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The Journal of Immunology, 2007, 179, 2774-2786
Copyright © 2007 by The American Association of Immunologists, Inc.
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CXCR3 Signaling Reduces the Severity of Experimental Autoimmune Encephalomyelitis by Controlling the Parenchymal Distribution of Effector and Regulatory T Cells in the Central Nervous System1

Marcus Müller*, Sally L. Carter*, Markus J. Hofer*, Peter Manders*, Daniel R. Getts{dagger}, Meghan T. Getts{dagger}, Angela Dreykluft*, Bao Lu{ddagger}, Craig Gerard{ddagger}, Nicholas J. C. King{dagger} and Iain L. Campbell2,*,{dagger}

* School of Molecular and Microbial Biosciences and {dagger} Department of Pathology and Bosch Institute, School of Medical Sciences, University of Sydney, Sydney, Australia; and {ddagger} Children’s Hospital and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The chemokine receptor CXCR3 promotes the trafficking of activated T and NK cells in response to three ligands, CXCL9, CXCL10, and CXCL11. Although these chemokines are produced in the CNS in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), their role in the pathogenesis of CNS autoimmunity is unresolved. We examined the function of CXCR3 signaling in EAE using mice that were deficient for CXCR3 (CXCR3–/–). The time to onset and peak disease severity were similar for CXCR3–/– and wild-type (WT) animals; however, CXCR3–/– mice had more severe chronic disease with increased demyelination and axonal damage. The inflammatory lesions in WT mice consisted of well-demarcated perivascular mononuclear cell infiltrates, mainly in the spinal cord and cerebellum. In CXCR3–/– mice, these lesions were more widespread throughout the CNS and were diffused and poorly organized, with T cells and highly activated microglia/macrophages scattered throughout the white matter. Although the number of CD4+ and CD8+ T cells infiltrating the CNS were similar in CXCR3–/– and WT mice, Foxp3+ regulatory T cells were significantly reduced in number and dispersed in CXCR3–/– mice. The expression of various chemokine and cytokine genes in the CNS was similar in CXCR3–/– and WT mice. The genes for the CXCR3 ligands were expressed predominantly in and/or immediately surrounding the mononuclear cell infiltrates. We conclude that in EAE, CXCR3 signaling constrains T cells to the perivascular space in the CNS and augments regulatory T cell recruitment and effector T cell interaction, thus limiting autoimmune-mediated tissue damage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis (MS)3 is the most common chronic demyelinating inflammatory disorder of the CNS that leads to a serious disability in a significant number of affected patients. MS is a heterogeneous disorder for which the pathogenesis is still uncertain. However, current evidence implicates in some forms of MS the involvement of a T cell-mediated autoimmune process in which encephalitogenic CD4+ and CD8+ T cells infiltrate the CNS and coordinate an inflammatory response that leads to demyelination, oligodendrocyte loss, and eventual neurodegeneration (1, 2, 3). The animal model experimental autoimmune encephalomyelitis (EAE) is generally accepted to recapitulate and provide experimental support for this basic disease mechanism in MS (4). EAE can be induced by active immunization of rodents with myelin-specific proteins or peptides, which stimulates the generation of encephalitogenic, myelin-reactive, CD4+ effector T cells. Encephalitogenic T cells coordinate an immune response in the CNS that leads to the formation of well-demarcated perivascular cuffs of mononuclear cells primarily in the cerebellum and spinal cord. The infiltration of encephalitogenic T cells into the brain, crossing the blood-brain barrier is a crucial step for the initiation and progression of EAE. Although it is clear that adhesion molecules, cytokines, and chemokines are involved and contribute to T cell migration into the brain, the exact mechanisms and the interplay of the different molecules remain unresolved (5, 6).

Chemokines are a family of small proteins that are well known for their function to orchestrate immune responses (7, 8), and it is strongly assumed that they have a critical impact on the pathogenesis of MS and EAE (6, 9). Chemokine functions are mediated by G protein-coupled, seven-transmembrane receptors, which, on the whole, display promiscuously for their chemokine ligands. CXCR3 is a chemokine receptor that is expressed by activated CD4+ and CD8+ T cells, NK cells, microglia/monocytes, and dendritic cells (10, 11, 12). High levels of CXCR3 are found on activated Th1 T cells but not on Th2-polarized cells (13). Three chemokines, CXCL9, CXCL10, and CXCL11, bind to and activate CXCR3 (10, 14, 15). The induction of all three CXCR3 ligands is mediated by IFN-{gamma} (15, 16, 17). These three chemokines have been shown to stimulate the chemotaxis of CXCR3+-activated T cells and NK cells in vitro (10, 15, 18, 19, 20, 21). Moreover, CXCL9 and CXCL10 have been shown to be pivotal for T cell migration in various experimental disease models including transplant rejection (22, 23, 24, 25), infectious disease, autoimmunity and tumor immunity (26, 27, 28, 29). Taken together, these observations support the notion that CXCR3 signaling is fundamental to T and NK cell trafficking in cell-mediated immunity.

Previous studies have suggested a role for CXCR3 and its ligands in MS and EAE. CXCL10 is detectable early and at abundant levels in EAE and is also found at high levels in active MS plaques, suggesting a role in the migration of encephalitogenic T cells into the brain (30, 31, 32, 33). CXCL10 and CXCR3 are colocalized in active MS plaques on perivascular T cells (CXCR3) and surrounding astrocytes (CXCL10) (32). Activated T cells, which cross or have crossed the blood-brain barrier, are CXCR3+ (34, 35, 36). Furthermore, CXCL9 and CXCL10 protein are increased in the cerebrospinal fluid of patients with an acute MS attack. whereas cerebrospinal fluid T cells are enriched for CXCR3+ cells (35).

Although these descriptive studies suggest a key role of CXCR3 signaling in the pathogenesis of EAE, functional studies aimed at defining the role of CXCL10 in EAE have produced conflicting results. Blocking of CXCL10 has been reported to either suppress or exacerbate EAE (37, 38, 39, 40). Moreover, CXCL10-deficient mice display similar induction and severity of EAE to wild-type (WT) controls when using standard immunization protocols and were even more susceptible to EAE when suboptimal immunization protocols were used (41). These conflicting results might be explained in part by the three CXCR3 ligands having redundant functional properties with CXCL9 and/or CXCL11 compensating for the loss of CXCL10.

The objective of this study was to use CXCR3–/– mice to exclude the possible complication of ligand redundancy and to clarify the role of CXCR3 signaling in myelin oligodendrocyte glycoprotein peptide (MOG)-induced EAE. During the course of our studies, Liu et al. (42) reported that CXCR3–/– mice develop more severe EAE with unaltered leukocyte migration but decreased IFN-{gamma} production. In contrast to these findings, an earlier report found that blocking CXCR3 function was associated with less severe EAE and diminished T cell infiltration of the CNS (43). Here our findings confirm those of Liu et al. (42) in demonstrating that CXCR3–/– mice develop a more severe chronic clinical course of EAE. However, our findings extend these recent observations and point to novel disease-limiting roles for CXCR3-signaling in EAE, which include: 1) corralling effector T cells to the perivascular space, thus limiting the spread of these tissue-damaging cells into the parenchymal white matter of the CNS; and 2) facilitating regulatory T cell accumulation in the perivascular lesions favoring the interaction of these suppressor T cells with target effector T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

The generation and characterization of the CXCR3–/– mice were described previously (23). The mice used in this study were backcrossed for 12 generations to the C57BL/6J strain. WT C57BL/6J mice were purchased from the Animal Resources Centre (Canning Vale, Australia) and served as controls. Animals were kept under pathogen-free conditions in the Blackburn animal facility of the University of Sydney (Sydney, Australia). Ethical approval for the use of all mice in this study was obtained from the University of Sydney Animal Care and Ethics Committee.

Induction of EAE and clinical evaluation

EAE was induced by active immunization with MOG35–56. On day 0, CXCR3–/– and WT animals were given injections s.c. into the hind flanks of an emulsion of 100 µl of MOG35–56 (3 mg/ml) and 100 µl of CFA (Sigma-Aldrich) supplemented with 4 mg/ml Mycobacterium tuberculosis H37RA (Difco). In addition, animals received an i.p. injection of 500 ng of pertussis toxin (Sigma-Aldrich), which was repeated 2 days after immunization. As controls, another cohort of mice from each genotype were immunized similarly with the exception that MOG peptide was replaced with BSA. Animals were examined daily until day 60, with some being euthanized earlier to collect tissue (day 5/pre-EAE, day 15/peak EAE, day 25 late peak/early chronic phase) for analysis. Clinical scores were assessed for each animal according to the following: 0 = no signs of disease; 1 = loss of tail tonus; 2 = hind limb paraparesis; 3 = hind limb paralysis; 4 = moribund; and 5 = death. For each animal with a clinical score of ≥1, time to disease onset, time to peak, peak-score, cumulative score (sum of all scores from disease onset to day 60), outcome (final score at day 60) and grade of remission (difference between peak score and outcome) were determined.

Tissue processing for histology

Brain and spinal cord were removed for routine histology and immunohistochemical examination. Tissues were placed immediately in PBS-buffered 4% paraformaldehyde (pH 7.4; Sigma-Aldrich) for 24 h at 4°C and were subsequently embedded in paraffin. The remaining tissue was embedded without prior fixation in Tissue Tek (Sakura Finetek) and snap frozen in liquid nitrogen-cooled isopentane (Sigma-Aldrich). For further procedures, 5-µm sections were prepared from paraffin-embedded tissue, and 10-µm sections were prepared from the cryoblocks.

Routine histology and immunohistochemistry

Primary Abs and corresponding protocols for immunohistochemistry are summarized in Table I. Paraffin sections were deparaffinized, and some were pretreated with proteinase K (Sigma-Aldrich; 10 µg/ml, 15 min at 37°C). Slides were then incubated for 1 h at room temperature with primary Abs. After a washing in PBS, a biotinylated secondary Ab (Vector Laboratories; 1/200, 45 min) and HRP-coupled streptavidin (Vector Laboratories; 1/200, 30 min) was used. Nova Red (Vector Laboratories) was applied as the immunoperoxidase substrate according to the manufacturer’s instructions. Sections were counterstained with hematoxylin (Sigma-Aldrich).


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  		Table I. Ab and lectin reagents used for immunohistochemistry

 
Dual-label staining on paraffin sections was performed using an additional primary Ab of a different species, which was detected with a secondary alkaline phosphatase-coupled Ab and Vector blue (Vector Laboratories) as the second color substrate. Counterstaining was omitted for dual-label immunohistochemistry.

Fluorescent immunohistochemistry was applied to frozen sections as described previously (44). In brief, frozen sections were fixed with ice-cold methanol-acetone (50:50) for 45 s. Primary Abs were incubated for 1 h at room temperature. After a washing in PBS, an OD594 or OD488 fluorescence-conjugated secondary Ab (Invitrogen Life Technologies; 1/200, 45 min) was used to visualize the primary Ab. The Foxp3 primary Ab was visualized by a biotinylated secondary Ab, followed by OD594-coupled streptavidin (Invitrogen Life Technologies; 1/200, 30 min.). Sections were mounted and counterstained with 4',6'-diamidino-2-phenylindole (DAPI) Vectamount (Vector Laboratories).

Analysis of demyelination and cell counting

Conventional and immunofluorescence-stained sections were examined under a DM4000B bright field and fluorescence microscope (Leica). Bright field images and monochrome fluorescent images were acquired using a Leica DFC480 camera and Leica Firecam 1.7.1 software (Leica). The acquired monochrome fluorescence signals were merged using SPOT Advanced 4.5 software (Diagnostic Instruments) to create a color image. For the immunofluorescence detection of T cell subsets, due to intercellular variation of the fluorochrome signal intensity, some double-labeled cells appeared single-labeled on low magnification images. Double labeling of these cells was confirmed by examining the separate monochrome images of each color channel.

To quantify demyelinated areas of lumbar spinal cord cross-sections, images of Luxol fast blue (LFB) stains were examined with Image analysis software AnalySIS 3.0 (Softimaging). The total area of the spinal cord cross-section and demyelinated areas were measured by an observer blinded to the sample identity. Demyelination was correlated with the total spinal cord area. Four animals from each group with a clinical outcome score close to the average group outcome score were used for this analysis (WT average clinical score, 1.3; CXCR3–/– average clinical score, 2.6). To count CD3+Foxp3+ regulatory T cells, images of stained WT and CXCR3–/– spinal cord sections were randomized and coded. The cells were counted by an observer blinded to the code using AnalySIS 3.0 and corrected to the white matter area examined (CD3+Foxp3+ regulatory T cells/mm2 white matter). A double staining for CD25 and Foxp3 was used to determine the percentage of CD25+Foxp3+ cells of all CD25+ cells in the spinal cord. Cells were again quantified observer blinded with AnalySIS 3.0.

RNA isolation and RNase protection assays

Tissue was removed, immediately snap frozen in liquid nitrogen and stored at –80°C pending RNA extraction. Polyadenylic acid-positive RNA was isolated according to a previously published method (45). RNase protection assays (RPA) were performed as described previously (46) using multiprobe sets for the detection of cytokine (47) and chemokine (48) RNAs or with a probe set containing probes for CXCR3 and its ligands CXCL9, CXCL10, and CXCL11. The probes for CXCL9, CXCL10, and CXCR3 were described previously (49). A new probe was constructed for CXCL11 (GenBank accession number NM_019494, base pairs 121–351) and IL-17 (GenBank accession number NM_010552, base pairs 78–405) as described previously (46). For all probe sets, a fragment of the RPL32-4A gene (50) served as an internal loading control.

Dual label in situ hybridization/immunohistochemistry

Paraffin-embedded sections were incubated with 33P-labeled cRNA probes transcribed from linearized plasmid constructs containing the CXCL9, CXCL10, CXCL11, and IFN-{gamma} cDNA inserts and processed for in situ hybridization histochemistry as described previously (51, 52). Sections for immunohistochemistry were reacted with Abs to detect astrocytes (rabbit anti-glial fibrillary acidic protein; DAKO Cytomation), and T lymphocytes (rabbit anti-CD3; DAKO Cytomation). Microglia were detected with biotinylated lectin from Lycopersicon esculentum (Sigma-Aldrich). Bound Ab or lectin was detected using Vectastain ABC kits (Vector Laboratories), and diaminobenzidine-H2O2 reagent (Vector Laboratories) as the immunoperoxidase substrate.

CNS leukocyte isolation and flow cytometry

Spinal cords from animals with late peak disease (day 25 postimmunization) were excised and placed in ice-cold PBS buffer solution before extrusion through a coarse 100-µm pore size metal cell strainer. The subsequent homogenate was passed through a 70-µm pore size cell strainer (BD Biosciences) before enzymatic digestion for 60 min in PBS with DNase (0.005 g/ml; Sigma-Aldrich) and collagenase IV (0.05 g/ml; Sigma- Aldrich). Digested samples were incubated at 37°C in a humidified atmosphere of 5% CO2 and were resuspended every 15 min. Digestion was stopped with 10% FCS. A pellet was obtained after 10 min of centrifugation at 340 x g and then dissolved in 30% Percoll (Amersham). Subsequently, the 30% Percoll homogenate mix was layered over 80% Percoll. Leukocytes were collected from the 30%/80% interface after a 1140 x g centrifugation for 25 min at room temperature. The collected cells were washed in PBS and blocked with CD16/CD32 (BD Biosciences) Abs. Viable cells were counted using trypan blue exclusion. Isolated leukocytes were incubated with fluorochrome-conjugated Abs to detect CD8 (PE-Cy7; BD Biosciences), CD4 (PE; BD Biosciences), CD45 (PerCp; BD Biosciences), GR1 (FITC; BD Biosciences), CD11b (APC-Cy7; BD Biosciences), CD25 (APC; BD Biosciences), CD44 (APC-Cy7; eBioscience), CD62L (FITC; BD Biosciences), CD69 (PE; BD Biosciences), and CXCR3 (APC; BD Biosciences). Intracellular staining of FoxP3 was done according to the manufacturer’s instructions (BioLegend). Cells were isolated as above from spleen, brain, or spinal cord tissue and stained for surface expression of CD4 (PerCP; BD Biosciences) and CD25 (FITC; BD Biosciences). Cells were then fixed with Foxp3 Fix/Perm solution (BioLegend), washed with Foxp3 Perm buffer (BioLegend), and stained with anti-Foxp3 Ab (PE; BioLegend). After a washing, bound Ab was detected using a FACS ARIA (BD Biosciences), and the acquired data were analyzed using the flow cytometry software, FlowJo (TreeStar).

In vitro production of IL-17 by splenocytes

Cells from spleens and draining lymph nodes were isolated from MOG-immunized WT and CXCR3–/– mice. Cells were incubated for 72 h in RPMI + 10% FCS with or without MOG peptide in 24-well plates (5 x 105 cells/ml). Supernatant was collected, and IL-17 protein was determined by ELISA (eBioscience), according to the manufacturer’s instructions.

Determination of serum IFN-{gamma}

Serum was prepared after cardiac puncture of six WT and six CXCR3–/– animals with peak EAE. Three additional serum samples were prepared from normal WT mice. Serum IFN-{gamma} was determined by ELISA (eBioscience). A standard curve was generated according to the manufacturer’s protocol.

Statistical analysis

For statistical analysis, the Wilcoxon test (Mann-Whitney U test) was applied to compare data from noninterval scales, like clinical EAE scores. Normally distributed data from interval scales were analyzed with a one-tailed Student t test with p < 0.05 considered to be statistically significant (clinical data like disease onset and time to peak, RPAs, demyelinated areas, results from FACS analysis, and IFN-{gamma} ELISA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Similar onset time and acute phase disease but more severe chronic phase disease without recovery in CXCR3–/– mice

To determine whether the clinical course of EAE was altered in the absence of CXCR3 signaling, CXCR3–/– and WT mice were immunized with MOG35–55 peptide. The clinical data from >60 days of observation for two independent experiments are summarized in Fig. 1D; the clinical course for one of these studies is shown in Fig. 1A. Although control mice immunized with BSA showed no clinical signs, the incidence of clinical EAE was 100% for both CXCR3–/– and WT animals. No differences in mortality or time to onset of initial clinical signs were found. The time to peak disease in CXCR3–/– was slightly delayed, but this was not statistically significant. The differences in peak score (3.5 in CXCR3–/– vs 2.9 in WT animals) and the accumulative score (121.5 in CXCR3–/– vs 93 in WT animals) were not significant. The differences in the clinical outcome 60 days after immunization (2.6 in CXCR3–/– vs 1.3 in WT) and the grade of remission (0.9 in CXCR3–/– vs 1.7 in WT) were significant. The scatter diagrams for grade of remission (Fig. 1B) and clinical outcome (Fig. 1C) revealed a higher clinical variability for the CXCR3–/– group. Overall, the results of these studies indicated that while CXCR3 signaling is not required for the development of EAE, it does play a protective role in the recovery phase of the disease.


Figure 1
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  		FIGURE 1. Clinical course of MOG-induced EAE in WT vs CXCR3–/– mice. A, Clinical score of one representative EAE experiment. Scatter diagrams show the distribution of the grade of remission (B) and the clinical outcome (C) of the individual CXCR3–/– mice and the WT controls. Animals were immunized with 300 µg of MOG peptide emulsified in CFA and injected i.p. with 500 ng of pertussis toxin at days 0 and 2. Five independent experiments were performed with a total number of 54 CXCR3–/– and 60 WT animals to obtain clinical data at the various time points. D, Statistical analysis of two representative experiments. Whereas WT controls show clinical recovery, CXCR3–/– mice develop a more chronic disease course with worse clinical outcome and reduced grade of recovery (remission). D, Summary of the clinical data of MOG-induced EAE experiments with an observation period of 60 days. The induction and peak phase disease did not differ between CXCR3–/– mice and the WT controls, whereas the clinical outcome was worse and the clinical recovery (remission) reduced in the CXCR3–/– mice. *, p < 0.05.

 
More severe and widespread demyelination and axonal damage in CXCR3–/– mice correlates with the clinical outcome

To correlate the clinical outcome of EAE with neuropathological alterations in the CXCR3–/– and WT mice, we examined brain and spinal cord by routine histology and immunohistochemistry. At peak disease, only minimal demyelination and tissue destruction was found. This was comparable for both genotypes (Fig. 3, A and D). In both CXCR3–/– and WT mice, white matter lesions and axonal damage were observed 60 days after immunization that were typical for MOG-induced EAE (4, 53). However, in CXCR3–/– mice, more extensive demyelination was found in the spinal cord (Fig. 2, C and F) and cerebellum (Fig. 3, J and M) 60 days after immunization. The demyelinated area at the lumbar level of the spinal cord (Fig. 2, B and D) was 2.5-fold higher in CXCR3–/– mice compared with WT mice (**, p < 0.01; Fig. 2A). The same degree of demyelination found in LFB routine staining was observed by immunohistochemistry against myelin basic protein (data not shown). In addition, demyelination in CXCR3–/– mice was accompanied by more severe axonal loss when compared with WT controls. This finding was most prominent in the dorsal column (Fig. 2, D–G) but also was observed in the brainstem (Fig. 2, H and J) and cerebellum (not shown).


Figure 3
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  		FIGURE 3. Colocalization of inflammatory lesions and demyelination in WT and CXCR3–/– mice. Colocalization of demyelination, T cells and microglia/macrophages by LFB staining and immunohistochemistry (CD3 to detect T cells and L. esculentum lectin to detect microglia/macrophages) on serial sections (5 µm) in the cerebellum (A–O, single-labeled) and spinal cord (P and Q, double-labeled). In WT controls, T cells accumulated in the perivascular space (B, H, and P) and were accompanied by activated microglia/macrophages (C, I, and P). At early peak disease and during remission, demyelination is limited in WT controls (A and G). In contrast, T cell distribution in CXCR3–/– mice is more widespread (E, K, N, and Q) and accompanied by diffuse microglia/macrophage activation in the white matter (F, L, O, and Q). Whereas demyelination is not observable at the early peak stage (D), CXCR3–/– mice have a diffuse demyelination later during the course of EAE in the areas of widespread T cell distribution (J and M). Bar, 400 µm (A, D, G, J, and M), 250 µm (B, C, E, F, H, I, K, L, N, and O), and 100 µm (P and Q).

 

Figure 2
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  		FIGURE 2. More severe demyelination and axonal damage in CXCR3–/– mice. To quantify demyelinated areas, images of LFB stains from lumbar spinal cord cross-sections from animals 60 days after immunization were examined with Image analysis software. The total area of the spinal cord cross-section and demyelinated areas were measured by an observer blinded to the sample identity. Four animals from each group with a clinical outcome score close to the average group outcome score were used for this analysis (WT average clinical score 1.3: 1, 1, 2, 2; CXCR3–/– average clinical score 2.6: 2, 3, 3, 3). LFB staining of cross-sections of the lumbar spinal cord revealed 2.5-fold increase of demyelination 60 days after EAE induction in CXCR3–/– mice vs WT controls (A, B, and arrows in C). The areas in B and C surrounded by a black square are displayed in higher magnification in D–G for colocalization of demyelination (D and F) and axonal loss (E and G). Demyelinated lesions with axonal pathology (I, arrows) were frequently observed in CXCR3–/– mice (I, brainstem) but not in WT controls (H, brainstem). **, p < 0.01.

 
In severely affected CXCR3–/– mice, but not WT controls, inflammatory lesions, detected by routine staining and immunohistochemistry against T cells and macrophage Ags, were not limited to the spinal cord and cerebellum but were also found in the paraventricular and midbrain white matter (not shown).

In summary, these findings indicated that the more severe chronic EAE seen in the CXCR3–/– mice was paralleled by increased and more widespread loss of myelin and axonal injury.

T cells in CXCR3–/– mice are less organized in perivascular infiltrates but rather are distributed throughout the white matter and accompanied by activated microglia/macrophages

Because EAE is mediated by MOG-reactive CD4+ T cells that likely express CXCR3 (54), we next examined the distribution of T cells in the brain and spinal cord from CXCR3–/– and WT mice in the acute and chronic stages of EAE.

At the peak (acute) stage of disease, T cells in WT controls were found typically in well-organized perivascular cuffs (Fig. 3, B and H). These T cell accumulations were closely associated with activated microglia/macrophages (Fig. 3, C and I). In contrast, T cells in CXCR3–/– mice were less organized in perivascular accumulations but, rather, were more widely scattered throughout the parenchyma (Fig. 3, E, K, and N). In addition, together with the scattered T cell distribution, a more widespread and pronounced activation of microglia/macrophage was observed (Fig. 3, F, L, and O). At the peak stage of disease, the T cell inflammatory lesions were not accompanied by demyelination in either WT or CXCR3–/– mice (Fig. 3, A, D, G, J, and M). At a later chronic disease stage, WT animals still displayed well-organized and mainly perivascular T cell accumulation with localized microglia/macrophage activation and limited demyelination (Fig. 3, G–I). At this stage, the T cell distribution and microglia/macrophage activation in CXCR3–/– mice remained diffuse but was now accompanied by widespread demyelination (Fig. 3, J–L), which in severely affected animals involved the complete cerebellar white matter (Fig. 3, M–O).

Perivascular clustering of CD4+ T cells but not CD8+ T cells is impaired in CXCR3–/– mice

Recent studies suggest a differential role of CXCR3 signaling on the recruitment of CD4+ vs CD8+ T cells to the CNS in viral disease models (55, 56). To determine whether the same might be true in EAE, we next examined the distribution of CD4+ and CD8+ T cells in spinal cord (Fig. 4) and cerebellar (not shown) lesions of the CXCR3–/– and WT mice at peak disease.


Figure 4
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  		FIGURE 4. Distribution of CD4+ and CD8+ T cells in WT controls and CXCR3–/– mice. Distribution of CD4+ and CD8+ T cells in spinal cord tissue from WT controls and CXCR3–/– mice at peak EAE. CD3+ T cells (green signal) were colocalized with CD4+ (red signal in A and C) and CD8+ (red signal in B and D) cells by fluorescent immunohistochemistry. Whereas most of the CD3+CD4+ T cells were clustered in WT controls (A), the distribution was much more disperse in CXCR3–/– mice (C). CD3+CD8+ T cells were more widespread in both WT controls (B, arrows) and CXCR3–/– mice (D, arrows). Nuclear counterstaining with DAPI (blue signal). Some cells appear CD3– due to the weaker signal detection for the green vs red fluorochrome. These cells were confirmed as CD3+ by colocalizing the separate monochrome signals for the red and green fluorochromes in single images. Bar, 150 µm.

 
CD8+ T cells were scattered throughout the parenchyma in both WT (Fig. 4B) and CXCR3–/– mice (Fig. 4D). Only a minority (<30%) of perivascular CD3+ T cells in WT and CXCR3–/– mice were CD8+. In contrast, in WT mice, CD4+ T cells were organized mainly in perivascular clusters (Fig. 4A), whereas in CXCR3–/– mice they were more diffusely distributed throughout the white matter with less perivascular accumulation (Fig. 4C).

In summary, in CXCR3–/– mice, the formation of perivascular CD4+ T cell accumulation and their distribution is strikingly more impaired than the distribution of CD8+ T cells, which was unaltered.

Regulatory but not effector T cells are diminished in the CNS of CXCR3–/– mice with EAE

CXCR3 expression may differ between the various T cell populations involved in EAE. To further characterize the leukocyte populations involved in the more severe course of EAE in CXCR3–/– mice, we enumerated by flow cytometry the different leukocyte phenotypes after isolation from the CNS of mice 25 days after immunization (Fig. 5, A and B).


Figure 5
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  		FIGURE 5. Differentiation of leukocyte subpopulations and detection of regulatory T cells. Flow cytometric analysis of CNS leukocyte populations in the CNS of WT controls vs CXCR3–/– mice at peak (acute) disease (day 20 postimmunization) was performed as described in Materials and Methods. No significant differences for the CD4+:CD8+ ratio or granulocyte population were found. In contrast, the fraction of CD45+CD11b+ macrophages/microglia was significantly increased in CXCR3–/– mice, whereas CD4+CD25+ T cells were significantly less in CXCR3–/– mice. No differences in activated T cells, detected by a CD44+CD62L– phenotype, could be found (A and B). Foxp3, as an unequivocal marker for regulatory T cells, was detected using a monoclonal anti-Foxp3 Ab (red signal in C and D). In WT controls, CD3+/Foxp3+ T cells (C, arrows) were mainly localized within perivascular clusters of CD3+ T cells (green fluorescence) and in close contact with CD3+Foxp3– T cells (C). In CXCR3–/– mice, CD3+Foxp3– T cells were reduced and scattered throughout the white matter (D, arrows). Nuclear counterstaining with DAPI (blue signal). Quantification of these CD3+Foxp3+ T cells revealed a significant (p < 0.05) reduction of regulatory T cells in the white matter of CXCR3–/– mice compared with WT mice (E, EAE peak). In contrast, CD3+Foxp3+ T cells were not observed in spinal cord of either non-MOG-immunized CXCR3–/– or WT mice (E, control). In both CXCR3–/– mice and WT controls, the majority of CD25+ cells were also Foxp3+ (F). Bar, 50 µm.

 
The overall number of CD4+ and CD8+ T cells and their relative proportions did not differ significantly between CXCR3–/– mice and WT controls (Fig. 5, A and B). The presence of activated CD4+ and CD8+ T cells, as reflected by a CD44highCD62L– phenotype, was similar in both WT and CXCR3–/– mice (Fig. 5B). Gr1+ granulocytes were slightly increased in CXCR3–/– mice, but this difference did not reach statistical significance (Fig. 5, A and B). However, CD45+CD11b+ microglia/macrophages were increased significantly in CXCR3–/– mice (Fig. 5, A and B) corroborating the immunohistochemical observations. No significant difference was found in the total CD45+ cell population (WT 88% vs CXCR3–/– 85%).

Further characterization of the CD4+ T cells revealed a significant reduction of the CD4+CD25high T cell subpopulation in the CXCR3–/– mice (1.4%) vs WT (2.9%) animals (Fig. 5, A and B). The CD4+CD25high T cell marker delineates regulatory T cells as well as some other CD4+ T cell phenotypes (57). To evaluate more definitively whether regulatory T cells were altered in the CNS of CXCR3 KO mice with EAE, we colocalized the T cell marker CD3 with Foxp3 by fluorescence immunohistochemistry. Foxp3 is a transcription factor that is specific for regulatory T cells and is used to identify these cells (58, 59). In WT controls, the majority (>90%) of Foxp3+ T cells were localized to perivascular T cell clusters and were in close contact with surrounding mononuclear cells. In CXCR3–/– mice, the number of Foxp3+ T cells was significantly reduced (173/mm2 vs 318/mm2 in WT; p < 0.05; Fig. 5E), and isolated Foxp3+ T cells were scattered throughout the parenchyma. In both CXCR3–/– and WT spinal cord, the majority of CD25+ cells were Foxp3+ T cells, and the relative proportions of these cells did not differ between WT and CXCR3–/– mice (Fig. 5F). To exclude the possibility that there was a more general reduction of Foxp3+ T cells in the periphery of CXCR3–/– mice, we analyzed the CD4+CD25+Foxp3+ cell population in spleen from CXCR3–/– or WT mice. The results showed that the numbers of CD4+CD25+Foxp3+ T cells did not differ significantly between the CXCR3–/– or WT mice (0.83% of CD4+ T cells in CXCR3–/– and 0.72% in WT mice, data not shown).

In summary, whereas CXCR3 deficiency in EAE did not alter the overall numbers of effector T cells in the CNS, Foxp3+ regulatory T cells were diminished in number and were distributed diffusely and in isolation throughout the parenchymal white matter.

Similar cytokine and chemokine profiles in CXCR3–/– and WT mice with EAE

We next determined whether the more severe clinical outcome of EAE in CXCR3–/– mice was associated with changes in expression of genes for the proinflammatory cytokines or chemokines (Table II). In both WT and CXCR3–/– mice, the mRNA levels for the proinflammatory cytokines IFN-{gamma}, TNF-{alpha}, IL-1{alpha}, and IL-1β was increased significantly and to similar levels both at peak disease and to a lower extent during chronic disease (Table II). Additional analysis of IFN-{gamma} mRNA in the brain revealed similar levels during pre EAE (WT 0.3 vs CXCR3–/– 0.4), peak EAE (WT 1.8 ± 0.5 vs CXCR3–/– 1.5 ± 0.3) and in the remission/chronic phase (WT 0.8 ± 0.1 vs CXCR3–/– 0.8 ± 0.1; Table II, Fig. 6E).


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  		Table II. Proinflammatory cytokine and chemokine RNA levels in the spinal cord during EAE in WT and CXCR3–/–mice

 

Figure 6
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  		FIGURE 6. Analysis of IFN-{gamma} and IL-17 production in WT vs CXCR3–/– mice. IFN-{gamma} gene expression and systemic IFN-{gamma} protein levels in CXCR3–/– and WT mice. IFN-{gamma} gene expressing T cells (arrows in A and B) were detected by ISH at peak EAE, using a P33-labeled riboprobe, and were more clustered in WT controls (A, arrows). The overall number of IFN-{gamma} gene-expressing T cells was slightly diminished in CXCR3–/– but this did not reach statistical significance (C, two separate experiments, each n = 3). Detection of IFN-{gamma} RNA by RPA did not reveal significant differences either. A trend toward a lower IFN-{gamma} RNA level at peak disease was found (E, n = 3, two separate experiments from spinal cord tissue). Serum IFN-{gamma} protein was detected by ELISA (n = 6) at peak EAE and did not differ between CXCR3–/– and WT (D). Cultured splenocytes isolated from MOG35–55 peptide-immunized mice after exposure to MOG35–55 peptide produced significant amounts of IL-17 protein as detected by ELISA (F). No statistical difference in the amount of IL-17 was found between CXCR3–/– and WT mice. Bar, 30 µm (A and B).

 
In addition to absolute CNS tissue levels, IFN-{gamma} RNA was localized in the brain and spinal cord by in situ hybridization. IFN-{gamma} RNA was detectable only in CD3+ T cells, which were found to be mainly either associated with perivascular T cell infiltrates in WT animals or dispersed through the white matter in CXCR3–/– mice (Fig. 6, A and B). Enumeration of the IFN-{gamma} RNA-producing T cells revealed a slightly lower number in spinal cord of CXCR3–/– mice, but this difference was not statistically significant (Fig. 6C). To determine further whether there was any gross alteration in IFN-{gamma} production in the CXCR3–/– mice compared with WT controls, we examined serum IFN-{gamma} by levels by ELISA. The average serum level of IFN-{gamma} during peak stage disease was 264 pg/ml in WT mice and 331 pg/ml in CXCR3–/– mice (Fig. 6D).

T cells belonging to the Th17 subset are crucial for the induction of MOG-EAE (60, 61). Therefore, we examined the expression of the IL-17 gene in the spinal cord of WT and CXCR3–/– mice with EAE. No significant difference was found in the level of IL-17 mRNA between either genotype (Table II). Furthermore, although MOG peptide restimulation induced the production of IL-17 from splenic and lymph node cells isolated from MOG-immunized WT or CXCR3–/– animals, no significance difference was observed between the two genotypes (Fig. 6F).

To determine the impact of CXCR3 on the chemokine gene expression profile in the CNS in EAE, we examined the range of chemokine mRNAs by RPA (Table II). With the exception of CCL6 mRNA, which was reduced at peak disease in CXCR3–/– mice, no significant differences in the levels of the other chemokine mRNAs were observed between WT and CXCR3–/– mice.

In summary, these findings showed that with the exception of CCL6, there was not a significant difference in the level of expression of a number of proinflammatory cytokine or chemokine genes in the CNS or in IFN-{gamma} protein in the periphery in CXCR3–/– vs WT mice with EAE.

The spatial distribution of the CXCR3 ligands differs between WT and CXCR3–/– mice

The altered distribution of CD4+ T cells seen in the CNS of CXCR3–/– mice during EAE suggests a role for this chemokine receptor and its ligands in regulating the local tissue trafficking and positioning of the CD4+ T cells. Because the presence of the CXCR3 ligands would be crucial in this function, we next defined the spatial and cellular localization of the CXCL9, CXCL10, and CXCL11 RNA transcripts (Fig. 7).


Figure 7
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  		FIGURE 7. Cellular localization of CXCL9, CXCL10, and CXCL11 RNA. Cellular localization of the CXCL9, CXCL10, and CXCL11 RNA transcripts was performed by in situ hybridization histochemistry using P33-labeled riboprobes combined with immunohistochemistry to detect T cells (A and D using a CD3 Ab) or astrocytes (B, C, E, and F using a GFAP Ab). In WT controls, CXCL9 RNA and a lower level of CXCL11 RNA were found in clustered infiltrating T cells (arrows in A and C) and glial cells in close proximity (C, arrowheads). In contrast, CXCL10 was expressed to a much lesser extent by infiltrating T cells (B, arrows) but mainly by astrocytes surrounding small vessels (B, arrowheads). In CXCR3–/– mice, CXCL9 RNA was less clustered and more dispersed following the distribution of CD3+ T cells (D). The localization pattern of CXCL10 RNA remained unchanged with high expression in perivascular astrocytes (E, arrowheads). The distribution pattern of CXCL11 RNA remained unchanged (F: arrows, T cell cluster; arrowhead, glial cell expressing CXCL11). Bar, 50 µm.

 
In brain from WT mice with peak EAE, high levels of CXCL9 RNA (Fig. 7A) and much lower levels of CXCL11 RNA (Fig. 7C) were located within and immediately surrounding lymphocyte clusters and perivascular lesions. By contrast, CXCL10 RNA, which was also found at high levels, was localized predominantly to astrocytes in close proximity to the perivascular T cell infiltrates (Fig. 7B). In CXCR3–/– mice, CXCL9 RNA was more widespread and less organized in clusters, which correlated well with the more scattered distribution of the T cells (Fig. 7D). The pattern of CXCL10 RNA was altered to a much lower extent and was as in WT controls, expressed by astrocytes near the vessels (Fig. 7E). The CXCL11 RNA was barely detectable and mainly expressed by vessel associated leukocytes (Fig. 7, C and F, arrows) and only a few astrocytes (Fig. 7, C and F, arrowheads). No differences in the CXCL11 RNA expression between WT and CXCR3–/– were observed.

In all, these findings show that in WT mice with EAE there is a unique spatial and cellular localization of the CXCL9, CXCL10, and CXCL11 genes in the CNS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CXCL9, CXCL10, and CXCL11 have been shown to be crucial for T cell trafficking in vitro and in a variety of systemic models of cell-mediated immunity (10, 15, 18, 19, 20, 21, 22, 23, 24). Descriptive studies in EAE and MS also suggested an important role for CXCR3 and CXCL10 in attracting encephalitogenic T cells into the brain (32, 34, 36). However, functional studies in EAE that have focused mostly on CXCL10 have produced conflicting and in some cases unexpected findings (37, 38, 39, 40, 41). Our study here examined the course of MOG-EAE in mice that were deficient for the common receptor for CXCL10, CXCL9, and CXCL11. An early study reported that Ab-mediated blockade of CXCR3 produced a drastic reduction in the incidence of PLP-induced EAE in SJL mice and was accompanied by a reduction of infiltrating inflammatory cells (43). However, rather than a detrimental role in the pathogenesis of EAE, the findings from our study point to an important protective role for CXCR3 signaling in EAE and confirm a recent report indicating that mice lacking this chemokine receptor develop a more severe form of MOG-EAE (42). Using a standard MOG immunization protocol, our findings indicated that although the peak disease phase of EAE is unaltered in the absence of CXCR3, the animals largely fail to recover from disease, exhibiting more severe chronic physical signs of disease. This observation together with the finding that overall numbers of activated effector T cells infiltrating the CNS is similar in the presence or absence of CXCR3 confirm that this chemokine receptor has a nonessential role in effector T cell trafficking to the brain during the development of EAE.

The concept that CXCR3 is not critically involved in the recruitment of autoreactive effector CD4+ T cells to the CNS in EAE even though activated T cells of this phenotype express CXCR3 in the mouse (23) has indirect support from previous findings. Although CXCR3 was found to be a marker for memory CD4+ T cells capable of migrating through the blood-brain barrier in vitro, this receptor was dispensable for transendothelial cell migration of these cells (62). Furthermore, transgenic mice developed by us with constitutive production of CXCL10 exhibit only modest CNS accumulation of CXCR3+ T cells that are retained largely in the meningeal and ventricular compartments, despite the parenchymal production of the chemokine by astrocytes (63).

The absence of a significant alteration in the recruitment of activated effector T cells to the CNS and the development of a more severe chronic physical EAE phenotype in the absence of CXCR3 suggested that CXCR3 modulated the ongoing disease process in the CNS. In support of this, we observed that the inflammatory lesions in the CNS of mice lacking CXCR3 were more widespread and associated with significantly increased demyelination and axonal injury. Inflammatory lesions could be found in the paraventricular and midbrain white matter of severely affected CXCR3–/– mice. These areas of the brain were rarely, if at all, involved in WT mice. Therefore, the threshold for developing inflammatory lesions in different white matter areas of the CNS is reduced in CXCR3–/– mice. The exact mechanism that controls the regional distribution of inflammation in the CNS in EAE is not known. A recent study reported that regional differences in the astrocyte response during EAE might contribute to the distribution pattern of inflammatory lesions (53). Our data suggest another possibility, that CXCR3 signaling may provide a molecular mechanism for controlling the distribution of inflammatory lesions in the CNS during EAE.

An important question arises as to what mechanism underlies the more severe inflammatory lesion development and tissue destruction in EAE that occurs in the CNS when CXCR3 is absent. Studies by Liu et al. (42) reported that compared with WT controls, IFN-{gamma} production and tissue levels were reduced significantly in CXCR3–/– animals. This further correlated with increased proliferation of MOG-reactive CXCR3–/– T cells suggesting a major defect in immunoregulatory mechanisms in these animals (42). In support of this potential mechanism, it was shown previously that deficiency in IFN-{gamma} lead to the exacerbation of EAE through a process involving increased proliferation and accumulation of autoreactive T cells in the CNS (64). However, we failed to observe a significant difference, either in the CNS levels of IFN-{gamma} gene expression, or in circulating levels of this cytokine in CXCR3–/– vs WT mice with EAE. CXCL9, CXCL10, and CXCL11 were all originally identified as IFN-{gamma}-inducible chemokines (15, 65, 66). Consistent with this, CXCL10 (67) as well as CXCL9 and CXCL11 (S. L. Carter, M. Müller, and I. L. Campbell, unpublished observation) gene expression in the CNS during EAE is completely ablated in the absence of IFN-{gamma} signaling. The finding in this study that the expression of the CXCR3 ligand genes showed a similar increase in the CNS during EAE in CXCR3null and WT animals further reflects unaltered local IFN-{gamma} gene expression in the CXCR3null mice. In their studies, Liu et al. (42) found the most striking differences between the CXCR3null and WT animals occurred at lower doses of immunizing MOG. It is conceivable that the IFN-{gamma} levels might differ more at lower but not higher MOG doses. In support of this, in the current study we did observe a trend toward a decreased amount of IFN-{gamma} at peak disease in CXCR3null mice. In any event, at the immunizing dose used in the current study, the findings do not support a crucial role for CXCR3 signaling in modulating IFN-{gamma} production by T cells as a protective mechanism in MOG-EAE.

We observed in the spinal cord and cerebellum of WT mice with EAE that typically the CD4+ T cells but not the CD8+ T cells formed well-organized perivascular cuffs with limited parenchymal infiltration. These T cell cuffs were surrounded by activated microglia/macrophages and were associated with localized demyelination. However, in CXCR3–/– mice with EAE, the CD4+ T cells failed to form perivascular cuffs but rather were spread diffusely throughout the white matter. Coincident with the more diffuse CD4+ T cell distribution, we observed more widespread activation of the microglia/macrophages and a corresponding increase in white matter areas affected by demyelination. The finding of a gross abnormality in the architecture of the inflammatory lesions in the CXCR3–/– state suggests a key role for this receptor in organizing the mononuclear cells into perivascular cuffs by regulating the localized migration of these cells. Such a mechanism would depend on the presence of corresponding ligand. Previously, it was established that astrocytes represent a major source of CXCL10 in EAE (30). Moreover, the same workers found that the temporal induction of CXCL10 in the CNS in EAE follows the arrival and entry of the T cells (31). Our findings here confirmed the astrocyte expression of the CXCL10 gene and further revealed that the expression of the genes for CXCL9 and to a lesser degree CXCL10 and CXCL11 is strongly associated with the perivascular mononuclear cell lesions in the CNS of WT mice with EAE. A high level of CXCL9 gene expression localized directly within the lesions to the infiltrating T cells and most likely was induced in response to IFN-{gamma} production by these cells. On the basis of these findings, we would argue that CXCR3 signaling in EAE has a protective role, in part, by retaining the T cells to the perivascular compartment. By preventing the spread of these cells to the parenchymal white matter, inflammation is limited and consequent tissue damage reduced.

Besides its primary receptor CCR7, which is present on activated T cells (13, 68), the chemokine CCL21 can also interact with CXCR3 (69, 70, 71). The functional expression of CCL21 by endothelial cells of the BBB during EAE suggested an involvement of this chemokine in T cell recruitment into the CNS (72). However, transgenic mice with a CNS-specific CCL21 expression developed polymorphonuclear infiltrates rather than lymphocytic accumulation (73), whereas CCR7– mice exhibit unaltered EAE (74). It remains to be established whether CCL21 can stimulate CCR7-independent, CXCR3-dependent T cell migration and might contribute to the perivascular accumulation of lymphocytes during EAE, which we found disrupted in CXCR3–/– mice.

We have observed that the distribution of CD4+ but not CD8+ T cells was perturbed by CXCR3 deficiency. Although CD8+ T cells were more scattered throughout the parenchyma, CD4+ T cells were organized in well-defined perivascular clusters during EAE in WT mice. These perivascular clusters were also focal points for the expression of the CXCR3 ligands, particularly CXCL9, pointing toward a more likely functional impact of CXCR3 signaling on CD4+ rather than CD8+ T cells in EAE. This is in contrast to a previous study in the lymphocytic choriomeningitis (LCM) viral model where it was reported that CD8+ T cells were most affected by CXCR3 deficiency (75). It is difficult to draw comparisons here as the two models are fundamentally different in etiopathogenesis with LCM being exclusively viral-induced, anti-LCM virus-CD8+ T cell driven, whereas EAE is autoimmune induced, anti-MOG CD4+ T cell driven. Beside differences in the pathogenic T cells involved, these models also differ substantially in the distribution of the target Ag with LCM virus being located predominantly outside the parenchyma whereas MOG is located deep in the parenchymal white matter. We feel that it is likely that these as well as numerous other differences between these models likely account for the differing roles of CXCR3. However, in another viral immunopathology model, mouse hepatitis virus encephalomyelitis, blocking of CXCR3 caused a reduction of infiltrating CD4+ T cells but not CD8+ T cells in the CNS (76). Thus, the function of CXCR3 in neuroinflammation is likely complex and might be determined by the type of immune pathology involved.

Regulatory T cells represent a subset of CD4+ T cells that have the ability to potently down-regulate effector T cell function (reviewed in Ref. 77). Regulatory T cells interact with effector T cells by direct cellular contact and/or through the localized secretion of counterregulatory cytokines such as TGF-β and IL-10. Recent reports highlight a key role for endogenous CD4+ regulatory T cells in suppressing the development of MOG-EAE (78, 79, 80). In our studies, Foxp3+ regulatory T cells were embedded in perivascular T cell accumulations in the CNS of WT mice with EAE. The close association of the regulatory T cells with effector CD4+ T cells in the perivascular cuffs would be expected to favor the interaction of these cells. However, in the absence of CXCR3, the Foxp3+ regulatory T cells were reduced in number and were scattered throughout the parenchyma. It has been reported that CXCR3 is dispensable for the regulatory functions of these cells (81). Regulatory T cells are CXCR3-positive (82) and are capable of migrating toward CXCR3 ligands (83). Thus, the recruitment of these cells to the CNS in EAE may in part be driven by CXCR3. Alternatively, CXCR3 signaling may be required for optimal survival of regulatory T cells in the CNS during EAE. The reduced number and loss of spatial organization of the regulatory T cells with effector CD4+ T cells would reduce the likelihood of a suppressive interaction between these cells. This may explain why there is increased tissue damage and failure of recovery from EAE in the absence of CXCR3. In support of a protective role of CD4+CD25+ regulatory T cells in EAE, recovery from EAE in WT mice was shown to correlate with the local accumulation of CD4+CD25+ regulatory T cells within the brain (78). In addition, CD4+CD25+ regulatory T cells generated in vitro are capable of suppressing EAE induced by adoptive transfer of MOG-reactive T cells (80).

In summary, we have shown that in EAE, CXCR3 signaling plays a major protective role that is independent of the recruitment of effector T cells to the CNS. We conclude that major functions of CXCR3 signaling in EAE is not to attract T cells into the brain but to constrain CD4+ T cells to the perivascular space in the CNS, to promote regulatory T cell accumulation and facilitate interaction of these cells with effector T cells thus limiting autoimmune-mediated tissue damage.


   Acknowledgments
 
We thank Jane Radford and Barbara Hernandez (Department of Pathology, University of Sydney, Sydney, Australia) for expert technical assistance with tissue processing and routine histology. Professor Hans Lassmann (Center for Brain Research, University of Vienna, Vienna, Austria) is gratefully acknowledged for fruitful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by U.S. Public Health Service National Institutes of Health Grant NS044905 and a start up grant from the University of Sydney (to I.L.C.). S.L.C. was supported by an Endeavour International Postgraduate Award and International Postgraduate Award from the University of Sydney. D.G. was supported by an Australian Postgraduate Award. M.J.H. was a postdoctoral fellow from the Deutsche Forschungsgemeinschaft (HO3298/1-1). M.M. was a postdoctoral fellow from the Deutsche Forschungsgemeinschaft (Mu17-07/3-1) and was also supported by the Innovative Medical Research Fund of the University of Munster Medical School, Munster, Germany. Back

2 Address correspondence and reprint requests to Dr. Iain L. Campbell, School of Molecular and Microbial Biosciences G08, University of Sydney, Sydney, Australia. E-mail address: icamp{at}mmb.usyd.edu.au Back

3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; WT, wild type; MOG, myelin oligodendrocyte glycoprotein; DAPI, 4',6-diamidino-2-phenylindole; LFB, Luxol fast blue; RPA, RNase protection assay; LCM, lymphocytic choriomeningitis. Back

Received for publication January 24, 2007. Accepted for publication June 22, 2007.


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 Materials and Methods
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 Discussion
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