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Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50–60 days
postinfection, appear in an ordered progression associated with their
relative encephalitogenic dominance, and also persist. Consistent with
disease initiation by virus-specific CD4+ T cells, CNS
mononuclear cells from TMEV-infected SJL mice endogenously process and
present virus epitopes throughout the disease course, while myelin
epitopes are presented only after initiation of myelin damage (>50–60
days postinfection). Activated F4/80+ APCs expressing high
levels of MHC class II and B7 costimulatory molecules and ingested
myelin debris chronically accumulate in the CNS. These results suggest
a process of autoimmune induction in which virus-specific T
cell-mediated bystander myelin destruction leads to the recruitment and
activation of infiltrating and CNS-resident APCs that process and
present endogenous myelin epitopes to autoreactive T cells in a
hierarchical order. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A primary example of the complexity of human autoimmune disease is MS, a disease characterized by CD4+ T cell-mediated demyelination of the CNS and autoimmune responses to myelin proteins such as MBP, PLP, and myelin oligodendrocyte glycoprotein (MOG) (1, 2, 3, 4). Epidemiological evidence strongly supports the hypothesis that human MS is initiated by a viral infection (5), although no one virus has been consistently found in MS lesions. Therefore, exploring the mechanisms by which infectious agents trigger autoimmune diseases is of great interest and importance. Three mechanisms have been suggested to explain how a viral infection could lead to autoimmunity: molecular mimicry between the pathogen and self Ags, which leads to direct activation of T cells that are cross-reactive with self epitopes (6); viral superantigens that nonspecifically lead to activation of autoreactive T cells (7); and epitope spreading evoked by virus-specific T cells that result in bystander damage to self tissue with consequent autoantigen release (8) or direct virus-induced release of self Ags (9), resulting in de novo activation of autoreactive T cells. We have demonstrated that epitope spreading plays a prominent pathologic role in the progression of two clinically distinct T cell-mediated demyelinating diseases that resemble the two major clinical presentations of MS, relapsing experimental autoimmune encephalomyelitis (R-EAE) and chronic-progressive Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) (10, 11, 12, 13, 14).
TMEV-IDD is the most relevant of the available virus-induced animal models of immune-mediated demyelination (10) and serves as an excellent system in which to assess the potential contribution of anti-myelin autoimmune responses to initiation and progression of clinical demyelination. TMEV, a picornavirus and natural mouse pathogen, induces a life-long persistent infection of CNS-resident APCs (15, 16, 17) and results in a chronic immune-mediated CNS demyelinating disease when inoculated intracerebrally into susceptible strains of mice. Infected SJL mice develop progressive symptoms of gait disturbance, spastic hind limb paralysis, and urinary incontinence (18), histologically related to perivascular and parenchymal mononuclear cell infiltration and demyelination of white matter tracts within the spinal cord (19, 20, 21). In the highly susceptible SJL mouse strain, we have recently shown that the myelin damage is initiated by TMEV-specific CD4+ T cells targeting persistent virus Ag in the CNS (22, 23, 24, 25, 26), while the chronic stage of the disease also involves the activity of CD4+ myelin epitope-specific T cells primed via epitope spreading (8). In addition to the role of T cells in TMEV-IDD, we have recently demonstrated for the first time that the CNS-resident APCs from mice with severe TMEV-IDD endogenously present self epitopes to myelin-specific Th1 lines in a B7-dependent, MHC class II-restricted manner (14). Moreover, CNS APCs isolated from mice at disease initiation or microglia from naive brain are unable to endogenously present self myelin epitopes. These results suggest that epitope spreading results from a process in which localized bystander myelin destruction, caused by virus-induced inflammatory immune reactivity, results in the processing and presentation of endogenous self myelin epitopes by CNS APCs. Thus, presentation of myelin epitopes by CNS APCs, either locally and/or in peripheral lymphoid organs, leads to the de novo activation of autoreactive T cells.
In this study we present a comprehensive characterization of the temporal course of epitope spreading in TMEV-infected mice by following the development of peripheral T cell responses (delayed-type hypersensitivity (DTH) and T cell proliferation) specific for a panel of virus and myelin epitopes and the endogenous presentation capabilities of APCs in the target organ (i.e., the spinal cord). The results support the hypothesis that the initial anti-viral response in TMEV-IDD results in the influx and activation of mononuclear cells leading to the gradual accumulation of degraded myelin Ags within and on CNS APCs. This inflammatory environment results in the ability of CNS APCs to efficiently present endogenous self myelin epitopes in a B7-dependent manner and to activate and maintain myelin-specific autoreactive T cells. Interestingly, multiple myelin epitopes are displayed on CNS APCs well before specific T cells can be demonstrated in the peripheral immune system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL/J mice, 6–7 wk old, were purchased from Harlan Laboratories (Indianapolis, IN). All mice were housed in the Northwestern animal care facility and maintained on standard laboratory chow and water ad libitum. Severely paralyzed mice were afforded easier access to food and water.
Peptides
Peptides used in this study were synthesized using a Synergy Peptide Synthesizer (ABI, Columbia, MD). The sequences were as follows: VP270–86 (WTTSQEAFSHIRIPLPH), VP324–37 (PIYGKTISTPSDY), PLP56–70, (DYEYLINVIHAFQYV), PLP104–117 (KTTICGKGLSATVT), PLP139–151 (HSLGKALGHPDKF), and PLP178–191 (NTWTTCQSIAFAPSK). The amino acid composition and purity (>97%) of these peptides was confirmed by mass spectroscopy at the University of North Carolina-Chapel Hill Biotechnology Center.
Virus
The BeAn 8386 strain of TMEV is a tissue culture-adapted strain of TMEV that has been plaque purified and passaged in BHK-21 cells grown in DMEM (27). Working stocks of virus were purified by polyethylene glycol precipitation of total BHK-21 cell lysates, sonication in the presence of SDS, and centrifugation over successive sucrose and CsSO4 gradients.
Induction and clinical evaluation of TMEV-IDD
Mice were anesthetized with methoxyflurane (Burns Veterinary Supply, Farmers Branch, TX) and inoculated in the right cerebral hemisphere with 9 x 107 PFU of virus in 30 µl of DMEM. Mice were examined two or three times per week for the development of chronic gait abnormalities and spastic paralysis indicative of demyelination (28) and were assigned a clinical score of 0–6 as follows: 0 = asymptomatic; 1 = mild waddling gait; 2 = severe waddling gait, intact righting reflex; 3 = severe waddling gait, spastic hind limb paralysis, impaired righting reflex; 4 = severe waddling gait, spastic hind limb paralysis, impaired righting reflex, mild dehydration and/or malnutrition; 5 = total hind limb paralysis, severe dehydration and/or malnutrition; and 6 = death. The data are plotted as the mean clinical score for one group of infected animals that displayed representative clinical signs.
DTH responses
DTH responses to murine and myelin peptides were quantitated using a 24-h ear swelling assay at varying times postinfection (PI). Prechallenge ear thickness was determined using a Mitutoyo model 7326 engineer’s micrometer (Schlesinger’s Tools, Brooklyn, NY). DTH responses were elicited by injecting 10 µg of peptide (in 10 µl of saline) into the dorsal surface of the ear using a 100 µl of Hamilton syringe (Reno, NV) fitted with a 30-gauge needle. Twenty-four hours after ear challenge, the increase in ear thickness over prechallenge measurements was determined. Results are expressed in units of 10-4 inches ± SEM.
T cell proliferation and IFN-
assays
At varying times PI, spleens of infected animals were removed
and teased into single-cell suspension over wire mesh in HBSS. RBC were
lysed by treatment with Tris-NH4Cl solution for 5
min at 37°C. Cells were washed with HBSS and resuspended in HL-1
medium (BioWhittaker, Walkersville, MD) supplemented with 1%
L-glutamine and 1% penicillin/streptomycin (Life
Technologies, Grand Island, NY). Bulk splenocytes were plated in
flat-bottom, 96-well plates (Costar, Corning, NY) at
106 cells/well and stimulated with viral or
myelin peptides in a range of concentrations (1–200 µM). Cell
cultures were pulsed with 1 µCi of [3H]TdR
(ICN, Costa Mesa, CA) after 72 h and harvested 18–20 h thereafter
on 96-well filter plates. [3H]TdR incorporation
was measured on a TopCount-NXT (Packard, Meriden, CT), and results are
expressed as the mean of triplicate cultures ± SEM (background
counts subtracted). Supernatants collected at 24 and 48 h from
replicate cultures were assayed for IFN- using Endogen Minikits
(Woburn, MA) following the manufacturer’s protocol.
Isolation of CNS-resident mononuclear cells
Mice were anesthetized with methoxyflurane and perfused through the left ventricle with cold PBS until the effluent ran clear. Spinal cords were extruded by flushing the vertebral canal with cold PBS and were rinsed in PBS. The spinal cords were forced through 60-mesh stainless steel screens to give a single-cell suspension, in a balanced salt solution containing type 4 Clostridial collagenase (Worthington, Freehold, NJ; 300 U/ml/cord) and incubated for 75 min (37°C). The spinal cord homogenate was resuspended in 30% Percoll (Pharmacia, Piscataway, NJ), divided into tubes (equivalent to five spinal cords/7 ml/tube), and underlaid with 70% Percoll (5 ml/tube). The gradients were centrifuged at 500 x g at 24°C for 20 min. CNS mononuclear cells were collected from the 30:70% interface, washed, and resuspended in RPMI 1640 and 10% FBS. Enrichment of the macrophage/microglia population was accomplished by allowing the cells to adhere to 100-cm2 plastic tissue culture dishes (75 min at 37°C in a humidified, CO2 incubator). Thereafter, the nonadherent cells were removed, and the dish was washed gently with RPMI 1640 and 10% FBS. Cold RPMI was then added to the dish; it was placed on ice for 10 min, and the adherent cells were removed by scraping. The cells were centrifuged, and cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin (DMEM-10, all from Sigma, St. Louis, MO).
Isolation of splenic APCs
Spleens were removed from naive mice, placed in balanced salt solution, and forced through 60-mesh stainless steel screens to yield a single-cell suspension. Erythrocytes in the spleen cell preparations were lysed by hypotonic shock in Tris-NH4Cl for 5 min at 37°C; thereafter, isotonic buffered saline was added, and the cells were washed and resuspended in DMEM-10.
Maintenance of T cell lines
Long term Th1 lines specific for four encephalitogenic myelin peptides (PLP56–70, PLP104–117, PLP139–151, and PLP178–191) were established from the lymph nodes of SJL/J mice primed 10 days earlier with 100 µg of the respective peptide emulsified in IFA supplemented with 200 µg of Mycobacterium tuberculosis H37Ra. Every 3–4 wk, live T cells were isolated on Ficoll/Histopaque (Pharmacia) by centrifugation at 500 x g at 24°C for 15 min and propagated by in vitro stimulation of 106 T cells with 5 x 106 irradiated syngeneic splenic cells with 25 µM of the respective peptide for 72 h. All stimulation assays were performed in DMEM (Sigma) supplemented with 10% FBS (Sigma), 2 x 10-3 M L-glutamine (Life Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), 5 x 10-5 M 2-ME, and 0.1 mM nonessential amino acids (Sigma). Between expansions, T cell lines were maintained in DMEM supplemented with 10% FBS, 2 x 10-3 M L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10-5 M 2-ME, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (Life Technologies), MEM essential vitamins (Life Technologies), 0.1 mM asparagine (Life Technologies), 0.1 mg/ml folic acid (Life Technologies), 0.8% T-Stim (Collaborative Biomedical Research, Bedford, MA), and rIL-2 (0.2 U/ml; Roche, Indianapolis, IN). All Ag presentation assays using T cell lines were conducted 14–20 days poststimulation.
Maintenance of T cell hybridomas
For generation of T cell hybridomas from virus-immunized mice, a single-cell suspension of the lymph nodes from SJL/J mice immunized twice with 50 µg of purified UV-inactivated TMEV (5 µg/ml) was used. Resulting hybridomas were selected in hypoxanthine-aminopterin-thymidine medium, as previously described (29). Virus-specific hybridomas were subcloned by limiting dilution to ensure clonality.
Ag presentation assays
The plastic-adherent fraction of isolated CNS-infiltrating mononuclear cells was assayed for the ability to stimulate the virus- and myelin epitope-specific T cell lines compared with naive SJL splenocytes. Irradiated (3000 rad) CNS adherent cells (2.5–3.5 x 104/well) isolated from spinal cords of mice with TMEV-IDD or irradiated SJL splenocytes (2.5–3.5 x 104/well) were cultured with 2–4 x 104 T cells from the different lines in the presence or the absence of 10 µM of the appropriate peptide. In all experiments triplicate cultures at each condition were conducted in flat-bottom 96-well microtiter plates in DMEM-10 supplemented with aminoguanidine (1 mM) to suppress NO synthetase activity. Proliferative responses were determined by [3H]TdR (0.1 µCi/well) incorporation during the final 16–24 h of the 48- to 72-h culture period. Cultures were harvested on 96-well filter plates (Packard, Downers Grove, IL) for liquid scintillation counting, and the results were expressed as cpm ± SEM.
Preparation, storage, and sectioning of tissues
Mice were anesthetized and perfused with 1x PBS. Spinal cords were removed by dissection, and 2- to 3-mm spinal cord blocks were immediately frozen in OCT (Miles Laboratories, Elkhart, IN) in liquid nitrogen. The blocks were stored at -80°C in plastic bags to prevent dehydration. Five- to 6-µm-thick sections from the lumbar region (approximately L2–L3) were cut on a Reichert-Jung Cryocut 1800 cryotome (Leica Instruments, Deerfield, IL), mounted on SuperFrost Plus electrostatically charged slides (Fisher, Fairlawn, NJ), air-dried, and stored at -80°C.
Immunohistochemistry
Slides were stained using the Tyramide Signal Amplification
Direct Kit (NEN Life Science Products, Boston, MA) according to the
manufacturer’s instructions. Lumbar sections from each group were
thawed, air-dried, fixed in acetone at room temperature, and rehydrated
in 1x PBS. Endogenous peroxide activity was inhibited with 3%
H2O2. Nonspecific staining
was blocked using anti-CD16/CD32 (Fc III/II receptor, 2.4G2;
PharMingen, San Diego, CA) and an avidin/biotin blocking kit
(Vector, Burlingame, CA) in addition to the blocking reagent provided
by the Tyramide Signal Amplification kit. Slides were stained with the
following biotin-conjugated Abs: anti-macrophage (F4/80; Caltag,
South San Francisco, CA); anti-CD4 (H129.19), anti-CD8a
(53-6.7), anti-CD45R/B220 (RA3-6B2),
anti-I-Ak,q,r,s (10-3.6), anti-B7-1
(16-10A1), and anti-B7-2 (GL1; PharMingen). Nile Red (Molecular
Probes, Eugene, OR) was used to localize lysosomal lipid droplets
within cells. Sections were counterstained with
4',6'-diamidino-2-phenylindole (Sigma) or propidium iodide (Molecular
Probes) and then coverslipped with Vectashield mounting medium
(Vector). Slides were examined by epifluorescence using a chroma
triple-band filter (Chroma Technology, Brattleboro, VT). Four serial
lumbar sections from each sample per group were analyzed at x100 and
x400 magnification throughout the entire spinal cord section, which
included gray and white matter and dorsal, ventral, and lateral
regions.
Cytokine PCR
Total RNA was isolated from 106 CNS-infiltrating plastic adherent cells on days 48 and 88 PI using the Qiagen RNeasy Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s protocol. First-strand cDNA was generated from 2 µg of total RNA with the Advantage-RT Kit (Clontech, Palo Alto, CA) using 20 pmol of oligo(dT) primer according to the manufacturer’s provided protocol in a total volume of 20 µl. Following first-strand synthesis each cDNA sample was brought to a final volume of 100 µl with distilled water. Final cytokine PCR conditions included 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 2.5 mM MgCl2, 2 mM dNTPs, 100 pmol of each 5' and 3' gene-specific primers, 1 U of Taq polymerase (Qiagen), and 5–10 µl of diluted cDNA. Cycling conditions for the GeneAmp 9600 (Perkin-Elmer, Norwalk, CT) were 94°C for 40 s, 60°C for 20 s, and 72°C for 40 s for a total of 30 cycles, linked to a final 72°C extension program for 3 min and then to a final 4°C soak program. PCR products were run on an ethidium bromide-containing 2% agarose gel and illuminated using a UV light source, then photographed using Polaroid type 667 film (Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The temporal appearance of T cell responses to viral and myelin
epitopes was determined by measuring T cell responses to a panel of
virus and myelin epitopes in TMEV-infected SJL mice displaying a
typical chronic-progressive disease course with disease onset occurring
between 25 and 30 days PI (Fig. 1
A). Fig. 1B shows
that as early as 6 days PI, DTH responses to the immunodominant viral
epitope, VP270–86, were demonstrable, followed
shortly thereafter by DTH reactivity to
VP324–37. However, infected mice displayed no
DTH responses at these early time points to any of six different
encephalitogenic myelin epitopes contained within the MBP, PLP, or MOG
proteins (Fig. 1, C and D). Anti-myelin DTH
responses in the TMEV-infected mice were first observed 2 mo PI,
about 3–4 wk after the onset of clinical disease. The first observed
anti-self DTH response was directed toward the immunodominant
epitope PLP139–151. Approximately 1 mo later, T
cell reactivity specific to PLP56–70 and to
MOG92–106 could also be found. It took 165 days
for the infected mice to develop T cell responses specific to
PLP178–191 and as long as 230 days to react
significantly to MBP84–104. The temporal
appearance of these DTH responses was consistent in terms of time and
order over many different experiments. In addition, DTH responses
toward VP270–86 and
PLP139–151 in TMEV-infected mice persisted >2
years PI (data not shown).
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production as assays for T cell responsiveness. Splenic T cell
proliferative responses to VP270–86 and
VP324–37 were readily demonstrable before the
onset of clinical disease, i.e., within 14 days PI and were still
evident at 90 days PI (Fig. 2A). In contrast, T cell
proliferative responses to the immunodominant myelin
PLP139–151 epitope were first observed on day 54
PI and increased through day 90 PI, but were not demonstrable before
clinical disease onset (Fig. 2A). Similarly, IFN-
production was seen in response to stimulation with
VP270–86 at both preclinical (25 days PI) and
postclinical (day 54 PI) time points, but cytokine production in
response to PLP139–151 was only observed 3 wk
after the onset of clinical disease on day 54 PI (Fig. 2B)
and at later points PI (data not shown). Interestingly, IFN-
production on day 54 PI was also seen in response to stimulation with
PLP178–191, while significant DTH responses to
this peptide were evident only much later in the disease course (Fig. 1
D). This is consistent with our recent findings showing
that measurement of T cell precursor frequency using IFN-
production is much more sensitive than using
[3H]thymidine incorporation or peripheral DTH
(30).
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We have recently reported (14) that CNS-derived APCs
purified from mice with ongoing TMEV-IDD are able to present TMEV and
endogenously acquired myelin epitopes ex vivo, as evidenced by their
ability to stimulate peptide-specific T cell lines/hybridomas to
proliferate and/or secrete IL-2. These studies showed that TMEV
epitopes were presented by CNS APCs both early (days 35–40) and late
(days 85–100) in the demyelinating process, while several
encephalitogenic epitopes on PLP were presented only at the late stage
after significant myelin destruction had occurred. To directly test the
possibility of temporal spreading of Ag presentation in the CNS and to
attempt to correlate it with the temporal appearance of myelin
epitope-specific T cells (Fig. 1), we tested the Ag-presenting capacity
of CNS APCs isolated from TMEV-infected mice at multiple time points
PI: disease onset (days 35–50), acute disease (days 60–80), early
chronic disease (days 85–100), and late chronic disease (days
130–150).
CNS-derived, plastic adherent mononuclear cells were isolated from the
spinal cords of TMEV-infected mice, irradiated, and tested for their
ability to activate a panel of T cell lines/hybridomas specific to
different myelin and viral determinants both with and without the
addition of exogenous Ag. Fig. 3
demonstrates the ability of these CNS APCs to endogenously present the
immunodominant viral epitope, VP270–86, and the
immunodominant myelin epitope, PLP139–151.
Using rather limited numbers ofCNS-derived APCs (2.5–3.5 x
104/well), it is evident that the
VP270–86 epitope is endogenously presented on
CNS-resident APCs as early as 35 days PI (the first time point when
sufficient cells can be isolated), and this ability persists through
day 139 PI, a profound demonstration of CNS virus persistence. We have
previously reported that activation of virus-specific T cells is
specific, in that CNS APCs from TMEV-infected mice failed to activate
an SJL horse myoglobin-specific T cell line (31) and CNS
APCs from mice in remission from
PLP139–151-induced failed to activate
TMEV-specific T cells (14). In contrast, endogenous
presentation of PLP139–151 was not evident on
days 25 and 67 PI, but developed by day 98 PI and persisted through day
139 PI (Fig. 3). Table I presents a
summary of four to six experiments that examined the temporal course of
endogenous presentation of multiple virus and myelin protein epitopes.
It can be seen that hybridomas specific for the viral epitopes
VP270–86 and VP324–37
secreted a large amount of IL-2 when cultured with CNS APCs isolated at
all four time periods, reflecting significant endogenous presentation
of these viral Ags in the CNS starting as early as 1 mo PI and
continuing throughout the disease. In contrast, the ability of the same
CNS APCs to stimulate T cell lines specific for various myelin epitopes
changed over time. CNS APCs taken before day 80 did not trigger
myelin-specific T cell lines either to proliferate or to secrete IL-2.
Nevertheless, CNS APCs taken at later disease stages could stimulate T
cell lines specific for PLP56–70,
PLP104–117, PLP139–151
(both Th1 and Th2 lines), and PLP178–191. This
ability of the CNS APCs to simultaneously present endogenous viral and
myelin epitopes emerges around 3 mo PI.
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, B and D). To
determine whether the failure of the CNS APCs harvested before
80–90 days PI to activate
PLP139–151-specific T cells was related to a low
density of peptide/MHC complexes, we asked whether increasing the
numbers of APCs would permit T cell activation. Thus, CNS APCs were
harvested from mice 64 days PI and irradiated, and varying numbers
(104–105/well) were
cultured together with a constant number of T cells (2 x
104) specific for either virus or myelin
epitopes. As few as 104 CNS APCs were sufficient
to stimulate significant levels of IL-2 production from hybridomas
specific for the two immunodominant TMEV epitopes,
VP270–86 (Fig. 4A) and
VP324–37 (Fig. 4B). In contrast, at
least 105 CNS APCs were required to stimulate
significant proliferation of a
PLP139–151-specific T cell line (Fig. 4D). Significant activation of the
PLP139–151 T cell line was seen in two of four
experiments using 105 CNS APCs harvested on days
62–65 PI. Even 105 CNS APCs were not sufficient
to stimulate a T cell line specific for cryptic
PLP56–70 epitope (Fig. 4C). These
data support the hypothesis that myelin-specific responses arise as the
number of activated APCs in the CNS and the amount of myelin debris
available to these APCs increase to a threshold level.
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The temporal development of endogenous myelin presentation by CNS
APCs could be due to a qualitative difference in the phenotype of the
APCs (i.e., changes in surface expression of MHC class II,
costimulatory molecules, cytokine production, etc.), a quantitative
difference in the number of cells, and/or the extent of myelin damage
(i.e., the amount of myelin epitopes expressed within MHC class II
molecules on the APCs). In an attempt to distinguish between these
possibilities, a phenotypic characterization of the CNS APCs was
performed. Using conventional RT-PCR, we examined relative mRNA levels
of various cytokines from CNS APCs purified from the spinal cords of
perfused mice 48 and 88 days post-TMEV infection. As shown in Fig. 5, mRNA levels of various cytokines
(IL-12, IFN-, TNF-
, IL-10, IL-1ß, and TGF-ß) were elevated in
the plastic adherent CNS microglia/macrophages at both time points. The
mRNA levels of IL-12 and TNF- appeared to be slightly decreased on
day 88. The presence of IFN- message is probably due to a small
contaminating population of T cells in the plastic-adherent
population.
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65%) and CD45high (activated
macrophages, 35%) subpopulations (14). TMEV-infected
mice were perfused at different time points pi: day 26 (just before
clinical signs), day 65 (early chronic disease when peripheral
anti-myelin responses emerge), and day 95 (late chronic disease).
Frozen sections of the lumber region of the spinal cords were stained
with Abs specific to F4/80, I-As, B7-1, B7-2,
B220, CD4, and CD8. Significant numbers of CD4+ T
cells and lesser numbers of CD8+ T cells
accumulated over time in TMEV-infected mice (Fig. 6). While B220 staining was minimal
compared with CD4, there was an increase in B220+
cells over the duration of the disease course. Neither B nor T cells
were found in the spinal cords of naive mice.
F4/80+ cells (microglia) were found in the spinal
cords of naive SJL mice, but these cells lacked observable expression
of MHC class II (Fig. 7). At the
preclinical time point the numbers of F4/80+
cells increased compared with those in naive mice, and the number and
size of the population continued to increase dramatically as the
disease progressed. The F4/80+ population in
TMEV-infected mice, unlike that in the naive spinal cord, coexpressed
high levels of I-As molecules and the
costimulatory molecules B7-1 and B7-2, suggesting that the population
was highly activated (Fig. 8). These
activated microglial cells and/or infiltrating macrophages initially
accumulated perivascularly and were later present in the parenchyma.
This progressive accumulation of F4/80+ cells
correlates with the number of plastic-adherent CNS APCs that can be
recovered from spinal cords of infected mice, which increases
significantly over time as the disease progresses. The similar
activation state of these cells, as indicated by their cytokine profile
(Fig. 5) and by their expression of MHC class II and costimulatory
molecules (Fig. 8), suggests that the quantitative differences in
endogenous presentation of myelin Ag by CNS APCs isolated in the early
(days 35–75) vs late (days 85–150) stages of disease is a consequence
of their increasing number and the increased availability of myelin
debris. As a direct demonstration that F4/80+
microglia/macrophages in the CNS acquire myelin debris, we performed an
additional immunohistochemical analysis. Fig. 9A shows the presence of
toluidine blue-staining myelin within phagocytic microglia/macrophages
in a cervical spinal cord section of a TMEV-infected SJL mouse
sacrificed 80 days PI. Fig. 9B shows the presence of Nile
red staining-myelin within several F4/80+ cells
in the spinal cord of a TMEV-infected SJL mouse at 100 days PI. The
localization of the Nile red-staining material within the
F4/80+ cells was confirmed by deconvolution
microscopy.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myelin damage in TMEV-infected SJL mice is initiated by TMEV-specific
CD4+ T cells targeting virus persisting in
CNS-resident APCs and leads to up-regulation of proinflammatory
cytokines in the CNS (22, 23, 24, 25, 26, 43). The chronic stage of
TMEV-IDD is associated with the activation of
CD4+ myelin-specific T cells. These autoreactive
T cells appear to be primed via epitope spreading as determined by
their late appearance in disease (
50–60 days PI) and by the fact
that there are no apparent viral epitopes that are shared with the
major encephalitogenic myelin epitopes on PLP, MBP or MOG, i.e., there
is no evidence for molecular mimicry in this system (8).
We used the TMEV-IDD model of CD4+ T
cell-mediated CNS demyelination to study the epitope spreading
processes in both the periphery and the CNS target organ. The spreading
process in the periphery was demonstrated in vivo by temporal changes
in the specificity of DTH, T cell proliferative, and IFN- responses
to viral and myelin epitopes. Anti-viral DTH and in vitro T cell
responses appear within a few days PI (Figs. 1 and 2), and these
responses continue throughout the disease course consistent with the
persistent virus infection of CNS APCs. In contrast, myelin-specific
responses can be detected beginning only 50–60 days PI, i.e., 3–4 wk
after disease onset. Most interestingly, T cell responses against
myelin epitopes arise in an ordered progression (Fig. 1, C
and D). The autoreactive response is initially directed
toward the immunodominant myelin epitope in the SJL mouse,
PLP139–151, and reactivity toward this peptide
continues throughout disease (Fig. 1C). As disease
progresses, T cell responses to PLP178–191
followed by responses to MBP84–104 arise,
paralleling the relative order of their appearance in
PLP139–151-induced R-EAE in SJL mice
(30). In addition, reactivity toward additional myelin
epitopes, e.g., PLP56–70 and
MOG92–106, are observed in chronic TMEV-IDD. The
similar kinetics of epitope spreading in TMEV-IDD and R-EAE might imply
a hierarchy in the processing and presentation of these different
myelin epitopes released during the T cell-mediated destruction of the
myelin, and/or it may correlate with the precursor frequency of T cells
specific for the various myelin epitopes. Although the T cell precursor
frequency of PLP139–151 >
PLP178–191 > MBP84–104
in SJL mice (30) does correlate with the order of the
appearance of specific T cell responses in the periphery of
TMEV-infected SJL mice, the kinetics of the processing and presentation
of these different epitopes on APCs in the CNS target organ are not
known.
The epitope spreading process in the target organ was established by ex
vivo demonstration of temporal changes in endogenous myelin epitope
presentation by CNS APCs, assayed by specific responses of T cell
lines/hybridomas. As expected in a chronic CNS infection, endogenous
presentation of viral envelope peptides was demonstrated on day 35 (the
earliest time point at which enough CNS APCs could be collected), and
endogenous expression of these epitopes persisted through 150 days PI
(Fig. 3A and Table I). In contrast, the endogenous
presentation of all myelin epitopes assayed could be demonstrated
(using 2.5–3.5 x 104 APC/well) only >80
days PI (Fig. 3, B–H, and Table I). Thus, there was a
significant delay in the endogenous presentation of myelin epitopes as
opposed to the early presentation of viral epitopes. These results are
in agreement with the later appearance of myelin epitope-specific DTH
(Fig. 1) and T cell proliferation and IFN- (Fig. 2) responses,
although our ability to detect the endogenous presentation of myelin
epitopes was delayed compared with the appearance of DTH responses.
This is probably due to the fact that the in vivo DTH assay is more
sensitive than the ex vivo method used for detection of endogenous Ag
presentation. This is supported by the observation that
endogenous presentation of PLP139–151 was
observed with CNS APCs isolated on 64 days PI if increased numbers of
cells (105/well) were employed (Fig. 4D).
The ability of CNS APCs to endogenously present multiple myelin
epitopes around the same time PI, considered in light of the ordered
progression of DTH responses to myelin epitopes, which in many cases
was significantly delayed, suggests that T cell precursor frequency
governs the hierarchy of epitope spreading. However, these data also
suggest that a threshold of myelin damage is required (Fig. 4) together
with the time-dependent accumulation and increased activation state of
APCs. Therefore, we characterized the spinal cord-infiltrating
mononuclear cells using immunohistochemical analysis of frozen
sections. Although B220+,
CD4+, and CD8+ cells are
not seen in spinal cord of SJL naive mice, nonactivated
F4/80+ cells (most likely residential microglia)
are readily observed (Fig. 7). However, after infection, the number of
CD4+ T cells and activated
F4/80+ increase dramatically. The
F4/80+ cells also increase in size and
up-regulate the requisite molecules required for activation of naive
CD4+ T cells (i.e., I-As,
B7-1, and B7-2), penetrate into the parenchyma, and accumulate in
the CNS. This progressive accumulation correlates with the disease
severity and the increasing number of CNS APCs that can be recovered
from spinal cords of infected mice (26) and is in
agreement with our previous immunohistochemistry (31) and
CNS APC FACS staining (14). Our previous analyses revealed
that the F4/80+ population was composed of two
subpopulations (resident microglia and infiltrating peripheral
macrophages) based on levels of expression of CD45, and that the
ability of these CNS APCs to activate
PLP139–151-specific T cells could be blocked by
both anti-I-As and CTLA-4 Ig, indicating that
the presentation of endogenous myelin epitopes was B7 dependent and MHC
class II restricted (14).
Within the normal CNS, a variety of cells demonstrate Ag presentation
potential: astrocytes, microglia, and macrophages. IFN--treated
primary astrocytes (44, 45) and microglia (46, 47) cultured from neonatal mouse brain up-regulate MHC class II
and can present Ags to T cells in vitro, but this may not faithfully
reproduce the in vivo state in adult animals. Microglia directly
isolated from adult rats can more efficiently present MBP to T cell
lines in vitro compared with neonatally derived microglia
(48). In our hands, naive CNS mononuclear cells are
inefficient in endogenously activating myelin-specific T cells,
although they are capable of processing and presenting exogenously
added myelin proteins/peptides, albeit with less efficiency than
irradiated splenic APCs (14). Studies using allogeneic
bone marrow chimeras have supported the idea that cells of hemopoietic
origin, i.e., microglia and macrophages, are the principle APCs in the
CNS during the initiation of EAE (49, 50, 51). Although they
are much more abundant than microglia, astrocytes are significantly
less potent when inducing EAE in chimeras (51). Here we
demonstrate that F4/80+,
I-As+, B7-1,2+ CNS APCs,
consisting of both activated CNS-resident microglia and activated
infiltrating macrophages, accumulate in the spinal cords of
TMEV-infected mice and endogenously present viral epitopes throughout
the disease together with self myelin epitopes that become available
following chronic tissue destruction. These results together with the
similarity of cytokine production by CNS APCs isolated at 48 vs 88 days
PI (Fig. 5) suggest that the difference in endogenous presentation of
myelin Ag by CNS APCs early and late in disease is a consequence of the
increasing number of activated APCs and the increased amount of myelin
debris.
It is of major interest whether T cells involved in the epitope spreading process that are specific for endogenous myelin epitopes become activated in the periphery (draining lymph nodes and spleen) and/or the CNS. It is possible that following inflammatory disruption of the blood-brain barrier, myelin debris and/or macrophages/microglia that have ingested myelin gain access to the cervical lymph nodes that drain the cerebrospinal fluid (52) or to the spleen, which concentrates blood-borne material. In support of this, it has been reported that donor cells from alloantigen-disparate solid CNS grafts placed intracerebrally can be later identified in the host spleen and lymph nodes (53). We are currently assessing the ability of APCs purified from the spleen of mice with chronic disease to endogenously present self epitopes. In contrast, it is also possible that T cells specific for endogenous myelin epitopes are activated in the local inflammatory environment within the CNS. In TMEV-IDD, the inflammatory infiltrate is composed of T and B lymphocytes, activated microglia derived from the CNS-resident pool, and macrophages infiltrating from the peripheral blood (31, 54, 55). Macrophages/microglia within the demyelinated areas contain phagocytized myelin debris (56) and, as we have shown, are capable of processing and presenting myelin epitopes. Therefore, any myelin-specific T cells that enter the CNS during the anti-viral inflammatory response, whether already primed in the periphery or not, could potentially be induced to proliferate and/or to secrete proinflammatory cytokines in response to myelin epitopes.
The current results expand our understanding of the epitope spreading process in TMEV infected mice. Intracerebral infection of susceptible SJL mice results in the induction of potent virus-specific Th1 responses which arise within 5–6 days and persist for many mo (24, 57, 58). Virus-specific Th1 cells then traffic to the CNS and upon encounter with infected microglia release chemokines and pro-inflammatory cytokines which results in the activation of resident microglia and the attraction and activation of circulating monocytes/macrophages. These activated mononuclear cells initiate myelin destruction via their phagocytic capacity and their ability to produce proinflammatory cytokines, NO, oxygen radicals, etc. This initial nonspecific myelin destruction leads to endogenous presentation of myelin constituents by the activated CNS APCs and the subsequent activation of myelin-specific CD4+ T cells, which may then play a major pathologic role in the chronic stages of disease. The continuous presence of the virus within the CNS perpetuates this chronic inflammatory process in which epitope spreading leads to the induction of autoreactive T cells.
These findings also enhance our understanding of the pathogenesis of human MS. MHC class II-bearing macrophages, astrocytes, and endothelial cells have been observed in or near MS lesions (59, 60, 61), together with expression of B7 costimulatory molecules (62, 63, 64). Therefore, multiple cells in MS lesions have the potential to fully activate both naive and memory T cells within the CNS. In addition, the epidemiology of MS strongly suggests a role for an infectious agent, perhaps a virus, that is widespread, chronic, and usually subclinical (5). Presentation of viral Ags within the CNS (leading to bystander demyelination), of neuroantigens cross-reactive with viral Ags (molecular mimicry), or of neuroantigens liberated by virus-induced CNS damage (epitope spreading) are all possible mechanisms by which pathogenic immune reactions could be initiated by viruses within the CNS. Our results demonstrate the temporal availability of viral and myelin epitopes on CNS APCs and thus support the possible role of a chronic CNS infection in the initiation of the autoimmune pathogenesis in MS.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611.
3 Abbreviations used in this paper: MBP, myelin basic protein; DTH, delayed-type hypersensitivity; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; PI, postinfection; PLP, proteolipid protein; R-EAE, relapsing experimental autoimmune encephalomyelitis; TMEV, Theiler’s murine encephalomyelitis virus; TMEV-IDD, TMEV-induced demyelinating disease.
Received for publication May 15, 2000. Accepted for publication August 4, 2000.
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) in ear swelling in units of
10-4 (mean value of three to five virus-infected mice
- the mean value of two or three naive age-matched mice challenged the
same day with the same peptide preparation). SEM was 10% or
less.
