From Neuroscience Research Laboratories, Department
of Psychiatry and Behavioral Sciences, Stanford University School of
Medicine, Stanford, California 94305-5485 and ¶ Scios,
Incorporated, Sunnyvale, California 94086
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ABSTRACT |
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In Alzheimer's disease (AD), a chronic cerebral
inflammatory state is thought to lead to neuronal injury. Microglia,
intrinsic cerebral immune effector cells, are likely to be key in the
pathophysiology of this inflammatory state. We showed that macrophage
colony-stimulating factor, a microglial activator found at increased
levels in the central nervous system in AD, dramatically augments
-amyloid peptide (
AP)-induced microglial production of
interleukin-1, interleukin-6, and nitric oxide. In contrast,
granulocyte macrophage colony-stimulating factor, another hematopoietic
cytokine found in the AD brain, did not augment
AP-induced
microglial secretory activity. These results indicate that increased
macrophage colony-stimulating factor levels in AD could magnify
AP-induced microglial inflammatory cytokine and nitric oxide
production, which in turn could intensify the cerebral inflammatory
state by activating astrocytes and additional microglia, as well as
directly injuring neurons.
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INTRODUCTION |
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According to the inflammatory hypothesis of Alzheimer's disease
(AD),1 chronic cerebral
inflammation results in injury to neurons, contributing over time to
cognitive decline. Neuronal injury is hypothesized to result from the
direct effects of inflammatory effectors such as cytokines and
activated complement, or indirect effects such as increased production
of neurotoxic reactive oxygen and nitrogen species in response to
cytokines or other inflammatory stimuli (1, 2). This hypothesis is
supported by epidemiological studies, which indicate that
anti-inflammatory medications may protect against AD (3-6). In the
present study, we demonstrate that macrophage colony-stimulating factor
(M-CSF), a cytokine which is increased in the central nervous system in
AD (7), dramatically augments -amyloid peptide (
AP)-induced
production of pro-inflammatory interleukin-1 (IL-1), interleukin-6
(IL-6), and nitric oxide (NO) by microglial cells.
Microglia are likely to have a pivotal role in inflammatory neuronal
injury in AD. As intrinsic immune effector cells of the brain,
microglia are potent mediators of cerebral inflammation in a variety of
disease states (8, 9). AP induces cultured microglia to produce
agents with the potential to directly or indirectly injure neurons,
including inflammatory and chemotactic cytokines (10, 11), nitric oxide
(12-14), and reactive oxygen species (12, 15). However, previously
reported
AP-induced increases in microglial production of these
factors have been of limited magnitude, on the order of only 2-5-fold
greater than control levels. It is difficult to reconcile this weak
in vitro microglial response to
AP with the hypothesis
that
AP activation of microglia is important in AD
pathophysiology.
One reason for the limited response of cultured microglia to AP may
be that important costimulatory agents present in AD brain have not
been taken into consideration in prior reports. The extracellular
environment surrounding neuritic plaques in AD brain is rich in a
variety of pro-inflammatory agents including cytokines (2), which are
likely to augment the effects of
AP on microglia. It has been shown
that interferon-
, phorbol ester, and lipopolysaccharide all augment
the effects of
AP on microglia and monocytic cells (13-16).
However, none of these augmenting stimuli has a physiologic role in AD.
Results showing large synergistic increases in
AP-induced microglial
activity in cultures cotreated with these agents may have no direct
relevance to AD.
We examined the effect of M-CSF (also called colony-stimulating factor
1 (CSF-1)) on AP-induced cytokine and NO production by cultured
microglia. M-CSF is an important regulator of mononuclear phagocyte
development and function throughout the body (17). In the brain, M-CSF
is expressed by neurons, astrocytes, and endothelial cells (7, 18-22),
where it induces proliferation, migration, and activation of microglia
(23-26). M-CSF treatment of microglia also induces increased
expression of macrophage scavenger receptors (7), which mediate
microglial interactions with
AP (27, 28).
AP binds to neuronal
receptors for advanced glycation end products to increase neuronal
M-CSF expression (7), which causes further microglial activation. In AD
brain, there is increased immunoreactivity for the M-CSF receptor
(c-fms) on microglia (29), neurons in AD show labeling with
M-CSF antibodies, and M-CSF levels in AD cerebrospinal fluid are 5-fold
greater than in controls (7). Thus, M-CSF represents a potent
microglial activator relevant to AD pathophysiology.
We hypothesized that in AD, M-CSF activates microglia to augment
AP-induced production of inflammatory cytokines and NO, which in
turn promote additional inflammation and may directly injure nerve
cells. To test this hypothesis, we examined the effects of combined
M-CSF and
AP treatment on production of interleukin-1, interleukin-6, and NO by the BV-2 immortalized murine microglial cell
line.
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EXPERIMENTAL PROCEDURES |
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-Amyloid Peptides--
Synthetic
AP 1-40 and
AP 40-1
were purchased from Bachem California (Torrance, CA). Peptides were
aggregated by resuspending at 2 mg/ml in endotoxin-free water (Sigma),
holding at 4 °C for 60 h, incubating at 37 °C for 8 h
with gentle mixing every 2 h, and then storing at 4 °C until
use.
Cytokines--
Recombinant mouse M-CSF, recombinant mouse
granulocyte macrophage-CSF (GM-CSF), and recombinant mouse
interleukin-3 (IL-3) were purchased from R & D (Minneapolis, MN).
Cytokines were resuspended in sterile tissue culture-grade
phosphate-buffered saline (Sigma) with 0.1% tissue culture-grade
bovine serum albumin (Sigma), aliquoted, and stored at 80 °C until
ready for use.
Cell Culture--
The BV-2 immortalized microglial cell line was
cultured as described previously (30). BV-2 cells were detached from
the substrate by gentle pipetting and reseeded at 1 × 105 cells in 500 µl of medium per well in a 48-well
tissue culture dish. After an additional 24 h in culture, cells
were used for experimentation by washing two times in serum-free medium
and then applying fresh serum-free medium containing AP and/or
M-CSF. All treatments were performed for 24 h, after which
conditioned media were collected, centrifuged at 600 × g at 4 °C for 10 min, and stored at
80 °C until
ready for analysis. Each experiment included triplicate cultures for
each treatment condition, and each experiment was replicated on
separate occasions a minimum of two additional times.
Cell Counting-- After harvesting conditioned medium for cytokine or NO assays, BV-2 cells were detached by trypsinization and resuspended in fresh medium. Aliquots of cells from each well were counted three times in a hemocytometer using trypan blue exclusion, and counts were averaged. For each treatment condition, triplicate wells were counted, and values were averaged. All cytokine and NO results were adjusted for the number of viable BV-2 cells present for each treatment condition.
IL-1 and IL-6 ELISA--
Mouse IL-1
and IL-6 in
conditioned media were determined using ELISA kits according to the
manufacturer's instructions (Endogen, Woburn MA). Each sample was
assayed in duplicate, and values from duplicates were averaged. Means
for each treatment condition were calculated, along with standard
errors. To increase signal intensity, poly-horseradish peroxidase (RDI,
Flanders, NJ) was substituted for streptavidin-horseradish peroxidase
in the IL-1
ELISA. Absorbency at 450 nm was determined using a
Molecular Devices (Sunnyvale, CA) microplate reader. Data were analyzed
using the SOFTmax 2.32 program (Molecular Devices).
Nitrite Assay-- Nitrite, an end product of NO oxidation, was used as an indicator of NO production by microglial cells (31). Nitrite in conditioned media was determined using the Griess assay according to the manufacturer's instructions (Promega). Absorbency was determined at 550 nm using a Dynatech Laboratories MR700 microplate reader (Dynex, West Sussex, UK).
Reverse Transcription and Polymerase Chain Reaction (RT-PCR) for
Inducible Nitric Oxide Synthase mRNA--
RT-PCR was used to
determine the effects of M-CSF and AP on inducible nitric oxide
synthase (iNOS) mRNA in BV-2 cells. Total RNA was extracted from
BV-2 cells using the Trizol reagent (Life Technologies, Inc.) according
to the manufacturer's instructions. Reverse transcription was
performed using 1 µg of total RNA and Superscript II RNase
H
reverse transcriptase (Life Technologies, Inc.) primed
with random hexamers according to the manufacturer's instructions. PCR
was performed on cDNA using primers for mouse iNOS (32) and 28 cycles of PCR amplification consisting of 94 °C for 30 s,
57 °C for 30 s, and 72 °C for 45 s. To control for
differences in total RNA concentration among samples, mRNA levels
for mouse hypoxanthine phosphoribosyl transferase were determined with
RT-PCR as described previously (33). PCR products were visualized on
2.5% agarose gels with ethidium bromide staining.
M-CSF Receptor Blocking--
To demonstrate specificity of the
M-CSF effect, BV-2 cells were reacted for 1 h with a monoclonal
blocking antibody against the mouse M-CSF receptor, c-fms,
at a concentration of 20 µg/ml (gift from Drs. R. Shadduck and G. Gilmore). This reagent has been shown previously to specifically block
the effects of mouse M-CSF on macrophages (34, 35). Sister cultures
were reacted with a subclass-matched mouse IgG1 control
antibody (Sigma) also at 20 µg/ml. After 1 h, medium was
removed, and the cells were treated with 22 µM
AP
1-40 or 50 ng/ml M-CSF, or 22 µM
AP 1-40 plus 50 ng/ml M-CSF, with or without the c-fms antibody or the
control antibody. After 24 h, conditioned media were harvested and
cleared by centrifugation, and IL-1
was quantified using ELISA as
described above.
GM-CSF Receptor Phenotyping--
To demonstrate the expression
of GM-CSF receptor and
subunits by BV-2 cells, RT-PCR was
performed on BV-2 cell total mRNA. For the
subunit, primers
were designed using the Genbank cDNA sequence MUSCLNYSIM (36) for
the mouse GM-CSF low affinity receptor subunit. The forward primer was
a 21-mer, which spanned nucleotides 508-528, whereas the reverse
primer was a 21-mer spanning nucleotides 931-951. Thirty-five cycles
of PCR amplification were performed consisting of 95 °C for 1 min,
61 °C for 1 min, and 72 °C for 2 min, 20 s. This resulted in
a PCR product of 444 bp. For the
subunit (AI2CB cDNA), the
primers and PCR protocol of Fung et al. (37) were used,
resulting in a PCR product of 325 bp. PCR products were visualized on
1.5% agarose gels using ethidium bromide staining. As a positive
control for GM-CSF receptor expression, total RNA harvested from mouse
bone marrow cells, which had been stimulated with 50 ng/ml GM-CSF for
24 h, was subjected to the same RT-PCR phenotyping protocol as the
BV-2 cell RNA.
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RESULTS |
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The BV-2 immortalized microglial cell line has been extensively
characterized and has many of the features of primary microglia (38,
39), but it is devoid of immunologically active cells such as
astrocytes commonly found in primary microglial cultures. Further, BV-2
cells express receptors for advanced glycation end products, which bind
AP and induce signal transduction, and BV-2 cells treated with M-CSF
show chemotaxis and other indications of activation (7, 40).
In the present study, treatment of BV-2 microglial cells for 24 h
with 11 µM AP 1-40 resulted in an increase in IL-1
production of about three times control levels (Fig.
1). However, when BV-2 cells were
simultaneously treated with M-CSF (25 or 50 ng/ml) and 11 µM
AP 1-40, there was a large increase in IL-1
production (approximately 70 times control levels in the experiment
illustrated in Fig. 1; these results were replicated in three other
independent experiments). M-CSF alone had little effect on BV-2 IL-1
production. A similar augmenting effect of
AP 1-40 and M-CSF on
IL-1
production was obtained with a
AP concentration of 22 µM (Fig. 4).
AP 40-1, a reverse sequence control
peptide that was prepared in the same manner as
AP 1-40, had little
effect either alone or in combination with M-CSF. The augmenting effect
of M-CSF on
AP-induced IL-1
production by BV-2 cells was
inhibited by a monoclonal antibody to the mouse M-CSF receptor,
c-fms (Table I), but not by a
subclass-matched control antibody.
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Simultaneous treatment of BV-2 cells with M-CSF and AP 1-40 also
induced a very large increase in IL-6 production (Table II). Treatment of BV-2 cells with M-CSF
alone or
AP alone resulted in modest increases in mouse IL-6 in
conditioned media. However, the combination of M-CSF and
AP 1-40
(22 µM) resulted in an increase in IL-6 production by
BV-2 cells that was over 200-fold greater than control values.
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M-CSF also augmented AP effects on NO (nitrite) production.
Treatment of BV-2 cells with
AP 1-40 (11 µM) or M-CSF
(50 ng/ml) alone had little effect on nitrite in conditioned medium
(Fig. 2). In contrast, simultaneous
treatment of BV-2 cells with
AP 1-40 and M-CSF resulted in nitrite
levels in conditioned medium that were about 30-fold greater than
control values. The control peptide
AP 40-1, either alone or in
combination with M-CSF, had little effect on nitrite in conditioned
medium. The augmenting effect of combined
AP and M-CSF treatment on
microglial NO production was also detected at the mRNA level.
Treatment with 22 µM
AP 1-40 in combination with 50 ng/ml M-CSF for 18 h resulted in a larger increase in iNOS
mRNA than either agent alone (Fig.
3). The control peptide
AP 40-1 did
not augment M-CSF effects on iNOS expression.
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To further test for the specificity of M-CSF in augmenting AP
effects on microglia, BV-2 cells were treated with the hematopoietic cytokine GM-CSF alone or in combination with
AP 1-40 (22 µM). Unlike M-CSF, GM-CSF did not augment
AP-induced
IL-1
expression (Fig. 4). Likewise,
cotreatment of BV-2 cells with
AP and the microglial activator IL-3
did not result in an increase in IL-1
production (data not shown).
Although GM-CSF did not augment
AP-induced cytokine secretion by
BV-2 cells, this was not because of a lack of GM-CSF receptors. RT-PCR
phenotyping of BV-2 cells showed the expression of mRNAs for both
the
and
subunits of the GM-CSF receptor (Fig.
5).
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Neither M-CSF nor GM-CSF alone or in combination with AP resulted in
proliferation of BV-2 cells at the doses, cell density, and treatment
duration used in the present study. At the end of a representative 24-h
experiment, the mean number of control cells was 1.7 × 105/ml (S.E. = 0.2), whereas for 50 ng/ml M-CSF, the mean
number was 1.5 × 105/ml (S.E. = 0.2), and for 1000 units/ml GM-CSF, the mean number was 1.3 × 105/ml
(S.E. = 0.2). For M-CSF plus 11 µM
AP 1-40, the mean
number of cells was 1.2 × 105/ml (S.E. = 0.2),
whereas for GM-CSF plus
AP 1-40, the mean number was 1.4 × 105/ml (S.E. = 0.2).
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DISCUSSION |
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The results presented here suggest that M-CSF augments
AP-induced microglial inflammatory cytokine and NO production in AD. Simultaneous treatment of BV-2 microglia with M-CSF and
AP resulted in large synergistic increases in IL-1
, IL-6, and NO in conditioned media, which were greater than increases due to either agent alone. Interleukin-1 is expressed early in AD (41, 42) primarily by microglia
(43), and through autocrine and paracrine mechanisms could further
augment microglia-mediated inflammation and neuronal injury in AD.
Interleukin-6, another microglial cytokine (44), is also increased in
AD brain (45) and in the serum of AD patients (46). Increased IL-6
expression may induce inflammatory changes, which injure neurons (47).
There is evidence that NO, an important inflammatory effector produced
by rodent and human microglia (48), is present at increased levels in
AD brain, resulting in nitration of proteins and other abnormal
cellular changes (49).
Our results and the results of prior studies indicate that in AD there
is a self-amplifying pathophysiologic cascade involving microglia,
astrocytes, and neurons and the key AD cytokines M-CSF, IL-1, and IL-6
(Fig. 6). M-CSF levels are increased in
the cerebrospinal fluid of AD patients, and M-CSF antibodies label
neurons in AD brain (7). Further, expression of the M-CSF receptor
c-fms is increased on microglia in AD brain (29), which may
sensitize these cells to M-CSF effects. Astrocytes, an important source of M-CSF in the brain, can be induced to secrete M-CSF by IL-1 (20). We
hypothesize that in AD, AP induces microglia to secrete small
amounts of IL-1, as our results and the results of others indicate (10,
16, 50). IL-1 then induces astrocytes to express M-CSF, which augments
AP-induced expression of IL-1 by microglia, resulting in further
M-CSF expression by astrocytes. In addition, microglial IL-1
self-activates microglia via autocrine and paracrine effects. Neurons
themselves may also secrete M-CSF in response to
AP (7), which may
further activate microglia.
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IL-6 promotes astrogliosis (51) and activates microglia (47). Increased
IL-6 found in AD brain could come from microglia, astrocytes, or both.
Our results suggest that M-CSF and AP would induce microglial IL-1
and IL-6 production in AD. IL-1 causes astrocytes to express IL-6 (52),
so microglial IL-1 induced by M-CSF and
AP would promote astroglial
IL-6 expression. Through pro-inflammatory effects, IL-6 is thought to
contribute to neurodegeneration in AD (47, 53).
Although AP alone may increase microglial NO production (13, 54), in
the presence of M-CSF
AP-induced microglial NO production is
dramatically augmented. Contrary to prior findings, recent evidence
indicates that human microglia can produce NO (48), so results obtained
with murine cells are likely to closely model the human system.
Microglial NO, either directly or through its highly toxic derivative
peroxynitrite, would injure neurons in AD (14, 49, 55). NO may also
induce additional IL-1 expression (56, 57), which in turn would promote
astroglial M-CSF expression, ultimately resulting in further
AP-induced NO production. Astrocytes, too, produce NO, and IL-1 can
induce astrocyte iNOS (58, 59). Thus, in AD, microglial IL-1 induced by
AP and M-CSF would augment NO neurotoxicity by activating astrocyte
iNOS. Finally, microglia are likely to generate neurotoxic reactive
oxygen species in response to
AP (12).
In contrast to M-CSF, the hematopoietic cytokines GM-CSF, present in
the brain in AD (60), and IL-3 did not augment AP-induced microglial
cytokine and NO expression in our studies. Both GM-CSF and IL-3 can
induce microglial activation (61). Thus, the synergistic effect of
M-CSF and
AP cannot be due to nonspecific microglial activation.
Whereas GM-CSF and IL-3 share a common receptor subunit (
c) and elements of signal transduction (62), the M-CSF
receptor, c-fms, is distinct (63). Indeed, our results
indicate that blockade of c-fms attenuates M-CSF
augmentation of
AP effects on microglia. Differences in receptor
function and signal transduction between M-CSF and other hematopoietic
cytokines may account for the unique effects of M-CSF on
AP-treated
microglia. The absence of an augmenting effect of GM-CSF cannot be the
result of a receptor deficiency, as BV-2 cells were shown to express
mRNA for both subunits of the GM-CSF receptor. M-CSF augmentation
of
AP effects is not secondary to proliferation, as neither M-CSF
nor GM-CSF induced BV-2 proliferation at the doses, cell density, and
treatment duration we employed.
In conclusion, our results indicate that M-CSF may have an important
role in the pathophysiology of AD by augmenting the microglial response
to AP. Further, these results support the hypothesis that
inflammatory effectors are an integral part of neuropathologic change
in AD rather than being nonspecific signs of brain injury. Future
studies should further clarify the relative roles of astrocytes and
neurons in generating M-CSF in AD, fully phenotype microglia activated
by combined M-CSF and
AP treatment, and determine the effects of
microglia activated by
AP and M-CSF on neurons.
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ACKNOWLEDGEMENTS |
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Drs. Richard Shadduck and Gary Gilmore generously provided the c-fms blocking antibody. We thank Edward Kao, Karen Schmidt, Fei Fei Zhao, and Angela Nguyen for technical assistance. The late Dr. Virginia Bocchini provided the BV-2 cells. The mouse bone marrow cells were a gift from Dr. Yafei Hou.
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FOOTNOTES |
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* This work was supported by the National Institute of Mental Health and Eli Lilly.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 650-725-0565; Fax: 650-498-7761; E-mail: greer.murphy{at}stanford.edu.
The abbreviations used are:
AD, Alzheimer's
disease; M-CSF, macrophage colony-stimulating factor; AP,
-amyloid peptide; IL-1, interleukin-1; IL-6, interleukin-6; NO, nitric oxide; GM-CSF, granulocyte macrophage colony-stimulating factor; iNOS, inducible nitric oxide synthase; bp, base pair(s); ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse
transcriptase-polymerase chain reaction.
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REFERENCES |
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