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(The FASEB Journal. 2006;20:896-905.)
© 2006 FASEB

The neuronal chemokine CX3CL1/fractalkine selectively recruits NK cells that modify experimental autoimmune encephalomyelitis within the central nervous system

DeRen Huang*,1, Fu-Dong Shi{dagger},1, Steffen Jung{ddagger}, Gary C. Pien§, Jintang Wang*, Thais P. Salazar-Mather§, Toby T. He*, Jennifer T. Weaver*, Hans-Gustaf Ljunggren||, Christine A. Biron§, Dan R. Littman{ddagger} and Richard M. Ransohoff*,2

* Neuroinflammation Research Center, Department of Neurosciences, Lerner Research Institute, The Cleveland Clinic Foundation, Ohio, USA;

{dagger} Barrow Neurological Institute, Phoenix, Arizona, USA;

{ddagger} Skirball Institute of Biomolecular Medicine and Howard Hughes Medical Institute, New York University Medical Center, New York, New York, USA;

§ Department of Molecular Microbiology and Immunology, Division of Biology and Medicine, Brown University, Providence, Rhode Island, USA; and

|| Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden

2Correspondence: Neuroinflammation Research Center, Department of Neurosciences NC30, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: ransohr{at}ccf.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte trafficking to the central nervous system (CNS), regulated in part by chemokines, determines severity of the demyelinating diseases multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). To examine chemokine receptor CX3CR1 in EAE, we studied CX3CR1GFP/GFP mice, in which CX3CR1 targeting by insertion of Green Fluorescent Protein (GFP) allowed tracking of CX3CR1+ cells in CX3CR1+/GFP animals and cells destined to express CX3CR1 in CX3CR1GFP/GFP knockouts. NK cells were markedly reduced in the inflamed CNS of CX3CR1-deficient mice with EAE, whereas recruitment of T cells, NKT cells and monocyte/macrophages to the CNS during EAE did not require CX3CR1. Impaired recruitment of NK cells in CX3CR1GFP/GFP mice was associated with increased EAE-related mortality, nonremitting spastic paraplegia and hemorrhagic inflammatory lesions. The absence of CD1d did not affect the severity of EAE in CX3CR1GFP/GFP mice, arguing against a role for NKT cells. Accumulation of NK cells in livers of wild-type (WT) and CX3CR1GFP/GFP mice with cytomegalovirus hepatitis was equivalent, indicating that CX3CL1 mediated chemoattraction of NK cells was relatively specific for the CNS. These results are the first to define a chemokine that governs NK cell migration to the CNS, and the findings suggest novel therapeutic manipulation of CX3CR1+ NK cells. Huang, D., Shi, F.-D., Jung, S., Pien, G. C., Wang, J., Salazar-Mather, T. P., He, T. T., Weaver, J. T., Ljunggren, H. G., Biron, C. A., Littman, D. R., Ransohoff, R. M.


Key Words: autoimmune disease • chemokine • chemokine receptor • NK • T cell


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MULTIPLE SCLEROSIS (MS) is an inflammatory demyelinating disease of the human central nervous system (CNS) that causes significant disability for the majority of patients and is characterized pathologically by abundant leukocyte infiltrates in the CNS. Pathways by which hematogenous cells accumulate in the inflamed CNS are incompletely understood (1 , 2) . However, general mechanisms of leukocyte trafficking have been clarified (3) and require interaction between leukocytes and endothelial adhesion molecules, governed in part by chemokines, which act through high-affinity receptors on leukocytes. The combined actions of ~ 100 adhesion molecules, chemokines, and chemokine receptors influence leukocyte trafficking, challenging investigators to identify specific determinants of migration of subpopulations of leukocytes.

Analysis of leukocyte trafficking during inflammation carries practical implications: natalizumab, a humanized monoclonal antibody (mAb) directed against alpha-4 integrins, markedly reduced clinical relapses and inflammatory brain lesions in phase III MS clinical trials (4) , and produced benefit for patients with inflammatory bowel disease in other trials (5) , but caused progressive multifocal leukoencephalopathy in some recipients (6 ,7) , underlining the promise and pitfalls of such treatments (8) . Antiadhesion molecule therapy targets multiple leukocyte subsets equally. However, lymphocytes, NK cells, and dendritic cells can be either pathogenic or regulatory, indicating a need to identify selective trafficking determinants that direct the movements of specific leukocyte subpopulations. Chemokine receptors are expressed more narrowly than adhesion molecules, and there has been considerable success in dissecting the functions of these receptors to gain insight into the specific trafficking patterns of leukocyte populations.

One of very few chemokines present at high levels in the normal CNS is CX3CL1 (fractalkine), a type I transmembrane glycoprotein that contains an N-terminal chemokine domain presented to receptor-bearing cells by a rigid mucin-like stalk (9 ,10) . CX3CL1 functions as an adhesion molecule or, after proteolytic release, as a soluble chemoattractant. Both CX3CL1 and its receptor (CX3CR1) were initially cloned from CNS sources (9 ,10) . The ligand-receptor pair are monogamous and considered true orthologs in human and rodent species.

The receptor, CX3CR1 is expressed by monocytes, lymphocytes, and NK cells. CD56bright NK cells are selectively responsive to CX3CL1 (11) . CX3CR1 and CX3CL1 are required for physiological trafficking of circulating monocytes to organs such as lung, and for the morphogenesis of CCR6-negative dendritic cells in the intestinal lamina propria (12 ,13) . In the CNS, CX3CR1 is expressed by microglia, the resident macrophage population (14 15 16 17) .

EAE is an autoimmune disease model that has been widely used to delineate mechanisms in MS. Studies in gene-targeted mice demonstrated that the CCR2 chemokine receptor and its ligand, CCL2/MCP-1, are required for monocyte accumulation in the CNS (18 19 20) . No corresponding data about infiltration of the CNS by lymphocyte subpopulations have emerged. In the present study, mutant mice were used in which the CX3CR1 locus was disrupted by insertion of a reporter gene encoding enhanced green fluorescence protein (GFP), permitting analysis of the distribution and phenotype of in vivo-labeled cells, that retained functional CX3CR1, in mice heterozygous for CX3CR1 (CX3CR1+/GFP) (21) . For comparison, cells destined to express CX3CR1, but lacking its function, were tracked in CX3CR1 knockout (GFP/GFP) animals. We found a selective deficiency of NK cells in the CNS of CX3CR1GFP/GFP mice with EAE, accompanied by very severe disease. Accumulation of other leukocyte populations was equivalent in the CNS of wild-type and CX3CR1-deficient mice. These results delineate for the first time a specific role for CX3CL1/CX3CR1 in the recruitment of NK cells to the inflamed CNS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Animal experimental procedures were carried out in accordance with the NIH guidelines on animal care, under oversight by the local Animal Research Committees at the Cleveland Clinic Foundation, Barrow Neurological Institute, and Brown University. The generation of CX3CR1 mutant mice was described previously (21) . Mice were originally on C57BL/6 x 129/Ola background and had been backcrossed to C57BL/6J mice for 5 generations. One F5 C57BL/6 x 129/Ola CX3CR1GFP/GFP mouse was bred to a C57BL/6J mouse (Jackson Laboratory, Bar Harbor, ME, USA) and their offspring were intercrossed to generate CX3CR1+/+, +/GFP and GFP/GFP mice. F7 and F8 C57BL/6 CX3CR1+/+, +/GFP and GFP/GFP mice were generated in a similar manner. NKT cell-deficient mice (22) on B6 background (backcrossed onto B6 for more than 10 generations) were provided by Dr. Luc Van Kaer (Vanderbilt University School of Medicine). CX3CR1GFP/GFP and NKT double deficient (CD1d-/-CX3CR1GFP/GFP) mice were generated by crossing F10 CX3CR1GFP/GFP and F10 CD1d-/- mice in a similar manner described above. CX3CR1 genotype was determined using polymerase chain reaction (PCR) based genomic DNA analyses and flow cytometric analyses of expression of the inserted GFP gene (ref (21) ., and see below). Primers X, 5'-TTC ACG TTC GGT CTG GTG GG-3' and Y, 5'-GGT TCC TAG TGG ACG TAG GG-3' (annealing temperature: 60°C), amplify the wild-type allele, while primers Y and Z, 5'-GAT CAC TCT CGG CAT GGA CG-3' (annealing: 60°C), the mutant allele.

Induction of EAE and murine cytomegaloviral hepatitis
Active EAE and adoptive-transfer EAE were induced and scored as described previously (20) . Mice were weighed and examined daily for clinical EAE in a blinded manner.

Murine cytomegalovirus hepatitis was induced, and cell populations found in liver, spleen, and peripheral blood were determined as described (23 ,24) .

Histology
Brains and spinal cords were rapidly dissected after intracardiac perfusion with ice-cold PBS followed by 4.0% paraformaldehyde solution. Thirty µm-thick vibratome sections were cut and used for the examination of GFP expression in the CNS tissues of normal or EAE mice, and 8 µm-thick paraffin sections were stained with hematoxylin and eosin (H&E). Polyclonal anti-MBP antibodies (DAKO, 1:2000 dilution) were used to evaluate the myelin structure in mice with EAE. Spinal cord sections were incubated with anti-MBP antibodies at 4°C overnight. Biotinylated anti-goat IgG antibodies (Vector Laboratories, CA, USA) were applied after extensive washing. Sections were examined after incubation with avidin-peroxidase and 3,3'-diaminobenzidine (Sigma-Aldrich, MO, USA).

T cell proliferation assay, cell cultures, and cytokine assay
T cell proliferation assay and cytokine studies were performed as described previously (20) . QuantikineTM kits (R&D system, MN, USA) were used for the determinations of levels of cytokines. The sensitivities of the methods were 2.0, 6.0, 2.0, 4.0, and 4.0 pg/ml for IFN-{gamma}, interleukin (IL)-2, IL-4, IL-5, and IL-10, respectively. All samples were measured in duplicate and diluted if necessary.

Flow cytometry
Cells from the CNS, draining lymph nodes, spleens, and livers were isolated as described previously (20 ,24) . Single cell suspensions were washed in FACS buffer (1% FCS and 0.1% sodium azide in PBS). After blocking with CD16/CD32 Fc Block (PharMingen, San Diego, CA, USA), cells were stained for surface markers with directly conjugated antibodies in FACS buffer. Antibodies used were CD4-PE, CD8-Cy, CD11b-PE, Gr1-Cy, CD45-Cy, NK 1.1-PE, CD11a-Cy, CD49-Cy, TCRß-Cy, and CD3-Cy. All antibodies were obtained from BD PharMingen (San Diego, CA, USA). For staining of V{alpha}14 NKT cells, mononuclear cells were treated with unlabeled neutravidin and CD16/CD32 Fc Block, and then incubated with FITC-labeled antibodies against TCR-ß and PE-labeled CD1d/{alpha}-GalCer tetramers, as described (25) .

NK cell depletion
To delete NK1.1+ cells (NK cells and NKT cells), B6 mice were treated with anti-NK1.1 mAb (PK136) or isotype control antibody (Ab) (IgG2a). For initial depletion, 500 µg anti-NK1.1 mAb was injected (i.p.) at day –2 postimmunization (p.i.) (26) . Every 5–7 days thereafter, 50 µg anti-NK1.1, was injected i.p. until the termination of experiments.

CNS fractalkine expression determined by ELISA and real time RT-polymerase chain reaction
Spinal cords were manually homogenized in 1 ml lysis buffer (150 mM NaCl, 0.01 M Tris, 1.0 mM EDTA, 1.0 µg/ml aprotinin, 100 µg/ml PMSF) and centrifuged at 500 g for 10 min. Protein concentration in the supernatants was measured and levels of CX3CL1 determined. For detection of CX3CL in the preparations, antimouse CX3CL1 polyclonal antibodies and conjugated anti-CX3CL1 (R&D Systems, Minneapolis, MN, USA) were used as capture and detection antibodies. Goat anti-C-terminal CX3CL1 antibodies (Santa Cruz, CA, USA) were used as capture antibodies to determine the amount of membrane-bound CX3CL1 in the samples. Standard curves were established on the same plate, using 2-fold serial dilutions of recombinant mouse CX3CL1 (ChemoCentryx, San Carlos, CA, USA).

RNAs were extracted from spinal cords of healthy or EAE mice using RNAzol (Life Technologies, Grand Island, NY, USA) and quantitative reverse transcription-coupled PCR assays were performed using LightCycler (Roche) as described previously (20) . Primers FKNF: ATT GTC CTG GAG ACG ACA CAG C and FKNR: TTG CCA CCA TTT TTA GTG AGG G were used for the detection of CX3CL1 expression. GAPDH expression was used as an internal control for each sample.

Migration assay
Brain and spinal cord tissues of C57BL/6 mice with EAE scores of 3.5 to 4.0 were manually homogenized in 1 ml RPMI 1640 medium containing 1.0 µg aprotinin (Sigma-Aldrich, St Louis, MI, USA) and centrifuged at 500 g for 10 min. Supernatants containing soluble CNS proteins were collected and kept at –80°C until assay. Transwell chemotaxis assay was performed as published (27 ,28) . Briefly, cells (2x106/ml, 0.5 ml/well) were added to the 3.0 µm pore transwell culture inserts of the 12-well Transwell plates (Costar, Cambridge, MA, USA). Chemotaxis buffer (RPMI 1640 with 0.1% BSA, 1.5 ml/well) was added to the lower chamber in the presence or absence of CNS protein extracts or mouse recombinant CX3C chemokine domain (a gift from Dr. Jen Gosling, ChemoCentryx) at different concentrations ranging from 0.05 to 10.0 µg/ml, or CNS protein extract (1:4 to 1:32 dilution). SDF-1/CXCL12 (ChemoCentryx, San Carlos, CA, USA) was used as a control in the current study as CXCL12 attracts essentially all peripheral blood lymphocytes with equal efficiency. Plates were incubated at 37°C for 4 h. Cells in the upper and lower chambers were collected and counted by flow cytometry.

Statistical analyses
The Mann-Whitney U test was used for the comparisons of disease severity, cytokine, chemokine gene expression and accumulations of subpopulations of leukocytes in the target organs. A {chi}2 test was used for the comparison of the frequencies of CNS hemorrhage and rates of death. All P values were two-sided. A P value < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CX3CR1-deficient mice develop severe EAE with persistent spastic paralysis and increased mortality
Previous studies showed earlier onset of EAE, with slightly worse severity of disease in C57BL/6 x 129SJ CX3CR1-deficient mice (29) . To extend this analysis, we characterized EAE severity and CNS-infiltrating leukocyte populations in CX3CR1GFP/GFP mice, their heterozygous and WT littermates after extensive breeding onto a C57BL/6 background. All mice developed clinical EAE regardless of CX3CR1 genotype. CX3CR1GFP/GFP mice developed EAE earlier and with higher mortality than WT mice (Table 1 ). CX3CR1+/GFP mice showed an intermediate concentration of EAE severity. CX3CR1GFP/GFP mice with EAE frequently manifested nonremitting spastic paralysis (Fig. 1 a), in contrast to wild-type and heterozygous mice that manifested transient flaccid paresis, indicating extensive CNS lesions in the CX3CR1-deficient mice with EAE. These results were confirmed using mice from 3 different generations during backcrossing onto the C57BL/6J strain (Table 1) . CNS tissues from CX3CR1GFP/GFP mice sacrificed at day 65 p.i. demonstrated more extensive demyelination than did tissues from CX3CR1+/+ mice (data not shown).


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  		Table 1. CX3CR1GFP/GFP mice showed increased severity of MOG35–55-induced EAE


Figure 1
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  		Figure 1. Spastic paralysis and hemorrhagic inflammation in CNS tissues in CX3CR1GFP/GFP mice with EAE. a) CX3CR1-deficient mice with MOG35–55 induced EAE manifest spastic paralysis. Note the rigid posture of legs and tail. b, c) Brain stem hemorrhage was associated with inflammatory reaction (original: ax100, bx400). BS denotes brain stem; C, cerebellum; H, hemorrhage; IR, inflammatory reaction. d) Frequent thoracic and lumbar spinal cord hemorrhage was found in mice lacking CX3CR1.

At necropsy, we detected a brainstem hemorrhage in one moribund CX3CR1GFP/GFP mouse. Subsequent neuropathological evaluation excluded vascular malformation or vasculitis, and demonstrated that this brainstem hemorrhage was associated with an intense inflammatory reaction (Fig. 1b, c ). Thereafter, CNS tissues of all mice sacrificed during acute EAE were examined for gross evidence of hemorrhage (Fig. 1d ). This analysis revealed a significantly higher incidence of CNS hemorrhage in CX3CR1GFP/GFP (18%, 7/38) than in either CX3CR1+/GFP (4%, 1/23) or CX3CR1+/+ (0/36) littermate control mice (P=0.004, CX3CR1GFP/GFP vs. CX3CR1+/+, {chi}2 test). Thus, EAE was more severe, both neurobehaviorally and pathologically, in mice lacking CX3CR1.

Equivalent priming of encephalitogenic T cells in CX3CR1GFP/GFP and wild-type mice
We considered whether the severe phenotype of EAE in CX3CR1GFP/GFP mice could be caused by altered priming of encephalitogenic T cell responses. Lymphocyte recall responses in CX3CR1 wild-type and knockout mice revealed similar antigen-driven proliferation. CX3CR1GFP/GFP lymphocytes showed MOG35–55 stimulation index [SI] ± SE of 4.5 ± 2.1 as compared with CX3CR1+/+ SI: 4.7 ± 2.0 (n=5 per group). Lymphocyte recall responses also showed equal production of IFN-{gamma}, IL-2, IL-4, IL-5, and IL-10 at 24, 48, and 72 h postinitiation of tissue culture (supplemental Fig. 1 , Fig. 1s ). Primed T cells from CX3CR1+/+ and CX3CR1GFP/GFP mice equally transferred EAE to WT mice (data not shown). CX3CR1+/GFP and CX3CR1GFP/GFP mice exhibited similar kinetics of accumulation of GFP+ cells in lymph nodes (data not shown), reflecting unaltered trafficking of GFP+ dendritic cells and lymphocytes to these sites. We concluded that severe EAE in CX3CR1GFP/GFP mice was not caused by altered priming of encephalitogenic T cells.

NK cells are selectively reduced in the CNS of CX3CR1-deficient mice with EAE
Given that encephalitogenic T cells were equivalent in WT mice and CX3CR1-deficient mice in the periphery, we reasoned that severe EAE in CX3CR1-deficient mice might stem from reduced recruitment of regulatory cells or increased accumulation of pathogenic/effector cells in the CNS. To address this issue, we analyzed the composition of CNS inflammatory infiltrates in CX3CR1+/GFP and CX3CR1GFP/GFP F8 mice with equivalent EAE clinical scores, after excluding mice with grossly visible CNS hemorrhage to avoid contamination by leukocytes associated with hemorrhagic lesions. The total numbers of cells isolated from CNS tissues of CX3CR1+/GFP and CX3CR1GFP/GFP mice with EAE were equivalent. Compared with CX3CR1+/+ littermates, CX3CR1GFP/GFP mice with EAE exhibited significantly reduced CNS GFP+NK1.1 cells (Fig. 2 ). This deficit was selective, as significant differences of other PB or CNS lymphocyte populations were not observed (Fig. 2 and Table 2 ). Mononuclear phagocytes (monocytes and macrophages) express CX3CR1 (Fig. 2s ) and constitute the predominant leukocyte population in the CNS of mice with EAE and their presence correlates tightly to disease severity. The combination of anti-Gr-1 and CD11b antibodies were used to evaluate the accumulation of CNS monocytes during EAE (21) . Gr-1intermediateCD11b+ monocytes but not neutrophils expressed high levels of GFP (21) . The percentages of Gr-1intermediate CD11b+ cells in CNS infiltrates were indistinguishable in CX3CR1+/+, +/GFP and GFP/GFP mice with EAE (data not shown), and few neutrophils were present in EAE tissues from mice bearing any of the three genotypes. We concluded that the only population whose migration to the CNS was significantly altered in CX3CR1GFP/GFP mice with EAE were NK1.1+ cells.


Figure 2
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  		Figure 2. Selective reduction of NK1.1+ cell accumulation in CNS tissues from CX3CR1-deficient mice with EAE. Cell suspensions from the CNS of CX3CR1+/+ and CX3CR1GFP/GFP mice with EAE of equivalent severity were analyzed for green fluorescence (GFP) and NK1.1 expression, using flow cytometry.


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  		Table 2. GFP+ cells in CX3CR1+/GFP and GFP/GFP mice with EAE (%)

GFP+/NK1.1+ cells are composed of NK cells and NKT cells, both of which have been reported to play regulatory roles in EAE (30 ,31) . We addressed whether the reduction of NK1.1+ cells in the CNS of CX3CR1GFP/GFP mice was due to selective loss of NK cells or NKT cells from that population. To address this issue directly, we bred NKT cell-deficient CD1d–/– F10 mice (32) with CX3CR1GFP/GFP F10 mice to generate double mutant mice, reserving single mutants and WT mice as controls. EAE was induced in CD1d+/+CX3CR1GFP/GFP, CD1d–/–CX3CR1GFP/GFP, CD1d–/–CX3CR1+/GFP, and CD1d+/+CX3CR1+/GFP mice. Leukocyte populations in peripheral blood and CNS compartments were monitored by flow cytometry. Results from these experiments are summarized in Table 2 . A significant reduction of NK1.1+/TCRß- cells in EAE CNS tissues from CX3CR1GFP/GFP mice with EAE was evident, consistent with data using mice from the F8 generation (Fig. 2) . At the peak of EAE, there was no difference in the percentages of NK1.1+/TCRß- cells in the CNS tissues of CD1d–/–CX3CR1+/GFP mice and CD1d+/+CX3CR1+/GFP mice (Table 2) . This finding suggested that NK cell, not NKT cell, migration to the CNS was impaired in CX3CR1GFP/GFP mice with EAE. To address this issue further, we determined the frequency of CD1d-restricted NKT cells by flow cytometry, using {alpha}-galactosylceramide/CD1d tetramers (25) , and found that NKT cells were present at comparable levels in blood and CNS in CX3CR1+/GFP and CX3CR1GFP/GFP mice with EAE (Table 2) . Therefore, the reduction of NK1.1+ cells in CX3CR1-deficient mice was due to impaired migration of NK cells, rather than NKT cells.

Anti-NK 1.1 Ab-treated CX3CR1+/GFP mice exhibit equally severe EAE as CX3CR1GFP/GFP mice
Tabira and colleagues showed that C57BL/6 mice depleted of NK1.1+ cells (26) exhibited markedly increased mortality from EAE. Given our finding of severe EAE in CX3CR1GFP/GFP mice and reduced NK cell recruitment to the CNS of these mice, we addressed a role for NK cells in the severe EAE phenotype of CX3CR1-null mice. In initial experiments, CX3CR1+/+ mice received 500 µg anti-NK1.1 antibodies (PK136) before MOG35–55 immunization, and weekly injections thereafter to maintain NK cell depletion, with the efficacy of NK depletion being monitored by flow cytometry. The results confirmed previous observations: NK-depleted CX3CR1+/+ mice had significantly increased mortality due to EAE, comparable to that of CX3CR1GFP/GFP animals (data not shown). We next compared the severity of EAE in CX3CR1GFP/GFP mice treated with control antibodies, and NK-depleted CX3CR1+/GFP mice. Depletion of NK1.1+ cells in CX3CR1+/GFP mice resulted in highly lethal disease, with 4/7 (56%) of mice dying of EAE. In this experiment, 2/6 (33%) of the CX3CR1GFP/GFP mice died during the EAE attack, and survivors manifested nonremitting disease. These results supported the hypothesis that severe EAE in CX3CR1GFP/GFP mice resulted from failure of NK cells to access the inflamed CNS.

CD1d-/-CX3CR1GFP/GFP and CD1d+/+CX3CR1GFP/GFP mice exhibit equally severe EAE
Since the PK136 anti-NK1.1 mAb depletes NK cells and a portion of NKT cells, it was important to determine whether enhanced EAE severity in PK136-treated mice was due to depletion of NK cells, NKT cells, or both. Initially, we immunized NKT cell-deficient CD1d–/– mice with MOG35–55 and CFA as above. CD1d–/– mice developed EAE of similar severity as wild-type controls (Fig. 3 ). This result suggested that CD1d-restricted NKT cells did not exert significant regulatory function in this model.


Figure 3
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  		Figure 3. CD1d–/–CX3CR1GFP/GFP double deficient mice and CX3CR1GFP/GFP mice showed comparable clinical severity and course of MOG35–55 induced EAE. EAE was induced in WT (CD1d+/+CX3CR1+/+), CD1d-deficient (CD1d–/–CX3CR1+/+), CX3CR1-deficient (CD1d+/+CX3CR1GFP/GFP), CD1d, and CX3CR1 double deficient (CD1d–/–CX3CR1GFP/GFP) mice, and daily clinical EAE scores were recorded in a blinded manner. CD1-deficient mice had similar clinical course and disease severity of EAE to that in wild-type control mice, while CD1d and CX3CR1 double deficient mice showed comparable clinical manifestation as did CX3CR1-deficient mice. There was no difference in EAE severity between CD1d+/+ and CD1d-/- mice.

To determine whether the absence of NKT cells would affect the course of EAE in CX3CR1GFP/GFP mice, we compared EAE phenotype in CD1d–/– mice and CD1d–/–CXCR3GFP/GFP double mutant mice. Immunization with MOG35–55 induced a more severe form of EAE in CD1d-/-CX3CR1GFP/GFP mice as compared to wild-type control mice and CD1d–/– mice, respectively. EAE in CD1d–/–CX3CR1GFP/GFP mice had early onset and nonremitting spastic paralysis (Table 3 ). The EAE course in double mutant mice was similar to that observed in CX3CR1GFP/GFP single mutant mice (Fig. 3) . Thus, the absence of NKT cells did not significantly influence the course of EAE in CX3CR1GFP/GFP mice. These data further supported the proposal that the severe phenotype of EAE in CX3CR1GFP/GFP mice resulted from the absence of regulatory NK cells during EAE attack.


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  		Table 3. CD1d–/–CX3CR1GFP/GFP and CD1d+/+CX3CR1GFP/GFP mice showed equivalent severity of MOG35–55-induced EAE

CNS recruitment of NK cells by CX3CL1 is regulated at the posttranslational concentration and is relatively selective for the CNS
Neurons in the CNS can release CX3CL1, providing one potential source for CX3CR1-specific chemoattraction. Levels of soluble but not membrane-bound CX3CL1 were markedly increased in CNS tissues from mice with EAE (Fig. 3s , panel a). Consistent with previous reports by others, CX3CL1 mRNA levels in CNS tissues from mice at the peak of EAE were equal to those found in healthy mice (Fig. 3s , panel b), suggesting posttranslational regulation of soluble CX3CL1 levels. In vitro migration assays revealed that CNS protein extracts contained chemoattractant activities toward CX3CR1+/GFP cells, which were diminished either by preincubating extracts with anti-CX3CL1 antibodies, or by desensitizing CX3CR1 through exposure of CX3CR1+/GFP cells to recombinant mouse CX3CL1 (Table 1s).

To address whether NK cell recruitment was globally impaired in CX3CR1GFP/GFP mice, we examined the course of murine cytomegalovirus (mCMV) infection (23 ,33) , a well-characterized NK-dependent hepatic inflammatory response. Compared to littermate controls, CX3CR1GFP/GFP mice exhibited identical disease clinical course and hepatic inflammatory focus formation. FACS analyses of dynamic changes of T cell and NK populations in blood, spleen and liver showed no differences between CX3CR1GFP/GFP and +/+ mice with mCMV infection (Fig. 4s).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We studied EAE in CX3CR1-deficient and corresponding control mice, with the following findings: 1) disease was more severe in mice lacking CX3CR1; 2) migration of NK cells to the CNS of CX3CR1GFP/GFP mice with EAE was impaired; 3) NKT cells were not implicated in the occurrence of severe EAE in CX3CR1GFP/GFP mice, since their recruitment to the CNS was not impaired and CD1d-/-CX3CR1GFP/GFP mice exhibited equally severe disease as CX3CR1GFP/GFP mice. We also found that soluble CX3CL1, the CX3CR1 ligand, was greatly increased in the CNS of mice with EAE, while levels of CX3CL1 mRNA were not changed. These findings suggested that CX3CL1 was released from neurons during the course of EAE. We also found that the NK recruitment defect in CX3CR1GFP/GFP mice was not global, by showing efficient recruitment of NK cells to the livers of mice infected with mCMV. Taken together, these results revealed a unique pathway by which CX3CL1 mediates the selective recruitment to the CNS of NK cells, through posttranslational generation of a soluble chemokine. This mechanism stands in contrast to the cytokine-driven transcriptional up-regulation of chemokine expression that typifies most inflammatory reactions (2) . Transient focal chemokine expression, a pattern most commonly associated with inflammation, also directs cell migration through the developing spinal cord (34) and cerebellum (35) .

Mice lacking either CX3CL1 or CX3CR1 (21 ,29 ,36) exhibited normal responses to a number of experimental inflammatory and infectious challenges. Additional studies showed that CX3CR1–/– recipients of MHC-mismatched heterotopic cardiac allografts exhibited identical graft survival as their WT littermates, but greatly reduced infiltration of NK1.1+ cells into the graft (29) . Although CX3CR1 is expressed on multiple leukocyte populations, CX3CL1 elicited a highly selective chemoattractant response in vitro from murine and human NK cells (11 ,29) .

In the heart and peripheral vasculature, transmembrane CX3CL1 is expressed by vascular endothelium and acts as an adhesion ligand for receptor-bearing circulating leukocytes. CX3CL1 is expressed on neurons but not blood vessels in the CNS (16 ,17 ,37) . As reported here and recently shown by others, total CX3CL1 levels do not increase during acute inflammation (38) , including EAE (37) . Damage to neurons causes release of CX3CL1, through the action of the ubiquitous metalloprotease ADAM17/TNF-{alpha} converting enzyme (39 ,40) . Stereotactic injections of soluble CX3CL1 activated the resident microglia, without causing hematogenous leukocyte infiltration (41) . Therefore, two steps are needed for CX3CL1 to chemoattract circulating cells to the CNS. First, CX3CL1 must be proteolytically released from neurons. Second, soluble CX3CL1 needs to be presented to circulating cells in the context of a disrupted blood-brain barrier (BBB).

Given the high constitutive expression of CX3CL1, the CNS may be well equipped for the efficient recruitment of NK cells in response to CNS tissue injury. CX3CL1 exerts selective and efficient chemoattraction of both murine (29) and human (11) NK cells, further supporting the concept that CX3CL1/CX3CR1 interactions account for selective recruitment of NK cells into the brain. It was impressive that CX3CR1-mutant mice exhibited such a selective impairment of NK cell recruitment, given that all CNS leukocyte populations expressed CX3CR1 (Fig. 2s ). These findings support a model of chemokine action in which wide arrays of chemokine receptors on individual cells support flexible, specific but nonredundant recruitment pathways to varied organs.

Monophasic MOG-induced EAE in C57BL/6 mice most nearly resembles the human demyelinating disorder acute disseminated encephalomyelitis (ADEM). CX3CR1GFP/GFP mice developed a form of EAE that closely mimicked the clinical and pathological presentation of hemorrhagic leukoencephalitis, an exceptionally severe variant of ADEM. CX3CL1 has been proposed to augment vascular pathology (42) in the kidney. However, the occurrence of hemorrhagic inflammatory EAE lesions in CX3CR1GFP/GFP mice indicates that CX3CL1 is protective rather than pathogenic in the setting of vascular pathology in the CNS.

In the mCMV hepatitis model reported here, deficiency of CX3CR1 had no effect. This observation can be rationalized, based on minimal-to-absent expression of CX3CL1 in the target organ and by the prior demonstration that accumulation of NK cells in the livers of mice with mCMV is regulated by CCL3 (23) . It has been reported that EAE in C57BL/6 x 129/SV CX3CR1-deficient mice was more severe than in WT animals, with earlier onset, but the results did not reach statistical significance (29) . We suspect that the more distinctive phenotype observed in the current experiments resulted from comparing extensively back-crossed CX3CR1-/- mice with their littermate controls that shared relevant disease modifier genes. Analysis of the facial nerve axotomy and laser lesion models in CX3CR1GFP/GFP mice showed no alteration in the kinetics or extent of microglial activation, and no leukocyte infiltration, despite a dramatic local increase in soluble CX3CL1 (14 ,21) . Taken with prior work, our results suggest that breach of the BBB is critical for released CX3CL1 chemokine domain to exert its chemoattractant activity toward hematogenous target cells. It is unlikely that impaired regulation of microglial activation in the absence of CX3CR1 plays the dominant role in determining the severity of EAE in CX3CR1GFP/GFP mice. This conclusion comes from our finding that NK cell depletion induced very severe EAE, even in CX3CR1+/GFP mice with CNS microglia bearing functional CX3CR1. These results suggest that loss of the regulatory influence of NK cells accounts for the severe EAE phenotype in CX3CR1GFP/GFP mice.

Emerging evidence indicates that NK cells can regulate inflammation and intervene in the loss of self-tolerance at multiple steps. Some studies suggest that NK cells may prevent or curb autoimmune responses by killing of DCs, regulation of cell cycle components related to T cell proliferation, or production of regulatory cytokines. Other authors assign a permissive role to NK cells for autoimmunity, perhaps because they promptly release proinflammatory cytokines, activate antigen-presenting cells (APCs), and favor differentiation of Th1 cells (43 44 45 46 47 48 49 50 51) . Our findings from studying CX3CR1GFP/GFP mice caution against assigning a uniform role for NK cells in all types of autoimmune phenomena or during different stages of the same disease. Mechanisms that account for the apparently conflicting roles of NK cells in regulating autoimmunity in the periphery remain to be identified. Further, it seems likely that NK cells regulate inflammatory responses differently at varying anatomical sites.

There are several avenues for exploring the clinical relevance of the current report. Polymorphisms of CX3CR1 have been reported, and have been proposed to alter receptor efficiency (52) , with potential effects on human clinical disorders. Evaluation of CX3CR1 polymorphisms may provide insight into pathogenesis of inflammatory diseases. Further, daclizumab (humanized anti-CD25 antibodies), a promising new treatment for MS and uveitis, appears to function in part by inducing a population of regulatory NK cells (53 ,54) and examination of chemokine receptors and trafficking patterns of these cells would be of interest. Finally, it was recently reported that the frequency of circulating CX3CR1-positive NK cells correlates with disease activity in MS patients (55) , suggesting that the CNS accumulation of inhibitory NK cells by a CX3CR1-dependent mechanism may be important for limiting immune-mediated inflammation in the CNS. Therefore, understanding the trafficking of regulatory NK cells carries the potential of modulating their accumulation to enhance the treatment of inflammatory CNS disorders.


   ACKNOWLEDGMENTS
 
We thank Dr. J. Gosling (ChemoCentryx, San Carlos, CA, USA) for providing recombinant mouse CX3CL1 and CXCL12, Dr. L. Van Kaer, Vanderbilt University School of Medicine for providing the CD1d-deficient mice. Dr. M. Kronenberg, La Jolla Institute for Immunology and Allergy, for providing CD1d-tetramers; Drs. J. J. Campbell (Harvard Medical School) and J. J. Lafaille (Skirball Institute of Biomolecular Medicine, NY University Medical Center) for stimulating discussion, and Drs. P. Kivisäkk, C. Trebst, Y. Han, R. Liu, Y. Jee for technical support.


   FOOTNOTES
 
1 These authors contributed equally to this research.

Received for publication December 1, 2005. Accepted for publication December 23, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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