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* Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute,
Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115;
Department of Pathology, Albert Einstein College of Medicine, New York, NY 10461;
Department of Neurology, Justus-Liebig University, Giesen, Germany;
¶ Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy; and
|| Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two hemopoietic CSFs, M-CSF and GM-CSF, are thought to play important
roles in neonatal and postnatal microglial differentiation
(9, 10, 11). M-CSF and GM-CSF both stimulate proliferation of
neonatal microglia (12), but differently affect their
morphology, phenotype, and Ag-presenting function. Morphologically,
neonatal microglial cells cultured in M-CSF appear amoeboid, whereas
those cultured in GM-CSF assume a dendriform shape (13).
Phenotypically, neonatal microglial cells treated with M-CSF display
some characteristics of macrophage
(m
)3 whereas those cultured
in GM-CSF assume some of the markers associated with an immature
dendritic cell (DC)-like phenotype (11, 13, 14).
Importantly, the cytokine production capacity and competence as APCs
are also differentially regulated by exposure to M-CSF and GM-CSF
(13, 14, 15, 16, 17). Priming of microglial cells with GM-CSF enables
them to become fully competent as APCs, upon contact with activated T
cells or proinflammatory cytokines (18, 19). Although the
competency of microglial cells for Ag presentation under
noninflammatory conditions is still a matter of debate; it is apparent
that GM-CSF endows both neonatal and adult microglial cells with an
enhanced ability to process and present Ags (18, 19).
M-CSF is constitutively produced by astrocytes from embryo to adulthood (20). However, although GM-CSF is produced during the neonatal period, it is scarcely present in the adult CNS (20, 21). In the normal adult CNS, the only source of GM-CSF is from peripherally primed, infiltrating T cells, which patrol the brain parenchyma (22, 23). Because extravasation of activated T cells into the CNS is non-Ag dependent, the majority of T cells will exit without meeting their specific Ag. In contrast, if the T cells encounter a cognate self ligand, a potential autoimmune process could begin (23) with massive production of proinflammatory mediators. Under such conditions, microglial cells, already primed by T cell-released GM-CSF, will become fully competent APCs. Activated astrocytes and endothelial cells could also function as an additional source of GM-CSF (23, 24). Thus, it appears that M-CSF plays an important role in shaping the adult resident parenchymal microglia, whereas GM-CSF could have an important role in promoting their proinflammatory function. In this respect, the functional states of microglial cells elicited by M-CSF or GM-CSF have been proposed to correspond to the resting or "activated" form of microglia, respectively (11).
DNA microarray analysis can provide a very broad transcriptional
profile of cells under different culture conditions or differentiation
states. In this study, the transcript levels of 
6000 genes have been
analyzed, in primary murine neonatal microglial cells either untreated
or cultured with GM-CSF or M-CSF. Both cytokines led to large-scale
changes in the microglia expression profile. M-CSF treatment primarily
induced transcription of genes involved in tissue organization and
remodeling, maintenance, and regulation of brain homeostasis. By
contrast, GM-CSF treatment induced transcription of genes important for
T cell stimulation, chemotaxis, Ag processing, innate immunity, and
immunosuppression, thus readying microglia for Ag presentation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neonatal microglia was prepared from newborn SJL/J mice as
previously described (13). Briefly, after careful removal
of the meninges, brains were mechanically disrupted and passed through
a 100-µm filter. Cells were seeded in MEM (Life Technologies,
Rockville, MD) with the addition of 10% FCS, 5 µg/ml insulin (Life
Technologies), and 2.0 mg/ml L-glucose (Sigma-Aldrich, St.
Louis, MO) for 12–14 days. Mixed glial cultures were shaken overnight
on an orbital shaker (first shake). Adherent glial cells were
trypsinized, split, and reseeded for an additional 10–12 days of
culture. The procedure was repeated twice (second and third shakes).
The purity of each preparation was assessed by CD11b staining and was
always >93%. GM-CSF (10 ng/ml) or M-CSF (5 ng/ml) were added at the
beginning of the culture and again every 3 days. Microglial cells from
the second shake were used for global gene expression analysis. In some
experiments (Fig. 3
, a and b), IFN-
was added
to the untreated control for 48 h. Surface staining was performed
as previously described (13) using the following mAbs,
hamster anti-mouse CD11c (clone HL-3), rat anti-mouse CD11b
(clone M1/70), rat anti-mouse CD45 (clone 30-F11), rat
anti-mouse CD24 (clone 30-F1), hamster anti-mouse B7-1 (clone
16-10A1), and rat anti-mouse B7-2 (clone GL1), all from BD
PharMingen (San Diego, CA); rat anti-mouse DEC-205 (clone NLDC-145;
American Type Culture Collection, Manassas, VA); goat anti-Tweak
(Research Diagnostics, Flanders, NJ); and rat anti-mouse MARCO
(clone ED31; a gift from P. Ricciardi-Castagnoli).
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Total RNA was isolated from 10 million cells using TRIzol reagents (Life Technologies), and biotinylated cDNA was prepared and hybridized to Affimetrix microarrays using the manufacturer’s protocols. Transcripts exhibiting an average difference (between specific and control oligonucleotides) of 1500 were considered to be expressed: the percentages calculated using the Affimetrix "absolute call" were similar. Transcripts with average difference below this level were considered below the level of detection.
RNase protection assay
Total RNA was isolated using TRIzol reagents (Life Technologies). RNase protection assay was performed using 4–6 µg of total RNA using BD PharMingen Riboquant kit rCK-2 multiprobe template set following the manufacturer’s recommendations.
Immunostaining
Spinal cord frozen sections (10 µm thick) were fixed in acetone at -20°C, washed in PBS, and blocked with BSA. The following primary Abs diluted in PBS/1% BSA were added to the slides and incubated overnight at 4°C: goat anti-Tweak, used at 5 µg/ml (Research Diagnostics), in combination with anti-mouse CD45-FITC conjugated (BD PharMingen). Sections were washed in PBS and incubated with rabbit anti-goat IgG conjugated to Alexa 594 (Molecular Probes, Eugene, OR) to detect Tweak. To amplify the signal from the green fluorescence, the amplification kit, Alexa Fluor 488 signal amplification system and Oregon Green Dye-conjugated probes (Molecular Probes) was used. Fluorescent microscopy was performed on an Olympus IX70 (Olympus, Melville, NY) with x60 N.A. 1.4 infinity corrected optics and a PXL cold CCD camera (Roper Scientific, Tucson, AZ).
Western blot analysis
Microglial cells untreated or treated with GM-CSF (10
ng/ml), M-CSF (5 ng/ml), or IFN- (10 ng/ml) were pelleted and lysed
in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris (pH 8), and
supplemented with the protease inhibitor mixture (Complete Mini Roche),
for 1 h at 4°C. Supernatants were collected by centrifugation
and protein content was normalized. Proteins were run on a SDS-PAGE gel
and transferred to polyvinylidene difluoride membrane. After
transfer, the membrane was probed with CD74 (clone In-1; BD PharMingen)
specific for the MHC-associated invariant chain or the anti-MHC
class II mAb KL-304 (ATCC), followed by 1/5000 dilution of the
secondary HRP-conjugated mAb and chemiluminescence detection.
Mixed lymphocyte reaction
MLR was performed combining different amount (from 25 to 200 x 103 cells) of microglial cells either untreated or treated with GM-CSF or M-CSF, previously irradiated at 600 rad (Cs137), with 200 x 103 splenocytes from either B10BR, C57BL/6, or SJL/J (The Jackson Laboratory, Bar Harbor, ME). Proliferation was tested by [3H]thymidine incorporation. Bone marrow-derived DCs prepared as previously described (25) were used as positive control in each MLR.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microglial cells were isolated from neonatal brain tissue of SJL
mice, and cultured in the presence or absence of GM-CSF (10 ng/ml) or
M-CSF (5 ng/ml) (13). Expression of RNA transcripts was
analyzed for 6000 murine genes of known or predicted function from
the National Center for Biotechnology Information Unigene
database, using Affimetrix U74A microarrays. Marked differences were
observed in the transcriptional profiles of the microglia after
treatment with GM-CSF or M-CSF, as compared with each other or as
compared with untreated cells. Over 600 transcripts were uniquely
expressed under each of these conditions, with more than one-third of
the transcripts exhibiting differential expression in largely
nonoverlapping sets (Fig. 1a).
Large differences were observed in the transcript levels of expressed
genes, with 16.5% of expressed genes exhibiting up- or
down-modulation of at least 2.5-fold after cytokine treatment (Fig. 1b). Separate experiments using independent primary
microglia isolates showed a good correlation in expression levels for
both GM-CSF and M-CSF treatments (R = 0.85, data not
shown). In addition, many gene products were analyzed individually by
flow cytometry or Western blotting to validate the transcript analysis
(see
Figs. 2–4
and 6). The high percentage of transcripts
differentially expressed after treatment suggest that neonatal
microglial cells undergo true lineage differentiation in response to
M-CSF or GM-CSF, as opposed to simply a change in activation state.
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). These include genes
involved in myelination growth and development, such as tenascin C
(24-fold), ryudocan (21-fold), biglycan (9-fold), nerve growth factor
(8-fold), and epidermal growth factor-like growth factor
(36-fold) (26, 27, 28, 29); in processing of neuropeptides, such
as peptidylglycine-
-amidating monooxygenase (11-fold) and
carboxypeptidase H (10-fold) (30, 31); in tissue
remodeling, such as collagens type I, IV, V, VI, and XI (from 3- to
11-fold); or in the heat shock response (hsp), for example, hsp47
(11-fold) and hsp105 (4-fold; Table I). In contrast, many of the gene
products up-regulated in GM-CSF-treated cells appear to be involved in
cellular processes related to immune cell activation, Ag presentation,
and innate immunity. These are discussed in detail.
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and DC markers
Although no proteins are known to be expressed uniquely on m or
DCs, the pattern of expression of the integrins CD11b (Mac-1), CD11c,
and the lectin-like receptor DEC-205 is considered characteristic, with
expression of CD11c+,
CD11blow, and DEC-205+
associated with an immature DC phenotype, and a higher surface
expression of CD11bhigh observed for m. As
previously reported (13), treatment of neonatal microglial
cells with GM-CSF or M-CSF alters the expression of these markers
toward a DC-like or m-like phenotype, respectively. These results
matched well with the levels of the corresponding transcripts from the
microarray analysis (data not shown).
Low-affinity receptors for GM-CSF are present on untreated microglial
cells and are slightly up-modulated by GM-CSF and down-modulated by
M-CSF (Table II). Interestingly, the
high-affinity GM-CSFR (common 
-chain among IL-3, IL-5, and GM-CSFR)
is absent on untreated cells, but is up-regulated >26-fold upon GM-CSF
treatment.
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Consistent with their key role as sensors of CNS injury, neonatal
microglia express mRNA encoding receptors for several proinflammatory
cytokines including IL-6R, IFN-R, and TNF-R (Table II). Neither
of the receptors appeared to be regulated by M-CSF or GM-CSF (Table II)
with the exception of IL-6R, which is slightly down-regulated by both
the hemopoietic factors.
Microglial cells also expressed receptors for anti-inflammatory
cytokines, particularly the IL-10R and the secreted form of the IL-4R
(Table II); both of which are slightly down-regulated by M-CSF but not
GM-CSF.
The role of cytokine in the CNS goes beyond the regulation of microglia immune function. In this respect, cytokines like IL-7 and IL-11 have been shown to induce proliferation on cultured astroglia (32) as well as to induce glial differentiation (33).
Overall, the cytokine receptors profile of microglial cells do not
appear to be significantly changed by treatment with both hemopoietic
factors, with exception of IL-7R and IL-11R, absent in untreated
microglial cells, but up-regulated by GM-CSF (Table II).
Cytokines and chemokines
Under inflammatory degenerative and traumatic conditions in the
brain, microglial cells are the resident primary source of both pro-
and anti-inflammatory cytokines. Untreated microglial cells display
transcripts for strongly immunosuppressive and/or proapoptotic
cytokines like TGF-1 and Tweak (Fig. 2, b and c),
reflecting a strategy that appear to limit immune-mediated events
within the CNS. Transcripts for TGF- and Tweak were both decreased
by M-CSF treatment. Tweak was also down-regulated by GM-CSF.
Transcripts encoding for several chemokines were detected in untreated
microglia, including m inflammatory protein (MIP)-1, MIP-1,
MIP-1, MIP-2, monocyte chemoattractant protein (MCP)-1,
MCP-3, and MCP-5. Treatment with GM-CSF markedly increased transcripts
for chemokines C10, MIP-1, MIP-1, and MIP-2, while at the
same time down-regulating MCP-5. C10, MIP-1, and MIP-2
were also up-regulated by M-CSF treatment, albeit to a lower degree.
MCP-1 was up-regulated only by M-CSF. Both global transcript analysis
(Fig. 2b) and RNase protection assay (Fig. 2a)
demonstrated induction of the proinflammatory IL-1, IL-1, and the
IL-1R antagonist upon treatment with GM-CSF but not M-CSF. Both
hemopoietic factors up-regulate mRNA encoding for IFN- (Fig. 2b).
Ag presentation
Microglial cells are the primary APCs resident in the brain. In
the resting condition, they express very low levels of class II MHC
protein and the class-II-associated invariant chain, but these can be
up-regulated by treatment with the inflammatory cytokines such as
IFN-. Previously, we have reported that GM-CSF skewing of microglial
cells made them more inclined to up-regulate class II MHC expression in
response to proinflammatory cytokines (13). Due to a
mismatch between the MHC haplotype of the microglia cells
(H-2s) and the microarray target mRNA
(H-2b), MHC expression could not be monitored by
the global gene analysis. Therefore, class II MHC levels were monitored
by Western blotting. Class II MHC protein, which is almost undetectable
in untreated cells, was increased to a barely detectable level after
treatment with GM-CSF, but not M-CSF, although class II MHC surface
staining was not increased (data not shown). As expected, IFN- was a
much better inducer of class II MHC proteins (Fig. 3a). Similar results were
observed for the class II-associated invariant chain chaperone Ii, for
which both alternately spliced p31 and p41 variants as well as
partially processed p10 and p12 forms were observed after GM-CSF but
not M-CSF treatment (Fig. 3b). This pattern for invariant
chain was observed also by transcript analysis (Fig. 3c).
Transcript levels for the class II MHC-associated peptide exchange
factor H2-M were low but detectable in each of the samples, and were
not changed by either treatment. No significant change in class I MHC
protein was observed after treatment with either M-CSF or GM-CSF (data
not shown).
Activation of T cells requires costimulatory interactions in addition
to generation and recognition of MHC-peptide complexes. Several
costimulatory receptors are present on microglial cells. As previously
reported, neonatal microglia express CD80 (B7-1) and CD86 (B7-2; Fig. 4, a and b), which
provide a CD28-dependent costimulatory signal (18). The
expression of B7-1 was slightly up-regulated by GM-CSF, whereas B7-2
profile was not affected. The heat-stable Ag, CD24, has recently been
added to the list of costimulatory molecules involved in T cell
activation (34). CD24 play an important role in CNS
immunity since its targeted mutation completely abrogated development
of experimental allergic encephalitis (EAE), although its
mutation does not prevent the induction of autoreactive T cells
(35). In this study, we report that CD24 was dramatically
increased (122-fold) in GM-CSF-treated cells (Fig. 4, a and
b). CD24 up-regulation on microglial cells could represent a
novel, CD28-independent, costimulatory signal for expansion of
autoreactive T cells.
Altogether, GM-CSF-skewed cells were endowed with enhanced T cell
stimulatory function, as assessed in an MLR assay (Fig. 3d).
Proteases
Cathepsins are a large family of cysteine and aspartic proteases
implicated in endosomal protein degradation and generation of peptides
for Ag processing. Using global gene expression analysis, differential
expression of many of the cathepsins was found. Cathepsin F, L, and S,
which are involved in invariant chain degradation and peptide loading,
are differently distributed among APCs. Cathepsin S is present in B
cells, DCs, and m, whereas cathepsin F is more selectively expressed
in tissue m. Neonatal microglial cells have been reported to express
cathepsin S and L, but not F (13), while resting
microglial cells have been reported to express cathepsin E
(36). Global gene expression analysis confirms these
observations. Interestingly, both cathepsins L and F were up-regulated
by GM-CSF (Fig. 5a), while
cathepsin E was down-regulated (Fig. 5a). Neonatal
microglial cells expressed cathepsin B, C, D, and S, with levels not
significantly regulated by either GM-CSF or M-CSF (Fig. 5a).
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metalloelastase), MMP-9, and MMP-11 (stromelysin-3; Fig. 5b), whereas M-CSF selectively up-regulates MMP-9. These
proteases are potent collagenase, and may play an important role in
remodeling the brain microenvironment during disease processes as well
as affecting cell trafficking into the brain (39, 40). It
is also tempting to speculate that these proteases may mediate
extracellular Ag processing. Notably, antigenic determinants derived
from myelin basic protein have been reported to be presented in the CNS
through a pathway that does not involve internalization and endosomal
processing (41). Antimicrobial response
Microglial cells respond to foreign pathogens by phagocytosis
after receptor-mediated recognition of microbial Ags as well as
producing several antimicrobial factors. Untreated microglial cells
express mRNA encoding for several endocytic receptors, including Fc
and complement receptors, the m scavenger receptor, and CD14, a
receptor for LPS (Fig. 6a).
Among these, both FcRI and RIII appeared to be down-modulated by the
treatment with GM-CSF (Fig. 6a). MARCO is a receptor
expressed on both m and DC (F. Granucci and P. Ricciardi-Castagnoli,
manuscript in preparation), which is important in DC for
clearance of bacteria. MARCO transcripts are expressed in untreated
microglial cells and up-regulated >15-fold in GM-CSF, but not in
M-CSF-treated cells (Fig. 6a). Surface staining with an
anti-MARCO Ab confirmed the up-regulation observed by global
transcript analysis (Fig. 6b).
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protein 1 and 2, as well as Cryptidin and
cathelin-like peptides. The majority of these antimicrobial peptides
are down-regulated by GM-CSF treatment, but unaffected by M-CSF. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-like or DC-like phenotype by M-CSF or GM-CSF, respectively
(13). Using global gene expression analysis, we now show
that upon treatment of microglial cells with either GM-CSF or M-CSF, a
large proportion of expressed genes is up-regulated or down-regulated
>2.5-fold (>16%). Such a large-scale shift in gene expression
indicates that neonatal microglial cells undergo a marked
transformation by either GM-CSF or M-CSF, and is further evidence that
neonatal cells exist in a relatively undifferentiated state. It has
been previously shown that in T cells activated by a specific super Ag,
the number of genes differentially expressed is 4.4% at best
(42). Also, in the DC response to different pathogens, the
number of regulated genes is always <10% (43). Thus, it
is likely that microglial cell skewing with either GM-CSF or M-CSF
induces a true lineage differentiation as opposed to a different state
of activation.
Although skewing of microglia toward a m-like phenotype occurs
following exposure to M-CSF within the CNS, both growth factors are
needed for full maturation of other tissue m.
M-CSF-/- mice are largely devoid of alveolar
m and bone osteoclasts (although interestingly, they do retain near
normal CNS microglial populations; Refs. 44 and
45). In contrast, alveolar ms from
GM-CSF-/- animals are normal in number but
exhibit deficits in function. GM-CSF-/-
alveolar m are unable to effectively degrade surfactant and are
defective in their ability to clear pathogens (46).
The biological activity of GM-CSF is initiated by its binding to a
specific receptor formed by an and subunit. The subunit
binds GM-CSF with low affinity (47). The subunit does
not bind GM-CSF alone, but in combination with the subunit will
form a high-affinity GM-CSFR (48). The interplay between
the two GM-CSFRs currently is incompletely understood, although it is
thought that in humans both receptors mediate myeloid lineage
differentiation, but only the high-affinity one mediates cell growth
(49). The low-affinity GM-CSFR was present on
untreated microglial cells and was down-regulated by M-CSF, consistent
with a skewing toward a m phenotype. Interestingly, the
high-affinity receptor appeared only after GM-CSF treatment, again
consistent with a differentiation away from the resting phenotype.
Overall, the majority of transcripts regulated by M-CSF were involved
in lipid, carbohydrate, or protein metabolism (Table I). Of particular
interest was the up-regulation of a series of proteoglycans involved in
tissue remodeling, as well as molecules important in glial-neuron
interaction. In general, it appeared that M-CSF regulates functions
already evident in untreated cells, and related to the role of
microglial cells in brain homeostasis under physiological conditions.
This is in accord with the fact that M-CSF is constitutively present in
the brain parenchyma.
GM-CSF up-regulated a large number of transcripts strongly implicated
in CNS immunity. Interestingly, both neonatal and adult microglial
cells require a multistep activation process to reach full competency
as APCs, wherein a preactivation with GM-CSF is followed by a secondary
activation with a proinflammatory cytokine (19) or CD40
ligand (18). Under physiological conditions in the adult
brain, the only source of GM-CSF is activated T cells, which survey the
brain parenchyma (22, 23). The amount of GM-CSF released
by these cells could be sufficient to induce a preinflammatory state or
precompetency of local microglial cells. If this is followed by a
sufficient number of infiltrating T cells, which either specifically or
by cross-reaction recognize a cognate ligand, an autoimmune process
could ensue. In contrast, if no productive interaction is generated,
the activated T cells will leave the brain without inciting
inflammation (23). The interaction between infiltrating T
cells and microglia has been studied in a model of host vs graft
disease, without interference from infiltrating m/monocytes. In this
model, when a sufficient number of T cells crossed the blood brain
barrier, the cytokine milieu produced was sufficient to activate
resident microglial cells (50).
Costimulatory molecules play a fundamental role in the interaction between T cells and APCs. CD24, expressed on APC, provides a CD28-independent costimulation by interacting with either p-selectin, or in a homotypic interaction, with another CD24 molecule expressed on T cells (51). The functional significance of this newly described costimulatory pathway is as yet not understood, although it appears that CD24, different from members of the B7 family, is not required for the induction phase of an immune response (52). In the animal model of EAE, it was shown that targeted mutation of CD24 does not affect the priming and proliferation of self-reactive T cells; however, it does effect EAE development (35). Also, passive transfer of myelin oligodendrocyte glycoprotein 35-55-specific CD24+ T cells in CD24-/- mice is not sufficient for EAE induction, because a still not well understood homotypic CD24 interaction must occur for extravasation of self-reactive T cells into the CNS (35). Because CD24 expression on microglial cells is dramatically up-regulated by GM-CSF, it seems likely that upon entry of activated GM-CSF-secreting T cells into the brain, CD24 would be readily up-regulated on microglial cells, which in turn would acquire higher costimulatory capacity to sustain T cell expansion.
Chemokines are small chemoattractant molecules, which regulate cell trafficking and homing in every organ. Microglial cells produce a basal level of several chemokines essential for regulating T cell infiltration and patrolling of the brain parenchyma. GM-CSF, much more than M-CSF, up-regulated the transcripts for several of these chemokines, further establishing its role as priming factor for brain immunity.
Microglial cells play a pivotal role in protein degradation and tissue remodeling, which occurs during degenerative, traumatic, and inflammatory CNS conditions. A series of aspartic proteases, cysteine proteases, and metalloproteinases are involved in this process. MMP-9, MMP-11, and MMP-12 are strongly up-regulated with GM-CSF and may participate in tissue remodeling and microglial cell migration. Cathepsin S, L, and F are involved in Ag processing and invariant chain degradation (53, 54, 55), both important for the Ag-presenting role of microglial cells. Both cathepsin F and L were up-regulated by GM-CSF. However, neither GM-CSF nor M-CSF altered the expression of cathepsin S, which was previously shown to degrade myelin basic protein at both neutral and acidic pH, and to be up-regulated by neurotrophic factors in microglial cells (56). Other proteins important for Ag processing and presentation also were up-regulated by GM-CSF, including both class II MHC protein and its associated chaperone invariant chain. Although up-regulation of class II MHC proteins by GM-CSF does not result in a large increase in surface expression, the small degree of up-regulation that was observed was sufficient to promote much stronger T cell proliferation in an MLR assay.
Several substances that are produced in the normal brain account for
the maintenance of an immunosuppressive environment. Normal neuronal
activity and secreted neurotrophin inhibit the microglia expression of
MHC class II and costimulatory molecules, and this tends to counteract
the activation of microglia and astrocytes by IFN- and LPS (7, 8). As observed in this study, microglial cells also produce
TGF- and Tweak, immunosuppressive cytokines that have been shown to
be strongly apoptotic toward activated T cells. Consistent with this
observation, activated T cells entering the brain parenchyma are known
to be more likely to undergo apoptosis than to proliferate. Consistent
with its role as preactivator of microglial cells, GM-CSF decreases
expression of Tweak, and up-regulates proinflammatory cytokines like
IL-1 and along with several chemokines that can promote
leukocyte migration into the brain.
In summary, the global gene expression analysis reported in this study
establishes the molecular basis for the previously recognized role of
GM-CSF in promoting microglial participation in adaptive immune
responses. GM-CSF treatment leads to up-regulation of molecules
involved in T cell costimulation (CD24), chemotaxis (C10, MIP-1,
MIP-1), Ag processing class II MHC, invariant chain, cathepsin F and
L), innate immunity (MARCO), and to down-regulation of
apoptosis-inducing factors (Tweak, TGF-), which together constitute
a differentiation process that prime microglia cells for Ag
presentation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Laura Santambrogio, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail address: Laura_Santambrogio{at}dfci.harvard.edu
3 Abbreviations used in this paper: m, macrophage; DC, dendritic cell; hsp, heat shock response; MIP, m inflammatory protein; MCP, monocyte chemoattractant protein; MMP, matrix metalloproteinase; EAE, experimental allergic encephalitis.
Received for publication April 15, 2002. Accepted for publication June 18, 2002.
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References |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-amyloid-induced interleukin-1, interleukin-6, and nitric oxide production by microglial cells. J. Biol. Chem. 273:20967., IL-10, and CSF-1. J. Leukocyte Biol. 60:502.[Abstract]
-independent antigen presentation function. J. Neuroimmunol. 42:87.[Medline]
-amidating monooxygenase, a neuropeptide processing enzyme. Brain Res. 596:202.[Medline]
chain (hc) responsible for human GM-CSF-induced myeloid cell differentiation. J. Biol. Chem. 273:19411. peptide. Mol. Med. 5:334.[Medline]This article has been cited by other articles:
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  M. S. Arredouani, F. Franco, A. Imrich, A. Fedulov, X. Lu, D. Perkins, R. Soininen, K. Tryggvason, S. D. Shapiro, and L. Kobzik Scavenger Receptors SR-AI/II and MARCO Limit Pulmonary Dendritic Cell Migration and Allergic Airway Inflammation J. Immunol., May 1, 2007; 178(9): 5912 - 5920. [Abstract] [Full Text] [PDF]
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E. D. Ponomarev, L. P. Shriver, K. Maresz, J. Pedras-Vasconcelos, D. Verthelyi, and B. N. Dittel GM-CSF Production by Autoreactive T Cells Is Required for the Activation of Microglial Cells and the Onset of Experimental Autoimmune Encephalomyelitis J. Immunol., January 1, 2007; 178(1): 39 - 48. [Abstract] [Full Text] [PDF]
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J. I. Saada, I. V. Pinchuk, C. A. Barrera, P. A. Adegboyega, G. Suarez, R. C. Mifflin, J. F. Di Mari, V. E. Reyes, and D. W. Powell Subepithelial Myofibroblasts are Novel Nonprofessional APCs in the Human Colonic Mucosa J. Immunol., November 1, 2006; 177(9): 5968 - 5979. [Abstract] [Full Text] [PDF]
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 T. L. Bonfield, C. M. Swaisgood, B. P. Barna, C. F. Farver, M. S. Kavuru, and M. J. Thomassen Elevated gelatinase activity in pulmonary alveolar proteinosis: role of macrophage-colony stimulating factor J. Leukoc. Biol., January 1, 2006; 79(1): 133 - 139. [Abstract] [Full Text] [PDF]
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S. Jozefowski, M. Arredouani, T. Sulahian, and L. Kobzik Disparate Regulation and Function of the Class A Scavenger Receptors SR-AI/II and MARCO J. Immunol., December 15, 2005; 175(12): 8032 - 8041. [Abstract] [Full Text] [PDF]
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 R. Alarcon, C. Fuenzalida, M. Santibanez, and R. von Bernhardi Expression of Scavenger Receptors in Glial Cells: COMPARING THE ADHESION OF ASTROCYTES AND MICROGLIA FROM NEONATAL RATS TO SURFACE-BOUND {beta}-AMYLOID J. Biol. Chem., August 26, 2005; 280(34): 30406 - 30415. [Abstract] [Full Text] [PDF]
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I. Potolicchio, G. J. Carven, X. Xu, C. Stipp, R. J. Riese, L. J. Stern, and L. Santambrogio Proteomic Analysis of Microglia-Derived Exosomes: Metabolic Role of the Aminopeptidase CD13 in Neuropeptide Catabolism J. Immunol., August 15, 2005; 175(4): 2237 - 2243. [Abstract] [Full Text] [PDF]
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 F. Granucci, F. Petralia, M. Urbano, S. Citterio, F. Di Tota, L. Santambrogio, and P. Ricciardi-Castagnoli The scavenger receptor MARCO mediates cytoskeleton rearrangements in dendritic cells and microglia Blood, October 15, 2003; 102(8): 2940 - 2947. [Abstract] [Full Text] [PDF]
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A. Monsonego, J. Imitola, V. Zota, T. Oida, and H. L. Weiner Microglia-Mediated Nitric Oxide Cytotoxicity of T Cells Following Amyloid {beta}-Peptide Presentation to Th1 Cells J. Immunol., September 1, 2003; 171(5): 2216 - 2224. [Abstract] [Full Text] [PDF]
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T. M. C. Hornell, G. W. Beresford, A. Bushey, J. M. Boss, and E. D. Mellins Regulation of the Class II MHC Pathway in Primary Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor J. Immunol., September 1, 2003; 171(5): 2374 - 2383. [Abstract] [Full Text] [PDF]
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