j pharmacol exp ther 2005 iruretagoyena 366 72

7/25/2019 J Pharmacol Exp Ther 2005 Iruretagoyena 366 72

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Andrographolide Interferes with T Cell Activation and ReducesExperimental Autoimmune Encephalomyelitis in the Mouse

Mirentxu I. Iruretagoyena, Jaime A. Tobar, Pablo A. Gonzalez, Sofa E. Sepulveda,Claudio A. Figueroa, Rafael A. Burgos, Juan L. Hancke, and Alexis M. Kalergis

Departamento de Genetica Molecular y Microbiologa, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica deChile, Chile (M.I.I., J.A.T., P.A.G., S.E.S., C.A.F., A.M.K.); and Instituto de Farmacolog a, Facultad de Ciencias Veterinarias,Universidad Austral de Chile, Chile (R.A.B., J.L.H.)

Received June 8, 2004; accepted August 26, 2004

ABSTRACT

Andrographolide is a bicyclic diterpenoid lactone derived fromextracts of Andrographis paniculata, a plant indigenous toSouth Asian countries that shows anti-inflammatory properties.The molecular and cellular bases for this immunomodulatorycapacity remain unknown. Here, we show that andrographolideis able to down-modulate both humoral and cellular adaptiveimmune responses. In vitro, this molecule was able to interferewith T cell proliferation and cytokine release in response toallogenic stimulation. These results were consistent with theobservation that T cell activation by dendritic cells (DCs) wascompletely abolished by exposing DCs to andrographolide dur-ing antigen pulse. This molecule was able to interfere withmaturation of DCs and with their ability to present antigens to Tcells. Furthermore, in vivo immune responses such as antibody

response to a thymus-dependent antigen and delayed-typehypersensitivity were drastically diminished in mice by an-drographolide treatment. Finally, the ability of andrographolideto inhibit T cell activation was applied to interfere with the onsetof experimental autoimmune encephalomyelitis (EAE), an in-flammatory demyelinating disease of the central nervous sys-tem that is primarily mediated by CD4 T cells and serves as ananimal model for human multiple sclerosis. Treatment withandrographolide was able to significantly reduce EAE symp-toms in mice by inhibiting T cell and antibody responses di-rected to myelin antigens. Our data suggest that andrographol-ide is able to efficiently block T cell activation in vitro, as well asin vivo, a feature that could be useful for interfering with detri-mental T cell responses.

Andrographis paniculata is a plant indigenous to South-east Asian countries that has been used as an official herbalmedicine in China for many years. Whole-plant extracts havebeen used as a popular remedy for the treatment of variousdisorders and recently shown to have antitumoral (Rajagopalet al., 2003), anti-inflammatory (Gabrielian et al., 2002), andantiviral properties (Calabrese et al., 2000). Diterpenoidchemicals are the primary constituents present in the ex-tracts of A. paniculata, where andrographolide, a bicyclic

diterpenoid lactone, is the major constituent. Andrographol-ide has been reported to be particularly efficient at regulat-ing immune responses (Calabrese et al., 2000; Rajagopal etal., 2003). This molecule has recently been shown to work asan anti-inflammatory agent by reducing the generation ofreactive oxygen species in human neutrophils (Shen et al.,2002), as well as preventing microglia activation (Wang etal., 2004). However, the molecular and cellular mechanismsresponsible for the immunomodulatory properties of an-

drographolide remain unknown, as well as the potential invivo anti-inflammatory effects resulting from treatment withthis drug.

Andrographolide could exert its immunomodulatory ef-fects at different levels on the immune system. Consider-ing that dendritic cells (DCs) play an important role inregulating adaptive immune responses, they represent apotential target for andrographolide. DCs are professionalantigen-presenting cells (APCs) that have the unique abil-

This work was supported by Fondo Nacional de Desarrollo Cientfico yTecnologico Grant 1030557, Direccion General de Postgrado, InvestigacionGrant 2002/11E, Fondo de Investigacion en Areas Prioritarias Grant13980001, and Fondo de Fomento al Desarrollo Cientfico y Tecnologico GrantDO1I1024. A.M.K. is a Helen Hay Whitney Foundation fellow, C.A.F. is aComite Nacional de Investigacion, Ciencia y Tecnologia fellow, and M.I.I. andJ.A.T. are Programa de Mejoramiento de la Calidad y la Equidad de la Edu-cacion Superior fellows.

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.104.072512.

ABBREVIATIONS: DC, dendritic cell; APC, antigen-presenting cell; MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis;

MOG, mouse myelin oligodendrocyte glycoprotein; PBS, phosphate-buffered saline; OVA, chicken egg ovalbumin; ELISA, enzyme-linked

immunosorbent assay; BSA, bovine serum albumin; NP-BSA, 4-hydroxy-3-nitrophenyl-acetyl conjugated to bovine serum albumin; NP-CGG,

4-hydroxy-3-nitrophenyl-acetyl conjugated to chicken gamma globulin; DTH, delayed-type hypersensitivity.

0022-3565/05/3121-366372$20.00THEJOURNAL OFPHARMACOLOGY ANDEXPERIMENTALTHERAPEUTICS Vol. 312, No. 1Copyright 2005 by The American Society for Pharmacology and Experimental Therapeutics 72512/1182828JPET 312:366372, 2005 Printed in U.S.A.

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ity to capture and present antigens to prime nave CD4

and CD8 T cells and are critical for the initiation of theadaptive immune response against infectious agents(Banchereau et al., 2000; Lanzavecchia and Sallusto,2001). DCs have also been recently implicated in thepathogenesis of several autoimmune diseases (Hart andvan Kooyk, 2004; Waldner et al., 2004). Besides their ca-pacity to initiate adaptive immune response, DCs can also

control immunity through their ability to induce antigen-specific lymphocyte unresponsiveness or tolerance to self-antigens (Yamazaki et al., 2003). Given the central rolethat DCs play as regulators of adaptive immunity, theyrepresent interesting therapeutic targets for pharmacolog-ical modulation of immune responses. Recent evidence in-dicates that several established immunosuppressive drugscould interfere with immune responses by altering DCactivity (Hackstein and Thomson, 2004). Thus, pharmaco-logical modulation of DC function could be beneficial forinterfering with deleterious immune responses such ashypersensitivity reactions and autoimmunity. Accordingto this notion, it is possible that the immunomodulatory

properties of andrographolide could be mediated by aneffect of this molecule on DC function.

Importantly, therapeutic benefits have been observed inresponse to the administration of plant-derived moleculesfor several inflammatory diseases, such as rheumatoidarthritis and multiple sclerosis (MS) (Killestein et al.,2003; Shin et al., 2003; Soeken et al., 2003), providingencouragement for the potential use of these preparationsto treat autoimmune disorders. MS is a chronic neuroin-flammatory demyelinating disorder of the central nervoussystem that predominantly affects young adults (Nosewor-thy et al., 2000). Although the etiology of the progressiveneurological loss has not yet been fully elucidated, evi-

dence points toward an autoimmune pathogenesis, wheremyelin-specific CD4 and CD8 T cells are thought to playa central role by reacting against and destroying the my-elin sheath (Wingerchuk et al., 2001). Due to the fact thatit shows close similarity to clinical and histopathologicalaspects of human MS, experimental autoimmune enceph-alomyelitis (EAE) represents a suitable animal model fortesting efficacy of potential therapeutic agents for MS (Sunet al., 2001; Kuchroo et al., 2002). In C57BL/6 mice, EAEcan be induced by injection of a peptide derived frommouse myelin oligodendrocyte glycoprotein (MOG), whichleads to chronic spinal cord demyelination and paralysis.EAE is characterized by focal areas of demyelination

throughout the central nervous system, with axonal lossthat results in ascending paralysis (Iglesias et al., 2001).Because DC function and T cell priming may possibly be

targets of the immunomodulatory activity of andrographol-ide, in this study we evaluated whether this molecule couldinterfere with these processes. Our data suggest that an-drographolide can interfere with the ability of DCs to processand present antigens to T cells. Consistent with these find-ings, we observed that andrographolide was able to reduce Tcell activation in vitro and in vivo. When this feature wasapplied for a potential favorable immune modulation of au-toimmune diseases, using EAE as a model, it was observedthat treatment with andrographolide reduced the severity of

this disease. Our results support the notion that the immu-

nosuppressive properties of this molecule could be consideredfor the treatment of autoimmune diseases.

Materials and Methods

Animals.Six- to eight-week-old female C57BL/6 mice were usedin these experiments and kept under pathogen-free conditions at theanimal core facility of the Pontificia Universidad Catolica de Chile.

All animal work was performed according to institutional guidelines.Reagents and Synthetic Peptides. Andrographolide was

kindly provided by Amsar Private (Maharashtra, India). A stocksolution for this molecule was prepared by dissolving andrographol-ide in dimethyl sulfoxide at 50 mM, which was then serially dilutedin PBS immediately prior to experiments. Myelin oligodendrocyteglycoprotein-derived peptide (MOG35-55, MEVGWYRSPFSRVVH-LYRNGK), chicken egg ovalbumin (OVA) peptide SIINFEKL(OVA257-264, for presentation on H-2K

b) and OVA peptide TEWTSS-NVMEERKIKV (OVA265-280, for presentation on I-A

b) were synthe-sized by solid-phase method using Fmoc chemistry on an automated433A peptide synthesizer (Applied Biosystems, Foster City, CA) atthe Peptide Synthesis Facility of the Albert Einstein College ofMedicine. All peptides were purified to 98% homogeneity by re-

versed-phase high-performance liquid chromatography on a Vydac

C-18 column (2.1 or 4.6 mm

25 cm, 300 ) using HP-1090Mhigh-performance liquid chromatography (Hewlett Packard, PaloAlto, CA). The identity of the purified peptide was determined by atandem quadrupole mass spectrometer (TSQ700; Thermo Finnigan,San Jose, CA).

EAE Induction. Six- to eight-week-old female C57BL/6 micewere injected subcutaneously with 50 g of MOG35-55peptide emul-sified in Complete Freunds Adjuvant (Invitrogen, Carlsbad, CA)supplemented with heat-inactivated Mycobacterium tuberculosisH37 RA (Difco, Detroit, MI). In addition, mice received intraperito-neal injections with 500 ng of pertussis toxin (Calbiochem, SanDiego, CA) at the time of sensitization and 48 h later. Clinical signsof disease were seen usually between days 15 and 18 after sensiti-zation and assessed daily according to the following scoring criteria:0, no detectable signs of EAE; 1, flaccid tail; 2, hind limb weakness or

abnormal gait; 3, complete hind limb paralysis; 4, paralysis of foreand hind limbs; and 5, moribund or death. To prevent unnecessaryanimal suffering, mice severely affected by the disease were eutha-nized with the supervision of a veterinarian. Mean clinical score wascalculated by adding every day clinical score for all mice in a groupand then divided by the total number of mice. Data shown arerepresentative of four independent experiments.

Andrographolide Treatment.Mice were treated intraperitone-ally with a daily dose equal to 4 mg/kg of andrographolide in PBS(total volume of 100 l). This dose is not maximal and it is consid-erably under the LD50for intraperitoneally administered androgra-pholide (11.6 g/kg) (Handa and Sharma, 1990). Treatment started 1week before MOG sensitization and continued through all the exper-iment. As controls, age-matched female mice were sensitized withMOG but not treated with andrographolide. Treated and controlmice were clinically evaluated on a daily basis. At the doses used,andrographolide was well tolerated by mice, and no evidence oftoxicity was observed.

DCs, Antigen Presentation Assay, and T Cell Hybridoma

Activation.Bone marrow-derived DCs were prepared as previouslydescribed (Inaba et al., 1992; Lopez et al., 2000; Kalergis andRavetch, 2002). Briefly, DCs were grown from bone marrow progen-itors in RPMI 1640 containing 5% fetal calf serum (Hyclone Labora-tories, Logan, UT) supplemented with granulocyte/macrophage col-ony-stimulating factor (50 U/ml) (BD Biosciences PharMingen, SanDiego, CA). Day 5 DCs were treated with 10 M andrographolide for24 h. After this time, DCs were pulsed for 16 h either with OVAprotein or OVA peptide (OVA257-264 for presentation on H-2K

b orOVA265-280 for presentation on I-A

b). After the pulse, DCs were

washed and cocultured at different ratios with either 1 105

B3Z or

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1 105 OT4H T cell hybridomas. B3Z and OT4H are specific forH-2Kb/OVA257-264and I-A

b/OVA265-280, respectively, and secrete IL-2upon T cell receptor stimulation (Shastri and Gonzalez, 1993). After20 h of DC-T cell coculture, IL-2 from supernatants was measured bycytokine ELISA as previously described (Kalergis and Nathenson,2000; Kalergis et al., 2000, 2001). DC viability was determined bytrypan blue exclusion. For anti-CD3 T cell activation, B3Z and OT4HT cell hybridomas (2 105 cells/well) were stimulated with plate-bound anti-CD3, 500 ng/ml (clone 145-2C11, BD Biosciences

PharMingen) in the presence of increasing concentrations of an-drographolide (010 M). After 24 h, culture supernatants wereanalyzed to determine IL-2 secretion by ELISA (Kalergis et al.,2001).

MOG-Specific T Cell Cytokine Release Assays. Inguinal andmesenteric lymph nodes were obtained on day 21 after EAE induc-tion from control or andrographolide-treated mice (average clinicalscore was 2 for controls and 1 for andrographolide-treated mice).Cellular suspensions from these lymph nodes were cultured (5 105

cells/well) in RPMI 1640 containing 5% fetal calf serum with differ-ent concentrations of MOG35-55peptide. Cultures were incubated in96-well round bottom plates for 48 h at 37C in a cell culture incu-bator. IL-2 release in response to MOG35-55peptide was determinedon culture supernatants by cytokine ELISA as previously described

(Kalergis and Nathenson, 2000; Kalergis et al., 2000, 2001). IFN-release was also determined by cytokine ELISA but using purifiedanti-mouse IFN-(clone R46A2, BD Biosciences PharMingen) ascapture antibody and biotin rat anti-mouse IFN- (clone XMG1.2,BD Biosciences PharMingen) as detection antibody.

Measurement of Anti-MOG Antibody Response. Mice serawere obtained on days 7 and 21 after sensitization with MOG 35-55peptide. At day 7, average clinical score for both groups was 0; at day21, average clinical score was 3 for controls and 1.5 for androgra-pholide-treated mice. Mice sera were analyzed for the presence ofMOG-specific IgG by ELISA. Briefly, ELISA plates (Falcon, Cowley,UK) were coated at 4C overnight with 10 g/ml MOG35-55peptide in0.1 M NaHCO3buffer (pH 8.4) and then blocked with PBS-BSA 1%for 2 h at room temperature. Serum samples were diluted in PBS-BSA 1% starting at 1:60 and incubated for 3 h at room temperature.

IgG was detected with rabbit anti-mouse IgG antibody conjugated tohorseradish peroxidase (Amersham Biosciences Inc., Piscataway,NJ). After extensive washing, horseradish peroxidase substrate wasadded (3,3,5,5 tetramethylbenzidine; Sigma-Aldrich, St. Louis,MO), and plates were read at OD450nm on a microplate reader.

DC Maturation Assays. DCs were treated with 10 M androgra-pholide for 24 h and then incubated with 1 g/ml LPS (Sigma-

Aldrich) for 36 h. Untreated control DCs were included in all theexperiments. After LPS treatment, cells were analyzed for expres-sion of surface markers I-Ab, CD86, and CD40 on a FACScan flowcytometer (BD Biosciences, San Jose, CA). To evaluate DC matura-tion, DCs were double-stained with anti-CD11c-PE (clone HL3; BDBiosciences PharMingen) plus anti-I-Ab-FITC (clone AF6-120.1; BDBiosciences PharMingen), anti-CD86-FITC (clone GL1, BD Bio-sciences PharMingen), or anti-CD40-FITC (clone 3/23, BD Bio-sciences PharMingen), fixed in paraformaldehyde (1% in PBS), andanalyzed by fluorescence-activated cell sorting. To determinate thedensities of H-2Kb/OVA complexes on the surface of DCs, OVA-pulsed cells were stained with anti-CD11c-PE and 150l of 25-D1.16supernatant (mouse -IgG1 mAb specific for the H-2Kb/SIINFEKLcomplex). After washing, goat anti-mouse IgG-FITC (BD BiosciencesPharMingen) was added to DCs. Cells were washed in PBS, fixed inparaformaldehyde (1% in PBS), and analyzed by fluorescence-acti-

vated cell sorting.Mixed Lymphocyte Reaction. Lymph node cell suspensions

obtained from C57BL/6 and BALB/c mice were cocultured in 96-wellround bottom plates with increasing concentrations of andrographol-ide (07.5 M) at 1 105 cells per strain on each well for 72 h. Afterthis time, supernatants were harvested and analyzed for IL-2 release

by cytokine ELISA as described above. T cell proliferation was as-

sessed using CellTiter Cell Proliferation Assay (Promega, Madison,WI) following the methodology provided by the manufacturer.

NP-Specific Antibody Response.Mice were immunized subcu-taneously with 50 g of 4-hydroxy-3-nitrophenylacetyl conjugated tobovine serum albumin (NP17-BSA; Biosearch Technologies, Inc., No-

vato, CA) in alum (Pierce Chemical, Rockford, IL). Mice were treatedintraperitoneally with a daily dose equal to 4 mg/kg andrographolidein PBS (total volume of 100 l) since the day of immunization andcontinued for the duration of the experiment. Seven days after im-

munization, NP-specific IgG antibodies were measured in mice seraby ELISA. Briefly, plates were coated at 4C overnight with NP23-CGG (0.5 g/well) in 0.1 M NaHCO3buffer (pH 8.4), and anti-NP IgGantibodies were detected as described above.

Delayed-Type Hypersensitivity Reaction. Mice were immu-nized subcutaneously with 100 g of OVA emulsified in CompleteFreunds Adjuvant (Invitrogen). Mice were treated intraperitoneallywith a daily dose equal to 4 mg/kg andrographolide in PBS since theday of sensitization. One week after immunization, animals wereintracutaneously challenged in the ear with 50 g of OVA dissolvedin 20 l of PBS. Ear thickness was measured at different times afterchallenge with a micrometer (Mitutoyo, Tokyo, Japan). Increased earthickness was expressed as the means of at least three measure-ments per mouse in millimeters 102 S.E.

Results

In Vitro T Cell Activation Is Inhibited by Androgra-

pholide. The ability of andrographolide to interfere with Tcell activation was evaluated in a mix lymphocyte reactionbetween C57BL/6 and BALB/c splenocytes. As shown in Fig.1A, in this assay, T cell proliferation and IL-2 release wereinhibited by andrographolide in a dose-dependent fashion.No measurable effect on background proliferation and IL-2release was observed (data not shown).

To determine whether this was an effect on the T cells orthe APCs, an antigen presentation assay was set up withbone marrow-derived DCs pulsed in vitro with OVA andcocultured either with H-2Kb/OVA257-264- or I-Ab/OVA265-280-specific T cell hybridomas (B3Z and OT4H, respectively). Asshown in Fig. 1, B and C, treating DCs with andrographolidebefore OVA pulse prevented them from activating both CD4

and CD8 OVA-specific T cell hybridomas. This inhibitionwas only observed when andrographolide-treated DCs werepulsed with whole OVA protein and not when OVA257-264orOVA265-280 peptides (for presentation on H-2K

b and I-Ab,respectively) were exogenously added to these cells (Fig. 1, Band C). Consistent with these findings, andrographolidetreatment had no effect on APC-independent T cell activationwith anti-CD3 (Fig. 1D). In addition, trypan blue exclusionassay shows that viability of DCs remains unaffected after

treatment with 10 M andrographolide (Fig. 1E). These dataare supported by measurements of mitochondrial functionshowing that concentrations up to 50 M andrographolidedid not affect cell viability (Habtemariam, 1998; Chiou et al.,2000).

Thus, our results suggest that, at the concentration tested,andrographolide inhibits the ability of DCs to process OVA andgenerate the peptide-MHC complexes required for T cell acti-vation. To test this notion, the ability of andrographolide-treated DCs to process and present OVA-derived peptides onMHC-I was evaluated using an H-2Kb/OVA257-264-specificmonoclonal antibody (Porgador et al., 1997). In these assays,andrographolide was able to prevent processing and presenta-

tion of OVA peptides on the MHC-I molecule H-2Kb

(Fig. 2A).

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To further evaluate the effect that andrographolide couldhave on DC function, maturation of DCs was induced by LPStreatment in the presence of andrographolide. As shown inFig. 2, B and C, andrographolide also inhibited up-regulationof the maturation markers I-Ab, CD40, and CD86 (B7.2) inresponse to LPS.

In Vivo T Cell Function Is Suppressed by Androgra-

pholide Treatment. Data shown above suggested that an-

drographolide could be able to interfere with the initiation of an

immune response by inhibiting antigen presentation by DCs,which is required for T cell priming. To test whether androgra-pholide was also able to affect in vivo immune responses,C57BL/6 mice were treated with the molecule and immunizedwith NP17-BSA (a thymus-dependent antigen) adsorbed toalum. Seven days postimmunization, anti-NP IgG titers weredetermined by ELISA using NP23-CGG as antigen. Comparedwith untreated controls, significantly reduced anti-NP IgG ti-

ters were observed for andrographolide-treated mice (Fig. 3A).

Fig. 1.In vitro T cell activation is inhibited by andrographolide. A, mixed lymphocyte reaction is inhibited by andrographolide. Lymph node cellsuspensions from C57BL/6 and BALB/c mice were coincubated as described underMaterials and Methods. Andrographolide was added at the indicatedconcentrations to the cells. Proliferation (filled squares) and IL-2 release (empty circles) were determined after 72 h of culture. B and C, T cellactivation by antigen-pulsed DCs is suppressed by andrographolide. Control DCs (filled squares) and andrographolide-treated DCs (empty squares)were pulsed with OVA protein and cocultured either with H-2Kb/OVA257-264-specific (B) or I-A

b/OVA265-280-specific (C) T cell hybridomas. As controls,andrographolide-treated DCs were pulsed with OVA-peptides SIINFEKL or TEWTSSNVMEERKIKV for presentation on H-2Kb or I-Ab, respectively(empty triangles) (,p 0.01 when compared with controls, Students ttest). D, andrographolide treatment did not affect antigen-independent T cellactivation. B3Z (filled squares) or OT4H (filled circles) T cell hybridomas were stimulated with plate-bound anti-CD3. Plates treated with vehicle wereincluded as controls (empty squares for B3Z and empty circles for OT4H). Andrographolide was added at the indicated concentrations, and IL-2 releasewas determined after 24 h of culture. E, DC viability remains unaffected after treatment with andrographolide, based on trypan blue exclusion assay.Data shown are means of at least four independent experiments.

Fig. 2.Antigen processing by DCs and DC maturation are inhibited by andrographolide. A, ability of DCs to process and present OVA-derived peptideson H-2Kb was abolished by andrographolide (top, untreated DCs; bottom, treated DCs). Histograms show fluorescence intensity from binding of25-D1.16 mAb to H-2Kb/SIINFEKL on CD11c cells (shaded and clear histograms show control or OVA-pulsed DCs, respectively). B, up-regulationof maturation markers I-Ab, CD40, and CD86 in response to LPS is inhibited by andrographolide. Histograms show surface expression of maturationmarkers on CD11c cells. Shaded histograms indicate immature DCs, and white histograms represent LPS-treated DCs (top, control DCs; bottom,treated DCs). C, bar graphs show the increase of mean fluorescence intensity for I-Ab, CD86, and CD40 (CD11c gate). Black bars represent controlDCs, and white bars indicate andrographolide-treated DCs. Data shown are means of at least three independent experiments.


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To evaluate the effect of andrographolide directly on T celleffector function, delayed-type hypersensitivity (DTH)against OVA was induced in control and andrographolide-treated mice. As shown in Fig. 3B, andrographolide almostcompletely suppressed DTH induced by OVA immunizationand challenge in C57BL/6 mice.

Andrographolide Treatment Significantly Reduces

Severity of Experimental Autoimmune Encephalomy-

elitis in the Mouse.To evaluate whether inhibition of T cellactivation and antibody production by andrographolide couldmodulate an autoimmune response, C57BL/6 mice were

treated with andrographolide and induced to develop EAE byinjection of MOG, as described under Materials and Methods.On day 14 after sensitization, control (solvent injected)C57BL/6 mice showed signs of disease (Fig. 4) and a progres-sion of EAE symptoms equivalent with the observed in pre-vious reports (Bright et al., 2003; Gilgun-Sherki et al., 2003).

In contrast, treatment with andrographolide not only de-layed the onset of EAE but also significantly reduced theseverity and incidence of disease (Fig. 4; Table 1).

Reduced Antimyelin T Cell and Antibody Response

in Andrographolide-Treated Mice.The diminished EAEincidence and severity resulting from andrographolide treat-ment in the mouse could be due either to an interference withautoreactive T cell activation and antibody production or to a

nonspecific anti-inflammatory effect of this compound. Toapproach this issue, 3 weeks after EAE induction, lymphnodes were obtained from control and andrographolide-treated mice to evaluate cytokine release in response to MOGpeptide. As shown in Fig. 5A, IFN-and IL-2 secretion wasobserved only in lymph node suspensions obtained from un-treated mice suffering from EAE. In contrast, neither IFN-nor IL-2 could be detected in supernatants from MOG-stim-ulated lymph node cell suspensions derived from androgra-pholide-treated mice. Consistent with these observations, an-ti-MOG IgG could only be measured in sera from controlanimals suffering from EAE, whereas andrographolide-treated animals showed an almost complete absence of anti-

MOG antibody titers (Fig. 5B). Thus, it seems likely thatandrographolide treatment reduced EAE severity by impair-ing T cell priming by DCs, which could also indirectly affectantibody production against myelin antigens.

Discussion

New immunomodulatory therapeutic strategies are re-quired to prevent or treat autoimmune diseases such asmultiple sclerosis. Here, we provide evidence for a potentialrole of a bicyclic diterpenoid lactone, known as andrographol-ide, as an inhibitor of DC function able to down-modulate Tcell-mediated immunity and ameliorate EAE in the mouse.When tested on a series of in vitro and in vivo assays, an-

drographolide was shown to interfere with the capacity ofantigen-pulsed DCs to activate T cells.

Andrographolide was able to prevent OVA-pulsed DCsfrom activating either CD4 or CD8 T cell hybridomas. Thisresult is consistent with the absence of H-2Kb/OVA257-264complexes on the surface of DCs treated with andrographol-ide at the time of OVA pulse. Thus, it seems likely thattreatment with this molecule prevented processing and pre-sentation of OVA peptides on MHC molecules to OVA-spe-cific T cells.

In addition to impairing generation of peptide-MHCcomplexes, andrographolide reduced the efficiency of DCmaturation in response to LPS. Thus, when fold increase in

surface expression was evaluated, reduced LPS-induced up-

Fig. 3. In vivo T cell function is suppressed by andrographolide treat-ment. A, T cell-dependent antibody production is diminished by an-drographolide. Control (filled circles) and andrographolide-treatedC57BL/6 (empty circles) were immunized with NP

17-BSA as described

under Materials and Methods. Seven days postimmunization, anti-NPIgG titers were determined by ELISA using NP

23-CGG as antigen

(,p 0.01; ,p 0.001, Studentst test). B, delayed-type hypersen-sitivity is reduced by andrographolide treatment. Control (filled circles)and andrographolide-treated C57BL/6 (empty circles) mice were immu-nized with OVA as described under Materials and Methods. One weeklater, mice were challenged on the ear with OVA dissolved in PBS. Earthickness was measured at different times after challenge (, p 0.01;

, p

0.001, Studentst test). Data shown are means of two indepen-dent experiments.

Fig. 4. Experimental autoimmune encephalomyelitis is reduced by an-drographolide treatment. EAE was induced by immunizing control (filledcircles) or andrographolide-treated (empty circles) mice with MOG

35-55

peptide as described under Materials and Methods. Clinical score wasdetermined according to the criteria described under Materials and Meth-ods (p 0.05, from Mann-Whitney rank sums two-tailed analysis, ap-plied to the entire data set). The analyses for each time period afterimmunization between the two groups show statistically significant dif-ferences during peak severity (weeks 36,p 0.001, Students t test) andno significant differences in residual clinical signs (after 6 weeks). Datashown represent average weekly clinical scores of all animals in the group

and are means of four independent experiments.

TABLE 1Summary of EAE disease parametersEAE was induced by immunizing control or andrographolide-treated mice withMOG35-55 peptide, as described under Materials and Methods. Clinical score wasevaluated on a daily basis, and mean maximum score was calculated by addingclinical scores at the peak of disease for animals that develop EAE in each group andthen divided by the total number of animals.

Group Incidence MaximumScore

MeanMaximum

Score

Day ofOnset

mean S.E.

Control 14/14 (100%) 5 3.7 0.1 14.7 0.2Andrographolide 8/17 (47%) 3 1.9 0.1* 17.6 0.5*

* p 0.05, compared with the control, unpaired Students t test.


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regulation of maturation markers I-Ab, CD40, and CD86(B7.2) was observed as result of andrographolide treatment.Although it was apparent that andrographolide slightly mod-ified basal expression of I-Ab, CD86, and CD40, these differ-ences were not statistically significant. The ability to inter-fere with DC maturation suggests an explanation for theinhibition of T cell activation observed in vivo after androgra-pholide treatment (see below).

Accordingly, the in vitro inhibition of T cell activationcaused by andrographolide is consistent with the suppressionof the immune response in the mouse, as shown on threedifferent experimental assays designed to measure immune

system function in vivo. Thus, antibody (IgG) secretionagainst the T cell-dependent antigen NP17-BSA was signifi-cantly reduced by andrographolide treatment. Similarly, theDTH response against the antigen OVA was diminished tobackground levels by treatment with andrographolide. Theseresults support the notion that T cell-mediated immune re-sponses can be effectively impaired by this molecule.

Finally, we evaluated whether the capacity of androgra-pholide to impair T cell activation could be applied to preventthe onset of EAE in mice. As shown in Fig. 4 and Table 1,andrographolide treatment significantly reduced both theincidence and clinical severity of EAE in C57BL/6 mice dur-ing early phase of disease. Residual clinical signs were not

significantly changed by treatment with this molecule. Clin-

ical data were consistent with the observation that lymphnode cellular suspensions derived from andrographolide-treated mice showed reduced IFN-and IL-2 release in re-sponse to MOG (Fig. 5A), two important pro-inflammatorycytokines that participate in EAE pathogenesis (Lassmannet al., 2001; Lucchinetti et al., 2000; Wingerchuk et al., 2001).These data support the notion that the beneficial effects ofandrographolide are mediated preferentially by specific in-

terference with antigen presentation by DCs and, thus, withT cell activation, and they correlate with the clinical scoresshown by the animals. Further research is required to eval-uate the potential therapeutical capacity of andrographolidewhen administered after symptoms of EAE have started.

The reduced in vivo T cell priming, which is probablyresponsible for the decreased DTH and EAE responses ob-served in andrographolide-treated mice, could result fromthe impairment on DC maturation and generation of peptide-MHC complexes caused by andrographolide. However,whether this molecule is directly altering DC function in vivoremains to be evaluated.

In addition to activation of autoreactive T cells during the

sensitization phase, EAE pathogenesis involves several in-flammatory mediators, which are also responsible for myelindamage. Recent studies provide evidence suggesting thatandrographolide could also interfere with the function ofinflammatory cells such as neutrophils and microglia (Bat-khuu et al., 2002; Shen et al., 2002; Wang et al., 2004).Because these inflammatory cells have been implicated inthe pathogenesis of inflammation in MS (Calabrese et al.,2002; Smith and Lassmann, 2002; Hill et al., 2004), therelative contribution of andrographolide to diminish adaptiveversus nonspecific inflammation needs to be defined. Fur-thermore, an antiapoptotic activity has been shown for an-drographolide (Chen et al., 2004), which could also contributeto reduce severity of EAE in the mouse by increasing neuro-nal resistance to cell death induced by local inflammation.Thus, it is likely that andrographolide interferes with EAEby preventing activation of autoreactive T cells and by reduc-ing inflammatory damage.

In summary, the data presented here suggest that an-drographolide is able to modulate T cell activation both invitro as well as in vivo. The exact mechanism by whichandrographolide exhibits its beneficial effect on EAE is stillnot fully elucidated, but we provide evidence it could preventinitial T cell priming by interfering with DC maturation andantigen presentation capacity. Therefore, andrographolidemay have utility as a therapeutic agent for the treatment ofautoimmune diseases, such as multiple sclerosis.

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Address correspondence to:Dr. Alexis M. Kalergis, Departamento de Ge-

netica Molecular y Microbiologa, Facultad de Ciencias Biologicas, PontificiaUniversidad Catolica de Chile, Alameda 340, Santiago, Chile. E-mail:[email protected]


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