Amelioration of experimental autoimmune encephalomyelitis in Lewis rats treated with fucoidan

Amelioration of Experimental Autoimmune Encephalomyelitis in Lewis Rats Treated with Fucoidan

Heechul Kim,1† Changjong Moon,2† Eun-jin Park,1 Youngheun Jee,1 Meejung Ahn,1 Myung Bok Wie3 and Taekyun Shin1*1Department of Veterinary Anatomy, College of Veterinary Medicine and Applied Radiological Science Research Institute, Jeju National University, Jeju 690-756, South Korea2Department of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, South Korea3Department of Veterinary Medicine, Kangwon National University, Chuncheon 200-701, South Korea

We examined whether fucoidan affected the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in rats. EAE was induced in Lewis rats that were immunized with guinea-pig myelin basic protein (MBP) and complete Freund’s adjuvant. Fucoidan (50 mg/kg, daily) was administered to rats with EAE intraperitone-ally, either in the EAE induction phase from either 1 day before immunization to day 7 post-immunization (PI), or the effector phase from day 8 to 14 PI, to test which phase of rat EAE is affected by fucoidan treatment.

The onset, severity and duration of EAE paralysis in the fucoidan-treated group in the days 8–14 PI-treated rats, but not in days -1–7 PI-treated rats, were signifi cantly delayed, suppressed and reduced, respectively, compared with the vehicle-treated controls. Treatment with fucoidan reduced the encephalitogenic response and TNF-a production during EAE. Moreover, the clinical amelioration coincided with decreased infi ltration of infl ammatory cells in the EAE-affected spinal cord. The ameliorative effect of fucoidan on clinical paralysis in EAE-affected rats may be mediated, in part, by the suppression of the autoreactive T cell response and infl ammatory cytokine production. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: experimental autoimmune encephalomyelitis; fucoidan; T cell; TNF-α.

INTRODUCTION

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system (CNS), used in the study of human demyelinating diseases, such as multiple sclerosis (MS). EAE is characterized by an infi ltration of T cells and macrophages into the subarachnoid space during the early stage and the activation of microglia and astro-cytes during the peak (symptomatic) stage of the disease (Shin et al., 1995).

Cell activation events, such as increased expression of cell adhesion molecules on vascular endothelial cells (Shin et al., 1995), astrocyte proliferation, activation of brain macrophages and apoptosis of infl ammatory cells, are typical fi ndings in both EAE and MS lesions (Schönrock et al., 1998). In addition, the extent of infi l-trating infl ammatory cells in EAE lesions is associated with the accelerating disease processes and the severity of clinical signs (Ahn et al., 2001). The regulation of infl ammatory cells is the primary target in ameliorating or treating EAE and MS (Moon et al., 2004, 2005; Stan-islaus et al., 2005).

Fucoidan comprises sulfated polysaccharides extracted from brown seaweeds, such as Laminaria saccharina, L. digitata, Fucus distichus, F. evanescens, F. serratus, F. spiralis, F. vesiculosus, Ascophyllum nodosum and Cladosiphon okamuranus (Cumashi et al., 2007). Fucoidan has diverse physiological activi-ties, including antiinfl ammatory, antiangiogenic, anti-coagulant, antiadhesive and neuroprotective effects (Jhamandas et al., 2005; Cumashi et al., 2007). Further, fucoidan reduces infl ammatory responses in experimen-tal pneumococcal meningitis (Granert et al., 1998), intracerebral hemorrhage (Del Bigio et al., 1999) and perinatal hypoxic–ischemic encephalopathy (Uhm et al., 2003). These fi ndings indicate that fucoidan may have the potential to inhibit infl ammatory responses involved in EAE, but little is known about the effects of fucoidan in the pathogenesis of EAE.

In this study, it was confi rmed that fucoidan amelio-rated the clinical symptoms of EAE in rats and the effects of fucoidan on the pathogenesis of EAE were examined in a rat model.

MATERIALS AND METHODS

Animals. Lewis rats were obtained from Harlan (Indianapolis, IN) and were bred in our animal facility. Rats of both sexes (7–8 weeks old; 160–200 g) were used. All experiments were performed in accordance with accepted ethical guidelines.

* Correspondence to: Dr T. Shin, Department of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju 690-756, South Korea.E-mail: [email protected]† The fi rst two authors equally contributed to this work.

Received 12 July 2009Revised 10 May 2009

Copyright © 2009 John Wiley & Sons, Ltd. Accepted 04 June 2009

PHYTOTHERAPY RESEARCHPhytother. Res. 24: 399–403 (2010)Published online 4 August 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2959

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 24: 399–403 (2010)DOI: 10.1002/ptr

400 H. KIM ET AL.

Induction of EAE. The footpads of both hind feet of rats in the EAE group were injected with 100 μL of an emulsion containing equal parts of guinea-pig myelin basic protein (MBP; 1 mg/mL) and complete Freund’s adjuvant (CFA), supplemented with Mycobacterium tuberculosis H37Ra (3 mg/mL; Difco, Detroit, MI). After immunization, the rats were observed daily for clinical signs of EAE. The progression of EAE was divided into eight clinical stages: grade 0 (G.0), no sign; G.0.5, mild fl oppy tail; G.1, complete fl oppy tail; G.2, mild paraparesis; G.3, severe paraparesis; G.4, tetrapa-resis; G.5, moribund condition or death; and R.0, recovery.

Fucoidan treatment. Two different treatment protocols were applied in Lewis rats with EAE to determine whether fucoidan can prevent or ameliorate EAE. Rats with EAE (10 rats/group) were given 50 mg/kg fucoidan (Sigma, St Louis, MO) daily, intraperitoneally from either 1 day before immunization to day 7 post- immunization (PI) to examine a preventive effect or from days 8–14 PI to test a therapeutic effect. Control animals for fucoidan-treated EAE rats received vehicle (saline) only according to the same protocol. In addi-tion, three normal rats were injected with fucoidan intraperitoneally (50 mg/kg). Immunized rats were observed daily for clinical signs of EAE, which were categorized according to the aforementioned stages. The progression of EAE was compared between fucoi-dan-treated and vehicle-treated control animals, using Student’s unpaired, two-tailed t-test. In all cases, p < 0.05 was taken to indicate statistical signifi cance.

T cell culture and proliferation. Spleen mononuclear cells (MNCs) from experimental animals (three rats/group at day 12 PI) described above were dissoci-ated and suspended in culture medium containing Dulbecco’s modifi ed Eagle’s medium (Gibco, Paisley, UK), supplemented with 1% (v/v) minimum essential medium (Gibco), 2 mm glutamine (Flow Laboratory, Irvine, CA), 50 IU/mL penicillin, 50 mg/mL streptomy-cin and 10% (v/v) fetal calf serum (Gibco). MNCs were isolated and incubated (4 × 105 MNCs with 200 μL of culture medium) in 96-well, round-bottomed microtiter plates (Nunc, Copenhagen, Denmark). MBP (Sigma, St Louis, MO) was added to wells at fi nal concentrations of 10 μg/mL MBP. After 4 days of incubation, the cells were pulsed for 18 h with 10 μL portions containing 1 μCi of 3H-methylthymidine (specifi c activity 42 Ci/mmol; Amersham, Arlington Heights, IL). The cells were harvested on glass fi ber fi lters, and thymidine incorporation was measured.

ELISA. Single-cell suspensions of MBP-primed spleen cells were cultured in the presence or absence of MBP (10 μg/mL). The supernatants were collected after 48 h in culture. Cytokines (TNF-α, IL-10) were detected in culture supernatants using commercially available immunoassay kits in accordance with the manufactur-er’s instructions (Biosource, Camarillo, CA). The absor-bance was read at 450 nm using a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA). Cytokine levels were calculated with standard curves using recom-binant rat cytokines.

Histological examination. Rats were killed at day 21 PI under ether anesthesia. The spinal cords were separated and dissected. Samples of the spinal cords were pro-cessed for embedding in paraffi n wax after fi xing in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). Paraffi n sections were used for hematoxylin and eosin staining and immunohistochemistry (Moon et al. 2004, 2005).

RESULTS

To examine whether fucoidan prevents EAE, fucoidan was administered to rats intraperitoneally beginning 1 day before immunization to day 7 PI. The course of EAE paralysis in this group was not signifi cantly differ-ent from that of the vehicle-treated group (Table 1).

In the test of the therapeutic effects of fucoidan in EAE, fucoidan treatment given on days 8–14 PI pre-vented the paralysis of rats immunized with MBP (20% suppression, 4/5 rats) compared with control EAE rats treated with vehicle (vehicle treatment on days 8–14 PI; 100% incidence, 5/5 rats). The fi rst onset of EAE paral-ysis in fucoidan-treated rats with EAE on days 8 to 14 PI (13.5 ± 0.3) was signifi cantly delayed in comparison with vehicle-treated controls on days 8–14 PI (11.4 ± 0.2; p < 0.05). Further, fucoidan treatment on days 8–14 PI signifi cantly ameliorated the clinical severity of EAE and the duration of the paralysis in EAE rats compared with vehicle treatment on days 8–14 PI (p < 0.05).

The study further examined whether fucoidan treat-ment affected the expansion of MBP-specifi c T cell responses. As shown in Fig. 1, the proliferation of anti-gen-specifi c T cells increased signifi cantly in vehicle-treated rats. In marked contrast, fucoidan-treated rats showed a signifi cant decrease in antigen-specifi c T cell proliferation in response to MBP compared with vehicle-treated controls (p < 0.001), indicating that fucoidan could alter myelin-specifi c T cells and their autoreactivity.

Table 1. Effects of fucoidan on the clinical signs of EAE

Incidence of EAE (paralysed/total animals)

First onset of paralysis

Average of max. clinical score

Duration of paralysis (days)

Treatment from day −1 to +7 PIVehicle control 6/6 12.3 ± 0.6 1.5 ± 0.4 5.2 ± 0.5Fucoidan 6/6 12.3 ± 0.7 1.7 ± 0.4 4.7 ± 1

Treatment from day +8 to +14 PIVehicle control 5/5 11.4 ± 0.2 1.8 ± 0.4 6 ± 0.5Fucoidan 4/5 13.5 ± 0.3a 0.6 ± 0.2a 2.6 ± 1a

Data are expressed as the mean ± SEM.a p < 0.05 vs vehicle-treated control (Student’s t-test).

AMELIORATIVE EFFECT OF FUCOIDAN ON EAE 401


Taken together, these data indicate that the capacity of fucoidan to suppress the encephalitogenic T cell response may underlie its clinically benefi cial effects in EAE.

Then the cytokine responses in EAE rats after fucoi-dan treatment were determined. Splenocytes were obtained from vehicle- or fucoidan-treated rats and then cytokine levels were determined by ELISA. The supernatants of these splenocytes contained low levels of cytokines without specifi c antigen stimulation. As shown in Fig. 2A, there was a signifi cant decrease in TNF-α in encephalitogenic T cells in response to MBP in fucoidan-treated EAE rats compared with their untreated counterparts (p < 0.05). On the other hand, fucoidan treatment did not alter the production of IL-10 by MBP-stimulated splenocytes (Fig. 2B). The Th1 cytokine production of myelin-reactive T cells was sig-nifi cantly reduced after fucoidan treatment.

Based on the behavioral improvement in the fucoidan-treated group, spinal cord tissues were exam-

ined in EAE rats with fucoidan treatment from days 8 to 14 PI. Infl ammatory cells, including ED1-positive macrophages, in the spinal cords of fucoidan-treated rats were fewer than those in vehicle-treated controls (Fig. 3).

Figure 1. Effects of fucoidan on MBP-reactive T cell responses. Proliferative responses to the antigen (MBP) were assessed in triplicate wells for each experiment (n = 3). Data are mean ± SEM of cpm. Statistical evaluation was performed to compare the experimental groups and corresponding control groups. * p < 0.001.

Figure 2. Effects of fucoidan on TNF-α (A) and IL-10 (B) produc-tion. The levels (pg/mL) of TNF-α (A) and IL-10 (B) in culture supernatants were measured using ELISA kits. Data are mean ± SEM. Statistical evaluation was performed to compare the experimental groups and corresponding controls. * p < 0.05.

Figure 3. Histopathological examination of the spinal cords of rats with EAE (day 21 PI) that were treated with either vehicle (A, C) or fucoidan (B, D) from days 8 to 14 PI. The spinal cords of vehicle-treated rats contained many infl ammatory cells (A) and ED1-positive macrophages (C) in the parenchyma, whereas there were fewer infl ammatory (B) and ED1-positive (D) cells in the spinal cords of fucoidan-treated rats. A and B: Hematoxylin-eosin staining. C and D: Immunostained with ED1 and counterstained with hematoxylin. Scale bars: in panels A and B, 100 μm; in panels C and D, 50 μm.


402 H. KIM ET AL.

DISCUSSION

This study examined the effects of fucoidan in actively induced Lewis rat EAE to determine whether fucoidan has preventive or therapeutic effects in rat EAE. The fi ndings indicate that the administration of fucoidan during the effector stage of EAE (days 8–14 PI), but not during the induction stage of EAE (days −1–7 PI), ame-liorated EAE paralysis, at least in Lewis rat EAE, com-pared with vehicle-treated EAE controls. These fi ndings suggest that fucoidan has a therapeutic effect, but not a preventive effect, in rat EAE with this treatment pro-tocol. The precise mechanism of EAE amelioration needs further study.

In cytokine assays, TNF-α and IL-10 were examined, which are representative proinfl ammatory and antiin-fl ammatory cytokines, respectively, in the course of rat EAE. In this study, it was found that fucoidan inhibited the production of TNF-α in encephalitogenic T cells in response to MBP. In support of this fi nding, it is known that the proinfl ammatory cytokine TNF-α affects the pathogenesis of acute EAE (Farias et al., 2007). Fur-thermore, a signifi cant increase of TNF-α mRNA was observed in pertussis toxin-induced hyperacute EAE, compared with the EAE-control, and the increase in TNF-α was correlated with increased infi ltrating infl am-matory cells in the spinal cord (Ahn et al., 2001). These fi ndings all suggest that the suppression of TNF-α in the fucoidan-treated group was partly associated with the amelioration of rat EAE.

The study further examined whether fucoidan stimu-lates the production of IL-10, which is important for the recovery from autoimmune diseases, including EAE (Bettelli et al., 1998; Cua et al., 1999; O’Neill et al., 2006; Fitzgerald et al., 2007). However, fucoidan did not alter the production of the Th2 cytokine IL-10 in our study, implying that IL-10 is less involved in the fucoidan-induced amelioration of rat EAE paralysis. Overall, the ability of fucoidan to regulate T cell proliferation and cytokine production that was observed suggests its

potential usefulness in suppressing infl ammatory responses and inhibiting the migration of infl ammatory cells into the CNS.

The mechanism by which fucoidan mediates its effect on EAE pathogenesis in vitro and in vivo remains to be elucidated. There is a consensus that fucoidan is associ-ated with inhibiting the function of L-selectin (Nasu et al., 1997), which is an adhesion molecule involved in the infl ammatory process in both lymphocyte recircula-tion through lymphoid tissues and leukocyte recruit-ment to sites of infl ammation (Patarroyo, 1994; Khan et al., 2003). In the pathogenesis of EAE, macrophages with autoimmune T cells are activated in peripheral immune organs and fi nally infi ltrate the spinal cord or brain. L-Selectin plays a major role in the progression of EAE pathogenesis (Archelos et al., 1998; Grewal et al., 2001). L-Selectin-defi cient myelin basic protein-specifi c TCR transgenic mice failed to develop antigen-induced EAE, and despite the presence of leukocyte infi ltration damage to myelin in the central nervous system was not seen (Grewal et al., 2001). In addition, treatment with HRL3, a monoclonal antibody to L-selectin, and its F(ab’)2 fragments suppressed rat EAE (Archelos et al., 1998). Therefore, it is suggested that fucoidan, an inhibitor of L-selectin, ameliorates EAE, possibly via the inhibition of its target, L-selectin, an important mediator in treatment strategies for auto-immune diseases.

In conclusion, the ability of fucoidan to inhibit the induction of a proinfl ammatory cytokine (TNF-α) and the suppression of T cell proliferation, and possibly inhibit the transmigration of infl ammatory cells into the CNS of EAE-induced rats indicates that fucoidan may be useful in the treatment of autoimmune central nervous system diseases, as well as other Th1-mediated autoimmune diseases.

Acknowledgement

This project was supported by the Honam Sea Grant R&D Program Fund for 2009.

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Amelioration of experimental autoimmune encephalomyelitis in Lewis rats treated with fucoidan

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