews et al., 1997). Whether NO may also
participate in oxidative stress and nerve cell death in PD remains to
be determined. Experimental evidence suggests that this may indeed be
the case: (1) the concentration of nitrites is increased in the
cerebrospinal fluid of patients with PD (Qureshi et al., 1995); (2) the
density of glial cells expressing inducible NO-synthase (iNOS/NOS-II)
is greatly increased in the SN of parkinsonian patients (Hunot et al.,
1996); and (3) 3-nitrotyrosine, an index of protein nitrosation induced
by the NO-derived oxidizing molecule peroxynitrite, has been detected in nigral dopaminergic neurons from parkinsonian patients (Good et al.,
1998). Taken together, these data support the possibility that
increased levels of NO, having reached a toxic threshold, may
participate in nerve cell death in PD. Nevertheless, the mechanism of
iNOS induction in PD remains to be elucidated.
Recent data indicate that functional iNOS expression can be obtained in
human macrophages after ligation of the cell surface antigen
FcRII/CD23 (Vouldoukis et al., 1995; Dugas et al., 1998). CD23 is a
low-affinity IgE receptor consisting of a 45 kDa transmembrane type-II
glycoprotein and is a member of the C-type lectine family (Delespesse
et al., 1991). It is expressed at the cell surface of various cell
types after stimulation by different cytokines (Dugas et al., 1995).
Conversely, it has been shown that the engagement of the CD23 molecule
also regulates the production of proinflammatory cytokines such as
tumor necrosis factor (TNF)- and interleukin-6 (Il-6) (Arock et al.,
1994; Mossalayi et al., 1994). Although activation of the NO pathway
through ligation of CD23 has now been described in human monocytes
(Mossalayi et al., 1994), keratinocytes (Bécherel et al., 1996),
and eosinophils (Arock et al., 1994), its role in the CNS has yet to be
analyzed. To determine whether such a mechanism could also be involved
in iNOS activation in the CNS, we first analyzed the implication of
various cytokines on CD23 expression and the subsequent induction of
iNOS in the 1321N1 astrocytoma cell line in vitro. Second,
because the density of both iNOS- and TNF--expressing cells has been
shown to be increased in the SN of parkinsonian patients (Boka et al.,
1994; Hunot et al., 1996), we also examined CD23 and cytokine-producing cells in postmortem samples of patients with PD and matched control subjects.
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MATERIALS AND METHODS |
In vitro experiments. 1321N1 astrocytoma cell
line was cultured at 2 × 106 cells/ml in
Iscove's modified Dulbecco's complete medium (Bioproduct, Gagny,
France) supplemented with 5% decomplemented fetal calf serum and
incubated at 37°C in an atmosphere of 5% CO2. The
culture medium was shown to be free of endotoxin, as assessed by the
limulus amoebocyte lysate assay (E-toxate, Sigma, St Louis, MO). The
cells were cultured for 48 hr in the presence or absence of various doses of recombinant human IFN- (Roussel Uclaf, Romainville, France)
from 1 to 1000 U/ml to induce the CD23 antigen, in the presence or
absence of 10 ng/ml of recombinant human TNF- (Immunogenex, Los
Angeles, CA) and/or 1 ng/ml of recombinant human IL-1 (Immunogenex). After this incubation period, the cells were analyzed by immunochemical staining for CD23 expression using a previously characterized (Rector
et al., 1985) monoclonal antibody (mAb) directed against the human CD23
antigen (135 mAb, IgG1 isotype; kindly provided by Dr. E. Kilchherr, Novartis, Basel, Switzerland). The percentage of
CD23-positive cells was evaluated on randomly distributed fields view.
This percentage was estimated by counting a total number of 200 cells
per slide. Nonspecific staining was determined using an isotype-matched
(IgG1) anti-CD19 mAb (Immunotech, Marseille, France)
as negative control. In our experimental conditions, the CD19 staining
never exceeded 2% of the cells.
To investigate the consequences of CD23 activation on TNF- and NO
production, cells were preincubated for 48 hr with IFN- (1000 U/ml)
to induce CD23 expression. They were then harvested from the culture
flasks by gentle scraping, resuspended at 2 × 106 cells/ml in fresh Iscove's modified Dulbecco's
medium supplemented with antibiotics and 5% decomplemented fetal calf
serum, and seeded in 6- or 12-well plastic culture plates. To trigger
CD23, the cells were then stimulated for 48 hr by a previously defined
optimal concentration of the anti-CD23 135 mAb (20 µg/ml)
(Paul-Eugène et al., 1995), or by anti-CD19 mAb or MOPC-21 mAb as
isotype-matched (IgG1) negative controls. Some
cultures were treated with the NOS inhibitor L-NAME (1 mM, Sigma) during anti-CD23 treatment. Cell-free
supernatants were then collected to determine TNF- and nitrite
contents. TNF- concentrations were measured using a specific
commercial ELISA kit (Medgenix, France). Nitrites
(NO2 ), the stable end-products of NO
degradation, were determined using the Griess reaction, as described
previously (Kolb et al., 1994). Briefly, 100 µl of the supernatant
were dispensed in 96-well microplates followed by the addition of 100 µl of a reactive solution composed of 1% sulfanilamide in 30%
acetic acid and 0.1% N-(1-naphtyl)ethylenediamine dihydrochloride in 60% acetic acid (1:1 v/v). A standard calibration curve was generated with sodium nitrite (Sigma) diluted in complete Iscove's medium. Optical density was measured at 540 and 622 nm using
an autoreader (Dynatech Laboratories, Alexandria, VA). Analysis of iNOS
mRNA was performed on 1321N1 astrocytoma cells by RT-PCR. Forty cycles
of amplification were performed, using the following specific primers: iNOS mRNA sense (5'-ATGCCAGATGGCAGCATCAGA-3', exon 8)
and iNOS mRNA antisense (5'-ACTTCCTCCAGGATGTTGTA-3', exon 11).
Hypoxanthine phosphoribosyltransferase (HPRT) mRNA sense (5'-TATGGACAGGACTGAACGTCTTGC-3') and HPRT mRNA antisense
(5'-GACACAAACATGATTCAAATCCCTGA-3') primers were used as controls. As a
positive control, additional RT-PCRs were performed on cDNA from
1321N1 astrocytoma cells treated by LPS and IFN-. The
PRC product was then sequenced, and a 99% homology was found with the
known sequence of the human iNOS. iNOS protein analysis was performed
by Western blot. Briefly, cell lysates prepared from culture cells were
loaded on an 8% SDS-PAGE gel under reducing conditions. Proteins were
then electroblotted onto a nitrocellulose membrane (Hybond ECL,
Amersham, Buckinghamshire, UK) and incubated with monoclonal anti-human
iNOS antiserum (kindly provided by Dr. R. K. Webber, R&D, Skokie,
IL; 1/1000 dilution) for 48 hr at 4°C. Immunoblots were developed by
enhanced chemiluminescence (Super Signal, Pierce, Rockford, IL). As
control, 1321N1 cells were stimulated for 48 hr with 10 µg/ml LPS
(Escherichia coli serotype 0111:B4, Sigma) in combination
with IFN- (500U/ml) and IL-1 (5 ng/ml).
Human brain tissue. The study was performed on autopsy
brainstem tissue from 12 control subjects (seven for the cytokine
analysis and five for the CD23 analysis) and 12 parkinsonian patients
(five for the cytokine analysis and seven for the CD23 analysis) who were well characterized clinically and neuropathologically. For each
experiment, PD patients and control subjects did not differ significantly in terms of their mean age at death and the mean interval
from death to freezing of tissue (Table
1). Autopsy striatum tissue from a
patient with Huntington's disease and two control subjects was used as
non-PD neurological control for CD23 analysis. Brainstem and striatum
tissue were dissected as described previously (Hunot et al., 1996;
Gourfinkel-An et al., 1997). Because we found in preliminary
experiments that CD23 and cytokine immunodetection were highly
sensitive to the fixation procedure, the tissue was fixed in 4%
paraformaldehyde and 15% picric acid from a saturated solution for
CD23 immunohistochemistry, and in 4% paraformaldehyde and 2.5%
glutaraldehyde for cytokine immunohistochemistry, as described
previously (Boka et al., 1994; Hunot et al., 1996).
Immunohistochemistry. Immunohistochemical labeling was
performed on free-floating 40-µm-thick sections of the mesencephalon including the SN pars compacta. Sections were incubated at 4°C for
48-96 hr under gentle agitation in the following primary antisera: anti-TNF (1:500; Sigma), anti-IFN- (1:1000; Genzyme Cambridge, MA), anti-IL-1 (1:250; Genzyme), and anti-CD23 (clone 135, 1-10 µg/ml). The primary antibodies were revealed using the avidin-biotin complex method (Vector Laboratories, Biosys, Compiègne, France) with appropriate secondary antibodies (Hunot et al., 1996) and developed using 0.04% (w/v) diaminobenzidine in 0.25 M
Tris buffer. To test the specificity of the antisera used for cytokine
detection, some sections were incubated with the primary antisera
preadsorbed for 6 hr at room temperature with a 2 × 104 excess of the corresponding antigen. Because a
sufficient amount of purified CD23 antigen was not available, the
specificity of the antibody was analyzed by Western blotting performed
on tissue homogenates of human SN, as described by Zhang et al. (1994). To analyze the molecular weight of the peptidic portion of CD23, some
homogenates were subjected to enzymatic deglycosylation using a
commercial kit (Bio-Rad, Ivry/Seine, France). Briefly, protein extracts
(100 µg) from parkinsonian substantia nigra were incubated with
NANase II (10 U/ml; 1:8 dilution) and O-glycosidase DS (1 U/ml; 1:8
dilution) for 1 hr at 37°C and then denatured for 5 min at 100°C in
denaturating solution (2% SDS and 1 M -mercaptoethanol; 1:15 dilution). After addition of 6.5% NP-40 (v/v), protein extracts were incubated with PNGase F (2.5 U/ml, 1:20 dilution) for 3 hr at
37°C.
Double-staining experiments were performed to determine the type of
glial cells expressing CD23, using glial fibrillary acidic protein
(GFAP) as a marker of astrocytes and ferritin as a marker of microglial
cells (Yoshioka et al., 1992). In brief, free-floating sections of the
mesencephalon were mounted on gelatin-double-coated slides and dried
for 2 hr at room temperature. After being washed in 0.1 M
PBS, tissue sections were successively incubated in 0.2% Triton X-100
for 5 min and 1:30 normal goat serum for 30 min. Sections were then
exposed simultaneously to the primary antibodies for 96 hr at 4°C
under gentle agitation in the following combinations: mouse anti-CD23
(135 mAb, 25 µg/ml) and rabbit anti-GFAP (1/100, Z0334; Dako,
Glostrup, Denmark); mouse anti-CD23 (25 µg/ml) and rabbit
anti-ferritin (1/1000; Sigma); mouse anti-GFAP (1/100; M0761, Dako) and
rabbit anti-ferritin (1/1000; Sigma). Sections were rinsed three times
in 0.1 M PBS containing 0.01% Triton X-100 (PBS-T),
incubated in biotinylated sheep anti-mouse IgG (1/50; Amersham) for 1 hr at room temperature, and revealed with fluorescein avidin (1/50;
Vector Laboratories) for 30 min at room temperature. Amplification of
this staining was obtained by incubating the sections in biotinylated
anti-avidin D antibodies (1/50; Vector Laboratories) for 30 min at room
temperature and fluorescein avidin DCS for 30 min, successively. After
extensive washing in PBS-T, the other primary antiserum was detected by
incubating tissue sections in biotinylated goat anti-rabbit IgG (1/50;
Vector Laboratories) for 1 hr and revealed with rhodamine 600-avidin D
(1/50; Vector Laboratories) for 30 min. Tissues were then washed in
PBS-T and mounted with Vectashield medium (Vector Laboratories).
Image and data analysis. The density of glial cells
expressing cytokines and CD23 in the SN pars compacta was determined on each stained section using a computer-based image analysis system (Biocom, Les Ulis, France). These densities were compared between control subjects and PD patients using a nonparametric statistical test
because distributions differed significantly from normality (Mann-Whitney U test; SigmaStat Statistical Software,
Jandel, St. Raphael, CA).
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RESULTS |
CD23 expression in cultured glial cell lines
Treatment of the 1321N1 astrocytoma cell line using IFN-
provoked a dose-dependent expression of CD23 reaching ~30% of the cells labeled for the maximal dose of IFN- used (1000 U/ml;
p < 0.01) (Fig. 1).
Co-incubation with IFN- and TNF- or Il-1 increased the
proportion of CD23-positive cells compared with cultures treated with
IFN- alone (Fig. 1). The extent of this additive effect of TNF-
and Il-1 on IFN--induced CD23 expression was comparable for both
cytokines. Furthermore, co-incubation with the three cytokines provoked
a further increase in the proportion of cells expressing CD23
(p < 0.05) (Fig. 1). By contrast, incubation with Il-1 or TNF- alone was unable to induce CD23 expression (data not shown).
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Figure 1.
Induction of CD23 expression in 1321N1 astrocytoma
cells by various cytokines. Cells were treated for 48 hr with various
doses of interferon- (IFN-) (1-1000 U/ml) alone
or in combination with interleukin-1 (Il-1) (1 ng/ml), and/or tumor necrosis factor (TNF-) (10 ng/ml). CD23-positive cells were detected by immunocytochemical
methods. Results are expressed as the mean percentage of CD23-positive
cells ± SEM in three independent experiments. *Significantly
different from cells with culture medium alone
(p < 0.01); **significantly different from
cells treated with IFN- alone (p < 0.05); two-tailed t test.
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TNF- and nitrite production by 1321N1 astrocytoma cells after
CD23 ligation
Stimulation of CD23 using the anti-CD23 135 mAb in cells
pretreated with IFN- provoked a 7.7- and 53-fold increase in
nitrites (Fig. 2A) and
TNF- (Fig. 2B) levels in the culture medium,
respectively (p < 0.01). No significant
increase in nitrites and TNF- production was observed when the cells
were treated with isotype-matched (IgG1) negative controls (anti-CD19
and anti-MOPC-21). However, a moderate level of nitrites was detectable
in the unstimulated and isotype-matched negative control
(IgG1)-stimulated cultures as a result of the treatment with IFN-
that was necessary to induce the expression of CD23. Furthermore, the
addition of a specific NOS inhibitor (L-NAME) to the
culture medium blocked the production of both nitrites
(p < 0.05) and TNF-
(p < 0.01) because of the ligation of CD23
(Fig. 2C). Finally, ligation of CD23-positive 1321N1 cells
obtained after IFN- treatment resulted in iNOS mRNA transcription
(Fig. 2D) and iNOS protein expression (Fig.
2E). iNOS mRNA transcription was not observed using
isotype-matched (IgG1) negative controls.
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Figure 2.
Ligation of CD23 induces production of nitrites
and TNF- in 1321N1 astrocytoma cells. After CD23 induction by
IFN- (1000 U/ml for 48 hr), cells were incubated for 48 hr with
10-20 µg/ml of anti-CD23 135 mAb to trigger the CD23 antigen, or
with an isotype-matched negative control IgG1. Nitrite
(A) and TNF- (B) levels
were determined in the supernatant 48 hr after the beginning of the
treatment. Some of the experiments were performed in the presence of 1 mM L-NAME (C). Results
are expressed as the mean ± SEM in three independent experiments.
*Significantly different compared with untreated cells
(p < 0.01, two-tailed t
test). Significantly different from cultures treated with anti-CD23 mAb
alone:  p < 0.05; p < 0.01, two-tailed t test. ND,
Nondetectable. D, After CD23 ligation in IFN--treated
1321N1 glial cells, total mRNA was extracted and assayed for iNOS mRNA
expression by RT-PCR. HPRT amplification was used as a control. As a
positive control some cells were treated with IFN- and LPS to induce
iNOS. E, Western blot analysis of treated cells using
mouse anti-human iNOS antiserum. In D and
E, Control indicates untreated cells,
IFN- indicates cells stimulated by IFN-, and
IFN-+anti-CD23mAb indicates cells
stimulated by IFN- and subsequently treated by a monoclonal antibody
raised against CD23 or an isotype-matched immunoglobulin
(IgG1) as a negative control.
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CD23 and cytokine expression in PD
On tissue sections, staining intensity decreased with lower
antibody concentrations, and no staining was observed when the primary
antisera were omitted and when preadsorption tests were performed with
a large excess of the homologous antigen for the antibodies directed
against the cytokines (data not shown). As a further test of the
specificity of the anti-CD23 antiserum, Western immunoblotting of
proteins extracted from the SN of parkinsonian patients showed several
bands with molecular weights ranging from 41 to 67 kDa with two major
bands at 45 and 43 kDa (Fig. 3), as expected from previous studies (Nakajima and Delespesse, 1986; Bonnefoy
et al., 1987; Yukawa et al., 1987). After enzymatic deglycosylation, the intensity of most of these bands was decreased, and a 36 kDa band
appeared, corresponding to the molecular weight deduced from the
peptidic sequence (Ludin et al., 1987) (Fig. 3).
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Figure 3.
Western blot of CD23 from SN of a patient with PD
after SDS-PAGE, blotting on nitrocellulose membranes, and
immunodetection with 135 mAb against CD23. After revelation by enhanced
chemiluminescence, two major bands at 45 and 43 kDa were observed
together with minor bands ranging from 41 to 67 kDa (lane
1). Treatment of tissue homogenate by enzymatic deglycosylation
revealed a new band at 36 kDa, corresponding to the molecular weight
deduced from the peptidic sequence of CD23 (lane
2).
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Cytokines were observed exclusively in glial cells in the human SN
(Fig.
4A-C). For
the three cytokines analyzed, the labeled perikarya were round, with a
diameter ranging between 8 and 10 µm. They displayed numerous thin
immunostained processes, except for IFN--stained sections, in which
the processes were hardly visible. These cells were located throughout
the parenchyma of the SN and often in the vicinity of blood vessels.
Their density was very low in the SN of control subjects and
significantly higher in the SN of patients with PD (IFN-,
p < 0.01; Il-1, p < 0.05; TNF-,
p < 0.01) (Fig.
5A).
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Figure 4.
Immunohistochemical detection of pro-inflammatory
mediators in sections of the parkinsonian SN. Sections were
immunostained to reveal TNF- (A), IL-1
(B), IFN- (C), and CD23
(D). Arrows, Immunostained glial
cells; arrowhead, melanized dopaminergic neuron. Scale
bar, 20 µm.
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Figure 5.
Density of glial cells expressing TNF-,
Il-1, and IFN- (A) and CD23
(B) in the SN of patients with PD and matched
control subjects. Each value represents the mean density of
immunostained cells ±SEM. Significant differences between parkinsonian
patients and control subjects according to Mann-Whitney
U test: *p < 0.05;
**p < 0.01. ND,
Nondetectable.
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CD23 immunoreactivity was observed in glial cells with variable size
(diameter ranging between 8 and 15 µm) in the SN of patients with PD
(Fig. 4D). Their distribution was extremely variable
within the SN and, in some patients, mostly concentrated in the
vicinity of blood vessels. Some of these cells displayed numerous long immunostained processes reminiscent of astrocytes. Double-staining experiments confirmed the heterogeneous populations of the CD23-labeled cells (Fig. 6). Indeed, although
colocalization of GFAP and ferritin was never observed (Fig.
6A,B), confirming the specificity
of the markers used, colocalization of CD23 with both GFAP and ferritin was observed (Fig. 6C-F). Yet, at simple
visual inspection, the proportion of astrocytes (GFAP-positive cells)
expressing CD23 was higher than that of microglial cells
(ferritin-positive cells). The quantitative analysis of CD23-positive
cells confirmed their absence in the SN of control subjects and their
presence in that of patients with PD (p = 0.001)
(Fig. 5B). Finally, numerous glial cells were found to
weakly express CD23 in the striatum (putamen and caudate nucleus) of a
patient with Huntington's disease but not of control subjects.
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Figure 6.
Double-immunofluorescence staining of CD23 and
glial markers in the SN of patients with PD. GFAP and ferritin were
used as markers of astrocytes and microglial cells, respectively.
Positive astrocytes are indicated by arrows in
A, and microglial cells are indicated by
arrowheads in B. On sections stained
simultaneously for GFAP (A) and ferritin
(B), no cells were stained for both markers. On
double-stained sections, CD23 (D,
F) was detected in astrocytes
(C) and some microglial cells
(E). Microglial cells expressing CD23 are
indicated by an arrowhead in E and
F. Scale bar, 20 µm.
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DISCUSSION |
The major finding of our study is the implication of a
cytokine/CD23-dependent activation pathway of iNOS and of
pro-inflammatory mediators in human glial cells and their possible role
in the pathophysiology of PD. This pathway has already been described for cells of the immune system (Arock et al., 1994; Mossalayi et al.,
1994; Vouldoukis et al., 1995) and keratinocytes (Bécherel et
al., 1996) in human and rat macrophages (Alonso et al., 1995). Human
CD23 is expressed as two isoforms, a and b, that differ in their
cytoplasmic N-terminal tail (Delespesse et al., 1991). The a isoform is
expressed constitutively in normal B lymphocytes, whereas the b isoform
is expressed after appropriate activation in several other cell types
(Dugas et al., 1995). Our results show that the CD23b isoform can also
be expressed by glial cells of the CNS. Similar to human
monocytes/macrophages, the expression of CD23b in glial cells requires
the activation of these cells by various cytokines. In our cell line,
IFN- was shown to be sufficient to induce CD23 expression. However,
an additive effect was obtained by the addition of other cytokines, as
already shown for other cell types (Bieber et al., 1989), indicating a
potentiation effect of both TNF- and Il-1 on IFN--mediated
CD23 expression. However, neither TNF- nor Il-1, used alone, had
an effect, suggesting that they represent activators of the
IFN--mediated expression of CD23 rather than direct inducers of CD23
expression. Nevertheless, the precise mechanism by which IFN-
induces CD23 expression remains to be identified.
CD23 appears to mediate various biological activities, including
cell-cell adhesion, B-cell survival in germinal centers, histamine
release from basophils, and regulation of IgE synthesis (Bonnefoy et
al., 1993). Our in vitro experiments suggest that it can
induce NO production in the CNS. Indeed, ligation of CD23 antigen in
our astrocytic cell line induces the release of nitrites in the culture
supernatant (Fig. 2A). This production of nitrites is
abolished by addition in the culture medium of L-NAME, an
inhibitor of NOS, suggesting that the engagement of CD23 at the surface of astrocytes leads to the induction of iNOS, as shown by RT-PCR mRNA
and protein immunoblot analysis. These data are compatible with a
previous demonstration of iNOS induction after activation of CD23 in
human macrophages (Vouldoukis et al., 1995). Furthermore, in the later
system, NO production has been shown to be particularly high and thus
capable of inducing the death of the parasite Leischmania major (Vouldoukis et al., 1995). If these data could be
extrapolated to our system, they would suggest that CD23-induced NO
production by astrocytes in the CNS could play a deleterious role for
surrounding cells, especially nigral dopaminergic neurons, the relevant
cells in PD.
Another issue raised by our in vitro data is that the
engagement of CD23 is capable not only of inducing iNOS expression but also of stimulating the production of pro-inflammatory cytokines such
as TNF- (Fig. 2B). A CD23-induced production of
TNF- has already been reported in human eosinophils (Arock et al.,
1994) and purified monocytes, in which a concomitant production of Il-6 has also been shown (Mossalayi et al., 1994). In addition, NO release
seems to play a major role in CD23-induced TNF- production, because
it is inhibited by the NOS inhibitor L-NAME (Fig.
2C). Because TNF- has also been shown to induce iNOS in
glial cells and to stimulate the expression of CD23 by IFN-, our
results suggest a mutual potentiation of the pro-inflammatory reaction in glial cells by these molecules. Taken as a whole, our data indicate
that CD23 may represent a possible regulator of the inflammation mediated by glial cells in the CNS, as proposed for immune cells (Fig.
7).
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Figure 7.
Schematic representation of CD23-mediated
iNOS induction and TNF- production pathway in glial cells.
Pro-inflammatory cytokines, such as IFN-, induce the expression of
CD23 in glial cells. This effect is potentiated by TNF- and IL-1.
Triggering of CD23 antigen by an appropriate ligand results in iNOS
induction and the subsequent release of NO, which in turn upregulates
the production of TNF-. The NOS inhibitor L-NAME
completely inhibits the production of both NO and TNF- in
vitro. This pathways results in an auto-amplification of NO
production, which could reach a toxic threshold. NO toxicity would in
part involve the formation of peroxynitrite, which has been shown
to initiate lipid peroxidation and protein oxidation and inactivation.
All of these events could contribute to the death of dopaminergic
neurons in PD.
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Given that the density of iNOS- and TNF--containing glial cells is
increased in the SN of patients with PD, one may speculate that such a
pro-inflammatory pathway is also activated in this disease. This
hypothesis cannot be tested directly on the basis of experiments
performed on postmortem human brain. However, interestingly, glial
cells expressing CD23 were detected in the SN of patients with PD but
not of control subjects, suggesting that CD23 may also play a role in
the pathophysiology of this disorder. Double-staining experiments with
specific markers indicated that CD23 is expressed by both astrocytes
and microglial cells, suggesting that both cell types may participate
in the inflammatory process during the progression of PD. The
expression of CD23 by microglial cells is in agreement with in
vitro studies using human embryonic microglial cells, in which
Il-4 stimulation has been shown to induce CD23 (N. Dugas, unpublished
results). The mechanism of CD23 induction in PD is not known.
Nevertheless, the in vitro experiments suggest that the
pro-inflammatory cytokines may represent potential candidates. This
concept is supported by the huge increase in the density of IFN--,
Il-1-, and TNF--expressing glial cells in the SN of patients with
PD (Fig. 5A). From a more general point of view, these data
suggest a role for glial cells in the pathophysiology of PD. In line
with this, McGeer et al. (1988) reported an increased expression of
HLA-DR-positive glial cells in the SN of parkinsonian patients, which
may be related to the fact that HLA-DR and CD23 antigens have been
shown to be spatially associated on B lymphocyte cell membrane and to
be regulated in a coordinated manner. (Bonnefoy et al., 1988; Rousset
et al., 1988; Flores-Romo et al., 1990).
The increased density of cytokine-producing glial cells in the SN of
patients with PD may have several implications for the pathophysiology
of the disease. Although the possibility that these cytokines have a
neuroprotective effect cannot be excluded (Maeda et al., 1994; Barger
et al., 1995), it seems very likely that, when chronically produced in
high amounts, they play a deleterious role (for review, see Campbell,
1998). This concept is supported by the fact that anti-inflammatory
agents have been shown to protect dopaminergic neurons against
degeneration in animal models of the disease (Matsuura et al., 1996;
Boireau et al., 1997). Such a deleterious effect may be mediated either
by a direct action involving receptors for the cytokines or by more
indirect mechanisms. Indeed, TNF- through interaction with its
receptor can activate a ceramide-dependent pro-apoptotic pathway;
evidence for such an activation has been reported in nigral
dopaminergic neurons from patients with PD (Hunot et al., 1997). The
cytokines may also play an indirectly deleterious role by inducing the
expression of iNOS in astrocytes and microglial cells, as shown
previously (Chao et al., 1996; Ding et al., 1997). Our results suggest
that CD23 participates in this induction and could positively regulate this activation, thus further increasing the production of NO, which
could then reach a toxic threshold for the surrounding neurons (Fig.
7). NO is thought to exert its toxic effect mainly by an interaction
with superoxide radicals, leading to the formation of peroxynitrite, a
highly oxidizing molecule (Beckman et al., 1990; Dawson and Snyder,
1994). The presence of nitrotyrosine in the SN of patients with PD, and
especially in Lewy bodies, supports this hypothesis (Good et al.,
1998). NO could also induce deleterious effects by releasing iron from
ferritin and by its action on iron metabolism through its interaction
with iron regulatory proteins (Reif and Simmons, 1990; Pantopoulos and
Hentze, 1995). This may explain in part some alterations in iron
metabolism observed in PD (Hirsch and Faucheux, 1998) and the
subsequent oxidative stress.
It seems highly likely that the increased production of inflammatory
mediators, and in particular of CD23, in PD is not specific to the
disease, because it has also been observed in Huntington's disease and
also in patients with Alzheimer's disease, multiple sclerosis, and HIV
encephalitis (N. Dugas and A. Calenda, unpublished results). However,
it probably represents a major step in the pathophysiology of PD that
could potentially perpetuate the pathological process. In this context,
interrupting the inflammatory reaction in this disease may represent a
therapeutic intervention that could reduce the progression of the
pathological process.
| |
FOOTNOTES |
Received Sept. 1, 1998; revised Feb. 16, 1999; accepted Feb. 19, 1999.
This study was supported by the Fondation pour la Recherche
Médicale (S.H.), Institut National de la Santé et de la
Recherche Médicale, the Association Claude Bernard pour le
Développement des Recherches Biologiques et Médicales dans
les Hôpitaux de l'Assistance Publique à Paris (B.A.F.),
and the National Parkinson Foundation (Miami, FL) (Y.A.). We thank Drs.
H. Beck, J. Y. Beinis, A. M. Bonnet, J. P. Bouchon, C. Duyckaerts, J. J. Hauw, M. Laurent, R. Moulias, F. Piette, A. Sachet, O. Saint Jean, and M. Verny for their clinical investigations
and cooperation in providing the brain specimens, and C. Betard for his
technical assistance.
Correspondence should be addressed to Dr. Etienne C. Hirsch, Institut
National de la Santé et de la Recherche Médicale U289, Hôpital de la Salpêtrière, 47 Boulevard de
l'Hôpital, F-75013 Paris, France.
| |
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ABSTRACT
INTRODUCTION
