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The Journal of Immunology, 1998, 161: 5586-5593.
Copyright © 1998 by The American Association of Immunologists

Suppressive Effect on Theiler’s Murine Encephalomyelitis Virus-Induced Demyelinating Disease by the Administration of Anti-IL-12 Antibody

Atsushi Inoue*, Chang-Sung Koh2,*, Masashi Yamazaki*, Hiroyuki Yahikozawa*, Motoki Ichikawa{dagger}, Hideo Yagita{ddagger} and Byung S. Kim§

* Third Department of Medicine and {dagger} Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan; {ddagger} Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and § Departments of Microbiology-Immunology and Pathology, Northwestern University Medical School, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the role of IL-12, a cytokine critical to the evolution of cellular responses, in the development of Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to IL-12, especially during the effector phase, resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of inflammatory and Th1-derived cytokines such as TNF-{alpha} and IFN-{gamma} in the spleen cells was decreased, and that of Th2-derived cytokines such as IL-4 and IL-10 was increased. The delayed type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. These data suggest that IL-12 is critically involved in the pathogenesis of TMEV-IDD and that Abs to IL-12 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Multiple sclerosis (MS)3 is an immune mediated chronic inflammatory and demyelinating disease in which lesions in the central nervous system (CNS) result in varying degrees of neurologic deficit. While the etiology of MS is unknown, it has been speculated that demyelination is triggered by an infectious agent, possibly a virus (1). Thus, a similar demyelinating disease induced by a virus could be one of the most attractive animal models in the study of the pathogenesis of MS.

Intracerebral infection of susceptible mouse strains with Theiler’s murine encephalomyelitis virus (TMEV) induces a chronic progressive demyelinating disease characterized histologically by perivascular inflammatory cell infiltrates and primary demyelination of the CNS and clinically by progressive hind limb paralysis (2, 3, 4). TMEV-induced demyelinating disease is considered an infectious mouse model for MS because the disease displays similar histopathologic, genetic, and clinical similarities to human MS (3, 5, 6, 7). Persistent CNS virus infection in susceptible mouse strains triggers clonal expansion and differentiation of TMEV-specific, MHC class II-restricted effector DTH (Th1) cells that are poorly controlled by normal immunoregulatory mechanisms. Proinflammatory cytokines produced by virus-specific Th1 cells lead to the recruitment, accumulation, and activation of additional monocytes and macrophages within the CNS that cause demyelination through a terminal nonspecific bystander response (8, 9). IFN-{gamma} plays a particularly key role in the TMEV-IDD inflammatory response, and a perturbation in the level of this cytokine results in acceleration of demyelinating disease (10).

IL-12 is a recently discovered cytokine produced predominantly by macrophages and specific Ag-presenting cells such as dendritic cells and Langerhans cells (11). This cytokine is required for effective Th1 cell generation (12). One of the central mechanisms by which IL-12 induces differentiation of Th1 cells is its ability to prime T cells during clonal expansion for high IFN-{gamma} production (13). IL-12 also influences the efficacy of Ag presentation by T cells by providing a costimulatory signal for T cell activation (14, 15). These findings suggest that IL-12 plays an important role in the induction of clonal expansion and differentiation of TMEV-specific, MHC Class II-restricted effector DTH (Th1) cells. In the present study, we examined whether IL-12 is involved in the pathogenesis of virally induced demyelination and whether systemic administration of Abs to IL-12 could be an effective treatment of virus-induced demyelinating disease.


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

Female pathogen-free SJL/J mice from The Jackson Laboratory (Bar Harbor, Maine) and female BALB/c mice from Japan SLC (Shizuoka, Japan) were housed and cared for in an approved facility, in accordance with the National Institutes of Health guidelines.

Virus

The BeAn 8386 strain of TMEV was propagated in baby hamster kidney-21 cells grown in DMEM supplemented with 7.5% donor calf serum and purified by isopycnic centrifugation on Cs2SO4 gradients as previously described (16).

Monoclonal Abs

The mAbs used in this study were C17.8 and M18/2, rat IgG2a mAbs. C17.8 is a neutralizing rat anti-mouse IL-12 Ab (17, 18). Because M18/2 does not block cell-mediated target cell lysis in vitro (nonneutralizing mAb) (19), we used M18/2 as control mAb. Hybridoma cells that produce these mAbs were cultured in RPMI 1640 supplemented with 10% FBS and 0.1% gentamicin. These cells were injected into nude mice; mAbs harvested as ascites were purified with the use of a protein G affinity column.

Injection of mice with TMEV

For i.c. inoculation of virus, 1.3 x 106 plaque-forming units (PFU) of virus in 30 µl were administered into the right cerebral hemisphere of mice anesthetized with methoxyflurane. This inoculum consistently induces neurologic signs in susceptible mouse strains (20).

Treatment with mAbs

Six- to eight-wk-old female SJL/J mice were separated into groups (A-G). Group A mice (control) were treated with PBS. In each experiment, TMEV was injected into SJL/J mice i.c. at day 0. mAbs (C17.8, M18/2 (non specific rat IgG2a mAb)) were injected i.p. into mice on days -2, 0, and 4 after i.c. infection with TMEV at induction phase or 20, 22, and 26 after i.c. infection with TMEV at the effector phase at a dose of 500 µg at a volume of 100 µl/mouse per injection. Details of the experimental design are given in Table IGo. We performed this mAb treatment experiment three times. In one experiment, 7 groups of mice were under investigation (n = 20 for each group). Before experiments, five mice were blindly selected from each group for histologic study, and another 5 mice were also blindly selected from each group for immunologic studies, such as TMEV-specific DTH, TMEV-specific T cell proliferation assay, and enumeration of cytokine-producing cells assay. Other mice were clinically observed until 80 days post i.c. infection (n = 10 for each group).


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  		Table I. Experimental design

 
Assessment of demyelinating disease

TMEV-infected mice were examined daily for clinical symptoms of demyelination. Mice were allowed to walk on a polyethylene (Dynalab) walking board and were observed for exhibition of symptoms, including a waddling gait, extensor spasms, paralysis, loss of righting reflex, incontinence, and hunched posture. Neurologic signs were recorded using the following grading system: normal = 0, slight waddling gait = 1, waddling gait = 2, spastic hind limb paralysis = 3, severe hind limb paralysis accompanied by incontinence = 4 (21). These clinical scores have been shown to be indicative of demyelination (2). A clinical score was recorded daily for each mouse in each experiment using this grading system. The mean clinical score for each group of mice on each day was calculated by dividing the sum of all clinical scores of the mice in a given group by the number of mice in that group.

Histology

In each experiment, mice were blindly selected from each group (n = 5 from each group) beforehand for histologic examination and sacrificed on day 40 post i.c. infection. Since we had repeated this experiment three times, 15 mice from each group were histologically examined. Mice were perfused under anesthesia by the intraventricular route with 4% paraformaldehyde in PBS, pH 7.4. Spinal cords were removed, fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in PBS, and embedded in epoxy resin. These epoxy-embedded (1 µm thick) sections were stained with toluidine blue. These sections from 12 segments/mouse were read under light microscopy, and grading was done in a blinded fashion by two independent investigators who were unaware of the treatment each animal had received. The score for inflammation was determined according to the following criteria: 0, none; 1, a few inflammatory cells; 2, numerous scattered cells with an occasional perivascular cuff; 3, many perivascular cuffs; 4 and 5, increasing perivascular infiltration and subarachnoid inflammation. The extent of demyelination was determined according to the following scoring system: 0, no demyelination; 1, a few scattered naked axons; 2, small groups of naked axons; 3, large groups of demyelinated axons with confluent plaques of demyelination (22).

Ag-specific DTH

A 24-h ear swelling assay was used to quantitate delayed type hypersensitivity (DTH) (23). Before experiments, five mice were also blindly selected from each group for immunologic studies. At 36 days post i.c. infection, prechallenge ear thickness of these mice was determined using a Mitutoyo digimatic micrometer (Mitutoyo, Tokyo, Japan). Subsequently, 5 µg of purified TMEV in 10 µl of saline was injected into the dorsal surface of the ear using a Hamilton syringe fitted with a 30-gauge needle. Twenty four hours later, ear thickness was again measured, and the increase in thickness was expressed in units of 10-4 inches. Ear swelling reactions were due to mononuclear cell infiltration and showed typical DTH kinetics (i.e., minimal swelling at 4 h, maximal swelling at 24 to 48 h).

T cell proliferation assay

After DTH measurement, the same mice were sacrificed. Spleen cells were harvested from three animals in each group and pooled. Cells (5 x 105) were cultured in 96-well flat-bottom microculture plates in RPMI 1640 containing 0.5% syngeneic mouse serum, 5 x 10-5 M 2-ME, and antibiotics. Triplicate cultures were stimulated with three different concentrations of UV-inactivated TMEV (0.5, 5, and 10 µg) and were incubated for 72 h in a humidified atmosphere of 5% CO2 and 95% air. Cultures were then pulsed with 1.0 µCi of [3H]dThd and harvested 24 h later. Measurement of [3H] dThd incorporation was determined using a scintillation counter and expressed as cpm. Background proliferation was less than 1/7 of TMEV-specific proliferation.

Anti-TMEV Ab and anti-TMEV subclass titration

TMEV-specific Ab titers were determined using ELISA as described earlier (24), utilizing sera from individual mice. Briefly, 0.3 µg of purified virus was used to coat microtiter plates. A BSA solution (0.3 µg) was also used to coat the plates, to serve as a negative control. Unless otherwise stated, twofold serial diluents of sera starting from a 1:100 (20 x 100) dilution were reacted with the Ags on the microtiter plates and then with goat anti-mouse secondary Ab conjugated with alkaline phosphatase (KPL, Gaithersburg, MD). For anti-TMEV subclass Ab titration, sera were reacted with the Ag on the microplates and then with biotinylated rat monoclonal anti-mouse IgG subclass Ab (Zymed, San Francisco, CA). After the plates were washed, streptavidin-alkaline phosphatase was added to each well, and the plates were incubated. After the plates were again washed, substrate (p-nitrophenyl phosphate) for the enzyme was added, and the enzyme reaction was colorimetrically measured by an ELISA reader at 410 nm. The Ab titers of ELISA represent log2 x 100.

Cytokine assay by ELISA

The concentration of circulating cytokines such as TNF-{alpha}, IFN-{gamma}, IL-4, or IL-10 were measured using commercially available ELISA kits (Genzyme, Cambridge, MA).

Enumeration of cytokine-producing cells

On day 40 post i.c. infection, spleen cells from the same mice used for Ag-specific DTH and T cell proliferation assay (n = 5 from each group) were harvested from animals in each group. The levels of TNF-{alpha}, IFN-{gamma}, IL-4, or IL-10-producing spleen cells were examined using enzyme-linked immunospot assay (ELISPOT). The original reverse ELISPOT assay (25) was modified by using nitrocellulose membrane (Bio-Rad, Hercules, CA). Wells were filled (50 µl/well) with monoclonal hamster anti-murine TNF-{alpha}, IFN-{gamma}, IL-4, or IL-10 mAbs (Genzyme) at a concentration of 10 µg/ml in 0.5% BSA in PBS overnight at 4°C. Unabsorbed Abs were removed, and wells were washed with PBS. The plates were then blocked with 1% BLOTTO (nonfat dry milk) for 2 h at 37°C. The outer surface of the nitrocellulose membrane was carefully dried. Spleen cells (1 x 105/well) in the culture medium (RPMI 1640 supplemented with 10% FBS and 0.1% gentamicin) were dispensed among individual wells (100 µl/well). Plates were then incubated for 48 h at 37°C in a humidified, 5% CO2 atmosphere and were washed three times with Tris-buffered saline with Tween 20 (TBST). Fifty microliters of a 1:250 dilution of polyclonal rabbit anti-murine TNF-{alpha}, IFN-{gamma}, IL-4, or IL-10 Abs (Genzyme) was added to each well followed by incubation for 2 h at 37°C. Plates were washed with TBST again and were treated with 50 µl of 500 µg/ml alkaline phosphatase-conjugated goat anti-rabbit IgG (KPL) for 2 h at 37°C. After another washing with TBST, cytokines secreted by single cells were visualized by adding a mixture of nitro-blue tetrazolium and 5-bromo-4-chloro-3-indole phosphate (Life Technologies, Inc., Grand Island, NY). The color reaction of the enzyme was halted after 30 min by washing with water, and spots were enumerated under x40 magnification.

Statistical analysis

Clinical scores were analyzed using the Mann-Whitney U test; other results were statistically evaluated using the Student t test (StatView program, Abacus Concepts, Berkeley CA). A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protection from demyelinating disease by administration of mAbs specific to IL-12

The results of experiments are summarized in Fig. 1Go and Table IIGo. Control animals (group A, nontreated; Groups C, E, and G, treated with control Ab M18/2) showed the typical disease course of TMEV-IDD. On day 40, about 70% of mice showed clinical signs such as waddling gait, extensor spasm, and hind leg paralysis; mean clinical scores were 2.7 in group A, 2.4 in group C, 2.5 in group E, and 2.8 in group G, respectively. On day 80, all mice from groups A, C, E, and G developed TMEV-IDD; the mean clinical scores were 3.5 in group A, 3.6 in group C, 3.7 in group E, and 3.8 in group G, respectively. In contrast, on day 40, no mice treated with anti-IL-12 mAb in the effector phase (group D) had clinical signs (the mean clinical score was 0), while 33% of mice treated with anti-IL-12 mAb in the induction phase (group B) had clinical signs, with a mean clinical score of 0.9. Fifteen percent of mice treated with anti-IL-12 mAb in both induction and effector phases (group F) had clinical signs; the mean clinical score was 0.6. These results demonstrate that clinical symptoms of demyelinating disease are significantly suppressed (footnotes a, d, and e, p < 0.01; footnote b, p < 0.005) in animals treated with anti-IL-12 mAb (groups B, D, and F), as compared with those in control groups (groups A, C, E, and G). On day 80 post i.c. infection, as shown by the mean clinical score, severities of neurologic signs were significantly lower (footnote c, p < 0.01) in mice treated with neutralizing anti IL-12 mAb in the effector phase (group D) than in control groups (groups A, C, E, and G). However, we could not detect any significant differences in other treatment groups (groups B and F).



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  		FIGURE 1. Effect of treatment with mAbs specific for IL-12 on the clinical course of TMEV-IDD. This figure shows the summary of the clinical course of anti-IL-12 treatment experiments. Since we performed this experiment three times (n = 10 from each group), the initial data points were representative of 30 mice per group. All mice were clinically observed from day 0 to day 80 post i.c. infection. Mice were infected i.c. at day 0. mAbs (group A, none; groups B, D, and F, anti-IL-12 mAb; groups C, E, and G, nonspecific IgG mAb (M18/2)) were injected i.p. into mice on days -2, 0, and 4 in the induction phase and days 22, 24, and 26 in the effector phase after i.c. infection of TMEV at a dose of 500 µg in a volume of 100 µl/mouse each time. On day 40 post i.c. infection, clinical signs of demyelinating disease were significantly suppressed (*, p < 0.01) in animals treated with anti-IL-12 mAb (groups B, D, and F), as compared with those in control groups (groups A, C, E, and G). On day 80 post i.c. infection, the percentage with clinical signs was significantly lower (*, p < 0.01) in mice treated with neutralizing anti-IL-12 mAb in the effector phase (group D) than in control groups (groups A, C, E, and G).

 

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  		Table II. Clinical and pathologic evaluation of anti-IL-12 mAb-treated and control mice6

 
Representative mice were blindly selected from each group (n = 5 from each group) for histologic examination and sacrificed on day 40 post i.c. infection. The characteristic perivascular and parenchymal mononuclear cell infiltration and extensive demyelination in the white matter of spinal cord were observed in mice from groups A, C, E and G (Fig. 2GoA). The inflammation scores and demyelination scores were significantly lower in mice from groups B, D, F (footnotes d and e, p < 0.01) than those from control groups (Fig. 2GoB; Table IIGo).



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  		FIGURE 2. Histologic findings. Epon-embedded section, 1 µm thick, stained with toluidine blue (x150). In each experiment, mice were blindly selected from each group (n = 5 from each group) beforehand for histologic examination and sacrificed on day 40 post i.c. infection. Since we repeated this experiment three times, 15 mice from each group were histologically examined. A, Cross-section of a spinal cord from a mouse treated with nonspecific IgG mAbs (M18/2). This mouse showed severe neurologic signs. Extensive demyelinative lesion with parenchymal mononuclear cell infiltrates in white matter is observed. B, Cross-section of a spinal cord from a mouse treated with anti-IL-12 mAbs. This mouse showed no clinical sign. This section shows almost normal myelinated spinal cord white matter.

 
TMEV-specific DTH

Virus-specific DTH, as measured by the ear-swelling assay, is known to correlate strongly with susceptibility to TMEV (23); DTH has been shown to be mediated by the Th1 lymphocyte subset (26). To compare the clinical signs and the level of TMEV-specific DTH, we assessed the level of DTH 36 days after viral infection in mice selected for immunologic examination (n = 5 from each group). The levels of DTH at 36 days postinfection in mice of control groups (groups A, C, E, and G) were increased at almost the same levels previously reported (27). Conversely, they were significantly lower (p < 0.01) in mice treated with neutralizing anti IL-12 Abs (groups B, D, and F). These results show that administration of these mAbs inhibits the level of TMEV-specific DTH (Fig. 3GoA). We also examined the level of TMEV-specific DTH at 78 days post i.c. infection. Mice were chosen blindly from each group (n = 5 from each group). In this stage, the DTH level was decreased only in mice treated with anti-IL-12 during effector phase (group D).



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  		FIGURE 3. Before experiments, five mice were blindly selected from each group for TMEV-specific DTH, and TMEV-specific T cell proliferation assay. Since these studies were examined three times, the initial data points are representative of 15 mice per group. A, The level of TMEV-specific DTH in mice treated with neutralizing mAb specific for IL-12 is low. On day 36 post viral infection, five SJL/J mice from groups A, B, C, D, E, F, and G and a group mock-infected with baby hamster kidney cell lysate were examined for ear-swelling response to UV-inactivated purified TMEV. Responses from this experiment are expressed as mean {Delta} ear swelling in units of 10-4 inches ± SEM. Average background ear-swelling responses (from mock-infected mice) were subtracted from the individual specific ear-swelling responses. The average background ear-swelling was 3.2 ± 1.2. The responses of groups B, D, and F were significantly lower (*, p < 0.01) than those of other groups. B, The level of TMEV-specific T cell proliferative response was significantly lower in mice treated with mAbs to IL-12 than control groups treated with either PBS (none) or a nonspecific IgG mAb (*, p < 0.01). On day 40 post viral infection, the same mice from DTH study were sacrificed and examined for TMEV-specific T cell proliferative response. Spleen cells (5 x 105) were cultured with 10 µg of intact UV-inactivated TMEV for 72 h and then pulsed with 1 µCi of [3H]TdR for the final 24 h. Data represent specific proliferation minus nonspecific proliferation of spleen cells. Results are expressed as mean cpm ± SEM from triplicate cultures. Treatments were as follows: group A, none (PBS) treated; group B, anti-IL-12 mAb (induction); group C, nonspecific IgG mAb (induction); group D, anti-IL-12 mAb (effector); group E, nonspecific IgG mAb (effector); group F, anti-IL-12 mAb (induction + effector); and group G, nonspecific IgG mAb (induction + effector).

 
TMEV-specific T cell proliferation

T cell proliferative responses have been used frequently to assess the level of response of CD4+ T helper cells to the virus (28). After DTH measurement, the same mice were sacrificed on day 40 post i.c. infection. Spleen cells were taken out and used for examination for TMEV-specific T cell proliferation and ELISPOT assay. When the TMEV-specific T cell proliferative responses of mice treated with neutralizing anti-IL-12 Abs were compared with those of control groups, we observed a distinct difference (Fig. 3GoB). These results may indicate that administration of anti-IL-12 mAb inhibits the ability of TMEV-specific T cell proliferation of TMEV-specific mouse spleen cells or inhibits the generation of TMEV-specific T cells at the precursor level.

We also examined the level of TMEV-specific T cell proliferation at 80 days post i.c. infection. The same mice that were used for TMEV-specific DTH at 78 days post i.c. infection were examined (n = 5 for each group). In this stage, TMEV-specific T cell proliferative response was decreased only in mice treated with anti-IL-12 during effector phase (group D).

TMEV-specific Ab responses

We examined the Ab responses in the experimental groups of mice to determine a possible effect of anti-IL-12 Abs on the production of TMEV-specific Abs. Sera were taken from all mice of each group (n = 10 from each group) on day 56 and day 80 post i.c. infection. On day 56, there was no significant difference in the TMEV-specific Ab levels among the six groups. We also explored whether Ab isotypes were affected. We detected IgG1 and IgG2b Abs in all groups, with no significant difference among the 7 groups. We also detected IgG2a Ab in control groups (groups A, C, E, and G), but not in the anti-IL-12 mAb-neutralizing Ab treated groups (groups B, D, and F) (Fig. 4Go). These results indicate that, despite an unaltered level of total Abs, there is a lack of IgG2a component in animals treated with anti-IL-12-neutralizing Abs.



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  		FIGURE 4. Analysis of TMEV-specific Abs. Since small amounts of sera were enough for Ab titration and we did not have to sacrifice mice, we took out sera from all mice from clinical course observation group (n = 10 for each group). Since these studies were examined three times, the initial data points are representative of 30 mice per group. SSDs represent values obtained from individual mice from different groups of mice, i.e., sera from each individual were assayed separately. We detected TMEV-specific IgG Ab in all groups. We could detect IgG2a Ab in control treated groups (groups A, C, E, and G) but not in anti-IL-12-neutralizing mAb-treated groups (groups B, D, and F).

 
We also examined the level of TMEV-specific Ab at 80 days post i.c. infection. In this stage, we also detected TMEV-specific IgG2a Ab in mice treated with anti-IL-12 mAb-neutralizing Ab during induction phase or induction and effector phase (groups B and F). We could not detect TMEV-specific IgG2a Ab in mice treated with anti-IL-12 during effector phase (group D).

Assessments of cytokine production

After DTH measurement, the same mice (n = 5 from each group) were sacrificed on day 40 post i.c. infection. Spleen cells were taken out and used for ELISPOT assay. We could not detect any TNF-{alpha}, IFN-{gamma}, IL-4, or IL-10 in the sera from animals of any group. The levels of cytokine-producing cells in the spleens of animals were also assessed using the ELISPOT method. TNF-{alpha} production by spleen cells from mice treated with neutralizing Abs to IL-12 was significantly suppressed (p < 0.01) compared with control groups (Fig. 5GoA). IFN-{gamma} production by spleen cells from mice treated with anti-IL-12-neutralizing Abs was also significantly suppressed (p < 0.01) compared with control groups (Fig. 5GoB). Production of IL-4 and IL-10 by spleen cells from mice treated with anti-IL-12-neutralizing Abs was significantly increased (p < 0.01) compared with control groups (Fig. 5Go, C and D). Since we could not detect any cytokine spots in uninfected mice and uninfected mice treated with anti-IL-12 mAb, these results suggest that production of Th1 cell-derived, inflammatory cytokines was down-regulated and Th2 cell-derived cytokine production was somewhat up-regulated in mice treated with anti-IL-12-neutralizing Abs.



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  		FIGURE 5. ELISPOT assays for cytokine production. The same mice used for TMEV-specific DTH, TMEV-specific T cell proliferation assay were examined this assay. Since these studies were examined three times, the initial data points are representative of 15 mice per group. SSDs represent values obtained from individual mice from different groups of mice, i.e., splenocytes from each individual were assayed separately. A, Results of TNF-{alpha} assay by ELISPOT. TNF-{alpha} production of spleen cells was significantly suppressed in the mice of anti-IL-12-neutralizing mAb-treated groups (groups B, D, and F) (*, p < 0.01) compared with control treated groups. B, Results of IFN-{gamma} assay. The number of IFN-{gamma}-producing spleen cells was significantly decreased in the mice of anti-IL-12-neutralizing mAb-treated groups (groups B, D, and F) (*, p < 0.01) compared with control treated groups (groups A, C, E, and G). C, Results of IL-4 assay. The number of IL-4-producing spleen cells was significantly increased in the mice of anti-IL-12-neutralizing mAb-treated groups (groups B, D, and F) (*, p < 0.01) compared with control treated groups (groups A, C, E, and G). D, Results of IL-10 assay. The number of IL-10-producing spleen cells was significantly increased in the mice of anti-IL-12-neutralizing mAb-treated groups (groups B, D, and F) (*, p < 0.01) compared with control treated groups (groups A, C, E, and G).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study revealed that administration of Ab to IL-12, especially during effector phase, suppresses the clinical signs of TMEV-IDD. Furthermore, infiltration of the CNS with mononuclear cells and demyelination were dramatically reduced by treatment with this mAb. Insofar as we are aware, the effects of anti-IL-12-neutralizing Abs have not been studied in TMEV-IDD, one of a few available experimental model of MS. After i.c. inoculation with TMEV, an initial viremia is followed by a persistent, low-level CNS infection (29). TMEV infects neurons, glial cells, and macrophages in the spinal cords (8, 30). This stage may be called the "induction phase." Antigen presentation is mediated by either macrophages or glial cells, which contain TMEV Ags. As a consequence of the immunologic process, MHC class II-restricted TMEV-specific DTH T cells are activated, which in turn participate in immune mediated inflammatory responses. It has been proposed that virus-specific DTH results in the recruitment and accumulation of macrophages in the CNS, producing demyelination via a nonspecific "bystander" mechanism (31). This stage may be called the "effector phase." Myelin damage induced following TMEV infection is an immune mediated process. In susceptible mice, the persistence of viral Ags within the CNS may result in continuous activation of T cells that can mediate pathogenesis through locally produced cytokines or direct cytolytic mechanisms. The role of T lymphocytes in immune mediated demyelination has been confirmed by the fact that thymectomized, irradiated, and bone marrow-restored SJL/J mice fail to develop any clinical signs of TMEV-IDD (9). It has been shown that treatment of susceptible mice with Abs to the CD4 molecule or its restricting MHC class II molecules can significantly reduce the development of inflammation and demyelination after infection with TMEV (9, 32-34). In contrast, depletion of the CD8+ T cells has no inhibitory effect on demyelination induced by the BeAn strain of TMEV (9, 35). In addition, demyelination correlates well with the development of a chronic, virus-specific DTH response (31). These studies strongly suggest that virus-specific CD4+, class II-restricted Th1 cells are a major component in myelin destruction by TMEV infection. The presence of IL-12 during Ag presentation has been shown to favor the expansion of Th1-type CD4+ T cells, which are characterized by IFN-{gamma} production (12, 36). Th1 cells produce IFN-{gamma} and TNF-ß, which activate macrophages and are involved in DTH reactions. IL-12 is critically involved in the development of naive Th cells into Th1 cells (37). As a result of this ability to drive T cell responses to the Th1 phenotype, IL-12 has been shown to have exacerbating effects on several autoimmune disease models. Administration of IL-12 induced a) earlier onset of insulin-dependent diabetes mellitus in female NOD mice (38); b) more severe and prolonged disease in adoptively transferred experimental autoimmune encephalomyelitis (EAE), another animal model of human MS (39); c) destructive collagen-induced arthritis (40); and d) accelerated glomerulonephritis in young MRL/lpr mice (41). The suppressive effects of this mAb on several immune mediated diseases have also been reported. Monoclonal Abs against IL-12 have been shown to abrogate experimental chronic inflammatory colitis in mice, which is an animal model of Crohn’s disease, mediated by Th1 T cells (42). Furthermore, Abs to IL-12 have been shown to prevent EAE. (39). EAE is an autoimmune disease of the CNS that can be induced in susceptible strains of mice by immunization with CNS myelin Ags, or, alternatively, the disease can be passively transferred to naive susceptible mice via Ag-specific CD4+Th1 cells (43). In EAE, treatment of mice with Ab to murine IL-12 after cell transfer completely prevents paralysis (39). In our present study, treatment with anti-IL-12-neutralizing Abs significantly suppressed the occurrence and severity of demyelinating disease, but not completely.

Virus-specific DTH and T cell proliferative responses were also decreased by this treatment. Our ELISPOT study additionally showed that in anti-IL-12 mAb-treated groups, spleen cell production of TNF-{alpha} and IFN-{gamma} was decreased and production of IL-4 and IL-10 was increased, compared with nontreated groups and nonspecific IgG-treated groups. Our data suggest that treatment with anti-IL-12 mAbs shifted Th1-Th2 balance to Th2 dominance. Decreased production of Th1-derived cytokines such as TNF-{alpha} and IFN-{gamma} and increased production of Th2-derived cytokines such as IL-4 and IL-10 might suppress CD4+ virus-specific DTH cells, which correlated well with the disease severity of TMEV-IDD, and suppressed demyelinating disease. In Ab study, though titers of virus-specific Abs were increased in mice from all groups, IgG2a isotypes were decreased in anti-IL-12 mAb-treated groups. Since IgG2a Abs are produced by the stimulation of Th1 cells, decreased levels of virus-specific IgG2a Abs suggest the suppression of Th1 cells. Taken together, induction of encephalitogenic CD4+ Th1 cells might be suppressed by the treatment of anti-IL-12 mAbs. The suppressive effect on demyelinating disease was prominent in groups of mice treated with anti IL-12 mAbs during the effector phase. During this period, induction of encephalitogenic CD4+ Th1 cells is prominent; inhibition of Th1 cells by anti IL-12 treatment may be effective for the suppression of demyelinating disease. In contrast, in anti-IL-12 mAb treatment during the induction or induction and effector phases, though on day 40 post i.c. infection, the suppressive effect was prominent; on day 80, there was no significant difference compared with control treated groups. IL-12 plays an important role in immune response to viral infections, as seen in lymphocytic choriomeningitis virus infection of C57BL/6 mice, an acute viral infection model of the CNS, in which administration of low dose IL-12 is protective and high dose IL-12 is detrimental (44). IL-12 injection at the time of vaccination stimulates an antiviral type 1 cytokine response and increases immunity against a neurotrophic herpes virus infection of mice (44). IL-12 treatment decreases viral titer of vesicular stomatitis virus (VSV)-infected mice and inhibits productive vesicular stomatitis virus infection of the CNS (46). These findings suggest that, in the induction phase, anti-IL-12 treatment may reduce the antiviral activity of Th1 cells. Whether anti-IL-12 mAb treatment had any effect on virus replication or persistence in the CNS would be a primary factor in assessing the usefulness of this anti-IL-12 therapy. Possibly, the anti-IL-12 therapy in the induction phase may cause dramatic elevations in CNS virus that would augment a direct mechanism of infectious pathogenesis. Then, increased levels of virus in CNS may lead the progression of demyelinating disease. Change of immunologic parameters such as TMEV-specific DTH, T cell proliferation, and Ab subtype from day 40 to day 80 may support this hypothesis. Alternatively, the anti-IL-12 therapy only in the effector phase may have little effect on CNS virus levels/persistence, and the inhibition of encephalitogenic Th1 cells may lead the suppression of TMEV-IDD. Finally, since methods of clinical therapy of MS are still incomplete, it is important to examine the possibility of anti-cytokine therapy, including mAb to IL-12.


   Footnotes
 
1 This study was supported by Grants 06670647, 07670707, 07457155 and 09670649 from the Ministry of Education and Culture, by a research grant for neuroscience, and by a research grant for allergy and immunity from the Ministry of Health and Welfare, Japan. Back

2 Address correspondence and reprint requests to Dr. Chang-Sung Koh, Third Department of Medicine (Neurology), Shinshu University School of Medicine, 3–1-1 Asahi, Matsumoto 390-8621, Japan. E-mail address: Back

3 Abbreviations used in this paper: MS, multiple sclerosis; TMEV-IDD, Theiler’s murine encephalomyelitis virus-induced demyelinating disease; EAE, experimental autoimmune encephalomyelitis; DTH, delayed-type hypersensitivity; CNS, central nervous system; i.c., intracerebral; ELISPOT, enzyme-linked immunospot. Back

Received for publication April 8, 1998. Accepted for publication July 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Booss, J., J. H. Kim. 1990. Evidence of a viral etiology of multiple sclerosis. S. D. Cook, ed. Handbook of Multiple Sclerosis 41. Marcel Dekker, New York.
  2. Lipton, H. L.. 1975. Theiler’s virus infection in mice: an unusual biphasic disease process leading to demyelination. Infect. Immun. 11:1147.[Abstract/Free Full Text]
  3. Dal Canto, M. C., H. L. Lipton. 1975. Primary demyelination in Theiler’s virus infection: an ultrastructural study. Lab. Invest. 33:626.[Medline]
  4. Lehrich, J. R., B. G. W. Arnason, F. Hochberg. 1976. Demyelinative myelopathy in mice induced by the DA virus. Neurol. Sci. 29:149.
  5. Nathanson, N., A. Miller. 1978. Epidemiology of multiple sclerosis: critique of evidence for a viral etiology. Am. J. Epidemiol. 107:451.[Free Full Text]
  6. Kurtzke, J. F., K. Hyllested. 1986. Multiple sclerosis in the Faroe Islands. II. Clinical update, transmission, and the nature of MS. Neurology 36:307.[Abstract/Free Full Text]
  7. Kappel, C. A., M. C. Dal Canto, R. W. Melvold, B. S. Kim. 1991. Hierarchy of effects of the MHC and T cell receptor ß-chain genes in susceptibility to Theiler’s murine encephalomyelitis virus-induced demyelinating disease. J. Immunol. 147:4322.[Abstract]
  8. Clatch, R. J., S. D. Miller, R. Metzner, M. C. Dal Canto, H. L. Lipton. 1990. Monocytes/macrophages isolated from the mouse central nervous system contain infectious Theiler’s murine encephalomyelitis virus (TMEV). Virology 176:244.[Medline]
  9. Gerety, S. J., M. K. Rundell, M. C. Dal Canto, S. D. Miller. 1994. Class II-restricted T cell responses in Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease. VI. Potentiation of demyelination with and characterization of an immunopathologic CD4+ T cell line specific for an immunodominant VP2 epitope. J. Immunol. 152:919.[Abstract]
  10. Pullen, L. C., S. D. Miller, M. C. Dal Canto, P. H. Van der Meide, B. S. Kim. 1994. Alteration in the level of interferon-{gamma} results in acceleration of Theiler’s virus-induced demyelinating disease. Neuroimmunology 55:143.[Medline]
  11. Trinchieri, G.. 1995. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen specific adaptive immunity. Annu. Rev. Immunol. 13:251.[Medline]
  12. Trinchieri, G.. 1993. Interleukin-12 and its role in the generation of Th1 cells. Immunol. Today 14:335.[Medline]
  13. Manetti, R., F. Gerosa, M. G. Giudizi, R. Biagiotti, P. Paronchi, M. Piccinni, S. Sampognaro, E. Maggi, S. Romagnani, G. Trinchieri. 1994. Interleukin-12 induces stable priming for interferon-{gamma} (IFN-{gamma}) production during differentiation of human T helper (Th) cells and transient IFN-g production in established Th2 cell clones. J. Exp. Med. 179:1273.[Abstract/Free Full Text]
  14. Murphy, E. E., G. Terres, S. E. Macatonia, C. S. Hsieh, J. Mattson, L. Lanier, M. Wysocka, G. Trinchieri, K. Murphy, A. O’Garra. 1994. B7 and interleukin 12 cooperate for proliferation and interferon {gamma} production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223.[Abstract/Free Full Text]
  15. Correale, J., M. McMillan, S. Li, K. McCarthy, T. Le, L. P. Weiner. 1997. Antigen presentation by autoreactive proteolipid protein peptide-specific T cell clones from chronic progressive multiple sclerosis patients: roles of co-stimulation by B7 molecules and IL-12. Neuroimmunology 72:27.[Medline]
  16. Lipton, H. L., A. Friedmann. 1980. Purification of Theiler’s murine encephalomyelitis virus and analysis of the structural virion polypeptides: correlation of the polypeptide profile with virulence. Virology 33:1165.
  17. Trinchieri, G.. 1994. Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 84:4008.[Free Full Text]
  18. Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, G. Trinchieri. 1995. Interleukin-12 is required for interferon-{gamma} production and lethality in LPS-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
  19. Sanchez-Madrid, F., P. Simon, S. Thompson, T. A. Springer. 1983. Mapping of antigen and functional epitopes on the {alpha}- and ß-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and MAC-1. J. Exp. Med. 158:586.[Abstract/Free Full Text]
  20. Crane, M. A., C. Jue, M. Mitchell, H. L. Lipton, B. S. Kim. 1990. Detection of restricted predominant epitopes of Theiler’s murine encephalomyelitis virus capsid proteins expressed in the {lambda}gt11 system: different patterns of antibody reactivity among different mouse strains. Neuroimmunology 27:173.[Medline]
  21. Karpus, W. J., J. G. Pope, J. D. Peterson, M. C. Dal Canto, S. D. Miller. 1995. Inhibition of Theiler’s virus-mediated demyelination by peripheral immune tolerance induction. J. Immunol. 155:947.[Abstract]
  22. Cannella, B., A. H. Cross, C. S. Raine. 1993. Anti-adhesion molecule therapy in experimental autoimmune encephalomyelitis. J. Neuroimmunol. 46:43.[Medline]
  23. Clatch, R. J., R. W. Melvold, S. D. Miller, H. L. Lipton. 1985. Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease in mice is influenced by the H-2D region: correlation with TMEV-specific delayed-type hypersensitivity. Immunology 135:1408.
  24. Inoue, A., Y.-K. Choe, B. S. Kim. 1994. Analysis of antibody response to predominant linear epitopes of Theiler’s murine encephalomyelitis virus. J. Virol. 68:3324.[Abstract/Free Full Text]
  25. Czerinsky, C., A. Tarkowski, L.-A. Nilsson, O. Ouchterlony, H. Nygren, C. Gretzer. 1984. Reverse enzyme-linked immunospot (RELISPOT) assay for detection of cells secreting immunoreactive substances. J. Immunol. Methods 72:489.[Medline]
  26. Li, L., J. F. Elliot, T. R. Mosmann. 1994. IL-10 inhibits cytokine production, vascular leakage, and swelling during T helper 1 cell- induced delayed type hypersensitivity. J. Immunol. 153:3967.[Abstract]
  27. Pullen, L. C., S. D. Miller, M. C. Dal Canto, B. S. Kim. 1993. Class I-deficient resistance mice intracerebrally inoculated with Theiler’s virus show an increased T cell response to viral antigens and susceptibility to demyelination. Eur. J. Immunol. 23:2287.[Medline]
  28. Miller, S. D., R. J. Clatch, D. C. Pevear, J. L. Trotter, H. L. Lipton. 1987. Class II-restricted T cell responses in Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Cross- specificity among TMEV substrains and related picornaviruses, but not myelin proteins. J. Immunol. 138:3776.[Abstract]
  29. Lipton, H. L., J. Kratochvil, P. Sethi, M. C. Dal Canto. 1984. Theiler’s virus antigen detected in mouse spinal cord 2 1/2 years after infection. Neurology 34:1117.[Abstract/Free Full Text]
  30. Brahic, M., W.G. Stroop. 1981. Theiler’s virus persists in glial cells during demyelinating disease. Cell 26:123.[Medline]
  31. Clatch, R.J., H.L. Lipton, S.D. Miller. 1986. Characterization of Theiler’s murine encephalomyelitis virus (TMEV)-specific delayed-type hypersensitivity responses in TMEV-induced demyelinating disease: correlation with clinical signs. J. Immunol. 136:920.[Abstract]
  32. Welsh, C., P. Tonks, A. A. Nash, W. F. Blakemore. 1987. The effect of L3T4T cell depletion on the pathogenesis of Theiler’s murine encephalomyelitis virus infection in CBA mice. J. Gen. Virol. 68:1659.[Abstract/Free Full Text]
  33. Rodriguez, M., W. P. Lafuse, J. Leibowitz, C. S. David. 1986. Partial suppression of Theiler’s virus-induced demyelination in vivo by administration of monoclonal antibodies to immune-response gene products (Ia antigens). Neurology 36:964.[Abstract/Free Full Text]
  34. Friedmann, A., G. Frankel, Y. Lorch, L. Steinman. 1987. Monoclonal anti-I-A antibody reverses chronic paralysis and demyelination in Theiler’s virus-infected mice: critical importance of timing of treatment. J. Virol. 61:898.[Abstract/Free Full Text]
  35. Borrow, P., P. Tomks, C. J. Welsh, A. A. Nash. 1992. The role of CD8+ T cells in the acute and chronic phases of Theiler’s murine encephalomyelitis virus-induced disease in mice. J. Gen. Virol. 73:1861.[Abstract/Free Full Text]
  36. Morris, S. C., K. B. Madden, J. J. Adamovicz, W. C. Gause, B. R. Hubbard, M. K. Gately, F. D. Finkelman. 1994. Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection. Immunology 152:1074.
  37. Romangi, S.. 1997. The Th1/Th2 paradigm. Immunol. Today 18:263.[Medline]
  38. Trembleau, S., G. Penna, E. Bosi, A. Mortara, M. K. Gately, L. Adorini. 1995. Interleukin-12 administration induces T-helper type-1 cells and accelerates autoimmune diabetes in NOD mice. J. Exp. Med. 181:817.[Abstract/Free Full Text]
  39. Leonard, J. P., K. E. Waldburger, S. J. Goldman. 1995. Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J. Exp. Med. 181:381.[Abstract/Free Full Text]
  40. Germann, T., J. Szeliga, H. Hess, S. Storkel, F. J. Podlaski, M. K. Gately, E. Schmitt, E. Rude. 1995. Administration of interleukin-12 in combination with type-II collagen induces severe arthritis in DBA/1 mice. Proc. Natl. Acad. Sci. USA 92:4823.[Abstract/Free Full Text]
  41. Huang, F.-P., G.-J. Feng, G. Lindop, D. I. Stott, F. Y. Liew. 1996. The role of interleukin 12 and nitric oxide in the development of spontaneous autoimmune disease in MRL/MP-lpr/lpr mice. Exp. Med. 183:1447.
  42. Neurath, M. F., I. Fuss, B. L. Kelsall, E. Stüber, W. Strober. 1995. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J. Exp. Med. 182:1281.[Abstract/Free Full Text]
  43. Pettinelli, C. B., D. E. McFarlin. 1981. Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt-1+2- T lymphocytes. J. Immunol. 127:1420.[Abstract]
  44. Orange, J. S., S. F. Wolf, C. A. Biron. 1994. Effects of IL-12 on the response and susceptibility to experimental viral infections. Immunology 152:1253.
  45. Schijins, V. E. C. J., B. L. Haagmans, M. C. Horzinek. 1995. IL-12 stimulates an antiviral type 1 cytokine response but lacks adjuvant activity in IFN-{gamma}-receptor-deficient mice. J. Immunol. 155:2525.[Abstract]
  46. Reiss, C. S., T. Komatsu, M. Barna, Z. Bi. 1996. Interleukin-12 promotes enhanced recovery from viral infection of neurons in the central nervous system. Ann. NY Acad. Sci. 795:257.[Medline]



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