Different Neurotropic Pathogens Elicit
Neurotoxic CCR9- or Neurosupportive
CXCR3-Expressing Microglia
This information is current as
of June 18, 2017.
He Li, Zhou Gang, He Yuling, Xie Luokun, Xiong Jie, Lei
Hao, Wei Li, Hu Chunsong, Liu Junyan, Jiang Mingshen, Jin
Youxin, Gong Feili, Jin Boquan and Tan Jinquan
J Immunol 2006; 177:3644-3656; ;
doi: 10.4049/jimmunol.177.6.3644
http://www.jimmunol.org/content/177/6/3644
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Copyright © 2006 by The American Association of
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References
The Journal of Immunology
Different Neurotropic Pathogens Elicit Neurotoxic CCR9- or
Neurosupportive CXCR3-Expressing Microglia1
He Li,2*† Zhou Gang,2*‡ He Yuling,2*‡ Xie Luokun,2* Xiong Jie,*¶ Lei Hao,储# Wei Li,储
Hu Chunsong,** Liu Junyan,§ Jiang Mingshen,† Jin Youxin,¶ Gong Feili,†† Jin Boquan,‡‡ and
Tan Jinquan3*‡§
M
icroglia make up the innate immune system of CNS
and are key cellular mediators of neurodegenerative
and neuroinflammatory processes. Microglia are now
recognized as the prime components of an intrinsic brain immune
system (1). Although their immune functions are “down-regulated” in healthy CNS, microglia are rapidly activated upon in-
*Department of Immunology and †Department of Parasitology, Institute of Allergy and Immune-Related Diseases, ‡Laboratories of Neuroscience, and §Allergy
and Clinical Immunology, Center for Medical Research, Wuhan University
School of Medicine, Wuhan, People’s Republic of China; ¶The State Key Laboratory of Molecular Biology and Neuroscience, Institute of Biochemistry and
Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, People’s Republic of China; 储The State Key Laboratory of Magnetic
Resonance and Atomic and Molecular Physics, Institute of Physics and Mathematics,
Chinese Academy of Sciences, Wuhan, People’s Republic of China; #Key Laboratory of
Colloid, Interface Science and Chemical Thermodynamics, Institute of Chemistry, Chinese Academy of Sciences, Beijing, People’s Republic of China; **Department of Immunology, College of Basic Medical Sciences, Anhui Medical University, Hefei, People’s
Republic of China; ††Department of Immunology, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan, People’s Republic of China; and ‡‡Department of Immunology, Fourth Military Medical University, Xian, People’s Republic of
China
Received for publication December 21, 2005. Accepted for publication July 4, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
The study was supported by the National Key Basic Research Program of China
from the Ministry of Science and Technology of People’s Republic of China (Grants
2001CB510004 and 2001CB510008), the National Natural Science Foundation of
China (Grants 39870674, 30572119, 30030130, and 30471509), a special grant from
the Personnel Department of Wuhan University, China, and the Research Foundation
of Health Department of Hubei Provincial Government, China (Grant 301140344).
T.J. is a Chang Jiang Scholar supported by Chang Jiang Scholars Program from
Ministry of Education, People’s Republic of China, and Li Ka Shing Foundation,
Hong Kong, People’s Republic of China.
2
flammatory processes, chronic brain diseases, or brain injury (2,
3). Activated microglia, the first cells in the CNS to respond to
neuronal damage, are able to exert two opposing functions—promoting neuronal regeneration and killing neurons— by means of a
variety of mechanisms (2, 4). Exerting one or the other function is
largely determined by the particular circumstances that evoke microglial activation (2). However, the specific nature of any such
constructive or destructive mechanisms remains nebulous.
Unlike others, the CNS is an immunologically privileged organ
because of a relatively impermeable blood-brain barrier and an
immunosuppressive microenvironment to limit immune cells entry
and function. However, microglia may direct initial responses to
neurotropic pathogenesis (5). The proximity of microglial and neuronal membranes is necessary to facilitate cell-cell communication
by means of signaling factors— diffusible molecules and surface
receptors such as chemokines and their receptors (6). A number of
CC and CXC chemokines/receptors are believed as mediators of
CNS development and pivotal players in the pathogenesis of immune-mediated neurodegenerative neuroinflammatory and diseases in the CNS (7). Importantly, CXCR3/CXCL10 signaling is
crucial in microglia recruitment and an essential element for neuronal reorganization (6, 8). Although CCR9/CCL25 signaling is
important for the homing, development, homeostasis, and resistance to apoptosis of T cells (9), there is so far no report on functional expression of CCR9 on microglia.
The immunoresponsive gene 1 (Irg1),4 an LPS-inducible gene
(10), remains unclear in its expression pattern and biological functions (11).
H.L., Z.G., H.Y., and X.L. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Tan Jinquan, Department of
Immunology, Wuhan University School of Medicine, Dong Hu Road 115, Wuchang
430071, Wuhan, People’s Republic of China. E-mail address: jinquan_tan@
hotmail.com
Copyright © 2006 by The American Association of Immunologists, Inc.
4
Abbreviations used in this paper: Irg1, immunoresponsive gene 1; STAg, soluble
tachyzoite Ag; rLCMVNP, recombinant nucleoprotein immunodominant domain of
lymphocytic choriomeningitis virus; siRNA, small interfering RNA.
0022-1767/06/$02.00
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What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We
report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9ⴙIrg1ⴙ
(immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-␣; priming and
rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3ⴙIrg1ⴚ microglia, which are sensitive
to apoptosis and produce a high level of IL-10 and TGF-␤. Administration of CCR9 and/or Irg1 small interfering RNA alters
the frequency and functional profiles of neurotoxic CCR9ⴙIrg1ⴙ and neurosupportive CXCR3ⴙIrg1ⴚ microglia in vivo.
Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria,
toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of
microglia has been confirmed as neurotoxic CCR9ⴙIrg1ⴙ and neurosupportive CXCR3ⴙIrg1ⴚ cells in these mouse models.
These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement
in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer’s disease and AIDS dementia. The Journal of
Immunology, 2006, 177: 3644 –3656.
The Journal of Immunology
3645
Materials and Methods
Animal models
Flow cytometry
For microglia analysis, the cortex or hippocampus was microdissected
from mice at indicated specific time intervals. Single-cell suspensions of
cortex or hippocampus were prepared using a 70-␮m mesh, followed by
collagenase/DNase (Sigma-Aldrich) digestion and Percoll (Pharmacia)
gradient separation. To avoid contamination of lymphocytes during flow
cytometry, CD3⫹ and CD19⫹ lymphocytes were positively depleted from
cell suspensions using anti-CD3 and anti-CD19 mAb-labeled magnetic
Dynabead assay (Dynal Biotech). Both anti-mouse CD11b (Mac-1; Leinco
Technologies) and CD45 (DakoCytomation) were used to stain the microglia (6, 8). Cells were stained with appropriate secondary fluorescencelabeled Abs. For detection of CCR9, CXCR3, or Irg1, a third color was
stained by goat anti-mouse CCR9 pAb (E-15; Santa Cruz Biotechnology)
Histological analysis
Cerebral cortex and hippocampal tissues of different mouse models were
fixed in 10% formalin and paraffin processed. Five-micrometer histological
sections were stained with H&E and photographed. For immunohistochemistry staining (6, 8), free-floating vibratome sections were permeabilized.
Endogenous peroxidases were blocked by 0.3% H2O2. Sections were incubated in blocking solution containing 5% BSA, followed by overnight
incubation at 4°C with primary Ab (anti-CD11b/Mac-1, anti-CCR9, antiCXCR3, or anti-Irg1). Appropriate biotinylated secondary Ab was applied
for 2 h at room temperature. Abs were then visualized with avidin-biotinperoxidase complex (DakoCytomation). For quantification of positive
cells, at least nine sections per animal and four randomly selected regions
in each section were analyzed. All slides were viewed on an Olympus Van
Ox microscope and evaluated in a blinded fashion by two independent
experts. For immunofluorescence digital confocal microscopy (9), the purified cells were spun down on a slide, fixed, immersed with 1% BSA
blocking buffer, added with primary Ab, and incubated overnight at 4°C,
followed by fluorescence-labeled secondary Ab fluorescence labeling.
Confocal microscopy analysis was performed using a confocal laser scanning system and an inverted microscope (LSMSIO; Zeiss).
Table I. The sequences of senses and antisenses for mRNA detection in real-time quantitative RT-PCR assay
Target
Sense
Irg1
T. gondii
LCMV
IL-2
IFN-␥
TNF-␣
IL-4
IL-10
TGF-␤
CX3CR1
CCR1
CCR2
CCR3
CCR4
CCR5
CCR6
CCR7
CCR8
CCR9
CXCR1
CXCR2
CXCR3
CXCR4
CXCR5
CX3CR1
XCR1
CX3CL1
CCL5
CCL25
CXCL10
CXCL12
5⬘-CGTGAGAAAGCACCTTGTGA-3⬘
5⬘-GGAACTGCATCCGTTCATGAG-3⬘
5⬘-GCATTGTCTGGCTGTAGCTTA-3⬘
5⬘-ATTACAGCATGCAGCTCGCAT-3⬘
5⬘-GAACGCTACACACTGCATC-3⬘
5⬘-ATGAGCACAGAAAGCATGATC-3⬘
5⬘-ACAAAAATCACTTGAGAGAGATCAT-3⬘
5⬘-ATGCCTGGCTCAGAC-3⬘
5⬘-GACCTCCATAGAAGACACC-3⬘
5⬘-TCAGCATCGACCGGTACCTT-3⬘
5⬘-AGGCCCAGTGGGAGTTCAC-3⬘
5⬘-TCATCCACGGCATACTATCAACA-3⬘
5⬘-GCTTTGAGACCACACCCTATGAA-3⬘
5⬘-AGAAAATTCATGCGAGGGAGAA-3⬘
5⬘-AGGCCATGCAGGCAACAG-3⬘
5⬘-GAACTGCCCACTTCCCTTTCT-3⬘
5⬘-GCTCCAGGCACGCAACTTT-3⬘
5⬘-TGACCGACTACTACCCTGATTTCTT-3⬘
5⬘-TCTCAGTTCCCCTACAACTCCATT-3⬘
5⬘-CCCTCTTTAAGGCCCACATG-3⬘
5⬘-ACCCTCTTTAAGGCCCACATG-3⬘
5⬘-CAGCCTGAACTTTGACAGAACCT-3⬘
5⬘-TCAGCCTGGACCGGTACCT-3⬘
5⬘-GCCTGCTCGTGGCCTGTA-3⬘
5⬘-TCAGCATCGACCGGTACCTT-3⬘
5⬘-GCCTTCTCTCATTGCTGTTTCA-3⬘
5⬘-ATGACCTCACGAATCCCAGTGG3⬘
5⬘-GCAAGTGCTCCAATCTTGCA-3⬘
5⬘-AGCACAGGATCAAATGGAATGTT-3⬘
5⬘-GGACGGTCCGCTGCAA-3⬘
5⬘-CACCTCGGTGTCCTCTTG-3⬘
Antisense
5⬘-CTGTGGAAGGATGGGACAGT-3⬘
5⬘-TCTTTAAAGCTTCGTGGTC-3⬘
5⬘-CAATGACGTTGTACAAGCGC-3⬘
5⬘-AGTCAAATCCAGAACATGCCG-3⬘
5⬘-GAGCTCATTGAATGCTTGG-3⬘
5⬘-GTCTGGGCCATAGAAC-3⬘
5⬘-AGTAATCCATTTGCATGATGCTCTT-3⬘
5⬘-GTCCTGCATTAAGGAGTCG-3⬘
5⬘-AACCCGTTGATGTCCACTTGC-3⬘
5⬘-CTGCACTGTCCGGTTGTTCA-3⬘
5⬘-TCCACTGCTTCAGGCTCTTGT-3⬘
5⬘-GTGGCCCCTTCATCAAGCT-3⬘
5⬘-GACCCCAGCTCTTTGATTCTGA-3⬘
5⬘-TGTCACTCAAGGGTCGTGTCA-3⬘
5⬘-TCTCTCCAACAAAGGCATAGATGA-3⬘
5⬘-CAGGCTGGCGTGGTTCTCT-3⬘
5⬘-GACTACCACCACGGCAATGA-3⬘
5⬘-GCTGCCCCTGAGGAGGAA-3⬘
5⬘-CAGTTGGAGATGAACATGGCATA-3⬘
5⬘-AAGGACGACAGCGAAGATGAC-3⬘
5⬘-CAAGGACGACAGCGAAGATG-3⬘
5⬘-GCAGCCCCAGCAAGAAGA-3⬘
5⬘-GCAGTTTCCTTGGCCTTTGA-3⬘
5⬘-CGGCGGTAGGCGTGAAC-3⬘
5⬘-CTGCACTGTCCGGTTGTTCA-3⬘
5⬘-TTTAGGTGTCTGCGGAACTTGA-3⬘
5⬘-CCGCCTCAAAACTTCCAATGC-3⬘
5⬘-GATGTATTCTTGAACCCACTTCTTCTC-3⬘
5⬘-GGTTGCAGCTTCCACTCACTT-3⬘
5⬘-GCTTCCCTATGGCCCTCATT-3⬘
5⬘-GGTCAATGCACACTTGTCTG-3⬘
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Five- to 6-wk-old female C57BL/6 were obtained from The Jackson Laboratory and maintained in a pathogen-free environment in the Animal Research Institute, Medical Research Center, Wuhan University School of
Medicine. Unless otherwise stated, the following animal models were used
throughout in the study: mice were primed with three intracranially injections of 8 ␮g of Toxoplasma gondii (DX strain) soluble tachyzoite Ag
(STAg) (termed Tp) (12) or primed with three intracranially injections of
0.1 ␮g of recombinant nucleoprotein immunodominant domain of lymphocytic choriomeningitis virus (rLCMVNP) (termed Lp) (13) in biweekly
intervals. Two weeks after the last priming, Tp mice were intracranially
infected (rechallenged) with a single dose of five cysts of T. gondii (sublethal, low-virulent DX strain, termed TpTi); Lp mice were intracranially
infected with a single dose of 100 PFU of LCMV (sublethal, clone 13,
termed LpLi) (14, 15); some Tp mice were cross-infected with LCMV
(termed TpLi); some Lp mice were cross-infected with T. gondii (termed
LpTi); and unprimed mice were intracranially infected with a single dose
of T. gondii (termed Ti) or LCMV (termed Li). The mortality of mice was
monitored semidiurnally for 55 days and was used to evaluate the clinical
severity of infections of neurotropic pathogens. All animals were deeply
anesthetized with the ketamine mixture before sacrifice. The experimental
procedures were approved by the Animal Care and Use Committee of
Wuhan University.
or goat anti-mouse CXCR3 pAb (SC-6226; R&D Systems) or rabbit antimouse Irg1 (generated in-house), respectively. In some cases, four-color
staining of CD11b-CD45-CD3-CCR9 or -CXCR3 in cell suspension was
used to distinguish microglia populations from T cells and detection CCR9
and CXCR3 expression on different cell populations. An intracellular immunofluorescence staining procedure of IntraPrep (Coulter-Immunotech)
was used to permeabilize cells in Irg1 staining. For detection of microglial
and neuronal apoptosis, the microglia were purified using the high-gradient
magnetic CD11b/Mac1-labeling cell separation system (MACS; Miltenyi
Biotec) (8). Apoptosis of microglia was evaluated by binding assay of
propidium iodide (1 ␮g/ml) and FITC-labeled annexin V (BD Clontech)
(16). Neuron cultures were derived from fetal mouse cerebral cortices (17).
Neuron-microglia cocultures (culture inserts) were maintained for 48 h
(17). After treatment, culture inserts containing microglia were removed,
and neurons were stained by the TUNEL method (Phoenix Flow Systems)
(18). Data were acquired using a flow cytometer (COULTER XL; Coulter).
3646
DIFFERENT MICROGLIA
Western and Northern blotting
For coimmunoprecipitations and phosphorylated-protein detection, cells
were washed extensively and lysed in lysis buffer (19). Cell lysates were
centrifuged (10,000 rpm for 10 min, 4°C), and the supernatants were recovered. Cell lysates were precleared three times with 20 ␮l of protein
A-Sepharose beads and were mixed with specified Ab (anti-Irg1) for 3 h at
4°C under constant agitation. Immune complexes were allowed to bind to
20 ␮l of protein A-Sepharose beads overnight, beads were washed three
times with lysis buffer, and immunoprecipitates were separated in 12%
SDS polyacrylamide gels and transferred to nitrocellulose membranes. Filters were blocked with 5% nonfat milk in blocking buffer and incubated
with specific rabbit anti-phosphorylated proteins Ab (Zymed Laboratories)
for 2 h, followed by HRP-labeled secondary Ab (Amersham Biosciences)
visualizing procedure and autoradiography. For Western blot analysis (20),
protein concentration in cell lysates was measured by Bio-Rad protein
assay. Protein (⬃40 ␮g) was loaded onto 16% SDS-PAGE, transferred
onto polyvinylidene difluoride membranes after electrophoresis, and incubated with the appropriate Abs (CCR9, CXCR3, and Irg1) at 0.5
␮g/ml. The membrane was blocked in 5% BSA-TBS, followed by secondary Ab visualizing procedure and autoradiography (19). For mRNA
detection, 5 ␮g of pooled total RNA samples was electrophoresed under
denaturing conditions, followed by blotting onto Nytran membranes,
and cross-linked by UV irradiation as described previously (21). Specific appropriate cDNA probes labeled by [␣-32P]dCTP were hybridized
FIGURE 2. CCR9 and CXCR3 expression on microglia from different mouse models. A, Triple-color flow cytometric analyses of CCR9 and CXCR3
on microglia from NML, Tp, Ti, TpTi, TpLi, Lp, Li, LpLi, and LpTi mice. The animals were sacrificed as described in Fig. 1B. Red curves, CCR9 staining;
blue curves, CXCR3 staining; gray curves, isotype Ab controls. The numbers were indicating the percentages of the positive cells. Data were representatives
of eight animals per group. B, Morphology and distribution of CCR9- or CXCR3-positive (brown) microglia-infiltrating inflammation-reactive cortex
determined by immunohistochemistry (n ⫽ 6). Magnifications, ⫻400. The insets were isotype controls. C, The microglia-infiltrating inflammation-reactive
cortex were photographed by immunofluorescence digital confocal microscopy (n ⫽ 6). Magnification, ⫻1200. Bar, 12 ␮m. The arrows: negative (N),
CCR9⫹, CXCR3⫹, and CCR9⫹CXCR3⫹ (DP) cells (all left panels). The insets were isotype controls. The percentages (all right panels) of different
microglia: negative, gray; CCR9⫹, red; CXCR3⫹, green; and CCR9⫹CXCR3⫹, yellow. The experiments in each group were independently repeated twice.
D, Expression levels of CCR9 and CXCR3 in microglia infiltrating inflammation-reactive cortex determined by Northern and Western blot analyses (left
panels) and real-time quantitative RT-PCR (right panels) in purified microglia from cortex (n ⫽ 6). ⴱ, p ⬍ 0.001, Tp or TpTi vs others (CCR9); Lp or LpLi
vs others (CXCR3). E, Chemotaxis of microglia infiltrating inflammation-reactive cortex toward CCL25 (left panel) and CXCL10 (right panel). Tp (f)
or Lp mice (䡺) were scarified at time described in Fig. 1B. All results were expressed as chemotactic index (C.I. ⫾ SD; n ⫽ 6). ⴱ, p ⬍ 0.001, Tp vs Lp.
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FIGURE 1. Analyses of microglial behaviors from different animal models. A, Mortality
of mice with different treatments (n ⫽ 10). 䡺,
Tp; f, TpTi; E, Ti; or F, TpLi (left panel); 䡺,
Lp; f, LpLi; E, Li; or F, LpTi (right panel). B,
Flow cytometric analysis of microglia from
normal (NML), Tp, TpTi, Ti, TpLi, Lp, LpLi,
Li, and LpTi mice. The animals were sacrificed
either at end of the priming procedure or at day
10 postinfection. Lymphocyte-depleted singlecell suspensions from cortexes were prepared
and stained. Microglia were identified on the
basis of expression of CD11b/Mac1 and CD45
double positive. Data were representatives of
eight animals per group. C, Analyses of the
number of microglia in slices from cortex of
NML (⽧), TpTi (f), TpLi (F), LpLi (䡺), or
LpTi (E) mice and sacrificed at days as indicated. CD11b/Mac1⫹ microglial numbers
were represented as mean ⫾ SD per highpower field (hpf) and n ⫽ 5. ⴱ, p ⬍ 0.001, TpTi
vs TpLi, LpTi, or LpLi at day 6; ⴱ, p ⬍ 0.01,
LpLi vs TpLi or LpTi at day 30. D, Morphology and distribution of CD11b/Mac1⫹ microglia (brown) infiltrating inflammation-reactive
cortex determined by H&E staining and immunohistochemistry (n ⫽ 6). Arrows, Necrotic
neurons and “cap-like” microglia-neuron bodies (TpTi) and lymphocyte-infiltrating and microglia-lymphocyte “rosette-like” bodies
(LpLi). The insets were isotype controls. E,
Levels of T. gondii DNA and LCMV RNA in
the inflammation-reactive cortex of Ti, TpTi,
or LpTi mice (left panel), Li, LpLi, or TpLi
mice (right panels) determined by real-time
quantitative PCR. Mice were sacrificed at day
10 postinfection with different neurotropic
pathogens. Data were mean ⫾ SD (n ⫽ 5). ⴱ,
p ⬍ 0.001, LpTi vs Ti or TpTi; LpLi vs Li or
TpLi. LCMV RNA levels were verified by
Northern blot assay (lower right panel).
3647
The Journal of Immunology
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3648
DIFFERENT MICROGLIA
Table II. The real-time detection of mRNA of chemokine receptors on microglia from different mouse
modelsa
Chemokine Rb
CX3CR
XCR1
CCR1
CCR2
CCR3
CCR4
CCR5
CCR6
CCR7
CCR8
CCR9*e
CXCR1
CXCR2
CXCR3*e
CXCR4
CXCR5
NML
Tp
6.8 ⫻ 10
-d
5.3 ⫻ 102
4.9 ⫻ 103
6.5 ⫻ 103
1.5 ⫻ 102
6.5 ⫻ 103
2.3 ⫻ 102
1.1 ⫻ 103
1.8 ⫻ 102
0.4 ⫻ 102
3.1 ⫻ 102
2.3 ⫻ 102
1.8 ⫻ 103
9.5 ⫻ 103
2.1 ⫻ 103
3c
TpTi
7.1 ⫻ 10
4.8 ⫻ 102
4.5 ⫻ 103
7.5 ⫻ 103
1.2 ⫻ 102
7.8 ⫻ 103
2.4 ⫻ 102
1.3 ⫻ 103
2.4 ⫻ 102
UPf
2.3 ⫻ 102
ND
NOg
9.8 ⫻ 103
2.3 ⫻ 103
3
Lp
6.9 ⫻ 10
4.4 ⫻ 102
5.1 ⫻ 103
7.1 ⫻ 103
1.1 ⫻ 102
7.1 ⫻ 103
3.0 ⫻ 102
1.2 ⫻ 103
2.8 ⫻ 102
UPf
2.1 ⫻ 102
ND
NOg
9.1 ⫻ 103
1.8 ⫻ 103
3
LpLi
6.5 ⫻ 10
5.1 ⫻ 102
5.2 ⫻ 103
7.2 ⫻ 103
1.4 ⫻ 102
7.5 ⫻ 103
ND
1.6 ⫻ 103
2.5 ⫻ 102
NOg
1.8 ⫻ 102
ND
UPf
8.5 ⫻ 103
1.9 ⫻ 103
3
6.9 ⫻ 103
4.6 ⫻ 102
5.0 ⫻ 103
7.8 ⫻ 103
1.7 ⫻ 102
8.5 ⫻ 103
0.1 ⫻ 102
1.5 ⫻ 103
2.2 ⫻ 102
NOg
1.6 ⫻ 102
ND
UPf
9.6 ⫻ 103
2.4 ⫻ 103
overnight with membranes followed by intensively washing with 0.2⫻
SSC before being autoradiographed.
Real-time quantitative RT-PCR
Total RNA was purified from microglia or brain tissues. All real-time
quantitative RT-PCR were performed as described elsewhere (22).
Briefly, total RNA was purified from microglia or brain tissues. Total
RNA was prepared by using the Quick Prep total RNA extraction kit
(Pharmacia Biotech). The RNA was reverse transcribed by using oligo(dT)12–18 and Superscript II reverse transcriptase (Invitrogen Life
Technologies). DNA in the tissues from T. gondii-infected animals tissues were extracted using the Qiamp tissue kit (Qiagen). The real-time
quantitative PCR was performed with an ABI PRISM 7700 Sequence
Detector Systems (Applied Biosystems) by using the SYBR Green PCR
Core Reagents kit (Applied Biosystems) according to the manufacturer’s instructions. Primers used for real-time quantitative PCR were
shown in Table I.
was anesthetized and then given intranasal HO-1 siRNA (8 mg/kg/day) or
equivalent doses of mismatched control siRNA duplex in a volume of 50
␮l. The administration began 2 wk before other experimental procedures
and continued till the end of experiments.
Chemotaxis and nitrite quantification assays
Migration of microglia in response to CCL25 and CXCL10 (R&D Systems) was determined as described earlier (25). NO⫺
2 in culture supernatants was measured to assess total NO production in microglial cells as
described earlier (16). Briefly, 50 ␮l of sample aliquots of culture supernatants from microglial cells was mixed with 50 ␮l of Griess reagent (1%
sulfanilamide-0.1% naphthylethylenediamine dihydrochloride-2% phosphoric acid) with adding nitrate reductase (0.003 U of Aspergillus species;
Sigma-Aldrich) in plates and incubated at 25°C for 10 min for measurement of for total NO levels. The OD550 was measured on a microplate
reader.
ELISA analysis
Briefly, cytokine and chemokine proteins were measured in conditioned
medium from cultures of microglia using ELISA kits (Promo Cell) (23).
Culture supernatant was collected after incubation for the time indicated
from microglia culture. Media samples were first cleared at 2000 rpm for
5 min and then assayed for cytokine and chemokines according to the
manufacturer’s instructions. The experiments were performed six times for
each group, and each sample was assayed in triplicate.
Cell transient transfection
Purified microglia were transiently transfected with plasmids encoding
CCR9, CXCR3, and Irg1 using the Amaxa nucleofection technology
(Amaxa) with optimization according to the manufacturer’s instructions.
Briefly, cells were resuspended in solution from nucleofector kit V, following the Amaxa guidelines for cell transfection. One hundred microliters
of 3 ⫻ 106 cell suspension mixed with 0.25 or 2.5 ␮g of cDNA was
transferred to the provided cuvette and nucleofected with an Amaxa
Nucleofector apparatus (Amaxa).
Small interfering RNA (siRNA) gene knockdown assay
The design of siRNA was based on the characterization of siRNA (24). The
siRNA were synthesized in 2⬘-deprotected, duplexed, desalted, and purified
form by Dharmacon Research. The sense of mouse HO-1 siRNACCR9 was
as follows: 5⬘-GTCATCCAAGCACAAGGCCCT-3⬘; and siRNAIrg1, 5⬘GTATCATTCGGAGGAGCAAGA-3⬘. For in vivo studies, each mouse
Results
T. gondii- and LCMV-priming differentially elicit microglia
To explore which factor(s) determined differential behavior of activated microglia for neuronal regeneration or degeneration, we
established different animal models to induce activated microglia
in vivo (see Materials and Methods). Unexpected mortalities in
TpTi and TpLi mice were observed after a sublethal dose infection.
There was no death case in Tp, Ti, Lp, Li, and LpLi mice and a
minor mortality in LpTi mice (Fig. 1A). Significant increased microglial frequencies in cortex in TpTi and LpLi mice were observed (Fig. 1B). There was a significant and rapid increase of
microglia at the early stage in TpTi mice and a late increase in
LpLi mice during pathogenesis (Fig. 1C). By H&E staining and
immunohistochemistry, a higher frequency of microglia, severe
edema, a large number of necrotic neurons, and many “cap-like”
microglia-neuron bodies were seen observed in cortex of TpTi
mice. Meanwhile, a higher frequency of microglia, a large number
of infiltrating lymphocytes, and microglia-lymphocyte “rosettelike” bodies were found in the cortex of LpLi mice (arrows, Fig.
1D). T. gondii DNA was not significantly altered in the brain of
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a
Mouse models were established as described in Materials and Methods, sacrificed, and the microglia were purified from
cortex as described in the legend to Fig. 1.
b
CX3C, XC, CC, and CXC chemokine receptors examined were listed.
c
The procedures for total RNA isolation and real-time quantitative RT-PCR were described in Materials and Methods. The
mRNAs of chemokine receptors were expressed as copies per 25 ng cDNA. The showing data were mean ⫾ SD of six
experiments conducted in each group.
d
-, under detectable level.
e
There were significant changes in the levels of mRNA expressions. Concrete data were presented in Fig. 2. *, p ⬍ 0.001.
f
UP, significantly up-regulation vs NML mice.
g
NO, no significantly change vs NML mice. The data for f and g were presented in Fig. 2. ND, no determination.
The Journal of Immunology
3649
Microglia from different animal models differentially express
CCR9 and CXCR3
To further characterize the microglia elicited from different mouse
models, we analyzed the all known chemokine receptor expressions in microglia from different mouse models (Table II). The
microglia in cortex in Tp and TpTi mice expressed a high level of
CCR9, whereas the microglia elicited from Lp and LpLi mice expressed a high level of CXCR3 (Fig. 2A). T. gondii infection
FIGURE 3. Functions of microglia from different mouse models. A.
Flow cytometric analysis of apoptotic microglia following stimulation with
(red curves) or without LPS (green curves) (n ⫽ 8). The microglia were
first purified from mice as described Fig. 1B, and cultured for 2 days and
stained. T. gondii inhibitor pyrimethamine (1.0 ␮g/ml, Sigma) and LCMV
inhibitor ganciclovir (GCV, 1.0 ␮g/ml, Roche Diagnostics) were added
into the cultures to avoid direct effects of pathogens. The microglia were
analyzed by flow cytometry for PI and FITC-conjugated annexin V
(shown, left panel). Data for apoptotic microglia were mean ⫾ SD with
LPS stimulation (right panel). ⴱ, p ⬍ 0.001, NML, Lp, or LpLi vs Tp or
TpTi. B. Flow cytometric analysis of apoptotic neurons. The neurons were
derived from cerebral cortices of fetal (embryonic day 17) mice (n ⫽ 8),
and cocultured with purified microglia infiltrating inflammation-reactive
cortex from mice in the presence (red curves) or absence of LPS (green
curves) for 48h with pyrimethamine and ganciclovir in culture. The neurons were analyzed for FITC-conjugated BrdU. The data for apoptotic
neurons were from a single experiment (left panel) or mean ⫾ SD (right
panel) with LPS stimulation. ⴱ, p ⬍ 0.001, Tp or TpTi vs NML, Lp, or
LpLi. C, Levels of cytokine (upper panels) and chemokine (lower panels)
mRNAs of microglia-infiltrating inflammation-reactive cortex determined
by real-time quantitative RT-PCR. The microglia were purified as described in Fig. 1B (n ⫽ 8) and cultured at presence of LPS for 6 h with
pyrimethamine and ganciclovir in system. Upper leftmost panel, 䡺, IL-2;
f, IFN-␥. ⴱ, p ⬍ 0.001, Tp or TpTi vs NML, Lp, or LpLi for TNF-␣; ⴱ,
p ⬍ 0.01– 0.001, NML, Lp, or LpLi vs Tp or TpTi for IL-10 and TGF-␤;
ⴱ, p ⬍ 0.001, Lp or LpLi vs NML, Tp or TpTi for CX3CL1 and CXCL10;
ⴱ, p ⬍ 0.001, Tp or TpTi vs NML, Lp, or LpLi for CCL25. D, Cytokine
and chemokine production of microglia from different mouse models
(NML, Tp, TpTi, Lp, and LpLi mice). Levels of cytokine (upper panels)
and chemokine (lower panels) in culture supernatant of microglia infiltrating inflammation-reactive cortex determined by ELISA as described in
Materials and Methods. The animals were sacrificed either at end of priming procedure or at day 10 postinfection. The microglia were purified using
the high-gradient magnetic CD11b/Mac1-labeling cell separation system
(n ⫽ 4) and cultured at the presence of LPS for 6 h with pyrimethamine and
ganciclovir in system. Upper leftmost panel, 䡺, IL-2; f, IFN-␥. ⴱ, p ⬍
0.001, Tp or TpTi vs NML, Lp, or LpLi for TNF-␣; ⴱ, p ⬍ 0.01– 0.001,
NML, Lp, or LpLi vs Tp or TpTi for IL-10 and TGF-␤; ⴱ, p ⬍ 0.001, Lp
or LpLi vs NML, Tp or TpTi for CX3CL1 and CXCL10; ⴱ, p ⬍ 0.001, Tp
or TpTi vs NML, Lp, or LpLi for CCL25. E, Total NO production of
microglia following stimulation with (f) or without LPS (䡺) (n ⫽ 6). ⴱ,
p ⬍ 0.001, Tp or TpTi vs NML, Lp, or LpLi in absence of LPS; ⴱ, p ⬍
0.001, presence vs absence of LPS in Lp or LpLi microglia.
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TpTi mice in comparison with Ti mice but significantly decreased
in LpTi mice. Similar data were obtained in the microglia-infiltrating inflammation-reactive hippocampus region from different
mouse models (data not shown). LCMV mRNA significantly decreased in the brain of LpLi mice in comparison with that of Li
mice, whereas LCMV mRNA slightly increased in TpLi mice (Fig.
1E). T. gondii DNA and LCMV mRNA burden were examined in
other vital organs (liver, spleen, kidney, lung, heart, and gut). The
pathogens were at low or undetectable levels in these organs, indicating that the animal death was not contributed by spreading to
other organs of pathogen infection (data not shown). Thus, T. gondii Ag intracranial priming exacerbated brain damage and caused
death (TpTi and TpLi mouse models). LCMV intracranial priming
showed significant protective effects (LpLi and LpTi mouse models). T. gondii priming-induced microglia were rapidly reactive,
directly attacked neurons, and were unable to clean pathogens
when the CNS was infected; LCMV priming-induced microglia
were relatively slowly reactive, attracted lymphocytes, and were
able to clean infected pathogens. These clues led us to search for
possibility of distinctive subsets of microglia.
3650
Microglia from Tp and TpTi mice selectively express Irg1
A report on that ⬎63-fold up-regulation of Irg1 in activated microglia in vitro (26) led us to examine the Irg1 expression in cortex
from different mouse models. Microglia from Tp and TpTi mice intracellularly expressed very high levels of Irg1 but not in the cells
from NML, Lp and LpLi mice (Fig. 4A). The observations were confirmed by immunohistochemistry (Fig. 4B), by immunofluorescence
digital confocal microscopy (Fig. 4C, Tp mice data not shown), and
by Northern and Western blots, and real-time quantitative RT-PCR
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mainly induced CCR9 expression (Ti mice), whereas LCMV infection mainly induced CXCR3 expression (Li mice). Similar data
were obtained in the microglia-infiltrating inflammation-reactive
hippocampus region from different mouse models (data not
shown). As it is presented in Fig. 2, B–D, by real-time RT-PCR,
Western and Northern blots confirmed that at both mRNA and
protein levels, CCR9 expression was dominant in microglia of Tp
and TpTi mice, whereas CXCR3 was expressed at a high level in
Lp and LpLi mice. CCL25 induced strong chemotactic migration
of microglia from Tp and TpTi mice but not from Lp and LpLi
mice. CXCL10 induced strong chemotaxis of microglia from Lp
and LpLi mice but not from Tp and TpTi mice (Fig. 2E). We
applied four-color flow cytometry in cell suspension with combined staining of CD11b-CD45-CD3-CCR9 or -CXCR3 to distinguish microglia populations from T cells. Within CD11b⫹CD45⫹
microglia populations, CCR9 was highly up-regulated in Tp and
TpTi mice, and CXCR3 was highly up-regulated in Lp and LpLi
mice, in comparison with normal mice. Meanwhile, within
CD3⫹CD45⫹ T cell populations, both CCR9 and CXCR3 were
expressed at very low levels (data not shown). The results confirmed that up-regulation of CCR9 and CXCR3 expression was
indeed on microglia.
DIFFERENT MICROGLIA
Microglia from different mouse models function differentially
In culture, the number of apoptotic microglia was very low in Tp
and TpTi mice but very high in Lp and LpLi mice in comparison
with those in NML mice (Fig. 3A). The numbers of apoptotic microglia in cortex in Lp and LpLi mice were significantly higher
than that in Tp and TpTi mice detected by TUNEL assay in vivo,
indicating that Lp and LpLi microglia were sensitive to apoptosis
(data not shown). In coculture of neuron-microglia, the numbers of
apoptotic neurons were very high in Tp and TpTi mice and very
low in Lp and LpLi mice in comparison with NML mice (Fig. 3B).
Lp microglia inhibited apoptotic effect of Tp microglia on neurons
in vitro (data not shown). Microglia from different mouse models
(Tp, TpTi, Lp, and LpLi mice) produced similar levels of IL-2,
IFN-␥, and IL-4 in culture at mRNA level (Fig. 3C). Microglia
from Tp and TpTi mice expressed significant higher level of
TNF-␣, whereas microglia from Lp and LpLi mice produced significant higher levels of IL-10 and TGF-␤ at mRNA level (Fig.
3C). Microglia from Tp and TpTi mice expressed significant
higher level of CCL25. Microglia from Lp and LpLi mice produced significant higher level of CX3CL1 and CXCL10 at mRNA
level (Fig. 3C). There was no difference in production of CCL5
and CXCL12. Using ELISA, we confirmed the similar pattern of
cytokine and chemokine expressions in microglia from different
mouse models at protein level (Fig. 3D). In culture without LPS,
microglia from Tp and TpTi mice produced 3- to 5-fold higher
nitrite than those in NML, Lp, and LpLi mice. In culture with LPS,
microglia from Tp, TpTi, Lp, and LpLi mice secreted identical
amounts of nitrite (Fig. 3E). NO inhibitor N␻-nitro-L-arginine
methyl ester significantly inhibited apoptotic effect of Tp and TpTi
microglia on neurons in vitro (data not shown). Moreover, mRNA
of brain-derived neurotrophic factors, a member of the neurotrophin family, was at a low level in NML microglia and did not alter
in Tp, TpTi, Lp, and LpLi mice (data not shown). Thus, Tp and
TpTi mouse model elicited the microglia that were expressing
CCR9 at a high frequency, resistant to apoptosis, neurotoxic, expressing a high level of TNF-␣ and autoligand CCL25, and high
NO productive; the microglia from Lp and LpLi mice were frequently highly expressed CXCR3, sensitive to apoptosis, neurosupportive, producing a high level of IL-10, TGF-␤, autoligand
CXCL10, and a low level of NO.
FIGURE 4. Expression of Irg1 in microglia from different mouse models. A, Triple-color flow cytometric analysis of Irg1 in microglia from
NML, Tp, TpTi, Lp, and LpLi mice. Lymphocyte-depleted single-cell suspensions from cortices were prepared and stained as described in Materials
and Methods. Gray curves, isotype Ab controls. The numbers showed
Irg1⫹ cells. Data were representatives of eight animals per group. B, Distribution of Irg1-positive microglia-infiltrating inflammation-reactive cortex determined by immunohistochemistry. The arrows were indicating
Irg1-expressing microglia (brown). The experiments in each group (n ⫽ 6)
were independently repeated twice. Magnifications, ⫻1000. The insets
were isotype controls. C, Distribution of Irg1⫹ microglia-infiltrating inflammation-reactive cortex determined by immunofluorescence digital
confocal microscopy. The microglia in cortex from TpTi mice were purified and stained. Magnification, ⫻1200. Bar, 12 ␮m. (i) was isotype control. D, Expression levels of Irg1 in microglia-infiltrating inflammationreactive cortex from mice determined by Northern and Western blot
analyses (upper and middle panels) and real-time quantitative RT-PCR
(lower panel). The illustrated data are from a single representative experiment of each group animals (n ⫽ 6). The mRNA of Irg1 in purified
microglia from cortex was determined by real-time quantitative detection
of RT-PCR. The showing bars were mean ⫾ SD of six experiments in each
group. ⴱ, p ⬍ 0.001, Tp or TpTi vs NML, Lp, or LpLi.
The Journal of Immunology
(Fig. 4D). Irg1-expressing microglia were also found in hippocampus
region of Tp and TpTi mice (data not shown).
Neurotoxic microglia function via CCL25/CCR9/Irg1 pathway
activation
We cotransfected CCR9, CXCR3, and Irg1 into purified microglia
from fetal mice cerebral cortices. Levels of phosphorylated-Irg1
proteins were dependent on transfected amount of pan Irg1 proteins and ligation of CCR9/CCL25. CXCR3/CXCL10 ligation induced no phosphorylated-Irg1 proteins (Fig. 5A1). The levels of
phosphorylated-Irg1 were dependent on the transfected amount
of CCR9 proteins (Fig. 5A2) but not on the transfected amount of
CXCR3 protein (Fig. 5A3). CCL25 could also directly induce Irg1
phosphorylation in freshly isolated CCR9⫹Irg1⫹ microglia from Tp
and TpTi mice (data not shown). We cotransfected CCR9 and
CXCR3 into purified microglia in the absence or presence of Irg1
transfection. Real-time RT-PCR data revealed that CCL25 ligation
caused increased levels of TNF-␣ mRNA (Fig. 5B1) and reduced
levels of IL-10 and TGF-␤ mRNA in an Irg1-transfection-dependent
manner (Fig. 5B2). CCR9/CCL25 ligation could induce higher NO
production in transfected microglia than CXCL10/CXCR3 in an Irg1transfection-dependent manner (Fig. 5C). CCR9/CCL25 ligation
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FIGURE 5. CCL25/CCR9 selectively and functionally activates Irg1
protein in microglia. A, Normal microglia purified from fetal mice cerebral cortices were cotransfected with
vectors encoding CCR9, CXCR3,
and Irg1 (600 ng each constantly; or
200, 400, and 600 ng, respectively)
as indicated (A1, A2, and A3). Cells
were pretreated at absence or presence of CCL25 and/or CXCL10
(each 100 ng/ml) before cell lysis.
Irg1 proteins were immunoprecipitated from the cell lysates for detection of levels of phosphorylation using a rabbit anti-phosphorylated
proteins Ab. B. The mRNA levels of
TNF-␣ (B1), IL-10 (䡺), and TGF-␤
(f) (B2) in microglia-transfected
CCR9 and CXCR3 at absence (upper
panels) or presence (lower panels) of
Irg1 transfection determined by realtime quantitative RT-PCR. Cells
were pretreated at absence or presence of CCL25 and/or CXCL10
(each 100 ng/ml) for 24 h before cell
lysis. LPS was used as positive controls. The showing bars were mean ⫾
SD (n ⫽ 8). ⴱ, p ⬍ 0.001. C, NO
production of microglia-transfected
CCR9 and CXCR3 at absence (upper
panel) or presence (lower panel) of
Irg1 transfection (n ⫽ 8). Cells were
stimulated without or with CCL25
and/or CXCL10 (each 100 ng/ml).
LPS was used as positive controls. D
and E, Flow cytometric analysis of
apoptotic microglia (D) and neurons
(E). The procedures were described
in Fig. 3, A and B. The microglia
transfected CCR9 and CXCR3 at absence (upper panels) or presence
(lower panels) of Irg1 transfection.
Exogenous TNF-␣ (20 ng/ml) was
used as an apoptotic inducer. Data for
apoptotic microglia (D) and apoptotic
neurons (E) were mean ⫾ SD (n ⫽ 8,
ⴱ, p ⬍ 0.001).
3651
3652
enhanced the resistance to TNF-␣-induced apoptosis of microglia in
an Irg1 transfection-dependent manner (Fig. 5D). CCR9/CCL25 ligation enhanced the neurotoxicity of microglia to cause neuron apoptosis in an Irg1 transfection-dependent manner (Fig. 5E). Thus, by
means of CCR9/CCL25/Irg1 pathway, activated microglia produce
higher levels of TNF-␣ and lower IL-10 and TGF-␤, along with enhanced resistance to apoptosis and increased neurotoxicity.
CNS-resident microglia and blood-derived macrophages
function differentially in vivo
To determine which cells, CNS-resident microglia or CNS-infiltrating macrophages (8, 27), might be a major responsive population in different mouse models, purified resting microglia and
blood-derived macrophages (28) were transfected with CCR9 plus
Irg1 or CXCR3 and subsequently primed with TSAg and rLCMVNP, respectively. CCR9⫹Irg1⫹-transfected microglia (intracranially) or CXCR3-transfected macrophages (i.v.) were infused into
adult mice, respectively. Mice were then intracranially infected
with T. gondii (Ti mice) or LCMV (Li mice). The results of cell
frequency (Fig. 6, A and B) and proliferation (Fig. 6, C and D)
showed that TSAg-primed CNS-resident CCR9⫹Irg1⫹ microglia
dominantly functioned and highly proliferated in Ti mice, whereas
both CXCR3⫹ CNS-resident microglia and blood-derived macrophages rapidly infiltrated into CNS and proliferated in a specific
Ag priming-dependent manner in Li mice. Thus, CCR9⫹Irg1⫹
neurotoxic microglia, often seen in Tp and TpTi mice, were mainly
generated from CNS-resident resting microglia; CXCR3⫹Irg1⫺
neurosupportive microglia, often seen in Lp and LpLi mice, would
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FIGURE 6. Analysis of CNS-resident or blood-derived CCR9⫹Irg1⫹ or CXCR3⫹ microglia/macrophages
in Ti or in Li mice. Normal purified microglia from fetal
mice cerebral cortices and purified F4/80⫹ blood-derived macrophages (27) (CI:A3-1; Abcam) were cotransfected with vectors encoding CCR9 and Irg1 (A and
C) or CXCR3 (B and D) (600 ng each), then cultured at
absence or presence of TSAg (1 ␮g/ml) or rLCMVNP
(0.5 ␮mol; Ref. 17) for 48 h indicated as TSAg⫺,
TSAg⫹, LCMV⫺, or LCMV⫹. Cells were labeled with
CFSE (Molecular Probes). The 106 of transfected microglia (intracranially) or macrophages (i.v.) infused
were into adult mice, respectively, indicated as (Microglia i.c.) and (Macrophage i.v.). After 24 h of infusion,
the mice were intracranially infected with T. gondii DX
strain (five cysts, Ti mice) (A and C) or LCMV clone 13
(100 PFU, Li mice) (B and D). Mice sacrificed at day as
indicated. A and B, Triple-color flow cytometric analyses of CCR9 or CXCR3 on microglia/macrophage from
Ti mice (A) or Li mice (C). Black curves, CCR9 or
CXCR3 staining; gray curves, isotype Ab controls. The
shown numbers were percentages of CCR9 or CXCR3positive cells. Data were representatives of eight animals per group. The experiments in each group were
independently repeated twice. B and D, Flow cytometric
analyses of the intensity of CFSE on microglia/macrophage from Ti mice (C) or Li mice (D). Data were representatives of eight animals per group.
DIFFERENT MICROGLIA
The Journal of Immunology
3653
be generated from both these infiltrating blood-derived vascular
macrophages (might be also perivascular macrophages) (29) and
CNS-resident resting microglia.
CCR9 and Irg1 siRNA alter neurotoxic and neurosupportive
profiles of microglia in vivo
TpTi mice administered with sufficient doses (2 ␮g) of hairpin
siRNACCR9, siRNAIrg1, or siRNAMIX were significantly
knocked down expression of CCR9 and Irg1 during the process of
establishment of TpTi mouse model (Fig. 7A) but not administrating low doses (0.02 ␮g) of the substances (data not shown). The
high (Fig. 7A) or low (data not shown) dose of mismatched siRNA
sequence (siRNAMIS) did not show any knocking-down effect.
There were “cross-knocking-down” phenomena, e.g., siRNACCR9 also inhibited Irg1 expression, and vice versa (Fig. 7A).
The possible explanation could be that CCR9 and Irg1 in the pathway had a close cross-taking, the expression and function of one
would interfere with those of the other. The exact mechanism of
the reciprocal inhibition should be subjected to further investigation. Data of flow cytometry (Fig. 7B) and immunohistochemistry
(Fig. 7C) revealed that siRNAMIX administration significantly
knocked down CCR9 expression and up-regulated CXCR3 expression on microglia in cortex from TpTi mice. The similar results
were also seen in TpTi mice administered with siRNACCR9 or
siRNAIrg1 (data not shown). Mortality of TpTi mice with different
administration revealed (Fig. 7D) that NS (data not shown) and
siRNAMIS did not change the pattern of TpTi mice death. The
administrations of siRNACCR9, siRNAIrg1, or siRNAMIX significantly decreased mortality in TpTi mice. Moreover, administrations of siRNACCR9, siRNAIrg1, or siRNAMIX suppressed
the levels of T. gondii DNA in cortex of TpTi mice (Fig. 7E).
Two types of microglia in CNS by priming with different
neurotropic pathogens
We further examined effects on microglial elicitation in CNS using
a series of vaccines (Ags) of neurotropic pathogens, including different intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury. The intracranial priming with T. gondii
STAg (virulent RH strain), HIV vaccine (30), rTMEV-VP3, and
A␤25–35 significantly caused an elicitation of neurotoxic
CCR9⫹Irg1⫹ microglia in CNS (Table III). Generally, in mice
administered with the neurotropic pathogens causing chronic but
irreversible neurodemyelinative or neurodegenerative diseases,
such as AIDS, Alzheimer’s disease, Theiler’s murine encephalomyelitis virus-induced demyelinating disease, experimental autoimmune encephalomyelitis model, and some of the intracellular
parasitic (T. gondii) infection, microglial elicitation was oriented
to neurotoxic. In mice administered with the neurotropic pathogens
causing acute but reversible neuroinflammatory diseases, such as
most of viral and bacterial encephalitis and reversible brain lesions, the microglia were apparently developing into neurosupportive cell type.
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FIGURE 7. Effects of siRNACCR9 and/or siRNAIrg1 on TpTi mice. A, CCR9 and Irg1 expression in
microglia from TpTi mice determined by Northern and
Western blot analyses (left panels) and real-time quantitative RT-PCR (right panels). Mice were treated with
normal saline (NS), siRNACCR9, siRNAIrg1, siRNACCR9 plus siRNAIrg1 (siRNAMIX), or mismatch
siRNA (siRNAMIS) along with priming and infection
with T. gondii and scarified at day 10 of infection (n ⫽
10). The showing bars for CCR9 and Irg1 mRNA were
mean ⫾ SD. ⴱ, p ⬍ 0.01– 0.001, siRNACCR9, siRNAIrg1, or siRNAMIX vs NS or siRNAMIS for CCR9
and Irg1. B, Triple-color flow cytometric analysis of
CCR9 and CXCR3 on microglia from individual TpTi
animals (n ⫽ 8). The animals were treated with siRNAMIX and sacrificed at day 14 postinfection. Red curves,
CCR9 staining; blue curves, CXCR3 staining; gray
curves, isotype Ab controls. C, Distribution of CCR9⫹
or CXCR3⫹ microglia-infiltrating inflammation-reactive cortex from TpTi mice (n ⫽ 8) determined by immunohistochemistry. The animals were treated with
siRNAMIX and sacrificed at day 14 postinfection. Magnifications, ⫻400. The insets were isotype controls. D,
Mortality of mice with different treatments. The animals
were treated with NS (data not shown), siRNACCR9
(F), siRNAIrg1 (䡺), siRNAMIX (E), or siRNAMIS
(f) along with priming and infection with T. gondii.
Mice (n ⫽ 10) were monitored daily for development of
clinical symptoms and survival. E, Levels of T. gondii
DNA in cortex of TpTi mice treated with NS, siRNACCR9, siRNAIrg1, siRNAMIX, or siRNAMIS. T.
gondii DNA determined by real-time quantitative PCR
were mean ⫾ SD (n ⫽ 10). ⴱ, p ⬍ 0.001, siRNACCR9,
siRNAIrg1, or siRNAMIX vs NS or siRNAMIS.
3654
DIFFERENT MICROGLIA
Table III. Effects on microglial derivation in CNS by priming with antigens or other procedures of different neurotropic
pathogensa
Vaccineb
NS
Toxoplasma STAg*
Leishmania SLAg
Malaria vaccine
HIV vaccine*
iLCMVd
rVSV
iJEV
rHSV
rTMEV-VP3*
Meningococcal vaccine
EAE*
A␤25–35*
Brain lesion
Dosec
Microgliae (%)f
CCR9g
CXCR3g
Irg1g
10 ␮l ⫻ 3
10 ␮g ⫻ 3
10 ␮g ⫻ 3
50 ␮g ⫻ 3
50 ␮g ⫻ 3
100 pfu ⫻ 3
50 ␮g ⫻ 3
100 pfu ⫻ 3
50 ␮g ⫻ 3
50 ␮g ⫻ 3
50 ␮g ⫻ 3
EAE model
25 ␮g ⫻ 3
Axotomy
2
15
8
6
17
5
6
6
8
20
9
19
20
7
UD; 1.1 ⫻ 102
62; 7.6 ⫻ 103
7; 8.4 ⫻ 102
4; 3.7 ⫻ 102
62; 6.7 ⫻ 103
6; 1.1 ⫻ 103
7; 1.8 ⫻ 103
4; 6.8 ⫻ 102
⬍2; 4.3 ⫻ 102
57; 8.9 ⫻ 103
⬍2; 3.0 ⫻ 102
55; 8.2 ⫻ 103
58; 6.8 ⫻ 103
1; 5.2 ⫻ 102
2; 2.1 ⫻ 103
⬍2; 1.2 ⫻ 102
38; 4.5 ⫻ 103
37; 4.7 ⫻ 103
⬍2; 1.5 ⫻ 102
62; 8.1 ⫻ 103
46; 6.2 ⫻ 103
35; 5.5 ⫻ 103
48; 5.7 ⫻ 103
⬍2; 1.8 ⫻ 102
52; 6.5 ⫻ 103
⬍2; 1.9 ⫻ 102
⬍2; 1.5 ⫻ 102
63; 7.1 ⫻ 103
UD; 1.4 ⫻ 102
57; 5.8 ⫻ 103
⬍2; 1.9 ⫻ 102
UD; 1.5 ⫻ 102
64; 6.1 ⫻ 103
⬍2; 5.2 ⫻ 102
ND; 1.0 ⫻ 102
ND; 5.1 ⫻ 102
ND; 5.9 ⫻ 102
65; 8.1 ⫻ 103
⬍2; 6.1 ⫻ 102
61; 8.4 ⫻ 103
53; 7.7 ⫻ 103
⬍2; 4.2 ⫻ 102
Discussion
Microglia are now recognized as the prime and major components
of an intrinsic brain immune system in the CNS (1). Many researchers now consider this innate immune response in the brain to
be a potentially pathogenic factor in a number of CNS diseases that
lack the prominent leukocytic infiltrates of adaptive immune responses, but that do have activated microglia to initiate neuroinflammation and neurodegeneration (1, 4, 31). An important but yet
unresolved question is whether microglial activation in vivo can be
triggered by pathophysiological stimuli that are not neuron derived
and whether such idiopathic activation of microglia can result in
microglial neurotoxicity exerted toward healthy neurons (4). This
issue is particularly relevant to understanding neurodegenerative
diseases such as AIDS and Alzheimer’s disease, in which chronic
CNS inflammation is believed to play a role in the pathogenesis
(31). In the present study, we have used different Ags (pathogens)—STAg from T. gondii low-virulent DX strain, a representative of neurotropic intracellular parasite, and rLCMVNP of
LCMV low-virulent clone 13, a representative of neurotropic
chronic virus—to directly (intracranially injection) and differentially prime microglia. After priming, a single dose of T. gondii or
LCMV have sublethally and intracranially been infected (rechallenged) in mice. These approaches allow us to investigate preactivated (primed) microglial behaviors in different neurotropic
pathogenesis in vivo. T. gondii Ag and LCMV Ag priming have
indeed elicited two different types of microglia (Figs. 1– 4). We
have further confirmed the observation by intracranially priming
and rechallenging in mice with a series of different neurotropic
pathogens, including intracellular parasites, chronic virus, bacteria,
toxic substances, and CNS injury (Table III). We therefore tentatively suggest to term the neurotoxic CCR9⫹Irg1⫹ cell pattern as
killer microglia (kM) and the neurosupportive CXCR3⫹Irg1⫺ cell
pattern as supporter microglia (sM).
We have found that microglia in normal animals appear at very
low frequency (Fig. 1, B–D). Most of the microglia in normal
animals are CXCR3⫺CCR9⫺Irg1⫺ (Figs. 2 and 4). They seem to
be inactive with respect to their functions of production of cytokines and chemokines in vitro (Fig. 3). There is so far no significant marker found on their surface with regard to expression of
chemokine receptors (Table. II). We have also obtained similar
data in peripheral macrophages in normal animals (data not
shown). However, are two subsets of microglia in CNS differentiated from a third phenotypic subset of inactive/undifferentiated
microglia? Or, do they proliferate or migrate to the site of pathology and differentiate into one or the other subsets by the specific
pathogen? We have found indirect evidence that CCR9⫹Irg1⫹ microglia are mainly derived from CNS-resident resting microglia;
CXCR3⫹Irg1⫺ microglia are derived from both these infiltrating
blood-derived vascular macrophages and CNS-resident resting microglia (Fig. 6). The direct evidence is warranted for further confirmation of these hypotheses.
The microglial immune functions are controlled and elaborated
by intrinsic factors in the CNS neurons. Both membrane-bound
and secreted factors, such as cytokines and chemokines, possess
the potential of controlling endogenous immune activity within the
CNS (6, 8, 32–35). Cytokines and chemokines constitute a substantial fraction of the microglial communication and effector system. TNF-␣, IL-6, and IFN-␥ have been shown to be critical for
protective actions as well as harmful outcomes of microglial engagement (33, 36). Chemokine receptor CCR9 expression is
highly regulated during T cell development. Most immature DN
thymocytes express no or low levels of CCR9 on their surface (37,
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a
Mice were primed intracranially three times with indicated dose at 2-wk intervals. After 2 wk, the last priming mice were sacrificed, and experiments
were carried out as described in Materials and Methods.
b
The priming of different neurotropic pathogens used in this study was as follows: NS, normal saline (as negative control). Toxoplasma STAg, soluble antigen
from T. gondii RH strain tachyzoites; Leishmania SLAg, soluble Leishmania Ag from Leishmania major (MRHO/IR/75/ER) promastigotes; malaria vaccine,
malaria DNA ME-TRAP (malaria pre-erythrocytic Plasmodium falciparum Ags vaccine; Qiagen; HIV vaccine, HIV DNA vaccine mFLex:gp120, which is DNA
plasmid expressing mFLex:gp120 (Ref. 30); iLCMV, inactivated LCMV Armstrong clone 53b using short-wavelength UV radiation (UVC, 254 ␮m); rVSV, the
recombinant vesicular stomatitis virus; iJEV, inactivated Japanese encephalitis virus Beijing-1 strain using short-wavelength UV radiation (UVC, 254 ␮m); rHSV,
recombinant vaccinia HSV (rVVESgB498 –505); rTMEV-VP3, recombinant A3A capsid protein (VP324 –37) of Theiler’s murine encephalomyelitis virus BeAn
8386 strain; meningococcal vaccine, meningococcal serogroup A/C/W135/Y polysaccharide vaccine (50 ␮g of each of the four polysaccharides in a single dose);
EAE, experimental autoimmune encephalomyelitis induction by myelin oligodendrocyte glycoprotein peptide35–55 plus mycobacterium tuberculosis H37Ra
immunization subcutaneously and an i.v. challenge with B. pertussis toxin. The mice were scarified at day 14 post-onset; A␤25– 35, ␤-amyloid protein fragment
25–35; brain lesion, the facial axotomy model (day 3 postaxotomy).
c
The volume in each priming was 10 ␮l.
d
Inactivated virus were used dose of equivalent to 100 (pfu ␮l⫺1) ⫻ 3.
e
Microglia from temporal cortex were determined by flow cytometry (FL, % of CD11b/Mac1⫹CD45⫹ cells) as described in Materials and Methods.
f
Data were mean ⫾ SD (each group n ⫽ 5– 8). For simplification of the table, SDs were not shown. UD, undetectable. ND, no determination. *,
Percentage of microglia in cortex and CCR9 and Irg1 expression in microglia were significantly higher than other groups, meanwhile, CXCR3 expression
in microglia was significantly lower; p ⬍ 0.001.
g
CCR9, CXCR3, and Irg1 in microglia from cortex were determined by flow cytometry (FL, % of positive cells) and real-time quantitative RT-PCR
(RT-PCR, mRNA copies per 25 ng cDNA) as described in Materials and Methods.
The Journal of Immunology
Acknowledgments
We thank Chen Lang, Guo Hui, Zhang Ying, Cai Guobin, and Chen Zhonghua for their constructive scientific discussions, excellent technical assistance, and animal husbandry.
Disclosures
The authors have no financial conflict of interest.
References
1. Medzhitov, R., and C. Janeway Jr. 2000. Advances in immunology: innate immunity. N. Engl. J. Med. 343: 338 –344.
2. Streit, W. J., S. A. Walter, and N. A. Pennell. 1999. Reactive microgliosis. Prog.
Neurobiol. 57: 563–581.
3. Fischer, H. G., and G. Reichmann. 2001. Brain dendritic cells and macrophages/
microglia in central nervous system inflammation. J. Immunol. 166: 2717–2726.
4. Kreutzberg, G. W. 1996. Microglia: a sensor for pathological events in the CNS.
Trends Neurosci. 19: 312–318.
5. Carson, M. J., and J. G. Sutcliffe. 1999. Balancing function vs self defense: the
CNS as an active regulator of immune responses. J. Neurosci. Res. 55: 1– 8.
6. Babcock, A. A., W. A. Kuziel, S. Rivest, and T. Owens. 2003. Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS. J.
Neurosci. 23: 7922–7930.
7. Karpus, W. J., and R. M. Ransohoff. 1998. Chemokine regulation of experimental
autoimmune encephalomyelitis: temporal and spatial expression patterns govern
disease pathogenesis. J. Immunol. 161: 2667–2671.
8. Rappert, A., I. Bechmann, T. Pivneva, J. Mahlo, K. Biber, C. Nolte, A. D. Kovac,
C. Gerard, H. W. Boddeke, R. Nitsch, and H. Kettenmann. 2004. CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion. J.
Neurosci. 24: 8500 – 8509.
9. Qiuping, Z., X. Jei, J. Youxin, J. Wei, L. Chun, W. Jin, W. Qun, L. Yan,
H. Chunsong, Y. Mingzhen, et al. 2004. CC chemokine ligand 25 enhances resistance to apoptosis in CD4⫹ T cells from patients with T-cell lineage acute and
chronic lymphocytic leukemia by means of livin activation. Cancer Res. 64:
7579 –7587.
10. Lee, C. G., N. A. Jenkins, D. J. Gilbert, N. G. Copeland, and W. E. O’Brien.
1995. Cloning and analysis of gene regulation of a novel LPS-inducible cDNA.
Immunogenetics 41: 263–270.
11. Cheon, Y. P., X. Xu, M. K. Bagchi, and I. C. Bagchi. 2003. Immune-responsive
gene 1 is a novel target of progesterone receptor and plays a critical role during
implantation in the mouse. Endocrinology 144: 5623–5630.
12. Bliss, S. K., Y. Zhang, and E. Y. Denkers. 1999. Murine neutrophil stimulation
by Toxoplasma gondii antigen drives high level production of IFN-␥-independent
IL-12. J. Immunol. 163: 2081–2088.
13. Von Herrath, M. G., D. P. Berger, D. Homann, T. Tishon, A. Sette, and
M. B. Oldstone. 2000. Vaccination to treat persistent viral infection. Virology
268: 411– 419.
14. Ahmed, R., A. Salmi, L. D. Butler, J. M. Chiller, and M. B. Oldstone. 1984.
Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of
persistently infected mice: role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160: 521–540.
15. Matloubian, M., R. J. Concepcion, and R. Ahmed. 1994. CD4⫹ T cells are required to sustain CD8⫹ cytotoxic T-cell responses during chronic viral infection.
J. Virol. 68: 8056 – 8063.
16. Lee, H., S. Cha, M. S. Lee, G. J. Cho, W. S. Choi, and K. Suk. 2003. Role of
antiproliferative B cell translocation gene-1 as an apoptotic sensitizer in activation-induced cell death of brain microglia. J. Immunol. 171: 5802–5811.
17. Xie, Z., M. Wei, T. E. Morgan, P. Fabrizio, D. Han, C. E. Finch, and
V. D. Longo. 2002. Peroxynitrite mediates neurotoxicity of amyloid ␤-peptide1–
42- and lipopolysaccharide-activated microglia. J. Neurosci. 22: 3484 –3492.
18. Venters, H. D., Q. Tang, Q. Liu, R. W. VanHoy, R. Dantzer, and K. W. Kelley.
1999. A new mechanism of neurodegeneration: a proinflammatory cytokine inhibits receptor signaling by a survival peptide. Proc. Natl. Acad. Sci. USA 96:
9879 –9884.
19. Sanna, M. G., C. S. Duckett, B. W. Richter, C. B. Thompson, and R. J. Ulevitch.
1998. Selective activation of JNK1 is necessary for the anti-apoptotic activity of
hILP. Proc. Natl. Acad. Sci. USA 95: 6015– 6020.
20. Massari, P., Y. Ho, and L. M. Wetzler. 2000. Neisseria meningitidis porin PorB
interacts with mitochondria and protects cells from apoptosis. Proc. Natl. Acad.
Sci. USA 97: 9070 –9075.
21. Sica, A., A. Saccani, A. Borsatti, C. A. Power, T. N. Wells, W. Luini,
N. Polentarutti, S. Sozzani, and A. Mantovani. 1997. Bacterial lipopolysaccharide
rapidly inhibits expression of C-C chemokine receptors in human monocytes. J.
Exp. Med. 185: 969 –974.
22. Kruse, N., M. Pette, K. Toyka, and P. Rieckmann. 1997. Quantification of cytokine mRNA expression by RT-PCR in samples of previously frozen blood. J.
Immunol. Methods 210: 195–203.
23. Mitrasinovic, O. M., G. V. Perez, F. Zhao, Y. L. Lee, C. Poon, and
G. M. Murphy, Jr. 2001. Overexpression of macrophage colony-stimulating factor receptor on microglial cells induces an inflammatory response. J. Biol. Chem.
276: 30142–30149.
24. Takei, Y., K. Kadomatsu, Y. Yuzawa, S. Matsuo, and T. Muramatsu. 2004. A
small interfering RNA targeting vascular endothelial growth factor as cancer
therapeutics. Cancer Res. 64: 3365–3370.
25. Dijkstra, I. M., S. Hulshof, P. Van der Valk., H. W. Boddeke, and K. Biber. 2004.
Cutting edge: activity of human adult microglia in response to CC chemokine
ligand 21. J. Immunol. 172: 2744 –2747.
26. Paglinawan, R., U. Malipiero, R. Schlapbach, K. Frei, W. Reith, and A. Fontana.
2003. TGF-␤ directs gene expression of activated microglia to an antiinflammatory phenotype strongly focusing on chemokine genes and cell migratory genes. Glia 44: 219 –231.
27. Raivich, G., and R. Banati. 2004. Brain microglia and blood-derived macrophages: molecular profiles and functional roles in multiple sclerosis and animal
models of autoimmune demyelinating disease. Brain Res. Brain Res. Rev. 46:
261–281.
28. Smith, P. 2004. Schistosoma mansoni worms induce anergy of T cells via selective up-regulation of programmed death ligand 1 on macrophages. J. Immunol.
173: 1240 –1248.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
38). A considerable body of investigations has shown that CCR9/
CCL25 is important for the homing, development, and homeostasis of T cells, particularly, mucosal T cells (39, 40). In the present
study, we have documented that CCR9-expressing microglia is the
dominant phenotype in Tp and TpTi mice, and CXCR3-expressing
microglia is the predominant cell population in Lp and LpLi mice.
To our knowledge, this is the first observation on expression of
CCR9 on microglia in CNS. Furthermore, we have demonstrated
that Tp and TpTi mouse model-induced CCR9⫹Irg1⫹ killer microglia express a high level of TNF-␣, CCL25, and NO by means
of CCR9/CCL25/Irg1 pathway activation; Lp and LpLi mouse
model-induced CXCR3⫹Irg1⫺ supporter microglia produce high
levels of IL-10, TGF-␤, CXCL10, and relatively lower levels of
NO (Figs. 3 and 5). Our data suggest that distinctive microglia can
be induced by different neurotropic pathogens. Of different profiles
in cytokine and chemokine release and chemokine receptor expression, killer and supporter microglia play distinctive roles in
pathogenesis to degenerate or to regenerate CNS neuron. The direct evidence is still lacking to demonstrate neurotoxic and neurosupportive effects of CCR9⫹Irg1⫹ and CXCR3⫹Irg1⫺ microglia in vivo. It is worthwhile to investigate whether there is
interaction between killer and supporter microglia cells in vivo.
Moreover, how microglia contribute to neuronal death and regeneration in vivo should be also subjected to further investigation.
It has been discussed for a long time whether activated microglia are beneficial or harmful to neurons. The challenge is to determine what sort of pathological scenarios can transform microglia into autoaggressive effector cells that attack healthy neurons
and cause neuroinflammation and neurodegeneration (2, 5, 32, 35,
36, 41). We are the first to document that Irg1 selectively and
functionally expressed in killer microglia in vivo (Fig. 4). Neurotoxicity of killer microglia is exerted by means of the CCL25/
CCR9/Irg1 pathway (Fig. 5). Under normal and especially under
pathological conditions, neuronal well being and proper functioning are highly dependent on the presence of large numbers of glial
cells that sustain an abundance of neurosupportive functions. Microglial facilitation of cell death is likely to also play an important
role in the elimination of superfluous cells during CNS development. In some chronic CNS diseases, such as Alzheimer’s disease,
the neurodegeneration in Alzheimer’s disease is caused by autoaggressive microglia that produce neurotoxins in response to continued amyloid (A␤) exposure (42). HIV is known to target primarily microglia in the CNS. Persistent infection of microglia with
HIV can result in depleting and/or disabling microglia, leading to
opportunistic CNS infections, neurodegeneration, and dementia.
We have shown that microglia are capable of performing both
neuroconstructive and neurodestructive functions. CCR9 and/or
Irg1 siRNA significantly knock down target expressions in killer
microglia and optimistically reduce mortality and change the expressive and functional profiles of killer and supporter microglia in
vivo in TpTi mice (Fig. 7). The observation is indicating that
CCR9 and/or Irg1 are potential therapeutic targets for gene therapy
of some neurodegenerative and neuroinflammatory diseases.
3655
3656
29. Williams, K., X. Alvarez, and A. A. Lackner. 2001. Central nervous system
perivascular cells are immunoregulatory cells that connect the CNS with the
peripheral immune system. Glia 36: 156 –164.
30. Sailaja, G., S. Husain, B. P. Nayak, and A. M. Jabbar. 2003. Long-term maintenance of gp120-specific immune responses by genetic vaccination with the
HIV-1 envelope genes linked to the gene encoding Flt-3 ligand. J. Immunol. 170:
2496 –2507.
31. Streit, W. J., and D. L. Sparks. 1997. Activation of microglia in the brains of
humans with heart disease and hypercholesterolemic rabbits. J. Mol. Med. 75:
130 –138.
32. Priller, J., A. Flugel, T. Wehner, M. Boentert, C. A. Haas, M. Prinz,
F. Fernandez-Klett, K. Prass, I. Bechmann, B. A. de Boer, et al. 2001. Targeting
gene-modified hematopoietic cells to the central nervous system: use of green
fluorescent protein uncovers microglial engraftment. Nat. Med. 7: 1356 –1361.
33. Hanisch, U. K. 2002. Microglia as a source and target of cytokines. Glia 40:
140 –155.
34. Thomas, W. E. 1999. Brain macrophages: on the role of pericytes and perivascular cells. Brain Res. Rev. 30: 42–57.
35. Qiusheng, S., M. L. Zhao, A. C. A. Morgan, C. F. Brosnan, and S. C. Lee. 2004.
15-Deoxy-␦12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications. J.
Immunol. 173: 3504 –3513.
DIFFERENT MICROGLIA
36. Streit, W. J. 2002. Microglia as neuroprotective, immunocompetent cells of the
CNS. Glia 40: 133–139.
37. Uehara, S., K. Song, J. M. Farber, and P. E. Love. 2002. Characterization of
CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T-cell development: CD3hiCD69⫹ thymocytes and ␥␦TCR⫹ thymocytes
preferentially respond to CCL25. J. Immunol. 168: 134 –142.
38. Carramolino, L., A. Zaballos, L. Kremer, R. Villares, P. Martin, C. Ardavin,
C. Martinez-A, and G. Marquez. 2001. Expression of CCR9␤-chemokine receptor is modulated in thymocyte differentiation and is selectively maintained in
CD8⫹ T cells from secondary lymphoid organs. Blood 97: 850 – 857.
39. Mora, J. R., M. R. Bono, N. Manjunath, W. Weninger, L. L. Cavanagh,
M. Rosemblatt, and U. H. Von Andrian. 2003. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 424: 88 –93.
40. Papadakis, K. A., C. Landers, J. Prehn, E. A. Kouroumalis, S. T. Moreno,
J. C. Gutierrez-Ramos, M. R. Hodge, and S. R. Targan. 2003. CC chemokine
receptor 9 expression defines a subset of peripheral blood lymphocytes with
mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile. J. Immunol.
171: 159 –165.
41. Guillemin, G. J., and B. J. Brew. 2004. Microglia, macrophages, perivascular
macrophages, and pericytes: a review of function and identification. J. Leukocyte
Biol. 75: 388 –397.
42. Streit, W. J. 2004. Microglia and Alzheimer’s disease pathogenesis. J. Neurosci.
Res. 77: 1– 8.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017