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 Nanki, T.
Right arrow Articles by Miyasaka, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nanki, T.
Right arrow Articles by Miyasaka, N.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
Medline Plus Health Information
*Joint Disorders
The Journal of Immunology, 2004, 173: 7010-7016.
Copyright © 2004 by The American Association of Immunologists

Inhibition of Fractalkine Ameliorates Murine Collagen-Induced Arthritis1

Toshihiro Nanki2,*, Yasuyo Urasaki*,{dagger}, Toshio Imai{ddagger}, Miyuki Nishimura{ddagger}, Kenzo Muramoto§, Tetsuo Kubota{dagger} and Nobuyuki Miyasaka*

* Department of Medicine and Rheumatology, Graduate School, {dagger} Graduate School of Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan; {ddagger} KAN Research Institute, Kyoto, Japan; and § Eisai Co., Ltd., Ibaraki, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-{gamma} by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA)3 is characterized by chronic inflammation of multiple joints. Large numbers of inflammatory cells, including T cells, macrophages, and dendritic cells, accumulate in the RA synovium (1). Infiltrated inflammatory cells, especially macrophages, produce inflammatory cytokines such as TNF-{alpha} and IL-1{beta} and are thought to contribute to the pathogenesis of RA. Interaction between chemokines and chemokine receptors is necessary for the accumulation of inflammatory cells (2). Chemokines induce directional migration of leukocytes and are generally classified into C, CC, CXC, and CX3C chemokines based on conserved cysteine motifs (3). Chemokine receptors are also classified into C, CC, CXC, and CX3C chemokine receptors. At present, >40 chemokines and 16 chemokine receptors have been identified (4), and the chemokine and chemokine receptor expression in RA synovium and their roles in RA pathogenesis have been intensively analyzed (2).

Fractalkine (FKN/CX3CL1) is a unique chemokine that contains three amino acids between the first two cysteine residues and exists in both soluble and membrane-anchored forms. The membrane-anchored form of FKN comprises a chemokine head tethered to the cell surface by a mucin-like stalk, followed by a single transmembrane-spanning domain (5, 6). FKN is expressed on activated endothelial cells, dendritic cells, and intestinal epithelial cells (6, 7, 8, 9, 10). In contrast, CX3CR1, the unique receptor for FKN, is expressed on subsets of peripheral CD4+ and CD8+ T cells, NK cells, and monocytes (11, 12). Recent studies have suggested that the interaction of FKN and CX3CR1 contributes to the firm adhesion of leukocytes to endothelial cells (13, 14), NK cell activation (15), cardiac allograft rejection (16, 17), monocyte recruitment and the formation of atherosclerotic lesions (18, 19), pathogenesis of kidney diseases (20), pulmonary hypertension (21), proliferation of microglia (22), and circulating effector-killer lymphocytes trafficking (23). With regard to RA, studies from our laboratories (24) and those of other groups (25, 26) showed that FKN, which is expressed on fibroblast-like synoviocytes (FLS) and endothelial cells in the RA synovium, may contribute to the accumulation of type-1 cytokine-producing T cells, macrophages, and dendritic cells, which express CX3CR1. These results suggested that inhibition of FKN and CX3CR1 interaction might be useful for RA treatment.

In the present study, we extended the previous studies and examined the therapeutic effects of FKN inhibition on the murine collagen-induced arthritis (CIA). The results showed that inhibition of FKN ameliorated CIA most likely by inhibition of inflammatory cell migration into the synovium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Induction of CIA

Male 8-wk-old DBA1/J mice were purchased from Oriental Yeast Co. (Tokyo, Japan). Bovine collagen type II (CII; Collagen Research Center, Tokyo, Japan) was dissolved in 0.05 M acetic acid at 4 mg/ml and emulsified in an equal volume of CFA (Difco Laboratories, Detroit, MI). Mice were immunized intracutaneously with 100 µl of the emulsion at the base of the tail (at day –21). After 21 days (day 0), the same amount of bovine CII emulsified into CFA was injected intracutaneously at the base of the tail as a booster immunization. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.

Treatment with anti-mouse FKN mAb

Five hundred micrograms of hamster anti-mouse FKN mAb or control Ab (hamster IgG; ICN Pharmaceuticals, Aurora, OH) was injected into the mouse peritoneal cavity three times per week from day 0 for 14 days. The anti-mouse FKN mAb inhibited migration of mouse CX3CR1-transfected B300.19 pre-B cells induced by mouse FKN (data not shown). Mice were observed for signs of arthritis. The disease severity was recorded for each limb as follows: score 0, normal; 1, erythema and mild swelling confined to the mid-foot (tarsals) or ankle joint; 2, erythema and mild swelling extending from the ankle to the mid-foot; 3, erythema and moderate swelling extending from the ankle to the metatarsal joint; 4, erythema and severe swelling of the ankle, foot, and digits. The clinical arthritis score was defined as the sum of the scores of all four paws of each mouse. At day 15, ankle joints were harvested and histologically examined after H&E staining.

Serum anti-CII IgG levels

At day 15, serum anti-mouse CII Ab (all IgG) levels in the CIA mice were measured using an ELISA kit (Chondrex, Redmond, WA) according to the protocol provided by the manufacturer.

IFN-{gamma} production by CII-stimulated splenic T cells

Thy1.2-positive splenic T cells of CIA mice treated with anti-FKN mAb or control Ab and normal mice at day 7 were purified using MACS microbead-coupled mAbs and magnetic cell separation columns (Miltenyi Biotec, Auburn, CA). An APC-enriched population was prepared from normal splenocytes depleted of Thy1.2-positive T cells and B220 (CD45R)-positive B cells using MACS. Purified splenic T cells (4 x 105) and 1 x 105 APC-enriched splenocytes were cocultured in 96-well culture plates in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) with 10% FCS (Sigma-Aldrich) supplemented where indicated with 50 µg/ml bovine CII. After 3-day incubation, the concentration of IFN-{gamma} in the culture supernatant was measured using an ELISA kit (BioSource International, Camarillo, CA).

Migration of Mac-1-positive splenocytes into the synovium

Mac-1-positive splenocytes from CIA mice were purified using MACS. The purified Mac-1+ cells were labeled with CellTracker Orange 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Molecular Probes, Eugene, OR) according to the protocol supplied by the manufacturer. The CMTMR-labeled 1.2 x 107 cells were i.v. injected into the tail vein of CIA mice at day 7. The recipient mice were injected with 500 µg of anti-FKN mAb or control Ab i.p. 24 and 2 h before the transfer. After 24 h, ankle joints were harvested, embedded in glycol methacrylate, and sagittal 3-µm-thick microtome sections were prepared (SRL, Tokyo, Japan). The numbers of CMTMR-labeled cells that migrated into the synovium between tibiotalar and tarsometatarsal joints were counted under fluorescent microscopy (Fluoview300; Olympus, Tokyo, Japan). Four slides from each joint were independently evaluated by two observers, and the mean cell count was calculated. An additional two slides from each joint were dried at 37°C for 2 h, incubated with 0.1% trypsin at 37°C for 20 min, and then washed with water for 5 min. The sections were incubated for 12 h at 4°C with rat anti-F4/80 mAb (C1:A3-1; Serotec, Raleigh, NC) or normal rat IgG as a control Ab at 45 µg/ml in the presence of 1% BSA in PBS. The samples were then washed twice for 5 min in PBS and stained with Alexa Fluor 488-conjugated goat anti-rat IgG (Molecular Probes) at 20 µg/ml for 1 h at room temperature. The number of Alexa Fluor 488-labeled cells in CMTMR-labeled cells that migrated into the synovium was counted under fluorescent microscopy.

FACS analysis

PBMC and spleen cells from normal mice or CIA mice were adjusted to 1 x 105 cells and incubated with hamster anti-CX3CR1 mAb raised against a peptide corresponding to the N-terminal portion of mouse CX3CR1 for 30 min, rinsed with PBS-3% FCS. PE-conjugated rabbit anti-hamster IgG (Southern Biotechnology Associates, Birmingham, AL) was used as a second Ab. Then, the cells were stained with FITC-conjugated anti-CD4 mAb (H129.19; Becton Dickinson, San Jose, CA), FITC-conjugated anti-CD8 mAb (KT15; Beckman Coulter, Fullerton, CA), or FITC-conjugated anti-Mac-1 mAb (M1/70; Beckman Coulter) and analyzed with FACScaliber (Becton Dickinson).

RT-PCR

At day 15, inflamed ankle joints and the surrounding skin were harvested. Tissues around the ankle joint were cut into small pieces. Total RNA was prepared from the tissue using Isogen (Qiagen, Valencia, CA) and treated with DNase I (Life Technologies, Gaithersburg, MD). First-strand cDNA was synthesized using oligo(dT)12–18 primers (Pharmacia Biotech, Piscataway, NJ) and SuperScript II reverse transcriptase (Life Technologies). The amount of cDNA for amplification was adjusted to the amount of RNA measured by OD meter and also {beta}2-microglobulin ({beta}2m) PCR products using serially diluted cDNA. The cDNA was amplified with primers for {beta}2m (5',CTG ACC GGC CTG TAT GCT ATC C; 3', CAT GTC TCG ATC CCA GTA GAC GG) or FKN (5', TTC ACC TTC AAC CCC AGA GG; 3', CGG GGA CAG GAG TGA TAA GC). The PCR conditions were described previously (27). The PCR products were then separated by electrophoresis through 1.5% agarose.

Immunohistochemistry

Ankle joints of CIA mice were resected at day 15. Immunohistochemistry was conducted on carboxymethyl cellulose-embedded sections of frozen synovial samples. Briefly, 8-µm-thick cryostat sections were prepared using CryoFilmTransfer kit (Finetec, Tokyo, Japahn) according to the protocol provided by the manufacturer. The sections were fixed in 4% paraformaldehyde for 10 min, and then the samples were rehydrated in PBS for 5 min three times. Nonspecific binding was blocked with 1.5% H2O2 in methanol for 15 min, and then with 5% normal donkey serum in PBS for 40 min. Serial sections were then incubated for 2 h at room temperature with the primary Abs: rabbit anti-mouse CX3CR1 Ab raised against a peptide corresponding C-terminal portion of mouse CX3CR1 or normal rabbit IgG as a control Ab at 5 µg/ml in the presence of 5% normal donkey serum in PBS. The samples were then washed twice for 5 min in PBS, and expression was detected using the Envision+ kit (Dako, Carpinteria, CA). For staining with F4/80, CD4, and CD8, sections were incubated with 10 µg/ml rat anti-mouse F4/80 mAb (C1:A3-1), 2 µg/ml rat anti-mouse CD4 mAb (GK1.5; Santa Cruz Biotechnology, Santa Cruz, CA), 5 µg/ml anti-mouse CD8 mAb (53-6.7; Becton Dickinson), or normal rat IgG. Subsequently, the samples were washed twice for 5 min in PBS and incubated with biotin-conjugated rabbit anti-rat IgG (Dako) for 30 min at room temperature. After washing twice in PBS for 5 min, sections were incubated with peroxidase-conjugated streptavidin (Dako) for 30 min at room temperature. Diaminobenzidine chromogen and buffered substrate (Dako) were used for visualization. Sections were counterstained with hematoxylin for 5 s and washed in tap water for 10 min.

For double staining with F4/80, CD4 or CD8, and CX3CR1, the sections were incubated for 2 h at room temperature with rabbit anti-mouse CX3CR1 Ab or normal rabbit IgG as a control Ab at 10 µg/ml in PBS-1% BSA. The samples were then washed twice for 5 min in PBS and incubated with Alexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes) at 20 µg/ml for 1 h at room temperature. For staining with F4/80, after washing twice in PBS for 5 min, sections were incubated with rat anti-mouse F4/80 mAb (C1:A3-1) or normal rat IgG at 10 µg/ml. Subsequently, the samples were washed twice for 5 min in PBS and incubated with Alexa Fluor 488-conjugated goat anti-rat IgG at 20 µg/ml for 1 h at room temperature. For staining with CD4 or CD8, sections were incubated with 2 µg/ml rat anti-mouse CD4 mAb (GK1.5), 5 µg/ml anti-mouse CD8 mAb (53-6.7), or normal rat IgG. Subsequently, the samples were washed twice for 5 min in PBS and incubated with biotin-conjugated rabbit anti-rat IgG. After washing in PBS twice for 5 min, the samples were incubated with Alexa Fluor 488-conjugated streptavidin (Molecular Probes). The numbers of Alexa Fluor 568-labeled CX3CR1-positive cells in Alexa Fluor 488-labeled F4/80-, CD4-, or CD8-positive cells were counted under fluorescent microscopy.

Statistical analysis

Differences in arthritis score, anti-CII IgG production, IFN-{gamma} production, and numbers of migrated Mac-1- or F4/80-positive splenocytes between control Ab and anti-mouse FKN mAb-treated mice were examined for statistical significance using the Mann-Whitney’s U test. The incidence of arthritis was examined using the {chi}2 test. All data were expressed as mean ± SEM. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of anti-mouse FKN mAb on CIA mice

To analyze the effect of inhibition of FKN and CX3CR1 interaction on CIA mice, 500 µg of anti-mouse FKN mAb or control Ab was injected into the peritoneal cavity three times per week after the second booster immunization (day 0). Clinical scores of arthritis were recorded for 2 wk. Treatment with anti-FKN mAb significantly reduced the clinical arthritis score compared with treatment with control Ab (Fig. 1, p < 0.05 from day 8 to day 15). A similar result was obtained in another independent set of experiments. The incidence of arthritis in control Ab-treated mice was 100% (n = 16, total of two separate experiments) at day 15. Furthermore, treatment with anti-FKN mAb significantly reduced the incidence of arthritis (73%, n = 15, p < 0.05).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 1. Inhibition of clinical arthritis score in CIA mice by treatment with anti-FKN mAb. Five hundred micrograms of hamster anti-mouse FKN mAb or control Ab was injected into the peritoneal cavity three times per week from second booster immunization (day 0) for 2 wk. The disease severity was recorded as an arthritis score. Each point represents the mean ± SEM value of eight animals. *, p < 0.05.

 
Histopathological examination of the ankle joints was performed to determine the mechanism of action of anti-FKN mAb, focusing on the effect of FKN inhibition on cellular infiltration. For this purpose, the ankle joints harvested at day 15 were stained with H&E. Fig. 2 shows representative histopathological changes. The joints of control Ab-treated mice showed massive mononuclear cell infiltration and synovial hyperplasia (Fig. 2, A and B). There was also significant pannus formation and bone erosion of the joint. In comparison, mice treated with anti-FKN mAb showed milder histological changes (Fig. 2, C and D). These results showed that treatment with anti-FKN mAb reduced clinical arthritis score, incidence of arthritis, and histological changes in CIA mice.



View larger version (165K):
[in this window]
[in a new window]
  		FIGURE 2. Histological changes induced by treatment with anti-FKN mAb. Ankle joints harvested at day 15 were stained with H&E. Representative photomicrographs of histology from experiment in Fig. 1 are shown. Mice treated with control Ab (A and B) showed mononuclear cell infiltration (MC), synovial hyperplasia (SH), pannus formation (P), and bone erosion (BE). Mice treated with anti-FKN mAb exhibited reduction of these changes (C and D). T, Talus; N, naviculare; C, cuneiform; M, metatarsal. Original magnification, x40 in A and C; x200 in B and D.

 
Effect of FKN inhibition on humoral and cellular immunity against CII

We also examined alternative possible mechanisms of the protective effect of anti-FKN mAb on CIA by studying the humoral and cellular immunity against CII. To evaluate the effect on humoral immunity, serum anti-CII Ab (total IgG) levels were measured by ELISA at day 15. Although production of serum anti-CII IgG was not detected in normal mice (<5 U/ml, n = 3), serum anti-CII IgG was produced in CIA mice. However, the levels of serum anti-CII IgG in mice treated with anti-FKN mAb were not significantly different from those treated with control Ab (Fig. 3).



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of anti-FKN mAb on serum anti-CII IgG levels. Serum anti-CII Ab (total IgG) levels in CIA mice treated with control Ab or anti-FKN mAb at day 15 were measured using ELISA. Data represent the mean ± SEM values of eight animals. N.S., Not significant.

 
To evaluate the effect of FKN inhibition on cellular immunity, we measured IFN-{gamma} production by splenic T cells stimulated with CII. Splenic T cells from normal mice with or without CII stimulation did not produce IFN-{gamma} (<6 pg/ml, n = 3). Although IFN-{gamma} was produced by nonstimulated splenic T cells of CIA mice, treatment with anti-FKN mAb did not change the levels of IFN-{gamma} production. Moreover, stimulation with CII up-regulated IFN-{gamma} production by the splenic T cells from CIA mice; however, the level of production was not significantly different between mice treated with control Ab and anti-FKN mAb (Fig. 4). These results suggest that treatment with anti-FKN mAb does not affect humoral and cellular immunity against CII in CIA mice.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of anti-FKN mAb on IFN-{gamma} production by CII-stimulated splenic T cells. Splenic T cells (4 x 105 cells/well) from control Ab- or anti-FKN mAb-treated CIA mice and APC-enriched splenocytes (1 x 105 cells/well) were cocultured in 96-well plates in RPMI 1640 with 10% FCS supplemented where indicated with 50 µg/ml bovine CII. After 3-day incubation, the concentration of IFN-{gamma} in the culture supernatant was measured using ELISA. Data represent the mean ± SEM values of six animals analyzed in duplicates. N.S., Not significant.

 
Inhibition of macrophage migration into the synovium by anti-FKN mAb

Next, we examined the effect of anti-FKN mAb treatment on inflammatory cell migration into the synovium. Mac-1 (CD11b)-positive splenocytes from CIA mice at day 7 without Ab treatment were purified. The purity of the cells was >98%. The purified Mac-1+ splenocytes were labeled with CMTMR. Subsequently, the labeled splenocytes, which were probably a mixture of macrophages, neutrophils, and dendritic cells, were i.v. transferred to normal or CIA mice. The recipient mice were not treated with either anti-FKN mAb or control Ab until day 5, and were injected with 500 µg of anti-FKN mAb or control Ab i.p. 24 and 2 h before the transfer. This double Ab injection did not reduce arthritis score (data not shown), indicating the transient effect on, but not depletion of, arthritis-promoting environment of recipient mice. Twenty-four hours later, the number of cells that migrated into the synovium was counted. Very few labeled Mac-1+ splenocytes migrated into the synovium of normal mice (8.0 ± 2.0, n = 3). In contrast, a significant number of labeled Mac-1+ splenocytes migrated into the CIA synovium (Fig. 5A). Treatment with anti-FKN mAb significantly reduced the migration of Mac-1-positive splenocytes into the synovium compared with that with control Ab (Fig. 5B). Frequencies of F4/80-positive cells, the other macrophage marker, in the migrated Mac-1+ cells were 77.4 ± 1.7% and 73.7 ± 2.7% from the mice treated with control Ab and anti-FKN mAb, respectively. Calculated number of migrated F4/80-positive cells ((number of migrated CMTMR-positive cells) x (frequency of F4/80-positive cells in the CMTMR-positive cells)) was also significantly reduced by treatment with anti-FKN mAb (252.6 ± 19.4 and 142.1 ± 23.2, p < 0.005, treated with control Ab and anti-FKN mAb, respectively). These results suggest that a majority of migrated Mac-1-positive splenocytes is F4/80-positive macrophages and that the migration of splenic macrophages into the synovium is reduced by treatment with anti-FKN mAb. In contrast, migration of Thy1.2 (CD90)-positive splenic T cells into the synovium of CIA mice was not detected by this method.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition of Mac-1-positive splenocyte migration into the synovium by anti-FKN mAb. Mac-1-positive splenocytes from CIA mice were purified and subsequently labeled with CMTMR. The labeled macrophages were adoptively transferred into CIA mice. Control Ab or anti-FKN mAb was injected i.p. 24 and 2 h before the adoptive transfer. Twenty-four hours later, ankle joints were harvested and examined for migrated cells under fluorescent microscopy (A). Numbers of labeled cells in the synovial tissue between tibiotalar and tarsometatarsal joints were counted in four slides (n = 8) (B).

 
CX3CR1 expression in the CIA mice

Expression of CX3CR1 on monocytes/macrophages and T cells in the peripheral blood and the spleen of CIA mice was analyzed by flow cytometry. More than half of Mac-1-positive monocytes or macrophages expressed CX3CR1 (peripheral blood, 61.0 ± 11.3%, n = 3; spleen, 54.9 ± 7.5%, n = 5). In contrast, the expression level of CX3CR1 on peripheral blood and splenic T cells was low (CD4, 0.7 ± 0.2% and 0.6 ± 0.1%; CD8, 0.5 ± 0.3% and 2.1 ± 0.2%, peripheral blood and spleen, respectively, n = 3).

CX3CR1 expression in synovial tissue was analyzed by immunohistochemistry. CX3CR1 was expressed on synoviocytes in CIA mice (Fig. 6A), but not in normal mice (Fig. 6B). Abundant F4/80-positive macrophages, moderate number of CD4-positive T cells, and a few CD8-positive T cells were detected in synovial tissue in CIA mice (Fig. 6, C–E). Double staining with F4/80 and CX3CR1 showed that most of CIA synovial macrophages expressed CX3CR1 (Fig. 6, F–H). Frequency of CX3CR1-positive cells in F4/80-positive macrophages was 79.4 ± 1.8% (n = 3). Significant number of CD4 and CD8 T cells also expressed CX3CR1 (35.4 ± 1.5% (n = 3) and 16.4 ± 1.9% (n = 4), respectively) (Fig. 6, I–N).



View larger version (98K):
[in this window]
[in a new window]
  		FIGURE 6. CX3CR1 expression in CIA synovium. CX3CR1 expression in synovial tissue samples from three CIA mice (A) and three normal mice (B) were examined by immunohistochemistry. Sections of CIA synovial tissues were stained with F4/80 (C), CD4 (D), and CD8 (E). All sections were counterstained with hematoxylin. Synovial tissues from CIA mice were double stained with F4/80, CD4 or CD8, and CX3CR1 and analyzed with fluorescent microscopy (F, F4/80; G, CX3CR1; H, merged F with G; I, CD4; J, CX3CR1; K, merged I with J; L, CD8; M, CX3CR1; N, merged L with M). Arrows indicate double-positive cells. Original magnification, x200.

 
FKN expression in the synovium of CIA mice

FKN expression in CIA synovial tissue was analyzed using RT-PCR. FKN mRNA was expressed in normal synovium, but was up-regulated in the synovial tissues of CIA mice compared with that of normal mice (Fig. 7).



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 7. FKN expression in CIA synovium. FKN mRNA expression in the synovium was analyzed in three normal mice and three CIA mice using RT-PCR. PCR products were separated by electrophoresis through 1.5% agarose.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study were: 1) inhibition of FKN reduced clinical symptoms and incidence of arthritis, with amelioration of histopathological features of chronic arthritis in CIA mice; and 2) inhibition of FKN reduced macrophage migration into the synovium, although the treatment did not affect humoral and cellular immunity against CII. These results suggest that abrogation of FKN and CX3CR1 interaction ameliorates murine CIA most likely by inhibition of inflammatory cell infiltration into the synovium.

We and other groups reported previously that FKN was expressed by endothelial cells and FLS in human RA synovium, and synovial T cells, macrophages, and dendritic cells expressed CX3CR1, the receptor for FKN, suggesting that these inflammatory cells may migrate into the synovium by interaction of FKN and CX3CR1 (24, 25, 26). In this study, we have clearly shown that inhibition of FKN activity in vivo reduces macrophage migration into the CIA synovium. It has been thought that macrophages in RA synovium produce proinflammatory cytokines and chemokines, which up-regulate expression of chemokines expression by FLS (28). These chemokines provoke migration of inflammatory cells, such as T cells, B cells, macrophages, and dendritic cells. It was also reported that synovial macrophages could differentiate into osteoclasts (29), which cause bone resorption in the joint, resulting in joint destruction. Thus, reduction of macrophage infiltration could suppress the inflammatory cytokine production, inflammatory cell migration, and osteoclastgenesis. Therefore, inhibition of macrophage migration might be useful for RA treatment. In fact, recent study has shown that a CCR1 antagonist, which reduces macrophage accumulation in the RA synovium, is effective for RA (30).

Treatment with anti-FKN mAb prevented macrophage migration into the synovium up to 40%, but not completely (Fig. 5). Other chemokine and chemokine receptor interactions such as MCP-1/CCL2 and CCR2 and IL-8/CXCL8 and CXCR2 may also contribute to the macrophage migration (31). In this regard, each of the gene deletions of MCP-1 (32, 33), CCR2 (34, 35), IL-8 receptor (36), or CX3CR1 (18, 19) resulted in ~50% decrease in atherosclerotic lesion formation or numbers of infiltrated macrophages in the lesion in experimental atherosclerosis mice. These three chemokines might be involved in macrophage migration.

We have already shown that peripheral blood CX3CR1-positive T cells expressing type-1 cytokines and cytotoxic molecules are increased in RA patients and migrate into RA synovium, suggesting that migrated CX3CR1-positive T cells might contribute to the pathogenesis of RA (23, 24). As similar to RA, significant number of CD4- and CD8-positive T cells expressed CX3CR1 in synovial tissue of CIA. Moreover, the frequencies of CX3CR1 expression of CD4- and CD8-positive T cells in the CIA synovial tissue were much higher than those of peripheral blood or spleen. These results suggest that FKN and CX3CR1 interaction might induce T cell migration as well as macrophage into the synovium of CIA. It is also possible that T cells and macrophages acquire CX3CR1 expression after migrating in the synovial tissue and are retained in the tissue by FKN.

Serum anti-CII IgG production in CIA mice and IFN-{gamma} production by CII-stimulated splenic T cells was not changed by anti-FKN mAb. These results suggest that FKN inhibition does not affect humoral and cellular immunity against CII. Furthermore, our preliminary data indicated that stimulation with FKN did not up-regulate IL-6 or matrix metalloproteinase-2 production by FLS, IL-1{beta} production by splenic macrophages, and IFN-{gamma} production by splenic T cells in CIA mice. Taken together, it is tempting to speculate that FKN inhibition reduces arthritis probably by hampering inflammatory cell infiltration.

It was reported that inhibition of MIP-1{alpha} or RANTES, the ligands for CCR1 and CCR5, suppressed arthritis of murine CIA (37, 38), although the severity of arthritis was not changed in the CCR5-deficient mice (39). It is indicating that CCR1 may be an important chemokine receptor for the arthritis. Matthys et al. showed that CXCR4 antagonist reduced CIA in the IFN-{gamma} receptor-deficient mice, but there is no data in the intact IFN-{gamma} mice (40). Blockade of CCR2 was only beneficial during the initiation phase in the CIA mice, while in the progression phase it aggravated the arthritis (41). Interference of CCR2 on the regulatory T cells might be responsible for the increased arthritis in the progression phase. Moreover, CIA was worsened in the CCR2-deficient mice (39). These results suggest that CCR2 blockade may not be good for RA treatment. In contrast, inhibition of FKN during the progression phase reduced arthritis, suggesting that the FKN might be an additional target for RA treatment.

We have established the method to analyze migration of CMTMR-labeled cells into the synovium of CIA mice in vivo. Using this method, we could clearly show the role of FKN on macrophage migration into the synovium. It has been reported that several kinds of inhibitors for chemokines and chemokine receptors can ameliorate animal models of arthritis (37, 38, 40, 41, 42, 43, 44, 45, 46, 47). However, as far as we know, this is the first report that directly shows the prevention of inflammatory cell migration into the synovium by a chemokine inhibitor. This method might allow us to analyze the migration of other cell types such as dendritic cells and B cells into the synovium, as well as the role of molecules involved in leukocyte trafficking such as chemokines and adhesion molecules, on intraarticular cell migration in vivo.

In conclusion, we have demonstrated in the present study that inhibition of FKN significantly improved clinical arthritic score and histopathology of CIA mice most likely by the suppression of inflammatory cell migration into the synovium. Thus, blockade of FKN and CX3CR1 interaction might be useful for RA treatment.


   Acknowledgments
 
We thank Fumiko Inoue, Keiko Mizuno, and Kohei Arita for the excellent technical support.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by grants-in-aid from the Japan Health Sciences Foundation (Research on Health Sciences focusing on Drug Innovation), Uehara Memorial Foundation, Suzuken Memorial Foundation, the Fujisawa Foundation, Ichiro Kanehara Foundation, and the Ministry of Health, Labor, and Welfare and the Ministry of Education, Science, Sports, and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Toshihiro Nanki, Department of Medicine and Rheumatology, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519 Japan. E-mail address: nanki.rheu{at}tmd.ac.jp Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; FKN, fractalkine; FLS, fibroblast-like synoviocytes; CIA, collagen-induced arthritis; CII, collagen type II; CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; {beta}2m, {beta}2-microglobulin. Back

Received for publication June 23, 2004. Accepted for publication August 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kinne, R. W., R. Brauer, B. Stuhlmuller, E. Palombo-Kinne, G. R. Burmester. 2000. Review: Macrophages in rheumatoid arthritis. Arthritis Res. 2:189.[Medline]
  2. Szekanecz, Z., J. Kim, A. E. Koch. 2003. Chemokines and chemokine receptors in rheumatoid arthritis. Semin. Immunol. 15:15.[Medline]
  3. Yoshie, O., T. Imai, H. Nomiyama. 2001. Chemokines in immunity. Adv. Immunol. 78:57.[Medline]
  4. Zlotnik, A., O. Yoshie. 2000. Chemokines: A new classification system and their role in immunity. Immunity 12:121.[Medline]
  5. Pan, Y., C. Lloyd, H. Zhou, S. Dolich, J. Deeds, J. A. Gonzalo, J. Vath, M. Gosselin, J. Ma, B. Dussault, E. Woolf, G. Alperin, J. Culpepper, J. C. Gutierrez-Ramos, D. Gearing. 1997. Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature 387:611.[Medline]
  6. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, D. R. Greaves, A. Zlotnik, T. J. Schall. 1997. A new class of membrane-bound chemokine with a CX3C motif. Nature 385:640.[Medline]
  7. Kanazawa, N., T. Nakamura, K. Tashiro, M. Muramatsu, K. Morita, K. Yoneda, K. Inaba, S. Imamura, T. Honjo. 1999. Fractalkine and macrophage-derived chemokine: T cell-attracting chemokines expressed in T cell area dendritic cells. Eur. J. Immunol. 29:1925.[Medline]
  8. Papadopoulos, E. J., C. Sassetti, H. Saeki, N. Yamada, T. Kawamura, D. J. Fitzhugh, M. A. Saraf, T. Schall, A. Blauvelt, S. D. Rosen, S. T. Hwang. 1999. Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up-regulated upon dendritic cell maturation. Eur. J. Immunol. 29:2551.[Medline]
  9. Muehlhoefer, A., L. J. Saubermann, X. Gu, K. Luedtke-Heckenkamp, R. Xavier, R. S. Blumberg, D. K. Podolsky, R. P. MacDermott, H. C. Reinecker. 2000. Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa. J. Immunol. 164:3368.[Abstract/Free Full Text]
  10. Fraticelli, P., M. Sironi, G. Bianchi, D. D’Ambrosio, C. Albanesi, A. Stoppacciaro, M. Chieppa, P. Allavena, L. Ruco, G. Girolomoni, F. Sinigaglia, A. Vecchi, A. Mantovani. 2001. Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses. J. Clin. Invest. 107:1173.[Medline]
  11. Imai, T., K. Hieshima, C. Haskell, M. Baba, M. Nagira, M. Nishimura, M. Kakizaki, S. Takagi, H. Nomiyama, T. J. Schall, O. Yoshie. 1997. Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91:521.[Medline]
  12. Combadiere, C., K. Salzwedel, E. D. Smith, H. L. Tiffany, E. A. Berger, P. M. Murphy. 1998. Identification of CX3CR1. A chemotactic receptor for the human CX3C chemokine fractalkine and a fusion coreceptor for HIV-1. J. Biol. Chem. 273:23799.[Abstract/Free Full Text]
  13. Fong, A. M., L. A. Robinson, D. A. Steeber, T. F. Tedder, O. Yoshie, T. Imai, D. D. Patel. 1998. Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow. J. Exp. Med. 188:1413.[Abstract/Free Full Text]
  14. Haskell, C. A., M. D. Cleary, I. F. Charo. 1999. Molecular uncoupling of fractalkine-mediated cell adhesion and signal transduction. Rapid flow arrest of CX3CR1-expressing cells is independent of G-protein activation. J. Biol. Chem. 274:10053.[Abstract/Free Full Text]
  15. Yoneda, O., T. Imai, S. Goda, H. Inoue, A. Yamauchi, T. Okazaki, H. Imai, O. Yoshie, E. T. Bloom, N. Domae, H. Umehara. 2000. Fractalkine-mediated endothelial cell injury by NK cells. J. Immunol. 164:4055.[Abstract/Free Full Text]
  16. Robinson, L. A., C. Nataraj, D. W. Thomas, D. N. Howell, R. Griffiths, V. Bautch, D. D. Patel, L. Feng, T. M. Coffman. 2000. A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection. J. Immunol. 165:6067.[Abstract/Free Full Text]
  17. Haskell, C. A., W. W. Hancock, D. J. Salant, W. Gao, V. Csizmadia, W. Peters, K. Faia, O. Fituri, J. B. Rottman, I. F. Charo. 2001. Targeted deletion of CX3CR1 reveals a role for fractalkine in cardiac allograft rejection. J. Clin. Invest. 108:679.[Medline]
  18. Lesnik, P., C. A. Haskell, I. F. Charo. 2003. Decreased atherosclerosis in CX3CR1–/– mice reveals a role for fractalkine in atherogenesis. J. Clin. Invest. 111:333.[Medline]
  19. Combadiere, C., S. Potteaux, J. L. Gao, B. Esposito, S. Casanova, E. J. Lee, P. Debre, A. Tedgui, P. M. Murphy, Z. Mallat. 2003. Decreased atherosclerotic lesion formation in CX3CR1/apolipoprotein E double knockout mice. Circulation 107:1009.[Abstract/Free Full Text]
  20. Segerer, S., E. Hughes, K. L. Hudkins, M. Mack, T. Goodpaster, C. E. Alpers. 2002. Expression of the fractalkine receptor (CX3CR1) in human kidney diseases. Kidney Int. 62:488.[Medline]
  21. Balabanian, K., A. Foussat, P. Dorfmuller, I. Durand-Gasselin, F. Capel, L. Bouchet-Delbos, A. Portier, A. Marfaing-Koka, R. Krzysiek, A. C. Rimaniol, G. Simonneau, D. Emilie, M. Humbert. 2002. CX3C chemokine fractalkine in pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 165:1419.[Abstract/Free Full Text]
  22. Hatori, K., A. Nagai, R. Heisel, J. K. Ryu, S. U. Kim. 2002. Fractalkine and fractalkine receptors in human neurons and glial cells. J. Neurosci. Res. 69:418.[Medline]
  23. Nishimura, M., H. Umehara, T. Nakayama, O. Yoneda, K. Hieshima, M. Kakizaki, N. Dohmae, O. Yoshie, T. Imai. 2002. Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression. J. Immunol. 168:6173.[Abstract/Free Full Text]
  24. Nanki, T., T. Imai, K. Nagasaka, Y. Urasaki, Y. Nonomura, K. Taniguchi, K. Hayashida, J. Hasegawa, O. Yoshie, N. Miyasaka. 2002. Migration of CX3CR1-positive T cells producing type 1 cytokines and cytotoxic molecules into the synovium of patients with rheumatoid arthritis. Arthritis Rheum. 46:2878.[Medline]
  25. Ruth, J. H., M. V. Volin, G. K. Haines, 3rd, D. C. Woodruff, K. J. Katschke, Jr, J. M. Woods, C. C. Park, J. C. Morel, A. E. Koch. 2001. Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant-induced arthritis. Arthritis Rheum. 44:1568.[Medline]
  26. Blaschke, S., M. Koziolek, A. Schwarz, P. Benohr, P. Middel, G. Schwarz, K. M. Hummel, G. A. Muller. 2003. Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis. J. Rheumatol. 30:1918.[Medline]
  27. Nanki, T., K. Hayashida, H. S. El-Gabalawy, S. Suson, K. Shi, H. J. Girschick, S. Yavuz, P. E. Lipsky. 2000. Stromal cell-derived factor-1-CXC chemokine receptor 4 interactions play a central role in CD4 T cell accumulation in rheumatoid arthritis synovium. J. Immunol. 165:6590.[Abstract/Free Full Text]
  28. Nanki, T., K. Nagasaka, K. Hayashida, Y. Saita, N. Miyasaka. 2001. Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis. J. Immunol. 167:5381.[Abstract/Free Full Text]
  29. Itonaga, I., Y. Fujikawa, A. Sabokbar, D. W. Murray, N. A. Athanasou. 2000. Rheumatoid arthritis synovial macrophage-osteoclast differentiation is osteoprotegerin ligand-dependent. J. Pathol. 192:97.[Medline]
  30. Haringman, J. J., M. C. Kraan, T. J. Smeets, K. H. Zwinderman, P. P. Tak. 2003. Chemokine blockade and chronic inflammatory disease: Proof of concept in patients with rheumatoid arthritis. Ann. Rheum. Dis. 62:715.[Abstract/Free Full Text]
  31. Hayashida, K., T. Nanki, H. Girschick, S. Yavuz, T. Ochi, P. E. Lipsky. 2001. Synovial stromal cells from rheumatoid arthritis patients attract monocytes by producing MCP-1 and IL-8. Arthritis Res. 3:118.[Medline]
  32. Gu, L., Y. Okada, S. K. Clinton, C. Gerard, G. K. Sukhova, P. Libby, B. J. Rollins. 1998. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol. Cell 2:275.[Medline]
  33. Gosling, J., S. Slaymaker, L. Gu, S. Tseng, C. H. Zlot, S. G. Young, B. J. Rollins, I. F. Charo. 1999. MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B. J. Clin. Invest. 103:773.[Medline]
  34. Boring, L., J. Gosling, M. Cleary, I. F. Charo. 1998. Decreased lesion formation in CCR2–/– mice reveals a role for chemokines in the initiation of atherosclerosis. Nature 394:894.[Medline]
  35. Dawson, T. C., W. A. Kuziel, T. A. Osahar, N. Maeda. 1999. Absence of CC chemokine receptor-2 reduces atherosclerosis in apolipoprotein E-deficient mice. Atherosclerosis 143:205.[Medline]
  36. Boisvert, W. A., R. Santiago, L. K. Curtiss, R. A. Terkeltaub. 1998. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice. J. Clin. Invest. 101:353.[Medline]
  37. Kasama, T., R. M. Strieter, N. W. Lukacs, P. M. Lincoln, M. D. Burdick, S. L. Kunkel. 1995. Interleukin-10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis. J. Clin. Invest. 95:2868.
  38. Plater-Zyberk, C., A. J. Hoogewerf, A. E. Proudfoot, C. A. Power, T. N. Wells. 1997. Effect of a CC chemokine receptor antagonist on collagen induced arthritis in DBA/1 mice. Immunol. Lett. 57:117.[Medline]
  39. Quinones, M. P., S. K. Ahuja, F. Jimenez, J. Schaefer, E. Garavito, A. Rao, G. Chenaux, R. L. Reddick, W. A. Kuziel, S. S. Ahuja. 2004. Experimental arthritis in CC chemokine receptor 2-null mice closely mimics severe human rheumatoid arthritis. J. Clin. Invest. 113:856.[Medline]
  40. Matthys, P., S. Hatse, K. Vermeire, A. Wuyts, G. Bridger, G. W. Henson, E. De Clercq, A. Billiau, D. Schols. 2001. AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-{gamma} receptor-deficient mice. J. Immunol. 167:4686.[Abstract/Free Full Text]
  41. Bruhl, H., J. Cihak, M. A. Schneider, J. Plachy, T. Rupp, I. Wenzel, M. Shakarami, S. Milz, J. W. Ellwart, M. Stangassinger, D. Schlondorff, M. Mack. 2004. Dual role of CCR2 during initiation and progression of collagen-induced arthritis: evidence for regulatory activity of CCR2+ T cells. J. Immunol. 172:890.[Abstract/Free Full Text]
  42. Gong, J. H., L. G. Ratkay, J. D. Waterfield, I. Clark-Lewis. 1997. An antagonist of monocyte chemoattractant protein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model. J. Exp. Med. 186:131.[Abstract/Free Full Text]
  43. Ogata, H., M. Takeya, T. Yoshimura, K. Takagi, K. Takahashi. 1997. The role of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of collagen-induced arthritis in rats. J. Pathol. 182:106.[Medline]
  44. Barnes, D. A., J. Tse, M. Kaufhold, M. Owen, J. Hesselgesser, R. Strieter, R. Horuk, H. D. Perez. 1998. Polyclonal antibody directed against human RANTES ameliorates disease in the Lewis rat adjuvant-induced arthritis model. J. Clin. Invest. 101:2910.[Medline]
  45. Halloran, M. M., J. M. Woods, R. M. Strieter, Z. Szekanecz, M. V. Volin, S. Hosaka, G. 3. Haines, S. L. Kunkel, M. D. Burdick, A. Walz, A. E. Koch. 1999. The role of an epithelial neutrophil-activating peptide-78-like protein in rat adjuvant-induced arthritis. J. Immunol. 162:7492.[Abstract/Free Full Text]
  46. Yang, Y. F., T. Mukai, P. Gao, N. Yamaguchi, S. Ono, H. Iwaki, S. Obika, T. Imanishi, T. Tsujimura, T. Hamaoka, H. Fujiwara. 2002. A non-peptide CCR5 antagonist inhibits collagen-induced arthritis by modulating T cell migration without affecting anti-collagen T cell responses. Eur. J. Immunol. 32:2124.[Medline]
  47. Salomon, I., N. Netzer, G. Wildbaum, S. Schif-Zuck, G. Maor, N. Karin. 2002. Targeting the function of IFN-{gamma}-inducible protein 10 suppresses ongoing adjuvant arthritis. J. Immunol. 169:2685.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Rheumatology (Oxford)Home page
G. Murphy, N. Caplice, and M. Molloy
Fractalkine in rheumatoid arthritis: a review to date
Rheumatology, May 21, 2008; (2008) ken197v1.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
J. Kezic, H. Xu, H. R. Chinnery, C. C. Murphy, and P. G. McMenamin
Retinal Microglia and Uveal Tract Dendritic Cells and Macrophages Are Not CX3CR1 Dependent in Their Recruitment and Distribution in the Young Mouse Eye
Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1599 - 1608.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Lu, L. Li, K. Kuno, Y. Wu, T. Baba, Y.-y. Li, X. Zhang, and N. Mukaida
Protective Roles of the Fractalkine/CX3CL1-CX3CR1 Interactions in Alkali-Induced Corneal Neovascularization through Enhanced Antiangiogenic Factor Expression
J. Immunol., March 15, 2008; 180(6): 4283 - 4291.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Yamaguchi, K. Fujio, H. Shoda, A. Okamoto, N. H. Tsuno, K. Takahashi, and K. Yamamoto
IL-17B and IL-17C Are Associated with TNF-{alpha} Production and Contribute to the Exacerbation of Inflammatory Arthritis
J. Immunol., November 15, 2007; 179(10): 7128 - 7136.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S.-J. Lee, S. Namkoong, Y.-M. Kim, C.-K. Kim, H. Lee, K.-S. Ha, H.-T. Chung, Y.-G. Kwon, and Y.-M. Kim
Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways
Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2836 - H2846.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S.-O. Moon, W. Kim, M. J. Sung, S. Lee, K. P. Kang, D. H. Kim, S. Y. Lee, J.-N. So, and S. K. Park
Resveratrol Suppresses Tumor Necrosis Factor-{alpha}-Induced Fractalkine Expression in Endothelial Cells
Mol. Pharmacol., July 1, 2006; 70(1): 112 - 119.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Suzuki, T. Nanki, T. Imai, H. Kikuchi, S. Hirohata, H. Kohsaka, and N. Miyasaka
Inhibition of CX3CL1 (Fractalkine) Improves Experimental Autoimmune Myositis in SJL/J Mice
J. Immunol., November 15, 2005; 175(10): 6987 - 6996.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J.-C. Gevrey, B. M. Isaac, and D. Cox
Syk Is Required for Monocyte/Macrophage Chemotaxis to CX3CL1 (Fractalkine)
J. Immunol., September 15, 2005; 175(6): 3737 - 3745.
[Abstract] [Full Text] [PDF]

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 Nanki, T.
Right arrow Articles by Miyasaka, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nanki, T.
Right arrow Articles by Miyasaka, N.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Substance via MeSH
Medline Plus Health Information
*Joint Disorders

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS