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*
Department of Molecular Biology and Biochemistry and
Reeve-Irvine Research Center, University of California, Irvine, CA 92612; and
Department of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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0.04) T
cell and macrophage/microglial (determined by F4/80 Ag expression,
p 0.004) infiltration into the CNS. Moreover,
MHV-infected CCR2-/- mice displayed a significant
decrease in Th1-associated factors IFN-
(p
0.001) and RANTES/CCL5 (p 0.002) within the CNS
as compared with CCR2+/+ mice. Further, peripheral
CD4+ and CD8+ T cells from immunized
CCR2-/- mice displayed a marked reduction in IFN-
production in response to viral Ag and did not migrate into the CNS of
MHV-infected recombination-activating gene
(RAG)1-/- mice following adoptive transfer. In
addition, macrophage/microglial infiltration into the CNS of
RAG1-/- mice receiving CCR2-/- splenocytes
was reduced (p 0.05), which correlated with a
reduction in the severity of demyelination (p
0.001) as compared with RAG1-/- mice receiving
splenocytes from CCR2+/+ mice. Collectively, these results
indicate an important role for CCR2 in host defense and disease by
regulating leukocyte activation and trafficking. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/CXCL9, CCL2,
macrophage inflammatory protein-1
/CCL4, CCL5, and CCL7, suggesting
an important role for these molecules in participating in leukocyte
infiltration (9). Indeed, recent studies from our
laboratory have demonstrated that administration of neutralizing
antisera specific for either CXCL10 or CXCL9 to MHV-infected mice
resulted in increased mortality accompanied by decreased T lymphocyte
infiltration and IFN- expression as well as delayed viral clearance
from the brain (10, 11). In addition, anti-CCL5
treatment of MHV-infected mice resulted in reduced macrophage
infiltration into the CNS that correlated with a significant decrease
in the severity of demyelination (7). Collectively, these
studies indicate an important role for chemokines in host defense and
demyelination by attracting T lymphocytes and macrophages into the
CNS. Chemokines activate leukocytes by binding to seven transmembrane receptors coupled to G-proteins (for review, see Refs. 12 and 13). Among the chemokines expressed within the CNS of MHV-infected mice are CCL2 and CCL7 (9). Previous studies have indicated that these chemokines are potent chemoattractants for T cells as well as macrophages, presumably through binding and signaling through CCR2 expressed on the surface of these cells (14, 15, 16, 17, 18, 19). Based on experiments that have established that T cells and macrophages are important in both host defense and demyelination, it would be of great interest to determine the functional contributions of CCR2 in these separate processes. To this end, we have infected CCR2-/- mice with MHV and assessed the severity of disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MHV-4 (wild type) and MHV-V5A13.1 (containing a 142-aa deletion in the surface glycoprotein) were kindly provided by M. Buchmeier (The Scripps Research Institute, La Jolla, CA) (20). CCR2+/+ mice (B6129F2/J, obtained from The Jackson Laboratory, Bar Harbor, ME), CCR2-/- (129B6F2-Cmkbr2tm1Kuz), and recombination-activating gene (RAG)1-/- mice (B6129S-Rag1tm1Mom, obtained from The Jackson Laboratory) were housed under specific pathogen-free conditions in enclosed filter-top cages (14). RAG1-/- mice do not produce mature T or B lymphocytes (21). Age-matched (5–8 wk) mice were anesthetized by inhalation of methoxyflurane (Pitman-Moore, Washington Crossing, NJ), followed by intracerebral (i.c.) injection with 10 PFU of MHV-V5A13.1 suspended in 30 µl of sterile saline. Control (sham) animals were injected with sterile saline alone. Mice were sacrificed at scheduled time points, when one-half of each brain was used for plaque assay on the DBT astrocytoma cell line to determine viral burden, and the remaining halves were either fixed for histologic analysis, stored at -80°C for RNA isolation, homogenized in sterile PBS and stored at -20°C for ELISA, or used for FACS analysis (7, 22).
RNase protection assay (RPA)
Total RNA was extracted from brains of MHV-infected and sham animals using TRIzol reagent (Life Technologies, Rockville, MD) (9). Chemokine mRNA transcripts were determined using the mCK-5 multitemplate probe set (BD PharMingen, San Diego, CA). RPAs were performed with 15 µg total RNA using a previously described protocol (7, 9). For quantification of signal intensity, autoradiographs were scanned, and individual chemokine bands were normalized as the ratio of band intensity to the L32 control present in the probe set (7, 9). Analysis was performed using NIH Image 1.61 software (9, 23, 24).
Mononuclear cell isolation and flow cytometry
Mononuclear cells were obtained from brains of mice as previously described (7). FITC-conjugated rat anti-mouse CD4 and CD8 Abs were used to detect infiltrating CD4+ and CD8+ T cells (GK1.5 and Ly-2, respectively, obtained from BD PharMingen). FITC-conjugated rat anti-mouse F4/80 (C1:A3-1, Serotec, Oxford, U.K.) was used to detect activated macrophage/microglial cells (7). An isotype-matched FITC Ab was used as a control. Cells were incubated with Abs for 30 min at 4°C, washed, fixed in 1% paraformaldehyde, and analyzed on a FACStar (BD Biosciences, Mountain View, CA) (7). Data are presented as the percentage of positive cells within the gated population.
Intracellular cytokine staining
Intracellular cytokine staining was performed using a previously
described procedure (8). In brief,
CCR2+/+ and CCR2-/- mice
were infected i.p. with 2 x 105 PFU of
MHV-4. Splenocytes were isolated 8 days after infection, pooled
together, and 1 x 106 total cells were
stimulated with peptide corresponding to either the CD8 epitope in the
surface glycoprotein spanning residues 510–518 (25, 26)
or the CD4 epitope present in the membrane protein at residues 133–147
(27). After incubation for 6 h at 37°C in medium
containing GolgiStop (Cytofix/CytoPerm kit, BD PharMingen), cells were
washed and blocked with PBS containing 10% FBS and a 1/200 dilution of
CD16/32 (BD PharMingen). Cells were then stained for surface Ags with
either FITC-conjugated CD4, CD8, or rat IgG-2b (as control) for 45 min
at 4°C. Cells were fixed and permeabilized using the Cytofix/CytoPerm
kit and stained for intracellular IFN- using PE-conjugated
anti-IFN- (1:50; XMG1.2, BD PharMingen) for 45 min at 4°C.
Cells were analyzed on a FACStar (7). Data are presented
as the percentage of positive cells within the gated population. The
absolute numbers of Ag-specific CD4+ or
CD8+ T lymphocytes was calculated by multiplying
the fraction of dual-positive cells by the total number of cells
obtained from the spleen.
ELISA
Brains were homogenized in 1 ml sterile PBS and centrifuged at 400 x g for 5 min at 4°C. The supernatants were collected and stored at -20°C. Protein levels of cytokines and chemokines in brain samples of MHV-infected and sham mice at days 6 and 7 postinfection (p.i.) were assessed using specific Quantikine M murine immunoassay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s specifications (7, 10).
Adoptive transfer
Splenocytes from CCR2+/+ or CCR2-/- mice isolated 8 days p.i. (2 x 105 PFU MHV-4, i.p. injection) were adoptively transferred (5 x 106 cells suspended in 200 µl sterile HBSS) via injection into the retro-orbital sinus to RAG1-/- or CCR2-/- mice 3 days following i.c. infection with 10 PFU of MHV-V5A13.1 (8, 28). Mice were sacrificed 7 days p.i., and brains and spinal cords were removed. One-half of the brains was used for flow analysis, and the remaining half was used to determine viral titers. Spinal cords were stained with luxol fast blue (LFB) to assess the severity of demyelination. Control animals included MHV-infected (i.c.), RAG1-/-, and CCR2-/- mice receiving sterile HBSS.
Histology
Spinal cords were fixed by immersion overnight in 10% normal buffered formalin, after which the tissues were embedded in paraffin. The severity of demyelination was scored on slides stained with LFB. Slides were coded and read blindly by three investigators. Scoring was as follows: 0, no demyelination; 1, mild inflammation accompanied by loss of myelin integrity; 2, moderate inflammation with increasing myelin damage; 3, numerous inflammatory lesions accompanied by significant increase in myelin stripping; and 4, intense areas of inflammation accompanied by numerous phagocytic cells engulfing myelin debris (7, 24). Scores are presented as mean ± SEM.
Statistical analysis
Statistically significant differences between groups of mice
were determined by t test using Sigma-Stat 2.0
software, (Jandel, San Rafael, CA), and p values of
0.05 were considered signficant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracranial infection of CCR2-/- mice
with 10 PFU of MHV-V5A13.1 resulted in a dramatic acceleration in
mortality as compared with CCR2+/+ mice (Fig. 1
). As early as day 5 p.i.,
CCR2-/- mice began to succumb to infection, and
by day 10 p.i., 100% of CCR2-/- mice
died. In marked contrast are the results of MHV infection of
CCR2+/+ mice, of which 100% survived. The
increased mortality in CCR2-/- mice correlated
with enhanced viral recovery from the brains at days 3 (5.5 ± 0.1
PFU/g, n = 4) and 6 p.i. (6.3 ± 0.2 PFU/g,
n = 8 and p 0.02) when compared with
titers present in the brains of infected CCR2+/+
mice (day 3, 5.3 ± 0.1 PFU/g, n = 3; day 6,
5.4 ± 0.4 PFU/g, n = 6) (Table I).
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Both CD4+ and CD8+ T
cells are important in clearance of MHV from the CNS
(4, 5, 6, 7). Therefore, one potential explanation for the
acceleration in mortality and increase in viral titers within the
brains obtained from MHV-infected CCR2-/- mice
is a reduction in T lymphocyte infiltration into the CNS following
viral infection. Immunophenotyping of mononuclear cell infiltrates into
the CNS by flow cytometric analysis revealed no differences in
leukocyte infiltration between the two populations of mice at day
2 p.i. (Table II). However, by day
4 p.i., CCR2-/- mice exhibited a 45 and
55% reduction in CD4+ and
CD8+ T lymphocyte infiltration, respectively, as
compared with CD4+ and CD8+
T lymphocyte levels present within the CNS of
CCR2+/+ mice. During acute disease (days 6 and
7 p.i.), there was a significant reduction
(p 0.04) in CD4+ T
lymphocyte entry into the CNS of CCR2-/- mice
(5.7% ± 1.6%, n = 6) when compared with levels
present in CCR2+/+ mice (14.6% ± 2.5%,
n = 6). In addition, analysis of
CD8+ T lymphocyte infiltration at this time
revealed an 
45% decrease in CCR2-/- mice
(8.3% ± 2.1%, n = 6) as compared with
CCR2+/+ mice (15.1% ± 2.2%, n
= 6). Macrophage/microglial staining (determined by F4/80 Ag
expression) was significantly decreased (p
0.004) in CCR2-/- mice (9.5% ± 2.2%,
n = 3) in comparison to CCR2+/+
mice (26.6% ± 2.0%, n = 3) during acute disease
(Table II). Although levels of F4/80+ cells were
lower in sham-infected CCR2-/- mice as compared
with sham-infected CCR2+/+ mice, this difference
was not significant (Table II). Therefore, it is unlikely that reduced
numbers of resident microglia are present within the CNS of
CCR2-/- mice and that this accounts for the
clinical disease phenotype.
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Chemokine mRNA transcripts were analyzed in MHV-infected
CCR2-/- and CCR2+/+ mice
at days 2, 4, and 6 p.i. by RPA, and the data are presented in
Fig. 2. Both
CCR2+/+ and CCR2-/- mice
exhibited a similar chemokine profile at all time points tested.
Semiquantitative analysis of chemokine mRNA signal intensity was
performed by densitometric scanning of the autoradiographs. These
results indicated that both strains of mice expressed similar levels of
chemokine transcripts following infection with virus (Fig. 2, A and B). The only differences detected were at
day 6 p.i., in which CCR2-/- mice
displayed reduced levels of CCL5 (p 0.02)
and CCL4 (p 0.002) transcripts in the brains
of CCR2-/- mice as compared with transcript
levels present in CCR2+/+ mice. Analysis of
chemokine and cytokine protein expression within the CNS of mice during
acute disease (days 6 and 7 p.i.) indicated >80% decrease
(p 0.002) in CCL5 within
CCR2-/- mice (155 ± 36 pg/ml,
n = 5) as compared with CCR2+/+
mice (836 ± 159 pg/ml, n = 5) (Table III). Furthermore, there was an
10-fold reduction in IFN- (p 0.001)
within the CNS of CCR2-/- mice (3.8 ± 0.8
pg/ml, n = 10) as compared with
CCR2+/+ mice (39.4 ± 7.2 pg/ml,
n = 8) (Table III). No differences in levels of CCL2
were detected between the two strains of mice, suggesting that the
absence of CCR2 did not alter production of the CCL2 ligand (Table III). To determine whether decreased production of the Th1-associated
factors IFN- and CCL5 correlated with increased levels of Th2
cytokines, CNS levels of IL-4 and IL-5 were determined. As shown in
Table III, there were no differences in CNS levels of any of these
cytokines between the two strains of mice, suggesting that a Th2
response does not predominate within the CNS in the absence of a robust
Th1 response.
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The experimental results presented above suggest that leukocyte
trafficking into the CNS is impaired in mice lacking CCR2. However, in
an attempt to more thoroughly characterize the contributions of CCR2 in
contributing to neuroinflammation following MHV infection of the CNS, a
series of adoptive transfer experiments were performed. Immune
splenocytes were obtained from CCR2+/+ mice and
transferred i.v. to either RAG1-/- or
CCR2-/- mice at 3 days after i.c. infection
with MHV. Transfer of immune splenocytes from
CCR2+/+ mice to MHV-infected
RAG1-/- mice resulted in T cell infiltration
into the CNS that correlated with a reduction in brain viral titers as
compared with MHV-infected RAG1-/- mice
receiving only HBSS (Table IV).
Surprisingly, transfer of immune CCR2+/+
splenocytes did not protect CCR2-/- mice from
MHV-induced disease, as all recipient mice (n = 6) died
by day 6 p.i. Furthermore, adoptive transfer of splenocytes
obtained from immunized CCR2-/- mice into
RAG1-/- mice resulted in 2- and 1.3-fold
reductions in numbers of CD4+ T cells
(p 0.05) and CD8+ T
cells, respectively, within the CNS, which correlated with increased
titers of virus (p 0.007) as compared with
RAG1-/- mice receiving immune
CCR2+/+ splenocytes (Table IV). In addition,
macrophage/microglia infiltration into the CNS was reduced
2-fold (p 0.05) in
RAG1-/-recipients of
CCR2-/- splenocytes (Table IV). Collectively,
these data suggest that expression of CCR2 by both host and donor cells
is important in allowing cells to migrate to the CNS and contribute to
viral clearance.
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Both T cells and macrophages have been shown to be important in
contributing to demyelination in MHV-infected mice (7, 8, 28, 29). To determine whether reduced macrophage entry into the CNS
of RAG1-/- recipients of
CCR2-/- splenocytes correlated with a reduction
in the severity of demyelination, spinal cords were stained with LFB,
and myelin damage was evaluated. As shown in Fig. 3, there is a marked reduction in
inflammatory foci accompanied by limited myelin stripping in
RAG1-/- mice receiving
CCR2-/- splenocytes as compared with spinal
cords obtained from RAG1-/- mice injected with
CCR2+/+ splenocytes. Quantitative analysis
revealed a reduction (p 0.001) in the
severity of demyelination in CCR2-/- recipients
(0.7 ± 0.1, n = 6) as compared with
CCR2+/+ recipients (1.9 ± 0.1,
n = 7) (Table IV
). Control mice showed limited myelin
stripping and inflammation (0.6 ± 0.2, n = 6)
(Table IV).
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In addition to trafficking, CCR2 may also contribute to the
development of an effective T cell response following MHV infection.
Previous studies have determined that the immunodominant
CD8+ T cell epitope is located in the surface
glycoprotein at residues 510–518 (25, 26). Xue et al.
(27) have mapped a CD4+ T cell
epitope to the membrane protein located at residues 133–147.
Splenocytes from immunized CCR2+/+ and
CCR2-/- mice were stimulated in vitro with
either the CD4 or CD8 viral epitopes, and intracellular staining for
IFN- was performed. Such analysis revealed an 6-fold reduction
(p 0.05) in the number of
CD8+ T cells responding to the surface 510–518
epitope in CCR2-/- mice (7.9 x
104 ± 5.1 x 104
cells) as compared with CCR2+/+ mice (4.6 x
105 ± 7.5 x 104
cells) (Fig. 4, A and
B). In addition, there was an 2.5-fold reduction
(p 0.05) in numbers of
CD4+ T cells responding to the membrane 133–147
epitope in CCR2-/- mice (4.7 x
104 ± 2.0 x 104
cells) when compared with CCR2+/+ mice (1.2
x 105 ± 2.0 x 104)
(Fig. 4, C and D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(36, 39). Collectively, these
studies strongly suggest that CCR2 expression is required for the
development of protective Th1 responses following microbial
challenge.
In the present study, we provide evidence that supports and extends
these previous observations. The major finding presented in this report
is that CCR2 signaling is important in regulating events contributing
to a protective immune response following MHV infection of the CNS.
These data are consistent with a recent report by Dawson et al.
(34) that demonstrated enhanced recovery of influenza A
virus from the lungs of infected CCR2-/- mice
as compared with wild-type mice, which correlated with a delayed T cell
response to virus. Our results indicate that mice lacking CCR2
exhibited accelerated mortality accompanied by enhanced recovery of MHV
from the brain. Although the reasons contributing to death in
MHV-infected CCR2-/- mice are unknown,
underlying factors include the dramatic reduction in numbers of
CD4+ and CD8+ T cells
infiltrating into the CNS. Numerous studies have documented the
importance of both populations of cells in controlling viral
replication within the CNS and protecting mice from death
(4, 5, 6, 7). Moreover, the dramatic decrease in IFN-
expression within the brains of infected
CCR2-/- mice may also contribute to the
increased viral burden, as this cytokine has been shown to contribute
to clearance of MHV from the brain (40). Collectively,
these results indicate that CCR2-/- mice are
not able to mount a protective Th1 response characterized by T cell
infiltration and IFN- production, and this may contribute to viral
spread within the brain and enhanced mortality.
Interestingly, the brains of infected CCR2-deficient mice did not show
increased levels of Th2 cytokines, and expression of the Th2-associated
chemokine receptors CCR3 and CCR4 was undetectable (data not shown).
The absence of a strong shift from a Th1 to a Th2 response in the
MHV-infected CCR2-deficient mice stands in sharp contrast to results
from the earlier infection studies with C. neoformans,
L. major, and L. donovani (35, 38, 39). Together, these data are consistent with the hypothesis
that there are organ-specific and/or pathogen-specific differences in
immune responses and that the response in the CNS differs markedly with
the response generated in the lungs and skin. It is likely that
differential expression of soluble factors underlies the development of
a Th2 response in the lungs and skin in the absence of CCR2. There is
compelling evidence, including information from CCL2-deficient mice,
that CCL2 itself is critical for the development of Th2 responses
(41, 42, 43), an idea that appears at odds with the strong Th2
responses exhibited by mice lacking the CCR2 receptor for CCL2
(35, 38). The fact that high exogenous amounts of CCL2
show biological activity in CCR2-deficient mice suggests that there
might be another receptor for CCL2 (14) and that the
Th2-inducing effects are mediated by this receptor. Although others
have also suggested the possibility of a second, functional CCL2
receptor (44, 45), such a CCL2 receptor has not been
unambiguously identified thus far. An alternative hypothesis follows
from the observation that significant accumulation of CCL2 occurs in
CCR2-deficient mice after infection or immunization (35, 46). Unnaturally high levels of CCL2 that build up in the
absence of CCR2 might allow promiscuous binding to one or more other
chemokine receptors, a situation that would not occur in wild-type mice
or in CCL2-deficient mice. The fact that we did not find a difference
in CCL2 levels in the MHV-infected CNS of wild-type mice or
CCR2-deficient mice may account for the lack of a switch to a Th2
response in the knockout animals (Table III).
The defining role of chemokines and their receptors is leukocyte
activation and trafficking to areas of infection and injury. In support
of this is the demonstration of the dramatic reduction in T cell and
macrophage/microglia infiltration into the CNS of
CCR2-/- mice following i.c. infection with MHV,
indicating that CCR2 is important in allowing migration of leukocytes
into the CNS following infection. Adoptive transfer experiments
confirmed these observations. Transfer of splenocytes obtained from
MHV-infected CCR2-/- mice into MHV-infected
RAG1-/- animals revealed a diminished ability
of T cells to enter the brain and eliminate virus (Table IV).
Surprisingly, similar results were obtained following adoptive transfer
of immune splenocytes from CCR2+/+ mice into
MHV-infected CCR2-/- mice, as all recipient
mice died by day 6 p.i., presumably due to an inability of T cells
to enter the brain and participate in viral clearance because these
mice exhibited similar mortality as CCR2-/-
mice that did not receive donor splenocytes. Although these data
indicate that CCR2 expression on cells present in both donor and
recipient mice is important in T cell trafficking, these results also
suggest that CCR2 may be important in the development of an effective T
cell response following MHV infection. In support of this possibility
is the demonstration of a pronounced decrease in the numbers of
CD4+ and CD8+ T cells
responding to defined viral epitopes, as determined by intracellular
staining for IFN- (Fig. 4). These results support and extend earlier
studies that implied that CCR2-dependent production of IFN- by T
cells is important in host defense following infection with C.
neoformans (35) or L. donovani
(39). Moreover, Izikson et al. (47) reported
that T cells from CCR2-/- immunized mice showed
decreased Ag-induced proliferation and IFN- production compared with
wild-type mice in the experimental autoimmune encephalomyelitis (EAE)
model following challenge with myelin oligodendrocyte glycoprotein
(MOG). Whether the reduction in Ag-specific T cells present within the
spleens of MHV-infected CCR2-/- mice is the
result of impaired CCR2 signaling by T cells or the result of impaired
trafficking of APCs, as has recently been reported, is unknown at this
time (39, 48).
In addition to CCR2, two other CC chemokine receptors, CCR1 and CCR5, have been shown to regulate the gateway for inflammatory cell entry into the CNS. CCR1-deficient mice have reduced CNS inflammatory responses in the MOG-induced EAE model (49), and CCR5-deficient mice have negligible inflammation after disseminated C. neoformans infection of the CNS (50). Although it may be assumed that the absence of CCR1, CCR2, or CCR5 from leukocytes is responsible for these phenotypic changes, a recent report by Andjelkovic and Pachter (51) suggests that the expression of CCR1, CCR2, and CCR5 on brain microvessels might also have a role in allowing leukocytes to traverse the blood-brain barrier. This idea is supported by the fact that adoptive transfer of immune splenocytes obtained from CCR2+/+ mice failed to protect MHV-infected CCR2-/- mice from death, suggesting that leukocytes were not able to enter the brain. It will be interesting to determine the relative roles of these chemokine receptors on leukocytes and on the brain microvascular endothelium in mediating inflammatory responses in the CNS.
Our present results with intracranial injection of MHV into
CCR2-deficient mice are consistent with the recent demonstration that
CCR2 is critical for CNS inflammatory responses induced by s.c.
immunization with MOG in a model of EAE (47, 52).
CCR2-deficient mice were strongly protected against disease induction,
and the animals demonstrated little evidence of CNS inflammation.
Moreover, Fife et al. (52) have correlated the reduction
in disease severity with reduced macrophage entry into the CNS. These
data are consistent with the data presented in this study showing that
CCR2 is important in macrophage trafficking into the CNS of
MHV-infected mice and contributing to demyelination (Fig. 3 and Table IV) (47, 52). The data from these two very different
models demonstrate that CCR2 is important in regulating CNS
inflammation. It also appears that CCR2 is important for inflammatory
cell invasion of the peripheral nervous system in a mouse model of
Wallerian degeneration (53). In addition to CCR2, recent
studies from our laboratory have documented the importance of CCR5 in
contributing to macrophage infiltration into the CNS and demyelination
in MHV-infected mice (54). Therefore, macrophage/microglia
expression of both CCR2 and CCR5 is important in allowing
macrophage/microglia to migrate to white matter tracts and participate
in myelin destruction. We have detected reduced expression of chemokine
receptor transcripts including CCR5 within the brains of
CCR2-/- mice following infection with MHV (data
not shown), suggesting that receptor cross-talk signaling may be
necessary for optimal expression of chemokine receptors.
In addition to CCL2, other chemokines contribute to host defense
following MHV infection of the CNS. Neutralization of the T cell
chemoattractant CXCL9 or CXCL10 by administration of specific antisera
to MHV-infected mice resulted in reduced T lymphocyte infiltration of
the CNS, a dramatic reduction in IFN- expression, and consequently,
an increased viral burden within the CNS accompanied by increased
mortality (10, 11). The data presented here extend this
work and demonstrate unambiguously that CCR2 is crucial for the
development of an effective host response against MHV infection of the
CNS. These data further illustrate the complexity of chemokine and
chemokine receptor interactions and expression patterns as they relate
to host defense mechanisms following viral challenge of the CNS. More
importantly, it is clear that these proinflammatory molecules exert
nonredundant roles in generating a protective Th1 response against MHV
infection of the CNS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas E. Lane, Department of Molecular Biology and Biochemistry, University of California, 3205 Biological Sciences II, Irvine, CA 92697-3900. E-mail address: tlane{at}uci.edu
3 Abbreviations used in this paper: MHV, mouse hepatitis virus; i.c., intracerebral; RAG, recombinase-activating gene; RPA, RNase protection assay; LFB, luxol fast blue; EAE, experimental autoimmune encephalomyelitis; p.i., postinfection; MOG, myelin oligodendrocyte glycoprotein.
Received for publication November 27, 2000. Accepted for publication August 17, 2001.
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References |
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L. N. Stiles, J. L. Hardison, C. S. Schaumburg, L. M. Whitman, and T. E. Lane T Cell Antiviral Effector Function Is Not Dependent on CXCL10 Following Murine Coronavirus Infection J. Immunol., December 15, 2006; 177(12): 8372 - 8380. [Abstract] [Full Text] [PDF]
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 T. H. Terwey, T. D. Kim, A. A. Kochman, V. M. Hubbard, S. Lu, J. L. Zakrzewski, T. Ramirez-Montagut, J. M. Eng, S. J. Muriglan, G. Heller, et al. CCR2 is required for CD8-induced graft-versus-host disease Blood, November 1, 2005; 106(9): 3322 - 3330. [Abstract] [Full Text] [PDF]
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A. A. Dandekar, K. O'Malley, and S. Perlman Important Roles for Gamma Interferon and NKG2D in {gamma}{delta} T-Cell-Induced Demyelination in T-Cell Receptor {beta}-Deficient Mice Infected with a Coronavirus J. Virol., August 1, 2005; 79(15): 9388 - 9396. [Abstract] [Full Text] [PDF]
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T. S. Kim and S. Perlman Viral Expression of CCL2 Is Sufficient To Induce Demyelination in RAG1-/- Mice Infected with a Neurotropic Coronavirus J. Virol., June 1, 2005; 79(11): 7113 - 7120. [Abstract] [Full Text] [PDF]
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 T. S. Kim and S. Perlman Virus-Specific Antibody, in the Absence of T Cells, Mediates Demyelination in Mice Infected with a Neurotropic Coronavirus Am. J. Pathol., March 1, 2005; 166(3): 801 - 809. [Abstract] [Full Text] [PDF]
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K. E. Peterson, J. S. Errett, T. Wei, D. E. Dimcheff, R. Ransohoff, W. A. Kuziel, L. Evans, and B. Chesebro MCP-1 and CCR2 Contribute to Non-Lymphocyte-Mediated Brain Disease Induced by Fr98 Polytropic Retrovirus Infection in Mice: Role for Astrocytes in Retroviral Neuropathogenesis J. Virol., June 15, 2004; 78(12): 6449 - 6458. [Abstract] [Full Text] [PDF]
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W. G. Glass, M. J. Hickey, J. L. Hardison, M. T. Liu, J. E. Manning, and T. E. Lane Antibody Targeting of the CC Chemokine Ligand 5 Results in Diminished Leukocyte Infiltration into the Central Nervous System and Reduced Neurologic Disease in a Viral Model of Multiple Sclerosis J. Immunol., April 1, 2004; 172(7): 4018 - 4025. [Abstract] [Full Text] [PDF]
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J. Melchjorsen, L. N. Sorensen, and S. R. Paludan Expression and function of chemokines during viral infections: from molecular mechanisms to in vivo function J. Leukoc. Biol., September 1, 2003; 74(3): 331 - 343. [Abstract] [Full Text] [PDF]
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D. Giunti, G. Borsellino, R. Benelli, M. Marchese, E. Capello, M. T. Valle, E. Pedemonte, D. Noonan, A. Albini, G. Bernardi, et al. Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS J. Leukoc. Biol., May 1, 2003; 73(5): 584 - 590. [Abstract] [Full Text] [PDF]
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M. J. Trifilo, C. C. Bergmann, W. A. Kuziel, and T. E. Lane CC Chemokine Ligand 3 (CCL3) Regulates CD8+-T-Cell Effector Function and Migration following Viral Infection J. Virol., April 1, 2003; 77(7): 4004 - 4014. [Abstract] [Full Text] [PDF]
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W. G. Glass and T. E. Lane Functional Expression of Chemokine Receptor CCR5 on CD4+ T Cells during Virus-Induced Central Nervous System Disease J. Virol., December 6, 2002; 77(1): 191 - 198. [Abstract] [Full Text] [PDF]
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