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*
Laboratory of Molecular Immunoregulation, Division of Basic Sciences, and
Intramural Research Support Program, Scientific Applications International Corporation-Frederick, National Cancer Institute, Frederick, MD 21702; and
Rega Institute, University of Leuven, Leuven, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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peptide which is a pathogenic agent in
Alzheimer’s disease. Primary murine microglial cells also expressed
FPR1 and FPR2 genes, but similar
to N9 cells, exhibited FPR2-mediated activation only after LPS
treatment. In contrast to its effect on the function of FPR2, LPS
reduced N9 cell binding and biological responses to the chemokine
stromal cell-derived factor-1
. Thus, LPS selectively modulates the
function of chemoattractant receptors in microglia and may promote host
response in inflammatory diseases in the CNS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the past few years, substantial interest has been generated by two
G protein-coupled receptors that were originally identified as
receptors for the bacterial chemotactic peptide fMLP
(5, 6, 7). In humans, the prototype receptor formyl peptide
receptor (FPR) is activated by low concentrations (in picomolar to
low-nanomolar range) of fMLP and is considered a high-affinity fMLPR.
An FPR variant, FPR-like 1 (FPRL1), interacts with high concentrations
(in the micromolar range) of fMLP and is defined as a low-affinity
fMLPR (5, 6, 7). FPR1 and FPR2, the murine counterparts of
human FPR and FPRL1, respectively, have been shown to interact
with fMLP in a similar pattern as human receptors (8, 9).
Although the biological role of these receptors has not been fully
defined, mice depleted of FPR1 were more susceptible to bacterial
infection (10). In contrast, FPRL1 has recently been found
to interact with a variety of novel and host-derived chemotactic
agonists, including peptide domains derived from HIV-1 envelope
proteins (7) and at least three amyloidogenic
polypeptides, the serum amyloid A (11), the 42 aa form of
amyloid (A42) (12), and a
peptide fragment of human prion protein (13). These
findings suggest that FPRs may play important roles in infection,
inflammation, and amyloidogenic diseases. Both FPR and FPRL1, or their
murine analogs FPR1 and FPR2, are highly expressed by peripheral blood
phagocytic leukocytes. In addition, FPRL1 appears to express in a
broader range of cell types including T lymphocytes and cells of the
nonhemopoietic origin (7). Activation of either FPR (FPR1)
or FPRL1 (FPR2) on the cells by agonists results in a series of
signaling events that lead to cell adhesion, chemotaxis, phagocytosis,
release of reactive oxygen intermediates, and production of
proinflammatory cytokines (5, 6, 7).
Microglial cells are of the myeloid lineage and, as major phagocytic
cells in the CNS, play a pivotal role in inflammation and
neurodegenerative diseases (14, 15). Like peripheral blood
monocytes, microglial cells express several chemokine receptors and the
receptor for C5a. Thus, these cells migrate in response to selected
chemokine ligands and C5a in vitro and may accumulate at sites of
inflammatory and immunological responses in the CNS, where
tissue-derived chemotactic factors are likely to be elevated
(16, 17, 18). However, unlike peripheral blood monocytes,
resting microglial cells lack the capacity to migrate in response to
the bacterial chemotactic peptide fMLP (19). The lack of
responsiveness to fMLP raises the question as to whether microglial
cells can contribute to host defense in the CNS by increasing their
motility toward bacterial chemotactic peptides. In contrast, we have
observed that A42, which is a pathogenic agent
in Alzheimer’s disease (AD), activates FPRL1 in human peripheral blood
mononuclear phagocytes. We also have detected FPRL1 gene expression in
CD11b+ cells infiltrating the brain lesions of AD
(12). However, it is not clear whether microglial cells
indeed express FPRL1 and can be chemoattracted by its agonists
including A42. Therefore, we studied the
expression and function of murine homologs of FPR (FPR1) and FPRL1
(FPR2) in mouse microglial cells. In this study, we report that
the bacterial endotoxin (LPS) stimulates microglial cells to develop a
potent chemotactic response to peptide agonists in the concentration
range that typically activated the low fMLPR FPR2. LPS concomitantly
down-regulated the microglial cell response to the stromal cell-derived
factor (SDF)-1, a chemokine that is crucial for homeostasis and
development, but is not implicated for inflammation (3, 4). Our results suggest that LPS may selectively stimulate
microglial response to noxious agents in the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fMLP, LPS, and genistein were purchased from Sigma-Aldrich (St.
Louis, MO). Mouse SDF-1 was purchased from PeproTech (Rocky Hill,
NJ). The chemotactic peptide WKYMVm (designated W peptide)
(20) was synthesized and purified by the Department of
Biochemistry, Colorado State University (Fort Collins, CO). The
synthetic 42 aa form of A42 peptide was from
California Peptide Research (Napa, CA).
125I-SDF-1 and
[3H]fMLP were obtained from DuPont NEN Life
Science (Boston, MA). V3 peptide (21) was kindly provided
by the National Institutes of Health AIDS Research and Reference
Reagents Program (Bethesda, MD) and was also synthesized by F-moc
chemistry on a 433-A peptide synthesizer (Applied Biosystems, Foster
City, CA) and purified by reversed phase-HPLC on a Resource RPC column
(Amersham Biosciences, Uppsala, Sweden).
Primary murine microglial cells were isolated from 1-day-old newborn
BALB/c mice (National Cancer Institute-Frederick Animal Facility,
Frederick, MD) according to the established procedures
(16).4
Typically, we were able to obtain 
1 million cells from 8 to 10
newborn mouse brains after first-round harvest. The cells were >85%
positive for MAC-1 (CD11b) (Table I
). By immunofluorescence, the primary
microglial cells were positive for staining with either DiI-acetylated
low-density lipoprotein (DiI-AcLDL) (Molecular Probes, Eugene, OR) or
FITC-isolectin B4 (Vector Laboratories, Burlingame, CA). A murine
macrophage cell line, RAW264.7, was positive only for DiI-AcLDL
(Fig. 1). The N9 murine microglial cell
line was a kind gift from Dr. P. Ricciardi-Castagnoli (Universita Degli
Studi di Milano-Bicocca, Milan, Italy). These cells express typical
markers of resting mouse microglia (Table I and Fig. 1) and have been
extensively used as representatives of mouse microglial cells
(22). The cells were grown in IMDM supplemented with 5%
heat-inactivated FCS, 2 mM of glutamine, 100 U/ml penicillin, 100
µg/ml streptomycin, and 50 mM of 2-ME. HEK 293 cells transfected with
murine FPR1 (FPR1/293) and FPR2 (FPR2/293) were kind gifts from Drs. P.
Murphy and J. Gao (National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD). The cells were
maintained in DMEM with 10% FCS, antibiotics, and 800 µg/ml
G418 (Life Technologies, Rockville,
MD).
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Chemotaxis assays for HEK 293 cells transfected with murine FPR and microglial cells were performed with 48-well chemotaxis chambers (NeuroProbe, Cabin John, MD) as described previously (20). Polycarbonate filters (8-µm pore size, incubated for 90 min at 37°C) were used for measurement of microglial migration. For receptor-transfected HEK 293 cells, 10-µm pore size, collagen type I-coated filters (incubated for 300 min) were used. The results are expressed as the mean ± SD of migrated cells in three high-power fields in triplicate samples, or as chemotaxis index (CI) which represents the fold increase in the number of migrated cells in response to chemoattractants over the spontaneous cell migration (to control medium).
Calcium mobilization
Calcium mobilization was measured by incubating 1 x 107 cells/ml in loading medium (RPMI 1640 containing 10% FCS, 2 mM of glutamine) with 2.5 µM of fura 2-AM (Molecular Probes) for 60 min at room temperature. The dye-loaded cells were washed and resuspended in saline buffer (138 mM of NaCl, 1 mM of KCl, 1 mM of CaCl2, 10 mM of HEPES (pH 7.4), 5 mM of glucose, 0.1% BSA) at a density of 2 x 106/ml. The cells were then transferred into cuvettes (4 x 106 cells in 2 ml, 0.4 x 106 for primary cells) which were placed in a fluorescence spectrometer (PerkinElmer, Beaconsfield, U.K.). Stimulants at different concentrations were added in a volume of 20 µl to each cuvette at the indicated time points. The intensity of fluorescence was calculated based on the ratio at 340 and 380 nm with a FL WinLab program (PerkinElmer).
RT-PCR
Total RNA was extracted from cells with an RNeasy Mini kit and
depleted of contaminating DNA with RNase-free DNase (Qiagen, Valencia,
CA) according to the manufacturer’s instructions. Sense
oligonucleotide primer, 5'-CAT GAA CAA GTC TGC AGT GAA CCT-3' and
antisense primer, 5'- AGG TTT ATG TCT ATT ACA GTA TAT-3' were designed
to amplify FPR1 with a 330-bp product. For amplification of FPR2 mRNA,
the following primers were used to yield a 268-bp product: sense,
5'-TCT ACC ATC TCC AGA GTT CTG TGG-3' and antisense, 5'-TTA CAT CTA CCA
CAA TGT GAA CTA-3'. Specific primers for mouse CXCR4 were: sense,
5'-GGC TGT AGA GCG AGT GTT GC-3' and antisense, 5'-GTA GAG GTT GAC AGT
GTA GAT-3', which yield a product of 390 bp. RT-PCR was performed with
0.5 µg of total RNA for each sample (High Fidelity ProSTAR HF system;
Stratagene, Kingsport, TN), consisting of a 15 min reverse
transcription at 37°C, 1 min inactivation of Moloney murine leukemia
virus reverse transcriptase at 95°C, 40 cycles of denaturing at
95°C (45 s), annealing at 55°C (52°C for CXCR4; 45 s), and
extension at 72°C (1 min), with a final extension for 10 min at
72°C. Primers for murine -actin gene were used as controls
(Stratagene).
Binding assays
Binding of SDF-1 to N9 cells was performed by incubating
duplicate samples of cells (2.5 x 106
cells/sample) with various concentrations of unlabeled SDF-1 in
binding medium (RPMI 1640, 1% BSA, 25 mM of HEPES, and 0.05%
NaN3), in the presence of a single concentration
of 125I-SDF-1. After incubation for 40 min at
room temperature, the cells were washed once with ice-cold PBS and
centrifuged through a 10% sucrose/PBS cushion. The tube tips
containing cell pellets were cut and the cell-associated radioactivity
was measured in a gamma counter. To determine binding of fMLP to
microglial cells, a single concentration of
[3H]fMLP (1 nCi) was added simultaneously with
different concentrations of unlabeled fMLP to 200 µl of cell
suspension (2.5 x 106 cells for N9,
0.1 x 106 for primary cells) in duplicate
samples. The samples were incubated under constant rotation for 20 min
at 37°C. After incubation, the samples were filtered onto GF/C fiber
discs (Whatman, Kent, U.K.) on a 12-well manifold followed by extensive
washing with ice-cold PBS. The paper discs were air-dried at 65°C and
were counted for beta emission in a liquid scintillation mixture. The
results were analyzed with a Mac-LIGAND program (courtesy of Dr.
P. J. Munson, National Institutes of Health).
Statistical analysis
All experiments were performed at least three times and the results presented are from representative experiments. The significance of the difference between test and control groups was analyzed using the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first examined the ligand specificity of murine FPR1 and FPR2
by using several peptide agonists known to activate human counterpart
receptors. As predicted, the bacterial fMLP induced significant
migration of FPR1/293 cells at concentrations in the picomolar to low
nanomolar range (Fig. 2A),
while micromolar concentrations of fMLP were required to activate
FPR2/293 cells. W peptide, which activates human FPR with preference
for the low-affinity fMLPR FPRL1 (20), also induced
migration of both FPR1/293 and FPR2/293 cells with a higher (100
fold) efficacy for FPR2/293 cells (Fig. 2B). In addition, V3
peptide, a chemotactic component of the HIV-1 envelope gp120
(21), and A42, an AD-associated
peptide (12), which are both agonists for human FPRL1,
only induced significant migration of FPR2/293 cells (Fig. 2, C and D). These results indicate that murine
counterpart receptors display similar agonist specificity as human FPR
and FPRL1 (12, 21).
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We next examined the expression of genes coding for FPR1 and FPR2
in a more readily available and well-established murine microglial cell
line, N9, by RT-PCR. As shown in Fig. 3A, nonstimulated N9 cells
expressed transcripts for FPR1 and FPR2, which were significantly
enhanced when the cells were exposed to LPS. The maximal effect of LPS
on receptor gene expression was obtained at 300 ng/ml with a 24 h
incubation period (Fig. 3A; data not shown). In parallel
experiments, we detected the expression of the gene encoding CXCR4, a
specific receptor for the chemokine SDF-1, in N9 cells. However,
unlike its effect on the expression of fMLPR genes, LPS did not
significantly affect the mRNA level of CXCR4 in N9 cells, suggesting
that LPS selectively enhanced the gene expression of fMLPRs.
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with high affinity
(Kd = 1 nM, 4000 sites/cell; Fig. 3
B). However, LPS-treated N9 cells developed a species of
specific fMLP binding sites with an estimated
Kd value of 200 nM (Fig. 3C), which is in the range comparable to that for the
low-affinity fMLPR FPRL1 in humans or FPR2 in mice (5, 6, 7).
These cells additionally expressed a large number of fMLP binding sites
with an extremely low affinity (Kd =
200 µM). In contrast, LPS treatment reduced SDF-1 binding sites on
N9 cells by >90% (280 sites/cell, Kd
= 5 nM; Fig. 3D). These results suggest that LPS
differentially regulated the expression of binding sites for fMLP and
SDF-1 on the surface of N9 cells. Function of the fMLPRs on N9 cells
To evaluate the function of fMLPR expressed by N9 cells, we
measured Ca2+ mobilization elicited by various
chemotactic agonists. The unstimulated N9 cells mobilized
Ca2+ only in response to the chemokine SDF-1,
but not to the fMLPR agonists tested (Fig. 4A). In contrast, while
SDF-1 failed to induce Ca2+ flux in
LPS-treated N9 cells, challenge of these cells with fMLP and W peptide
resulted in significantly increased mobilization of the
Ca2+ (Fig. 4B). However, the
concentrations required for fMLP to induce Ca2+
mobilization in LPS-treated N9 cells were in the micromolar range,
suggesting the activation of a low-affinity fMLPR. Moreover,
LPS-treated N9 cells also mobilized Ca2+ in
response to A42 and V3 peptide, which
specifically activated the low-affinity fMLPR FPR2 (Fig. 4B;
data not shown). These results suggest that although LPS-treatment
up-regulated mRNA for both high- and low-affinity fMLPR in N9 cells,
these cells respond to peptide agonists mainly through the low-affinity
fMLPR.
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, A and B).
However, these cells showed a potent chemotaxis response to the
chemokine SDF-1 (Fig. 5C), in agreement with previous
reports (17, 18, 19). In contrast, fMLP and W peptide induced
a markedly increased migration in N9 cells treated with LPS (Fig. 5, A and B), while the same cells were no longer
responsive to the chemotactic activity of SDF-1 (Fig. 5C). Kinetic studies revealed that LPS differentially
modulated N9 cell responses to fMLP (not shown) and W peptide (Fig. 6A) vs SDF-1 (Fig. 6B) at an optimal concentration range of 300–600 ng/ml
after a 24 h incubation, consistent with the effect of LPS on the
expression of fMLPR mRNA.
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) at concentrations
that optimally activated FPR2 in receptor-transfected HEK 293 cells
(Fig. 2). Furthermore, as illustrated in Fig. 6, C and
D, only LPS-treated N9 cells migrated in response to
A42 and V3 that specifically activated
FPR2/293 cells. Thus, results derived from both
Ca2+ mobilization and chemotaxis experiments
support the notion that LPS selectively up-regulated the function of
FPR2 in N9 cells. The effect of LPS on primary murine microglial responses
To validate the physiological relevance of the results obtained
with N9 cells, we examined the activity of LPS on the response of
primary murine microglial cells to SDF-1 and FPR agonists. By
RT-PCR, we detected FPR2 gene expression in primary
microglia (Fig. 7A), but these
cells in nonstimulated condition only migrated in response to SDF-1
(Fig. 7, B–E). In contrast, while LPS slightly
up-regulated the FPR2 gene in primary microglia (Fig. 7A), cells treated with LPS showed significantly increased
chemotactic response to fMLP, W peptide, and
A42 (Fig. 7, C–E) at
concentrations that optimally activated FPR2-transfected HEK 293 cells
and N9 cells preincubated with LPS. In contrast, LPS treatment
completely abolished SDF-1-induced migration of primary microglial
cells without significantly affecting the level of CXCR4
gene expression (Fig. 7B; data not shown). In addition,
although primary murine microglia expressed the gene coding for the
high-affinity fMLPR FPR1 (Fig. 7A), these cells did not show
any chemotactic response to picomolar or low nanomolar concentrations
of fMLP despite preincubation with LPS. In addition, in
Ca2+ flux experiments, nonstimulated primary
microglia responded to SDF-1, but not to fMLP or
A42 (Fig. 8A). In contrast, while
SDF-1 failed to elicit Ca2+ mobilization in
LPS-stimulated cells, these cells showed considerable levels of
Ca2+ flux (Fig. 8B) when challenged by
fMLP or A42 and these two chemotactic peptides
desensitized each other’s signaling (Fig. 8B). Furthermore,
only LPS-treated, but not resting, primary microglial cells exhibited a
number of specific binding sites for radiolabeled fMLP (Fig. 8, C and D), with an estimated
Kd value of 250 nM, comparable to that
detected on LPS-treated N9 cells. These results suggest that, compared
with N9 cells, although primary murine microglial cells expressed a
relatively higher constitutive level of mRNA for FPR2, these cells
similarly require prestimulation of LPS to promote the expression of
fMLP binding sites on the cell surface and activation by FPR2 agonists
with concomitant down-regulation of the cell response to the chemokine
SDF-1. Due to the lack of specific Abs against murine fMLPR, we were
unable to evaluate whether LPS promoted the surface expression of
intracellularly existing pools of FPR2. Nevertheless, our initial
effort to elucidate the mechanisms of the LPS action revealed that
murine microglial cells incubated with the tyrosine kinase inhibitor,
genistein, in addition to LPS had greatly reduced chemotaxis response
to the FPR2 agonists (Fig. 9A). In addition, by RT-PCR,
the FPR2 transcripts in cells treated with both genistein and LPS
remained at the baseline level in comparison to resting cells or cells
treated with LPS alone (Fig. 9B). These results suggest that
the activity of LPS on microglial cell expression and function of FPR2
is dependent on tyrosine kinase activation and may also involve an
increase in transcription of the FPR2 gene.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the cell response was abolished after LPS
treatment. To our knowledge, this is the first demonstration that LPS
was able to up-regulate the function of a FPR in murine microglial
cells with concomitant down-regulation of CXCR4 function. Microglial cells play important roles in the development, inflammation, and immunological responses in the CNS. In fact, it has been proposed that there is no pathology in the CNS without active participation of microglia (14, 15). Microglial cells are considered to be of mononuclear phagocyte lineage and reside in various areas of the CNS during fetal development (14, 15). Unlike peripheral blood monocytes, microglial cells under normal condition are quiescent, do not express high levels of activation markers, and lack phagocytic capacity. However, these cells are capable of rapidly reacting to even minor pathological insults in the CNS and become key phagocytic cells engaged in the defense of neuronal parenchyma against infection, inflammation, trauma, ischemia, and tumors (14, 15).
Similar to macrophages, microglial cells express chemoattractant
receptors that may account for the ability of these cells to migrate
and accumulate at sites of inflammation and infection. It has been
reported that unstimulated human or rodent microglial cells express the
receptors for C5a and a number of chemokines, including CXCR4, and
migrate in response to the ligands specific for these receptors
(16, 17, 18, 19). However, the expression and function of
receptors for the bacterial chemotactic peptide fMLP are less clear.
Gene expression of the high-affinity fMLPR was observed in normal adult
human microglia (23, 24), yet no functional observations
have been available and the level of receptor protein in human
microglial cells was reportedly low (23, 24). In contrast,
rodent microglia lack the capacity to migrate in response to fMLP
(19) suggesting that the fMLP receptor in these cells is
either not expressed or expressed at a low level. Our present study
showed that N9 cells expressed mRNA for FPR2 as detected by RT-PCR and
the receptor expression and function could be markedly enhanced when
the cells were stimulated by LPS. Primary microglial cells isolated
from newborn mice also expressed mRNA for FPR2, but these cells
migrated in response to FPR2 agonists only after LPS treatment. The
lack of an apparent FPR1-mediated high-affinity response to fMLP is
intriguing. However, this deficiency was similarly observed by an
earlier study with rat primary microglial cells (25) which
could be stained positively with an Ab against the human high-affinity
fMLPR FPR, but did not respond to fMLP by release of IL-1, even after
incubation with LPS. In contrast, only LPS-treated rat microglial cells
released IL-1 upon stimulation with A42, an
agonist for human FPRL1 and murine FPR2 as observed in our previous
(25) and the current studies. These results suggest that
LPS selectively up-regulates the function of the low-affinity fMLPR in
rodent microglial cells.
LPS is a major component of the outer membrane of Gram-negative
bacteria and plays a pivotal role in eliciting an overwhelming host
innate response to infection (26). It is well established
that LPS activates phagocytic leukocytes, including microglia, to
release proinflammatory mediators. Furthermore, although LPS rapidly
increases gene transcription and protein production of a number of
cytokines and chemokines, it down-regulates the expression and function
of several chemokine receptors, including CCR1, CCR2, and CCR5
(27, 28, 29, 30) in monocytes or CXCR1 and CXCR2 in neutrophils
(31, 32, 33, 34). This reciprocal up-regulation of the expression
of ligands vs down-regulation of receptors by LPS has been proposed as
a protective host reaction aimed at limiting excessive inflammatory
responses. The mechanisms for down-regulation of chemokine receptors by
LPS have not been fully elucidated. It has been reported that LPS
reduces chemokine receptor gene transcription or mRNA stability
(27, 33, 35). LPS also can induce rapid internalization of
the chemokine receptors, presumably by activating protein tyrosine
kinases and metalloproteinases (32, 34) without
affecting receptor gene expression (28). Our present study
showed that LPS treatment markedly reduced surface expression of
SDF-1 binding sites on murine microglial cells and abolished cell
migration to SDF-1 without a substantial effect on mRNA expression
of CXCR4, thus providing evidence that LPS modulation of CXCR4 may not
occur at the transcriptional level. The effect of LPS on the expression
and function of FPR appears more complicated and may be cell-type
dependent. For instance, LPS was reported to prime human neutrophil
response to fMLP presumably by increasing the surface expression of
intracellularly stored receptors (36, 37, 38, 39). It was also
reported that LPS decreased human monocyte response to fMLP (27, 40, 41). In our study, LPS clearly increased the expression of
FPR2 gene in N9 microglial cells, which occurred earlier (12
h) than the maximal display of the receptor function (24 h). Although
compared with N9 cells, nonstimulated primary microglial cells
expressed a relatively higher level of genes for FPR2, these cells also
required LPS-treatment to gain receptor function in response to the
agonists. Thus, LPS may up-regulate the function of FPR2 in microglial
cells, either by promoting the receptor gene transcription and protein
synthesis in N9 cells or by priming the responsiveness of the existing
receptors in primary murine microglial cells. The activity of LPS on
chemoattractant receptor expression by microglial cells was not
diminished by using neutralizing Abs against TNF- and IL-1 (data not
shown), suggesting that the effect of LPS was not dependent on the
production of proinflammatory cytokines by stimulated cells. However,
we have found that TNF-1 alone was also able to up-regulate the
expression and function of FPR2 in murine microglial cells (Y.-H. Cui,
W. Gong, H. Li, X. Zhang, K. Abe, R. Sun, Y. Le, J. Van Damme, P.
Proost, and J. M. Wang, manuscript in preparation), suggesting that
FPR2 can be modulated by both bacteria and host-derived proinflammatory
signals. In an attempt to study the mechanistic basis for the action of
LPS, we observed that the regulatory activity of LPS on the function of
FPR2 was blocked by genistein, a tyrosine kinase inhibitor. In
addition, genistein appears to inhibit LPS-induced increments of FPR2
transcripts in microglial cells. These results suggest that tyrosine
kinase activation is involved in the action of LPS on murine microglial
cells. Further research is being conducted to precisely delineate the
mechanisms by which LPS selectively modulate the expression and
function of distinct chemoattractant receptors.
The enhancement of FPR2 function by LPS with concomitant
down-regulation of CXCR4 in microglial cells may have considerable
biological significance. The fMLPR have been proposed to play important
roles in host response to bacterial products and amyloidogenic protein
fragments generated in inflammation and neurodegenerative diseases
(5, 6, 7, 11, 12, 13). The low responsiveness of nonstimulated
microglial cells to FPR2 agonists may be important for the homeostasis
of the CNS which, under normal conditions, is protected by the
blood-brain barrier and is not readily exposed to pathogens. However,
it has been reported that in endotoxemia, LPS was able to enter the
brain parenchyma by diffusion through specific regions in the brain,
where unique structures of microvessels form incomplete blood-brain
barriers (42), which enables systemically circulating LPS
to stimulate a variety of brain cells including microglia. Therefore,
microglial cells, by responding to the bacterial signal LPS, may become
activated to assume the full characteristics of tissue macrophages,
including the enhancement of the FPR2 function. Such a "gain of
function" by microglial cells may facilitate their accumulation at
the sites where an aberrant production of host-derived and bacterial
chemotactic agonists is elevated. In this context, the concomitant
down-regulation by LPS of microglial cells responses to SDF-1, a
chemokine implicated for hemopoiesis and development but not
inflammation (3, 4), may help mobilize the cells more
specifically toward proinflammatory chemoattractants. Moreover, FPR2
and its human homolog, FPRL1, are specific receptors for
A42 and a prion protein fragment (12, 13), thus the presence of LPS may also promote a microglial cell
response to these agonists associated with AD and prion diseases.
Whether an increased microglial response to amyloidogenic peptides
results in a beneficial clearance of noxious agents or exacerbates the
disease states by promoting inflammation is unknown and is under
further investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ji Ming Wang, Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick, Building 560, Room 31-40, Frederick, MD 21702-1201. E-mail address: wangji{at}mail.ncifcrf.gov
3 Abbreviations used in this paper: C5a, activated C component 5; FPR, formyl peptide receptor; A42, 42 aa form of amyloid ; AD, Alzheimer’s disease; SDF, stromal cell-derived factor; 125I-SDF-1, radioactive isotope of SDF-1; CI, chemotaxis index; DiI-AcLDL, DiI-acetylated low-density lipoprotein; DPBS, Dulbecco’s PBS; FPRL1, FPR-like 1.
4 Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23; 1985).
Received for publication March 21, 2001. Accepted for publication October 23, 2001.
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  K. Chen, L. Zhang, J. Huang, W. Gong, N. M. Dunlop, and J. M. Wang Cooperation between NOD2 and Toll-like receptor 2 ligands in the up-regulation of mouse mFPR2, a G-protein-coupled A{beta}42 peptide receptor, in microglial cells J. Leukoc. Biol., June 1, 2008; 83(6): 1467 - 1475. [Abstract] [Full Text] [PDF]
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 A. Afkhami-Goli, F. Noorbakhsh, A. J. Keller, N. Vergnolle, D. Westaway, J. H. Jhamandas, P. Andrade-Gordon, M. D. Hollenberg, H. Arab, R. H. Dyck, et al. Proteinase-Activated Receptor-2 Exerts Protective and Pathogenic Cell Type-Specific Effects in Alzheimer's Disease J. Immunol., October 15, 2007; 179(8): 5493 - 5503. [Abstract] [Full Text] [PDF]
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T. A. Hamilton, M. Novotny, S. Datta, P. Mandal, J. Hartupee, J. Tebo, and X. Li Chemokine and chemoattractant receptor expression: post-transcriptional regulation J. Leukoc. Biol., August 1, 2007; 82(2): 213 - 219. [Abstract] [Full Text] [PDF]
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P. Mandal and T. Hamilton Signaling in Lipopolysaccharide-Induced Stabilization of Formyl Peptide Receptor 1 mRNA in Mouse Peritoneal Macrophages J. Immunol., February 15, 2007; 178(4): 2542 - 2548. [Abstract] [Full Text] [PDF]
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K. Chen, P. Iribarren, J. Huang, L. Zhang, W. Gong, E. H. Cho, S. Lockett, N. M. Dunlop, and J. M. Wang Induction of the Formyl Peptide Receptor 2 in Microglia by IFN-{gamma} and Synergy with CD40 Ligand J. Immunol., February 1, 2007; 178(3): 1759 - 1766. [Abstract] [Full Text] [PDF]
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 K. Tahara, H.-D. Kim, J.-J. Jin, J. A. Maxwell, L. Li, and K.-i. Fukuchi Role of toll-like receptor signalling in A{beta} uptake and clearance Brain, November 1, 2006; 129(11): 3006 - 3019. [Abstract] [Full Text] [PDF]
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 K. Chen, P. Iribarren, J. Hu, J. Chen, W. Gong, E. H. Cho, S. Lockett, N. M. Dunlop, and J. M. Wang Activation of Toll-like Receptor 2 on Microglia Promotes Cell Uptake of Alzheimer Disease-associated Amyloid beta Peptide J. Biol. Chem., February 10, 2006; 281(6): 3651 - 3659. [Abstract] [Full Text] [PDF]
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P. Mandal, M. Novotny, and T. A. Hamilton Lipopolysaccharide Induces Formyl Peptide Receptor 1 Gene Expression in Macrophages and Neutrophils via Transcriptional and Posttranscriptional Mechanisms J. Immunol., November 1, 2005; 175(9): 6085 - 6091. [Abstract] [Full Text] [PDF]
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P. Iribarren, K. Chen, J. Hu, X. Zhang, W. Gong, and J. M. Wang IL-4 Inhibits the Expression of Mouse Formyl Peptide Receptor 2, a Receptor for Amyloid {beta}1-42, in TNF-{alpha}-Activated Microglia J. Immunol., November 1, 2005; 175(9): 6100 - 6106. [Abstract] [Full Text] [PDF]
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K. Kurosaka, Q. Chen, F. Yarovinsky, J. J. Oppenheim, and D. Yang Mouse Cathelin-Related Antimicrobial Peptide Chemoattracts Leukocytes Using Formyl Peptide Receptor-Like 1/Mouse Formyl Peptide Receptor-Like 2 as the Receptor and Acts as an Immune Adjuvant J. Immunol., May 15, 2005; 174(10): 6257 - 6265. [Abstract] [Full Text] [PDF]
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