Myelin/Oligodendrocyte Glycoprotein–Specific T-Cells Induce Severe Optic Neuritis in the C57Bl/6 Mouse

Abstract

purpose. The optic nerve is a common site of tissue damage in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). To determine the relationship between optic neuritis (ON) and EAE, we examined the incidence of ON in C57BL/6 (B6) mice immunized with a myelin oligodendrocyte/glycoprotein (MOG)–derived peptide or injected with MOG-specific T cells, which are known to induce EAE.

methods. Mice were immunized with MOG35-55 or MOG40-54 peptides emulsified in complete Freund’s adjuvant (CFA). Pertussis toxin (PTX) was injected intraperitoneally 1 day before and after immunization. For disease induction by adoptive transfer of primed cells, donor C57BL/6 mice were received with T-cell blasts (1–6 × 10⁶/mouse). Both EAE and ON were observed by either clinical signs or histology.

results. ON developed in a high proportion of B6 mice treated with either protocol. The most severe inflammation was observed in the adoptively transferred mice. The induced ON was most frequently bilateral. In either actively or adoptively transferred diseases, both association and dissociation of EAE and ON was observed.

conclusions. Different MOG-specific T-cell subsets might be involved in the pathogenesis of EAE and ON. A better understanding of the pathogenesis of ON after induction by MOG may have important diagnostic and therapeutic implications.

One of the most devastating complications of vaccination against viral pathogens and of viral infection is acute optic neuritis (ON). ^{1 2 3 4} This typically presents in young adults as sudden monocular loss of vision accompanied by eye pain and occurs more frequently in women than in men. ON is often observed in patients with multiple sclerosis (MS) ^{5 6} and in experimental autoimmune encephalomyelitis (EAE) in various animal species, including the mouse, ^{4 7 8 9 10} rat, ¹¹ guinea pig, ¹² and primates. ^{13 14} The development of ON after induction of EAE suggests an autoimmune origin of the optic nerve inflammation observed in MS. 

Recent studies have demonstrated that a large fraction of transgenic mice with a T-cell receptor (TCR) specific for a major myelin protein, myelin/oligodendrocyte glycoprotein (MOG), have spontaneous development of ON in the absence of clinical and histologic EAE, ¹⁰ implying that MOG-specific autoreactive T cells play a major role not only in encephalomyelitis, but also in ON. In the present study, we investigated optic neuropathy induced by encephalitogenic peptides derived from MOG. To establish a working model for ON in nontransgenic mice on a B6 genetic background (for which genetically compatible knockout mice are available), and to further determine the pathogenic association between ON and EAE, we investigated the optic neuropathy induced by MOG-derived encephalitogenic peptides and found that severe ON could be induced either by immunization of B6 mice with the encephalitogenic epitope MOG35-55 in complete adjuvant or by transfer of in vitro cultured MOG-specific T cells into naïve B6 mice. The availability of this experimental model of ON should greatly facilitate efforts to determine the pathogenesis of ON and identify possible novel therapies. 

Methods

Mice

C57BL/6 (B6) mice (8 to 10 weeks old), obtained from the Jackson Laboratory (Bar Harbor, ME), were housed and maintained at the animal facilities of the University of Louisville. Treatment of the animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Reagents

Peptides MOG35-55 (MEVGWYRSPFSRVVHLYRNGK), MOG40-54 (YRSPFSRVVHLYRNG), and human interphotoreceptor retinoid-binding protein (IRBP)1-20 (GPTHLFQPSLVLDMAKVLLD) peptide were synthesized by Sigma-Aldrich (St. Louis, MO). 

Induction and Assessment of EAE and ON

For disease induction by active immunization, groups of B6 mice (n = 6) were immunized by multisite injection (subcutaneous and footpads) with 50 to 200 μg of MOG35-55 or MOG40-54 in 75 to 100 μL of phosphate-buffered saline, (PBS; pH 7.35) emulsified with an equal volume of complete Freund’s adjuvant (CFA) made by adding 600 μg/mL of Mycobacterium tuberculosis to incomplete Freund’s adjuvant (Sigma-Aldrich). All animals were also injected intraperitoneally on days −1 and +1 with 400 ng of pertussis toxin (PTX; Sigma-Aldrich). 

For disease induction by adoptive transfer of primed cells, donor C57BL/6 mice were immunized with 200 μg MOG35-55, as just described, and 15 days later, lymph node and spleen cells were collected and pooled and the cell suspension adjusted to 10⁷ cells/mL in culture medium. The cell cultures in six-well plates were incubated for 48 hours with MOG35-55 (5 μM), then the activated T-cell blasts were separated by gradient centrifugation (Ficoll; Pharmacia Upjohn, Uppsala, Sweden), washed, and injected intraperitoneally (1–6 × 10⁶ cells/mouse) into naive C57BL/6 mice. 

Diagnosis of EAE was made on the basis of the clinical symptoms of the disease or by pathologic examination of the spinal cord of the same animals that were examined for ON. Clinical assessment of EAE was performed daily according to the following criteria: 0, no disease; 1, decreased tail tone; 2, hindlimb weakness or partial paralysis; 3, complete hindlimb paralysis; 4, front and hindlimb paralysis; and 5, moribund state. ON was diagnosed by pathologic examination of randomly sampled mice and was scored by using the following criteria, based on the degree of infiltration seen in sections of optic nerve: 0, no disease; 0.5, mild mononuclear cell infiltration of the optic nerve parenchyma, and/or optic nerve sheath; 1.0, moderate mononuclear cell infiltration of the optic nerve parenchyma and/or optic nerve sheath; 2.0, severe infiltration of the optic nerve parenchyma; and 3.0, massive or nodular infiltration of the optic nerve parenchyma. 

Limiting Dilution Analysis

B6 mice were immunized subcutaneously with 200 μg MOG peptides emulsified in CFA, and the spleen and draining lymph nodes were removed 15 days later and a single-cell suspension prepared. T cells were enriched by nylon wool adherence and seeded at 3 × 10⁴ to 5 × 10⁵ cells/well with 24 replicate wells for each cell density in two sets of 96-well flat-bottomed culture plates containing irradiated spleen cells (1 × 10⁵ per well), one set of which contained an optimal dose of immunizing peptide (20 μg/mL). Seventy-two hours later, the plates were pulsed for 6 hours with 0.5 μCi [³H]thymidine/well and the cells assessed for isotope incorporation (Packard Instruments, Meriden, CT). The number of T cells seeded in each well was based on preliminary limiting dilution analysis (LDA) estimates of MOG-reactive cell frequencies. 

Pathologic Examination

For study of EAE, mice were anesthetized, exsanguinated, and perfused with 25 mL PBS and 10 mL 4% paraformaldehyde in buffered PBS. Brains and spinal cords were dissected and fixed in 4% paraformaldehyde before embedding in paraffin. Paraffin sections were stained with hematoxylin/eosin and Luxol fast blue (for myelin; Fisher Scientific, Pittsburgh, PA). For histology study of ON, whole eyes were collected, immersed for 1 hour in 4% phosphate-buffered glutaraldehyde, and transferred to 10% phosphate-buffered formaldehyde until processed. The fixed and dehydrated tissue was embedded in methacrylate and 5-μm sections cut through the pupillary–optic nerve plane and stained with hematoxylin and eosin. The evaluation of the presence or absence of disease was masked, by examining six sections cut at different levels for each eye. 

Statistical Analysis

The data are expressed as the mean ± SD. Each experiment was repeated at least three times. Student’s t-test was used to analyze the results. 

Results

Induction of ON by Immunization of B6 Mice with MOG Peptides

More than 40 B6 mice were studied for ON and EAE after immunization with MOG35-55, a known encephalitogenic peptide derived from MOG. ^{15 16} Recipient mice were injected with 200 μg of MOG35-55 emulsified in an equal volume of CFA on day 0 and with two doses of 400 ng of PT on days −1 and +1. All animals were monitored daily for weight loss and clinical symptoms (paralysis) of EAE and randomly selected from day 15 (early disease phase) to day 60 (late phase of disease) for pathologic study. 

Paralytic symptoms characteristic of EAE were observed from day 15 to 20 post injection (pi), at which time mononuclear cell infiltrates were readily identifiable in both the spinal cord (Figs. 1A 1B) and the submeningeal region and/or parenchyma of the optic nerve (Figs. 1C 1D 1E 1F) . 

ON was an early event, because inflammation of the optic nerves was seen in mice with preclinical symptoms of EAE, such as weight loss, which developed a few days before clinical and pathologic EAE. However, some animals failed to show evidence of ON, despite extensive spinal cord lesions. Examination of the optic nerves from mice at different time points (15–60 days pi) showed that ON was also a late feature of the disease. More than 50% of the mice showed mononuclear cell infiltration of the optic nerve at more than 30 days pi (Table 1) . ON induction was antigen specific, as a similar injection of T cells specific for a uveitogenic peptide, IRBP1-20, ¹⁷ only induced retinal disease (Fig. 2) . 

Induction of ON by Adoptive Transfer of MOG-Specific T Cells

We have shown that adoptive transfer of a few million isolated MOG-specific T cells into naïve B6 mice induces severe EAE, ¹⁶ and we followed a similar protocol to study the incidence of ON after adoptive transfer. At 15 days pi with MOG35-55/CFA, the animals were killed, and an enriched splenic T-cell population was prepared by passage through nylon wool. These T cells were incubated for 48 hours with irradiated spleen cells (antigen-presenting cells; APCs) and 20 μg/mL MOG35-55, and activated T-cell blasts were separated on a gradient (Ficoll; Fisher Scientific). As shown in Figure 3 , in mice receiving 6 × 10⁶ newly activated MOG-specific T cells, severe ON developed at 8 to 10 days pi. The pathologic features of ON occurring after adoptive transfer of MOG-specific T cells differed from those occurring after active immunization. As shown in Figures 1C 1D 1E , the optic nerve inflammation observed after active immunization was characterized by a diffuse distribution of infiltrating cells; however, after adoptive transfer, inflammatory nodules were observed throughout the optic nerve, varying in size from a quarter of the diameter (Figs. 3C 3D) to the entire diameter (Figs. 3A 3B) of the optic nerve. In most instances, the induced ON was bilateral (Fig. 3E) . 

Association and Dissociation of EAE and ON

The majority of mice immunized with MOG35-55 (>60%) had both EAE and ON. However, ON occurred in mice without EAE and vice versa (Table 2) , showing that the two diseases were not always linked. This observation appears to disagree with that made in the MOG-specific TCR transgenic mouse, in which ON had a lower threshold of disease induction than EAE. ¹⁰  

To examine further the dissociation and association of EAE and ON, we used three additional approaches: (1) inducing EAE and ON with a suboptimal dose (50 μg) of MOG35-55, (2) inducing EAE and/or ON with a truncated MOG peptide, MOG40-54, and (3) adoptively transferring disease with different numbers of activated MOG-specific T cells. As shown in Table 2 , both EAE and ON developed in a significant percentage of B6 mice immunized with 50 μg MOG35-55, showing that a low dose of autoantigen induced both EAE and ON. In addition, a significant proportion of the animals with a normal optic nerve had encephalomyelitis (Table 2) . We have shown that the truncated MOG peptide, MOG40-54, is encephalitogenic in the B6 mouse. ¹⁵ To determine whether this truncated 15-mer peptide was more able to induce ON preferentially, rather than EAE, compared with MOG35-55, we immunized groups of B6 mice (n = 6) with 200 μg MOG40-54 or MOG35-55 and examined the time-course of ON and EAE induction. Immunization with the two peptides gave essentially the same relative frequency of ON and EAE induction (Table 3) . Finally, we determined whether the transfer of a low number (1 × 10⁶) of MOG-specific T cells into naïve mice induces ON more frequently than EAE. As summarized in Table 2 , this small number of MOG-specific T cells induced both ON and EAE. There was no statistically significant difference in the incidence or severity of ON and EAE in the groups of animals receiving a low or high number of MOG-specific T cells. However, MOG40-54 appeared to be more immunogenic than MOG35-55 in B6 mice, because immunization with the same dose of MOG40-54 resulted in the isolation of twice as many total T cells (Table 3) . Using the LDA, we compared the MOG-specific T cells among the total splenic T cells, between MOG35-55–and MOG40-54–immunized animals, as we reported previously. ^{19 20} Our results show that MOG-specific T cells increased by 50% (7.6 per million) in MOG40-54–immunized mice compared with MOG35-55–immunized mice (5 per million, see Table 3 ). 

Discussion

The clinical association between encephalomyelitis and ON is well documented. In approximately 30% to 60% of MS patients, ON is an early clinical manifestation. ²¹ A similar observation has been made in several different experimental animal models that mimic human MS. ^{11 12 13 22}  

Studies in patients with ON have provided clinically important information regarding the risk of, and progression to, MS. ^{21 23 24} However, the pathogenic relationship between ON and MS remains obscure. Consequently, MS-like disease has been studied in several different animal models by immunization of disease-prone rodents with myelin proteins or pathogenic peptide derived from these proteins. ^{9 16 25 26}  

Although involvement of the optic nerve has been ultrastructurally documented in EAE in the guinea pig ²⁷ and rat, ²⁸ detailed murine studies have been limited to reports of inflammation of the optic nerve in proteolipid protein (PLP)-induced EAE in SJL mice ²² and MOG-induced EAE in rats. ²⁸ Like EAE induced by myelin proteins, ON appears to be induced by an aberrant cellular, rather than humoral, immune response (e.g., the severity of ON does not parallel the level of anti-myelin protein antibody). ¹² In the present study, the transfer of MOG-specific T cells into naïve B6 mouse readily induced ON. The incidence of ON was as high as the incidence of EAE. Among 40 mice immunized with MOG peptide and 16 mice injected with activated MOG-specific T cells, more than 60% of the recipients had ON develop. Thus, MOG-induced ON is a reproducible murine model and should be important in further investigations of the association between encephalomyelitis and ON. 

Optic nerves express higher levels of MOG than the spinal cord and 30% of MOG-specific TCR transgenic mice have spontaneous development of ON. ¹⁰ In contrast, no MBP- or PLP-specific TCR transgenic mice were observed to have spontaneous development of ON. ^{29 30} Thus, we hypothesized that MOG-specific T cells may have a greater ability than MBP- or PLP-specific T cells to induce ON. Our results using active immunization or adoptive transfer support this hypothesis. 

It is important to mention that the diagnosis of ON was established solely by pathologic examination. Although this approach ensures the accuracy of diagnosis, it prevents determination of the absolute incidence of ON after immunization with MOG and examination of the relapsing nature of the disease. For example, it is likely that negative cases in each set of experiments included mice that would have had ON develop with a delayed onset. Alternatively, the inflammation of the optic nerve during the later phase of the disease (>60 days pi) may represent either persistent chronic inflammation or relapse of inflammation. In future experiments, we will try to correlate a functional abnormality (e.g., aberrant pupillary responses), with histologic evidence of ON. In addition, pathologic inflammation may precede the clinical symptoms. Because the diagnosis of ON has largely relied on pathologic examination, whereas the diagnosis of EAE exploits our experience with clinical expression, it is likely that the onset of the two diseases was not significantly different. 

As previously mentioned, more than 30% of MOG-specific TCR transgenic mice have spontaneous development of isolated ON in the absence of clinical or histologic evidence of EAE. ¹⁰ This suggests that ON may have a lower disease threshold than EAE or that a small number of activated autoreactive T cells may preferentially induce ON, rather than EAE. Our study showed that ON and EAE induced by immunization of MOG or MOG-specific T cells in wild-type B6 mice could be associated or occur independently. One of the explanations could be that the TCR of the reported transgenic mice derived from a MOG-specific T-cell clone that has biased ON/EAE inducing capability, whereas the disease was induced in wild-type mice by polyclonal responder T cells that may not show a similar biased functional activity of MOG-specific T cell subsets. We have also shown that more than 50% of the MOG-induced ON in B6 mice was bilateral, thus differing from MOG-induced ON in the rat, in which the disease is reported to be primarily unilateral. ²⁸  

To examine further the possible association and dissociation of EAE and ON and to determine whether ON and EAE were induced by the same autoreactive T-cell subset(s) with different disease thresholds, we examined induction of EAE and ON using a lower dose of autoantigen, a truncated autoantigen peptide, and a lower number of autoreactive T cells. Our results showed that, in each of these disease-inducing conditions, both association and dissociation of the two diseases could be seen. In particular, EAE, but not ON, developed in a significant proportion of the mice. These results demonstrated that the dissociation and association of EAE and ON was not simply due to a lower threshold for ON than for EAE induced by the same subset(s) of autoreactive T cells. In combination with the observations made in transgenic mice, our results suggest the alternative possibility that subset(s) of MOG-specific T cells may have distinct pathogenic activities that induce either EAE or ON. 

Preliminary results indicate that MOG40-54–stimulated T-cells survive better during culture and expansion in vitro, possibly because some residues in MOG35-55 are not mandatory for T-cell activation, but prevent the peptide from binding to MHC molecules on APCs. ¹⁵ Because the availability of only a limited number of MOG-specific T cells has been a major obstacle to the characterization of the pathogenic mechanism of MOG-induced disease, the use of MOG40-54, rather than the more commonly used MOG35-55, should have significant technical advantages. 

 Supported by Grants EY12974 and EY014-366 from the NIH and RG3413-A4 from the National Multiple Sclerosis Society, by Research to Prevent Blindness (RPB) and the Commonwealth of Kentucky Research Challenge Trust Fund (HJK). HS is a recipient of a career development award from RPB.

 Submitted for publication May 18, 2004; revised June 4 and June 14, 2004; accepted June 19, 2004.

 Disclosure: H. Shao, None; Z. Huang, None; S.L. Sun, None; H.J. Kaplan, None; D. Sun, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Figure 1.

 

EAE and ON induced in B6 mice by immunization with MOG peptides. Histologic analysis of spinal cord inflammation and optic nerve inflammation in B6 mice immunized with 200 μg of MOG35-55. Animals were killed on day 15 pi after intracardial perfusion through the left ventricle with ice-cold PBS, followed by 10% buffered formalin. (A, B) Central nervous system inflammation (arrows) induced by immunization with MOG35-55. Paraffin-embedded sections of spinal cord were stained with fast blue. (C–F) ON (arrows) induced by MOG35-55 immunization. Coronal (C, D) and longitudinal (E, F) sections of optic nerve from mice with ON (D, F) or control mice (C, E) stained with hematoxylin and eosin. Original magnification, ×40.

Table 1.

 

 

Induction of EAE and ON by Active Immunization or Adoptive Transfer

Table 1.

 

Induction of EAE and ON by Active Immunization or Adoptive Transfer

	Active Immunization	Adoptive Transfer
EAE
Incidence	18/22	6/8
Onset (Day)	15.0 ± 2.0	8.0 ± 1.0
Clinical score	2.0 ± 0.4	2.0 ± 0.5
Optic neuritis
Incidence	14/22	5/8
Onset	13.0 ± 2.0	8.0 ± 1.0
Pathological score	2.0 ± 0.5	3.0 ± 0.5

 B6 mice were immunized with 200 μg MOG35-55 or were injected ip with 6 × 10⁶ MOG-specific T cells. The clinical score of EAE was monitored by physical examination, and the disease score of ON was determined by pathologic examination. The results are the pooled data of four separate experiments.

Figure 2.

 

Induction of ON was antigen specific. Naïve B6 mice were injected intraperitoneally with 6 × 10⁶ newly activated IRBP1-20 (uveitogenic)–specific T cells. At 15 days pi, ON and EAU were determined by pathologic examination. Paraffin-embedded sections stained with hematoxylin and eosin. Original magnification, ×40.

Figure 3.

 

MOG-specific T cells induced severe ON in B6 mice. Naïve B6 mice were injected intraperitoneally with 6 × 10⁶ newly activated MOG-specific T cells and killed on day 15 pi, and the optic nerves were subjected to pathologic examination. Sagittal sections showed perivenular inflammatory infiltrates (A, B, arrows), parenchymal inflammation (C, D, arrows), and retrobulbar optic neuritis (E, F, arrows). Note the massive inflammation in the optic nerve. (A) and (B) were from one mouse and (C) and (D) from another. (A, C) Fast blue; (B–F) hematoxylin and eosin. Most of the induced ON was bilateral (see Fig. 3E). (F) Bilateral inflammation in the optic cross.

Table 2.

 

 

EAE/ON Induced by a Suboptimal Antigen Dose or Suboptimal Disease-Inducing Conditions

Table 2.

 

EAE/ON Induced by a Suboptimal Antigen Dose or Suboptimal Disease-Inducing Conditions

Treatment	Incidence	Onset (day)	Clinical Score
A. MOG35-55 (200 μg per mouse)
EAE	18/22	15.0 ± 2.0	2.0 ± 0.4
ON	14/22	13.0 ± 2.0	2.0 ± 0.5
EAE without ON	4/18
ON without EAE	3/14
B. MOG35-55 (50 μg per mouse)
EAE	8/12	15.0 ± 1.4	2.0 ± 1.0
ON	5/12	14.0 ± 1.3	1.5 ± 0.3
EAE without ON	4/8
ON without EAE	2/5
C. MOG40-54 (200 μg per mouse)
EAE	8/12	15.0 ± 1.2	3.0 ± 0.5
ON	6/12	13.0 ± 1.2	2.5 ± 0.3
EAE without ON	3/8
ON without EAE	2/6
D. MOG-specific T cells (6 × 106 per mouse)
EAE	6/8	8.0 ± 1.0	2.0 ± 0.5
ON	5/8	8.0 ± 1.0	3.0 ± 0.5
EAE without ON	2/6
ON without EAE	2/5
E. MOG-specific T cells (1 × 106 per mouse)
EAE	3/8	9.0 ± 1.0	2.2 ± 0.4
ON	2/8	8.0 ± 1.0	1.5 ± 0.2
EAE without ON	1/3
ON without EAE	1/2

 B6 mice were immunized with an optimal [A, 200 μg] or a suboptimal [B, 50 μg] dose of MOG35-55 or with a truncated MOG peptide [C, MOG40-54] or were injected with a high (6 × 10⁶ per mouse, D.) or a suboptimal (1 × 10⁶ per mouse, E.) dose of MOG-specific T cells. The results are the pooled data of four separate experiments.

Table 3.

 

 

Effectiveness of MOG40-54 Compared with MOG35-55 in Inducing Immune Cells in the Spleen

Table 3.

 

Effectiveness of MOG40-54 Compared with MOG35-55 in Inducing Immune Cells in the Spleen

	MOG35-55			MOG40-54
		Day 12 pi	Day 50 pi	Day 12 pi	Day 50 pi
Total splenic T cells (×106)	50.0 ± 7.2	60.0 ± 8.5	100.0 ± 8.5	60.0 ± 4.5
MOG-specific T cells (per 106 cells)	5.0 ± 0.4	NT	7.6 ± 0.3	NT

 B6 mice were immunized with MOG35-55 or MOG40-54 emulsified in CFA. At 15 days pi, splenic T cells were partially enriched by passage through a nylon wool column and MOG-specific T cells were estimated by limiting dilution assay as described previously.¹⁸¹⁹ NT, not tested.