International Immunology

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International Immunology, Vol. 12, No. 10, 1449-1457, October 2000
© 2000 Japanese Society for Immunology

Oral administration of cholera toxin B subunit conjugated to myelin basic protein protects against experimental autoimmune encephalomyelitis by inducing transforming growth factor-ß-secreting cells and suppressing chemokine expression

Jia-Bin Sun¹, Bao-Guo Xiao², Marianne Lindblad¹, Bin-Ling Li¹, Hans Link², Cecil Czerkinsky^1,3 and Jan Holmgren¹

¹ Department of Medical Microbiology and Immunology, Göteborg University, 413 46 Göteborg, Sweden
² Division of Neurology, Karolinska Institute, Huddinge Hospital, 141 86 Stockholm, Sweden

Correspondence to: J. Holmgren

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
The efficacy and mechanism of immunosuppression against experimental autoimmune encephalomyelitis (EAE) by oral low-dose administration of myelin basic protein (MBP) conjugated to cholera toxin B subunit (CTB) were investigated in Lewis rats immunized with MBP together with complete Freund's adjuvant 4 days before the start of treatment. Oral treatment with CTB–MBP conjugate gave almost complete protection against disease, an effect that was totally abrogated by including a low dose of cholera holotoxin (CT). The protection by CTB–MBP was associated with a dramatic reduction in the number of leukocytes staining for CD4, CD8, IL-2R or MHC class II in the spinal cord as examined by immunohistochemistry. The mRNA expressions of T_h1 cytokines IFN-, IL-12 and tumor necrosis factor-, as well as of chemokines monocyte chemotactic protein (MCP)-1 and RANTES in the spinal cord were also reduced by 76–94%, as assessed by in situ hybridization. In contrast, transforming growth factor (TGF)-ß mRNA-expressing cells were strongly increased in the spinal cord from animals treated orally with the CTB–MBP conjugate. In the draining peripheral lymph nodes, the number of MBP-specific TGF-ß mRNA-expressing cells was also increased, whereas there was a decrease in cells expressing T_h1 or T_h2 cytokine mRNA. Protection against EAE could be transferred by injection of cells from the mesenteric lymph nodes of animals fed with CTB–MBP into naive animals exposed to encephalitogenic T cells. The results indicate that the protective anti-inflammatory effect by oral treatment with CTB–MBP conjugate is, to a large extent, due to the induction of TGF-ß-secreting suppressive-regulatory T cells and to local down-regulation of MCP-1 and RANTES in the spinal cord.

Keywords: cholera toxin B subunit, experimental autoimmune encephalomyelitis, myelin basic protein, transforming growth factor-ß

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Experimental autoimmune encephalomyelitis (EAE), an inflammatory disease of the central nervous system (CNS), has been widely used as an animal model for testing novel therapeutic approaches for multiple sclerosis. EAE is mediated by myelin-reactive CD4⁺ T cells that develop after injection of myelin basic protein (MBP) emulsified in an appropriate adjuvant, e.g. complete Freund's adjuvant (CFA), in susceptible animals (1–3). Consistent with this, EAE can also be induced in naive animals by transfer of MBP-activated T cells (4).

Based on the concept of `oral tolerance', oral administration of MBP has been shown to protect against EAE in rats (5,6). However, to be effective, the treatment typically requires administration of multiple, relatively large (milligram) doses of MBP antigen. In recent clinical trials in patients with multiple sclerosis only modest and inconsistent therapeutic effects have been achieved by oral treatment with myelin antigens alone. It seems clear that for oral tolerance therapy to become clinically effective there will be a need to combine the oral antigen administration with a suitable tolerance-enhancing agent or formulation (7).

Cholera toxin B subunit (CTB) has emerged as a particularly promising system for increasing the oral tolerogenic potency of various antigens (8). We recently described that the conjugation of various antigens, including MBP, to CTB could dramatically enhance the efficacy of oral tolerance induction and with appropriate autoantigens also the level of protection against several experimental autoimmune diseases (9–11). Thus, we observed that a single feeding with microgram amounts of a CTB–MBP conjugate could effectively protect against both clinical and histopathological manifestations of EAE in Lewis rats, even after disease induction (9). Our previous work suggests that CTB acts both by promoting the uptake of effective amounts of the tolerizing antigen across the mucosa and by presenting it more efficiently to antigen-presenting cells (12 and unpublished manuscript). However, the specific effects of mucosal administration of CTB- conjugated antigen on different T cell subsets, cytokines and chemokines, and their relation to oral tolerance and protection, remain largely unknown.

In this study, we have examined the mechanisms of immunosuppression in EAE in Lewis rats. We show that oral treatment with CTB–MBP almost completely protects rats immunized with MBP/CFA against EAE. The protection was found to result in a dramatic reduction of leukocyte infiltration into the CNS, and to be associated with increased transforming growth factor (TGF)-ß mRNA expression and decreased expression of T_h1 cytokines IFN-, IL-12 and tumor necrosis factor (TNF)-, as well as of chemokines monocyte chemotactic protein (MCP)-1 and RANTES in the CNS. The increased expression of TGF-ß was also seen with cells from the draining lymph nodes, as well as with mesenteric lymph node (MLN) cells from CTB–MBP-treated animals. Furthermore, the protection against EAE could be transferred to naive animals by the i.v. injection of isolated MLN T cells from protected CTB–MBP-treated rats, indicating that regulatory TGF-ß-expressing T cells play an important role in oral tolerance and immunosuppression after oral treatment with CTB–MBP conjugate.

	Methods

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Animals and antigen preparations
Lewis rats were purchased from Harlan (Bicester, UK). The animals were used in experiments at the age of 7–8 weeks old. Guinea pig MBP and ovalbumin (OVA) were purchased from Sigma (St Louis, MO) and cholera toxin (CT) from List (Campell, CA). CTB was produced in a mutant strain of Vibrio cholerae deleted of the CT genes and transfected with a plasmid encoding CTB (13). The recombinant CTB was purified to apparent homogeneity from the culture filtrate by precipitation by hexametaphosphatate followed by gel filtration chromatography through Sephadex G-100 (Pharmacia, Uppsala, Sweden).

Preparation of CTB–MBP or CTB–OVA conjugates
MBP was covalently conjugated to CTB using N-succinimidyl (3-[2-pyridyl]-dithio)propionate (SPDP) as a bifunctional coupling reagent (14), largely according to the manufacturer's instructions (Pharmacia). Briefly, CTB and MBP were separately derivatized with SPDP by incubation (23°C, 30 min) at molar ratios of 1:2.5 and 1:5 respectively. Excess SPDP was then removed by gel filtration of the proteins through Sephadex G-25 columns (Pharmacia). The SPDP-derivatized MBP was reduced with dithiothreitol and the resulting preparation was freed of excess DTT and pyridine-2-thione by Sephadex G-25 chromatography. SPDP-derivatized CTB was mixed with the same amount of derivatized and reduced MBP, and incubated for 16 h at 23°C. The resulting CTB–MBP conjugate was purified by gel filtration through a Sephacryl S-300 column (Pharmacia). Purified conjugate was shown to have ganglioside GM₁-binding capacity and to retain both CTB and MBP serological reactivities as tested in a solid-phase ELISA using GM₁ as the capture system (15) and enzyme-labeled antibodies to CTB and MBP as detection reagents. Quantitation of free and bound MBP and free and bound CTB was done by reference to standard curves established by assaying known amounts of unconjugated antigens. The CTB–MBP conjugate contained <10% free CTB and negligible amounts of unconjugated MBP. In an analogous manner, OVA was coupled to CTB to provide a CTB–OVA conjugate in order to serve as a control.

Induction of EAE and clinical evaluation
EAE was induced by s.c. injection of 50 µg of guinea pig MBP in 100 µl PBS emulsified in an equal volume of CFA containing 5 mg/ml of Mycobacterium tuberculosis H37R (Difco, Detroit, MI) in one of the hind footpads of Lewis rats. The animals were then evaluated daily for signs of disease. Clinical severity was graded as follows: 0, no disease; 1, mild tail weakness; 2, tail paralysis and hind leg weakness; 3, complete hind leg paralysis; 4, hind leg paralysis or mild forelimb weakness; 5, death.

Oral tolerance induction
Using a baby catheter feeding tube, groups of animals were given three intragastric administrations—hereafter referred to as oral administration—with 50 µg per dose of CTB–MBP dissolved in 0.35 M sodium bicarbonate buffer 4, 6 and 8 days after immunization with MBP/CFA. Other groups of rats were treated either with three oral doses of PBS on the same days or with five doses of 1 mg free MBP per dose given every second day starting on the same day that the rats were sensitized with MBP/CFA. For cell transfer experiments, similar treatments with CTB–MBP, free MBP or PBS were given to rats that had not received any prior immunization with MBP/CFA.

Lymphocyte proliferative responses
Two weeks after immunization with MBP/CFA rats were sacrificed and the draining popliteal lymph nodes (PLN) excised. Single-cell suspensions were prepared by pressing the nodes through a stainless steel mesh. Triplicate aliquots (200 µl) of the PLN cell suspensions were added to flat-bottomed 96-well microtiter plates (Nunc, Roskilde, Denmark) at a cell concentration of 4x10⁵/ml medium. Then 10 µl aliquots of MBP were added to the wells to give a final concentration of 10 µg/ml MBP. After 60 h of incubation, the cells were pulsed for 12 h with [³H]thymidine (1 µCi/well) (Amersham, Stockholm, Sweden). Incorporation was measured with an argon-activated scintillation counter (Inotech, Basel, Switzerland) following cell harvesting. Results are expressed in terms of the stimulation index, defined as the ratio of the mean radionucleotide cell incorporation in MBP-stimulated cultures divided by the unstimulated cultures.

Enumeration of cytokine-secreting cells
Cells secreting IFN- production were detected by reverse ELISPOT assays (16) using pairs of unconjugated and biotinylated rat mAb to mouse IFN- antibodies (Genzyme, Cambridge, MA). Briefly, PLN cells (5x10⁵ cells/well) were incubated in the presence or absence of MBP (10 µg/ml) at 37°C in an atmosphere with 5% CO₂ for 24 h in nitrocellulose-bottomed wells (Millipore, Bedford, MA). These wells were previously coated with antibodies to the desired cytokine and blocked with 5% FCS medium. Plates were then washed with PBS and individual wells were exposed to the homologous biotinylated anti-cytokine reagent, appropriately diluted in PBS. After consecutive incubations with horseradish peroxidase conjugated anti-biotin antibodies (HRP; Vector, Burlingame, CA) and chromogen substrate (H₂O₂ and 3-amino-9-ethylcarbazole; H₂O₂-AEC), plates were thoroughly washed with tap water and examined for the presence of brown-colored spots; the brown-colored spots were enumerated under low magnification.

Adoptive transfer of tolerance
Lymphocytes were prepared from pooled MLN of previously unimmunized rats given three 50 µg doses of CTB–MBP orally 7, 10 and 13 days earlier. These putatively `tolerogeneic cells', consisting of 3x10⁷ in 200 µl saline, were transferred by i.v. injection into the tail veins of normal recipient rats on either the same day or 4 days before or 4 days after i.v. injection comprising 3x10⁷ encephalitogenic cells. These latter pathogenic MBP-activated T lymphocytes were prepared as previously described (3,17). Briefly, 10 days after immunization with MBP/CFA, popliteal and inguinal lymph nodes were collected and teased to yield single-cell suspensions. Cells were washed and cultured (5x10⁶/cells/ml in 2 ml volume) in 24-well plates for 4 days at 37°C with 6% CO₂ in RPMI medium supplemented with 10% FCS in the presence of 50 µg/ml MBP. At the end of the culture, cells were harvested, washed and resuspended in saline. Each recipient received 200 µl of cell suspension containing 3x10⁷ viable cells via a tail vein.

Immunohistochemistry
Spinal cords from sacrificed rats were dissected and segments of the lumbar spinal cord were snap-frozen in liquid nitrogen. Cryostat sections (5 µm) were exposed to appropriate dilutions of the following mouse mAb: W3/25 (anti-rat CD4, T_h and macrophages), OX8 (anti-rat CD8 and T cytotoxic/suppressor cells), CD25 (anti-rat IL-2 receptor) and OX6 (anti-rat MHC class II). All of these antibodies were purchased from Serolab (Crawley-Down, UK). Sections were stained according to the avidin–biotin technique (Vectastain Elite; Vector). Omission of the primary antibody served as a negative control. The specific stain infiltrates were counted in whole-tissue areas by using x20 magnification. The tissue areas were measured by Image analysis using the SeeScan Image Analysis System (Cambridge, UK). The numbers of the stained cells and infiltrates were calculated per 100 mm² of tissue area. CD4⁺, CD8⁺ and CD25⁺ cells, in addition to MHC class II⁺ cells, were semi-quantitatively scored using coded sections. The whole area of each section was examined at x20 magnification for each marker staining. The indicated scoring values are the mean values of the results from eight to 10 rats.

Detection of mRNA expression of TGF-ß in draining lymph node cells
Expression of TGF-ß mRNA was examined with in situ hybridization as described elsewhere (18). Briefly, 200 µl aliquots containing 4x10⁵ PLN mononuclear cells were applied to 96-well round-bottomed microtiter plates (Nunc) in triplicate. Then 10 µl aliquots of MBP or concanavalin A (Con A) were added to a final concentration of 10 µg/ml for MBP and 2 µg/ml for Con A in the cell culture, and 24 h later 1x10⁵ of the cells were washed, counted and dried onto electrically charged glass ProbeOn slides (Fisher Scientific, Pittsburgh, PA). Synthetic oligonucleotide probes (Scandinavian Gene Synthesis, Köping, Sweden) were labeled using [³⁵S]deoxyadenosine-5'--(thio)-triphosphate (New England Nuclear, Boston, MA) with terminal deoxynucleotidyl transferase (Amersham, Little Chalfont, UK). A mixture of two different oligonucleotide probes was employed for the cytokine. The oligonucleotide sequences were obtained from GenBank using MacVector software. Cells were hybridized with 10⁶ c.p.m. of labeled probe per 100 µl of hybridization mixture. After emulsion autoradiography, development and fixation, the coded slides were examined by dark field microscopy for positive cells defined as containing >15 grains per cells in a star-like pattern. The intracellular grains were calculated by light microscopy and the number of labeled cells was counted. A negative control probe was used in parallel with the specific cytokine probes on cells from each specimen and did not give any staining of cells.

Detection of cytokine and chemokine mRNA expression in CNS
The same snap-frozen segments of lumbar spinal cords were used as those for immunohistochemistry. Cryostat sections (14 µm) were prepared and thaw-mounted onto ProbeOn slides. The technique was carried out according to Dagerlind et al. (19). Mixtures of four different labeled synthetic oligonucleotide probes were employed for each of the tested chemokines and cytokines. Chemokines examined included MCP-1 and RANTES (regulated upon activation, normal T cell expressed and secreted chemokine) and examined cytokines IL-4, TNF-, IFN-, IL-12 and IL-10, as well as TGF-ß; for TGF-ß, a mixture of two rather than four probes was used. The oligonucleotide sequences were obtained from GenBank and probes were designed using MacVector software. In situ hybridization was performed as described above for detection of cytokine mRNA expression in PLN cells. Results are expressed as number of labeled cells per 100 mm² tissue section based on examination of whole tissue sections and using the Image analysis system to measure the tissue section. Results of chemokine expression were expressed as number of labeled cells per field of microscopy (x40 magnification) from spinal cord sections.

Statistics
Statistical significance between groups was, when not stated otherwise, tested using the Wilcoxon rank-sum test. Significance was reached at *P < 0.05 or **P < 0.01.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Oral low-dose treatment with CTB–MBP conjugate protects rats against EAE
Groups of Lewis rats (eight to 10 animals per group) were s.c. immunized with MBP emulsified in CFA to induce acute EAE. At 4, 6 and, 8 days after immunization, animals were given three oral doses of CTB–MBP (50 µg per dose), MBP alone (50 µg per dose) or PBS. Clinical signs of EAE were scored daily in a blinded fashion. All PBS-treated animals developed severe EAE associated with paralysis, whereas both the severity of the disease and the incidence of paralysis were markedly reduced in the animals treated with the CTB–MBP conjugate (Fig. 1). In contrast, feeding three similar doses of unconjugated MBP, whether given alone or mixed with free CTB or of an irrelevant conjugate (CTB–OVA), had no effect on EAE development in reference to the PBS-treated group (data not shown). However, more intense oral treatment with free MBP, five doses with 1 mg per dose, did reduce the clinical score of EAE to a level comparable to that achieved by the low-dose CTB–MBP treatment (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Oral treatment with CTB–MBP suppresses acute EAE. Rats were immunized with MBP/CFA and then (4, 6 and 8 days later) treated with three oral 50 µg doses of CTB–MBP conjugate either alone or together with 15 µg CT. Other groups of MBP/CFA immunized rats received oral treatment with five 1 mg doses of unconjugated MBP or as control three doses of PBS as described in Methods. Clinical scores of EAE were assessed daily as described in Methods.

 
Oral CTB–MBP conjugate treatment prevents leukocyte infiltration and MHC class II expression in CNS
The numbers and surface-marker phenotypes of infiltrating inflammatory cells in the spinal cord were determined in rats injected with MBP in CFA and subsequently orally treated with MBP, either conjugated to CTB, given alone or treated with PBS. Animals treated with either five doses of free MBP (1 mg per dose) or three doses of CTB–MBP conjugate (50 µg per dose) had a marked decrease in the number of leukocytes in CNS with only a few cellular infiltrates and a marked reduction in all T cell markers examined, as well as in MHC II-expressing cells, and this effect was especially pronounced with the conjugated antigen (Table 1). In the CNS from animals fed with PBS, the spinal cord contained abundant CD4⁺ and activated IL-2R⁺ T lymphocytes, as well as CD8⁺ T cells and MHC II-expressing cells (Table 1).

View this table: [in this window] [in a new window]   Table 1. Immunohistochemical staining for infiltrating lymphoid cells expressing different surface markers and MHC class II antigen in the spinal cord of EAE-induced rats after different oral treatmentsa

 
Oral treatment with CTB–MBP decreases cytokine IL-12, IFN- and TNF- mRNA-expressing cells in the spinal cord but increases TGF-ß mRNA-expressing cells
To study the expression of different cytokines in the target tissue for EAE, rats were first injected with MBP in CFA and then treated orally with 3x50 µg of CTB–MBP conjugate, 5x1 mg of MBP alone or PBS. Cells in the spinal cord specimens expressing mRNA for IL-12, IFN-, TNF- and TGF-ß were assayed by using in situ hybridization with complementary DNA oligonucleotide probes. A greater reduction of IL-12 and IFN- mRNA expression was observed in the spinal cord of rats treated with either oral MBP-CTB or MBP than in rats treated with PBS (Fig. 2). TNF- mRNA was also lower in the spinal cord from animals treated with CTB–MBP or MBP as compared with PBS-treated animals (Fig. 2). Most importantly, in comparison with the PBS-treated group, animals treated with CTB–MBP or MBP showed a much increased expression of TGF-ß mRNA (Fig 2). IL-10 mRNA-expressing cells, on the other hand, did not increase in CNS from animals fed with CTB–MBP and IL-4 mRNA expression was even significantly reduced in the CNS of animals treated with CTB–MBP (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effects of oral treatment with CTB–MBP and other regimens on the numbers of mRNA-expressing cells for cytokines IL-12, IFN-, TNF- and TGF-ß, and for chemokines MCP-1 and RANTES in spinal cords of Lewis rats with EAE. Each column indicates the mean + SD of the number of mRNA-expressing cells. The number of mRNA-expressing cells was expressed per 100 mm2 of spinal cord tissue section (eight to 10 animals per group).

 
Oral treatment with CTB–MBP decreases chemokines MCP-1 and RANTES mRNA-expressing cells in the spinal cord
Expression of mRNA for chemokines MCP-1 and RANTES in the spinal cord was investigated in EAE animals by in situ hybridization. A marked reduction in MCP-1 and RANTES mRNA expression was observed in the spinal cord of rats orally treated with 3x50 µg of CTB–MBP as compared with animals treated with PBS (Fig. 2). At variance with the cytokine mRNA expression pattern, treatment with five 1 mg doses of free MBP did not result in any reduction of these chemokines (Fig. 2).

Suppression of proliferative responses, IFN- production and up-regulation of TGF-ß mRNA-expressing cells in draining lymph nodes by oral CTB–MBP conjugate
Lymphocyte proliferative responses to MBP in vitro (Fig. 3A) and MBP-specific IFN--producing cells (Fig. 3B) were reduced in PLN cell cultures from rats primed by a s.c. injection with MBP in CFA and then treated orally with three 50 µg doses of CTB–MBP or five 1 mg doses of MBP as compared with corresponding cultures from rats treated with PBS. In contrast, the levels of MBP-induced TGF-ß mRNA were significantly higher in PLN cells from CTB–MBP-treated or MBP-treated rats than from PBS-treated rats (Fig. 3C), while the numbers of IFN--producing cells were reduced (Fig. 3B).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Oral treatment with CTB–MBP decreases T cell proliferation (A) and IFN- production (B), but increases TGF-ß mRNA expression (C) in draining PLN cells. The treatment groups correspond to those in Fig. 1 and cells were examined 14 days after immunization. Lymphocyte proliferative responses to MBP of cells from PLN were determined after 60 h of incubation and the cells were pulsed for 12 h with [3H]thymidine; the stimulation index results refer to ratio of radionucleotide uptake in MBP-stimulated as compared with unstimulated cultures. MBP-specific cells secreting IFN- were detected by a reverse ELISPOT assay. Data are expressed as mean + SD numbers of cytokine-producing cells per 106 PLN cells. TGF-ß mRNA-expressing cells were determined by in situ hybridization after 24 h of cell cultures in vitro in the presence of MBP. TGF-ß mRNA-expressing cells are expressed as mean + SD numbers per 106 PLN cells. *P < 0.05 and **P < 0.01 by Wilcoxon's rank sum test.

 
T cells can transfer tolerance induced by feeding CTB–MBP conjugate
To examine whether lymphocytes taken from orally CTB–MBP-treated rats can modulate EAE development, MLN lymphocytes taken after oral treatment with three 50 µg doses of CTB–MBP conjugate were transferred by i.v. injection into recipient rats at different times relative to the induction of EAE achieved by the co-transfer of in vitro MBP-activated T lymphocytes. The results show that the co-transfer of these `oral tolerance' T cells, in comparison with the co-transfer of `non-tolerance' T cells from PBS-treated animals, together with in vitro activated MBP-specific T cells (`pathogenic T cells') were found to markedly suppress the development of EAE (Fig. 4). Meanwhile, part of the MLN lymphocytes taken after the oral treatment with CTB–MBP conjugate were separately assessed for expression of TGF-ß mRNA following 24 h cell culture in the presence of MBP by in situ hybridization as described above. The results show that the `oral tolerance' immunosuppressive MLN T cells had a marked elevation of TGF-ß expression as compared with the concurrently tested MLN cells from PBS-treated rats (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Protective effect against EAE development by transfer of MLN cells induced by oral administration of CTB–MBP conjugate. EAE was induced by i.v. injection of pathogenic MBP-activated T cells (3x107/rat) from EAE animals into the recipients (four to six rats per group), who then also received regulatory-suppressive cells (3x107/rat) from MLN of animals fed with CTB–MBP on the same day or 4 days before the transfer of the EAE-inducing T cells, or as control, MLN cells from PBS-fed animals (`non-tol. cells').

 
Cholera toxin concomitantly blocks TGF-ß expression and oral tolerance
In early experiments, we observed that CT effectively blocked the oral tolerance induced by CTB–antigen conjugates (8). In our present work, when testing the effect of CT on EAE, CT (15 µg p.o.) was found to substantially accelerate the onset of EAE disease and completely abrogate the protective effect of the normally effective treatment with CTB–MBP conjugates (Fig. 1). Consistent with the clinical manifestations, a massive infiltration of T lymphocytes expressing CD4⁺, CD8⁺, IL-2R⁺ and/or MHC class II antigen in the spinal cord was also evident in the CT-treated animals as assessed by immunohistochemistry (Table 1). Moreover, oral administration of CTB–MBP could, when given together with CT, no longer suppress any of the inflammatory cytokines IL-12, IFN- or TNF-. Instead, when CTB–MBP was given together with CT (15 µg), the TGF-ß expression in the CNS was completely suppressed (Figs 2 and 3).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous work from our laboratory has shown that oral administration of even small amounts of MBP conjugated to the non-toxic mucosa-binding molecule CTB can both prevent and reverse the induction of EAE in Lewis rats (9). The findings presented here confirm and extend our previous work. Oral treatment with CTB–MBP conjugate gave, as expected from our previous work excellent, almost complete protection against development of EAE disease in Lewis rats. The protection was associated with a marked reduction in inflammatory cell infiltration into the CNS and with decreased expression of different pro-inflammatory T_h1- and T_h2-type cytokines as well as CC chemokines and various cell surface molecules. Immunohistological examination revealed that oral administration of CTB–MBP notably reduced the number of lymphoid cells of all examined phenotypes in the CNS, including CD4⁺ T cells and activated T cells expressing IL-2R molecules. Furthermore, animals orally treated with CTB–MBP exhibited a marked reduction in the expression of MHC class II antigen in comparison with placebotreated control animals with EAE. These effects were more pronounced after treatment with CTB–MBP than after treatment with MBP alone, even when the latter treatment comprised much larger and more frequent doses of antigen.

Several mechanisms have been described to explain oral tolerance associated with suppression of EAE disease. Feeding multiple very high doses (20 mg) of MBP can result in anergy or clonal deletion of encephalitogenic T cells (20); tolerance after feeding multiple lower doses of antigens (5 mg) is instead described to be largely mediated by active suppression by regulatory T cells (21). In the latter situation, the production of the cytokines TGF-ß, IL-4 and IL-10 is described to suppress the activity of the disease-inducing T cells (22–24). Our findings here show that oral immunotherapy with even very low doses (150 µg) of CTB–MBP effectively leads to the development of regulatory T cells associated with the production of elevated levels of TGF-ß. We found that TGF-ß mRNA-expressing cells were strongly elevated in the CNS of rats fed with CTB–MBP conjugate, whereas lymphoid cells of all other phenotypes and cytokine expression profiles, as well as cells expressing MHC class II antigen, were markedly decreased in comparison with placebo-treated animals with EAE. Of particular interest is our finding that the oral treatment with CTB–MBP did not give rise to any increase in putative regulatory cells producing T_h2 cytokines IL-4 and IL-10 in the CNS. Thus, the local production of T_h2 cytokines at effector sites, including IL-4 and IL-10, may not be involved in the prevention of EAE development in animals fed with CTB–MBP conjugate. Instead, a strong increase in T cells expressing TGF-ß in protected animals supports the notion that a special type of regulatory T cells selectively producing TGF-ß, the so-called T_h3 cells (21), are the key effector cells in tolerance induced by CTB–MBP treatment. Thus, these T cells can effectively suppress both T_h1 and T_h2 cytokine production in the CNS.

A major unresolved question is the site of action of CTB to induce tolerance. To resolve this issue, we examined the effect of oral treatment with CTB–MBP on cells expressing different cytokines in MLN, which are putative inductive and/or draining sites for oral tolerance. We found that this treatment induced a significant increase both in cells producing TGF-ß and in cells producing IL-4 as compared with control animals (data not shown). These findings imply that oral treatment with CTB–MBP activates a pre-regulatory IL-4-producing T_h2 cell population at mucosal inductive sites. This population, in turn, may promote the development of regulatory TGF-ß-producing cells that can migrate into the CNS and/or its draining peripheral lymph nodes to suppress T_h1 and T_h2 cells and cytokines. This theory concurs with the recent report that IL-4 facilitates the generation of T cells secreting TGF-ß (25).

The suppression in most cytokines except TGF-ß, including both T_h1 and T_h2 types of cytokines, noted after the protective treatment with CTB–MBP was seen not only in CNS but also in draining PLN. This contrasts with our earlier report that IFN- was instead elevated in PLN after protective CTB–MBP treatment (9). The reason for this discrepancy in IFN- findings is under investigation. Our preliminary data suggest that different CTB–MBP conjugates may have differential effects on IFN- production, although they have consistent effects on other cytokines and chemokines (A. George et al., unpublished). The findings anyway support our previous notion (9) and that of others (26) that IFN- may not be critical for mediating EAE. Thus, while early reports suggested a disease-promoting role of IFN- (27,28), recent studies have questioned such a role. Thus, mice with the disrupted IFN- gene have been shown to be susceptible to the induction of EAE (26) and it has also been found that treatment with mAb against IFN- led to worsening of EAE (29,30). It has been further reported that parenterally induced tolerance with model antigen was associated with decreased IL-2 and increased IFN- production in draining lymph nodes (31).

CT has been found to be a potent mucosal adjuvant and oral administration of tiny amounts of CT can also effectively abrogate mucosal tolerance induction (30,31). CTB comprises the same binding region as CT but lacks the toxic-active A subunit responsible for ADP-ribosylation of G proteins and cyclic AMP formation in cells exposed (32). In this study, we found that CT completely abrogated the protection against EAE and the associated increase in TGF-ß production in the CNS normally obtained by oral treatment with CTB–MBP. Consistent with these findings, co-administration of CTB–MBP and CT led to increased production of both T_h1 and T_h2 cytokines in the CNS. The results indicate that CT specifically interrupts the induction of TGF-ß-producing regulatory T cells normally achieved by oral CTB–MBP conjugate treatment and thus acts as a potent anti-TGF-ß induction factor to break oral tolerance.

To test the hypothesis that active suppression of pathologic T cells by regulatory TGF-ß-producing T cells is a key process in the protection and tolerance achieved by mucosal administration of MBP linked to CTB, we determined if the tolerance induced could be transferred by T cells to naive animals. The results show that transfer of MBP-activated T lymphocytes from EAE-induced animals to naive recipients normally induced EAE disease, but failed to do so when the pathogenic T cells were given together with `oral tolerance' lymphocytes collected from the MLN of animals fed with CTB–MBP. Consistent with the key role ascribed above to MLN cells producing TGF-ß, the protective MLN cells mediating oral tolerance contained a much higher number of TGF-ß-producing cells than non-protective MLN cells from control animals. Thus, the tolerance induced by oral CTB–MBP can be explained by the active suppression of MBP-reactive encephalitogenic T cells by mucosal antigen-induced regulatory T cells producing TGF-ß, even though additional mechanisms may also be at hand.

Local antigen presentation in the CNS is important for maintaining the EAE inflammatory process with MBP-specific T cells. It is likely that the observed reduction in MHC class II antigen expression by CTB–MBP contributes to the reduction in the number of inflammatory cells in the spinal cord. The chemokines MCP-1 and RANTES have also been shown to correlate with acute disease severity (33), and are known to be important for helping monocytes and lymphocytes to migrate into the CNS during EAE development (34). Our findings show that oral CTB–MBP treatment markedly reduced MCP-1 and RANTES expression in the CNS, and this probably contributes to the strongly reduced infiltration of inflammatory cells in the CNS. On the other hand, unlike the treatment with CTB–MBP, treatment with repeated oral doses of MBP alone (five 1 mg doses) did not reduce the chemokine mRNA expression in the CNS even though this latter treatment was also associated with protection of EAE, suppression of inflammatory T_h1 cytokines and increased expression of TGF-ß. These results suggest that modulation of MCP-1 and RANTES in the CNS is an additional pathway by which oral CTB–MBP protects against EAE disease. This modulation may be independent of the TGF-ß-producing regulatory T cell pathway induced by treatment with either CTB–MBP conjugate or MBP alone.

It is also worth mentioning that in preliminary studies (J.-B. Sun et al., accepted for publication) we have further shown that oral administration of CTB–MBP conjugate in mice significantly also reduces the disease progression of a chronic relapsing form of EAE (CREAE), a disease that may be more closely related to multiple sclerosis in humans. The results in mice with CREAE suggested that the effect of mucosal tolerance induced by CTB–MBP is long-lasting with a duration of many weeks. In other work, we have also shown that gene fusion proteins between CTB and selected MBP or PLP peptide epitopes are also effective in protecting mice against CREAE (unpublished data). This capacity of the gene fusion proteins, if extending to multiple sclerosis in humans, would greatly facilitate the effective large-scale production of a well-defined inexpensive therapeutic agent for clinical use.

In conclusion, our findings confirm and extend our previous report that oral treatment with CTB–MBP conjugate protects against development of EAE in the rat model more effectively than other regimens. Our findings further show that the protective, anti-immunopathological effect is associated with and can probably largely be explained by the induction of TGF-ß-secreting suppressive-regulatory T cells in MLN (and/or Peyer's patches) and by local down-regulation of ß-chemokines in the spinal cord.

	Acknowledgments

 
The authors gratefully thank Dr Tomas Olsson for providing the facilities to perform mRNA analysis. The studies were supported by the Swedish Medical Research Council (projects 16x-3382) and by the European Union (project BIO4-98-0503 and B104-CT-96-0374).

	Abbreviations

 

AEC 3-amino-9-ethylcarbazole

CFA Freund's complete adjuvant

CNS central nervous system

Con A concanavalin A

CREAE chronic relapsing EAE

CT cholera toxin

CTB cholera toxin B subunit

EAE experimental autoimmune encephalomyelitis

HRP horseradish peroxidase

MBP myelin basic protein

MCP monocyte chemotactic protein

MLN mesenteric lymph nodes

OVA ovalbumin

PLN popliteal lymph nodes

SPDP N-succinimidyl(3-[2-pyridyl]-dithio)propionate

TGF transforming growth factor

TNF tumor necrosis factor

	Notes

 
³ Present address: INSERM UNITE 364, Faculté de Médicine-Pasteur, Avenue de Vallombrose, 06107 Nice Cedex 02, France

Transmitting editor: H. Bazin

Received 6 March 2000, accepted 6 July 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Ortiz-Ortiz, L., Nakamura, R. M. and Weigle, W. O. 1976. T cell requirement for experimental allergic encephalomyelitis induction in the rat. J. Immunol. 117:576.[Abstract/Free Full Text]
2. Panitch, H. S. 1980. Adoptive transfer of experimental allergic encephalomyelitis with activated spleen cells: comparison on in vivo activation of concanvalin A. Cell. Immunol. 56:1163.
3. Ben-Nun, A., Wekerle, H. and Cohen, I. 1981. The rapid isolation of clonal antigen specific lines capable of mediating autoimmune encephalomyelitis. Eur. J. Immunol. 11:195.[Web of Science][Medline]
4. Ben-Nun, A. and Cohen, I. R. 1982. Spontaneous remission and acquired resistance to autoimmune encephalomyelitis (EAE) are associated with suppression of T cell reactivity: suppressed EAE effector T cells recovered as T cell lines. J. Immunol. 128:1450.[Web of Science][Medline]
5. Bitar, D. M. and Whitacre, C. C. 1988. Suppression of experimental autoimmune encephalomyelitis by the oral administration of myelin basic protein. Cell. Immunol. 112:364.[Web of Science][Medline]
6. Higgins, P. J. and Weiner, H. L. 1988. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments. J. Immunol. 140:440.[Abstract]
7. Weiner, H. L. 1999. Oral tolerance with copolymer 1 for the treatment of multiple sclerosis. Proc. Natl Acad. Sci. USA 96:3333.[Free Full Text]
8. Sun, J.-B., Holmgren, J. and Czerkinsky, C. 1994. Cholera toxin B subunit: an effective transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc. Natl Acad. Sci. USA 91:10795.[Abstract/Free Full Text]
9. Sun, J.-B., Rask, C., Olssson, T., Holmgren, J. and Czerkinsky, C. 1996. Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit. Proc. Natl Acad. Sci. USA 93:7196.[Abstract/Free Full Text]
10. Bergerot, I., Ploix, C., Petersen, J., Moulin, V., Rask, C., Fabien, N., Lindblad, M., Mayer, A., Czerkinsky, C., Holmgren, J. and Thovolet, C. 1997. A cholera toxoid–insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes. Proc. Natl Acad. Sci. USA 94:4610.[Abstract/Free Full Text]
11. Tarkowski, A., Sun, J.-B., Holmdahl, R., Holmgren, J. and Czerkinsky, C. 1999. Treatment of experimental autoimmune arthritis by nasal administration of a type II collagen–cholera toxoid conjugate vaccine. Arthritis Rheum. 42:1628.[Web of Science][Medline]
12. Holmgren, J., Czerkinsky, C., Sun J.-B. and Svennerholm, A.-M. 1996. Oral vaccination, mucosal immunity and oral tolerance with special reference to cholera toxin. In Kaufmann, S. H. E., ed., Concepts in Vaccine Development, p. 444. Springer, Berlin.
13. Sanchez, J. and Holmgren, J. 1989. Recombinant system for overexpression of cholera toxin B subunit in Vibrio cholerae as a basis for vaccine development. Proc. Natl Acad. Sci. USA 86:481.[Abstract/Free Full Text]
14. Czerkinsky, C., Russell, M. W., Lycke, N., Lindblad, M. and Holmgren, J. 1989. Oral administration of a streptococcal antigen coupled to cholera toxin B subunit evokes strong antibody response in salivary glands and extramucosal tissues. Infect. Immun. 57:1072.[Abstract/Free Full Text]
15. Svennerholm, A.-M. and Holmgren, J. 1978. Identification of Escherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM₁-ELISA) procedure. Curr. Microbiol. 1:19.
16. Sun, J.-B., Mielcarek, N., Lakew, M., Grzych, J. M., Capron, A., Holmgren, J. and Czerkinsky, C. 1999. Intranasal administration of a Schistosoma mansoni glutathione S-transferase–cholera toxoid conjugate vaccine evokes anti-parasitic and anti-pathological immunity in mice. J. Immunol. 163:1045.[Abstract/Free Full Text]
17. Richert, J. R., Driscoll, B. F., Kies, M. W. and Alvord, E. C., Jr. 1979. Adoptive transfer of experimental allergic encephalo- myelitis: incubation of rat spleen cells with specific antigen. J. Immunol. 122:494.[Abstract/Free Full Text]
18. Link, J., Fredrikson, S., Söderström, M., Olsson, T., Höjeberg, B., Ljungdahl, Å. and Link, H. 1994. Organ-specific autoantigens induce transforming growth factor-ß mRNA expression in mononuclear cells in multiple sclerosis and myasthenia gravis. Ann. Neurol. 35:197.[Web of Science][Medline]
19. Dagerlind, Å., Friberg, K., Bean, A. J. and Hökfelt, T. 1992. Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histo- chemistry. Histochemistry. 98:39.[Web of Science][Medline]
20. Whitacre, C. C., Bitar, D. M., Gienapp, I. E. and Orosz, C. G. 1991. Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy. J. Immunol. 147:2155.[Abstract]
21. Weiner, H.L. 1997. Oral tolerance immune mechanisms and treatment of autoimmune diseases. Immunol. Today. 18:335.[Web of Science][Medline]
22. Khoury, S. J., Hsancock, W. W. and Weiner, H. L. 1992. Oral tolerance to myelin basic protein and natural recovery from experimental autoimmune encephalomyelitis are associated with down-regulation of inflammatory cytokines and differential up-regulation of transforming growth factor beta, interleukin 4, and prostaglandin E expression in the brain. J. Exp. Med. 176:1355.[Abstract/Free Full Text]
23. Friedman, A. and Weiner, H. L. 1994. Induction of anergy and/or active suppression in oral tolerance is determined by frequency of feeding and antigen dosage. Proc. Natl Acad. Sci. USA 91:6688.[Abstract/Free Full Text]
24. Weiner, H. L., Friedman, A., Miller, A., Khoury, S. J., al Sabbagh, A., Santos, L., Sayegh, M., Nussenblatt, R. B., Trentham, D. E. and Hafler, D. A. 1994. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu. Rev. Immunol. 12:809.[Web of Science][Medline]
25. Inobe, J., Slavin, A. J., Komagata, Y., Chen, Y., Liu, L. and Weiner, H. L. 1998. IL-4 is a differentiation factor for transforming growth factor-beta secreting T_h3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalo- myelitis. Eur. J. Immunol. 28:2780.[Web of Science][Medline]
26. Ferber, I. A., Brocke, S., Taylor-Edwards, C., Ridgway, W., Dinisco, C., Steinman, L., Dalton, D. and Fathman, C. G. 1996. Mice with a disrupted IFN- gene are susceptible to induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156:5.[Abstract]
27. Panitch, H. S., Hirsch, R. L. and Schindler, J. and Johnson, K. P. 1987. Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system. Neurology 37:1097.[Abstract/Free Full Text]
28. Vartanian, T., Li, Y., Zhao, M. and Stefansson, K. 1995. Interferon-gamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis. Mol. Med. 1:732.[Web of Science][Medline]
29. Heremans, H., Dillen, C., Groenen, M., Martens, E. and Billiau, A. 1996. Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice: enhancement by monoclonal antibodies against interferon-. Eur. J. Immunol. 26:2393.[Web of Science][Medline]
30. Billiau A., Heremans, H., Vandekerckhove, F., Dijkmans, R., Sobis, H., Meulepas, E., Carton, H. 1988. Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-. J. Immunol. 140:1506.[Abstract]
31. Vidard, L., Colarusso, L. J. and Benaceraf, B. 1995. Specific T-cell tolerance may reflect selective activation of lymphokine synthesis. Proc. Natl Acad. Sci. USA 92:2259.[Abstract/Free Full Text]
32. Elson, C. O. and Ealding, W. 1984. Cholera toxin feeding did not induce oral tolerance in mice and abrogated oral tolerance to an unrelated protein antigen. J. Immunol. 133:2892.[Abstract]
33. Holmgren, J., Lycke, N. and Czerkinsky, C. 1993. Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems. Vaccine 11:1179.[Web of Science][Medline]
34. Holmgren, J. 1981. Actions of cholera toxin and the prevention and treatment of cholera. Nature 292:413.[Medline]
35. Kennedy, K. J., Strieter, R. M., Kunkel, S. L., Lukacs, N. W. and Karpus, W. J., 1998. Acute and relapsing experimental autoimmune encephalomyelitis are regulated by differential expression of the CC chemokines macrophage inflammatory protein-1alpha and monocyte chemotactic protein-1. J. Neuroimmunol. 92:98.[Web of Science][Medline]
36. Miyagishi, R., Kikuchi, S., Takayama, C., Inoue, Y. and Tashiro, K. 1997. Identification of cell types producing RANTES, MIP-1 alpha and MIP-1 beta in rat experimental autoimmune encephalomyelitis by in situ hybridization. J. Neuroimmunol. 77:17.[Web of Science][Medline]

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