Myelin/Oligodendrocyte Glycoprotein–Specific T-Cells Induce Severe Optic Neuritis in the C57Bl/6 Mouse
Hui Shao, Zhigang Huang, Sheher L. Sun, Henry J. Kaplan, and Deming Sun
PURPOSE. The optic nerve is a common site of tissue damage in multiple sclerosis (MS) and experimental autoimmune enceph- alomyelitis (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). Per- tussis 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.
From the Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, Ken- tucky.
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 Re- search Challenge Trust Fund (HJK). HS is a recipient of a career development award from RPB..
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 encephalo- myelitis, but also in ON. In the present study, we investigated optic neuropathy induced by encephalitogenic peptides de- rived 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 na¨ıve 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 Ophthal- mic and Vision Research.
Reagents
Peptides MOG35-55 (MEVGWYRSPFSRVVHLYRNGK), MOG40-54 (YR-
SPFSRVVHLYRNG), and human interphotoreceptor retinoid-binding protein (IRBP)1-20 (GPTHLFQPSLVLDMAKVLLD) peptide were syn- thesized 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 adju- vant (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 de- scribed, 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, Upp- sala, 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, com- plete hindlimb paralysis; 4, front and hindlimb paralysis; and 5, mori- bund 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 prelim- inary 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% paraformalde- hyde 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 pep- tide 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 mononu- clear cell infiltrates were readily identifiable in both the spinal cord (Figs. 1A, 1B) and the submeningeal region and/or paren- chyma of the optic nerve (Figs. 1C–F).
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 clin- ical and pathologic EAE. However, some animals failed to show evidence of ON, despite extensive spinal cord lesions. Exami- nation 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).
TABLE 1. Induction of EAE and ON by Active Immunization or Adoptive Transfer
FIGURE 2. Induction of ON was antigen specific. Na¨ıve B6 mice were injected intraperitoneally with 6 × 106 newly activated IRBP1-20 (uveitogenic)–specific T cells. At 15 days pi, ON and EAU were deter- mined by pathologic examination. Paraffin-embedded sections stained with hematoxylin and eosin. Original magnification, ×40.
ON induction was antigen specific, as a similar injection of T cells specific for a uveitogenic peptide, IRBP1-20,17 only in- duced 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 na¨ıve 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 popu- lation 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 devel- oped at 8 to 10 days pi. The pathologic features of ON occur- ring after adoptive transfer of MOG-specific T cells differed from those occurring after active immunization. As shown in Figures 1C–E, the optic nerve inflammation observed after active immunization was characterized by a diffuse distribution of infiltrating cells; however, after adoptive transfer, inflamma- tory nodules were observed throughout the optic nerve, vary- ing 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 differ- ent numbers of activated MOG-specific T cells. As shown in Table 2, both EAE and ON developed in a significant percent-
age 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 encephalito- genic 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 na¨ıve 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. How- ever, 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 re- ported previously.19,20 Our results show that MOG-specific T cells increased by 50% (7.6 per million) in MOG40-54 –immu- nized 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.11–13,22
Studies in patients with ON have provided clinically impor- tant 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 immuniza- tion of disease-prone rodents with myelin proteins or patho- genic peptide derived from these proteins.9,16,25,26
Although involvement of the optic nerve has been ultra- structurally documented in EAE in the guinea pig27 and rat,28 detailed murine studies have been limited to reports of inflam- mation of the optic nerve in proteolipid protein (PLP)-induced EAE in SJL mice22 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 na¨ıve 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 repro- ducible 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 spon- taneous 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 deter- mination 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 de- velop 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 re- sponses), with histologic evidence of ON. In addition, patho- logic inflammation may precede the clinical symptoms. Be- cause the diagnosis of ON has largely relied on pathologic examination, whereas the diagnosis of EAE exploits our expe- rience 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 iso- lated ON in the absence of clinical or histologic evidence of EAE.10 This suggests that ON may have a lower disease thresh- old 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 sub- sets. 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 dissocia- tion 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 autoan- tigen peptide, and a lower number of autoreactive T cells. Our results showed that, in each of these disease-inducing condi- tions, 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-spe- cific 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 tech- nical advantages.
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