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* Department of Immunobiology, Biomedical Primate Research Center, Rijswijk, The Netherlands;
Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands;
Department of Experimental In Vivo NMR, Image Sciences Institute, University Medical Center, Utrecht, The Netherlands;
Department of Research and Development, Centocor, Inc., Malvern, PA 19355;
¶ Division of Neuroimmunology, Brain Science Institute, University of Vienna, Vienna, Austria; and
|| Department of Pharmacology and Pathobiology, University of Utrecht, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 is the predominant cytokine for triggering Th1-mediated (autoimmune) responses; it is induced when CD4+ T cells and APC interact (8, 9, 10) and abrogated when CD40-CD154 ligation is disturbed (11, 12, 13). Evidence is accumulating that IL-12 plays a pivotal role in the induction of the critical autoimmune responses involved in the initiation of experimental autoimmune encephalomyelitis (EAE), lesion formation, and progression of the disease (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Therapies directed at the neutralization of IL-12 or prevention of production by abrogation of CD40-CD154 interaction have proven effective in rodent (20, 21, 23, 24, 25, 26, 27) as well as marmoset (28) models of EAE, while excess IL-12 reverses these effects and enhances EAE severity (15, 16, 17, 22, 29). Furthermore, local expression of IL-12 within the CNS of rodents (14, 30, 31) and common marmosets (32) during active EAE has been demonstrated.
Although the situation is less clear in MS, IL-12 is locally expressed
within the CNS of affected individuals (33, 34, 35), and
levels of IL-12 in cerebrospinal fluid and plasma are increased during
active disease (36, 37, 38). Moreover, it has been argued that
the beneficial effect of IFN-
on MS is exerted via suppression of
IL-12 production (39, 40, 41). Finally, lower baseline levels
of IL-12p35 and p40 mRNA seem to predict clinical responsiveness to
IFN- treatment (42, 43).
In its radiological and neuropathological presentation, the chronic progressive EAE model in the common marmoset approximates the most prevalent lesion type, pattern II, in MS patients (7, 44, 45). These aspects added to the MS-like clinical expression of the disease (7, 46) and the close immunological similarity with humans (47, 48, 49) make this model an excellent test system for preclinical evaluation of new therapies for chronic MS. The model is particularly important for the safety and effectivity testing of biotechnologically engineered reagents, which by their species specificity cannot be evaluated in rodent EAE models. The present study demonstrates that i.v. injected mAb directed against human IL-12p40 significantly reduces the lesion load, as detected by magnetic resonance imaging (MRI) and neuropathological examination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten nonrelated healthy common marmosets (Callithrix
jacchus) were selected from the experimental stock of the
Biomedical Primate Research Center (Rijswijk, The Netherlands). Before
the monkeys entered the experiment, a full physical, hematological, and
biochemical check-up was performed. Individual data of the monkeys,
which were identified with an implanted transponder, are summarized in
Table I
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).
Paired individuals were handled identically throughout the experiment.
The time points for collection of larger blood volumes for
immunological tests and MRI were determined on the basis of the
clinical stage of EAE in one individual of each pair. During the experiments the monkeys were individually housed in spacious cages with padded shelters provided at the bottom of the cage and were under constant veterinary care. The daily diet consisted of commercial food pellets for New World monkeys (Special Diet Services, Witham, U.K.), supplemented with rice, raisins, peanuts, marshmallows, biscuits, and fresh fruit. Drinking water was provided ad libitum. According to Dutch law on animal experimentation, the protocol of this study was reviewed and approved by the institute’s animal care and use committee.
Disease induction and clinical read-out
EAE was induced by a single immunization with 300 µl human myelin in water (10 mg/ml) emulsified with 300 µl CFA (Difco, Detroit, MI) under ketamine anesthesia (6 mg/kg; AST Farma, Oudewater, The Netherlands) as described previously (50). Bordetella pertussis was not used for reasons discussed previously (50).
Twice daily, clinical signs of EAE were scored blind by a trained observer using a previously described semiquantitative scale (50): 0, no clinical signs; 0.5, apathy, loss of appetite and altered walking pattern without ataxia; 1, lethargy and/or anorexia; 2, ataxia, sensory loss/blindness; 2.5, hemi- or paraparesis; 3, hemi- or paraplegia; 4, quadriplegia; and 5, spontaneous death attributable to EAE. Body weights were determined once weekly as a surrogate disease marker. Monkeys were sacrificed for ethical reasons once a monkey had reached an EAE score of 3.0 or on day 86 after immunization, which was the planned end point of the study.
Reactivity, dosing regimen, plasma levels, biodistribution, and immunogenicity of anti-IL-12p40 Ab
The neutralization of marmoset IL-12 by the anti-human
IL-12p40 mAb (IgG1k) was tested using LPS-stimulated cells. Briefly,
plastic-adherent cells from common marmosets and human PBMC were
stimulated for 24 h with LPS (1 µg/ml). Cell-free supernatant
was collected and titrated onto 4-day PHA-stimulated human T cell
blasts. After 24 h IFN-
levels were determined using standard
ELISA techniques. An 90% effective concentration stimulation level was
determined for each preparation and used at this concentration for
titration of Ab to determine the IC50 of
neutralization.
The animals were treated with Ab between days 14 and 86 after immunization. Five animals received once weekly i.v. injections of 10 mg/kg of Ab in saline into the vena saphena under ketamine anesthesia. Control animals (n = 5) received once weekly i.v. injections of sterile PBS (1 ml/kg) as a placebo treatment.
Three days after each treatment serum was collected and stored frozen until determination of Ab levels with ELISA. Briefly, recombinant human IL-12 (10 µg/ml) was coated on 96-well plates (Costar Corning, New York, NY). Thawed serum samples were incubated for 1 h and, after washing, Ab was detected by mouse-anti-human Fc Ab, grown from the cell line HP6017 (American Type Culture Collection, Manassas, VA). The Ab was purified via protein A, coupled with sulfo-N-hydroxysuccinimide LC biotin (Pierce, Rockford, IL), and detected using HRP-conjugated streptavidin (SA-HRP; Jackson ImmunoResearch Laboratories, West Grove, PA). Concentrations were calculated using a standard curve. Anti-Ab levels were determined by a double Ag enzyme immunoassay using bound anti-IL-12p40 Ab, detected with biotinylated anti-IL-12p40 Ab and SA-HRP.
Magnetic resonance imaging
High resolution MRI experiments were performed on a 4.7 T horizontal bore, nuclear magnetic resonance spectrometer (Varian, Palo Alto, CA), equipped with a high performance gradient insert (11-cm inner diameter; maximum gradient strength, 220 mT/m). For in vivo MRI animals were anesthetized with 30 mg/kg ketamine in combination with 1 mg/kg of valium (Diazepam; Kombivet, Etten-Leur, The Netherlands). MRI datasets were collected for T1-weighted (T1-w) and T2-w images. A bird cage volume coil (diameter, 9 cm) was used for radiofrequency transmission and signal reception. For all sequences the field of view was 4 x 4 cm (matrix, 128 x 128; zero filled to 256 x 256; in plane resolution 312 x 312 µm). Postcontrast T1-w images were made after i.v. injection of 0.3 mmol/kg gadolinium-diethylenetriaminepentaacetic acid (Magnevist, Schering, Berlin Germany). Postmortem high contrast T2-w images were made of formalin-fixed brains. A solenoidal coil (four windings; diameter, 35 mm) was used for radiofrequency transmission and signal reception (field of view, 3 x 3 cm; matrix, 128 x 128; zero filling, 256 x 256; in-plane resolution, 240 x 240 µm). The in vivo and postmortem MRI scores were calculated as proposed by Jordan and coworkers (51): score 0, no visible lesions; score 1, one to five lesions on T2 scans; score 2, six to 10 lesions on T2 scans; score 3, 11–20 lesions on T2 scans; and score 4, >21 lesions or diffuse white matter abnormalities on T2 scans. A score of 0.5 is added for one gadolinium-enhancing lesion and a score of 1 is added for two or more enhancing lesions. It should be emphasized that this scoring is based on the number of lesions rather than their size or pathomorphological aspects. The datasets were analyzed on a MacIntosh I-MAC G3 (Apple Computer, Cupertino, CA) using the public domain NIH program Image 1.52.
Neuropathological examination
After formalin fixation parts of the brain, spinal cord, and peripheral nerves were embedded in paraffin and processed as described previously (28). In brief, the cerebrum and cerebellum were divided into seven or eight coronally cut parts, and the spinal cord was dissected transversely. The extent of inflammation, demyelination, and axonal pathology was evaluated on 3- to 5-µm tissue sections stained with H&E to visualize infiltrated cells, with Klüver Barrera Luxol Fast Blue combined with periodic acid-Schiff for myelin and myelin degradation products, and with Bielschowsky silver impregnation for axons. The degree of inflammation was expressed as an index, representing the average number of inflamed blood vessels per spinal cord section (n = 10–15 sections). Furthermore, the surface area of demyelination was quantified on 10–15 spinal cord fields using a monomorphic grid. Macrophages were visualized using mouse anti-human mAb MRP14 (BMA Biomedicals, Augst, Switzerland), while mAb M4 was used to detect amyloid precursor protein (APP; Roche, Mannheim, Germany).
Expression profiles of pathogenic effector molecules in the CNS
Immunohistochemistry was performed essentially as previously described (32, 52) with minor modifications. Frozen sections of 6 µm thickness were cut, thaw-mounted on glass slides, and kept overnight at room temperature in a humidified atmosphere. After air-drying for 1 h slides were fixed in fresh acetone containing 0.02% H2O2 (v/v), air-dried for 10 min, washed with PBS, and incubated overnight at 4°C with primary Ab. Incubations with secondary and tertiary reagents were performed for 1 h at room temperature. Between the incubation steps slides were washed twice with PBS. Detection of primary unlabeled mouse Ab was performed with rabbit anti-mouse Ig HRP (DAKO, Glostrup, Denmark) or, in the case of three-step staining, with rabbit anti-mouse Ig biotin (DAKO) and HRP-labeled avidin-biotin complex (DAKO). Rabbit polyclonal Ab was detected with biotin-labeled donkey anti-rabbit Ig (Amersham, Little Chalfont, U.K.) as a second step. HRP activity was revealed by incubation for 10 min at room temperature with 3-amino-9-ethyl-carbazole (Sigma, Zwijndrecht, The Netherlands), resulting in a bright red translucent precipitate.
For detection of IFN- and TNF-
, mAb MD-2 and 61E71 were used,
respectively (U-Cytech, Utrecht, The Netherlands). mAb against IL-4 and
IL-6 were obtained from Genzyme (Cambridge, MA). C8.6, a mouse Ab used
for detection of IL-12p40, was obtained from BD PharMingen (San Diego,
CA). IL-10 was visualized using B-S10 mAb (Instruchemie, Hilversum, The
Netherlands). The Ab against IL-18, M318, was obtained from R&D Systems
(Abingdon, U.K.). 2D9, a mouse mAb against matrix metalloprotease 9
(MMP-9; gelatinase B), was a gift from Dr. G. Opdenakker (REGA
Institute, Leuven, Belgium). Rabbit polyclonal Ab against CD3 and
inducible NO synthase (iNOS) were obtained from DAKO and Calbiochem
(San Diego, CA), respectively. Finally, CD40 was detected using a
mouse anti-human CD40 mAb (mAb 5D12; Tanox Pharma, Houston, TX),
which is known to be cross-reactive with marmoset CD40 (28, 32).
In situ detection of i.v. administered anti-IL-12p40 Ab
To determine whether the Ab traverses the blood-brain barrier and gains access to lesions in the CNS, 1 mg of biotinylated anti-IL-12p40 Ab was i.v. injected into a PBS-treated monkey with an EAE score of 3.0 (Mi-031). One hour after injection of the Ab the monkey was sacrificed. Frozen tissue sections of brain and spleen were incubated with SA-HRP (Jackson ImmunoResearch) for 1 h at room temperature, followed by tyramide signal amplification (NEN Life Science Products, Boston, MA). HRP activity was revealed as described above. To assess whether the biotinylated Ab bound to IL-12p40 produced by astrocytes, tissue sections were double stained using the C8.6 mAb against IL-12p40 and donkey anti-human polyclonal Ab (Sanbio) directed toward glial fibrillary acidic protein characteristic for astrocytes. A combination of HRP and alkaline phosphatase-labeled conjugates was used, giving a red precipitate for 3-amino-9-ethyl-carbazole and a bright blue precipitate using Fast Blue BB base and napthol AS-MX phosphate for alkaline phosphatase, as described in detail previously (32, 52).
T and B cell functions
Just before necropsy, heparinized venous blood was drawn via a needle puncture from the vena saphena, after which PBMC were isolated using lymphocyte separation medium (ICN Biomedical, Aurora, OH). Cell suspensions were prepared from aseptically removed lymph node (LNC) and spleen (SC) and cultured in the presence of recombinant human myelin oligodendrocyte glycoprotein (rhMOG; 10 µg/ml) or human myelin basic protein (hMBP; 25 µg/ml) (46).
Serum was isolated from venous blood collected without coagulant. Sera were collected from paired animals at the preimmunization stage, when one of both monkeys in a pair displayed an EAE score of 2.0, and at the time of necropsy. Standard ELISAs were used to determine IgM and IgG Ab levels directed against MBP and MOG as previously described (28).
Statistics
The 
2 test was used to determine
statistically significant treatment-related effects on the progression
to an EAE score of 3.0; the Mann-Whitney U test was used for
anti-MOG and anti-MBP Ab levels and MRI scores. In all cases,
p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EAE course in placebo- and Ab-treated monkeys is given as the
day of disease onset, when the animal showed clear neurological signs
(EAE score of 2.0), and when the disease score of 3.0 was reached
(Table ). The percentages of maximal weight loss during the experiment
are depicted in the same table. The results show a beneficial effect of
Ab treatment on both aspects of EAE. Four PBS-treated monkeys developed
severe progressive EAE, and one had a period of mild EAE during the
observation period of 86 days. In the Ab-treated group only one monkey,
Mi-019, developed clinical signs of EAE (p <
0.001 vs PBS treatment, by 2 test). It should
be noted, however, that the time interval between disease onset and EAE
score of 3.0 in this animal was considerably longer than that in the
placebo-treated monkeys (see Table I).
Cross-reactivity, serum levels, and anti-Ab responses
As shown in Fig. 1, the
IFN--inducing properties of conditioned medium derived from
LPS-stimulated marmoset adherent cells were neutralized by
anti-IL-12p40 Ab. The IC50 was comparable
with conditioned medium from human cells. The Ab levels measured at 3
days after each administration were generally maintained at a
concentration of 50–75 µg/ml throughout the observation period of 86
days (Fig. 2). This is an 
50-fold
excess of the IC50 determined in vitro. The assay
used to measure anti-IL-12p40 Ab required the plasma Ab to
bind to IL-12p40s to be detected. Therefore, the assay was designed to
measure only active anti-IL-12p40, which may, in fact, be less than
the total plasma concentration. In one animal (Mi-026) an unexplained
disappearance and subsequent reappearance of anti-IL-12p40 Ab were
observed. No anti-Ab responses were detectable, and no alterations
in hematological and biochemical parameters were observed during the
study period.
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The a priori condition set before the experiment was to subject paired monkeys to MRI analysis once one of the animals had reached an EAE score of 2.0 (ataxia) regardless of the clinical condition of the second monkey. Because of the acute onset of the disease in Mi-032 and Mi-043, both animals were euthanized for ethical reasons before an in vivo MRI could be made.
The characteristic feature of myelin-induced EAE is a strong
periventricular inflammatory reaction that appears as a hyperintense
area in T2-w brain MRI images, which is hypointense on T1-w images.
Fig. 3 depicts as representative examples
for both test groups the MRI-detectable changes in the brains of pair
4. In monkey Mi-031, which had an EAE score of 2.0 at the time of
scanning, large hyperintense areas were found around the ventral horns
of both lateral ventricles (Fig. 3A). Such abnormalities,
which are typical for this model, were not found in the clinically
normal Ab-treated monkey Mi-024, which was scanned on the same day
(Fig. 3B). Instead multiple, very small, hyperintense foci
were found in the T2-w brain images of this monkey, yielding an in vivo
MRI score of 4.0 as defined by Jordan et al. (51). In the
postmortem scans, however, these foci were not visible, suggesting that
they may represent patches of edema rather than demyelinated lesions.
Scores of in vivo and postmortem MRI are given in tabular form. The
table shows that postmortem MRI scores of Ab-treated animals tended to
be lower compared with the scores of control animals
(p = 0.08, by Mann-Whitney U
test).
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All PBS-treated control animals displayed cellular infiltrates in
the brain and spinal cord. Four PBS-treated animals showed extensive
demyelination, which was mainly localized in the spinal cord, while in
the animal with the mild EAE (Mi-038) the CNS pathology was mainly
localized in the brain (see Table I). Although inflammation and
demyelination of the CNS were detected in two Ab-treated individuals,
three animals (Mi-003, Mi-023, Mi-026) were completely devoid of CNS
pathology in spinal cord and brain (p = 0.03
for inflammation and demyelination, by Mann-Whitney U test).
Fig. 4 shows monkey pair 2 as a
representative example. The PBS-treated animal Mi-032 displayed large
areas with infiltrated mononuclear cells and specific demyelination of
the CNS in the spinal cord (Fig. 4, A and C),
while no such pathological changes were observed in the Ab-treated
animal of this pair, Mi-026 (Fig. 4, B and D).
Macrophages actively involved in demyelination were present in the
lesions as shown in Fig. 4, E and F, while B and
T cells also could be detected (Fig. 4, G and H,
respectively). The immunoreactivity for APP points at degeneration of
the demyelinated axons (Fig. 4I).
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Immunological aspects of the brain lesions were assessed in
cryosections on the basis of the number and size of the infiltrates as
defined by hematoxylin counterstaining, as well as the expression of
the pan-T cell marker CD3 and of acid phosphatase as a marker of
infiltrating macrophages. Both the number and size of infiltrates in
the brains of Ab-treated animals were markedly reduced compared with
those in control animals (Table I). While CD3+ T
cells were present in the cellular infiltrates of all EAE-affected
monkeys, these were undetectable in the brains of three of four
Ab-treated animals that remained asymptomatic. Together these data
indicate that neutralization of IL-12p40 affects recruitment of
mononuclear cells from the circulation into the CNS.
Next we assessed whether IL-12p40 neutralization would interfere with
CNS expression of Th1 or Th2 cytokines (IFN-, IL-4, IL-10, IL-12p40,
IL-18, TNF-) or the inflammatory mediators iNOS and MMP-9. IL-12p40
was expressed by some mononuclear cells within brain infiltrates and by
many astrocytes (Fig. 5, A and
B). The number of IL-12p40-expressing astrocytes decreased
with distance from the infiltrates. Similar expression of IL-12p40 was
found in both groups of animals (Fig. 5, A vs B).
In control animals lacking CNS inflammation, naive animals, or monkeys
immunized with OVA emulsified in CFA, no IL-12p40 was detectable within
the CNS (Fig. 5C).
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D). Mononuclear cell
infiltrates in the brain of PBS-treated animals displayed clear
expression of IL-4, TNF-, and MMP-9 (Fig. 5, E,
G, and I). In asymptomatic Ab-treated animals CNS
expression of IL-4, TNF-, and MMP-9 was substantially reduced (Fig. 5, F, H, and J). IFN- was also
expressed at lower frequency in nonaffected mAb-treated animals (data
not shown). The two Ab-treated animals that showed CNS inflammation
with histology displayed staining patterns similar to those in the
EAE-affected animals in the PBS-treated group. No marked effect of Ab
treatment on the numbers of IL-6-, IL-10-, IL-18-, and iNOS-expressing
cells was observed (results not shown). Access of anti-IL-12p40 Ab to lesions within the CNS
We assessed whether i.v. administered Ab extravasates into CNS
lesions, allowing the capture of locally produced IL-12p40. To this
end, biotinylated anti-IL-12p40 Ab was injected i.v. into two
PBS-treated animals with clinically manifest EAE (score of 3.0) at
1 h before sacrifice. The subsequent immunohistochemical analysis
revealed granular staining adjacent to the ventricles as well as in the
cellular infiltrates within the brain of animals injected with the Ab
(Fig. 6, A and B).
No such staining was detected in the brains of naive animals (Fig. 6C), or OVA/CFA-immunized marmosets (not shown).
Biotinylated Ab was found attached to the astrocytes in the immediate
environment of the inflammatory infiltrates. This is in conformity with
the immunohistochemical demonstration of IL-12p40 by brain astrocytes
in EAE-affected animals (Fig. 5A).
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To investigate the effect of anti-IL-12p40 Ab on
anti-myelin T cell activity we determined the proliferative
responses of PBMC, SC, and LNC to rhMOG and hMBP in Ab- and
placebo-treated animals. The results in Fig. 7 show that in all cases the T cell
response to MBP was low, while high responses were found to rhMOG (MOG
vs MBP, p < 0.01, by Mann-Whitney U test).
In the animals that developed EAE relatively early after disease
induction, significant proliferative responses to rhMOG were detectable
in SC as well as LNC (Fig. 7, A–C). In contrast, in all
animals sacrificed at the end point of the study (both PBS- and
Ab-treated animals), proliferative responses were mainly restricted to
the spleen, while those in LNC were very low or undetectable (Fig. 7, E–J). As a similar profile was found in the placebo monkey
of pair 5, we assume that the number of autoreactive T cells in the
spleen is not affected by the Ab treatment.
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A critical role of anti-MOG Ab in the marmoset EAE model,
mediating demyelination in particular, has been reported (45, 53, 54, 55, 56). We therefore determined the serum levels of
anti-MBP and anti-MOG Ab in PBS- and Ab-treated animals using
ELISA. Anti-MOG and anti-MBP IgM Ab were hardly detectable in the
sera of the animals from both groups at any of the evaluated time
points. As shown in Fig. 8, increased
anti-MOG (A) and anti-MBP (B) IgG serum
levels were found in PBS-treated animals at an EAE score of 2.0
compared with IgG Ab levels in sera from the paired Ab-treated animals
(p = 0.05, by Mann Whitney U test).
At necropsy, only serum levels of MBP-specific IgG were significantly
reduced in the asymptomatic Ab-treated animals
(p = 0.03). Taken together, neutralization of
IL-12p40 seems to have a suppressive effect on the production of
autoantibodies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All PBS-treated monkeys in this study developed clinical EAE. In
contrast, four of five Ab-treated animals remained asymptomatic. The
disease progression in the one Ab-treated animal that developed
clinical EAE was substantially delayed compared with that in the
PBS-treated monkeys. Our results also show a diminution of
MRI-detectable changes in CNS white matter in Ab-treated monkeys
compared with those in paired placebo-treated monkeys. A clear effect
of the treatment was that the periventricular inflammatory reactions,
as observed in all PBS-treated monkeys, were absent in all
anti-IL-12p40 Ab-treated monkeys. The beneficial effect of Ab
treatment on in vivo MRI scores is less significant than that on
the scores based on postmortem images. This is probably because
T2-w MR images detect mainly altered water content of tissues, such as
due to vasogenic edema, while with T2-w images of fixed tissue areas of
demyelination can be visualized (50). Indeed, the
postmortem MRI scans showed clearly reduced MRI scores in the
Ab-treated monkeys (with the exception of Mi-019) compared with the
placebo group (Table I). More advanced MRI parameters are needed to
reveal the beneficial effects of IL-12p40 neutralization in vivo.
Quantitative MRI parameters are now being developed and implemented for
serial imaging, in particular T1, T2, and MTR maps (E. L. A. Blezer, H.
P. M. Brok, K. Nicolay, and B. A. t’Hart, manuscript in
preparation). The beneficial effect of anti-IL-12p40 Ab on
CNS white matter pathology was confirmed with histology; three of five
Ab-treated monkeys remained completely devoid of CNS infiltration by
CD3+ T cells, while inflammation and
demyelination were also prevented.
IL-12 is expressed at sites where T cells and APC interact (8, 9, 10). In the common marmoset EAE model, such sites are the secondary lymphoid organs and the developing lesions within the CNS white matter (32, 52). While microglia is an important source of IL-12 within the CNS (10, 59), astrocytes have also been found to produce IL-12p40 (60). Our present finding that astrocytes in EAE-affected marmosets indeed express intracellular IL-12p40 confirm this observation.
Intravenously injected biotinylated Ab was retrieved at
peripheral (spleen/lymph nodes) as well as central (brain) locations,
confirming our previous finding that during active disease the
blood-brain barrier is permeable for large molecules such as IgG
(28). This allows the local action of Ab within the CNS at
locations where infiltrating T cells interact with resident APC or
infiltrating macrophages. As the time needed for 1-mm diffusion of a
20-kDa protein into unaffected CNS parenchyma has been estimated to be
3 days (61), we assume that the activity range of i.v.
injected Ab (±50 kDa) inside the CNS is limited to the perivascular
space and the lesion.
In view of the 100% susceptibility of outbred common marmosets to myelin-induced EAE (7, 44), it is highly unlikely that the observed beneficial effects of anti-IL-12p40 Ab can be explained by the possibility that the Ab-treated monkeys that remained asymptomatic were all nonresponders to the disease induction. Hence, the conclusion is warranted that anti-IL-12p40 Ab treatment protects marmoset monkeys immunized with human myelin in CFA against the clinical and neuropathological expression of EAE. Our present results suggest that anti-IL-12p40 Ab may modulate autoimmune responses.
Anti-myelin Ab are considered a critical factor in the marmoset EAE model as mediators of CNS demyelination (44, 53, 54, 55, 62). It is therefore of great interest that during the development of EAE, circulating autoantibody levels were consistently lower in anti-IL-12p40 Ab-treated animals than in the paired PBS-treated animals. This suggests that besides preventing T cell recruitment into the CNS, autoantibody responses are also mediated by IL-12p40. Similar observations have been made in marmosets that were protected from EAE by anti-CD40 mAb treatment. In that study the protective effect of the mAb to clinical EAE was associated with abrogation of broadening of the epitope response against MOG (28).
The different reactivity patterns of T cells present in PBMC, LNC, and SC to MBP and MOG between PBS- and Ab-treated monkeys can be explained by the different disease durations. Our data are in line with observations by Targoni and coworkers (63) that T cell autoreactivity in mice wanes in time from draining lymph nodes and the circulation during EAE development, but persists in the spleen. All Ab-treated monkeys displayed a similar reaction pattern as the one PBS-treated monkey sacrificed at the same time point (day 86). We assume, therefore, that IL-12p40 modulates the development of autoreactive T cells, yet does not interfere with cell circulation kinetics.
A likely result of IL-12p40 neutralization is deviation of the encephalitogenic myelin-reactive T cells into the Th2 direction, as was observed in mice (21). However, we are presently unable to prove this, since reagents for specific detection of Th2 cytokines in common marmosets with ELISA are lacking. Instead, using immunostaining we determined cytokine expression patterns within the CNS of both groups of monkeys.
In three of five Ab-treated animals neither T cell infiltrates nor
IFN-- or IL-4-producing cells could be detected. Moreover,
inflammatory mediators such as TNF- and MMP-9 were not expressed in
the brains of these monkeys. In contrast, in all PBS-treated animals we
found brain infiltrates expressing each of the tested inflammatory
mediators. The patterns of intracellular IL-12p40 expression within the
CNS white matter did not differ between PBS- and Ab-treated animals. A
possible explanation for this lack of difference is that the
anti-IL-12p40 mAb acts on the effector mechanisms induced by
released IL-12, but not on the actual production of IL-12p40.
In conclusion, anti-IL-12p40 Ab has direct access to secondary lymphoid organs and the CNS. Since Ab treatment was initiated at a late stage (day 14 after immunization), it is less likely that the Ab interferes with initial activation of autoreactive T and B cells. However, by neutralization of APC-derived IL-12, it affects the influx of autoreactive T cells into the CNS, inhibits the inflammatory response, and suppresses the autoantibody response against myelin proteins, resulting in prevention of EAE development.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bert A. ’t Hart, Department of Immunobiology, Biomedical Primate Research Centre, Lange Kleiweg 139, 2288 GJ Rijswijk, The Netherlands. E-mail address: hart{at}bprc.nl
3 Abbreviations used in this paper: MS, multiple sclerosis; APP, amyloid precursor protein; EAE, experimental autoimmune encephalomyelitis; LNC, lymph node cell; iNOS, inducible NO synthase; (h)MBP, (human) myelin basic protein; MMP-9, matrix metalloprotease 9; MRI, magnetic resonance imaging; (rh)MOG, (recombinant human) myelin oligodendrocyte glycoprotein; ROI, region of interest; RT, room temperature; SA-HRP, HRP-conjugated streptavidin; SC, splenocytes, spleen cell; T1-w, T1-weighted.
Received for publication November 5, 2001. Accepted for publication October 8, 2002.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 subunit in the generation of pathogenic autoreactive Th1 cells. Springer Semin. Immunopathol. 21:249.[Medline]
and tumor necrosis factor . J. Neuroimmunol. 61:205.[Medline]
inhibits the development of epitope spreading and disease progression in murine autoimmune encephalomyelitis. J. Neuroimmunol. 111:55.[Medline]
mRNA expression in CSF and peripheral blood mononuclear cells from multiple sclerosis patients. J. Neuroimmunol. 91:198.[Medline]
and IL-12 by endogenous IL-10 in progressive MS. Neurology 55:192.-1b inhibits IL-12 production in peripheral blood mononuclear cells in an IL-10-dependent mechanism: relevance to IFN--1b therapeutic effects in multiple sclerosis. J. Immunol. 165:548. in multiple sclerosis: is IL-12 suppression the key?. Immunol. Today 21:24.[Medline]
-1b treatment in relapsing-remitting multiple sclerosis: does baseline interleukin-12p35 messenger RNA predict the efficacy of treatment?. Ann. Neurol. 48:313.[Medline]
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) or in the presence
of rhMOG (10 µg/ml; 
) or hMBP (25 µg/ml; 
). Animals
sacrificed early after disease induction showed proliferative responses
to MOG in both SC and LNC (A–C). In animals that were
sacrificed 2 wk before (D) or at the end point of the
study (E–J; both PBS- and Ab-treated animals), MNC
proliferation tend to localize primarily in the spleen, while LNC
responses wane. n.d., not done.
, 
,
•) and Ab-treated animals (
, 
, 
) were screened for the
presence of anti-MOG (A) and anti-MBP
(B) IgG Abs using ELISA. 
, pair 5. *, Mi-019 at EAE score of 2.0. Sera
were collected from paired animals just before immunization
(preimmune), when the PBS-treated animal of a pair presented a clear
neurological dysfunction EAE score of 2.0 (ataxia), and at time of an
EAE score of 3.0 or on day 86 after immunization (necropsy). Sera from
PBS-treated monkeys collected at the manifestation of a score of 2.0
contained significantly higher anti-MBP and anti-MOG IgG Ab
levels than sera from the paired Ab-treated animals
(p = 0.05, by Mann-Whitney U test).
At the time of an EAE score of 3.0 the levels of anti-MBP IgG, but
not of anti-MOG IgG, remained significantly higher than those in
the paired Ab-treated animals that were sacrificed much later
(p = 0.03).