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
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Abstract |
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Keywords: cholera toxin B subunit, experimental autoimmune encephalomyelitis, myelin basic protein, transforming growth factor-ß
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Introduction |
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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 (911). Thus, we observed that a single feeding with microgram amounts of a CTBMBP 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of the tolerizing antigen across the mucosa and by presenting it more efficiently to antigen-presenting cells (12 and unpublished manuscript).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 CTBMBP 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 Th1 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 CTBMBP-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 CTBMBP-treated rats, indicating that regulatory TGF-ß-expressing T cells play an important role in oral tolerance and immunosuppression after oral treatment with CTBMBP conjugate.
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Methods |
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Preparation of CTBMBP or CTBOVA 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 CTBMBP 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 CTBMBP conjugate contained <10% free CTB and negligible amounts of unconjugated MBP. In an analogous manner, OVA was coupled to CTB to provide a CTBOVA 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 administrationshereafter referred to as oral administrationwith 50 µg per dose of CTBMBP 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 CTBMBP, 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 CTBMBP 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, Th 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 avidinbiotin 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.
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Results |
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Suppression of proliferative responses, IFN- production and up-regulation of TGF-ß mRNA-expressing cells in draining lymph nodes by oral CTBMBP 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 CTBMBP 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 CTBMBP-treated or MBP-treated rats than from PBS-treated rats (Fig. 3C
), while the numbers of IFN-
-producing cells were reduced (Fig. 3B
).
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Discussion |
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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 (2224). Our findings here show that oral immunotherapy with even very low doses (150 µg) of CTBMBP 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 CTBMBP 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 CTBMBP did not give rise to any increase in putative regulatory cells producing Th2 cytokines IL-4 and IL-10 in the CNS. Thus, the local production of Th2 cytokines at effector sites, including IL-4 and IL-10, may not be involved in the prevention of EAE development in animals fed with CTBMBP 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 Th3 cells (21), are the key effector cells in tolerance induced by CTBMBP treatment. Thus, these T cells can effectively suppress both Th1 and Th2 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 CTBMBP 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 CTBMBP activates a pre-regulatory IL-4-producing Th2 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 Th1 and Th2 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 Th1 and Th2 types of cytokines, noted after the protective treatment with CTBMBP 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 CTBMBP treatment (9). The reason for this discrepancy in IFN-
findings is under investigation. Our preliminary data suggest that different CTBMBP 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 CTBMBP. Consistent with these findings, co-administration of CTBMBP and CT led to increased production of both Th1 and Th2 cytokines in the CNS. The results indicate that CT specifically interrupts the induction of TGF-ß-producing regulatory T cells normally achieved by oral CTBMBP 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 CTBMBP. 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 CTBMBP 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 CTBMBP 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 CTBMBP 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 CTBMBP, 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 Th1 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 CTBMBP protects against EAE disease. This modulation may be independent of the TGF-ß-producing regulatory T cell pathway induced by treatment with either CTBMBP 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 CTBMBP 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 CTBMBP 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 CTBMBP 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.
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Acknowledgments |
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Abbreviations |
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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 |
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Notes |
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Received 6 March 2000, accepted 6 July 2000.
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References |
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