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Regular Articles |
From the Neuroimmunology Branch,*
National Institute of
Neurological Disorders and Stroke, Division of Acquired
Immunodeficiency Syndrome,
National Institute
of Allergy and Infectious Diseases, Center for Biologics Evaluation and
Research,
Food and Drug Administration,
Bethesda, Maryland; the Department of
Neurology,§
Washington University School of
Medicine, St. Louis, Missouri; and the Departments of Pathology,
Neurology, and Neuroscience,¶
Albert Einstein
College of Medicine, Bronx, New York
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental allergic encephalomyelitis (EAE), an animal model with similarities to MS, exists in many forms, some of which present with clinical relapses and episodes of inflammatory demyelination.13-20 In the present adoptive transfer model of EAE in mice,20 young adults are commonly used and these develop a predictable clinical and pathological disease with a uniform and usually rapid onset 1 to 2 weeks after transfer of lymphoid cells sensitized to CNS antigen. Among the features of MS that have been difficult to replicate in animal models is the putative latent period of disease before clinical onset. Clinical presentation before adulthood is rare, although an early predisposition with minimal clinical expression during adolescence is postulated and at the time of initial diagnosis, pathological manifestations are usually well established. Occasionally at the time of diagnosis of MS, review of past history may show evidence of early transient clinical signs or preclinical MRI abnormalities can be found in individual cases, supportive of the possibility of a presumed early window of predisposition in childhood.21-23 In most animal models of MS, disease induction occurs in adults and this usually leads to limited, uniform delays in onset of signs.
The present study was undertaken to determine whether certain features of MS could be documented and investigated in EAE. These include developmental, age-related resistance to clinical disease and an early preclinical predisposition for autoimmune demyelination. Our findings showed the existence of a period of time during development when animals were resistant to acute clinical manifestations yet displayed fulminant CNS pathology during the silent latent period, after which a much delayed, chronic relapsing clinical course ensued. Understanding the immunological mechanisms underlying the opening and closing of this window of early clinical resistance to EAE may provide important insights into host factors operative during the preclinical or remission phases of MS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study protocol was reviewed and approved by an institutional animal care and use committee and all animals were maintained in animal facilities according to the NIH Animal Research Advisory Committee guidelines in NIH Manual 3040–2. Six-week-old SJL/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and housed for 4 to 5 weeks before they were used as lymph node cell (LNC) donors. For each experiment, 30 mice were used as donors. Recipient SJL mice were from timed pregnancies obtained from the National Cancer Institute/Division of Cancer Treatment colony (Frederick, MD), and were maintained in the National Institute of Neurological Disorders and Stroke animal facilities. Each EAE experiment involved 25 to 45 recipient mice that were followed for up to 120 days after transfer of sensitizing cells, vide infra.
Adoptive Transfer EAE
Ten-week-old mice were anesthetized with methoxyfluorane and immunized subcutaneously at four sites with 0.025 ml of an emulsion containing 400 µg guinea pig myelin basic protein (MBP), prepared as previously described,24 and complete Freund's adjuvant (Difco, Detroit, MI). Ten days later, animals were killed and lymph nodes draining the immunization sites aseptically removed and pressed through a wire screen to produce a single-cell suspension. Lymph node cells (LNC) were washed 4 times in Hanks' Balanced Salt Solution (HBSS, Biofluids, Rockville, MD) and cultured (5% CO2, 37°C, 95% humidity) for 4 days at a concentration of 4 x 106 cells/ml with 25 µg/ml of MBP in 24-well plates (Nunc, Roskilde, Denmark). Culture medium was RPMI-1640 (Biowhittaker, Walkersville, MD), with 10% heat-inactivated fetal bovine serum, 200 mmol/L L-glutamine, 10 mmol/L penicillin/streptomycin, 200 mmol/L nonessential amino acids, 100 mmol/L sodium pyruvate, 200 mmol/L Hepes buffer (Biowhittaker), and 50 mmol/L mercaptoethanol (Sigma, St. Louis, MO). After washing in phosphate buffered saline (PBS), 3 x 107 LNC in 200 µl were injected intraperitoneally (i.p.) into groups of naïve recipient mice aged 10 days to 12 weeks. For a single experiment, all age groups were injected at the same time. For consistency, the i.p. route was selected for all age groups because tail vein injection was not possible in the younger groups, whose small size (weights as low as 2 g) made venous access difficult and intravascular fluid volumes poorly tolerated.
On three occasions, instead of MBP, ovalbumin (OVA, Millipore, Freehold, NJ) was used for control purposes at the same concentrations for immunization, cell culture, and proliferation assay.
Proliferation Assay
To verify specificity to immunizing antigen, LNC were cultured at a concentration of 4 x 105 cells/well, plated in quadruplicate in 96-well flat-bottom plates (Nunc) with 100, 50, 25, 12.5, and 0 µg/ml MBP or 4 µg/ml Concanavalin A (Con A) (Sigma) for 4 days.25 3H-methyl thymidine (New England Nuclear, Boston, MA) (1 µCi/well), was added at 80 hours, plates were harvested with a Tomtec cell harvester (Wallac Inc., Gaithersburg, MD) onto glass filters at 96 hours, and counts per minute (cpm) were measured with a 1205 Betaplate (Wallac, Oy, Turku, Finland). The stimulation index (S.I.) of cpm to antigen/cpm to no antigen was used to confirm specific dose-response proliferation (S.I. 30–100) to immunizing antigen and for assessment of age differences in antigen presentation.
Clinical Evaluation
Mice were observed daily for signs of EAE.25 Neurological deficits were scored by a blinded individual as follows: 0.5 = slight tail weakness; 1.0 = weak floppy tail; 2.0 = mild hind limb weakness; 3.0 = mild hind limb and forelimb weakness; 4.0 = severe hind limb and forelimb involvement with or without paralysis; and 5.0 = moribund state or death. Because this is not a linear scale, differences between each grade were not equivalent.
Monoclonal Antibodies and Fluorescence-Activated Cell Sorter (FACS) Analysis
Spleens from animals of different ages were removed aseptically and pressed through wire screens to produce a single-cell suspension of antigen-presenting cells (APC). Spleen cells (SC) were washed in HBSS and cultured in 6-well plates (Costar, Cambridge, MA), at a concentration of 2 x 106 cells/ml in freshly prepared RPMI with additives, as described above.26 SC were removed from culture after 6, 18, 24, 48, and 72 hours and prepared for FACS analysis (FACScan, Becton Dickinson, Mountain View, CA). SC were washed, incubated for 20 minutes at 18°C with 2% normal mouse serum or rat IgG in PBS before incubation with mouse or rat monoclonal antibodies (mAb, 30 minutes at 0°C). SC were then washed twice and fixed with 2% paraformaldehyde. mAb OX-6 (anti-Ias), conjugated to fluorescein isothiocyanate (FITC, Serotec, Indianapolis, IN) at 1:100 was used for the staining of MHC class II; mAb L3T4 conjugated to phycoerythrin (PE, Becton Dickinson, Mountain View, CA) at 1:100 for CD4; mAb Lyt-2 conjugated to PE (Becton Dickinson, Bedford, MA) at 1:100 for CD8; and anti-Mac-1 (Boehringer Mannheim, Indianapolis, IN), conjugated to FITC at 1:15, was used to stain macrophages. Control Ab were rat IgG-FITC at 1:200 and rat IgG-PE at 1:200 (Zymed, South San Francisco, CA), mouse IgG-PE at 1:50, and mouse IgG-FITC at 1:50 (Becton Dickinson, Bedford, MA).
Antigen Presentation Analysis
Presentation of MBP to MBP-sensitized adult LNC by splenocyte APC from 2- and 10-week-old female mice was measured by 3H-methyl thymidine incorporation. LNC were obtained from immunized animals and cultured for 4 days in 24-well plates, as described above. The medium containing MBP was then replaced with fresh medium containing no MBP and 2 x 106/ml irradiated (3500 rads) splenocytes from 12-week-old animals. Cells were cultured for 10 days in 80-cm flasks (Nunc), washed, and replated (2 x 105 cells/well, 25 µg/ml MBP) in quadruplicate in 96-well flat-bottom plates (Costar) with irradiated splenocytes (1 x 105 cells/ml) from 2- or 10-week-old animals. Each well was pulsed with 1µCi 3H-methyl thymidine and radioactivity was measured as described above.
Neuropathology
Animals from each age group were studied from 7 days post-transfer (dpt), the usual time of clinical onset postsensitization in adult animals, to 69 dpt. At time of sampling, mice were anesthetized with ether and perfused via the left cardiac ventricle with 20 to 40 ml of cold 2.5% glutaraldehyde in phosphate buffer, pH 7.4. The entire CNS was removed and thin slices taken from the cerebrum, cerebellum, medulla/pons, and cervical, thoracic, and lumbar spinal cord. In addition, optic nerves and spinal nerve roots were sampled. The tissue was postfixed in 1% osmic acid, dehydrated in ethyl alcohol, and embedded in Epon 812. Epoxy sections were cut 1 µm thick and stained with toluidine blue for light microscopy. Thin sections for electron microscopy (EM) were contrasted with lead and uranium salts, carbon-coated, and scanned in a Siemens 101. Light microscope analysis was carried out by a blinded individual. Histological scores for demyelination and inflammation were scored from 1 to 5 based on the extent of white matter involvement.
Terminal Deoxyribonucleotidyl Transferase dUTP Nick End-Labeling (TUNEL)
Terminal deoxyribonucleotidyl transferase (TdT) labeling was performed to evaluate the incidence of apoptosis.27-29 MBP-specific LNC were transferred into groups animals (n = 5) of different ages (2, 4, 6, 10, 12 weeks). By 7 dpt, clinical signs (hind limb and tail weakness) were well developed in 10- and 12-week-old animals. At this time point, representative animals were sacrificed under methoxyfluorane and perfused with 60 ml PBS followed by Parafix (Molecular Histology, Gaithersburg, MD) via the left cardiac ventricle. The CNS was removed en bloc from 1 animal at each age point and from a normal 2-week-old and a 7-day-old animal. In addition, CNS tissue was taken 36 hours after 500 Gy total body irradiation of a normal 10-week-old animal. The other injected animals from the different age groups were followed to verify a remitting-relapsing clinical disease course. Brains and spinal cords were sectioned longitudinally as 5-µm sections, and a section adjacent to the central canal from each spinal cord was mounted on a sialinized slide. For positive controls, sections from the cervical and inguinal lymph nodes, thymus, and spleen of the 10-week-old irradiated animal were also mounted. Sections were dewaxed and hydrated in graded alcohols, washed in 2% H2O2 for 5 minutes, and digested in 20 µg/ml proteinase K. Slides were rinsed and washed in TdT buffer, 30 mmol/L Tris, pH 7.2, 140 mmol/L cacodylate, and 1 mmol/L biotinylated dUTP (Pharmacia, Piscataway, NJ) for 1 hour at 37°C. Reaction was terminated in 3x SSC (Biofluids, Rockville, MD) for 15 minutes. Sections were covered with 2% bovine serum albumin (Sigma, St. Louis, MO) for 20 minutes, rinsed in PBS, covered with 1:20 alkaline phosphatase, stained with Fast Red (Dako, Carpinteria, CA), and counterstained with hematoxylin (Sigma). Thirty light photomicrographs (each covering 0.75 mm in tissue depth) were taken of the entire length of a longitudinal section of spinal cord from each animal and the number of Fast Red-labeled cells (cells showing DNA fragmentation) per 0.75 mm recorded by a blinded individual.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinical Course Differs between Immature and Adult Mice
Adoptive transfer of 3 x 107
MBP-sensitized LNC into female mice of various ages produced an
age-related onset of clinical disease which was associated with the
youngest recipient animals expressing well-developed clinical disease
by the time of maturity or 7 to 10 weeks of life, and in animals mature
at the time of transfer, by 7 to 10 dpt. Thus, younger animals had
longer delays before well-developed signs were expressed as a clinical
event. With increase in recipient age at time of transfer, time to
development of clinical EAE decreased. The longest preclinical periods
were seen in younger animals. Experiments were repeated 4 times. A
representative experiment is presented in Figure 1
. All young female animal groups (1.5 to
4 weeks of age at time of transfer) had long delays before expression
of well-developed or consistent disease (Figure 1, A–C)
. Rarely, in
about 10% of animals in the intermediate young (3- and 4-week-old)
animal groups, there was the brief (1 day) appearance of very mild
disease (clinical score <1) in the 3 to 5 weeks after cell transfer.
However, most young animals up to 5 weeks of age at the time of
transfer showed no clinical signs for many weeks (Figure 1, A–C)
. The
duration of the preclinical phase was greatest in the youngest groups
(Figure 1, A and B)
, and became shorter as the age at time of LNC
transfer increased, as shown in another representative experiment
in Table 1
. Ultimately, all young age
groups manifested significant clinical disease (clinical scores
consistently 
2 to 5), by the time they reached adulthood.
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. Initial
signs began most often with a weak tail and progressed to hind limb,
then forelimb involvement. With increase in age at time of cell
transfer, delay in clinical onset declined such that in most mice,
regardless of age at time of transfer, full expression of clinical
disease occurred between 50 and 70 days of age. Despite the separation
of animals by age at time of transfer, the ages of the animals at the
time of peak clinical disease showed remarkable concordance, as shown
in Table 1
. The severity of the clinical disease reached as high as
grade 5 in some individual animals, although when compared to mean
scores for adult animal groups in the same experiments, mean scores
were most often 1 to 2 clinical points less than those of adult
animals. Maximum clinical disease occasionally varied from experiment
to experiment by about 1 clinical score point for all age groups, but
within a single experiment, the relationship of delayed clinical
disease in young versus adult animals remained constant.
In comparison to the youngest groups (1.5 to 2.5 weeks of age at time
of sensitization), transfer of adult LNC into intermediate-aged females
(5.5 to 7 weeks of age at time of sensitization), resulted in shorter
or no delays in clinical disease onset and a more consistent clinical
disease course (Figure 1, D and E
, and Table 1
), more closely
approximating the clinical course of adult (8- to 10-week-old) animals.
Although these intermediate-aged animals had onset of clinical signs at
7 to 10 days after transfer of LNC, the initial episode of clinical
signs was milder than in the oldest adult age groups and was often
separated in time by a return to normal baseline before onset of later
episodes which were marked by a chronic accumulation of baseline
neurological deficit, as seen in the oldest age groups.
Transfer of LNC into females at 8 to 12 weeks of age reproducibly
resulted in an onset of clinical disease 7 to 10 dpt, as seen in Figure 1F
. Initial signs were most often a weak tail at 7 dpt, followed by
ascending weakness involving the hind limbs and then the forelimbs by
10 dpt. Well-developed (clinical score 2) disease was attained 3 to 5
days after onset. The initial episode was marked by rapid onset and
rise in clinical score and more severe signs as compared with later
episodes. Later episodes had a more gradual onset and scores for the
episodes were often lower with less recovery for the following
remissions. During the first episode, adult animals maintained a high
clinical score for 5 to 7 days and then showed clinical improvement
which continued for 1 to 3 weeks. Of these mice, 90% displayed
residual signs during the recovery period; few recovered completely.
Four to five weeks after the initial signs, adult age groups showed new
clinical relapses with onset over 5 to 10 days. Early relapses were
followed by a period of clinical improvement and later episodes of mild
to moderate clinical worsening. Each period of remission was associated
with a higher level of baseline-accumulated neurological deficit.
Gender Effects Occur across All Ages
When clinical scores of male and female animals in each age group
(2 to 10 weeks at time of transfer) were compared, the clinical course
had a similar remitting-relapsing pattern for both genders. In each of
four experiments with both genders, within each age group, male mice
commonly displayed a greater delay in clinical onset in comparison to
females, though this delay varied in length from experiment to
experiment. Both male and female mice had chronic relapses, but the
relapses in females were more distinct and severe than those of the
males, which had more gradual onset and milder clinical changes. Within
mature age groups, in comparison to females, males showed resistance to
clinical disease with longer delays in onset and lower clinical scores,
as previously described.30,31
The same differences were
also seen in young age groups, when 3 x 107
LNC from adult female mice were transferred into 2- and 4-week-old male
and female recipients, as shown in two (of four) experiments in Table 2
. Although males were resistant to
clinical disease, once severe disease occurred, in comparison to female
animals, they retained substantial clinical disease with more rapid
accumulation of chronic neurological deficits in older age groups. The
clinical scores in male mice, as in females, could be increased in a
dose-related manner in each age group by increasing the number of
transferred female LNC into naïve male mice (Figure 2A)
. Interestingly, although male
recipient mice were resistant to clinical EAE, male donor LNC
transferred clinical EAE with a typical 7- to 8-day onset after
injection into susceptible 10-week-old female recipients (Figure 2A)
.
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To investigate whether T cell specificity was necessary or whether merely relatively large numbers of activated T-cells could induce EAE in the youngest age groups, 3 x 107 adult or 6 x 107 OVA-sensitized LNC were transferred into recipients. OVA-sensitized LNC did not produce clinical EAE in animals of any age followed up to 100 dpt; nor did they produce CNS lesions at times typical for acute (7 dpt) or chronic (62 dpt) EAE in animals of any age.
To evaluate whether T cells from the youngest animals could participate
in or prevent induction of EAE, 10-week-old females were injected with
3 x 107
young MBP-specific LNC produced by
immunizing 9-day-old female animals, followed by culture of the LNC as
for cells from adult animals. The transfer of LNC from the youngest
group of mice produced a clinical disease similar to that seen after
3 x 107
adult MBP LNC were injected into
10-week-old females at the same time (data not shown). Mixing 10-week-
and 2-week-old MBP-specific LNC 1:1 and injecting 3 x
107
LNC, had no effect on clinical outcome,
whereas 6 x 107
cells of the 1:1 mixture
gave higher clinical scores in 10-week-old adult female animals (Figure 2B)
. No inhibition of clinical disease in adult animals was observed
after injection of young LNC.
Discordant Pathological and Clinical Signs in Immature but Not Adult Mice
The CNS of animals from all groups were examined 7, 42, and 52 dpt
for histopathological changes. Surprisingly, younger animal groups (2
and 3 weeks of age at transfer) showed well-developed lesions typical
of acute EAE despite lack of clinical disease and delay in clinical
signs (Table 3)
. CNS tissue from 2- and
3-week-old mice sampled for pathology at 7 dpt displayed extensive
lesion activity. This comprised large areas of white matter from all
levels of the neuraxis, including cerebellum (Figure 3, A and B)
, cervical spinal cord (Figure 3, C and D)
, and lower lumbar cord (Figure 3, E and F)
, that contained
perivascular, meningeal, and parenchymal collections of small
lymphocytes, large mononuclear cells, polymorphonuclear leukocytes
(PMNs), macrophages containing myelin debris, and primary
demyelination. In contrast to the usual picture of adoptively
transferred EAE in older age groups, vide
infra,19,20
CNS lesions in 2- and 3-week-old animals
sensitized for EAE were diffuse, poorly circumscribed, and not
particularly limited to perivascular areas. Some of the diffuse nature
of the pathology was related to changes occurring in CNS tissue that
was still undergoing myelination, so that myelin sheath thickness
varied from negligible to mature dimensions, giving the tissue a more
edematous texture. By light microscopy, large numbers of mononuclear
cells with densely staining nuclei were readily observed in the CNS of
lesions from animals sensitized at 2 and 3 weeks of age and sampled 7
days later (Figure 3, C and D)
. These cells with dense nuclei were
suggestive of apoptosis, were more common in young sensitized animals,
and were frequently seen to have been internalized by other cells,
commonly astrocytes (Figure 3G)
. The presence of infiltrating cells
undergoing apoptosis was confirmed by EM and immunocytochemistry
(vide infra).
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. CNS lesions in these animals were
similar in distribution to those occurring in animals sensitized at a
later age and displayed a wide gamut of activity from recent
inflammation and demyelination (Figure 4A)
to long-term demyelination and
gliosis (Figure 4B)
and chronic inflammation and remyelination (Figure 4, C and D)
.
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6 weeks of age was identical
to that detailed in previous works on this model,18-20
with acute lesions occurring throughout the CNS white matter being
centered on perivascular (Figure 4, E and F)
and leptomeningeal
infiltrates, correlating with clinical signs that developed between 6
and 8 dpt. In older-sensitized animals sampled during the chronic
relapsing phase of the disease (eg, 52 dpt), lesions indicative of
long-standing disease containing chronically demyelinated axons,
fibrous astrogliosis, and remyelination were common, as described
previously.18
Also evident were recently active lesions
displaying recent inflammation, polymorphonuclear cells (PMNs),
demyelination, and macrophage activity occurring against an edematous
parenchyma containing little evidence of glial scarring (Figure 4, G and H)
. TUNEL Staining and EM Show Prominent Apoptosis in Immature Groups
Staining by TdT labeling for DNA fragmentation in fixed sections
of spinal cord sampled from animals at 7 dpt that had been sensitized
at 2, 4, 8, 10, and 12 weeks of age revealed an appreciable number of
TUNEL+ infiltrating cells in all animal groups. The number of TUNEL+
cells was greatest in the 2- and 4-week-old groups and lowest in the
12-week group (Table 5)
. A single normal
animal sampled at 2 weeks of age from which 30 sections of spinal cord
were examined revealed a total of 25 TUNEL+ cells (cell density of 1.28
per cubic millimeter). This figure compares with the highest value of
1125 cells in the same number of sections from a 4-week-old
recipient and a low value of 75 in the 12-week-old group. In heavily
infiltrated spinal cord tissue from the 2-week group, TUNEL+
infiltrating cells were seen scattered among cells in perivascular
cuffs (Figure 5, A and B)
, and throughout
the parenchyma (Figure 5C)
. These apoptotic TUNEL+ cells were almost
exclusively small lymphocytes. Glial cells, particularly
oligodendroglial cells, were not affected. Indeed, chains of
interfascicular oligodendrocytes could be readily distinguished by
nuclear size from infiltrating cells and were never seen to contain
apoptotic nuclei (eg, Figure 5B
). Apoptotic nuclear profiles were most
common, as stated above, in the younger animal groups sensitized for
EAE and sampled at 7 dpt.
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. On
the basis of cell shape, cytoplasmic
content, and glycogen granules, the investing cells were identifiable
as resident astroglia, as opposed to hematogenous macrophages or
microglia. Indeed, microglial cells were not a prominent feature of
lesions in the younger groups. The invested cells were usually small
lymphocytes, although on occasion, cells with features of PMNs were
also encountered undergoing apoptosis. Such appearances confirmed those
encountered by light microscopy (see Figure 3, B, D, and G
).
Oligodendrocytes were not seen to undergo apoptosis and sometimes lay
adjacent to astrocytes containing lymphocytes undergoing apoptosis,
apparently structurally normal, even to the extent of sharing
membrane specializations (gap junctions) with the astrocyte (Figure 6, B and C)
.
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MHC Class II expression and antigen presentation by APC of CNS origin were of interest in this model, because we reasoned that age-related differences might be associated with the observed age-related resistance to clinical EAE in young mice. CNS-derived APC from 10- and 12-week-old animals, however, were markedly less viable in culture (without the addition of growth factors) than those from younger animals, making direct comparisons of CNS APC difficult. Cultures of spleen-derived APC from animals of different ages had similar viability and cellular make-up by FACS analysis at 72 hours, with similar proportions of CD4, CD8, and Mac-1-positive cells and were therefore used as an approach to study age-related differences in APC function and MHC expression and to give insight into possible age-related events in the CNS.
The cellular components, both infiltrating and resident, of CNS lesions
from all age groups appeared similar despite the lack of clinical EAE
in the younger age groups. To evaluate whether MHC Class II expression
on APC might vary with age and correlate with clinical disease
expression, APC from normal animals at different ages were studied
using FACS analysis of splenocyte APC cultures in 6 separate
experiments. Constitutive MHC Class II expression by APC, as measured
by percentage of positive cells or mean fluorescence intensity at 6
through 72 hours in culture (Figure 7)
, was found to increase with age.
In addition, there was significantly less expression of Class II MHC by
APC from 2-week-old animals compared with APC from 4-, 6- and
12-week-old groups. Constitutive expression by 2-week APC decreased
over time in culture, in contrast to the older groups, which showed
increasing constitutive Class II MHC. The increase in constitutive MHC
Class II over time with increasing age paralleled the increased
susceptibility to clinical EAE with age seen in this model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to MS, most models of EAE exhibit a predictable rapid onset of clinical signs associated with CNS lesions, usually within 7 to 10 days after transfer of CNS antigen-sensitized cells. Although previous studies on actively sensitized juvenile guinea pigs also developed subclinical disease, interpretation of the studies was complicated by the ongoing presence of injected antigenic emulsion.16,17,34 The early subclinical phase, which may represent an early or age-related environmentally induced predisposition as well as an inherent genetic predisposition, was achieved here using juvenile mice from an EAE-susceptible strain that were sensitized for adoptive EAE at different ages. Our findings have shown that transfer of encephalitogenic cells into younger animals was typified by a latent period that was induced in animals up to 5 weeks of age and that preceded the expression of clinical signs and maximum clinical disease, the latter occurring during early adulthood. Prominent acute CNS lesions were present in all animals, including the animals that were youngest at time of transfer, despite the absence of overt clinical disease in young animals, whereas increased female clinical susceptibility was maintained at all ages. Regardless of the age at time of transfer and clinical disease expression, pathology was seen by 7 dpt in all age groups. This lack of correlation between clinical and pathological signs bespeaks a role for age-related compensatory mechanisms for CNS dysfunction resulting from autoimmune attack and is reminiscent of situations occasionally encountered in MS.
Although mice of all ages developed CNS pathology, some differences
between young and adult animals were noted. These comprised the
presence of higher numbers of apoptotic infiltrating cells, large
reactive astrocytes (some investing multiple apoptotic lymphocytes),
and normal myelination in the presence of ongoing demyelination, all
evident in the younger but not the older groups of mice. The higher
numbers of apoptotic cells were invariably associated with milder
clinical disease in younger (2 to 4 weeks of age at time of transfer)
animals. Whether or not widespread apoptosis of infiltrating effector
cells was functionally linked to effective clearance of activated T
cells from affected areas in the murine model has not been proven, but
supportive evidence for this exists from other
studies.28,35-37
Studies in the rat model both
support38-42
and refute41
this mechanism,
though the use of the immunosuppressant cyclosporin-A in some
studies43
may complicate interpretation of some
observations, especially in the absence of correlation with
pathological EAE. In other experimental settings, defective Fas- and
Fas ligand-associated decreases in target cell apoptosis have been
linked to improved clinical course in gld and lpr
mice.44,45
Although the precise role of apoptosis of
infiltrating cells in the present murine model remains to be defined,
the abundance of apoptotic cells would support the presence of an
intact mechanism for its induction. Developmental studies of cytokine
gene expression in normal mouse CNS suggest the cytokine milieu may be
supportive of apoptosis before 6 weeks of age and may change with
maturation to a more resistant environment. Our preliminary work on CNS
tissue from 2- to 12-week-old normal animals suggests that interleukin
(IL)-4 is present during the first 6 weeks of development and absent in
older animals. IL-2 and interferon (IFN)-
are absent during the
early weeks of life while IFN- is present for a short period in
adult animals46,47
(Smith ME, Scott DE, Ellen NE,
McFarland HF, manuscript in preparation). The presence of IL-4
was noted in both normal SJL/J and SCID mouse strains during
development without any experimental manipulation. This suggests a
brain-derived rather than an infiltrating lymphocyte-derived origin for
the cytokine message. IL-4 associated with an absence of IL-2 and
IFN- in development has been previously described to be associated
with delayed immune responses in neonatal lymphoid murine cells as
well, but in lymphocytes this pattern changes after 5 days of
age.48
IL-4 has also been shown to both activate and
deactivate macrophages and APCs, depending on the environment, and can
enhance IFN--induced (possibly of LNC origin) tumor necrosis factor
(TNF)-
production,49,50
which may be influential in the
CNS and play a role in the age-related apoptosis and resistance to
clinical expression of EAE shown here. The large number of cells not
undergoing apoptosis in the EAE lesions, however, suggested that
apoptosis formed but one component of the multifactorial resistance
mechanism that an animal might mount against autoimmune CNS disease
expression. In addition, chronic changes (42–69 dpt) in animals
originally sensitized with transferred cells at early ages, when
prominent acute CNS lesions were seen, consisted of fewer chronic
lesions, thus providing supportive evidence that the developing CNS
environment is more permissive to recovery from autoimmune inflammatory
demyelination.
These results suggest that the maturation process within the CNS itself may hold part of the key to understanding susceptibility to clinical disease expression in MS. In the EAE model, it is conceivable that as the animal matures, a CNS environment less resistant to the consequences of autoimmunity and more conducive to the rapid expression of clinical EAE disease may develop. The immature CNS is an environment conducive to ongoing myelination that functions normally in both young animals and humans with incomplete myelin Together, perhaps, with the normal developmental changes in axon and myelin sheath integrity, eg, phenomena like reorganization of axonal sodium channels,51,52 these features may contribute to the observed resistance to clinical signs. Interestingly, myelination in the CNS tapers off at approximately 6 weeks in mice and in the third decade in humans, and these time points correspond to the periods of increased susceptibility to clinically significant autoimmune demyelination.
Although sexual maturation was suspected to be related to increased EAE
expression, in the present model this topic could not be readily
approached because both females and males showed resistance to EAE both
before and after sexual maturation. Despite this, it may be
hypothesized that growth hormone as well as sex hormone levels during
development provide an age-related natural protection from clinically
relevant demyelination and TNF- toxicity,53,54
which
has been shown to be associated with clinical events during CNS
demyelination.55,56
In the present model, transfer of
encephalitogenic cells early in life ultimately produced clinical EAE,
albeit in a delayed fashion, suggesting that a change in susceptibility
to clinical EAE occurs during development as a process separate from
predisposing events, such that two steps, immune predisposition and
development of clinical susceptibility, are needed for complete
manifestation of EAE.
The present work also explored selected immune parameters, focusing on either APC or T cell function and whether one facet of the immune process might correlate more closely with young animals' resistant periods despite their genetic predisposition to EAE. The autoimmune basis of CNS lesions, including those in the youngest mice, was evidenced by lack of either clinical or pathological disease produced despite transferring large numbers of OVA-specific LNC and the requirement for CNS antigen-specific LNC. However, in comparison to the mature CNS, there was a disproportionately large number of lymphocytes within the immature CNS during EAE, and this may be related in part to the large number of MBP-specific cells transferred into very small animals. Of note, transfer of CNS antigen-specific LNC from either young or mature animals into susceptible 10-week-old mice consistently produced disease, showing that there was relatively little correlation between age-related delays in clinical EAE with age of LNC and young donor mice for the LNC. There was a similar ability to transfer disease with LNC from adult and young (2-week-old) mice, suggesting more mature T cell function with respect to CNS antigen-based autoimmunity, even at younger ages, and fewer grounds to implicate age-related differences in EAE expression. Moreover, the ability to transfer disease with LNC stimulated by whole-molecule MBP provides evidence of comparable antigen processing between the different age groups.
Age-related differences in APC, large scale effector cell apoptosis, and the close relationship of astrocytes with apoptotic cells in the immature CNS environment where ongoing myelination and resistance to chronic lesion accumulation were seen, seemed to correlate more closely with age-associated delays in expression of clinical EAE. MHC Class II expression in vitro by APC was most decreased in 2-week-old animals, and a trend of increased constitutive expression with increasing age was noted. An attempt was made to confirm this finding in vivo by immunocytochemistry on the CNS of very young animals, but the results were difficult to interpret because the CNS tissue was extremely delicate and did not withstand processing. Functionally, APC from the youngest animals presented antigen less well to MBP-specific LNC and baseline proliferation was decreased as compared to 10-week-old animals. The ages at which MHC Class II expression and antigen presentation approximated that of adult animals paralleled the increase in clinical expression of EAE as animals matured. This suggested that a similar situation might also occur in the CNS, where both infiltrating APC and resident CNS APC may contribute to the transient protection from clinical EAE seen in the younger animals. However, successful direct study of specific interactions of the APC from young animals was somewhat limited. In addition, attempts to study the effects of young APC by maintaining MBP-specific LNC beyond the usual culture period after removal from donor animals using 2-week-old APC were unsuccessful in producing longer term survival of the LNC.
Low levels of Class II MHC expression by peripheral APC from young mice and poor function of APC during development observed here and elsewhere57 may reflect a similar situation in the CNS of young mice. Class II expression by keratinocytes has been found to parallel similar expression on CNS astrocytes in vitro,58 and thus, poor function or decreased MHC expression by peripheral APC in these young age groups may correlate with immature function of astrocytes, microglia, or endothelial cells. The latter CNS cell types are known to possess the ability to express a number of immune system molecules in vivo and in vitro59-65 and to interact with lymphocytes in the adult animal.66-69 Astrocytes appeared actively involved in immunomodulation in CNS lesions, especially when cells undergoing apoptosis were in high concentration. Moreover, astrocytes in the youngest animal groups appeared most active in this role compared to the older groups. Oligodendroglia and microglia appeared to be less involved in these effector cell interactions.
The present developmental model of immune-mediated CNS demyelination affords yet another approach for the study of a clinically significant CNS demyelinating syndrome with adult onset, despite the induction of risk conferred by the transfer of CNS antigen-sensitized LNC during the process of maturation. These observations may have relevance to MS, for which a large number of immunosuppressant treatments is used in its management and its pathological expression and which is believed to have an immune-mediated pathogenesis.70,71 In MS, it is surmised that onset of immune dysfunction probably precedes clinical expression. Understanding the former event may uncover important predictive information of relevance to the therapeutic modulation of this perplexing human disease.
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Acknowledgements |
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Footnotes |
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Supported in part by U. S. Department of Health and Human Services grants NS 08952, NS 11920, and NS 07098, by National Multiple Sclerosis grant RG 1001-I-9 to C. S. R., by the Sol Goldman Charitable Trust, and by the Wollowick Family Foundation (to C. S. R.). M. K. R. is a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society and the Young Investigator in Multiple Sclerosis of the American Academy of Neurology Education and Research Foundation.
Accepted for publication June 14, 1999.
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