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The Journal of Immunology, 1998, 161: 5097-5104.
Copyright © 1998 by The American Association of Immunologists

Antibodies Against IL-12 Prevent Superantigen-Induced and Spontaneous Relapses of Experimental Autoimmune Encephalomyelitis1

Cris S. Constantinescu2,*, Maria Wysocka{dagger}, Brendan Hilliard*, Elvira S. Ventura*, Ehud Lavi{ddagger}, Giorgio Trinchieri{dagger} and Abdolmohamad Rostami3,*

* Department of Neurology, University of Pennsylvania, Philadelphia, PA 19104; {dagger} The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104; and {ddagger} Department of Pathology and Laboratory Medicine, Division of Neuropathology, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immunization of (PL/J x SJL/J)F1 mice with myelin basic protein (MBP) induces relapsing experimental autoimmune encephalomyelitis (EAE). Relapses occur 7 to 10 days after recovery from the initial paralysis. Staphylococcal enterotoxins (SE) A or B, administered after recovery from the initial paralysis, induce immediate relapses. IL-12 is involved in the induction of EAE. Here, we show that SEA and SEB induce IL-12 in splenocytes from (PL/J x SJL/J)F1 mice in vitro and increase the level of IL-12 in the sera of mice treated with these superantigens. IL-12 administration mimics SE in inducing spontaneous relapses and in enhancing the severity and frequency of spontaneous relapses. IL-12 neutralization blocks SE-induced and subsequent relapses of EAE, and, when instituted after recovery from the initial attack, prevents spontaneous relapse. This is the first report of prevention of relapses of EAE with anti-IL-12 Ab, an approach which may prove useful in the prevention of exacerbations in multiple sclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune encephalomyelitis (EAE)4, an animal model for the human disease multiple sclerosis (MS), is a T cell-mediated central nervous system (CNS) autoimmune disease. The autoreactive T cells, directed against neuroantigens including myelin basic protein (MBP), are of the Th1 type (producing IFN-{gamma} and TNF and promoting cell-mediated immunity). These cells preferentially use the Vß8 TCR in Lewis rats and H2u mice (1). The superantigen (SAG) staphyloccocal enterotoxin (SE) B (SEB) is a potent T cell activator stimulating a large proportion of Vß8 T cells and has been postulated to trigger autoimmunity by stimulating autoreactive T cells (2). SEB induces relapsing paralysis in PL/J (3, 4) and (PL/J x SJL/J)F1 mice (3) that have recovered from the first acute EAE attack. This property of SEB has been attributed to its ability to activate Vß8 T cells and has given rise to analogies with the frequent precipitation of MS relapses by infectious events (5).

(PL/J x SJL/J)F1 mice, when immunized with whole MBP, also have a high incidence of spontaneous relapses (6). These typically occur between days 23 and 32 after immunization, (7–10 days after recovery from the initial EAE episode) (6) (C.S.C. and A.R., unpublished observations). Thus, spontaneous relapses in (PL/J x SJL/J)F1 mice are distinguishable from the SEB-induced relapses (3), which occur within 1 to 3 days after SEB administration. Interestingly, staphyloccocal enterotoxin A (SEA), a SAG that does not activate Vß8 T cells, can induce similar relapses (3, 4), suggesting that the mechanisms of SAG-induced relapses are not strictly Vß8-dependent.

Staphylococcal SAG bind the MHC class II molecule of the APC outside of the binding groove and, subsequently, as a binary complex bind to the Vß region of the TCR (2). This process induces cytokines both in the T cell and in the APC (7). SAG binding to the MHC class II molecule on the APC induces signaling (8), cytokine gene transcription (9) and secretion (10), and nitrite production (11). Staphylococcal SAG preferentially induce Th1 cytokines (12, 13, 14). SAG binding is enhanced by IFN-{gamma}, suggesting that cooperation of T cells and MHC class II-positive APC is needed for optimal SAG-induced responses (15).

An important cytokine produced by activated APC is IL-12. This heterodimeric cytokine, consisting of the p35 and p40 subunits, induces IFN-{gamma} production by T and NK cells (16, 17) and is pivotal in Th1-type immune response development (18, 19). IL-12 is involved in the induction of the acute phase of EAE, as demonstrated by the ability of a neutralizing anti-IL-12 Ab to prevent both actively induced EAE (C.S.C. and A.R., unpublished observations) and adoptively transferred EAE (20), and by the increased encephalitogenicity of neuroantigen-reactive T cells stimulated with IL-12 (20, 21). In addition, exogenous IL-12 induces relapses in an otherwise typically monophasic form of EAE in Lewis rats (22). Thus, it is of interest to determine whether endogenous IL-12 plays a role in EAE relapse, characterized in our mouse model by both spontaneous and SAG-induced relapses. Moreover, because SAG, including staphylococcal SAG, can induce IL-12 production by APC in vitro (23, 24, 25), it is interesting to determine this ability in vivo (particularly because SAG may have different effects in vitro and in vivo) and to investigate whether this induction plays a role in the relapsing paralysis of EAE.

Staphylococcus aureus is a potent inducer of IL-12 (26). Moreover, recent evidence indicates that the ability of several bacteria-derived substances, including staphylococcal products, to overcome resistance to EAE is mediated through IL-12 induction (27). S. aureus SAG also synergize with IL-12 (28). In this study, we investigated the role of IL-12 in relapsing EAE in (PL/J x SJL/J)F1 mice. We analyzed the p40 subunit (the inducible component of IL-12) and the p70 heterodimer (generally p40 correlates well with the biologically active p70) (16, 26). We showed that SE induce IL-12 in splenocytes in vitro and in vivo. We also demonstrated that neutralizing anti-IL-12 Abs prevent SE-induced relapses. Further, spontaneous relapses are prevented by anti-IL-12 Abs. IL-12 administration mimics SE in inducing spontaneous relapses and enhancing the severity and frequency of spontaneous relapses.


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

Male or female (PL/J x SJL/J)F1 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The animals were sex and age matched within each experiment. Previous studies in our laboratory and others’ (6) showed no differences between male and female mice of this strain in terms of EAE susceptibility and frequency of relapses.

Reagents and Ag

MBP was prepared from guinea pig spinal cord (Rockland, Gilbertsville, PA) (29), lyophilized, and stored at -20°C until use. SEA and SEB were purchased from Sigma (St. Louis, MO). Recombinant murine IL-12 was a generous gift of Dr. Maurice Gately (Hoffmann-La Roche, Nutley, NJ). Murine rIFN-{gamma} was purchased from PharMingen (San Diego, CA). Monoclonal anti-mouse IL-12 Abs C17.8 (rat IgG2a), C15.1 (rat IgG1), and C15.6 (rat IgG1) were previously described (30). IFA and Mycobacterium tuberculosis H37 Ra were purchased from Difco (Detroit, MI). A 1:1 mixture resulted in CFA with 5 mg/ml M. tuberculosis. Bordetella pertussis toxin was obtained from List Biological Laboratories, (Campbell, CA). Rat Ig was purchased from Sigma.

Induction of EAE

Mice, 10 wk old, under anesthesia were immunized on day 0 in the hind footpads and at three sites on each side of the back with a total of 300 µl of 1:1 (v/v) mixture of MBP (2.67 mg/ml in PBS) and CFA containing 5 mg/ml M. tuberculosis H37Ra. On days 0 and 2, 400 ng B. pertussis toxin were given i.p.

Clinical scoring

Mice were weighed daily and observed for clinical signs of disease for more than 40 days postimmunization. A clinical scoring system with a scale of 0 to 5, with 0.5 points for intermediate signs, was used as follows: 0, normal; 1, flaccid tail, abnormal gait; 2, hind leg weakness or severe ataxia; 3, minimal hind leg movement; 4, hind leg and forelimb paralysis; 5, moribund due to EAE, with impaired breathing and little or no spontaneous movement. Mild disease had to be observed for 2 days or more and be confirmed by two independent, blinded observers to be considered positive. The score increment was defined as the maximal increase, during a relapse, in the clinical score from the prerelapse score.

Induction of relapses and treatment after recovery from first EAE episode

Mice that had recovered from the acute disease (day 18 or 19 postimmunization) were injected i.p. with a single dose of 50 µg SEB or 25 µg SEA, both in PBS, or with 100 ng murine rIL-12 in PBS with 1% mouse serum. On the day of the above treatment and on the subsequent 2 days, some of the mice received 1 mg/day of either anti-IL-12 mAb C.17.8 or control rat IgG.

The mice were observed daily for development of relapsing disease. Relapses occurred 1 to 3 days after SE administration. Relapses with onset occurring 3 to 5 days following the treatment were attributed to the intrinsically relapsing nature of EAE in these mice and coincided with relapses in mice that had been immunized on the same day but received no treatment following recovery from the initial episode. However, we cannot rule out the possibility that relapses occurring during this period may also be related to the effect of the SAG in the SAG-treated animals.

Measurement of cytokines in serum and splenocyte supernatants

Mice were given the above doses of SEA or SEB in 100 µl PBS via i.p. injection. Control mice were given 100 µl PBS. Blood was harvested in heparinized tubes by retro-orbital bleeding at 6 h and 24 h, centrifuged, sera removed and diluted at concentrations of 1:10 for IL-12 p40 and IL-12 p70 measurement.

For in vitro cytokine studies, spleens of mice were homogenized to single-cell suspensions by passage through a stainless steel mesh. RBC were removed by hypotonic lysis in NH4Cl-containing buffer. IL-12 p40 and IL-12 p70 assays were performed in total spleen cell populations stimulated either with medium alone (RPMI 1640 with 5% FBS with antibiotics) or with the same medium containing SEA or SEB, 2.5 µg/ml. IL-12 p40 was assayed in supernatants of spleen cells of mice using recombinant murine IL-12 as standard, following a two-site RIA as described (30). Briefly, samples were placed in 96-well plates (Dynatech Laboratories, Chantilly, VA) coated with 5 µg/ml of C17.15 anti-mouse IL-12 mAb and incubated overnight at 4°C. Plates were washed, and 125I-labeled C15.6 was added. Bound radioactivity after 6 h incubation at 4°C was measured in a microplate scintillation counter (Topcount, Packard, Meriden, CT). Samples were assayed in triplicate. IL-12 p70 was measured in a biologic assay based on IL-12-dependent induction of IFN-{gamma} in murine splenocytes, as described (30). The sensitivity of RIA detecting IL-12 p40 is 30 pg/ml, and the sensitivity of biologic assay detecting IL-12 p70 is 3 pg/ml. IFN-{gamma} was assayed in supernatants of spleen cells of mice using recombinant murine IFN-{gamma} as standard according to a two-site RIA as described (30).

Histopathology

Animals under anesthesia were perfused through the heart with 10% phosphate-buffered formalin. Brains and spinal cords were fixed in formalin, dehydrated through graded alcohols, and embedded in paraffin. Five-micrometer sections of brain and spinal cord were cut serially and mounted on poly-L-lysine-coated glass slides. Five spinal cord sections and five brain sections obtained at similar levels from each mouse were stained. For assessment of inflammation, sections were stained with hematoxylin-eosin. For evaluation of demyelination, sections were stained with Luxol fast blue and counterstained with cresyl violet. The extent of inflammation and demyelination was scored on a scale of 0 to 3, based on the fraction of tissue section quadrants containing lesions out of 20, as previously described (31): 0, absent; 1, mild (1–7 quadrants); 2, moderate (8–13 quadrants); 3, severe (14–20 quadrants); and 0.5 points for intermediate degrees of histologic severity.

Statistical analysis

The two-tailed Student’s t test was used to assess the significance of differences in clinical scores between groups. Incidences of EAE relapses were compared using the {chi}2 test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SE induce IL-12 in vitro

To determine whether SE induce IL-12 production by splenocytes (most likely APCs) in vitro, we harvested spleen cells from naive (PL/J x SJL/J)F1 mice and measured the production of IL-12 p40 in cell culture supernatants 6 and 24 h after stimulation with SE in vitro, with or without IFN-{gamma} (100 U/ml) pretreatment. Both SEA and SEB induced IL-12 p40 in (PL/J x SJL/J)F1 splenocytes without IFN-{gamma} pretreatment (data not shown), confirming results of studies using murine peritoneal macrophages (24). This induction was consistently higher when splenocytes were pretreated with IFN-{gamma} (100 U/ml) for 16 to 18 h before stimulation. Figure 1GoA shows the results of a typical experiment, in which induction of IL-12 p40 by SEA and SEB is demonstrated in murine splenocytes after pretreatment with IFN-{gamma}. Without IFN-{gamma} pretreatment, the corresponding IL-12 p70 measured by biologic assay was either just above the detection limit (<=2 pg/ml) or absent. In contrast, IFN-{gamma} pretreatment resulted in consistently detectable amounts of IL-12 p70 after SE stimulation. Figure 1GoB shows an example of IL-12 p70 induction by SEA and SEB after IFN-{gamma} pretreatment measured in the same supernatants as in Figure 1GoA. Stimulation with IFN-{gamma} alone did not induce significant levels of IL-12 p40 or IL-12 p70 (data not shown). The slight increase in IL-12 p40 after 24 h in culture (as seen in Fig. 1GoA) is also noted with splenocytes that were allowed to adhere for <=24 h without IFN-{gamma} or other stimuli; this increase is never associated with detectable IL-12 p70.



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  		FIGURE 1. Production of IL-12 p40 (A) and IL-12 p70 (B) by murine spleen cells prestimulated in vitro with IFN-{gamma} (100 U/ml) for 16 h and then stimulated with 2.5 µg/ml SEA or SEB for the designated durations. Results are shown as mean ± SEM of triplicate measurements of one experiment. Splenocytes were obtained and pooled from two to three (PL/J x SJL/J)F1 mice per experiment. Similar results were obtained in four additional experiments.

 
To determine whether IL-12 induction by SE correlates with induction of IFN-{gamma}, we measured IFN-{gamma} in supernatants of SE-stimulated spleen cells. Large amounts of IFN-{gamma}, up to 40 ng/ml, were detected after both SEA and SEB stimulation, the peak of IFN-{gamma} corresponding to that of IL-12 p40 and IL-12 p70 at 24 h after stimulation (data not shown).

SE induce IL-12 in vivo

The in vitro and in vivo effects of SAG can be different, particularly with respect to T cell activation vs anergy. In contrast, SE are known to induce cytokines, including TNF, both in vivo and in vitro (32). However, although neutralization of TNF alone significantly delayed SEB-induced relapses, it did not completely prevent them (3). IL-12 is involved in EAE induction (20) and synergizes with TNF (33, 34). Therefore, we investigated whether in vivo SE administration increases IL-12. We measured serum IL-12 p40 in mice 6 and 24 h after the i.p. administration of 25 µg/mouse SEA or 50 µg/mouse SEB. We demonstrated that this treatment induces increased levels of p40 IL-12 in the sera of these mice (Fig. 2Go). The induction kinetics is similar to that shown in murine endotoxic shock (30). We also measured the levels of IL-12 p70 heterodimer in these sera and observed consistently detectable levels only at the 6-h time point (data not shown).



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  		FIGURE 2. Induction of IL-12 p40 by SE in vivo. Mice were injected with SEA 25 µg or SEB 50 µg i.p. Heparinized blood was harvested at the designated time points, and IL-12 p40 levels were measured in diluted serum samples by RIA as described in Materials and Methods. Results are shown as mean ± SEM of three measurements per time point, each obtained from the serum of one mouse. Similar results were obtained in three additional experiments.

 
SE-induced relapses are prevented by anti-IL-12 Ab

Previous studies have shown that anti-IL-12 Abs prevent acute monophasic EAE, while IL-12 increases its severity (20). We examined whether the neutralizing anti-IL-12 mAb C17.8 could inhibit or prevent SEB- or SEA-induced EAE relapses. After recovering from the initial acute EAE episode (day 18 or 19), mice received SEA 25 µg/mouse or SEB 50 µg/mouse i.p. On the day of SE treatment and on the following 2 days, a group of mice received additional treatment with anti-IL-12 mAb (1 mg/mouse i.p. per treatment), while the control group received control rat IgG (same dose). As shown in Table IGo and Figure 3Go, the incidence and severity (measured as mean increment in clinical score and mean maximal score) of SAG-induced EAE relapses was significantly reduced (p < 0.001 for both SEA and SEB) by treatment with anti-IL-12 Ab, while treatment with control rat IgG was ineffective.


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  		Table I. Effect of anti-IL-12 on SE-induced EAE relapse1

 


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  		FIGURE 3. Prevention of SEA-induced (A), SEB-induced (B), or spontaneous EAE relapses (shown both in A and B in the No-treatment groups) by neutralizing anti-IL-12 mAb. After recovery from the initial EAE attack, mice were either given no further treatment or were treated as indicated in the figure by the arrow and the notation "Treatment." Results are shown as mean ± SD of clinical scores given as described in Materials and Methods. The differences between SE only or SE + control Ab- and SE + anti-IL-12 Ab-treated mice are statistically significant (p < 0.05). Further characteristics of EAE in each group are given in Tables I and II. The number of mice shown in the figure for each experimental group corresponds to the total number per group shown in Tables I and II.

 
SE enhance severity and frequency of EAE relapses

The above results confirm the ability of SE to induce clinical relapses in (PL/J x SJL/J)F1 mice. In addition, this strain of mice has a tendency toward spontaneous relapses, with incidence varying between 20% (3) and 100% (6), depending in part on the immunization protocol. Our unpublished results have shown a mean incidence of spontaneous relapses of 75% (range: 50–100%), with typical occurrence 7 to 10 days after recovery from the initial paralysis. In this paper, we use the term "spontaneous relapses" to denote relapses that occur in immunized mice that have received neither SE nor IL-12 treatment after the recovery from their initial EAE episode. We examined whether SE administration influences the occurrence or severity of spontaneous relapses. We found that animals treated with SE after recovery from the initial EAE event, in addition to experiencing an immediate SE-induced relapse, had an increased incidence and severity of later relapses (p < 0.0001) (Table IIGo and Fig. 3Go). Anti-IL-12 Ab treatment at the time of SE administration significantly decreased the incidence and severity of relapses, while administration of the control Ab rat IgG at the time of SE administration had no effect.


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  		Table II. Effect of anti-IL-12 on SE-enhanced spontaneous EAE relapses1

 
Histopathologic evaluation of the brain and spinal cord was performed on selected mice from the SE + control Ab- and SE + anti-IL-12-treated groups. Mice were sacrificed at day 33 postimmunization. SE + control Ab-treated mice exhibited mild perivascular and meningeal inflammation in the brain and severe inflammation involving the meninges, the perivascular spaces, and parenchyma, and accompanied by demyelinating changes in the spinal cords (Fig. 4Go, A and B). In contrast, histopathology in SE + anti-IL-12-treated mice revealed no inflammatory or demyelinating changes (Fig. 4GoC), with the exception of minimal meningeal infiltration in a single case out of three in SEB + anti-IL-12-treated mice (Table IIIGo). The differences between SE + control Ab and SE + anti-IL-12 were statistically significant (p < 0.05).



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  		FIGURE 4. Representative section of spinal cord of mouse with EAE after administration of SEB + control Ab (A and B) or SEB + anti-IL-12 Ab (C). Perivascular and parenchymal inflammation and demyelination are noted in A and B, while no significant histopathology is observed in C. Similar results were obtained in mice with SEA-induced relapses. The differences between SE + control Ab- and SE + anti-IL-12 Ab-treated mice were statistically significant (p < 0.05). This section was stained with Luxol fast blue and counterstained with cresyl violet. Magnification: A and C, x200; B, x400.

 

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  		Table III. Histopathology of EAE in SE + control Ab and SE + anti-IL-12 Ab-treated mice2

 
Severity and frequency of EAE relapses are enhanced by IL-12 and suppressed by anti-IL-12 Ab

We postulated that anti-IL-12 treatment may suppress relapses in mice not given SE treatment. We also wanted to determine whether systemic administration of murine rIL-12 can mimic SE effects by inducing EAE relapses and/or affecting the severity of relapses. After recovery from the initial bout of EAE, five mice received anti-IL-12 mAb C17.8 (1 mg/mouse/day i.p. for three consecutive days) while four control mice received the same quantities of rat IgG. None of the anti-IL-12-treated mice developed relapses, whereas three of four (75%) control Ab-treated mice had relapses. The mean severity of the relapse (±SD) in the rat IgG-treated group was 1.125 (±1.031); the mean increment was 1.125 (±0.25). The difference was statistically significant (p = 0.04 compared with the rat IgG-treated group) (Fig. 5Go). The protective effect of anti-IL-Abs was longer lasting than the ~15 days of persistence of these Abs in the circulation. None of the five animals followed >=50 days developed a relapse, in contrast to the average of 60% of untreated mice that developed at least a third attack.



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  		FIGURE 5. Effect of IL-12 and of neutralizing anti-IL-12 mAb on the course of relapsing EAE. After recovery from the initial attack of EAE, mice were treated as indicated in the figure by the arrow and the notation "Treatment." IL-12 mimics in part SE action by inducing immediate relapses and worsening later relapses. Anti-IL-12 Ab prevents spontaneous relapses. Results are shown as mean ± SD of clinical scores given as described in Materials and Methods. The course of EAE in mice treated with control rat IgG completely overlaps with that of mice receiving no treatment and is omitted for graphical clarity. The number of mice in each group is as follows: anti-IL-12 mAb, n = 5; rat IgG, n = 4; IL-12, n = 10; no treatment, n = 10). Further characteristics and statistical data are included in Results.

 
After recovery from the initial EAE episode (day 18 or 19), 10 mice received 100 ng i.p. of murine rIL-12. Six mice (60%) developed a relapse on the day following IL-12 administration (mean ± SD score increment, 1.08 ± 0.44). No relapses were noted immediately following the cytokine treatment in the remaining four mice. However, all mice receiving IL-12 treatment had a significantly more severe relapse. These relapses occurred at the same time (day 24–30) as the spontaneous relapses in mice not receiving treatment after recovery from the initial paralysis. However, the incidence (10/10 or 100%) was higher than that of spontaneous relapses in the animals without this treatment (6/9 or 67%). In addition, the mean (±SD) score increment was 2.61 (±1.53), as compared with 1.08 (±0.2) in the untreated mice. Also, the mean (±SD) maximal scores were enhanced: 2.71 (±1.44) as compared with 1.11 (±1.54) in mice receiving no IL-12 treatment. The difference was significant (p < 0.05 compared with mice receiving no treatment).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies indicate that IL-12 plays a role in spontaneous and SE-induced EAE relapses. In our investigation of expression of IL-12 during murine relapsing EAE in (PL/J x SLJ/J)F1 mice, we have found mRNA for IL-12 in the CNS only during the acute phases and its absence during remission phases (C.S.C. and A.R., unpublished observations). When we measured serum IL-12 during various stages of EAE in these mice, we did not find detectable levels of IL-12 p70. We believe that the amount of IL-12 in the serum of these animals is below the threshold of detection of our assay and will increase to detectable levels when mice are treated with SE or IL-12. The fact that neutralization of endogenous IL-12 prevented relapses argues for an important role of endogenous IL-12 in the course of EAE.

IL-12 is essential in the generation of Th1 responses and, therefore, plays a key role in the immune response to intracellular pathogens (19). Moreover, IL-12 is involved in the induction of T cell-mediated autoimmune diseases (35) including EAE (20, 21, 27). The role of IL-12 in the maintenance or recurrence of Th1 immune responses has also become a subject of recent investigations. The memory responses to certain infections appear to be IL-12-independent (36, 37). In the well-characterized model of murine leishmaniasis, we also recently demonstrated IL-12 independence of the Th1 response in the secondary infection (69). In contrast, the maintenance and chronicity of some, but not all, T cell-mediated autoimmunity appear to require IL-12. For example, the established chronic autoimmune reaction in experimental colitis is abrogated by neutralization of IL-12 (38). In contrast, however, administration of IL-12 during the established phase of autoimmune collagen-induced arthritis suppressed disease due to induction of IL-10 (39). Seder (40) has hypothesized that the differential IL-12 dependence of established Th1 responses in autoimmunity vs infectious diseases may reflect differences in the initial levels of endogenous IL-12 induction, with higher amounts of IL-12 produced during infection than during the response to an autoantigen. Because IL-12 also induces IL-10 (41, 42) in a potential autoregulatory loop, it is possible that the differential IL-12 dependence resides in the balance between IL-12 and IL-10. The role of IL-10 in EAE has been debated. A role in the recovery from EAE has been postulated based on the presence of IL-10 mRNA in the CNS of EAE animals in the recovery phase (43). With regard to the induction phase of EAE, the exogenous delivery of the cytokine in some studies was successful (44), while in other studies it was unsuccessful in preventing the disease (45). With regard to relapses, exogenous IL-10 could not prevent the SEB-induced relapses, while neutralization of the endogenous IL-10 worsened the relapses (46). This underscores the strength of cross-regulation in the endogenous cytokine network, which can determine the susceptibility, the course, and the cytokine dependence of autoimmunity. Recently, a unique IL-10/IL-12 immunoregulatory circuit controlling susceptibility to EAE has been demonstrated (47), and it is likely that perturbation of this loop (for example, as in our study) by microbial products powerfully changes the outcome and manifestations of EAE.

A clear possibility to consider in the case of relapsing autoimmunity is that IL-12 is important for the phenomenon of determinant (epitope) spreading. Evidence has accumulated indicating that propagation and reactivation of autoimmune diseases occur through the acquisition of autoreactivity to new self-determinants (48, 49, 50). Recently it has been shown that during the development of the determinant spreading cascade in murine relapsing EAE new sets of T cells are generated that exhibit a Th1 phenotype (50). Therefore, it is likely that the presence of IL-12 in the cytokine milieu during epitope spreading facilitates the development of these pathogenic neoautoreactive T cells.

Optimal Th1 responses require a synergy between IL-12 and the B7-CD28 interaction (51, 52). Blockade of B7 costimulation effectively prevented epitope spreading and clinical relapses in EAE (53, 54). Therefore, it is also important to characterize the role of IL-12, the other principal component required for optimal Th1 responses, in ongoing chronic or relapsing T cell-mediated autoimmunity. Here, we show that IL-12 neutralization can also prevent spontaneous relapses in EAE. In addition, we effectively prevented the occurrence of SE-induced relapses with an anti-IL-12 Ab. Although the assessment of the longevity of the protective effect of IL-12 neutralization was not the primary objective of this study, the fact that animals given anti-IL-12 Ab did not develop relapses for a long time following treatment while untreated animals did, provides further support to the hypothesis that once epitope spreading is prevented through blockade of costimulation and/or of IL-12, relapses are also prevented.

The mechanisms of SAG-induced and spontaneous relapses may be different. Activation of the residual SAG-responsive T cells with specific Vß TCR (for example, Vß8 for SEB) (3) is postulated and likely to be responsible for the SE-induced relapses. Epitope spreading, as discussed above, is involved in spontaneous EAE relapses (45). Our current findings implicate IL-12 in the mechanism of both processes of T cell reactivation and corresponding clinical relapse in EAE. With respect to the SE-induced relapses, we demonstrated that SE induces IL-12 in vitro, consistent with previous results (24), and, to our knowledge, documented for the first time induction of IL-12 by SAG in vivo. Moreover, we showed that exogenous IL-12 mimicked SE action, inducing rapid relapses, resembling effects recently shown in Lewis rats (22). Because staphylococcal SAG also induce IFN-{gamma} (55), it is possible that IFN-{gamma} production by T cells is stimulated by SE-induced IL-12, and reciprocal stimulation of IFN-{gamma} and IL-12 between T cells and APC is initiated. Although both SE induced similar levels of IL-12 in vitro and in vivo, the severity of SEA-induced relapses was lower than that of relapses induced by SEB. However, both SEA- and SEB-induced relapses exhibited significant dependence of this IL-12 induction. The basis of these differences is currently not completely elucidated. We used the same doses of SE that were shown in a previous study (3) to induce relapses in (PL/J x SJL/J) mice. Interestingly, in that study, SEA was also less efficient than SEB in inducing relapses. It is possible that, despite similar IL-12 inducibility, the lower frequency of SEA-responsive T cells makes this reciprocal stimulation between IL-12 and IFN-{gamma} less efficient, which may explain the lower severity of SEA-induced relapses.

Migration of T cells to the inflammatory compartment, in this case the CNS, may also be stimulated by SE via IL-12 in a manner similar to the demonstrated IL-12 mediation of skin-homing receptor induction by staphylococcal SAG (23). This can explain the absent or minimal inflammation or demyelination in the CNS of mice given SE + anti-IL-12 compared with the inflammatory infiltrates of mice given SE + control Ab observed in our study. IL-12 up-regulates the very late Ag (VLA)-4-dependent T-cell migration (56), a phenomenon known to be required for T cell entry into the brain parenchyma (57). We also demonstrated enhanced expression of VLA-4 on murine MBP-reactive T cells after exposure to IL-12 (C.S.C. and A.R., unpublished observations). Thus, the neutralization of IL-12 may have prevented the up-regulation of VLA-4 and the reentry of autoreactive T cells into the brain. In addition, IL-12 may provide a death-preventing signal to MBP reactive T cells, as shown for other Ag-specific T cells (58).

No or very few residual inflammatory cells were seen in the CNS of mice given SE and treated with anti-Il-12 Abs. Because the time elapsed after recovery from the initial attack was relatively short, one may have expected residual inflammatory infiltrates. Their absence could be interpreted as a stimulated efflux of inflammatory cells from the CNS of mice treated with anti-IL-12 Ab similar to that postulated in the case of EAE mice treated with altered peptide ligands in which relapses were also prevented (59). Another explanation, consistent with our observation that the first bout is relatively short and mild in these mice and with the fact that the clinical residual deficit after the first bout was also mild, is that the inflammation was almost completely resolved at the time of SE administration. In support of this explanation is the fact that histologic analysis of the CNS of mice with near-complete clinical recovery shortly after the first episode is usually normal or only minimally abnormal (C.S.C., A.R., and B.H., unpublished observations).

Because spontaneous relapses may occur through epitope spreading (49), our results suggest that IL-12 plays a role in this phenomenon. Because B7 blockade also prevents spontaneous relapses, this is additional evidence for the complementarity of these two costimulatory factors necessary for optimal Th1 responses in vivo and in vitro.

Previously, other cytokine-based immunologic manipulations have affected either SAG-induced or spontaneous EAE relapses. Administration of TGF-ß (46, 60, 61), IL-10 (46), or IFN-{tau} (62) prevented relapses, while anti-TGF-ß worsened them (63). Blockade of TNF decreased the incidence and severity of relapses (3, 63) and ameliorated EAE in a chronic/relapsing model (64). Interestingly, the cytokines that can prevent relapses antagonize IL-12, while cytokines implicated in relapse pathogenesis synergize with IL-12 (33, 65, 66). Thus, disease-suppressing effects in relapsing EAE may be mediated through IL-12 blockade, while relapses may involve IL-12 and synergistic factors.

Triggering and reactivation of autoimmunity by infectious products are documented in both spontaneous and experimental disease. Spontaneous EAE in MBP-specific transgenic mice occurred only in animals kept in a normal, pathogen-containing environment and not in animals kept in a specific pathogen-free facility (67). The effects of SAG on EAE exacerbations have been attributed either to restricted TCR-dependent mechanisms (3) or to nonspecific mechanisms, which include cytokine release (4). The present study supports the latter hypothesis that SAG also employ nonspecific mechanisms in reactivating EAE. The role of cytokines in the infection-induced reactivation of autoimmunity has been investigated, particularly with respect to TNF (3, 46, 68). Segal et al. (27) recently implicated IL-12 in the pathogenesis of this phenomenon as well. Our results extend these observations and bring them into the context of bacterial T cell SAG.

In conclusion, we show the ability of SE to induce IL-12 and demonstrate the involvement of IL-12 in SE-induced and spontaneous relapses of EAE. These findings may help to elucidate immunopathogenic mechanisms of relapsing autoimmunity and may provide clues to the immunopathology of MS. Neutralization of IL-12 may prove an effective therapy for autoimmune demyelination.


   Acknowledgments
 
We thank Dr. Maurice K. Gately (Hoffmann-La Roche, Nutley, NJ) for supplying murine rIL-12, Elsa Aglow (The Wistar Institute) for expert histologic assistance, and Dr. Peter J. Perrin for helpful discussions.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants 1K11 HD-01049, NS-11037, CA 10815, CA 20833, CA 32898, and AI 34412. Back

2 Current address: Departments of Neurology and Research, Laboratory of Neurobiology, University Hospital Basel, Basel, Switzerland. Back

3 Address correspondence and reprint requests to Dr. Abdolmohamad Rostami, Department of Neurology, University of Pennsylvania Medical Center, 3400 Spruce Street, Philadelphia, PA 19104-4283. E-mail: Back

4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; CNS, central nervous system; MBP, myelin basic protein; SAG, superantigen; SE, staphyloccocal enterotoxin; SEB, staphyloccocal enterotoxin B; SEA, staphyloccocal enterotoxin A. Back

Received for publication March 12, 1998. Accepted for publication July 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zamvil, S. S., L. Steinman. 1990. The T lymphocyte in experimental allergic encephalomyelitis. Annu. Rev. Immunol. 8:579.[Medline]
  2. Marrack, P., J. Kappler. 1990. The staphylococcal enterotoxins and their relatives. Science 248:705.[Abstract/Free Full Text]
  3. Brocke, S., A. Gaur, C. Piercy, A. Gautam, K. Gijbels, C. G. Fathman, L. Steinman. 1993. Induction of relapsing paralysis in experimental autoimmune encephalomyelitis by bacterial superantigen. Nature 365:642.[Medline]
  4. Schiffenbauer, J., H. M. Johnson, E. J. Butfiloski, L. Wegrzyn, J. M. Soos. 1993. Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. Proc. Natl. Acad. Sci. USA 90:8543.[Abstract/Free Full Text]
  5. Matthews, W. B., A. Compston, I. V. Allen, C. N. Martyn. 1991. McAlpine’s Multiple Sclerosis Edinburgh, Churchill Livingstone.
  6. Fritz, R. B., J. C.-H. Chou, D. E. McFarlin. 1983. Relapsing murine experimental allergic encephalomyelitis induced by myelin basic protein. J. Immunol. 130:1024.[Abstract]
  7. Miethke, T., C. Wahl, K. Heeg, B. Echtenacher, P. H. Krammer, H. Wagner. 1992. T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor. J. Exp. Med. 175:191.[Abstract/Free Full Text]
  8. Morio, T., R. Geha, T. Chatila. 1994. Engagement of MHC class II molecules by staphylococcal superantigens activates src-type protein tyrosine kinases. Eur. J. Immunol. 24:651.[Medline]
  9. Mourad, W., K. Mehindate, T. J. Schall, S. R. McColl. 1992. Engagement of major histocompatibility complex class II molecules by superantigen induces inflammatory cytokine gene expression in human fibroblast-like synoviocytes. J. Exp. Med. 175:613.[Abstract/Free Full Text]
  10. Pontzer, C. H., N. D. Griggs, H. M. Johnson. 1993. Agonist properties of a microbial superantigen peptide. Biochem. Biophys. Res. Commun. 193:1191.[Medline]
  11. Hauschildt, S., W. Bessler, P. Scheipers. 1993. Engagement of major histocompatibility complex class II molecules leads to nitrite production in bone marrow-derived macrophages. Eur. J. Immunol. 23:2988.[Medline]
  12. Schmitz, J., A. Radbruch. 1992. Distinct antigen presenting cell-derived signals induce Th cell proliferation and expression of effector cytokines. Int. Immunol. 4:43.[Abstract/Free Full Text]
  13. Cardell, S., I. Hoiden, G. Moller. 1993. Manipulation of the superantigen-induced lymphokine response: selective induction of interleukin-10 or interferon-{gamma} synthesis in small resting CD4+ T cells. Eur. J. Immunol. 23:523.[Medline]
  14. Nagelkerken, L., K. J. Gollob, M. Tielemans, R. L. Coffman. 1993. Role of transforming growth factor-ß in the preferential induction of T helper cells of type 1 by staphylococcal enterotoxin B. Eur. J. Immunol. 23:2301.
  15. Krakauer, T.. 1994. Costimulatory receptors for the superantigen staphylococcal enterotoxin B on human vascular endothelial cells and T cells. J. Leukocyte Biol. 56:458.[Abstract]
  16. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biological effects on human lymphocytes. J. Exp. Med. 170:827.[Abstract/Free Full Text]
  17. Stern, A. S., F. J. Podlaski, J. D. Hulmes, Y. E. Pan, P. M. Quinn, A. G. Wolitzky, P. C. Familletti, D. L. Stremlo, T. Triutt, R. Chizzonite, M. K. Gately. 1990. Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 87:6808.[Abstract/Free Full Text]
  18. Scott, P.. 1993. IL-12: initiation cytokine for cell-mediated immunity. Science 260:496.[Free Full Text]
  19. Trinchieri, G.. 1993. Interleukin-12 and its role in the generation of Th1 cells. Immunol. Today 14:335.[Medline]
  20. 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]
  21. Waldburger, K. E., R. C. Hastings, R. G. Schaub, S. J. Goldman, J. P. Leonard. 1996. Adoptive transfer of experimental allergic encephalomyelitis after in vitro treatment with recombinant murine interleukin-12. Am. J. Pathol. 148:375.[Abstract]
  22. Smith, T., A. K. Hewson, C. I. Kingsley, J. P. Leonard, M. L. Cuzner. 1997. Interleukin-12 induces relapse in experimental allergic encephalomyelitis in the Lewis rat. Am. J. Pathol. 150:1909.[Abstract]
  23. Leung, D. Y. M., M. Gately, A. Trumble, B. Fergusson-Darnell, P. M. Schlievert, L. J. Picker. 1995. Bacterial superantigens induce T cell expression of the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen, via stimulation of interleukin 12 production. J. Exp. Med. 181:747.[Abstract/Free Full Text]
  24. Skeen, M. J., M. A. Miller, T. M. Shinnick, H. K. Ziegler. 1996. Regulation of murine macrophage IL-12 production: activation of macrophages in vivo, restimulation in vitro, and modulation by other cytokines. J. Immunol. 156:1196.[Abstract]
  25. Sriskandan, S., T. J. Evans, J. Cohen. 1996. Bacterial superantigen-induced human lymphocyte responses are nitric oxide dependent and mediated by IL-12 and IFN-{gamma}. J. Immunol. 156:2430.[Abstract]
  26. D’Andrea, A., M. Rengaraju, N. M. Valiante, J. Chehimi, M. Kubin, M. Aste-Amezaga, S. H. Chan, M. Kobayashi, D. Young, E. Nickbarg, et al 1992. Production of natural killer cell stimulatory factor (NKSF/IL-12) by peripheral blood mononuclear cells. J. Exp. Med. 176:1387.[Abstract/Free Full Text]
  27. Segal, B. M., D. M. Klinman, E. M. Shevach.. 1997. Microbial products induce autoimmune disease by an IL-12-dependent pathway. J. Immunol. 158:5087.[Abstract]
  28. Kuge, S., Y. Miura, Y. Nakamura, T. Mitomi, S. Habu, T. Nishimura. 1995. Superantigen-induced human CD4+ helper/killer T cell phenomenon: selective induction of Th1 helper/killer cells and application to tumor immunotherapy. J. Immunol. 154:1777.[Abstract]
  29. Deibler, G. E., R. E. Martenson, M. W. Kies. 1972. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep. Biochem. 2:139.[Medline]
  30. Wysocka, M., M. Kubin, L. Vieira, L. Ozmen, G. Garotta, P. Scott, G. Trinchieri. 1995. Interleukin-12 is required for interferon-{gamma} production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
  31. Gombold, J. L., R. M. Sutherland, E. Lavi, Y. Paterson, S. R. Weiss.. 1995. Mouse hepatitis virus A-59-induced demyelination can occur in the absence of CD8+ T cells. Microb. Pathol. 18:211.
  32. Miethke, T., C. Wahl, K. Heeg, H. Wagner. 1995. Superantigens: the paradox of T-cell activation versus inactivation. Int. Arch. Allergy Immunol. 106:3.[Medline]
  33. D’Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, G. Trinchieri. 1993. Interleukin 10 (IL-10) inhibits human lymphocyte interferon-{gamma} production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041.[Abstract/Free Full Text]
  34. Tripp, C. S., S. F. Wolff, E. R. Unanue. 1993. Interleukin 12 and tumor necrosis factor are costimulators of interferon {gamma} production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725.[Abstract/Free Full Text]
  35. Trembleau, S., T. Germann, M. K. Gately. 1995. The role of IL-12 in the induction of organ-specific autoimmune diseases. Immunol. Today 16:383.[Medline]
  36. Gazzinelli, R. T., M. Wysocka, S. Hayashi, E. Y. Denkers, S. Hieny, P. Caspar, G. Trinchieri, A. Sher. 1994. Parasite-induced IL-12 stimulates early IFN-{gamma} synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533.[Abstract]
  37. Tripp, C. S., O. Kanagawa, E. R. Unanue. 1995. Secondary response to Listeria infection requires IFN-{gamma} but is independent of IL-12. J. Immunol. 155:3427.[Abstract]
  38. 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]
  39. Joosten, L. A. B., E. Lubberts, M. M. A. Hellsen, W. B. van den Berg. 1997. Dual role of IL-12 in early and late stages of murine collagen type II arthritis. J. Immunol. 159:4092.
  40. Seder, R. A., B. Kelsall, D. Jankovic. 1996. Differential roles for IL-12 in the maintenance of immune responses in infectious versus autoimmune disease. J. Immunol. 157:2745.[Abstract]
  41. Peritt, D., M. Aste-Amezaga, F. Gerosa, C. Paganin, G. Trinchieri. 1996. Interleukin-10 induction by IL-12: a possible modulatory mechanism?. Ann. NY Acad. Sci. 795:387.[Medline]
  42. Winghagen, A., D. E. Anderson, A. Carrizosa, R. E. Williams, D.A. Hafler. 1997. IL-12 induces human T cells secreting IL-10 with IFN-{gamma}. J. Immunol. 157:1127.[Abstract]
  43. Kennedy, M.K., D. S. Torrance, K. S. Picha, K. M. Mohler. 1992. Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. J. Immunol. 149:2496.[Abstract]
  44. Rott, O., B. Fleisher, E. Cash. 1994. Interleukin-10 prevents experimental allergic encephalomyelitis in rats. Eur. J. Immunol. 24:1434.[Medline]
  45. Cannella, B., Y. L. Gao, C. Brosnan, C. S. Raine.. 1996. IL-10 fails to abrogate experimental autoimmune encephalomyelitis. J. Neurosci. Res. 45:735.[Medline]
  46. Crisi, G. M., L. Santambrogio, G. M. Hochwald, S. R. Smith, J. A. Carlino, G. J. Thorbecke. 1995. Staphylococcus enterotoxin B and tumor-necrosis factor-{alpha}-induced relapses of experimental allergic encephalomyelitis: protection by transforming growth factor-ß and interleukin-10. Eur. J. Immunol. 25:3035.[Medline]
  47. Segal, B. M., B. K. Dwyer, E. M. Schevach. 1998. An Interleukin (IL)-10/IL-12 immunoregulatory circuit controls susceptibility to autoimmune disease. J. Exp. Med. 187:537.[Abstract/Free Full Text]
  48. Lehmann, P. V., E. E. Sercarz, T. Forsthuber, C. M. Dayan, G. Gammon. 1993. Determinant spreading and the dynamics of the autoimmune T-cell repertoire. Immunol. Today 14:203.[Medline]
  49. McRae, B. L., C. L. Vanderlugt, M. C. Dal Canto, S. D. Miller. 1995. Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. J. Exp. Med. 182:75.[Abstract/Free Full Text]
  50. Yu, M., J. M. Johnson, V. K. Tuohy. 1996. Generation of autonomously pathogenic neo-autoreactive Th1 cells during the development of the determinant spreading cascade in murine autoimmune encephalomyelitis. J. Neurosci. Res. 45:463.[Medline]
  51. Kubin, M., M. Kamoun, G. Trinchieri. 1994. Interleukin-12 synergizes with B7/CD28 interaction in inducing efficient proliferation and cytokine production of human T cells. J. Exp. Med. 180:211.[Abstract/Free Full Text]
  52. Murphy, E., G. Terres, S. 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]
  53. Miller, S. D., C. L. Vanderlugt, D. J. Lenschow, J. G. Pope, N. J. Karandikar, M. C. Dal Canto, J. A. Bluestone. 1995. Blockade of CD28/B7 interaction prevents epitope spreading and clinical relapses of murine EAE. Immunity 3:739.[Medline]
  54. Perrin, P. J., D. Scott, L. Quigley, P. S. Albert, O. Feder, G. S. Gray, R. Abe, C. H. June, M. K. Racke. 1995. Role of B7:CD28/CTLA4 in the induction of chronic relapsing experimental allergic encephalomyelitis. J. Immunol. 154:1481.[Abstract]
  55. Miethke, T., C. Wahl, D. Regele, H. Gaus, K. Heeg, H. Wagner. 1993. Superantigen-mediated shock: a cytokine release syndrome. Immunobiology 189:270.[Medline]
  56. Ogawa, M., T. Tsutsui, J.-P. Zou, R. Wijesuriya, W.-G. Yu, S. Herrmann, T. Kubo, H. Fujiwara, T. Harnaoka. 1997. Enhanced induction of very late antigen 4/lymphocyte function-associated antigen 1-dependent T-cell migration to tumor sites following administration of interleukin-12. Cancer Res. 57:2216.[Abstract/Free Full Text]
  57. Baron, J. L., J. A. Madri, N. H. Ruddle, G. Hashim, C. A. Janeway.. 1993. Surface expression of a4 integrin by CD4+ T cells is required for their entry into brain parenchyma. J. Exp. Med. 177:57.[Abstract/Free Full Text]
  58. Marth, T., W. Strober, B. L. Kelsall. 1996. High dose oral tolerance in ovalbumin TCR-transgenic mice: systemic neutralization of IL-12 augments TGF-ß secretion and T cell apoptosis. J. Immunol. 157:2348.[Abstract]
  59. Brocke, S., K. Gijbels, M. Allegretta, I. Ferber, C. Piercy, T. Blankenstein, R. Martin, U. Utz, N. Karin, D. Mitchell, et al 1996. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature 379:343.[Medline]
  60. Kuruvilla, A. P., R. Shah, G. M. Hochwald, M. Liggitt, A. Palladino, G. J. Thorbecke. 1991. Protective effect of transforming growth factor ß1 on experimental autoimmune diseases in mice. Proc. Natl. Acad. Sci. USA 88:2918.[Abstract/Free Full Text]
  61. Racke, M. K., S. Dhib-Jalbut, B. Cannella, P. Albert, C. S. Raine, D. E. McFarlin. 1991. Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-ß1. J. Immunol. 146:3012.[Abstract]
  62. Soos, J. M., P. S. Subramaniam, A. C. Hobeika, J. Schiffenbauer, H. M. Johnson. 1995. The IFN pregnancy recognition hormone IFN-{gamma} blocks both development and superantigen reactivation of experimental allergic encephalomyelitis without associated toxicity. J. Immunol. 155:2747.[Abstract]
  63. Santambrogio, L., G. M. Hochwald, B. Saxena, C.-H. Leu, J. E. Martz, J. A. Carlino, N. H. Ruddle, M. A. Palladino, L. I. Gold, G. J. Thorbecke. 1993. Studies on the mechanism by which transforming growth factor-ß (TGF-ß) protects against allergic encephalomyelitis: antagonism between TGF-ß and tumor necrosis factor. J. Immunol. 151:1116.[Abstract]
  64. Baker, D., D. Butler, B. J. Scallon, J. K. O’Neill, J. L. Turk. 1994. Control of established experimental allergic encephalomyelitis by inhibition of tumor necrosis factor (TNF) activity within the central nervous system using monoclonal antibodies and TNF receptor-immunoglobulin fusion proteins. Eur. J. Immunol. 24:2040.[Medline]
  65. Hunter, C., L. Bermudez, H. Beernink, W. Waegell, J. S. Remington. 1995. Transforming growth factor-ß inhibits interleukin-12-induced production of interferon-{gamma} by natural killer cells: a role for transforming growth factor-ß in the regulation of T-cell independent resistance to Toxoplasma gondii. Eur. J. Immunol. 25:994.[Medline]
  66. Scharton-Kersten, T., L. C. C. Afonso, M. Wysocka, G. Trinchieri, P. Scott. 1995. IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J. Immunol. 154:5320.[Abstract]
  67. Goverman, J., A. Woods, L. Larson, L. P. Weiner, L. Hood, D. M. Zaller. 1993. Transgenic mice that express a myelin basic protein-specific T cell receptor develop spontaneous autoimmunity. Cell 72:551.[Medline]
  68. Lichtman, S. N., J. Wang, R. B. Sartor, C. Zhang, D. Bender, F. G. Dalldorf, J. H. Schwab. 1995. Reactivation of arthritis induced by small bowel bacterial overgrowth in rats: role of cytokines, bacteria, and bacterial polymers. Infect. Immun. 63:2295.[Abstract]
  69. Constantinescu, C. S., B. D. Hondowicz, M. M. Ellosa, M. Wysocka, G. Trinchieri, P. Scott. 1998. The role of IL-12 in the maintenance of an established Th1 response in experimental leishmaniasis. Eur. J. Immunol. 28:2227.[Medline]



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