Immune regulatory effects of central nervous system antigens in culture

J. William Lindsey and Rui Jin

Department of Neurology, University of Texas-Houston, 6431 Fannin, Ste 7.044, Houston, TX 77030, USA

Correspondence to: J. W. Lindsey


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Evidence from several different experimental systems suggests that regulatory cells specific for self-antigens exist in the normal immune repertoire, and that these cells are necessary for maintenance of self-tolerance and prevention of autoimmune disease. We attempted to demonstrate the existence of regulatory cells specific for central nervous system (CNS) antigens in normal mice. We tested the effects of myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and a mixture of soluble brain proteins (SBP) on cultured splenocytes. MBP at 50 µg/ml inhibited antigen-driven proliferation and this suppressive effect could be partially blocked by neutralizing antibodies to transforming growth factor (TGF)-ß. MBP decreased expression of mRNA for the cytokines IL-2 and IFN-{gamma}, and slightly increased mRNA expression for TGF-ß. These effects did not appear to be mediated by regulatory cells specific for MBP, since MBP also suppressed proliferation in MBP-deficient shiverer mice and the suppressive effect could not be reproduced with selected MBP peptides. SBP at 250 µg/ml also inhibited antigen-driven proliferation, but this effect could not be blocked by neutralizing antibodies against IL-4, IL-10 or TGF-ß. SBP reduced expression of mRNA for IL-2, IL-10 and TGF-ß. These results are more consistent with the presence of a soluble inhibitory factor than with the action of SBP-specific regulatory cells. GFAP had no significant effect on proliferation. These results do not support the existence of regulatory cells specific for CNS antigens. Further investigation into non-antigen-specific mechanisms will be important in defining how autoimmune damage in the CNS is prevented.

Keywords: autoimmune disease prevention, central nervous system antigens, regulation, self-tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The immunologic mechanisms which maintain self-tolerance and prevent autoimmune disease are incompletely understood, but regulatory T lymphocytes may play an important role. Evidence supporting the existence of regulatory T cells in the normal immune repertoire has been demonstrated in several different experimental models. The most convincing evidence for the existence of regulatory T cells as a normal part of the immune repertoire has come from models of autoimmune diseases occurring in lymphocyte-depleted mice. CD4+CD25+ or CD4+CD45RBlow lymphocytes transferred from normal animals to lymphocyte depleted animals act as regulatory cells and prevent autoimmune disease (14). CD4+CD25+ T cells also have regulatory activity in culture and have been suggested to be a naturally occurring regulatory population (57). The antigen specificity and mechanism of action of these cells is under investigation. Regulatory T cells have also been demonstrated in mice transgenic for a TCR specific for myelin basic protein (MBP). A small number of T cells in these transgenic mice carry a non-transgenic TCR and these cells prevent the occurrence of spontaneous autoimmune disease (8,9). The regulatory cells are CD4, but their antigen specificity and mechanism of action is unknown.

The regulatory cells described above occur spontaneously, and their antigen specificity and mechanism of action is still under investigation. Regulatory T cells can also be experimentally induced and these cells are better characterized. After recovery from experimental autoimmune encephalomyelitis (EAE), regulatory CD4 cells are present in the spleen of the Lewis rat. These cells protect against the recurrence of EAE, and inhibit lymphocyte proliferation and IFN-{gamma} secretion through a mechanism involving transforming growth factor (TGF)-ß (10,11). Regulatory CD8 T cells specific for MBP can be induced by oral feeding of MBP (12). These cells also act through the secretion of TGF-ß (13).

Based on the above studies, we formed the hypothesis that regulatory T cells specific for self-antigens exist in normal adult animals and play an important role in maintaining self-tolerance. This should be particularly true for `immune-privileged' organs such as the central nervous system (CNS). These cells should act through secretion of regulatory cytokines such as TGF-ß or IL-10, and such cells should exist for most abundant self-antigens. These cells may be CD25+. To test the hypothesis, we investigated the effects of CNS self-antigens on antigen-driven proliferation and cytokine mRNA expression in culture. Since we expected that most CNS antigens should have similar effects, we tested diverse self-antigens with distinct physical properties. MBP is a major constituent of myelin, and as the name suggests is basic and positively charged at neutral pH. Glial fibrillary acidic protein (GFAP) is a structural protein found in astrocytes, and is acidic and negatively charged at neutral pH. As our third antigen, we used soluble proteins extracted from brain tissue (SBP). These proteins are expected to vary in charge and mol. wt, and should include proteins expressed throughout the body as well as proteins expressed primarily in the CNS.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
DBA/2J and PL/J female mice were obtained from Jackson Laboratories (Bar Harbor, ME) at age 6 weeks and maintained in microisolator cages. At age 7 weeks or later, mice were injected s.c. in two sites with a total volume of 0.2 ml of an emulsion containing 0.2 mg/ml ovalbumin (OVA; Sigma, St Louis, MO), 0.2 mg/ml Mycobacterium tuberculosis (Difco, Detroit, MI), and equal volumes of saline and incomplete Freund's adjuvant (Difco). MBP-deficient shiverer mice on the C3HeB/FeJ background were obtained from Jackson Laboratories at 4 weeks of age and immunized with OVA from age 5 to 8 weeks. Heterozygous mice and normal C3HeB/FeJ mice of the same age were used as controls.

CNS antigens and other reagents
MBP was prepared from mouse brains and spinal cords, and purified on a carboxymethylcellulose column as described (14,15). Column fractions were run on a polyacrylamide gel, and fractions containing MBP were combined, concentrated in an ultrafiltration cell using a membrane with a nominal mol. wt cut-off of 1 kDa (Amicon, Beverly, MA) and then dialyzed exhaustively against PBS in a dialysis cassette with a mol. wt cut-off of 2 kDa (Pierce, Rockford, IL) to remove urea. Forty grams of wet tissue usually yielded about 40 mg of purified protein. Bovine MBP was purified from bovine spinal cord using identical methods.

SBP was prepared from mouse brain. Mice were sacrificed by CO2 narcosis, perfused with saline and the brains were homogenized in 10 mM HEPES, pH 7.4. The homogenate was centrifuged at 10,000 g for 30 min and then the supernatant was further centrifuged at 100,000 g for 30 min to remove all non-soluble material. The proteins remaining in the supernatant were precipitated by adding ammonium sulfate to 60% saturation. The precipitated proteins were collected by centrifugation, resuspended in water and dialyzed against PBS.

GFAP was isolated using the procedure described for bovine tissue (16), with the salt gradient modified to 15– 100 mM. Fractions containing GFAP were identified on polyacrylamide gels, combined, concentrated and dialyzed against PBS.

After dialysis, all proteins were filtered through a 0.2 µm filter to remove aggregates and ensure sterility, and stored at –80°C in 1 ml aliquots. An equal number of aliquots of the final dialyzate were filtered and stored for use as controls. Protein concentration was determined using the BCA method (Pierce).

MBP1–11, MBP125–135 and MBP136–146 peptides were synthesized commercially (Biosynthesis, Lewisville, TX) using Fmoc chemistry according to the mouse MBP sequence, and were 85% pure.

Proliferation assays
Mice were sacrificed by CO2 narcosis 10–30 days after immunization and spleens were removed. Splenocytes were cultured at 2x105 cells/well in 0.2 ml of HL-1 serum-free media (Biowhittaker, Walkersville, MD) supplemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 5x10–5 M 2-mercaptoethanol (Gibco, Grand Island, NY). OVA was used at 200 µg/ml. In a typical experiment, cells were cultured with no antigen, OVA, OVA + one of the CNS proteins and OVA + dialyzate from the CNS protein. The dialysate from the CNS protein was used as a control for the possible effects on proliferation due to the PBS or contaminants and had only minimal effects on proliferation. Triplicate wells were done with each antigen. [3H]Thymidine (1 µCi) was added to each well after 2 days and the cells were collected on the third day using a semi-automated cell harvester. The incorporated radioactivity was counted on a scintillation counter.

Other tissue culture reagents
Other reagents used in tissue culture included purified protein derivative of M. tuberculosis (PPD; Parke-Davis, Rochester, MI), recombinant murine IL-2 and IFN-{gamma} (R & D Systems, Minneapolis, MN), and neutralizing antibodies against IL-4, IL-10 and TGF-ß (R & D Systems). Hen egg lysozyme (HEL) and BSA (fraction V; Sigma) were used as non-self-protein antigens.

FACS depletion of CD25+ cells
Spleens were taken from immunized PL/J mice and erythrocytes were removed by density gradient centrifugation over Lympholyte-M (Cedarlane, Hornby, Ontario, Canada). Leukocytes from the interface were washed and then suspended at 107/ml in HBSS containing 0.1% BSA, and incubated for 30 min on ice with rat anti-mouse CD25-conjugated to phycoerythrin (clone 3C7; PharMingen, San Diego, CA). Excess antibody was removed by washing with HBSS and stained cells were sorted on a Moflo cell sorter (Cytomation, Boulder, CO) to remove CD25+ cells. After sorting, an aliquot of cells was checked to ensure that CD25+ cells were depleted as intended. The remainder of the cells were used in cell culture as described above.

Preparation of cDNA
Cells intended for use in competitive PCR were incubated in 96-well plates with antigens at the same concentrations used in the proliferation assays. After 8 h, cells were resuspended by pipetting and cells from six wells cultured with the same antigen were combined. The cells were collected by centrifugation and lysed in Trizol (Gibco/BRL, Grand Island, NY). The wells in the culture plate were also washed with Trizol to extract mRNA from any adherent cells. Lysed cells were frozen at –80°C and total RNA was extracted after results of proliferation assays were available. After extraction, the RNA was dissolved in 20 µl of water. RNA was reverse transcribed to cDNA using 20 µl RNA, 400 U Superscript II reverse transcriptase (Gibco/BRL), 0.1 U random hexamers (Pharmacia, Piscataway, NJ), 2 µl of 10 mM dNTP (Pharmacia) and 4 µl of 0.1 M dithiothreitol in a total volume of 40 µl. The reverse transcriptase mixture was incubated at 42°C for 1 h and then at 95°C for 5 min.

PCR primers and constructs
The primers used and the sizes of the amplified target and construct DNAs were: actin sense CAT CGT GGG CCG CCC TAG GCA C, anti-sense CCG GCC AGC CAG GTC CAG ACG C, target 451 bp, construct 274 bp; IL-2 sense TCA AGC TCT ACA GCG GAA GC, anti-sense TGA CAG AAG GCT ATC CAT CTC C, target 401 bp, construct 315 bp; IL-10 sense GCT GGA CAA CAT ACT GCT AAC CG, anti-sense TCG GAG AGA GGT ACA AAC GAG G, target 482 bp, construct 316 bp; IFN-{gamma} sense TGT TTC TGG CTG TTA CTG CCA CGG C, anti-sense TTC CGC TTC CTG AGG CTG GAT TCC, target 405 bp, construct 320 bp; and TGF-ß sense CGA CAT GGA GCT GGT GAA ACG G, antisense CTG AAT CGA AAG CCC TGT ATT CCG, target 552 bp, construct 317 bp.

Constructs were prepared by two different methods. The actin construct is the same as described previously (17) and the other constructs were synthesized using the actin construct as a base. Intermediate primers containing the cytokine primer sequence followed by 10 bases of the actin construct sequence were used to amplify a new and slightly longer construct with the desired primers at the ends. In some cases several different primer pairs were tested to find one which would amplify both target and construct cleanly without secondary bands which would interfere with the quantification. Concentration of the undiluted construct DNA was determined by comparison of band density on agarose gel of known volumes of the construct with the band density of the 383 bp fragment of a BstNI digest of pBR322 (New England BioLabs, Beverly, MA). Constructs were serially diluted in 0.5 log unit increments down to 10–10 of the original concentration for use in competitive PCR.

Competitive PCR
The competitive PCR procedure for quantification of mRNA for cytokines was similar to the procedure previously described (17). The cDNA and construct were amplified in a 25 µl PCR reaction containing 1 µl cDNA, 5 µl construct, 1.25 U Taq polymerase (Perkin-Elmer, Foster City, CA), 12.5 pmol of each primer, 2.5 µl 10xPCR buffer and 0.5 µl 10 mM dNTP. Amplification was carried out for 35 cycles of 94°C for 60 s, then 55°C for 90 s, then 72°C for 90 s in a 96-well thermal cycler (Stratagene, La Jolla, CA). For each cytokine, three to four reactions were performed using serial dilutions of construct covering the expected target concentration and the resulting PCR products were resolved on an agarose gel containing ethidium bromide. The gel was photographed under UV illumination using Polaroid type 55 film, and the densities of the construct and target bands on the negative were measured with a densitometer (BioRad, Hercules, CA). Near the point where the construct and target bands were of equal density, the difference in target and construct density was approximately a linear function of the log of the construct concentration. The concentration of construct which would give a band equal in density to the target band was calculated by linear interpolation using the density values for the construct concentrations just higher and lower than the target. This concentration was then used to calculate the number of molecules of the target mRNA in the original sample. The lower limit for quantification was between 102–103 molecules.

The cell collection, RNA extraction and reverse transcription steps were done using a uniform methodology, but there could be variations in the efficiency of these steps which would affect the amount of cytokine mRNA detected. To correct for this possible variation, we also quantified the amount of actin mRNA present in each sample. The amount of the cytokine mRNA is expressed relative to the amount of actin cDNA present in the same sample.

Calculations and statistics
The stimulation index (SI) was calculated as: SI = c.p.m.st /c.p.m.ba, where c.p.m.ba is the background c.p.m. and c.p.m.st is the c.p.m. with the stimulating antigen. The percent suppression was calculated as: 100x(c.p.m.st – c.p.m.st + su)/ (c.p.m.st – c.p.m.ba), where c.p.m.st + su is the c.p.m. with the stimulating and suppressing antigens. Because of variations in amount of stimulation, percent suppression is used to combine data from different animals. For all data reported the SI > 4. In all cases where the effects of different suppressing antigens are compared, the two suppressing antigens were tested in the same animals. Values are expressed as the mean ± SD of at least three independent experiments.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effect of brain antigens on antigen-driven proliferation
The presence of MBP at a concentration of 50 µg/ml inhibited proliferation to OVA in a reproducible fashion (Figs 1 and 2GoGo). SBP also inhibited proliferation, with a greater effect at higher concentration. The effects of MBP and SBP were greater in PL/J mice than in DBA/2J mice, although the difference in the two strains is larger for MBP (P = 0.0008 for MBP and P = 0.017 for SBP 250, t-test). In contrast, GFAP at 100 µg/ml had only a minimal effect on proliferation in either strain.



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Fig. 1. Representative example of the effect of CNS proteins on antigen-driven proliferation. Proliferative response of cultured splenocytes from a single PL/J mouse to no antigen and to OVA + different CNS proteins. To ensure the effects on proliferation are due to the protein, the final dialyzate from each protein is used as a control. Each CNS protein is compared to an equal volume of its own dialyzate. As expected, the dialyzates had little effect on proliferation to OVA in the quantities used (5–13 µl/well). In this example, MBP at 50 µg/ml suppressed 57%, SBP at 250 µg/ml suppressed 80% and GFAP at 100 µg/ml suppressed 14%. Error bars indicate SD of triplicate wells. The dotted line indicates the background proliferation with no antigen of 2350 c.p.m.

 


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Fig. 2. Percent suppression of OVA-induced proliferation with CNS proteins. Combined results from several experiments such as the one shown in Fig. 1Go. The bars indicate mean percent suppression for each antigen with error bars indicating the SD. Concentration of each CNS antigen is given in µg/ml. MBP and SBP suppressed significantly, but GFAP did not. For the PL/J mice, SI with OVA was 8.8 ± 1.8, n = 12. For the DBA/2J mice, SI to OVA was 7.1 ± 1.4, n = 10.

 
The effects of MBP and SBP were not specific to OVA. Proliferation to PPD was also inhibited by a similar amount (Table 1Go). The suppressive effect was not simply due to the presence of additional protein, since addition of non-self-proteins had little effect on proliferation to OVA. HEL at 250 µg/ml suppressed proliferation by only 4 ± 14% (n = 5) and 250 µg/ml BSA suppressed proliferation by 0 ± 13% (n = 4). Neither MBP nor SBP alone had much effect on background proliferation. In the PL/J strain, the SI with MBP was 0.96 ± 0.44 (n = 9) and the SI with SBP was 1.51 ± 0.64 (n = 6).


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Table 1. Percent suppression with combinations of stimulating and suppressing antigens in PL/J mice
 
Murine MBP is more effective than bovine MBP
As an additional control for the comparison of self- and non-self-proteins, we compared the effects of murine and bovine MBP. In both species, MBP consists of several isoforms which are produced by differential splicing of the mRNA from a single gene. The major form of MBP in the mouse has 127 amino acids and the main form of bovine MBP has 169 amino acids. The proteins are very similar in amino acid composition and net positive charge, and the amino acid sequences are 91% identical. In spite of this similarity, murine MBP had a significantly greater suppressive effect than equimolar concentrations of bovine MBP (Table 2Go). Similar results were obtained with two additional independent preparations of bovine MBP.


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Table 2. Percent suppression with murine and bovine MBP
 
Neutralizing anti-TGF antibody partially blocks MBP suppression
We investigated whether the regulatory cytokines IL-4, IL-10 or TGF-ß were involved in the suppression of proliferation in PL/J mice. Anti-cytokine antibodies at 10 µg/ml were added to cells cultured with OVA alone or with OVA + one of the CNS antigens. The anti-TGF antibody partially blocked the suppressive effect of MBP, reducing the percent suppression from 67 ± 15 to 43 ± 11 (Fig. 3Go). The anti-TGF antibody also increased the mean SI to OVA from 9.8 ± 1.8 to 11.2 ± 2.1, suggesting that there is some baseline secretion of TGF by cultured cells which limits the proliferative response. A control antibody of the same isotype as the anti-TGF-ß had no effect on proliferation or MBP suppression. The percent suppression was 64 ± 9 with no antibody and 63 ± 8 with the isotype control (n = 6). In similar experiments with SBP, none of the antibodies blocked suppression. The percent suppression without antibody was 67 ± 11 and it did not change significantly with any of the antibodies (n = 5).



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Fig. 3. Effects of anti-cytokine antibodies on MBP suppression. Neutralizing anti-TGF-ß antibody partially blocks MBP suppression (P = 0.008, paired t-test). Antibodies to IL-4 or IL-10 had no effect. Bars indicate mean percent suppression, error bars indicate SD, n = 7.

 
Addition of IL-2 does not reverse suppression
Other investigators have suggested that self-antigen-specific regulatory cells might act by blocking the secretion of IL-2 and that their effect could be reversed by the addition of IL-2. We tested the effects of both IL-2 and IFN-{gamma} on MBP and SBP suppression. The addition of 10 ng/ml IL-2 approximately doubled the proliferative response to OVA. Proliferation with OVA and CNS antigen increased by a proportional amount, so the percent suppression did not change (Table 3Go).


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Table 3. Effect of cytokines on percent suppression with MBP and SBP
 
Competitive PCR for cytokine mRNA
To further investigate the effects of brain antigens, we used competitive PCR to measure the effects of brain antigens on the expression of cytokine mRNA. Cells were cultured with either no antigen, OVA, OVA + MBP or MBP for 8 h before they were collected for mRNA extraction. To correct for possible variations in the efficiency in the processing of the specimens to cDNA, the concentration of cytokine mRNA in each sample is expressed relative to the concentration of actin mRNA in the same sample. Cells cultured with no antigen had measurable amounts of mRNA for IL-2, IL-10 and TGF-ß; but IFN-{gamma} mRNA was undetectable in seven of the eight samples (Fig. 4Go). Addition of OVA markedly increased mRNA for IL-2 and IFN-{gamma}, but had no consistent effect on IL-10 or TGF-ß. Culture with MBP alone caused IL-2 to become undetectable in all eight samples, but had no consistent effect on the other cytokines in comparison to no antigen. IFN-{gamma} remained undetectable in the majority of samples cultured with MBP.



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Fig. 4. Effects of MBP on cytokine mRNA concentration. Cytokine mRNA concentration plotted as log (molecules cytokine mRNA/106 molecules actin mRNA). For example, a value of 2 indicates there are 100 molecules of cytokine mRNA for every 106 molecules actin mRNA in that sample. Cells were cultured with no antigen, OVA, OVA + MBP or MBP for 8 h. Data are arranged to facilitate the comparison of MBP with no antigen, OVA with no antigen and OVA with OVA + MBP. Results for cells from the same animal cultured with different antigens are connected with lines. <Ass. signifies less than assay, cytokine mRNA was not detectable with PCR. Individual results for the eight specimens tested are shown.

 
Comparison of mRNA concentrations in cells cultured with OVA or OVA + MBP reveals some interesting findings (Fig. 5Go). The addition of MBP to OVA-stimulated cells decreased the concentration of mRNA for IL-2 and IFN-{gamma}, and increased the concentration of mRNA for TGF-ß (P = 0.020 for IL-2, 0.005 for IFN-{gamma} and P = 0.015 for TGF-ß, paired t-test). There was no consistent change in IL-10.



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Fig. 5. Effect of MBP on OVA-induced cytokine expression. Change in cytokine mRNA concentration is expressed as log(concentration with OVA and MBP) – log(concentration with OVA). Negative values indicate that addition of MBP decreased that cytokine, positive values indicate that addition of MBP increased that cytokine. Small solid symbols are values for individual specimens, the larger open symbol indicates the mean ± SD. MBP decreases the expression of mRNA for IL-2 and IFN-{gamma}, and increases the expression of TGF-ß.

 
Similar experiments utilizing SBP gave slightly different results (Fig. 6Go). As expected, the differences between no antigen and OVA were similar to those seen in the prior experiment. Like MBP, SBP decreased the concentration of IL-2 mRNA when compared to no antigen; however, surprisingly, it increased the amount of IFN-{gamma} mRNA in five of the six specimens. Comparison of the cells stimulated with OVA and with OVA + SBP demonstrates a different effect of SBP than MBP (Fig. 7Go). Like MBP, SBP reduces IL-2 (P = 0.002, paired t-test), but the effects on the other cytokines differ. IFN-{gamma} shows no consistent change, IL-10 decreases (P = 0.032) and TGF-ß shows a trend towards a decrease (P = 0.059) rather than the increase seen with MBP.



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Fig. 6. Effects of SBP on cytokine mRNA concentration. Individual results for six specimens. Axes are the same as in Fig. 4Go.

 


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Fig. 7. Effect of SBP on OVA-induced cytokine expression. Axes and symbols are the same as in Fig. 5Go. SBP decreases the expression of mRNA for IL-2, IL-10 and TGF-ß.

 
Suppression is not mediated by CD25+ cells
Because of the reports of others that CD25+ cells might represent a naturally occurring regulatory population, we investigated whether the effects of MBP and SBP were mediated by CD25+ cells. CD25+ cells were depleted by cell sorting and the response of CD25 splenocytes was compared to that of unfractionated splenocytes from the same animal. Percent suppression by MBP was 83 ± 5 for unfractionated splenocytes and 90 ± 6 in CD25 cells (n = 3). Percent suppression by SBP was 79 ± 6 for unfractionated cells and 79 ± 15 for CD25 cells (n = 2).

The effect of MBP cannot be reproduced with selected peptides
If MBP suppresses proliferation through the activation of MBP-specific regulatory cells, then MBP peptides containing the suppressive epitope should also suppress proliferation. We tested the effect of three different peptides on proliferation to OVA. The MBP125–135 and MBP136–146 peptides are the immunodominant epitopes in MBP-deficient H-2u mice, and are known to bind stably to I-Au and induce tolerance in wild-type H-2u mice (18,19). Cultured T cells specific for these peptides cause EAE in wild-type H-2u mice, so these peptides must be presented in vivo. These two peptides are therefore leading candidates for regulatory epitopes of MBP. The MBP1–11 peptide is a subdominant epitope in MBP-deficient H-2u mice and the dominant epitope in wild-type H-2u mice. MBP1–11 is less effective in inducing tolerance, but it is also encephalitogenic.

None of the three peptides had any significant effect on proliferation. The percent suppression was –6 ± 14 with MBP1–11, 1 ± 14 with MBP125–135 and 5 ± 7 with MBP136–146 with the peptides at 100 µg/ml. In the same mice, MBP at 50 µg/ml suppressed proliferation by 65 ± 13%. In further experiments, none of the peptides had any effect on proliferation at 200 µg/ml nor was there any effect when the three peptides were used in combination.

MBP suppresses proliferation in MBP-deficient shiverer mice
To further investigate the mechanism of suppression, we tested the effect of MBP and SBP in shiverer mice which do not express MBP. We expected that SBP should suppress proliferation in these mice, but that MBP should not. SBP suppressed proliferation to OVA by 79 ± 28% as expected and MBP suppressed proliferation 65 ± 14% (n = 4). This was not significantly different from the suppression in heterozygote and wild-type controls assayed at the same time.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We began this work with the hypothesis that regulatory T cells specific for abundant CNS antigens should be present in the normal immune repertoire and should have demonstrable effects in culture. Our experimental results do not support this hypothesis. GFAP had no significant suppressive effect, and the suppressive effects of MBP and SBP do not appear to be mediated through regulatory T cells.

Our initial results with MBP seemed to support the existence of MBP-specific regulatory cells. MBP in relatively low concentrations inhibited proliferation, decreased expression of mRNA for the inflammatory cytokines IL-2 and IFN-{gamma}, and increased expression of mRNA for TGF-ß. The effect of MBP could be partially blocked by antibody against TGF-ß, providing further support for the role of this cytokine as a mediator of MBP suppression. The effect of murine MBP was greater than bovine MBP, suggesting that the exact amino acid sequence might be more important for the suppressive effect than the unique physical properties shared by bovine and murine MBP.

However, our subsequent investigations did not support the existence of MBP-specific regulatory cells. The strongest evidence against our hypothesis is the fact that MBP suppressed proliferation in shiverer mice. Since these mice do not express MBP, they should not have MBP-specific regulatory cells. This suggests that MBP suppresses proliferation through a non-antigen-specific mechanism. Our results with the MBP peptides also failed to support our hypothesis. Based on the available knowledge of the processing, binding and presentation of MBP epitopes in H-2u mice, the three peptides tested are the most likely regulatory epitopes. However, none of these three peptides had any effect on proliferation. This does not conclusively rule out the existence of a regulatory epitope in MBP since we did not test peptides covering the entire MBP molecule. The depletion of CD25+ cells had no effect on MBP suppression, suggesting that MBP does not selectively stimulate the putative natural regulatory population of CD4+CD25+ cells.

These results suggest that the effects of MBP are not mediated through the action of MBP specific regulatory cells. There are several plausible alternative explanations. MBP is known to bind to MHC class II molecules and could conceivably block the presentation of OVA to T cells (20). The positively charged MBP molecule might also bind non-specifically to negatively charged cell membranes and the tissue culture plate, and interfere with cell–cell interactions or cell migration. In support of this explanation, we have found that other molecules with a similar degree of net positive charge, including histones and glatiramer acetate (formerly known as copolymer-1), have a similar suppressive effect on proliferation in culture (data not shown).

The results with SBP also do not support our hypothesis. SBP does suppress proliferation, but the concentrations required are high and there is no evidence for the involvement of regulatory cytokines. One possible explanation is that SBP contains one or more of the soluble immunosuppressive factors reported to be present within the CNS. Further experiments in which SBP is separated into fractions will be required to verify this.

Taken together, our results with MBP, SBP and GFAP do not support the hypothesis that regulatory cells specific for self-antigens exist in the normal immune system. It is possible that such cells do exist, but that our methods were inadequate to demonstrate their presence. This could occur if these cells required some signal for activity which is present in vivo but not in tissue culture. For example, regulatory cells might require contact with a normal extracellular matrix for activity. It is also possible that such regulatory cells exist, but are specific for antigens other than MBP, SBP and GFAP. Another possibility is that effects on antigen-driven proliferation and cytokine mRNA expression in vitro are not relevant measures of in vivo regulatory activity.

It is also possible that regulatory cells specific for self-antigen are not a part of the normal immune repertoire. Although not the expected result, this conclusion is consistent both with the present work and with the work by others discussed in the Introduction. The spontaneously occurring regulatory cells described in various experimental systems are defined by their ability to prevent autoimmune disease in vivo, but their antigen specificity has not been defined. In some cases these cells appeared to be tissue specific (21,22), but the relevant antigen has never been directly demonstrated. In cases where the specific antigen is known, the regulatory cells are not present in normal animals, but are found only after experimental manipulation such as induction of EAE or oral tolerization. It is also possible that such cells exist for systemic antigens, but not for sequestered CNS antigens.

If CNS antigen-specific regulatory cells are not present in the normal immune repertoire, then there must be other regulatory mechanisms which maintain self-tolerance and prevent autoimmune disease. Delineation of these non-antigen-specific mechanisms is a promising avenue for future research. One possibility is that immunoregulatory molecules may be present in the extracellular microenvironment (23). Destruction of these molecules by an infectious process would permit immune responses, but the presence of these molecules in a normal tissue would suppress immune responses. Further investigation is needed to determine the relative importance of antigen-specific and non-antigen-specific mechanisms for self-tolerance.


    Acknowledgments
 
This work was supported in part by the Clayton Foundation for Research. We thank Steve Ullrich for critical review of the manuscript and Barbara Conner for technical assistance.


    Abbreviations
 
CNS central nervous system
EAE experimental autoimmune encephalomyelitis
GFAP glial fibrillary acidic protein
HEL hen egg lysozyme
MBP myelin basic protein
OVA ovalbumin
PPD purified protein derivative
SBP soluble brain proteins
TGF transforming growth factor

    Notes
 
Transmitting editor: L. Steinman

Received 10 April 2000, accepted 2 August 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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