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The Journal of Immunology, 1999, 162: 4300-4310.
Copyright © 1999 by The American Association of Immunologists

Golli-Induced Paralysis: A Study in Anergy and Disease1

Lise Clark, Laszlo Otvos, Jr., Paul L. Stein, Xiang-Ming Zhang, Annette F. Skorupa, Georgia E. Lesh, F. Arthur McMorris and Ellen Heber-Katz2

The Wistar Institute, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Golli-MBP transcription unit contains three Golli-specific exons as well as the seven exons of the classical myelin basic protein (MBP) gene and encodes alternatively spliced proteins that share amino acid sequence with MBP. Unlike MBP, which is a late Ag expressed only in the nervous system, Golli exon-containing gene products are expressed both pre- and postnatally at many sites, including lymphoid tissue, as well as in the central nervous system. To investigate whether Golli-MBP peptides unique to Golli would result in neurological disease, we immunized rats and observed a novel neurological disease characterized by mild paralysis and the presence of groups of lymphocytes in the subarachnoid space but not in the parenchyma of the brain. Disease was induced by Th1-type T cells that displayed an unusual activation phenotype. Primary stimulation in vitro induced T cell proliferation with increased surface CD45RC that did not become down-regulated as it did in other Ag-stimulated cultures. Secondary stimulation of this CD45RChigh population with Ag, however, did not induce proliferation or IL-2 production, although an IFN-{gamma}-producing population resulted. Proliferation could be induced by secondary stimulation with IL-2 or PMA-ionomycin, suggesting an anergic T cell population. Cells could adoptively transfer disease after secondary stimulation with IL-2, but not with Ag alone. These responses are suggestive of a chronically stimulated, anergic population that can be transiently activated to cause disease, fall back into an anergic state, and reactivated to cause disease again. Such a scenario may be important in chronic human disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental allergic encephalomyelitis (EAE)1 is thought to be the best animal disease model for the human disease multiple sclerosis. The paralysis seen in EAE is caused by an inflammatory response and demyelination due to the recirculation of myelin Ag-specific T cells from the peripheral circulation into the central nervous system (CNS) (1, 2, 3, 4, 5, 6). EAE is generally induced by immunization with myelin Ags emulsified in CFA; myelin basic protein (MBP) is the most well described (1), although there are many proteins produced by the oligodendrocyte that can serve as encephalitogens. Proof that EAE is in fact an autoimmune disease can be demonstrated by the adoptive transfer of MBP-reactive T cells into naive animals with resulting onset of disease and histologic evidence of white matter infiltrates and lesions. In both rats and mice, the predominant T cells are CD4+ and often bear Vß8.2 TCR (7).

In this paper we describe a new autoimmune disease induced by a peptide from a developmentally early form of MBP, known as Golli-MBP, shown to be present in the CNS as well as in lymphoid tissue. Campagnoni et al. (8) showed that the MBP gene, which spans 32 kb in the mouse and 45 kb in the human, is actually part of a larger transcription unit that spans 105 kb in the mouse and 179 kb in the human and includes four additional exons upstream of the classical seven MBP exons (8, 9). They named this larger transcription unit the Golli (gene of the oligodendrocyte lineage)-MBP gene and renumbered the seven classical MBP exons as exons 5–11 of the Golli-MBP sequence. In the mouse, three alternatively spliced Golli-MBP mRNAs have been identified that contain upstream Golli exons spliced to classical MBP exons. The 5'-most exons of the Golli transcripts encode amino acid sequences that are unique to Golli, whereas the 3' portions contain classical MBP exons, spliced in-frame in most cases, so that Golli and MBP amino acid sequences are identical over much of their lengths.

Different species and tissues vary in the Golli splice variants expressed. The transcript named BG21 in the mouse and HOG5 in the human includes exon 1 of MBP, the largest MBP exon. The J37 transcript of mouse Golli contains classical MBP exons 1, 3, 4, and 7 in the same reading frame as MBP, and the HOG7 transcript of the human contains classical MBP exons 1, 3, 4, 5, 6, and 7 in-frame. Only one Golli transcript, TP8 in the mouse, shares no amino acid sequence with MBP, because MBP exons 3, 4, and 7, although present in TP8, are spliced in a different reading frame. Both J37 and BG21 are expressed in murine CNS earlier than MBP; the latter is expressed at high activity at the earliest time examined (embryonic day 18) (8). Quite unlike classical MBP, Golli transcripts are expressed transiently in developing CNS neurons and later in developing oligodendroglial cells (10, 11). Moreover, at least one of these transcripts, BG21, is expressed outside the nervous system in heart, kidney, spleen, and lung (8) and in a T cell and a B cell line (S. Amur-Umerjee and A. T. Campagnoni, unpublished observations). Grima et al. (12) isolated a clone identical with BG21 and detected expression in mouse peritoneal macrophages, spleen, and thymus as well as brain. Independently, Mathisen et al. (13) described an MBP variant that they call embryonic or E-MBP. Like Golli, E-MBP transcripts contain classical MBP exons and additional upstream coding sequence, but the upstream E-MBP sequence is only partially known, and its relationship to the Golli exons is unclear. E-MBP, like Golli, is expressed early in development both within and outside the nervous system, including in lymphoid organs (13).

The finding that Golli mRNA is found in immune tissue has potential implications for immune reactivity to neuroantigens. Immunostaining of Golli protein using a rabbit Ab to a Golli-unique region, residues 1–133 from exons 2–5a (14), showed expression of Golli in human fetal thymic macrophages (15). Another myelin protein, DM20, an alternatively spliced form of the encephalitogenic CNS myelin proteolipid protein, was shown to be expressed as early as embryonic day 10 in the CNS and peripheral nervous system, long before the onset of myelination (16), and to be expressed outside the nervous system in heart and spleen (17). This, too, was found in human fetal thymic macrophages. Whether this is the site of synthesis or the protein is there due to phagocytosis may be irrelevant if Ag presentation is occurring.

Most recently, in the Lewis rat we have shown that Golli transcripts are found in fetal thymus, fetal liver, spleen, and gut4 (18). The predominant alternatively spliced form was found to use exons 1, 2, 3, 5a, 5b, and 5c and to be the same as BG21 in mouse tissue (8) and HOG-5 in human tissue (9). This molecule has been found not only in fetal CNS tissue but also in the developing immune system (8, 9, 12, 19). Whether the presence of Golli in the developing thymus results in the induction of tolerance has been a question of interest. We show herein that Golli peptides, and even more so glcosylated Golli peptides, induce an inflammatory response mainly in the meninges of Lewis rats and not in the parenchyma of the brain or spinal cord. The disease is T cell induced, can be adoptively transferred, and displays novel histopathology and symptomology. The T cells, however, show in vitro characteristics of chronic stimulation and anergy and provide a new model for self tolerance. Golli-induced paralysis is a new experimental system for studying neurologic autoimmune diseases and may have relevance to human disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Lewis rats were obtained from Charles River Production Area 13 (Canada) and bred in the Wistar Institute’s animal facility.

Antigens

MBP was purified from guinea pig spinal cords by a modification of the method of Diebler as previously described (20). OVA was obtained from Sigma (St. Louis, MO; catalogue No. A-2512).

Golli peptides were synthesized, purified, and analyzed as previously described (21). Briefly, NHSGKRELSAEK and N(ß-Gal-GlcNAc) derivatives were prepared by standard F-moc protocols (22). F-moc-Asn (Gal(ß1–3)-GalNAc(ß1-N)-OH) was prepared from one amino sugar derivative (23). A part of the peptide resin was N-terminally acetylated with pentafluorophenyl acetate (Aldrich, Milwaukee, WI) (24). The peptides were purified by reverse phase HPLC and analyzed by mass spectroscopy and sugar-sensitive amino acid analysis (21). The amino acid sequences of the peptides used in this study are as follows: mouse Golli exon 2 sequence, MGNHSGKRELSAEKASK; rat Golli exon 2 sequence, MGNHSGKRELASEKASK (18); synthetic mouse Golli peptide sequence, NHSGKRELSAEK; synthetic mouse glyGolli sequence, N(Gal-GlcNAc)HSGKRELSAEK; and synthetic mouse ac-Golli sequence, Ac-NHSGKRELSAEK.

Serum stabilization studies were conducted as follows. Ten microliters of an aqueous peptide stock containing 0.8 mg/ml peptide was added to 200 µl of 25% rat serum that had been heated to 37°C. After 30, 120, and 240 min, three samples of each peptide were precipitated by the addition of 40 µl of a 15% aqueous solution of TCA. The samples were stored at 4°C for 20 min and then centrifuged. Supernatants were immediately frozen on dry ice, and 220 µl of each were analyzed on reverse phase HPLC. The amount of peptide at 0 min was used as 100% (25, 26).

Disease induction

Adult Lewis rats received an intradermal injection in the hind footpads of Ag (Golli peptides, guinea pig MBP, or OVA) emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis, strain H37RA. All rats were monitored daily for clinical neurologic signs, which were scored as follows: distal tail paralysis, 0.5+; flaccid tail, 1+; ataxia, 2+, hind limb paralysis, 3+; and forelimb paralysis, 4+.

Analysis of T cells

Adoptive transfer of disease. T cell lines specific for guinea pig MBP (50 µg/animal), OVA (50 µg/animal), or Golli peptides (5 µg/animal) were stimulated with 5.0 µg/ml of Ag in vitro (106 cells/ml), and on day 3 blasts that had been recovered on a Ficoll-Hypaque gradient were injected i.p. into x-irradiated (350 rad from a 137Cs gamma ray source) naive Lewis rats. The Ag used to stimulate the cells was the same as the immunogen unless noted otherwise. Rats were observed daily from day 4 for neurological signs, and signs were scored based on clinical findings for actively induced disease.

Cytokine assays. ELISA plates for IFN-{gamma} detection were coated overnight at 4°C with a monoclonal mouse anti-mouse/rat IFN-{gamma} Ab (500 ng/well; BioSource International, Camarillo, CA; catalogue No. ARC 4033). Ab was removed by washing, and wells were blocked with PBS/1% BSA and 0.1% NaN3 for 2 h at room temperature. After washing, duplicate wells of culture supernatant as well as dilutions of rat IFN-{gamma} standard (Genzyme, Cambridge, MA), all containing 100 µl/well, were incubated overnight at 4°C. Wells were washed again and incubated with 500 ng in 50 µl of rabbit anti-rat IFN-{gamma} (BioSource International, catalogue No. ARC 4832) for 1 h at 37°C. After washing, 100 µl of a 1/1000 dilution of goat anti-rabbit Ig coupled to alkaline phosphatase (AP; Sigma, catalogue No. A-3687) was incubated in the wells at room temperature for 1 h. Wells were thoroughly washed before adding the AP substrate, PNPP (Sigma, catalogue No. 104-0), as the color reagent, and plates were read at a dual wavelength of 405/750.

To determine cytokine mRNA levels, total RNA was extracted from cultured T cells using a modification of the miniprep method described by Gough (27). After thawing at 37°C for 2 min, the cells from each sample were washed with PBS and centrifuged in a 1.5-ml microcentrifuge tube. Each pellet was resuspended in a 0°C solution containing diethylpyrocarbonate H2O, 10 mM Tris (pH 7.5), 0.15 M NaCl, 1.5 mM MgCl2, 0.65 M Nonidet P-40 (BDH, Poole, U.K.), and 20 mM vanadyl ribonucleoside complex (Sigma). The cells were lysed by rapid freezing and thawing, followed by vortexing. After centrifugation, the supernatant was mixed with a second solution containing diethylpyrocarbonate H2O, 7.0 M urea, 1% SDS, 0.35 M NaCl, 10 mM EDTA, and 10 mM Tris (pH 7.5). The RNA was then extracted with phenol/chloroform/isoamyl alcohol (1 part phenol mixed with 1 part 49/1 diluted chloroform/isoamyl alcohol mixture) and precipitated in 100% ethanol.

The cDNA titration quantitative RT-PCR was conducted by determining the cytokine mRNA level relative to ß-actin. Two micrograms of total RNA from each T cell sample was used to synthesize cDNA in a total of 20 µl using an oligo(dT) primer and SuperScript reverse transcriptase (Life Technologies, Gaithersburg, MD). Fivefold dilutions of the cDNA were performed in sterile water. Each dilution of the cDNA was then amplified with the rat actin primers in 20 µl of the RT-PCR reaction mixture (2 µl of 10x PCR buffer, 0.2 µl of 25 mM dNTP, 0.2 µl of 25 µM of each primer, and 0.1 µl of Taq polymerase; Boehringer Mannheim, Indianapolis, IN) using 0.2 µl of diluted cDNA. Following 42 cycles of amplification (94°C for 1 min, 55°C for 1.5 min, 72°C for 2 min), 6 µl of RT-PCR DNA products were run on a 2% agarose-ethidium bromide TBE buffered gel. The RT-PCR DNA band intensity was derived from scanning equivalent areas using the FluorImager SI (Molecular Dynamics, Sunnyvale, CA). The cDNA concentration in each sample was then normalized according to this actin RT-PCR titration. Next, similar cDNA titration and amplification were conducted for each cytokine. Finally, the relative amounts of RT-PCR products were calculated by the equation Q = (C - B)/(A - B) x 106 where A is actin RT-PCR DNA counts, B is gel background counts, C is cytokine RT-PCR DNA counts, and Q is the relative cytokine mRNA level.

The following PCR primer sequences were used: IL-2, 5'-GCG CAC CCA CTT CAA GCC CT, CCA CCA CAG TTG CTG GCT CA-3'; IL-4, 5'-ATG CAC CGA GAT GTT TGT ACC, CTT TCA GTG TTG TGA GCG TG-3'; IL-5, 5'-TGA CGA GCA ATG AGA CGA TG, TCA TCA CGC CAA GGA ACT CT-3'; IFN-{gamma}, 5'-CCC TCT CTG GCT GTT ACT GC, CTC CTT TTC CGC TTC CTT AG-3'; TNF-{alpha}, 5'-CGA GTG ACA AGC CCG TAG CC, GGA TGA ACA CGC CAG TCG CC-3'; ß-actin, 5'-GGT CAG AAG GAC TCC TAC GTG, CAG CAC TGT GTT GGC ATA GAG-3'.

Flow cytometric analysis of cell phenotypes. Cells to be analyzed were washed with HBSS twice and resuspended in PBS containing 1% rat serum. Cells (1 x 106) were incubated with 10 µl of mAb (Bioproducts for Science, Serotec, Oxford, U.K.) for 30 min at 4°C. mAbs directed against the following cell surface molecules were used: TCR Cb (R73), CD4 (W3/25), CD8 (OX-8), CD45RC (OX-22), MHC class I (OX-18), and MHC class II (OX-6 and -17). Cells were washed twice with PBS followed by incubation with FITC-conjugated F(ab')2 goat anti-mouse Ig (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at 4°C. Cells were washed twice with PBS, resuspended in 1 ml of 1% paraformaldehyde, and analyzed on an EPICS Profile II flow cytometer (Coulter, Hialeah, FL), using an excitation wavelength of 488 nm. Forward and right angle light scatter measurements were used for gating lymphocyte populations, and cursors were set based on fluorescence staining of isotype control-stained cells. Data were collected as relative fluorescence intensity counting 2500 gated cells and were represented on a 4-decade log scale.

Histology

For histopathologic assessment, spinal cords were removed and fixed in 10% formalin, after which the tissue sections were dehydrated and embedded in paraffin, and 5-µm sections were made. The sections were then stained with hematoxylin-eosin.

Proliferative cultures

Lymph nodes and spleens were removed from the rats 10 days after immunization with Ag in CFA, and single cell suspensions were prepared. The cells were placed in 24-well flat-bottom microtiter plates (Nunc, Copenhagen, Denmark) at 5 x 105 cells/well in RPMI 1640 supplemented with 10 mM HEPES, 5 x 10-5 M 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% 100x MEM vitamins, 1% 100x MEM nonessential amino acids, 250 µg gentamicin/ml, and 2% rat serum plus Ag. Proliferation was also induced with ionomycin (200 ng/ml; Sigma) and PMA (1 ng/ml; Sigma) or with rIL-2 (100 U/ml). Depending upon the experiment, after 2 or 3 days of culture 50 µCi/ml of [3H]thymidine was added, and the cultures were harvested 16 h later by transferring the contents of the 24-well plates into 96-well plates and adding all counts so that incorporation of radioactivity into DNA was determined. For secondary proliferative assays and assays to detect IL-2 in culture supernatants using HT-2 indicator cells, cultures were set up in standard 96-well plates. Secondary cultures were harvested at the times indicated.

Serum Ab

Ninety-six-well plates were incubated overnight at 4°C; their wells contained Ag in 50 µl of PBS (concentrations: Golli, 1 µg/ml; MPB, 10 ng/ml). Ag solution was then removed, and wells were blocked using 200 µl of 1% BSA for 2 h. After washing, duplicate wells containing 100 µl of rat serum at the dilutions given in Fig. 2Go were incubated overnight at 4°C. Wells were washed and incubated for 2 h at room temperature with a 1/200 dilution of goat anti-rat Ig coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories). ABTS was used as the color reagent, and plates were read at a wavelength of 600. Isotype analysis was conducted as described above except that, instead of using goat anti-rat Ig, mouse anti-rat isotype reagents were used (Serotec RMT RC1) followed by a rat anti-mouse mAb coupled to AP (PharMingen). The AP substrate used was PNPP (Sigma, catalogue No. 104-0) as the color reagent, and plates were read at a dual wavelength of 405/750.



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  		FIGURE 2. Serum binding activity by ELISA analysis. A, Serum Ab obtained from Lewis rats immunized with various doses of Golli, glyGolli, and acGolli peptides; 5 µg of whole guinea pig MBP (GP-MBP); or normal rat serum (NRS) was tested. All immunogens had been emulsified in CFA, and serum was obtained 10 days after Ag injection. The results presented show the binding of anti-MBP serum on wells coated with 10 ng/ml of GP-MBP or anti-Golli peptide (all forms) serum on wells coated with 1 µg/ml of Golli-peptide. The secondary Ab used was a goat anti-rat Ab labeled with horseradish peroxidase, and ABTS was used as a substrate. The ELISA plates were read at an OD of 600 nm. B, Serum Ab at a dilution of 1/50 from Lewis rats immunized with GP-MBP ({blacksquare}) or from normal controls () was isotyped by binding to GP-MBP (10 ng/ml)-coated plates. The secondary Abs used were mouse anti-rat isotype-specific mAbs followed by a rat anti-mouse Ab coupled to AP with PNPP as the substrate. The ELISA plates were read at a dual wavelength of 405/750. The experiment was repeated twice.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of paralysis in rats receiving Golli peptides

We chose the first expressed exon of the unique region of Golli to begin our examination for immunoreactivity. The peptide (12 mer) sequence derived from exon 2 of mouse Golli, residues 3–14, was synthesized. Although glycosylated forms of Golli proteins have not been described, Golli exon 2 (the first translated exon) contains two potential N-glycosylation sites, of which the first falls near the N-terminus of the protein product. Therefore, a glycosylated version of the peptide was also made. In the glycopeptide, the asparagine carried a ß-linked Gal-GlcNAc disaccharide. Finally, as there is precedent for only N-terminally acetylated versions of the conventional MBP1–11 in H-2u mice (28) and an N-terminally acetylated epitope of proteolipid protein to be encephalitogenic in the Lewis rat (29), a third peptide, an ac-Golli3–14, was made.

Initial studies were conducted in Lewis rats by comparing the ability of MBP vs Golli peptides to induce EAE. MBP-CFA given at 50 µg of Ag/animal induced disease on day 12, which peaked on day 15 with hind limb paralysis and rapidly subsided. The unsubstituted Golli peptide given at the same dose resulted in no clinical symptoms, whereas the glycosylated form of the Golli peptide at this dose led to tail paralysis (Fig. 1GoA).



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  		FIGURE 1. Neurologic symptoms induced by Golli peptides. A, Lewis rats were injected s.c. with 50 µg of MBP ({circ}), Golli peptide ({square}), and glycosylated Golli peptide (•) emulsified in CFA. B, Lewis rats were injected with either 5 µg ({circ}) or 50 µg (•) of glycosylated Golli peptide in CFA. Animals were followed for clinical signs of disease as follows: distal tail paralysis, 0.5+; flaccid tail, 1+; ataxia, 2+, hind limb paralysis, 3+; and full limb paralysis, 4+. Data represent the means of disease scores for two rats for each Ag.

 
An examination of the dose-response relationship showed that lower doses of Golli peptides were more effective at inducing disease. This was manifested as more animals with more severe symptoms at an earlier time of onset (Fig. 1GoB and Table IGo). Paralysis occurred, similar to that seen with MBP. In contrast to MBP (Fig. 1GoA), the symptoms appeared later, starting on approximately day 15 (Table IGo). Also, swelling of the hind feet developed with either Golli or glyGolli peptides. Swelling of the hind feet never occurred independent of other symptoms. Since such swelling was not seen with CFA or MBP in CFA (all immunogens were prepared with the same lot of CFA), the possibility that the peptides were inducing an inflammatory response due to toxicity independent of a specific immune response was tested by injecting the Golli peptides in saline s.c. Visual and histological examination found no swelling or local infiltrates (data not shown). This lack of response and the inability of the highest concentrations of Golli peptides to induce any symptoms strongly indicated that this swelling was not due to toxicity of the Ag preparation but instead was most likely due to an immune response.


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  		Table I. Golli-mediated CNS disease: active induction of disease with Golli peptides in adjuvant

 
Relative stability of Golli peptides

The initial finding that the glycosylated Golli peptide was a better inducer of disease than the nonmodified parent peptide suggested that the glycopeptide was a more stable molecule in vivo and/or that the glycosylated form of Golli was expressed in vivo. The fact that glyGolli T cells could be stimulated with Golli peptide and still cause disease did not help us distinguish between these possibilities (Table IIGo and data not shown). We found that the glycopeptide was indeed much more stable in 25% rat serum in vitro than the nonglycosylated parent analogue. While only 5% of the glycopeptide decomposed after 2 h and 33% decomposed after 4 h, 65% of the nonglycosylated peptide was degraded after 30 min, and the peptide was fully degraded after 2 h (Table IIIGo). The acetylated Golli-MBP peptide was also stabilized (10% decomposed after 30 min, 21% decomposed after 2 h, and 56% decomposed after 4 h) and gave a level of disease similar to that produced by glyGolli (Table IGo).


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  		Table II. Golli-mediated CNS disease: adoptive transfer of disease with Golli peptide-specific T cell lines

 

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  		Table III. Stability of Golli-MBP peptides in 25% rat serum1

 
Lack of an Ab response

Sera from rats injected with any of the Golli peptides in CFA at several doses (0.5–50 µg/animal) showed little or no detectable anti-Golli Ab (Fig. 2GoA). In contrast, anti-MBP Ab was clearly detectable after MBP-CFA immunization (Fig. 2GoA). Also, Golli-primed rats did not make Ab to MBP, nor did MBP-primed rats make Ab to the Golli peptides (data not shown). Isotype analysis of the anti-MBP Ab revealed a response composed of IgA, IgG1, and IgG2c (Fig. 2GoB). This is consistent with a mixed Th1 (IgG2c)/Th2 (IgG1 and no IgG2a) response (30).

T cells can transfer tail paralysis

Lymph node and spleen cells from Lewis rats injected with the nonglycosylated or glycosylated peptides proliferated in culture in response to the immunizing Ag. These cells were then used for adoptive transfer. Animals receiving activated Golli-, glyGolli-, or ac-Golli-specific cells showed signs of low level disease (Table IIGo); no differences in potency were obvious among the different cell populations. Adoptive transfer never resulted in the level of disease induced by Ag in CFA or the severe disease transferred by MBP-activated T cells (data not shown).

Histological evidence of T cell infiltration with adoptive transfer

The CNS histopathology of spinal cords from Lewis rats with adoptively transferred T cells specific for MBP, glyGolli, or OVA is shown in Fig. 3Go. Subarachnoid or meningeal plaques as well as lesions in adjacent submeningeal white matter could be seen in spinal cords of rats transferred with glyGolli-specific T cells but not in those transferred with OVA- or MBP-specific T cells. In contrast, transfer of MBP-specific T cells induced perivascular infiltrates not seen with OVA- or glyGolli-specific T cells. The Golli-specific cells were seen as tight ball of cells, and many such groups of cells were seen unassociated with or on the exterior of the spinal cord, presumably having been previously associated with meninges, which often do not remain intact upon sectioning.



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  		FIGURE 3. Histological cross-sections of infiltrating cells in the spinal cord of Lewis rats that had received adoptively transferred T cells. A, OVA-specific T cells (3 x 107) were adoptively transferred into 350R Lewis rats and represent a negative control. No perivascular infiltrates can be seen (x40 magnification). The same lack of infiltrates was observed with the same number of adoptively transferred Golli-specific T cells (data not shown). B, Perivascular cuffing (arrow) in the spinal cord can be seen when MBP-specific T cells are adoptively transferred (x40 magnification). C, No infiltrates can be seen in the subarachnoid space of the spinal cord, with the transfer of OVA-specific T cells used as a negative control (x10 magnification). The same lack of infiltrates is seen with the adoptive transfer of MBP-specific T cells (data not shown). D andE, With the adoptive transfer of Golli-specific or glyGolli-specific T cells, clumps of interacting cells (arrow) can be found in the subarachnoid space (x10 and x40 magnification, respectively). This clump of cells is at the margin of the basilar vein (BV), which is dorsal to the basilar artery (BA), and all these structures are found in the subarachnoid space. F and G, Tight cell clumping (arrow) seen in the white matter (F) and at the gray/white junction (G) with the adoptive transfer of Golli or glyGolli-specific T cells. One lesion per spinal cord was seen with Golli' cells transferred compared with three or four lesions per spinal cord with MBP' cells transferred.

 
Properties of T cells that cause disease

FACS. The phenotypes of Golli and glyGolli-stimulated cells were analyzed by FACS and compared with MBP- and OVA-stimulated T cell populations (Fig. 4GoA). All populations of cells were approximately 75% TCR{alpha}ß+ or greater and predominantly CD4+, although the glyGolli T cells showed a greater percentage of CD8+ T cells than the MBP-stimulated T cells. The phenotype of the Golli-specific T cells (not shown) was similar to that of the glyGolli-specific T cells (Fig. 4GoA). The level of CD45RC (OX-22) differentiated the glyGolli- and Golli-stimulated T cells from the OVA- and MBP-specific T cells. The Golli T cells were shifted to an intermediate-high OX-22+ population, whereas the majority of MBP- and OVA-reactive T cells were low-intermediate OX-22+ (OVA spleen was similar to MBP spleen; data not shown). Freshly isolated unstimulated T cells from immunized rats were OX-22low. Increased OX-22 staining intensity occurred within 3 days of in vitro stimulation for both MBP and glyGolli populations. The MBP population lost intensity within 3 days of activation, suggesting that CD45RC is an activation marker. GlyGolli T cells, however, remained OX-22high for 6 days (Fig. 4GoB), suggesting that these cells were still being activated.



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  		FIGURE 4. FACS analysis of stimulated T cell lines. A, Lymphocytes were obtained from spleens and lymph nodes of Lewis rats immunized 10 days previously with GP-MBP, glyGolli peptide, and OVA. Cells were stimulated with Ag in culture, blasts were isolated on a Ficoll gradient 3 days later, then the cells were placed in a resting culture supplemented with 5% Con A supernatant for 5 days before analysis by flow cytometry. The cells were stained with anti-CD4 mAb (W3/25), anti-CD8 mAb (OX-8), anti-TCRß constant region (R73), and anti-CD45RC (OX-22). The secondary Ab alone is shown as the solid area, and the specific binding is shown as the open area. B, Lymphocytes, 3 and 6 days after stimulation with Ag in bulk culture, were analyzed for the binding of OX-22. C, Lymphocytes were stimulated with Ag for 3 days in bulk culture, and then separated blasts were cultured in medium supplemented with 5% Con A supernatant plus B35 Ab (an isotype control specific for herpes simplex virus), OX-6 Ab (the anti-Ia Ab used that is specific for the I-A isotype), or no Ab. After 4 days of culture, the cells were washed and used for flow cytometry to assess their expression of CD45RC using OX-22 Ab.

 
To determine whether the long term activation of Golli T cells was due to class II-mediated stimulation, we added a blocking anti-MHC class II Ab. Golli-specific T cells lost CD45RC staining intensity upon addition of OX-6, an anti-MHC class II I-A Ab (Fig. 4GoC), but not with addition of OX-17, an anti-class II I-E Ab, or with OX-18, an anti-class I Ab (data not shown). This supports the contention that continual T cell activation is through MHC class II.

Cytokines. The T cells were also analyzed for cytokine-specific mRNA using RT-PCR. As shown in Fig. 5GoA, MBP-reactive T cells from day 3 primary Ag cultures made mRNA for IL-2 and IFN-{gamma} and low levels of mRNA for IL-4 and IL-5, supporting the possibility of a mixed Th1-Th2 or Th0 population (along with the presence of an IgG1 and IgG2c anti-MBP Ab; Fig. 2Go). Golli-specific T cells, on the other hand, made IL-2, low IFN-{gamma} mRNA, but no IL-4 or IL-5 mRNA, indicating a Th1-only response. Expression of cytokine protein was determined using a biological assay for IL-2 with HT-2 cells and for IL-3 and/or granulocyte-macrophage CSF with DA-1 cells, and an ELISA for IFN-{gamma}. In all Ag-stimulated primary cultures, significant proliferation and IFN-{gamma}, IL-2, and IL-3 production were detected (data not shown). However, backgrounds were often high, possibly due to Ag carryover on APC from the immunized rat; therefore, secondary Ag cultures were set up.



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  		FIGURE 5. Semiquantitation of RT-PCR cytokine products. The cytokine mRNA levels relative to internal ß-actin from T cells were determined by titration using the cDNA RT-PCR method (see Materials and Methods). Lymphocytes were removed from lymph nodes of Golli-primed ({blacksquare}) or MBP-primed (•) Lewis rats, stimulated once with Ag in culture for 3 days (upper panel, A), rested, and then restimulated (lower panel, B). Blasts were separated on Ficoll, and RNA was isolated.

 
Analysis of cytokine mRNA in secondary cultures (Fig. 5GoB) indicated that MBP-specific T cells expressed IL-2 and IFN-{gamma} mRNA, Golli T cells expressed only IFN-{gamma} mRNA. Consistent with these results, MBP-specific T cells proliferated and made detectable IL-2 in response to MBP, but Golli-specific T cells neither proliferated nor made IL-2 in response to Ag (Fig. 6GoA). IFN-{gamma} was produced by MBP-reactive T cells in response to Ag, whereas constitutive production was seen with Golli-specific T cells (Fig. 6GoB).



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  		FIGURE 6. Proliferation and cytokine production. A, Lymphocytes from lymph nodes of Lewis rats immunized 10 days previously with Ag in CFA were placed into culture with Ag. These bulk cultures were separated on Ficoll-Hypaque on day 3 after Ag exposure to select Ag-specific blasts. The cells were then cultured in medium supplemented with 5% Con A supernatant for 7 days. The cells were restimulated with Ag a second time in a 96-well plate, and after 48 h the plate was pulsed with [3H]thymidine for 18 h to assess proliferation. Supernatants (50 µl/well) from these secondary cultures were harvested at 24 h and cultured with HT-2 cells for 24 h; the plate was then pulsed with [3H]thymidine for 6 h ({blacksquare}, PROLIF; , IL-2). B, As described in A the same supernatants (50 µl/well) were analyzed by ELISA for IFN-{gamma}. Plates were coated with monoclonal anti-IFN-{gamma} Ab; supernatant or recombinant rat IFN-{gamma} as a standard (C) was then added, followed by goat anti-rabbit Ig coupled to AP. PNPP was used as the color reagent, and plates were read at a dual wavelength of 405/750.

 
Evidence of anergy

The inability to proliferate to Ag by the Golli T cells was further explored by the addition of PMA and ionomycin, as shown in Fig. 7Go, A and B. These cells, after primary Ag stimulation and a 7-day rest period, were able to respond vigorously to ionomycin and PMA. Addition of IL-2 also led to proliferation and supported the idea that these cells were still viable and had become nonresponsive to Ag but were still responsive to IL-2.



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  		FIGURE 7. Proliferative responses of secondary T cells. Lymphocytes from Lewis rats immunized with the Golli peptide or MBP were stimulated with Ag; on day 3 blasts were recovered and rested in medium plus 5% Con A supernatant for 8 days. These cells were then cultured in 96-well flat-bottom plates with the following: x-irradiated splenic APC with or without Ag (upper panel) or PMA (1 ng/ml), ionomycin (200 ng/ml), rIL-2 (100 U/ml), and DMSO (3.5 x 10-7 M) as a control for the diluent in the ionomycin (lower panel). After 72 h, plates were pulsed overnight with [3H]thymidine, harvested, and counted.

 
If these cells were anergic, could they cause disease? If not, could this be affected by the addition of IL-2? Golli T cells were obtained from immunized rats, stimulated with Ag for 3 days, rested, and then restimulated with Ag or IL-2 for 3 days and injected into rats. As shown in Fig. 8Go, MBP T cells stimulated with MBP induced EAE; these T cells stimulated with IL-2 alone induced less severe disease. In contrast, Golli T cells stimulated with Golli peptide induced little or no disease; however, when stimulated with IL-2, these cells induced clear paralysis.



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  		FIGURE 8. Adoptive transfer of paralysis by stimulated T cells. Lymph node cells from rats immunized with Golli peptide (5 µg/animal) or guinea pig MBP (50 µg/animal) were stimulated with 5.0 µg/ml of the immunizing Ag in a primary bulk culture. On day 3 blasts were recovered, and these cells were rested for 7 days in medium plus 5% Con A supernatant. Each population was then divided in half and stimulated with either Ag plus x-irradiated spleen cells as APCs (Golli-specific T cells stimulated with Golli peptide (•; n = 1) and MBP-specific T cells stimulated with MBP (•; n = 4)) or rIL-2 at a final concentration of 100 U/ml (Golli-specific T cells ({circ}; n = 3) and MBP-specific T cells ({circ}: n = 3)). Three days later, blasts were isolated from these cultures, and 2.4 x 106 cells were adoptively transferred into 350R naive Lewis rats, which were followed for disease symptoms. Similar results were obtained using various doses of both lymph node and splenic T cells.

 
Examination of Golli and MBP primary day 3 stimulated and 5-day rested cells (Fig. 4GoA) and secondary cells, both the resting and day 3 stimulated cells (data not shown) showed no change in TCR (R73) surface expression levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we used a synthetic peptide derived from Golli exon 2, the first translated exon of the Golli locus, which encodes an amino acid sequence present in all known Golli protein products but not in any of the MBPs. Various splice variants of Golli have been shown to be expressed in the developing and mature CNS and also in the developing immune system. We investigated whether this protein might serve as an autoantigen or a molecule that potentially regulates autoimmune responses. We used both glycosylated and nonglycosylated forms of the peptide due to the presence of a natural glycosylation site, although it is not known whether Golli proteins are glycosylated in vivo. Injection of the peptide in adjuvant into adult Lewis rats resulted in mild encephalitogenic disease, whereas injection of the glycosylated form resulted in more severe disease. T cells derived from immunized rats were able to transfer mild paralysis and lesions were found in the meninges. Although these cells caused disease, they also showed signs of nonreactivity after stimulation in culture. This may be why adoptive transfer resulted in less severe disease.

CHO determinants

Because a potential N-glycosylation site exists at the Asn-His-Ser of the Golli exon 2 sequence, we made both glycosylated and nonglycosylated peptides. The level of disease achieved was greatest with glyGolli, less with acetylated Golli, and least with Golli peptide. Several possible explanations are suggested by this observation. First, it may be that the determinant in vivo is actually glycosylated and that is the form in which it is presented. There is evidence for the presentation of glycosylated peptides to class I-restricted CTL (31) as well as to class II-restricted T cells (32, 33, 34, 35). Because only 30% of the predicted N-glycosylation sites are used in natural glycoproteins (36), it is tempting to speculate that the degree of glycosylation has a causative relationship with the severity of the induced disease. On the other hand, we have no evidence that this molecule is glycosylated.

Second, it may be that glycosylation stabilizes the molecule. Earlier, we have shown that glycosylation increases peptide stability in serum, especially when the sugar moiety is placed at an N-terminal position (25). In fact, glycosylated analogues of MHC class II binding Ags retain considerably more T cell stimulatory potency than the unglycosylated parent analogues after incubation in serum provided that the glycopeptides retain their ability to bind to MHC (21, 26). Support for this hypothesis in this study comes from the fact that the hierarchy of disease induction follows precisely the degree of stability in vitro in rat serum, but this does not rule out the possibility that the glycosylated form of Golli is preferentially recognized in vivo.

Third, it is possible that the glycosylated peptide stimulates T cells with a higher affinity for the Golli determinant. However, adoptive transfer does not support this idea, as Golli-specific and glyGolli-specific T cells are equally good at disease induction.

CD45RC

The Golli-specific T cells generated in this study were found to be CD45RChigh, as shown by the binding of the OX-22 mAb after in vitro stimulation. It has been shown in the rat that OX-22high T cells are Th1-like as they make IL-2 and IFN-{gamma}, are involved in graft-vs-host disease, and cause the down-regulation of autoimmune B cell responses to mercury, possibly by regulating Th2 responses. In contrast, OX-22 low T cells are Th2-like, as they make IL-4 and regulate graft-vs-host disease in both rat and mouse (13, 37, 38, 39, 40). Consistent with the cytokine characteristics of OX-22high T cells, the Golli-specific T cells in this study secreted IL-2 and IFN-{gamma} but expressed no IL-4 or IL-5 mRNA. Furthermore, the animals immunized with the Golli peptide do not appear to make specific Ab.

When we examined the kinetics of appearance of OX-22, we found that both MBP and Golli populations became OX-22high upon Ag stimulation in vitro. The MBP T cell population rapidly returned to OX-22low status, supporting the possibility that CD45RC is an activation molecule. That Golli T cells remained OX-22high suggest that these cells were being stimulated chronically in vitro. We found that the addition of anti-class II I-A specific Ab to a purified T cell 3-day blast population led to a decrease in OX-22 staining, which did not occur with anti-class I or anti-class II I-E. We believe that such a result is consistent with the chronic class II-restricted Ag stimulation not seen with MBP-reactive cells. Furthermore, we found that secondary stimulation with Golli Ag revealed constitutive IFN-{gamma} production. We also noted the lack of IL-2 production or proliferation, classic properties of anergic cells (41, 42, 43, 44, 45, 46, 47, 48). Stimulation of proliferation was demonstrated in this population using ionomycin and PMA or high concentrations of IL-2, indicating that the T cells had not undergone apoptosis.

What might be causing this chronic stimulation? It has been shown that T cells make Golli mRNA (S. Amur-Umerjee and A. T. Campagnoni, unpublished observation) (49). It is thus possible that T cells present Golli to each other through class II, expressed on the surface of activated rat T cells. This also provides an explanation for the appearance of cells as clusters in the CNS. That rat T cells present Ag and lead to anergy has been previously reported (50). Here, however, Ag is potentially being presented to self. The possibility that autoimmune cells are down-regulated through chronic stimulation is suggested in a recent study on anti-dsDNA B cells in nonautoimmune mice (51) in which the B cells are anergic and display an activated phenotype.

Cells in the meninges

It is not known why the OX-22high Golli-specific T cells might be found in the meningeal and submeningeal regions of the CNS. One possibility is that Golli is both present in the meninges and presented as Ag, and Golli-specific T cells migrate to this region. However, Golli determinants are also present in the white matter, so why are these cells not found in abundance there? A previous EAE study using guinea pig MBP in the Lewis rat examined OX-22 staining and the location of infiltrating lymphoid cells. It was found that OX-22-negative cells were seen in the spinal cord parenchyma, and OX-22-positive cells were seen in the subarachnoid space (49). Although it is not clear why this is so, it may be that the cells found in the subarachnoid space are in fact Golli specific. These could have been induced by a response to MBP exon 1 (Golli exon 5). A second study of interest performed in the mouse involved TCR transgenic T cells specific for MBP1–11, a determinant found in both classical MBP and Golli proteins. Here, T cells when injected i.v. migrated to the parenchyma of the brain, but when injected intrathecally migrated to the meninges and not the parenchyma. These cells are potentially both MBP and Golli specific, and intrathecal injection may bypass white matter and lead to meningeal migration (52). It is known that T cells specific for MBP68–88 from Lewis rats injected intracranially show accumulation in the parenchyma and not in the meninges, as these cells cannot recognize Golli since MBP68–88 is not present in Golli (53, 54).

Presence of Golli peptides in the thymus

As discussed above, Golli-MBP genes are expressed in the thymus of the mouse, human, and rat. Furthermore, Golli-MBP protein can be found in thymic macrophages. Whether these thymic macrophages are making or have scavenged the protein is not clear. Similar to what has been found with another CNS Ag, S100, in which interdigitating cells of the thymus express this Ag (55), preliminary studies with the rat thymus show that Golli has the same broad expression pattern as S100 (F. A. McMorris and E. Heber-Katz, unpublished observations). How does thymic expression affect immune responsiveness and the development of the T cell repertoire?

Shared determinants found in MBP exon 1 and Golli exon 5 do not seem to delete the murine response to the 1–11 dominant determinant in H-2u mice. Furthermore, it has been reported that MBP-specific murine T cells respond to an Ag in the thymus that could be Golli (56). It has also been shown that human T cells specific for MBP residues 11–29 recognize the human Golli proteins HOG5 and HOG7, and those recognizing the dominant determinant MBP83–99 recognize HOG7 (57, 58).

Conclusions

In the present study we show that the Golli exon 2 peptide can induce CNS autoimmune disease and that T cells can adoptively transfer disease to naive recipients. Clearly, in the rat such cells have not been eliminated by negative selection.

However, these cells are unusual, since they rapidly become nonresponsive in culture. It is possible that Ag in the thymus leads to chronic stimulation, which, in turn, leads to a nonresponsive state. Immunization with peptide in adjuvant could then activate such cells in the periphery, resulting in autoimmunity, although the cells might easily fall back into a nonresponsive state. On the other hand, as we suggest above, T cells themselves may be synthesizing and presenting this Ag, again leading to chronic stimulation and anergy. If this is so, then this is a novel way of regulating the immune response and may be operating for other T cell Ags, such as TCR determinants (59), as well as for B cells (51).


   Acknowledgments
 
We thank H. Quill, L. Spain, J. Erikson, M. Jenkins, and E. Blankenhorn for useful discussions and the review of this manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants NS11037 and NS33902 (to E.H.-K.), GM45011 (to L.O.), and CA72806 (to P.L.S.). P.L.S. is a recipient of an Arthritis Foundation Investigator Award; A.F.S., G.E.L., and F.A.M. are supported by National Institutes of Health Grant NS32122 and a National Multiple Sclerosis Society grant; F.A.M. is supported by an H. H. Smith Foundation grant. Back

2 Address correspondence and reprint requests to Dr. Ellen Heber-Katz, The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. E-mail address: Back

3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; CNS, central nervous system; MBP; myelin basic protein; Golli, gene of the oligodendrocyte lineage; E-MBP, embryonic myelin basic protein; glyGollli, glycosylated Golli; acGolli, acetylated Golli; AP, alkaline phosphatase; ABTS, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid; PNPP, {rho}-nitrophenyl phosphate. Back

4 A. Skorupa, S. Goldman-Brezinski, G. Lesch, E. Heber-Katz, and F. A. McMorris. Expression of Golli mRNA during development of immune cells in the rat. Submitted for publication. Back

Received for publication May 15, 1998. Accepted for publication December 10, 1998.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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