The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fife, B. T.
Right arrow Articles by Karpus, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fife, B. T.
Right arrow Articles by Karpus, W. J.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 2001, 166: 7617-7624.
Copyright © 2001 by The American Association of Immunologists

CXCL10 (IFN-{gamma}-Inducible Protein-10) Control of Encephalitogenic CD4+ T Cell Accumulation in the Central Nervous System During Experimental Autoimmune Encephalomyelitis1

Brian T. Fife*, Kevin J. Kennedy*, Mary C. Paniagua*, Nicholas W. Lukacs{dagger}, Steven L. Kunkel{dagger}, Andrew D. Luster{ddagger} and William J. Karpus2,*

* Department of Pathology, Immunobiology Center, Robert H. Lurie Cancer Center, and Institute for Neuroscience, Northwestern University Medical School, Chicago, IL 60611; {dagger} Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and {ddagger} Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Charlestown, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE) is a CD4+ Th1-mediated demyelinating disease of the CNS that serves as a model for multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific and Ag-nonspecific T lymphocytes into the CNS. In the present report, we investigated the role of the CXC chemokine CXCL10 (IFN-{gamma}-inducible protein-10) in the pathogenesis of EAE. Production of CXCL10 in the CNS correlated with the development of clinical disease. Administration of anti-CXCL10 decreased clinical and histological disease incidence, severity, as well as infiltration of mononuclear cells into the CNS. Anti-CXCL10 specifically decreased the accumulation of encephalitogenic PLP139–151 Ag-specific CD4+ T cells in the CNS compared with control-treated animals. Anti-CXCL10 administration did not affect the activation of encephalitogenic T cells as measured by Ag-specific proliferation and the ability to adoptively transfer EAE. These results demonstrate an important role for the CXC chemokine CXCL10 in the recruitment and accumulation of inflammatory mononuclear cells during the pathogenesis of EAE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE)3 is a CD4+ Th1 cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for the human demyelinating disease, multiple sclerosis (MS) (1). EAE can be induced in SJL mice by immunization with proteolipid protein (PLP) or the immunodominant, encephalitogenic peptide sequence 139–151 (PLP139–151) emulsified in CFA (2). Alternatively, EAE can be adoptively transferred to normal recipient mice by Ag-activated PLP139–151-specific T cells (3). Immunohistological analysis of mononuclear cell infiltration in the CNS has revealed that Ag-specific and -nonspecific CD4+ and CD8+ T cells as well as macrophages constitute the recruited cell population, with little or no polymorphonuclear cell infiltration (4). The mechanism by which these cells traffic to the CNS and accumulate before and during clinical disease is not well understood; however, several parameters have recently been identified. The entry of activated T cells into tissue compartments is a process governed by both integrin-mediated adhesions as well as chemokine-mediated migration. Very late Ag-4 (VLA-4) (5, 6), LFA-1 (7), and ICAM-1 (8) have all been demonstrated as important adhesion molecules regulating disease progression.

An essential step during the pathogenesis of tissue-specific inflammatory diseases is the chemokine-induced recruitment of leukocytes. Chemotactic cytokines (chemokines) are molecules that induce leukocyte accumulation in tissue sites of inflammation (9, 10) that can be divided into four highly conserved, but distinct families: the CXC, CC, C, and CX3C families, based on the position of the cysteines in the amino terminus portion of the molecule (11, 12). CC chemokine family members have been implicated as candidates in the immunopathology of EAE as T cell production of CCL3 (macrophage-inflammatory protein-1{alpha} (MIP-1{alpha})) and CCL1 (T cell activation protein-3 (TCA-3)) were shown to be associated with T cell clones that were able to induce the adoptive transfer of EAE (13). MIP-1{alpha}, CCL2 (monocyte chemotactic protein-1 (MCP-1)), and CXCL10 (IFN-{gamma}-inducible protein-10 (IP-10)) expression in the CNS has been associated with acute disease symptoms in both rat (14) and murine EAE models (15, 16, 17, 18). Functional significance of MIP-1{alpha} and MCP-1 in EAE pathogenesis was demonstrated using neutralizing Abs to inhibit disease onset with the administration of anti-MIP-1{alpha} (17) or ameliorate relapsing disease severity with the administration of anti-MCP-1 (18).

The important role chemokine receptors have in recruiting cells into the CNS during the induction of clinical EAE has recently been identified. CCR2-/- mice were shown to be protected from clinical EAE in an active disease model (19, 20). CCR2 was shown to be important for the recruitment of peripheral macrophages to the CNS in a series of adoptive transfer experiments using CCR2-/- or wild-type CD4+ T cells transferred to wild-type or CCR2-/- recipients (19). In the absence of CCR2, CD4+ T cells from wild-type animals were able to induce clinical disease (19). However, when wild-type CD4+ T cells were transferred to CCR2-/- recipients, clinical disease was not observed, due to a significant decrease of peripheral macrophages accumulating in the CNS (19). CCR1-/- mice have also been shown to have reduced clinical EAE in an active disease model (21), demonstrating partial disease protection. These findings demonstrate an important role for chemokine receptor expression and CNS mononuclear cell infiltration.

In the present study, we investigated the potential role IP-10 plays during Ag-specific T cell migration/accumulation in the pathogenesis of an organ-specific, T cell-mediated autoimmune disease. IP-10 is a member of a subset of chemokines including CXCL11 (IFN-inducible T cell {alpha}-chemoattractant (I-TAC)) and CXCL9 (monokine induced by IFN-{gamma} (Mig)), which are regulated by the expression of IFN-{gamma} (22, 23, 24). CXCR3, the receptor for these chemokines, is expressed on a subset of circulating T cells (25); however, expression of CXCR3 and IP-10 chemotaxis are increased dramatically after T cell activation (26), and CXCR3 expression has been shown to be associated with Th1 cell lines and clones (27, 28). CXCR3 expression has also been detected on infiltrating monocytes within demyelinating MS brain lesions where high levels of IP-10 are expressed (29). EAE is a CD4+ Th1-mediated demyelinating disease of the CNS, we therefore investigated the role IP-10 plays in the recruitment of CD4+ Th1-autoreactive cells into the CNS during the pathogenesis of autoimmune encephalomyelitis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female SJL mice (H-2s) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Congenic SJL.Thy1a mice were received from H. Tse (30) (Wayne State University, Detroit, MI) and subsequently bred and maintained at Northwestern University (Chicago, IL). Mice were 6–7 wk old at the initiation of the experiment and were maintained on standard laboratory chow and water ad libitum. Animals were housed under specific pathogen-free, barrier facility conditions. Animal care was provided in accordance with the Northwestern University and National Institutes of Health guidelines.

Antigens

PLP139–151 (HSLGKWLGHPDKF) was purchased from Peptides International (Louisville, KY). The amino acid composition was verified by mass spectrometry, and purity (>98%) was assessed by HPLC.

Priming of donor lymphocytes, cell culture, and transfer of EAE

Donor congenic SJL.Thy1a or normal SJL mice were primed by s.c. immunization with 50 µg of PLP139–151 emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco, Detroit, MI). Seven days later, draining lymph node cells were pooled and cultured in vitro for 72 h in complete DMEM (Life Technologies, Grand Island, NY) containing 5 x 10-5 M 2-ME (Life Technologies), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), 0.1 M nonessential amino acids (Life Technologies), and 10% FCS (HyClone, Logan, UT) at 6 x 106 cells/ml in the presence of 50 µg/ml PLP139–151. Cells were incubated at 37°C in a humidified atmosphere containing 7.5% CO2. The cells were harvested after 72 h culture, washed, and 5 x 106 viable T cell blasts were transferred i.v. to normal SJL recipients. Subsequent to cell transfer mice were evaluated for the development of EAE.

Clinical evaluation

Adoptive R-EAE was induced by the transfer of 5 x 106 in vitro-stimulated, PLP139–151-specific T cell blasts from PLP139–151 peptide-primed mice. Individual animals were observed daily and graded according to their clinical severity as follows: grade 0, no abnormality; grade 1, limp tail; grade 2, limp tail and hind limb weakness (waddling gait); grade 3, partial hind limb paralysis; grade 4, complete hind limb paralysis; grade 5, death.

Isolation of CNS-infiltrating mononuclear cells, lymph node cells, and splenocytes

Mice were anesthetized with methoxyflurane (Pitman-Moore, Mundelein, IL) and perfused through the left ventricle with ~60 ml of PBS. Spinal cords were extruded by flushing the vertebral canal with PBS and rinsed in PBS. Lymph nodes and spleens were removed from the same mice and placed in HBSS. Tissues were forced through 100-mesh stainless steel screens to yield a single-cell suspension. RBC in the spleen preparations were lysed by hypotonic shock in Tris-NH4Cl (pH 7.3), and the cells were washed and resuspended in HBSS. CNS mononuclear cells were isolated by centrifugation (500 x g) at 24°C on a 30–70% discontinuous Percoll (Pharmacia, Piscataway, NJ) gradient. Cells were collected from the interface, washed in HBSS, and resuspended in isotonic-buffered saline (IBS) (Baxter Diagnostics, McGaw Park, IL) containing 0.1% NaN3 and 0.2% BSA (Sigma, St. Louis, MO).

Antibodies

mAbs to murine Thy1a (CD90.1; HIS51), Thy1b (CD90.2; 53-2.1), CD4 (RM4-5), CD8a (Ly-2), CD19 (1D3), CD45 (Ly-5), and CD16/32 (2.4G2, anti-mouse FcR{gamma}II/III) were purchased from BD PharMingen (San Diego, CA). mAbs to murine F4/80 were purchased from Caltag Laboratories (Burlingame, CA). Isotype control Abs were purchased from BD PharMingen. Rabbit anti-human polyclonal IP-10 Ab was prepared by multiple-site immunization of New Zealand White rabbits with recombinant human IP-10 (PeproTech, Rocky Hill, NJ) in CFA (31, 32). The specificity of the IP-10 anti-serum recognized both murine and human forms of IP-10 and was confirmed by ELISA and Western blot analysis (31, 32). Polyclonal anti-IP-10 Abs were titered by direct ELISA (~106 titer), and specificity was verified by the failure to cross react with any of the following human or mouse CC or CXC family chemokines: growth-related oncogene (GRO)-{alpha}, GRO-{beta}, GRO-{gamma}, IL-8, epithelial cell-derived neutrophil-activating factor 78 amino acids, Mig, I-TAC, neutrophil-activating protein-2, granulocyte chemoattractant protein-2, mouse MIP-2, mouse KC, mouse Mig, RANTES, mouse C10, eotaxin, macrophage-derived chemokine, thymus- and activation-related chemokine, human and mouse MIP-1{alpha}, human and mouse MIP-1{beta}, and human and mouse MCP-1 (31, 32). The IgG portion of the serum was purified over a protein A column and used in a sandwich ELISA while whole serum (0.5 ml) was used for in vivo treatments to block IP-10 (31). Neutralizing hamster anti-murine IP-10 mAb was generated by immunizing Armenian hamsters with recombinant Escherichia coli-produced murine IP-10 in CFA as previously described (33). mAbs were tested for their specificity using available purified mouse chemokines, including Mig, I-TAC, MIP-1{alpha}, MIP-1{beta}, stromal cell-derived factor-1, KC, TCA-3, RANTES, eotaxin, MCP-1, MCP-3, and MCP-5 in a direct ELISA and immunoblot assay (33). The ability to inhibit IP-10-induced chemotaxis was demonstrated in vitro and in vivo using this mAb (33).

Flow cytometry

Cells (0.5–1 x 106) were incubated with anti-mouse FcR{gamma}II/III for 15 min at 4°C in to block Fc-mediated binding. Cells were washed in IBS followed by incubation with specific Abs CD90.1, CD90.2, CD4, CD8a, CD19, CD45, or F4/80 at a predetermined optimal concentration for 15 min at 4°C. As a control, parallel populations of cells were incubated in the presence of isotype-matched control Abs. Cells were washed and resuspended in 0.5 ml IBS. Data collection and analysis were performed on a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using CellQuest software with 5 x 104 events/analysis.

Cell sorting

Splenocytes and infiltrating mononuclear cells were isolated from the CNS as described above and were incubated with Fc Block (CD16/32 clone 2.4G2; BD PharMingen) at 1 x 106 cells/ml for 15 min at 4°C in IBS to block Fc-mediated binding. Cells were washed in IBS followed by incubation with directly conjugated CD90.1 FITC and CD4 PE mAbs for 15 min at 4°C in the dark at a predetermined optimal concentration. Cells were washed twice in IBS and resuspended in IBS at final concentration of 1 x 106 cells/ml for the CNS and 1 x 107 cells/ml for the spleen cells. CD4+CD90.1+ and CD4+CD90.1- cells were sorted from the CNS and spleen cell preparations using an EPICS Elite cell sorter (Beckman Coulter, Fullerton, CA). Cells were collected in HBSS supplemented with 2% FCS, pelleted, and lysed in TRIzol (Life Technologies) and stored at -70°C for RNA isolation. Following cell sorting, an aliquot of cells from each tube was analyzed for percent purity by flow cytometry. All sorted populations had >=98% purity.

RT-PCR

Total RNA was isolated from the sorted cells using TRIzol (Life Technologies) following the manufacturer’s suggested protocol. Total RNA was quantitated by measuring A260/A280 absorbance. Equal amounts of total RNA were used for the synthesis of cDNA using the first-strand cDNA synthesis kit from Clontech Laboratories (Palo Alto, CA) following the manufacturer’s suggested protocol. PCR was performed using a Perkin-Elmer 9600 thermocycler (Norwalk, CT). Chemokine receptor primers were designed using software provided by National Center for Biotechnology Information and synthesized by Life Technologies. PCR conditions for G3PDH and CXCR3 were as follows: 94°C for 3 min, followed by 40 cycles of 30 s at 94°C, 30 s at 62°C, and 1 min at 72°C, with a final extension at 72°C for 3 min. Primer sequences used in this study: CXCR3 sense, 5'-GAACGTCAAGTGCTAGATGCCTCG-3'; antisense, 5'-GTACACGCAGAGCAGTGCG-3'; and G3PDH sense, 5'-ACCACAGTCCATGCCATCAC-3'; antisense, 5'-TCCACCACCCTGTTGCTGTA-3'.

Histology

Histological evaluation was performed on representative mice from each experimental group. Mice were anesthetized with methoxyflurane (Pitman-Moore) and perfused through the left ventricle with ~60 ml of PBS. Spinal cords were extruded by flushing the vertebral canal with PBS. The most caudal 1 cm of the lumbar spinal cord was fixed in a phosphate-buffered 10% formalin solution and embedded in paraffin. Twelve 10-µm sections from each animal were stained with hematoxylin and eosin and examined for the presence of mononuclear cell infiltration. Histological scores were determined using the following scale: 0, no mononuclear cell infiltration; 1, 1–5 perivascular lesions per section with parenchymal infiltration; 2, 5–10 perivascular lesions per section with parenchymal infiltration; and 3, >10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score ± SD was calculated for each group. Representative photomicrographs were taken using a Nikon microscope (Fryer Company, Huntley, IL) equipped with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Images were created using Metamorph Meta Imaging Series 4.5 software (Universal Imaging Corporation, West Chester, PA) and printed with a Fujix pictrography 3000 (Fuji Photo Film USA, Elmsford, NY).

Cytokine and chemokine ELISA

Assessment of cytokine production was determined from lymph node culture supernatants harvested following 48 h stimulation with 0, 0.5, 5, or 50 µM PLP139–151 peptide. The supernatants were tested for the presence of IL-2, IL-4, and IFN-{gamma} by commercial ELISA kits (Endogen, Cambridge, MA). Assessment of IP-10 was quantitated from tissue samples using previously described noncommercial ELISA (31). Briefly, spinal cord samples were homogenized in 1 ml PBS and clarified by centrifugation (400 x g) for 10 min. Flat-bottom microtiter plates (Nunc, Naperville, IL) were coated with capture Ab diluted to 3.2 µg/ml in borate-buffered saline coating buffer and blocked with 2% BSA (Sigma) in PBS for 1 h at room temperature, and samples were subsequently added in triplicate and incubated overnight at room temperature. Biotinylated goat anti-rabbit detection Ab was added, and the plates were incubated for an additional 1 h at room temperature. The plate was developed using streptavidin-peroxidase (Zymed, South San Francisco, CA) and o-phenylenediamine substrate (Sigma), and absorbance was read at 490 nm using a Vmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA). Standard curves for the individual cytokines and chemokines were generated using a series of dilutions of purified recombinant protein (R&D Systems, Minneapolis, MN). Chemokine levels in spinal cords were quantitated by comparison to the standard curves and expressed as nanograms per milliliter. The individual chemokine Abs used in the ELISA are specific and do not cross-react with any other chemokine as described above. The detection limit of the cytokine ELISA kits was as follows: IL-2, 15.6 pg/ml, IL-4, 31.3 pg/ml, and IFN-{gamma}, 48.8 pg/ml. The detection limit for the chemokine IP-10 was 312.5 pg/ml.

In vitro T cell proliferation assays

Spleen and lymph node cells were obtained from mice at the peak of acute clinical disease for the control-treated group. Tissues were forced through 100 mesh stainless steel screens to yield a single-cell suspension. RBC in the spleen preparations were lysed by hypotonic shock in Tris-NH4Cl (pH 7.3), and the cells were washed and resuspended in HBSS. Cells were cultured in 96-well microtiter plates (Corning-Costar, Acton, MA) at 5 x 106 viable cells/ml in DMEM (Life Technologies) containing 5 x 10-5 M 2-ME (Life Technologies), 2 mM L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), 0.1 M nonessential amino acids (Life Technologies), and 5% FCS (HyClone, Logan, UT) in the presence of 0, 0.5, 5, and 50 µM PLP139–151. Cells were incubated at 37°C in a humidified atmosphere containing 7.5% CO2. The cells were pulsed with 1 µCi of [3H]TdR (ICN Radiochemicals, Irvine, CA) after 72 h, harvested after 96 h, and [3H]TdR uptake was detected using a Packard Topcount microplate scintillation counter (Packard Instruments, Meriden, CT). Results are presented as the mean ± SEM of triplicate wells.

Statistical analysis

Comparison of disease incidence were analyzed by {chi}2 test, using Fisher’s exact probability test. Statistical significance of cytokine levels, thymidine incorporation, disease onset, and disease severity was analyzed using Student’s t test for comparisons of two means. Values of p <= 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CNS IP-10 production and clinical EAE

We wanted to determine whether there was a relationship between CNS IP-10 protein production and development of clinical EAE. To analyze this, we examined organ-specific chemokine protein production by ELISA. EAE was induced in SJL mice by adoptive transfer of PLP139–151-specific, in vitro-activated T cells. Mice typically begin to show clinical signs of disease 5–14 days following transfer of activated T cells. A kinetic analysis of IP-10 induction was performed on groups of mice 0, 2, 6, 8, 9, and 11 days following transfer of encephalitogenic T cells. The spinal cords were snap frozen in liquid nitrogen, homogenized in PBS, and analyzed for the presence of IP-10 by ELISA. Fig. 1Go demonstrates that increasing IP-10 production in the CNS precedes the development of clinical disease symptoms as well as maintains high levels during peak clinical disease.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1. CNS IP-10 production correlates with development of clinical EAE. Disease was induced by adoptive transfer of 5 x 106 PLP139–151-specific T cell blasts on day 0. All mice were graded for clinical disease as described in Materials and Methods. At each time point, three mice from the group were sacrificed, spinal cords were harvested and homogenized, and clarified tissue homogenates from individual mice were analyzed for IP-10 by ELISA. The clinical disease severity data represent the mean score for the group at that particular time point (•). ELISA data represent the mean of three individual mice ± SD in nanograms per milliliter ({square}). The data shown are representative of two independent experiments.

 
CNS IP-10 and CXCR3 expression

We next wanted to determine whether there was a correlation between CNS IP-10 mRNA expression and development of clinical EAE. EAE was induced in SJL mice by adoptive transfer of PLP139–151-specific, in vitro-activated T cells. A kinetic analysis of IP-10 mRNA expression was performed on groups of mice 1, 2, 3, 4, and 9 days following transfer of encephalitogenic T cells. Total RNA from the spinal cords was isolated and analyzed for the presence of IP-10 by RT-PCR. Fig. 2GoA demonstrates IP-10 mRNA expression in the CNS precedes the development of clinical disease symptoms and increases at disease onset, day 9. From the observation that IP-10 is produced in the CNS following EAE induction, we would predict that T cells accumulating in the CNS following EAE induction would express CXCR3, the receptor for IP-10. To assess this possibility, EAE was induced in normal SJL (Thy1b) mice by the adoptive transfer of congenic SJL.Thy1a PLP139–151-specific T cell blasts. By inducing disease with congenic Thy1a cells, we were able to track the encephalitogenic CD4+ T cells in normal SJL (Thy1b) recipients using specific mAbs (CD90.1 and 90.2, respectively). At the peak of acute EAE, the CNS-derived mononuclear cells and splenocytes were labeled with Abs to CD4 and CD90.1. Double-positive CD4+CD90.1+ T cells (encephalitogenic donor cells) and single-positive CD4+CD90.1- T cells (host derived) were sorted from these tissues as described in Materials and Methods. Sorted cells were analyzed by RT-PCR for CXCR3 mRNA expression. CXCR3 was expressed by both donor- and host-derived CD4+ T cells isolated from the CNS during peak acute clinical disease, day 12 post adoptive transfer (Fig. 2GoB). In contrast, CXCR3 expression was not found on either CD4+CD90.1+ (encephalitogenic donor cells) or CD4+CD90.1- (host-derived) T cells from sorted splenic populations (Fig. 2GoB). Taken together, these data demonstrate the presence of both IP-10 and its receptor, CXCR3, in the CNS during the development of acute EAE.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 2. IP-10 mRNA production in the CNS and CXCR3 expression on CNS-infiltrating CD4+ T cells. A, Disease was induced by adoptive transfer of 5 x 106 PLP139–151-specific T cell blasts on day 0. Analysis of organ-specific chemokine mRNA expression was performed on groups of mice 1, 2, 3, 4, and 9 days following transfer of encephalitogenic T cells. Total RNA from the spinal cords of three mice was isolated at each time point and analyzed for the presence of IP-10 by RT-PCR. IP-10 mRNA expression from one representative mouse per time point is shown. IP-10 mRNA expression in the CNS precedes the development of clinical disease symptoms, days 1–4, and increases at disease onset, day 9. B, CXCR3 mRNA expression by RT-PCR was examined from PLP139–151-specific donor CD4+CD90.1+ and host CD4+CD90.1- cells isolated from the CNS or spleen during the peak of acute disease. EAE was induced by the adoptive transfer of SJL.Thy1a PLP139–151-specific T cell blasts to naive SJL (Thy1b) mice. Animals were monitored daily for disease symptoms to develop. At the peak of acute clinical disease, CD4+CD90.1+ and CD4+CD90.1- cells were sorted by flow cytometry from the CNS infiltrate or spleen. Encephalitogenic CD4+CD90.1+ (Thy1a+) and CD4+CD90.1- (Thy1b+) T cells from the CNS infiltrate expressed CXCR3 mRNA compared with CD4+CD90.1+ and CD4+CD90.1- cells sorted from the spleen. G3PDH was used in this figure as a loading control and also as a control for mRNA integrity. Data are representative of two independent experiments.

 
Decreased clinical disease severity during acute EAE by anti-IP-10 administration

To determine the biological significance of IP-10 expression during acute EAE, we tested the ability of Ab against IP-10 to affect adoptive EAE in recipient mice. Administration of 0.5 ml of anti-IP-10 i.p. on days 0 and 2 relative to adoptive transfer of encephalitogenic T cells significantly decreased clinical disease compared with normal rabbit serum (NRS) control-treated animals (Fig. 3Go). All NRS control-treated recipients developed severe EAE with a mean clinical score of 3.9, while only 2 of 6 (33%) of the recipients in the anti-IP-10-treated group developed disease with delayed onset, p < 0.001, and a significantly decreased mean clinical score of 0.8, p < 0.0001. The results presented here demonstrate that neutralization of IP-10 during disease initiation, but before the development of clinical symptoms, significantly reduces the severity of acute EAE. In a separate experiment, EAE was induced by the adoptive transfer of PLP139–151-specific T cells, and the ability of an independent hamster anti-murine IP-10 mAb to ameliorate clinical disease was tested. The hamster Ig-treated control group developed severe EAE (mean clinical score = 1.7; incidence = 14/14), while the anti-mIP-10 mAb-treated group developed significantly less (p < 0.003) severe disease (mean clinical score = 1.0; incidence = 11/14). These data provide an important confirmation of the biological role of IP-10 in the pathogenesis of EAE using a mAb specific for murine IP-10. Taken together, these results demonstrated that in vivo neutralization of IP-10 before the onset of clinical EAE significantly reduced the clinical disease severity.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 3. Anti-IP-10 treatment decreases acute EAE severity. Mice were monitored for the development of clinical disease following EAE induction by the adoptive transfer of PLP139–151-specific T cell blasts to naive SJL recipient mice. Mice were treated i.p. with 0.5 ml of either control NRS or anti-IP-10 antisera on days 0 and 2 relative to adoptive transfer of encephalitogenic T cells. The data are expressed as the mean clinical disease score for all mice in each group as a function of days post adoptive transfer. The mean clinical disease severity was significantly decreased in anti-IP-10-treated animals relative to rabbit control-treated animals (p < 0.001 by Student’s t test) over the entire course of the experiment. Disease incidence is shown in parentheses and was determined to be significantly decreased in anti-IP-10-treated mice compared with control mice (p < 0.001 by {chi}2 test). Data are representative of three independent experiments.

 
Decreased histological EAE by anti-IP-10 administration

To determine the requirement for IP-10 recruitment of mononuclear cell infiltration into the CNS, mice were examined for spinal cord pathology. Longitudinal lumbar spinal cord sections from anti-IP-10-treated mice and NRS control mice were prepared as described in Materials and Methods and evaluated for the extent of mononuclear cell infiltration of the meninges, perivascular areas, and parenchyma by hematoxylin and eosin staining. It should be emphasized that at the time of histological examination, the anti-IP-10-treated mice showed no signs of clinical EAE, while the NRS control mice showed severe clinical EAE (Fig. 3Go). Spinal cord sections from NRS control mice exhibited extensive meningeal, perivascular, and parenchymal infiltrates (Fig. 4GoA). In contrast, spinal cord sections from anti-IP-10-treated mice demonstrated no meningeal, parenchymal, or perivascular cell infiltration (Fig. 4GoB). These histological results were quantitated and as shown in Table IGo the anti-IP-10-treated mice lacked CNS mononuclear cell infiltration and had a significantly decreased number of lesions per section compared with the NRS control mice. These results suggest that IP-10 is required for mononuclear cell accumulation in the CNS.



View larger version (123K):
[in this window]
[in a new window]
  		FIGURE 4. Prevention of CNS mononuclear cell accumulation in mice treated with anti-IP-10. Spinal cords from two mice in each group were assayed for mononuclear cell infiltration in the meninges and white and gray matter by hematoxylin and eosin staining when control-treated mice showed peak clinical EAE. The photomicrographs are representative sections from the NRS control-treated (A) and anti-IP-10-treated groups (B). Perivascular lesions are indicated with arrows (A). A and B, Magnification, x100. Data are representative of two independent experiments.

 

View this table:
[in this window]
[in a new window]
  		Table I. Decreased CNS perivascular lesions in anti-IP-10-treated mice

 
Anti-IP-10 treatment decreases the accumulation of mononuclear cells in the CNS

Administration of neutralizing IP-10 antisera results in a significant reduction of clinical and histological EAE. We next examined whether there was a change in mononuclear cell numbers in the CNS and periphery as a result of anti-IP-10 treatment. When control-treated mice showed peak clinical disease, CNS, lymph node, and spleen mononuclear cells from control- and anti-IP-10-treated mice were enumerated. The results shown in Fig. 5Go demonstrate a decrease in total mononuclear cells in the CNS from anti-IP-10-treated mice compared with control-treated mice.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. Anti-IP-10 treatment decreased accumulation of mononuclear cells in the CNS. Disease was induced by the adoptive transfer of PLP139–151-specific T cell blasts. When control-treated mice showed peak clinical EAE, CNS mononuclear cells; pooled axillary, brachial, and inguinal lymph node cells (LNC); and splenocytes (SPL) were harvested and enumerated. Data are expressed as total cells per mouse for each organ (CNS, lymph node cells, or spleen). The data represent the average number of cells ± SD from three independent experiments, with six mice per experiment. Anti-IP-10 treatment resulted in a significant decrease in total mononuclear cells in the CNS compared with control-treated mice, p < 0.001. Anti-IP-10 administration resulted in a significant increase in total cells within the peripheral lymph nodes and spleen, p < 0.001.

 
Anti-IP-10 treatment decreases encephalitogenic T cell accumulation in the CNS during acute EAE

Flow cytometric analysis of the CNS was performed to determine whether anti-IP-10 treatment changed the cellular composition of the mononuclear cell infiltrate. EAE was induced in normal SJL (Thy1b) mice by the adoptive transfer of congenic SJL.Thy1a PLP139–151-specific CD4+ T cell blasts. The immunophenotyping results are shown in Table IIGo and reveal that control mouse spinal cord infiltrate was composed of T cells (CD4+ and CD8+) and F4/80+ cells as expected. Not only was there a decrease in the total number of CNS mononuclear cells in the anti-IP-10-treated cords (~20-fold, Table IIGo), but there was also a decrease in the percentage of T cells (<1%, Table IIGo). CD4 and CD90.1 staining allowed us to distinguish between the disease-inducing CD4+CD90.1+ (Thy1a+) cells and the host-derived CD4+CD90.1- (Thy1b+) cells. Anti-IP-10-treated mice had a significant reduction of both encephalitogenic Thy1a+ and Thy1b+ host-derived CD4+ cells accumulating in the CNS (Table IIGo). Differential CD45 staining intensity coupled with F4/80 staining has been used to distinguish between infiltrating macrophages and resident microglia. Infiltrating macrophages are CD45highF4/80+, while resident microglial cells are CD45lowF4/80+ (34). Our results indicate that most of the total CNS mononuclear cells from the spinal cords of anti-IP-10-treated mice were resident microglial cells, not infiltrating macrophages. In contrast, ~20% of the total CNS mononuclear cells from the control mice were CD45bright, corresponding to infiltrating macrophages. The relative increase in resident microglial cells for the anti-IP-10-treated group does not indicate an increase in microglial cell infiltration, rather it is indicative of an increase in relative percentage due to the lack of other infiltrating cell types.


View this table:
[in this window]
[in a new window]
  		Table II. Decreased CNS recruitment/accumulation of T cells and infiltrating monocytes in anti-IP-10-treated mice1

 
Administration of anti-IP-10 does not affect peripheral Ag recall immune responses

We asked whether disease protection in anti-IP-10-treated mice could result from a defect in T cell activation and differentiation. When control-treated mice showed peak clinical EAE, draining lymph node cells and splenocytes from both control- and anti-IP-10-treated mice were stimulated in vitro with PLP139–151 to determine their ability to produce cytokines. The results in Fig. 6Go indicate that there was no defect in anti-IP-10-treated cells isolated from peripheral lymph nodes (A and C) or spleen (B and D) to produce IFN-{gamma} (A and B), IL-2 (data not shown), or proliferate in response to specific Ag compared with control-treated cells (C and D). Collectively, these results indicate that IP-10 and its specific receptor, CXCR3, are expressed in the CNS during development of acute EAE and that inhibition of IP-10 with in vivo Ab therapy ameliorates clinical disease by preventing mononuclear cell accumulation in the CNS.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 6. Administration of anti-IP-10 does not affect peripheral Ag recall immune responses. Lymphocytes and splenocytes from anti-IP-10-treated mice produce proinflammatory cytokines upon Ag restimulation. NRS control-treated and anti-IP-10-treated mice were monitored for the development of clinical EAE. At the time the NRS control mice showed peak acute clinical disease, peripheral lymph node (A) and splenocytes (B) pooled from six representative mice for each group were cultured with 0–50 µM PLP139–151 in vitro. Cultured supernatant was harvested at 48 h and measured for the production of IL-2, IL-4, and IFN-{gamma} by specific ELISA. Pooled axillary, brachial, and inguinal lymph node cells (C) and splenic T cells (D) from anti-IP-10-treated mice showed PLP139–151 dose-dependent proliferation responses compared with controls. Data are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EAE is a CD4+ Th1 cell-mediated autoimmune disease characterized by CNS-infiltrating lymphocytes and macrophage/monocytes resulting in a chronic, relapsing remitting paralytic disease in SJL mice. Regulation of disease pathogenesis includes migration of activated T cells from peripheral lymphoid tissues into the CNS. Following T cell activation and Th1 differentiation, activated cells leave the lymphoid tissues and enter the blood. Once in the blood, activated cells interact via adhesion molecules with vascular endothelial cells lining the blood vessels. The initial interactions between activated T cells and cerebral vascular endothelial cells are not well understood; however, activated T cells express VLA-4 (CD49d/CD29) and LFA-1 (CD11a/CD18), which bind to VCAM-1 (CD106) and ICAM-1 (CD54) on activated endothelium (5, 6). The interactions of these integrins and adhesion molecules is important for the development of EAE, because disease pathogenesis can be inhibited by using blocking Abs to the integrins VLA-4 (5) and LFA-1 (7), as well as the adhesion molecule ICAM-1 (8).

Chemokine-induced recruitment of leukocytes into the tissue is thought to be an essential step in the pathogenesis of tissue-specific autoimmune inflammatory disease progression (35). We (17, 18) and others (14, 16, 36, 37) have demonstrated a relationship between production of chemokines in the CNS and development of acute and relapsing EAE. Here we report the biological significance of IP-10 expression in the CNS and the regulation of subsequent acute EAE. CD4+ encephalitogenic T cells can be detected in the CNS as early as 3–24 h posttransfer (38, 39) (our unpublished observation) and can stimulate resident cells, through TNF-{alpha} and IFN-{gamma}, to express CCL5 (RANTES), MIP-1{alpha}, MCP-1, and IP-10 (36, 37), resulting in additional mononuclear cell recruitment. IP-10 expression in the CNS of mice induced to develop EAE occurs well before the onset of clinical symptoms (Fig. 2GoA) and has been localized to astrocytes that form the perivascular-parenchymal boundary (36).

The expression of CNS IP-10 mRNA (Fig. 2GoA) and protein (Fig. 1Go), along with the expression of CXCR3 mRNA by encephalitogenic Th1 cells isolated from the target organ during clinical disease (Fig. 2GoB), led us to hypothesize that IP-10 was responsible for mononuclear cell accumulation resulting in subsequent disease development. By administering neutralizing IP-10 antisera, we were able to decrease clinical disease, lower disease incidence, and delay clinical onset. Anti-IP-10 treatment decreased mononuclear cell accumulation in the CNS (Fig. 5Go). We believe that because mononuclear cells did not receive IP-10-mediated signals to accumulate in the CNS there was an apparent increase in the total number of peripheral mononuclear cells (Fig. 5Go). This peripheral increase is most likely a result of the neutralization of a chemotactic gradient and the inability of cells to accumulate in the CNS rather than expansion of peripheral lymphocytes, as there is no evidence of an increase in peripheral lymphocyte proliferation or production of proinflammatory cytokines (Fig. 6Go). The mechanism of the disease and pathology inhibition appears to be reduction in the accumulation of PLP139–151-specific autoreactive Th1 cells in the CNS (Table IIGo). The prevailing model of CNS T cell entry suggests that activated T cells gain entry to the CNS, but only the Ag-specific T cells are retained (38). It is possible that initial CNS Ag-specific T cell migration does not require chemokine expression by the target organ. However, we hypothesize that IP-10 expression is required for Ag-specific T cell and other mononuclear cell accumulation. In the absence of disease-inducing T cell accumulation, the inflammatory signals that induce the accumulation of other cell types such as host-derived T cells and monocytes would not be present. This would potentially account for the failure to detect other CNS-infiltrating mononuclear cells in the anti-IP-10-treated mice (Table IIGo).

IP-10 chemokine expression has also been reported in cerebral spinal fluid of MS patients during MS attacks (29). In addition to chemokine expression, CXCR3, the receptor for IP-10, was found to be expressed on infiltrating lymphocytic cells in demyelinating MS brain lesions (29). Given the correlation of chemokine/chemokine receptor expression in active MS lesions and EAE, the production of IP-10 in the target organ early during disease initiation may be one of the important events regulating early trafficking and/or accumulation of autoreactive cells within the CNS. It was recently shown that infusions of IP-10 antisense oligonucleotides reduced acute EAE severity in Lewis rats, further demonstrating an important role for its production (40). However, IP-10 was not expressed in the CNS during clinical EAE in IFN-{gamma}-deficient BALB/c mice when disease was induced with whole myelin-basic protein (41). This result suggests IP-10 expression is not required for EAE induction in BALB/c mice; however, the requirement for IP-10 in various mouse strains may differ. A possible explanation may include the type of disease manifestation in individual mouse strains. Disease in BALB/c mice develops as widespread demyelination and disseminated leukocytic infiltration of the spinal cord, which differs from the focal perivascular infiltrates seen in SJL/J mice (42). The CNS infiltrate in BALB/c mice is primarily composed of Gr-1+ neutrophils, whereas SJL/J infiltrate is comprised of mononuclear cells with little or no polymorphonuclear infiltrate (4, 42). Therefore, production of IP-10 in the CNS and its role during disease initiation may differ between mouse strains. Alternatively, IP-10 may function as a chemokine that participates in CNS T cell accumulation, but does not necessarily function alone. The production of IP-10 in SJL EAE may be acting coordinately with other chemokines, especially MIP-1{alpha} given its known role in EAE pathogenesis (17).

Several other inflammatory models have demonstrated the important role chemokines have for directed cell migration. Administration of RANTES antisera to mouse hepatitis virus (MHV)-infected C57BL/6 mice resulted in a significant reduction in macrophage infiltration and demyelination during MHV infection (43). Polyclonal Ab directed against RANTES ameliorates disease in the Lewis rat adjuvant-induced arthritis model (44). Neutralization of CXCL1 (MIP-2) and MIP-1{alpha} was shown to attenuate neutrophil recruitment in the CNS during experimental bacterial meningitis (45). IP-10 was shown to be important for host antiviral responses following infection of the CNS with MHV, where treatment of mice with anti-IP-10 antisera led to increased mortality, delayed viral clearance, and decreased CD4+ and CD8+ T lymphocyte infiltration to the CNS (46).

Our present results suggest that temporal IP-10 expression governs the accumulation of autoreactive inflammatory T cells into the target organ, resulting in tissue damage and clinical progression of EAE. These findings open the possibility of using CXCR3 antagonists for organ-specific autoimmune disease therapy. We are currently assessing the function of other CXCR3 ligands, such as Mig and I-TAC, for their role in EAE development to understand the universality of a CXCR3 antagonist approach to the treatment of autoimmune disease.


   Footnotes
 
1 This work was supported by National Institutes of Health Training Grant AI07476 (to B.T.F.), National Institutes of Health Grant NS34510 (to W.J.K.), and National Institutes of Health Grant CA69212 (to A.D.L.). Back

2 Address correspondence and reprint requests to Dr. William J. Karpus, Department of Pathology, Northwestern University Medical School, 303 East Chicago Avenue, W127, Chicago, IL 60611. E-mail address: w-karpus{at}northwestern.edu Back

3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; PLP, proteolipid protein; MS, multiple sclerosis; MIP, macrophage-inflammatory protein; TCA-3, T cell activation gene-3; MCP, monocyte chemotactic protein; IP-10, IFN-{gamma}-inducible protein-10; I-TAC, IFN-inducible T cell {alpha}-chemoattractant; Mig, monokine induced by IFN-{gamma}; GRO, growth-related oncogene; VLA-4, very late Ag-4; IBS, isotonic-buffered saline; NRS, normal rabbit serum; MHV, mouse hepatitis virus. Back

Received for publication January 16, 2001. Accepted for publication April 11, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wekerle, H.. 1991. Immunopathogenesis of multiple sclerosis. Acta Neurologica 13:197.[Medline]
  2. Tuohy, V. K., R. A. Sobel, Z. Lu, R. A. Laursen, M. B. Lees. 1992. Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice. J. Neuroimmunol. 39:67.[Medline]
  3. Whitham, R. H., D. N. Bourdette, G. A. Hashim, R. M. Herndon, R. C. Ilg, A. A. Vandenbark, H. Offner. 1991. Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis. J. Immunol. 146:101.[Abstract]
  4. Hickey, W. F., N. K. Gonatas, H. Kimura, D. B. Wilson. 1983. Identification and quantitation of T lymphocyte subsets found in the spinal cord of the Lewis rat during acute experimental allergic encephalomyelitis. J. Immunol. 131:2805.[Abstract]
  5. Yednock, T. A., C. Cannon, L. C. Fritz, F. Sanchez-Madrid, L. Steinman, N. Karin. 1992. Prevention of experimental autoimmune encephalomyelitis by antibodies against {alpha}4{beta}1 integrin. Nature 356:63.[Medline]
  6. Baron, J. L., J. A. Madri, N. H. Ruddle, G. Hashim, Jr C. A. Janeway. 1993. Surface expression of {alpha}4 integrin by CD4 T cells is required for their entry into brain parenchyma. J. Exp. Med. 177:57.[Abstract/Free Full Text]
  7. Kobayashi, Y., K. Kawai, H. Honda, S. Tomida, N. Niimi, T. Tamatani, M. Miyasaka, Y. Yoshikai. 1995. Antibodies against leukocyte function-associated antigen-1 and against intercellular adhesion molecule-1 together suppress the progression of experimental allergic encephalomyelitis. Cell. Immunol. 164:295.[Medline]
  8. Morrissey, S. P., R. Deichmann, J. Syha, C. Simonis, U. Zettl, J. J. Archelos, S. Jung, H. Stodal, H. Lassmann, K. V. Toyka, A. Haase, H. P. Hartung. 1996. Partial inhibition of AT-EAE by an antibody to ICAM-1: clinico-histological and MRI studies. J. Neuroimmunol. 69:85.[Medline]
  9. Rossi, D., A. Zlotnik. 2000. The biology of chemokines and their receptors. Annu. Rev. Immunol. 18:217.[Medline]
  10. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 2000. The role of chemokine receptors in primary, effector, and memory immune responses. Annu. Rev. Immunol. 18:593.[Medline]
  11. Murphy, P. M., M. Baggiolini, I. F. Charo, C. A. Hebert, R. Horuk, K. Matsushima, L. H. Miller, J. J. Oppenheim, C. A. Power. 2000. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52:145.[Abstract/Free Full Text]
  12. Zlotnik, A., O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12:121.[Medline]
  13. Kuchroo, V. K., R. A. Sobel, J. C. Laning, C. A. Martin, E. Greenfield, M. E. Dorf, M. B. Lees. 1992. Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein: fine specificity and T cell receptor V{beta} usage. J. Immunol. 148:3776.[Abstract]
  14. Hulkower, K., C. F. Brosnan, D. A. Aquino, W. Cammer, S. Kulshrestha, M. P. Guida, D. A. Rapoport, J. W. Berman. 1993. Expression of CSF-1, c-fms, and MCP-1 in the central nervous system of rats with experimental allergic encephalomyelitis. J. Immunol. 150:2525.[Abstract]
  15. Glabinski, A. R., M. Tani, V. K. Tuohy, R. J. Tuthill, R. M. Ransohoff. 1995. Central nervous system chemokine mRNA accumulation follows initial leukocyte entry at the onset of acute murine experimental autoimmune encephalomyelitis. Brain Behav. Immun. 9:315.[Medline]
  16. Godiska, R., D. Chantry, G. N. Dietsch, P. W. Gray. 1995. Chemokine expression in murine experimental allergic encephalomyelitis. J. Neuroimmunol. 58:167.[Medline]
  17. Karpus, W. J., N. W. Lukacs, B. L. McRae, R. M. Strieter, S. L. Kunkel, S. D. Miller. 1995. An important role for the chemokine macrophage inflammatory protein-1{alpha} in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
  18. Kennedy, K. J., R. M. Strieter, S. L. Kunkel, N. W. Lukacs, W. J. Karpus. 1998. Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1{alpha} and monocyte chemotactic protein-1. J. Neuroimmunol. 92:98.[Medline]
  19. Fife, B. T., G. B. Huffnagle, W. A. Kuziel, W. J. Karpus. 2000. CC chemokine receptor 2 is critical for induction of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:899.[Abstract/Free Full Text]
  20. Izikson, L., R. S. Klein, I. F. Charo, H. L. Weiner, A. D. Luster. 2000. Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2. J. Exp. Med. 192:1075.[Abstract/Free Full Text]
  21. Rottman, J. B., A. J. Slavin, R. Silva, H. L. Weiner, C. G. Gerard, W. W. Hancock. 2000. Leukocyte recruitment during onset of experimental allergic encephalomyelitis is CCR1 dependent. Eur. J. Immunol. 30:2372.[Medline]
  22. Loetscher, M., P. Loetscher, N. Brass, E. Meese, B. Moser. 1998. Lymphocyte-specific chemokine receptor CXCR3: regulation, chemokine binding and gene localization. Eur. J. Immunol. 28:3696.[Medline]
  23. Cole, K. E., C. A. Strick, T. J. Paradis, K. T. Ogborne, M. Loetscher, R. P. Gladue, W. Lin, J. G. Boyd, B. Moser, D. E. Wood, B. G. Sahagan, K. Neote. 1998. Interferon-inducible T cell {alpha} chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187:2009.[Abstract/Free Full Text]
  24. Weng, Y., S. J. Siciliano, K. E. Waldburger, A. Sirotina-Meisher, M. J. Staruch, B. L. Daugherty, S. L. Gould, M. S. Springer, J. A. DeMartino. 1998. Binding and functional properties of recombinant and endogenous CXCR3 chemokine receptors. J. Biol. Chem. 273:18288.[Abstract/Free Full Text]
  25. Qin, S., J. B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, A. E. Koch, B. Moser, C. R. Mackay. 1998. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101:746.[Medline]
  26. Rabin, R. L., M. K. Park, F. Liao, R. Swofford, D. Stephany, J. M. Farber. 1999. Chemokine receptor responses on T cells are achieved through regulation of both receptor expression and signaling. J. Immunol. 162:3840.[Abstract/Free Full Text]
  27. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129.[Abstract/Free Full Text]
  28. Sallusto, F., D. Lenig, C. R. Mackay, A. Lanzavecchia. 1998. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187:875.[Abstract/Free Full Text]
  29. Sorensen, T. L., M. Tani, J. Jensen, V. Pierce, C. Lucchinetti, V. A. Folcik, S. Qin, J. Rottman, F. Sellebjerg, R. M. Strieter, J. L. Frederiksen, R. M. Ransohoff. 1999. Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J. Clin. Invest. 103:807.[Medline]
  30. Skundric, D. S., C. Kim, H. Y. Tse, C. S. Raine. 1993. Homing of T cells to the central nervous system throughout the course of relapsing experimental autoimmune encephalomyelitis in Thy-1 congenic mice. J. Neuroimmunol. 46:113.[Medline]
  31. Arenberg, D. A., S. L. Kunkel, P. J. Polverini, S. B. Morris, M. D. Burdick, M. C. Glass, D. T. Taub, M. D. Iannettoni, R. I. Whyte, R. M. Strieter. 1996. Interferon-{gamma}-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J. Exp. Med. 184:981.[Abstract/Free Full Text]
  32. Keane, M. P., D. A. Arenberg, J. P. Lynch, R. I. Whyte, M. D. Iannettoni, M. D. Burdick, C. A. Wilke, S. B. Morris, M. C. Glass, B. DiGiovine, S. L. Kunkel, R. M. Strieter. 1997. The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J. Immunol. 159:1437.[Abstract]
  33. Khan, I. A., J. A. MacLean, F. S. Lee, L. Casciotti, E. DeHaan, J. D. Schwartzman, A. D. Luster. 2000. IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 12:483.[Medline]
  34. Ford, A. L., A. L. Goodsall, W. F. Hickey, J. D. Sedgwick. 1995. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J. Immunol. 154:4309.[Abstract]
  35. Karpus, W. J., R. M. Ransohoff. 1998. Chemokine regulation of experimental autoimmune encephalomyelitis: temporal and spatial expression patterns govern disease pathogenesis. J. Immunol. 161:2667.[Abstract/Free Full Text]
  36. Ransohoff, R. M., T. A. Hamilton, M. Tani, M. H. Stoler, H. E. Shick, J. A. Major, M. L. Estes, D. M. Thomas, V. K. Tuohy. 1993. Astrocyte expression of mRNA encoding cytokines IP-10 and JE/MCP-1 in experimental autoimmune encephalomyelitis. FASEB J. 7:592.[Abstract]
  37. Glabinski, A. R., M. Tani, R. M. Strieter, V. K. Tuohy, R. M. Ransohoff. 1997. Synchronous synthesis of {alpha}- and {beta}-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am. J. Pathol. 150:617.[Abstract]
  38. Hickey, W. F., B. L. Hsu, H. Kimura. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res. 28:254.[Medline]
  39. Cross, A. H., B. Cannella, C. F. Brosnan, C. S. Raine. 1990. Homing to central nervous system vasculature by antigen-specific lymphocytes. I. Localization of 14C-labeled cells during acute, chronic, and relapsing experimental allergic encephalomyelitis. Lab. Invest. 63:162.[Medline]
  40. Wojcik, W. J., P. Swoveland, X. Zhang, P. Vanguri. 1996. Chronic intrathecal infusion of phosphorothioate or phosphodiester antisense oligonucleotides against cytokine responsive gene-2/IP-10 in experimental allergic encephalomyelitis of Lewis rat. J. Pharmacol. Exp. Ther. 278:404.[Abstract/Free Full Text]
  41. Glabinski, A. R., M. Krakowski, Y. Han, T. Owens, R. M. Ransohoff. 1999. Chemokine expression in GKO mice (lacking interferon-{gamma}) with experimental autoimmune encephalomyelitis. J. Neurovirol. 5:95.[Medline]
  42. Tran, E. H., E. N. Prince, T. Owens. 2000. IFN-{gamma} shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759.[Abstract/Free Full Text]
  43. Lane, T. E., M. T. Liu, B. P. Chen, V. C. Asensio, R. M. Samawi, A. D. Paoletti, I. L. Campbell, S. L. Kunkel, H. S. Fox, M. J. Buchmeier. 2000. A central role for CD4+ T cells and RANTES in virus-induced central nervous system inflammation and demyelination. J. Virol. 74:1415.[Abstract/Free Full Text]
  44. Barnes, D. A., J. Tse, M. Kaufhold, M. Owen, J. Hesselgesser, R. Strieter, R. Horuk, P. H. Daniel. 1998. Polyclonal antibody directed against human RANTES ameliorates disease in the Lewis rat adjuvant-induced arthritis model. J. Clin. Invest. 101:2910.[Medline]
  45. Diab, A., H. Abdalla, H. L. Li, F. D. Shi, J. Zhu, B. Hojberg, L. Lindquist, B. Wretlind, M. Bakhiet, H. Link. 1999. Neutralization of macrophage inflammatory protein 2 (MIP-2) and MIP-1{alpha} attenuates neutrophil recruitment in the central nervous system during experimental bacterial meningitis. Infect. Immun. 67:2590.[Abstract/Free Full Text]
  46. Liu, M. T., B. P. Chen, P. Oertel, M. J. Buchmeier, D. Armstrong, T. A. Hamilton, T. E. Lane. 2000. The T cell chemoattractant IFN-inducible protein 10 is essential in host defense against viral-induced neurologic disease. J. Immunol. 165:2327.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
R.-N. E. Dogan, A. Elhofy, and W. J. Karpus
Production of CCL2 by Central Nervous System Cells Regulates Development of Murine Experimental Autoimmune Encephalomyelitis through the Recruitment of TNF- and iNOS-Expressing Macrophages and Myeloid Dendritic Cells
J. Immunol., June 1, 2008; 180(11): 7376 - 7384.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. B. Su, R. S. Grajewski, D. Luger, R. K. Agarwal, P. B. Silver, J. Tang, J. Tuo, C.-C. Chan, and R. R. Caspi
Altered Chemokine Profile Associated with Exacerbated Autoimmune Pathology under Conditions of Genetic Interferon-{gamma} Deficiency
Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4616 - 4625.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Muller, S. L. Carter, M. J. Hofer, P. Manders, D. R. Getts, M. T. Getts, A. Dreykluft, B. Lu, C. Gerard, N. J. C. King, et al.
CXCR3 Signaling Reduces the Severity of Experimental Autoimmune Encephalomyelitis by Controlling the Parenchymal Distribution of Effector and Regulatory T Cells in the Central Nervous System
J. Immunol., September 1, 2007; 179(5): 2774 - 2786.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. S. Hansen, N. J. Bernard, C. Q. Nie, and L. Schofield
NK Cells Stimulate Recruitment of CXCR3+ T Cells to the Brain during Plasmodium berghei-Mediated Cerebral Malaria
J. Immunol., May 1, 2007; 178(9): 5779 - 5788.
[Abstract] [Full Text] [PDF]

Home page
 Mult SclerHome page
M J Bugeja, D R Booth, B H Bennetts, R N. Heard, and G J Stewart
An investigation of polymorphisms in the 4q13.3-21.1 CXC chemokine gene cluster for association with multiple sclerosis in Australians
Multiple Sclerosis, November 1, 2006; 12(6): 710 - 722.
[Abstract] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
Y.-N. Zhu, X.-G. Zhong, J.-Q. Feng, Y.-F. Yang, Y.-F. Fu, J. Ni, Q.-F. Liu, W. Tang, W.-M. Zhao, and J.-P. Zuo
Periplocoside E Inhibits Experimental Allergic Encephalomyelitis by Suppressing Interleukin 12-Dependent CCR5 Expression and Interferon-{gamma}-Dependent CXCR3 Expression in T Lymphocytes
J. Pharmacol. Exp. Ther., September 1, 2006; 318(3): 1153 - 1162.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
F. Nishimura, J. E. Dusak, J. Eguchi, X. Zhu, A. Gambotto, W. J. Storkus, and H. Okada
Adoptive Transfer of Type 1 CTL Mediates Effective Anti-Central Nervous System Tumor Response: Critical Roles of IFN-Inducible Protein-10.
Cancer Res., April 15, 2006;