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Institut National de la Santé et de la Recherche Médical,
*
Unité 25 and
Unité 345, Necker Hospital, Paris, France; and
Sanofi-Synthelabo, Bagneux, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and monocyte
chemoattractant protein-1, the prototypical pro-Th1 and pro-Th2 CC
chemokines, respectively. This chemokine imbalance was associated with
an immune deviation of the autoreactive response, with reduced IFN-
and increased IL-4 and TGF-
1 levels. Moreover, G-CSF limited the
production of TNF-, a cytokine also associated with early CNS
infiltration and neurological deficit. These findings support the
potential application of G-CSF in the treatment of human autoimmune
diseases such as multiple sclerosis, taking advantage of the wide
clinical favorable experience with this
molecule. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production
capacity (3). Recipients of G-CSF-mobilized
peripheral blood stem cells do not display higher incidence of
graft-vs-host disease than bone marrow recipients (4)
despite much larger numbers of T cells in unmanipulated stem cell
grafts. Murine studies have shown that G-CSF-induced protection from
acute graft-vs-host disease is associated with a shift toward Th2 of
the production of T cell-derived cytokines, with a reduction of
Th1-oriented cytokine production capacity, particularly IFN-
(5). A similar shift in cytokine production pattern has
been recently confirmed in human peripheral blood T cells
(6).
Here we address the hypothesis that these immunoregulatory properties
of G-CSF might be beneficial in autoimmune diseases. We investigated
the therapeutic potential of G-CSF in experimental autoimmune
encephalomyelitis
(EAE),2 a murine model
for the human demyelinating disease multiple sclerosis (MS). EAE
(7) is mediated by the activation of autoaggressive Th1
cells, which trigger an inflammatory reaction. Immunoprevention of EAE
has been hitherto achieved either by suppressing the production and/or
action of the neurological deficit-associated cytokines TNF- or
lymphotoxin- (8, 9, 10) or by eliciting regulatory Th2/Th3
cells that release anti-inflammatory cytokines, such as IL-4,
IL-10, and TGF- and impair activation of the Th1 pathogenic cells
(11, 12). More recently, therapeutic strategies have been
aimed at the neutralization of chemokines, based on reports showing
that expression of chemokines, particularly the CC chemokine macrophage
inflammatory protein-1 (MIP-1), and chemokine receptors in the
CNS correlated with EAE disease symptoms (13) and was
increased in patients with acute MS (14, 15). Treatments
of proteolipid protein- or myelin basic protein (MBP)-immunized
SJL/J mice with anti-MIP-1 was able to inhibit acute EAE
severity (16). Of particular relevance to the
role of chemokines in autoimmune settings is the fact that in
addition to driving leukocyteattraction and accumulation to tissue
sites of inflammation, chemokines play a regulatory role in T
cell proliferation, differentiation, and Th1/Th2 balance in cytokine
production (17, 18, 19). In vivo and in vitro studies have
identified MIP-1 and macrophage chemoattractant protein-1 (MCP-1) as
prototypical pro-Th1 and pro-Th2 chemokines, respectively
(20, 21, 22).
We now demonstrate that a short treatment with G-CSF at the onset of
clinical signs provides durable protection of SJL/J mice from EAE.
G-CSF reduces the T cell infiltration and autoimmune inflammation
within the CNS, an effect based on immunoregulatory events that take
place in the periphery and include an imbalance in the chemokine
(MIP-1/MCP-1) production ratio and an immune deviation toward Th2 of
the T cell autoreactive response together with a reduction of systemic
and lymphocyte TNF- production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice (8–10 wk of age, purchased from Elevage Janvier, Le Genest St. Isle, France) were immunized by s.c. injection at two sites at the tail base on day 0, and boosted on day 7 in the flanks, with 400 µg guinea pig MBP (Sigma, St. Louis, MO) emulsified in CFA containing 50 µg heat-inactivated Mycobacterium tuberculosis H37Ra (Sigma), in a volume of 50 µl/site. The disease developed with an acute phase characterized by loss of weight as well as progressive ascending clinical paralysis, followed by periods of remission with weight recovery, and subsequent relapses or chronic disease. Clinical symptoms were scored as follows: 0, no symptoms; 1, flaccid tail; 2, impairment of righting reflex or abnormal gait; 3, severe hind limb weakness; 4, complete hind limb paralysis; and 5, paraplegia, moribund. The mean clinical score was defined as the mean of all clinical scores in a group (including animals with no symptoms) at a given time point or as the mean of the mean clinical scores at all time points over the entire disease length, when specified. The mean disease incidence was also calculated as the mean of incidences at all disease time points.
G-CSF treatment
Recombinant human G-CSF (Amgen, Thousands Oaks, CA), which is active in mice (5), was injected s.c. at 200 µg/kg/day. Excipient consisted of 5% dextrose in sterile H2O.
Histology and immunohistochemistry
Mice were anesthetized with sodium pentobarbital and perfused intracardially through the left ventricle with ice-cold 10% formalin for OCT-embedded tissues. Five-micrometer sections of brain and spinal cord were stained with toluidine blue or Luxol Fast Blue to assess demyelination and with H&E to assess leukocyte infiltration. Histologic scores were determined on three mice per group on 20 sections per animal, using the following scale: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, >10 perivascular lesions per section with extensive parenchymal infiltration. Immunohistochemical staining was performed with Histomouse immunostaining kits (Zymed, South San Francisco, CA) according to the manufacturer’s instructions, except for the use of secondary Ab, which was a biotin-goat anti-rat IgG (H + L) (Zymed) used at 3.75 µg/ml in PBS buffer containing 1% BSA and 0.3% Triton X-100. The primary rat Abs used were anti-CD3 (clone KT3), anti-Mac-1/CD11b (clone M1/70), anti-Gr-1 (clone RA3-8C5), and anti-F4/80 (all from BD PharMingen, Le Pont de Claix, France).
TNF- concentration in serum
TNF- was measured in sera diluted 1/2 with commercial ELISA
from R&D Systems Europe (Abingdon, U.K.).
Assessment of T cell autoreactive response
Splenocytes were harvested on day 40 after MBP immunization.
Proliferation was assessed using 2 x 105
viable splenocytes/well in the presence of MBP (50 µg/ml, final
concentration) or PBS in RPMI Glutamax medium supplemented with 5% FBS
(Life Technologies, Cergy Pontoise, France), 1% penicillin and
streptomycin, and 5 x 10-5 M -ME in
96-well culture plates. T cell proliferative responses were quantified
at 96 h after a 14-h pulse with
[3H]thymidine. Supernatants for cytokine and
chemokine determinations were generated by 72-h culture in the
above-described conditions.
Cytokine and chemokine measurements
Cytokine and chemokine levels were determined by commercial
ELISAs for TNF-, MIP-1, and MCP-1 (R&D Systems
Europe) and by sandwich ELISA with mAbs prepared in our laboratory from
hybridoma or purchased from BD PharMingen for IFN- (R46A2 and
AN18), IL- 4 (11B11 and BVD6), and IL-10 (JES2.A5 and JES5-16E3) as
previously described (23).
Preparation of RNA
Total RNA was also extracted from PBS-perfused CNS spinal cords using RNAble (Eurobio, Les Ulis, France) following the manufacturer’s instructions. Splenocytes (4 x 106 cells/well in 24-well plates) were incubated with PBS or MBP (50 µg/ml) for 5 h, and total RNA was extracted with RNAble. Control RNA consisted of total RNA extracted from the spleen of BALB/c mice 60 min after i.v. injection of 5 µg anti-CD3 (clone 145 2C11) Ab in 150 µl PBS.
RNase protection assay (RPA)
mRNA levels for the cytokines, chemokines, and hemopoietic
factors were determined using multiprobe RPA III kit from Ambion
(Ambion, Austin, TX). [-32P]UTP-labeled
antisense RNA transcripts prepared from mCK5, mCK1b, mCK3b, or mCK4 DNA
templates (Riboquant, PharMingen, San Diego, CA) using MAXIscript in
vitro transcription kit (Ambion) were hybridized with total RNA (20
µg for spinal cords and 10–15 µg for splenocytes) overnight at
56°C according to the manufacturer’s instructions. Bands were
detected by phosphorimaging using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). Results were calculated as a ratio of the
volume of the band of interest to the mean of the volumes of the bands
for the housekeeping genes large ribosomal protein L32 and
GAPDH.
Macrophage isolation
Macrophages were isolated from the spleens of mice after 5 days of treatment with G-CSF or vehicle and were purified by 24-h adherence (98% pure as assessed by FACS analysis using Mac-1+ staining) in 24-well culture plates (1 x 106 cells/well). After incubation with anti-CD40 (5 µg/ml, clone 3/23, Valbiotech, Paris, France) or PBS for 24 h, supernatants were collected, and their contents of cytokines and chemokines were measured by ELISA. At the end of the incubation, cellular protein content was measured by the bicinchoninic acid colorimetric method (Pierce, Rockford, IL) in adherent cells disrupted in PBS/0.01% Tween after three freeze-thaw cycles.
FACS analysis
The cell surface phenotype of splenocytes was analyzed by flow
cytometry. All cells were incubated in cold PBS supplemented with 2%
FCS and 0.02% azide. One million cells per sample were preincubated in
20 µl for 15 min at room temperature under constant shaking with
anti-FcRIII/II (clone 2.4G2) to reduce nonspecific binding,
followed by mAbs (anti-CD4 (clone GK1.5), anti-CD8 (clone
53-6.7), anti-CD3 (clone 145-2C11), anti-TCR (clone
H57-597), anti-B220 (clone RA3-6B2), anti-CD19 (clone 1D3),
anti-Mac-1/CD11b (clone M1/70), anti-Gr-1 (clone RA3-8C5), and
anti-CD11c (clone HL3)). They were produced in our laboratory from
hybridomas or were purchased from PharMingen and used coupled either to
biotin and revealed by streptavidin-CyChrome (PharMingen) or to PE
or FITC.
Ten thousand events were acquired using the FACScan flow cytometer (BD Biosciences). Analysis of the acquired data was performed using CellQuest software (BD Biosciences).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EAE was actively induced in the genetically sensitive SJL/J mouse
by immunization on days 0 and 7 with MBP emulsified in CFA. We treated
mice s.c. with recombinant human G-CSF (200 µg/kg/day).
No effect was observed when the treatment was initiated at the
induction phase of the disease, i.e., from day -1 before immunization
to day 8 postimmunization (not shown). However, when treatment was
started on day 10 after the first immunization, approximately 1 wk
before the appearance of the first clinical signs, and continued daily
until day 30, a significant protection from the disease was observed
(Fig. 1
A), with a reduction in
the maximal mean clinical score as well as in the incidence of disease
(p < 0.01), thus demonstrating that G-CSF was
able to prevent EAE.
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C) or was given for a limited period of 7
consecutive days (Fig. 1E), a significant and durable
protection from the disease was observed until the end of the
experiment, up to days 60–80. The mean clinical score, calculated on
all mice per group over the entire disease length, was reduced in
G-CSF-treated mice relative to excipient-injected mice (Table I). This effect corresponded to a
reduction both in the mean incidence of EAE measured at the peak of the
disease (5/12 vs 8/12) and over the whole disease length and in the
average severity of the disease among mice with EAE (Table I). The
weight loss, which ranged from 5 to 20% of initial body weight and
preceded the development of paralysis, was also abrogated (Fig. 1, B, D, and F; p <
0.001). There was a trend to a reduction in the incidence of relapse,
but it was not significant. There was no difference between the groups
of mice in the mean time of disease onset, as expected given that
treatment started thereafter. Thus, both acute and subsequent chronic
phases of the disease were affected by G-CSF when it was administered
at a dose of 200 µg/kg/day during the effector phase of the disease.
A lower dose of 10 µg/kg/day was ineffective (not shown).
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CNS inflammation and demyelination were assessed in MBP-immunized
mice, treated with excipient or G-CSF on day 40 postimmunization.
Toluidine blue was used to stain myelin in Fig. 2, A and D. Loss of
myelin was extensive in the cerebellum of excipient-treated mice (Fig. 2A), but remained limited in G-CSF-treated mice (Fig. 2D). In excipient-treated mice, H&E staining (Fig. 2B) showed extensive parenchymal infiltration with
perivascular cuffs in cerebellum, that were not observed in
G-CSF-protected mice, which displayed few cells around the vessels
(Fig. 2E). The mean histologic scores (Table I) were
significantly reduced by G-CSF treatment. Immunohistochemical staining
revealed that infiltrating cells near these perivascular cuffs
consisted mainly in T cells (Fig. 2C) and
Mac-1+ cells (Fig. 2I), including
essentially macrophages and fewer granulocytes (Fig. 2G).
Gr1+ granulocytes were detected in the
perivascular zone and fewer in the surrounding parenchyma in both
excipient-treated (Fig. 2G) and G-CSF-treated (Fig. 2J) mice, but at much lower rate in the latter.
Mac-1+ staining (Fig. 2I) correlated
with F4/80+ staining (Fig. 2H) and was
also observed on activated microglia with dendritic morphology in the
surrounding parenchyma of excipient-treated mice, whereas G-CSF-treated
mice displayed almost no F4/80 label (Fig. 2K) and very
limited Mac-1 staining (Fig. 2L).
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Spinal cords of G-CSF-protected mice expressed no or very limited
amounts of mRNAs for cytokines, chemokines, and growth factors as
analyzed by RPA. G-CSF treatment prevented the induction of both
Th1-oriented cytokines (such as IFN- and IL-2) as well as of
regulatory cytokines, which are expressed in EAE undergoing SJL/J mice
treated with excipient, including IL-10 (Fig. 3A), as previously observed by
Tran et al. (24), and TGF-1 (Fig. 3B).
Levels of TNF-, lymphotoxin-, and TNF- mRNAs were also
significantly reduced by G-CSF treatment (Fig. 3B). Similar
findings were obtained for chemokines (Fig. 4). RANTES, and MCP-1, predominantly
expressed in spinal cords of excipient-treated mice, were barely
detectable in G-CSF-treated mice. No particular chemokine among those
analyzed was increased by G-CSF treatment.
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A) show that G-CSF mRNA,
although expressed at levels below quantitation limits in spinal cords
of mice developing EAE, was not apparently enhanced in mice treated
with G-CSF, thus suggesting that G-CSF treatment did not significantly
induce its own production at this site. Instead, M-CSF mRNA was
expressed constitutively at high levels in spinal cords from naive mice
and was further increased in mice injected with excipient and
presenting with EAE, yet returned to basal levels in mice treated with
G-CSF (Fig. 5B). Although expressed at much lower levels
than M-CSF, IL-6 and stem cell factor mRNA levels followed the
same trend.
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Cellular subsets affected by G-CSF
We first investigated the cellular changes induced by G-CSF in
EAE. The hemopoietic properties of G-CSF were confirmed by
extramedullary hemopoiesis, with an increase in total spleen cell
number (p < 0.05) in G-CSF-treated,
MBP-immunized mice (208 ± 28 x 106
cells/spleen; n = 3) relative to excipient-treated
animals (117 ± 12 x 106 cells/spleen;
n = 3) and to control nonimmunized animals (126 ±
15 x 106 cells/spleen; n =
3). Moreover, FACS analysis of spleens of animals on day 40 after
immunization essentially revealed that macrophages
(CD11bhigh Gr-1- cells),
which represented 2.44 ± 0.2% of total spleen cells in naive SJL
mice (n = 3) and 3.28 ± 0.3% in MBP-immunized,
excipient-treated mice (n = 3), increased to 3.92
± 0.2% in G-CSF-treated mice (n = 3), while
granulocytes (CD11bhigh
Gr-1+) changed from 1.5 ± 0.2 to
3.2 ± 0.3 and 6.8 ± 0.3% in the same cell populations,
respectively (Fig. 6). No major
alterations were observed in the percentages of the other immune cell
subsets, including T cells (CD3+,
CD4+, CD8+) and B cells
(CD19+B220+). G-CSF had no
particular effect at this stage of the disease on the respective
proportions of the dendritic cell subsets, i.e., myeloid, characterized
as CD11c+ CD11bhigh, vs
lymphoid, CD11c+ CD11blow,
at variance with the preferential pro-lymphoid effect reported in man
(29). Essentially similar results were obtained in lymph
nodes (not shown).
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/MCP-1 ratio in macrophages and
autoreactive splenocytes
Since the migration of T cells to the CNS is under the control of
chemokines, we measured chemokine production by 1) macrophages isolated
by adherence from spleens of excipient- or G-CSF-treated animals and
activated with anti-CD40 to mimic the T cell cognate interaction,
and 2) autoreactive splenocytes. We observed that the chemokine content
measured by specific ELISA in supernatants of macrophages from G-CSF-
compared with excipient-treated mice showed 2-fold reduced production
of MIP-1 (Fig. 7A), whereas
MCP-1 levels were 2-fold increased (Fig. 7B), leading to a
final 4-fold reduction in the MIP-1/MCP-1 balance (Fig. 7C). Similarly, in supernatants of MBP-stimulated
splenocytes, the levels of MIP-1 were 2.5-fold reduced, whereas
those of MCP-1 were enhanced 2.5-fold in G-CSF-treated mice relative to
mice injected with excipient. The overall MIP-1/MCP-1 protein ratio
was reduced approximately 5-fold (Fig. 7, D–F), close to
the ratio of the corresponding chemokine mRNA levels analyzed by RPA
(Fig. 7, G–I).
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production in anti-CD40-stimulated
macrophages and reduced systemic levels of TNF- in EAE
TNF- activates endothelial cells (30, 31) and
facilitates T cell migration to the CNS. We observed that production of
TNF- was 2-fold reduced in macrophages isolated from spleens of
G-CSF-treated animals and activated with anti-CD40 to mimic the T
cell cognate interaction (Fig. 8A), relative to cells
isolated from excipient-treated mice. Moreover, a dramatic reduction in
TNF- serum levels was observed, with 11.5 ± 1.26 vs 85.67
± 14.77 pg/ml TNF- (p < 0.002) at the
acute phase of the disease and 4.2 ± 1.53 vs 41 ± 16.34
pg/ml (p < 0.005) at the chronic phase, in
G-CSF-treated mice relative to excipient-treated animals, respectively
(Fig. 8B).
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The autoreactive T cell response to the recall Ag MBP was
substantially affected by G-CSF treatment. Although proliferation to
the autoantigen did not differ between the two groups of mice (not
shown), analysis of the cytokine contents on day 4 of incubation in
supernatants of MBP-stimulated splenocytes from G-CSF-treated mice,
relative to mice treated with excipient, revealed significant
inhibition of the production of Th1 cytokines, such as IFN- and
TNF-, whereas IL-4 was increased (Fig. 9A). IL-10 levels were
unaffected. By contrast, RPA analysis revealed an increase in TGF-1
mRNA levels in splenocytes of G-CSF- vs excipient-treated mice (Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which inhibits Th1 cytokine
production, but for which discontinuation of treatment after 10 days
caused rapid onset of EAE with enhanced clinical signs and prolonged
duration of the disease (32). Interestingly, the
therapeutic window for G-CSF treatment appears similar to that reported
for soluble TNF receptor-stabilized molecules or IL-1 receptor
antagonist (8, 9, 33). Peripheral administration of G-CSF was able to significantly reduce the EAE-associated CNS pathology. Whether this results from a local CNS effect of G-CSF, which may potentially cross the blood-brain barrier which is damaged at the onset of clinical signs when G-CSF s.c. administration is initiated, cannot be excluded. Analysis of mRNA in spinal cords does not support an endogenous role of brain-derived G-CSF, but a regulatory effect of G-CSF therapy on brain-derived M-CSF was observed. At variance with M-CSF, which has been found at increased levels in the cerebrospinal fluid of patients with a variety of neurological diseases, including brain tumors, bacterial meningitis, and AIDS-dementia complex (34), but not in MS patients, G-CSF was found only in patients with bacterial meningitis. Constitutive expression of M-CSF has been previously reported in normal mouse brain tissue; the transcripts are detected from embryonic day 13 through adulthood (35). In vivo expression of G-CSF has been reported in neoplastic astrocytes (36, 37, 38), but was lost at higher degrees of dedifferentiation. Inflammatory and viral stimuli enhance M-CSF expression and promote G-CSF production by human and murine astrocytes (25, 26). We report here that the constitutive, high level expression of M-CSF in spinal cords increases in mice with EAE, but returns to basal levels in mice given G-CSF, whereas G-CSF itself was not significantly induced by either EAE disease or G-CSF treatment.
All mediators of immune response that we analyzed in brains of mice
given G-CSF appeared to be inhibited, including proinflammatory
cytokines such as IFN-, IL-2, TNF- and -, and lymphotoxin-,
as well as regulatory cytokines such as IL-10 and TGF-. Chemokine
expression was also altered, with no enhancement of any particular
member of this family.
G-CSF treatment exerted a marked influence on cellular infiltration to
the CNS. Experiments conducted in rats (39) demonstrating
that increased absolute neutrophil count in rats given G-CSF did not
increase posttraumatic brain neutrophil accumulation, suggested that
the ability of G-CSF-stimulated neutrophils to migrate into injured
tissue may be impaired. Likewise, the expansion of the neutrophil and
macrophage subsets that we observed in spleens of our G-CSF-treated
mice, which illustrated the hemopoietic properties of G-CSF, did not
occur in CNS. Instead, T cell infiltration and the inflammatory
reaction that normally follows were remarkably limited. These processes
are under control of several sets of molecules, including the
chemokines and TNF- whose production in the periphery was shown to
be altered and/or reduced by G-CSF treatment.
The role of chemokines in EAE pathogenesis differs according to the
strains of mice and Ag used to induce disease. Whereas MOG-immunized
C57BL/6 mice show early CNS expression of MCP-1, and lack of MCP-1
expression results in protection from the disease (an effect that may
reflect the absence of recruitment of CCR2-bearing, inflammatory
macrophages), in the MBP/CFA and proteolipid protein/CFA-based
immunization models in SJL/J mice, MIP-1 and MCP-1 molecules appear
to be involved at different, i.e., acute vs chronic, stages of the
disease, respectively (13). Thus, treatment with
anti-MIP-1, but not anti-MCP-1, inhibited acute EAE.
Conversely, anti-MCP-1 moderately reduced relapsing EAE severity,
whereas anti-MIP-1 had no effect at this stage of the disease
(16). In MBP/CFA-immunized SJL mice, we show that the
protective effect of G-CSF correlated with a reduced MIP-1/MCP-1
production ratio in the autoreactive response at both mRNA and protein
levels.
This imbalance in MIP-1/MCP-1 production ratio in macrophages and
autoreactive splenocytes may first affect T cell migration to the CNS,
particularly via reduction of MIP-1 production, in keeping with the
inhibitory effect on the primary acute phase of EAE of
anti-MIP-1 Ab treatment observed by Karpus et al.
(16). Moreover, both MIP-1 reduction and MCP-1 increase
may participate in immune deviation of the T cell autoreactive response
in the periphery. A large body of data have demonstrated the existence
of cross-regulation between cytokines and chemokines (17).
MIP-1 was shown to be induced predominantly in a Th1 setting and to,
in turn, activate IFN- production, whereas MCP-1 is preferentially
produced in Th2 settings and promotes IL-4 production (20, 21). MCP-1-deficient mice are unable to mount Th2 responses and
are resistant to infection by Leishmania major
(40). G-CSF treatment of MBP-immunized SJL/J mice, by
reducing the production of MIP-1 and enhancing that of MCP-1 at both
protein and mRNA levels, creates a pro-Th2 imbalance. Interestingly,
the reduction of the MIP-1 /MCP-1 ratio matched the
reduction of the IFN-/IL-4 ratio measured in the same
splenocyte supernatants.
The production of TGF-1 was also increased by G-CSF treatment. While
the role of this potent immunoregulatory factor in promoting immune
deviation is well documented in autoimmune diseases, it should be noted
that it is also able to reduce MIP-1 production (41).
Whether immune deviation of the autoreactive response is effectively
associated with either reduced encephalitogenic potential of the
lymphocytes in G-CSF-treated mice and/or regulatory properties of these
cells deserves further investigation using adoptive transfer and
cotransfer experiments.
In addition to the imbalance in chemokine production and the immune
deviation toward Th2, the protection against EAE by G-CSF treatment was
associated with a reduced production of TNF-, a cytokine involved in
the disease pathogenesis, particularly at the early inflammatory phase
(10). This property may be related to the potent promotion
of chemokine synthesis by this cytokine, often exerted in synergy with
IFN-, on both endothelial cells (30, 31) as well as
astrocytes (42). Whether this reduction of TNF- levels
is a primitive anti-inflammatory effect of G-CSF, as reported in
experimental models of endotoxemia (1), or is secondary to
the anti-Th1 and pro-Th2 properties also displayed by this molecule
(3, 5, 6) remains to be investigated. Particularly
relevant to the latter hypothesis is the fact that IL-4 is known to
inhibit the macrophage production of IL-1 and TNF-
(43). The dual capacity of G-CSF to reduce the production
of TNF- by both macrophages and autoreactive splenocytes confers
high regulatory potential to this molecule in the context of
inflammatory reactions associated with cell-mediated autoimmune
diseases.
In conclusion, we demonstrate that a short treatment with G-CSF, which limits the potential risk of immunization against the molecule, exerts remarkable and long-lived protective effects on the clinical course of EAE, even at a late stage of the disease process, when clinical signs are first detected. The progression of the disease is inhibited in the CNS of G-CSF-treated animals, which display only limited demyelination and almost no inflammation. Whether remyelination takes place under the influence of G-CSF treatment remains to be investigated. No signs of immune activation, including the expression of regulatory cytokines or chemokines, are observed in the brain tissue of G-CSF-protected mice, suggesting that this protection from autoimmune disease is secondary to reduced T cell infiltration in the CNS. We have shown that G-CSF protection is strikingly associated with important alterations of the peripheral immune system, some of which were previously shown to be induced by this factor in alloreactive and inflammatory settings (1, 5).
G-CSF has been used in humans for >10 yr with a remarkable tolerance. It has been shown to decrease infectious episodes and acute inflammatory response in patients with a variety of brain pathologies, including brain tumors, acute traumatic brain injury, and cerebral hemorrhage (44). Together with recent data from this laboratory (23), in which treatment with G-CSF was able to protect mice from the development of another AID, spontaneous systemic lupus, the present results, which demonstrate that G-CSF targets mechanisms critical for the pathogenesis of EAE, constitute a rationale for the clinical evaluation of G-CSF in human AID, such as MS.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; AID, autoimmune disease; MBP, myelin basic protein; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage-inflammatory protein-1; MS, multiple sclerosis; RPA, RNase protection assay.
Received for publication August 31, 2001. Accepted for publication November 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Immunol. 149:918.[Abstract]
and IP-10 are expressed in demyelinating brain lesions. Proc. Natl. Acad. Sci. USA 96:6873. and MCP-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as Th1/Th2 lymphocyte differentiation. J. Leukocyte Biol. 62:681.[Abstract]
shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759. and tumor necrosis factor-. J. Immunol. 149:2358.[Abstract]
prevents or aggravates experimental autoimmune encephalomyelitis in Lewis rats. J. Immunol. 157:5721.[Abstract]
peptides, MIP-1 and MIP-1, by lipopolysaccharide is differentially regulated by immunomodulatory cytokines -IFN, IL-10, IL-4, and TGF-. Mol. Med. 4:648.[Medline]
, tumor necrosis factor , and IL-6 by human monocytes. Blood 76:1392.This article has been cited by other articles:
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 A. Oishi, A. Otani, M. Sasahara, H. Kojima, H. Nakamura, Y. Yodoi, and N. Yoshimura Granulocyte Colony-Stimulating Factor Protects Retinal Photoreceptor Cells against Light-Induced Damage Invest. Ophthalmol. Vis. Sci., December 1, 2008; 49(12): 5629 - 5635. [Abstract] [Full Text] [PDF]
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A. E. Anderson, B. L. Sayers, M. A. Haniffa, D. J. Swan, J. Diboll, X.-N. Wang, J. D. Isaacs, and C. M. U. Hilkens Differential regulation of naive and memory CD4+ T cells by alternatively activated dendritic cells J. Leukoc. Biol., July 1, 2008; 84(1): 124 - 133. [Abstract] [Full Text] [PDF]
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 S. Gaudreau, C. Guindi, M. Menard, G. Besin, G. Dupuis, and A. Amrani Granulocyte-Macrophage Colony-Stimulating Factor Prevents Diabetes Development in NOD Mice by Inducing Tolerogenic Dendritic Cells that Sustain the Suppressive Function of CD4+CD25+ Regulatory T Cells J. Immunol., September 15, 2007; 179(6): 3638 - 3647. [Abstract] [Full Text] [PDF]
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E. E. McCandless, Q. Wang, B. M. Woerner, J. M. Harper, and R. S. Klein CXCL12 Limits Inflammation by Localizing Mononuclear Infiltrates to the Perivascular Space during Experimental Autoimmune Encephalomyelitis J. Immunol., December 1, 2006; 177(11): 8053 - 8064. [Abstract] [Full Text] [PDF]
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M. Miyake, K. Sasaki, K. Ide, Y. Matsukura, K. Shijima, and D. Fujiwara Highly Oligomeric Procyanidins Ameliorate Experimental Autoimmune Encephalomyelitis via Suppression of Th1 Immunity J. Immunol., May 15, 2006; 176(10): 5797 - 5804. [Abstract] [Full Text] [PDF]
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E. Gonzalez-Rey, A. Fernandez-Martin, A. Chorny, J. Martin, D. Pozo, D. Ganea, and M. Delgado Therapeutic Effect of Vasoactive Intestinal Peptide on Experimental Autoimmune Encephalomyelitis: Down-Regulation of Inflammatory and Autoimmune Responses Am. J. Pathol., April 1, 2006; 168(4): 1179 - 1188. [Abstract] [Full Text] [PDF]
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 I. Solaroglu, J. Cahill, V. Jadhav, and J. H. Zhang A Novel Neuroprotectant Granulocyte-Colony Stimulating Factor Stroke, April 1, 2006; 37(4): 1123 - 1128. [Abstract] [Full Text] [PDF]
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 C. Teuscher, R. W. Doerge, P. D. Fillmore, and E. P. Blankenhorn eae36, a Locus on Mouse Chromosome 4, Controls Susceptibility to Experimental Allergic Encephalomyelitis in Older Mice and Mice Immunized in the Winter Genetics, February 1, 2006; 172(2): 1147 - 1153. [Abstract] [Full Text] [PDF]
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S. Rutella, F. Zavala, S. Danese, H. Kared, and G. Leone Granulocyte Colony-Stimulating Factor: A Novel Mediator of T Cell Tolerance J. Immunol., December 1, 2005; 175(11): 7085 - 7091. [Abstract] [Full Text] [PDF]
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 Y. Sugano, T. Anzai, T. Yoshikawa, Y. Maekawa, T. Kohno, K. Mahara, K. Naito, and S. Ogawa Granulocyte colony-stimulating factor attenuates early ventricular expansion after experimental myocardial infarction Cardiovasc Res, February 1, 2005; 65(2): 446 - 456. [Abstract] [Full Text] [PDF]
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 H. Kared, A. Masson, H. Adle-Biassette, J.-F. Bach, L. Chatenoud, and F. Zavala Treatment With Granulocyte Colony-Stimulating Factor Prevents Diabetes in NOD Mice by Recruiting Plasmacytoid Dendritic Cells and Functional CD4+CD25+ Regulatory T-Cells Diabetes, January 1, 2005; 54(1): 78 - 84. [Abstract] [Full Text] [PDF]
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 L. Zou, B. Barnett, H. Safah, V. F. LaRussa, M. Evdemon-Hogan, P. Mottram, S. Wei, O. David, T. J. Curiel, and W. Zou Bone Marrow Is a Reservoir for CD4+CD25+ Regulatory T Cells that Traffic through CXCL12/CXCR4 Signals Cancer Res., November 15, 2004; 64(22): 8451 - 8455. [Abstract] [Full Text] [PDF]
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 K. E. Lawlor, I. K. Campbell, D. Metcalf, K. O'Donnell, A. van Nieuwenhuijze, A. W. Roberts, and I. P. Wicks Critical role for granulocyte colony-stimulating factor in inflammatory arthritis PNAS, August 3, 2004; 101(31): 11398 - 11403. [Abstract] [Full Text] [PDF]
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) starting on day 10 until
day 30 (A), at the onset of clinical signs, from day 13,
until day 30 (C), or from day 17 until day 23 (day 7 of
treatment; E) after immunization. Mean clinical scores
and incidence of disease (numbers in parentheses) were measured in 12
mice/group (p < 0.001, vehicle-treated compared
with G-CSF-treated, by Student’s unpaired t test).
B, D, and F, Evolution of
body weight in animals treated with excipient (
