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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
-Dependent CXCR3 Expression in T Lymphocytes
Laboratories of Immunopharmacology (Y.-N.Z., X.-G.Z., Y.-F.Y., Y.-F.F., J.N., W.T., J.-P.Z.) and Natural Products Chemistry (J.-Q.F., Q.-F.L., W.-M.Z.), State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China; and Laboratory of Immunology and Virology, Shanghai University of Traditional Chinese Medicine, Shanghai, People's Republic of China (J.-P.Z.)
Received March 29, 2006; accepted June 1, 2006.
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Abstract |
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-dependent CXCR3 expression in T cells through suppressing T-cell receptor (TCR) ligation-induced IFN- production, whereas it inhibited interleukin (IL)-12-dependent CCR5 expression through suppressing IL-12 reactivity in TCR-triggered T cells. As a result, the initial influx of T cells into CNS was inhibited in PSE-treated mice. The consequent activation of macrophages/microglia cells was inhibited in spinal cord from PSE-treated mice as determination of chemokine expressions (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10). Consistently, the secondary influx of CD4+, CD8+, and CD11b+ cells was decreased in spinal cords from PSE-treated mice. These findings suggest the potential therapeutic effect of PSE on multiple sclerosis.
; Alvord et al., 1984). Leukocyte trafficking to the site of inflammation at the CNS was characterized as a sequential multistep event (Karin et al., 1993). At first, a very limited repertoire of T cells, which we named the primary influx, interacts with their target Ag at the site of inflammation, leading to the activation of the BBB to express various adhesion molecules and thus to increase its permeability to circulating leukocytes (Yednock et al., 1992; Karin et al., 1993). Enhanced permeability of this barrier allows a nonselective influx of leukocytes, which we named the secondary influx. This influx correlates with disease onset (Karin et al., 1993; Brocke et al., 1996).
Interleukin (IL)-12 stimulates activated myelin-reactive Th1 cells to selectively up-regulate CC chemokine receptor (CCR) 5 [receptor for CC chemokine ligand (CCL) 3/MIP-1
, CCL4/MIP-1
, and CCL5/regulated on activation normal T cell expressed and secreted], in direct correlation with the acquisition of CNS-infiltrating capacities by these cells (Iwasaki et al., 2001; Bagaeva et al., 2003). CXC chemokine receptor (CXCR) 3 [receptor for CXC chemokine ligand (CXCL) 9/monokine induced by interferon-, CXCL10/interferon--inducible protein-10, and CXCL11/interferon-inducible T-cell -chemoattractant] resembles CCR5 in that it is selectively expressed on activated myelin-reactive Th1 cells. Nonetheless, CXCR3, in contrast to CCR5, was modulated by interferon (IFN)- rather than IL-12 costimulation (Nakajima et al., 2002; Bagaeva et al., 2003).
In primary influx, only autoreactive T cells that have up-regulated CCR5 and/or CXCR3 expression in the periphery penetrated across the vascular wall (Bagaeva et al., 2003). Lymphocyte interaction with vascular endothelium under flow is mediated by the sequential interaction of cell adhesion molecules and signals provided by chemokine on the endothelial surface, which bind to their specific receptors on the leukocyte surface. Chemokine receptor signaling leads to an increase in integrin avidity on the leukocyte surface, allowing the leukocyte to firmly bind to the endothelium under flow and subsequently to follow chemokine concentration gradients across the vascular wall (Butcher et al., 1999).
These encephalitogenic T cells that migrated to CNS perivascular sites interacted with major histocompatibility complex class II-associated peptides of myelin oligodendrocyte glycoprotein (MOG) in the CNS-elicited proinflammatory cytokines, such as tumor necrosis factor- and IFN-, that have the capability to induce macrophages/microglial cells to express chemokines, which are key mediators in the recruitment of the secondary influx of leukocytes (Merrill et al., 1992; Shrikant et al., 1994; Renno et al., 1995; Sun et al., 1996). CCR5 and/or CXCR3 are expressed on T cells infiltrating EAE and MS lesions, as well as on T cells in the cerebrospinal fluid and periphery of MS patients during exacerbations (Balashov et al., 1999; Sorensen et al., 1999; Kennedy et al., 2001). The elevated expressions of CCL3, CCL4, and CCL5 (ligands for CCR5) and CXCL9, CLXL10, and CXCL11 (ligands for CXCR3) were essential for the secondary inflammatory infiltrates of CCR5+ cells and CXCR3+ cells (Kuchroo et al., 1992; Hulkower et al., 1993; Glabinski et al., 1995; Godiska et al., 1995; Karpus et al., 1995; Kennedy et al., 1998).
Periplocoside E (PSE), a pregnane glycoside, has been identified from Periploca sepium, which was used for treating rheumatoid arthritis in China. Recent studies have shown that PSE is an immunosuppressive compound, which directly inhibits T-cell activation in vitro and in vivo (Zhu et al., 2006). However, no previous study has examined the use of PSE in the treatment of EAE or other Th1 cell-mediated inflammatory diseases of the CNS. In this study, we have examined the effect and mechanisms of PSE on the pathogenesis of CNS inflammation and demyelination in EAE. Our results showed that PSE inhibits CNS demyelination by suppressing IL-12-dependent CCR5 expression and IFN--dependent CXCR3 expression in T cells, attenuating the primary influx of CCR5+ and/or CXCR3+ T cells into the CNS, and down-regulating chemokine expressions elicited by the primary influx and consequently the secondary influx of CCR5- and/or CXCR3-bearing CD4+ and CD8+ T cells and CD11b+ cells.
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Materials and Methods |
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and IL-12p40, recombinant-mouse IFN- and IL-12, anti-IFN- (R4–6A2), anti-CD3 (145–2C11), anti-CD28 (37.51), anti-CD4 (GK1.5), and anti-CD8 (2.43) monoclonal antibody (mAb) were purchased from PharMingen (San Diego, CA).
Induction, Treatment, and Clinical Evaluation of EAE. Female C57BL/6 mice, aged 6 to 8 weeks, were purchased from the Shanghai Experimental Animal Center, the Chinese Academy of Sciences. The animals were housed in specific pathogen-free conditions. Experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Bioethics Committee of the Shanghai Institute of Materia Medica. We used an active EAE model as described previously (Youssef et al., 2002; Fu et al., 2006). In brief, female C57BL/6 mice were immunized on day 0 by s.c. injection with 100 µl of an emulsion of MOG-(35–55) peptide in CFA, distributed over three sites: one along the midline of the back between the shoulders, and two on either side of the midline on the lower back. Each mouse was immunized by 200 µg of MOG-(35–55) peptide together with 200 µg of M. tuberculosis H37Ra and additionally received 400 ng of B. pertussis toxin by i.p. injection in 400 µl of phosphate-buffered saline (PBS) on day 0 and 72 h postimmunization (p.i.). To determine the effect of PSE on actively induced EAE, PSE was dissolved in PBS containing 1.6% ethanol and administered i.p. following MOG-(35–55) immunization and continued throughout the study (n = 15 mice). The dose of PSE (10 mg/kg/day) was chosen based on previous in vivo data (Zhu et al., 2006) and our own preliminary experiments. As a control, an equal volume of PBS containing 1.6% ethanol was injected daily into control mice i.p. (n = 15 mice). Clinical assessment of EAE was performed daily, and mice were scored for disease according to the following criteria: 0, no overt signs of disease; 1, limp tail or hind limb weakness but not both; 2, limp tail and hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; and 5, moribund state or dead (Sakurai et al., 2002). Confirmatory evidence of EAE onset was that the mean clinical score of mice was assessed as 1.
Histopathology Analysis. To assess the degree of CNS inflammation and demyelination, C57BL/6 mice treated with PSE following induction of active EAE were euthanized on day 17 (at the peak of the disease) by CO2 asphyxiation and perfused by intracardiac injection of PBS containing 4% paraformaldehyde and 1% glutaraldehyde. Five-micrometer-thick transverse sections were taken from cervical, upper thoracic, lower thoracic, and lumbar regions of the spinal cord (four sections per mouse). The sections were stained with Luxol fast blue to assess demyelination and with H&E to assess leukocyte infiltration and inflammation. The signs of inflammation and demyelination in the anterior, posterior, and two lateral columns (four quadrants) of the spinal cord sections were scored under microscope. Each quadrant displaying the infiltration of mononuclear cells was assigned a score of one inflammation, and the quadrants that showed perivascular lesion and loss of myelin staining were assigned a score of one demyelination. Thus, each animal had a potential maximal score of 16 points of inflammation and/or 16 points of demyelination, and this study represents the analysis of six representative mice from three different groups. The pathologic score (inflammation or demyelination) for each group was expressed as the percentage positive over the total number of quadrants examined (Calida et al., 2001).
Preparation of Purified T Cells and Enriched Antigen-Presenting Cells. T cells were purified by using immunomagnetic negative selection to delete B cells and I-A+ antigen-presenting cells (APC) as described previously (Zhu et al., 2006). Lymph node cells were allowed to react with anti-I-Ad/b mAb and then incubated with magnetic particles bound to goat anti-mouse immunoglobulin (Ig) (Polysciences, Inc., Eppelheim, Germany). A T-cell population depleted of anti-I-Ad/b-labeled and surface Ig+ cells was obtained by removing cell-bound magnetic particles with a rare earth magnet (Polysciences, Inc.). Purity of the resulting T-cell populations was examined by flow cytometry and found to be consistently >95%.
Splenic APC-enriched populations were separated using immunomagnetic negative selection to delete the surface Ig+ cells (B cells) and T cells as described previously; spleen cells were allowed to react with a mixture of rat anti-mouse CD4 (GK1.5) and rat anti-mouse CD8 (2.43) mAb and then incubated with a mixture of magnetic particles bound to goat anti-rat (Advanced Magnetics, Cambridge, MA) and goat antimouse Ig. An APC-enriched population was obtained by removing cell-bound magnetic particles. Purity of the resulting APC-enriched populations was examined by flow cytometry, and T cells and B cells were found to consistently remain <1%.
Cytokines Assay. In the MOG-immunized T-cell system, purified T cells (4 x 105/well) were cocultured with APC-enriched populations (1 x 105/well) in 96-well flat-bottomed tissue culture plates. In the TCR-trigged T-cell system, purified primary T cells (2 x 105/well) were stimulated with immobile anti-CD3 mAb (5 µg/ml) plus anti-CD28 mAb (2 µg/ml) in 24-well flat-bottomed tissue culture plates.
APC-enriched cells were obtained from MOG-immunized mice with or without PSE treatment. Purified T cells were obtained from MOG-immunized mice and cocultured in the presence or absence of 10 µg/ml MOG. Supernatants were harvested at 24 h to measure IL-12 levels by ELISA.
Purified T cells were obtained from MOG-immunized mice with or without PSE treatment. APC-enriched cells were obtained from normal mice and cocultured in the presence of 10 µg/ml MOG or 1000 pg/ml IL-12 as indicated. Supernatants were harvested at 48 h to measure IFN- levels by ELISA.
Purified primary T cells were stimulated with immobile anti-CD3/CD28 mAb for 48 h in the first culture. TCR-triggering T cells were harvested, washed, and then stimulated with 1000 pg/ml IL-12 in the presence or absence of PSE in the second culture for 24 h. IFN- level was measured by ELISA.
CXCR3 and CCR5 mRNA Expression Assay. Purified primary T cells were stimulated with immobile anti-CD3/CD28 mAb in the presence of anti-IFN- (20 µg/ml), PSE (1–4 µM), or PSE plus IFN- (5 ng/ml) for 48 h in the first culture. TCR-triggering T cells were harvested, washed, and then cultured without additional stimulation in the second culture for 24 h. Cells from the second culture were examined for the expression of CXCR3 mRNA (Nakajima et al., 2002).
Purified primary T cells were stimulated with immobile anti-CD3/CD28 mAb for 48 h in the first culture. TCR-triggering T cells were harvested, washed, and then stimulated with 1000 pg/ml IL-12 in the presence of PSE (1–4 µM) in the second culture for 24 h. Cells from the second culture were examined for the expression of CCR5 mRNA (Iwasaki et al., 2001).
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). cDNA were amplified. Samples were separated by a 1.2% agarose gel and stained with ethidium bromide. Relative quantitation with real-time polymerase chain reaction (PCR) was performed with SYBR Green PCR Reagents (QIAGEN, Valencia, CA) and a continuous fluorescence detection system (MJ Research, Watertown, MA) according to the manufacturer's instructions. The mRNA levels were normalized to those of -actin. Statistical Analysis. Student's t test was used to determine significance between groups where appropriate. A value of p < 0.05 was considered significant.
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Results |
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PSE Reduced Inflammation and Demyelination in the Spinal Cord Tissues of MOG-Immunized Mice. Spinal cord sections from MOG-immunized mice at the peak of EAE (day 17 p.i.) were analyzed for the infiltration of mononuclear cells (inflammation) and myelin loss (demyelination). PSE treatment significantly reduced both the infiltration of mononuclear cells (Fig. 2, A, B, and E) and the extent of lesion formation in the spinal cords of MOG-immunized mice (Fig. 2, C–E).
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). We used real-time reverse transcription-polymerase chain reaction (RT-PCR) to assess the levels of mRNA specific for CD4, CD8, and CD11b in spinal cord extracts as markers of infiltrating CD4+ T cells, CD8+ T cells, and macrophages/microglia cells, respectively. Compared with those of nonimmune mice, CD4, CD8, and CD11b mRNA levels were markedly increased in the spinal cord tissues of MOG-immunized, vehicle-treated mice (Fig. 2F). PSE treatment reduced the levels of CD4, CD8, and CD11b mRNA in the spinal cord tissues of MOG-immunized mice (Fig. 2F).
To obtain a quantitative assessment of the CNS inflammatory cell infiltration, we performed flow cytometric analysis on spinal cord mononuclear cells, which were isolated from representative mice at the peak of EAE (day 17 p.i.). The results revealed that infiltrating inflammatory cells in spinal cord of MOG-immunized mice were composed of T cells (CD4+ and CD8+) and CD11b+ cells (Supplemental Fig. 1A). Not only was there an increase in the total number of infiltrating inflammatory cells in the spinal cords, but there was also an increase in the number of infiltrating T cells and CD11b+ (Supplemental Fig. 1B).
CCR5 and CXCR3 Inductions on MOG-Immunized T Cells Were Decreased by PSE Treatment. The pathogenesis of EAE was thought to be associated with the initial migration of autoreactive T cells into CNS tissues (primary influx), which consequently caused a proinflammatory CNS environment and then a massive infiltration of inflammatory cells (second influx) (Hartung and Rieckmann, 1997). Thus, PSE may exert therapeutic effect in EAE by preventing the initial autoreactive T cells from accumulating in the CNS. CCR5 and/or CXCR3 are essential for the primary influx of T cells to bind to the chemokine on the endothelial surface. We examined whether CCR5 and CXCR3 expressions were induced on MOG-immunized T cells and, if so, whether the inductions of CCR5 and CXCR3 were influenced by PSE treatment. T cells were prepared from inguinal/axillary lymph nodes of MOG-immunized mice treated with vehicle or PSE at day 8 p.i. T cells from MOG-immunized mice expressed enhanced levels of CCR5 and CXCR3 mRNA compared with T cells from normal mice, indicating the presence of CCR5+ T cells and CXCR3+ T cells (Fig. 3). Decreased expressions of CCR5 and CXCR3 mRNA were observed for T cells from PSE-treated mice (Fig. 3). Thus, these results indicated that PSE treatment decreased the inductions of CCR5 and CXCR3 on T cells in lymphoid organs from MOG-immunized mice.
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PSE Suppressed IL-12-Dependent CCR5 Expression and IFN--Dependent CXCR3 Expression in T Cells. Previously we have suggested that PSE inhibited T-cell activation both in vivo and in vitro (Zhu et al., 2006). To confirm the effect of PSE on CCR5 and CXCR3 expressions and to study the mechanisms involved, we used both MOG-immunized T-cell system and TCR-trigged T-cell system.
IFN- signal produced as a result of TCR stimulation is an absolute requirement for CXCR3 induction (Nakajima et al., 2002). In vivo PSE treatment directly inhibited MOG-induced IFN- production in MOG-immunized T cells (Fig. 4A). Likewise, PSE (0.5–4 µM) concentration-dependently inhibited MOG-induced IFN- production in MOG-immunized T cells in vitro (Fig. 4B). Anti-CD3/CD28 mAb stimulation leads to the production of IFN- from T cells. Neutralization of IFN- with anti-IFN- mAb resulted in potent inhibition of CXCR3 induction (Fig. 4C). PSE inhibited anti-CD3-induced IFN- production (Zhu et al., 2006). PSE (1–4 µM) concentration-dependently inhibited the CXCR3 mRNA induction (Fig. 4C). The additive recombinant IFN- reversed the inhibitory effect of PSE (2 µM) on CXCR3 expression (Fig. 4C). It suggested that PSE inhibited IFN--dependent CXCR3 expression in T cells through suppressing IFN- production (Fig. 4C).
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). PSE treatment had no effect on IL-12 production from MOG-immunized APC (Fig. 5A). However, PSE treatment directly inhibited IL-12-induced responses in MOG-immunized T cells, as assessed by IL-12-induced IFN- production (Fig. 5B). Likewise, PSE concentration-dependently inhibited IL-12-induced IFN- production in TCR-trigged T cells (Fig. 5C). We used real-time RT-PCR to determinate whether PSE also inhibited IL-12-dependent CCR5 mRNA expression. The result suggested that PSE (1–4 µM) completely inhibited the IL-12-dependent CCR5 mRNA induction (Fig. 5D).
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PSE Inhibited the Primary T Cell Infiltration in the Spinal Cord Tissues of MOG-Immunized Mice. Because MOG-induced CCR5 and CXCR3 expressions in T cells were inhibited in PSE-treated mice, we next determined whether the primary influx of T cells into CNS was consistently decreased using RT-PCR. The expressions of CD4, CD8, CCR5, and CXCR3 mRNA were not detected before day 8 p.i. (data not shown). Compared with those of nonimmunized mice, CD4, CD8, CCR5, and CXCR3 mRNA levels were increased in the spinal cord tissues of MOG-immunized mice at day 8 p.i., when the mice showed no signs of disease (Fig. 6). PSE treatment reduced the expressions of CD4, CD8, CCR5, and CXCR3 mRNA in the spinal cord tissues of MOG-immunized mice (Fig. 6). However, CD11b mRNA has not been elevated by MOG immunization (Fig. 6). It suggested at this time point there was only primary T-cell infiltration without other inflammatory cells, such as macrophages. PSE showed a marked inhibition in the primary CCR5- and CXCR3-bearing CD4+, CD8+ T-cell influx.
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PSE Inhibited the Elevated Expressions of Chemokine in the Spinal Cord Tissues of MOG-Immunized Mice. These encephalitogenic T cells that migrated to CNS in the first influx have the capability to induce macrophages/microglial cells and astrocytes to express chemokine (Sun et al., 1997). The kinetics of chemokine expression in the spinal cord parenchyma was addressed from four different time points corresponding to preclinical when no mice were exhibiting disease symptoms (day 8 and day 11 p.i.), disease onset when mice showed first symptoms of EAE (day 13 p.i.), and peak EAE (day 17 p.i.). Chemokine expressions were never detected in the absence of primary infiltrates (data not shown). At day 11 p.i., CCL3, CXCL9, and CXCL10 showed a low level expression in CNS from some of MOG-immunized mice. After onset of clinical disease (day 13 p.i.), the expressions of CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10 all elevated to a higher level and remained elevated throughout the course of acute clinical disease of EAE (from day 13 to day 17 p.i. in our observation) (Fig. 7A). PSE treatment inhibited the up-regulation of chemokine expressions induced by the primary T-cell influx (from day 13 to day 17 p.i.) (Fig. 7B).
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PSE Inhibited the Secondary Influx in the Spinal Cord Tissues of MOG-Immunized Mice. Spinal cords from four different time points corresponding to preclinical (day 8 and day 11 p.i.), EAE onset (day 13 p.i.), and peak EAE (day 17 p.i.) were evaluated to show the kinetics of mononuclear cell infiltration. Compared with the expressions at day 8 and day 11 p.i., CD4, CD8, CCR5, and CXCR3 mRNA levels were increased in the spinal cords of MOG-immunized mice from day 13 to day 17 p.i. (Fig. 8A). CD11b mRNA has also been elevated compared with the expression at day 11 p.i. (Fig. 8A). It suggested there was not only secondary infiltration of T cells but also of other inflammatory cells, such as macrophages, which was different from the primary influx only involving T cells (Karin et al., 1993; Brocke et al., 1996). PSE treatment not only reduced the levels of CD4, CD8, CCR5, and CXCR3 mRNA but also decreased the levels of CD11b mRNA in the spinal cords of MOG-immunized mice (Fig. 8B). The results showed PSE inhibited the secondary influx.
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Discussion |
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). In this study, we showed that PSE exhibited a protective effect against EAE, which might be through multiple mechanisms. Here we found it might occur through suppressing IL-12-dependent CCR5 expression and IFN--dependent CXCR3 expression in T cells, thereby inhibiting the primary T-cell influx, accordingly decreasing elevated chemokine expressions and therefore inhibiting the secondary influx of inflammatory cells, including T cells and macrophages, and consequently attenuating CNS lesions.
The difference between the primary and secondary influx was that primary influx involved only T cells but secondary influx involved both T cells and macrophages (Karin et al., 1993; Brocke et al., 1996). As shown in Fig. 8A, at days 8 through 11 p.i., the expression of CD11b mRNA in MOG-immunized mice was held at baseline, whereas the expression of CD4 and CD8 mRNA elevated compared with normal mice. It suggested that days 8 through 11 p.i. belonged to the primary influx stage. From day 13 p.i., the expression of CD11b mRNA was elevated, as was the expression of CD4 and CD8 mRNA in MOG-immunized mice compared with that at days 8 through 11 p.i. It suggested that the secondary influx stage came, which was identical with the EAE onset.
All of the chemokines detected in the spinal cord parenchyma of MOG-immunized mice were expressed at least 3 days after the appearance of their receptors, CCR5 and CXCR3 (Fig. 7A). These results strongly suggested that chemokines expressed in the spinal cord parenchyma were not essential for the initial recruitment of CCR5+ and/or CXCR3+ T cells. When the CCR5+ and/or CXCR3+ T cells in the initial recruitment cross the BBB, CCR5 and CXCR3 bind to the chemokine expressed on the surface of vascular endothelium cells. In our study, we could not detect this kind of chemokine because the spinal cord tissue had been perfused by intracardiac injection of PBS. The chemokine expressed on the surface of vascular endothelium cells may not exist after perfusing by a large amount of PBS. However, several studies in different models have shown a correlation between the EAE onset and the expression of the chemokine in the parenchyma, such as CCL5, CCL3, CXCL10, CCL4, and CCL2 (Hulkower et al., 1993; Ransohoff et al., 1993; Godiska et al., 1995). The peak expression of chemokine paralleled that of chemokine receptors at day 13 p.i. (Figs. 7A and 8A). It implied that chemokines expressed in the parenchyma are key mediators in the recruitment of secondary influx of leukocytes at the inflamed spinal cord tissues.
PSE treatment reduced the accumulation of T cells and macrophages and the lesion formation in the spinal cords of MOG-immunized mice (Fig. 2F). The apparent decrease in inflammatory cell invasion was paralleled with reduced levels of mRNA specific for several inflammatory mediators implicated in the disease pathogenesis, including IL-18, tumor necrosis factor-, IFN-, inducible nitric-oxide synthase, and cyclooxygenase 2 (data not shown).
It has been shown that IFN- and IFN- receptor-deficient mice would develop more severe EAE than wild-type mice (Ferber et al., 1996; Chu et al., 2000). Likewise, administration of anti-IFN- to wild-type mice exacerbates EAE (Billiau et al., 1988; Lublin et al., 1993). These mice have a massive expansion of myelin-specific CD4+ cells, suggesting that the complete loss of IFN- results in a diminished capacity to regulate the autoreactive T cells (Chu et al., 2000) and reinforcing the notion that loss of a particular gene from all the cell types can have unforeseen side effects. Although these observations indicate that IFN- is not essential for the induction of EAE, they do not negate the fact that encephalitogenic T cells generated in vivo in wild-type mice produce significant amounts of IFN- or the fact that in vitro suppression of IFN- production during the stimulation of myelin-specific T cells reduces the encephalitogenic capacity of these cells (Olsson, 1992). Taken together, these studies would suggest that suppressing IFN- production in the encephalitogenic Th1 cells, while preserving IFN- expression in other cell types, might provide therapeutic benefit. In our study, PSE inhibited IFN- production from T cells both in vivo (Figs. 4A and 5B) and in vitro (Figs. 4B and 5C). However, whether PSE also showed inhibitory effects on IFN- production from natural killer cells, natural killer T cells, and even macrophages deserves further study.
Injection of MS patients with recombinant IFN- induced exacerbation of the disease (Panitch et al., 1987). Patients who received anti-IFN- mAb treatment showed statistically significant improvement in secondary progressive MS (Skurkovich et al., 2001). Furthermore, increase in IFN- production by peripheral blood mononuclear cells precedes clinical attacks (Dettke et al., 1997), and the inflammatory process in the cerebrospinal fluid of patients with MS is characterized by increased IFN- expression (Woodroofe and Cuzner, 1993). Taken together, the data are consistent with an important pathogenic role for IFN- in inflammatory demyelinating diseases like MS. Although so many treatments are effective in EAE, few of them have been effective in human MS. Benefiting from its potent inhibitory effect on IFN- production from T cells, PSE may exhibit a therapeutic effect on MS. We will continue to evaluate the pharmacological effects of PSE in future studies. Our recent results also showed that p.o. administration of PSE has therapeutic effects on this EAE model (data not shown).
In conclusion, this study highlights the fact that PSE inhibits EAE by suppressing IL-12-dependent CCR5 expression and IFN--dependent CXCR3 expression in T cells and suggests its therapeutic effect in the treatment of MS.
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Footnotes |
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ABBREVIATIONS: EAE, experimental allergic encephalomyelitis; CNS, central nervous system; MS, multiple sclerosis; BBB, blood-brain barrier; IL, interleukin; CCR, CC chemokine receptor; CCL, CC chemokine ligand; CXCR, CXC chemokine receptor; CXCL, CXC chemokine ligand; IFN, interferon; MOG, myelin oligodendrocyte glycoprotein; PSE, periplocoside E; CFA, Complete Freund's adjuvant; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; PBS, phosphate-buffered saline; p.i., postimmunization; APC, antigen-presenting cell(s); Ig, immunoglobulin; RT-PCR, reverse transcription-polymerase chain reaction; TCR, T-cell receptor; MIP, macrophage inflammatory protein.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
1 These authors contributed equally to this work.
Address correspondence to: Dr. Jian-Ping Zuo, Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China. E-mail: jpzuo{at}mail.shcnc.ac.cn
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