The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Croxford, J. L.
Right arrow Articles by Miller, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Croxford, J. L.
Right arrow Articles by Miller, S. D.
The Journal of Immunology, 2005, 174: 907-917.
Copyright © 2005 by The American Association of Immunologists

Viral Delivery of an Epitope from Haemophilus influenzae Induces Central Nervous System Autoimmune Disease by Molecular Mimicry1

J. Ludovic Croxford, Holly A. Anger and Stephen D. Miller2

Department of Microbiology-Immunology, and Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Multiple sclerosis (MS) is an autoimmune CNS demyelinating disease in which infection may be an important initiating factor. Pathogen-induced cross-activation of autoimmune T cells may occur by molecular mimicry. Infection with wild-type Theiler’s murine encephalomyelitis virus induces a late-onset, progressive T cell-mediated demyelinating disease, similar to MS. To determine the potential of virus-induced autoimmunity by molecular mimicry, a nonpathogenic neurotropic Theiler’s murine encephalomyelitis virus variant was engineered to encode a mimic peptide from protease IV of Haemophilus influenzae (HI), sharing 6 of 13 aa with the dominant encephalitogenic proteolipid protein (PLP) epitope PLP139–151. Infection of SJL mice with the HI mimic-expressing virus induced a rapid-onset, nonprogressive paralytic disease characterized by potent activation of self-reactive PLP139–151-specific CD4+ Th1 responses. In contrast, mice immunized with the HI mimic-peptide in CFA did not develop disease, associated with the failure to induce activation of PLP139–151-specific CD4+ Th1 cells. However, preinfection with the mimic-expressing virus before mimic-peptide immunization led to severe disease. Therefore, infection with a mimic-expressing virus directly initiates organ-specific T cell-mediated autoimmunity, suggesting that pathogen-delivered innate immune signals may play a crucial role in triggering differentiation of pathogenic self-reactive responses. These results have important implications for explaining the pathogenesis of MS and other autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
At present, the mechanism(s) of initiation of multiple sclerosis (MS),3 a CNS demyelinating disease considered to be mediated by myelin-specific autoreactive CD4+ T cells, is unknown. Evidence from clinical and epidemiological studies suggests that environmental factors, such as viruses, may play an important role in the etiology of MS (1). Other CNS demyelinating diseases, both in humans and animals, are known to be associated with viral infections (2, 3, 4, 5). There is substantial evidence that MS is an autoimmune disease, although normal, healthy individuals also possess peripheral T cells specific for the epitopes within the myelin Ags, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein, and proteolipid protein (PLP) (6, 7, 8). However, the mechanism(s) underlying the activation of myelin-specific autoreactive T cells is unknown. One putative mechanism for initiation of autoimmune demyelinating disease is molecular mimicry, whereby autoreactive T cells are activated by epitopes from infectious agents that share structural or sequence homology to self Ags (9, 10). A recent study has demonstrated molecular mimicry between MBP96–102, a candidate autoantigen for MS, and the U24 protein of human herpesvirus-6 (HHV-6; residues 4–10), a viral agent that may be associated with MS (11). A significant number of CD4+ T cells from MS patients could recognize either MBP93–105 or a synthetic HHV-6 peptide compared with cells from normal healthy controls. Importantly, cross-activation by the HHV-6 peptide induced Th1 differentiation of the autoreactive T cells (11).

Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model of MS mediated by CD4+ cells, and characterized by a chronic-progressive paralytic course in SJL/J mice (12, 13). TMEV is a natural neurotropic mouse pathogen and intracerebral (i.c.) infection of mice with TMEV induces an initial virus-specific Th1 response that initiates bystander myelin destruction, with subsequent activation of self-reactive myelin epitope-specific CD4+ Th1 cells via epitope spreading (14, 15, 16).

Although epitope spreading may explain how a persistent virus infection can lead to myelin-specific autoimmunity, the major postulated mechanism for initiation of viral-induced autoimmunity is molecular mimicry, although there is currently little direct evidence to support this. Previous studies have used mimic sequences from infectious pathogens, to modulate experimental autoimmune encephalomyelitis (EAE), or administered T cell lines specific for mimic epitopes to induce EAE (17, 18, 19, 20). To provide more compelling evidence for the initiation of CNS autoimmunity via molecular mimicry, we asked whether CNS disease could be initiated by infecting SJL mice with a neurotropic virus expressing a peptide mimic of the immunodominant PLP epitope (PLP139–151) derived from an infectious pathogen.

We demonstrate here a defined model of molecular mimicry, wherein infection of SJL mice with a nonpathogenic variant of the wild-type (WT)-TMEV (BeAn strain) expressing a mimic sequence (Haemophilus influenzae (HI)574–586) derived from the protease IV protein of HI, a natural bacterial pathogen of mice, leads to early-onset demyelinating disease (21). This early-onset clinical disease was associated with activation, proliferation, and differentiation of PLP139–151-specific autoreactive Th1 T cells. Furthermore, these data highlight the importance of studying molecular mimicry in the context of an infectious pathogen, because immunization with the HI574–586 peptide emulsified in CFA failed to induce clinical disease. However, immunization of mice preinfected with HI-BeAn with HI574–586/CFA induced a significantly more severe clinical disease. This suggests that severe clinical disease, due to HI-BeAn-induced molecular mimicry, requires both the presence of the mimic sequence and the persistent stimulation of innate immune system by the pathogen. This model of molecular mimicry will allow potential mimic epitopes from other infectious pathogens to be tested for their ability to induce autoimmunity, and has important implications for the etiology and pathogenesis of MS and other autoimmune diseases.


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

Five- to 6-wk-old female SJL mice were obtained from Harlan Sprague Dawley. Mice were housed under barrier conditions at the National Institutes of Health-approved Northwestern University Medical School animal facilities. All protocols were approved by the Northwestern University Animal Care and Use Committee. Paralyzed mice were afforded easier access to food and water.

Construction of the mimic-expressing virus

The cDNA encoding the BeAn strain of TMEV was modified by inserting ClaI sites at bp 1137 (Fig. 1A). This resulted in a 23-aa deletion in the leader sequence (L) of the BeAn genome. This virus was designated {Delta}Cla-BeAn as described previously (21). Briefly, ClaI sites were introduced by PCR to the PLP cDNA at either end of a 30-aa sequence PLP130–159, which encompassed the immunodominant encephalitogenic PLP139–151 sequence (Fig. 1B). A sequence from serine protease IV (HI574–586), naturally expressed in HI, which shares 6 of 13 aa with PLP139–151 (Fig. 1B), was constructed by PCR mutagenesis of the PLP139–151 sequence to introduce amino acid substitutions at positions 139 (H->E), 140 (C->Q), 142 (G->V), 147 (H->L), 149 (D->A), 150 (K->P), and 151 (F->I). Following an enzyme restriction cut with ClaI, the 30-aa piece containing the HI574–586 sequence flanked by the original PLP sequences was inserted into the ClaI site in the {Delta}Cla-BeAn virus cDNA. This was designated HI-BeAn cDNA (Fig. 1A). As a negative control, an OVA sequence, OVA317–346, encompassing the OVA323–339 epitope, with no homology to the PLP139–151 or HI574–586 sequence (Fig. 1B), was inserted into the {Delta}Cla-BeAn parental virus to yield OVA-BeAn (data not shown). Viral RNA was produced from the cDNA through the T7 promoter and transfected into BHK-21 cells, resulting in the production of infectious virus by cells as described previously (21). Viral titers were measured by viral plaque assay (22). Sequencing of the HI-BeAn cDNA confirmed that the HI sequence was correct.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 1. The construction of a PLP139–151 mimic expressing virus. A, A ClaI insertion in the leader sequence (L) of cDNA encoding the TMEV BeAn strain 8386 resulted in a 23-aa deletion in the leader sequence of the parent virus ({Delta}Cla-BeAn). ClaI sites were introduced by PCR into the PLP cDNA at either end of a 30-aa sequence PLP130–159, which encompassed the immunodominant encephalitogenic PLP139–151 sequence. The PLP139–151 mimic sequence from the protease IV protein of HI (HI574–586) was constructed by PCR amino acid substitutions of the PLP139–151 sequence. The 30-aa sequence containing the HI574–586 sequence flanked by the PLP sequences was inserted into the ClaI site in the {Delta}Cla-BeAn virus cDNA. This was designated HI-BeAn cDNA. Viral RNA was produced from the cDNA through the T7 promoter and transfected into BHK-21 cells resulting in the production of infectious virus. B, Peptide sequences of myelin and myelin mimic peptides. The mimic HI574–586 epitope shares 6 of 13 aa with the native PLP139–151 epitope (in bold type) including the primary (L145) and secondary (P148) I-As binding residues and the primary (W144) TCR contact residue. I-As-restricted VP270–86 and OVA323–339 peptides used as negative controls do not share sequences with PLP or HI peptides.

 
Infection of SJL mice with TMEV

SJL mice (n = 5–8 per group) were infected by i.c. injection of 3 x 107 PFU of either WT-TMEV (BeAn 8386 strain), HI-BeAn, OVA-BeAn, or {Delta}Cla-BeAn, and scored at daily intervals on a clinical scale of 0–5: 0, no signs of disease; 1, mild gait abnormalities; 2, severe gait abnormalities; 3, paralysis in one limb; 4, more than one paralyzed limb; 5, moribund.

Induction of active EAE

For actively induced relapsing-EAE, mice (n = 5) were immunized s.c. with 100 µl of a CFA emulsion containing 400 µg of Mycobacterium tuberculosis H37Ra (Difco) and 100 µg of PLP139–151 distributed over three sites on the lateral hind flanks and dorsally. For HI574–586 or OVA323–339 peptide priming, the same protocol was used with 100 µg of peptide per animal. Clinical scores were assessed on a 0–5 scale as follows: 1, lack of tail tone; 2, limp tail and hindlimb weakness; 3, partial hindlimb paralysis; 4, total hindlimb paralysis; and 5, moribund.

Peptides

PLP139–151 (HSLGKWLGHPDKF), the TMEV capsid peptide VP270–86 (WTTSQEAFSHIRIPLPH), OVA323–339 peptide (ISQAVHAAHAEINEAGR), and the HI peptide HI574–586 (EQLVKWLGLPAPI) (Fig. 1B) were purchased from Peptides International. The amino acid composition was verified by mass spectrometry, and purity was assessed by HPLC. Both VP270–86 and OVA323–339 induce immune responses (delayed-type hypersensitivity (DTH), proliferation, and IFN-{gamma} secretion) in SJL mice either following infection with WT-TMEV or when immunized in CFA, respectively (21) (data not shown).

DTH response

DTH responses were elicited by injecting mice s.c. with 10 µg of the challenge peptides, PLP139–151 or VP270–86, into alternate ears following measurement of ear thickness using a Mitutoyo model 7326 engineer’s micrometer (Schlesinger’s Tools). Twenty-four hours following peptide challenge, the ears were remeasured and differences in ear swelling over prechallenge thickness were expressed in units of 10–4 inches ± SEM.

T cell proliferation and cytokine analysis

Spleens were removed from infected mice (n = 2) at various times following infection. T cell proliferation and cytokine analysis were performed as described previously (23). Proliferation was determined from triplicate wells for each peptide concentration added in vitro and then expressed as {Delta}counts per minute. For IFN-{gamma} cytokine analysis, a duplicate set of proliferation wells were used to collect supernatants at 48 and 72 h, and cytokine concentrations were determined by ELISA (Endogen Minikits).

Immunohistochemistry and quantitation of cellular infiltrates

Five to 8 mice per experimental group were anesthetized and perfused with 1x PBS on the indicated days. Spinal cords and brains were removed by dissection, and multiple 2- to 3-mm spinal cord blocks were immediately frozen in OCT (Miles Laboratories) in liquid nitrogen. The blocks were stored at –80°C in plastic bags to prevent dehydration. Five-micrometer-thick cross-sections from the lumbar and thoracic region of the spinal cord, or longitudinal sections of the brain, were cut on a Reichert-Jung Cryocut CM1850 cryotome (Leica), mounted on Superfrost Plus electrostatically charged slides (Fisher), air dried, and stored at –80°C. Slides were stained using a Tyramide Signal Amplification Direct kit (NEN) according to manufacturer’s instructions. Sections from each group were thawed, air-dried, fixed in 2% paraformaldehyde at room temperature, and rehydrated in 1x PBS. Nonspecific staining was blocked using anti-CD16/CD32, (Fc{gamma}III/IIR, 2.4G2; BD Pharmingen), and an avidin/biotin blocking kit (Vector Laboratories) in addition to the blocking reagent provided by the Tyramide Signal Amplification kit. Tissues were stained with biotin-conjugated Abs anti-mouse CD4, anti-mouse CD8, anti-mouse B220, and anti-mouse F4/80 (BD Pharmingen). Sections were counterstained with 4',6'-diamidino-2-phenylindole (Sigma-Aldrich) and then coverslipped with Vectashield mounting medium (Vector Laboratories). Slides were examined and images were acquired via epifluorescence using the SPOT RT camera (Diagnostic Instruments) and Metamorph imaging software (Universal Imaging). Eight sections from each sample per group were analyzed at x100 magnification.

Photomicrographs of immunostained sections from spinal cord, cerebellum, and brainstem representative of the cellular infiltrates in the different groups were used to quantify the numbers of positive inflammatory cells per area of each tissue section. Data from photomicrographs were stored as 8-bit binary images in grayscale format. Quantification was determined using ImageJ software, version 1.32j (<(http://rsb.info.nih.gov/ij/)>). Threshold values of brightness and contrast were determined for the photomicrographs and were kept constant between each sample. Before analysis, the minimum and maximum size of pixels to be counted was determined whereby sections with no positive staining gave a measurement of 0.0 pixels. Data are presented as percentage of positive pixels per area of the photomicrograph.

Statistics

Clinical severity results were presented as the mean group clinical score, and the statistical difference calculated by the Mann-Whitney nonparametric ranking test. Analysis of DTH responses and IFN-{gamma} ELISA were performed using the two-tailed Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunization of mice with HI574–586 induces PLP139–151 T cell cross-reactivity but not clinical disease

Following the immunization of mice with PLP139–151 in CFA, 100% of mice exhibited a typical acute-phase disease course of EAE. In contrast, mice immunized with the PLP139–151 mimic peptide, HI574–586, did not exhibit clinical disease (Fig. 2A) even if primed twice with peptide/CFA (days 0 and +7 postinfection (p.i.)) and treated with pertussis toxin (200 ng/day, days 0 and +2 p.i.) (data not shown) (19). The ability of HI574–586 immunization to induce the cross-activation of PLP139–151-specific T cells was measured by T cell proliferation, DTH, and IFN-{gamma} secretion. Negative controls consisted of naive mice rechallenged with either HI574–586 or PLP139–151 peptides, or HI574–586- or PLP139–151-immunized mice rechallenged with PBS or an irrelevant TMEV capsid peptide, VP270–86 (Fig. 3).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2. Infection, but not immunization, of SJL mice with the myelin mimic, HI574–586, induces a rapid-onset, nonprogressive form of clinical CNS disease. A, Separate groups of SJL mice (n = 5) were immunized on day 0 with 100 µg of either the self-myelin peptide PLP139–151, the myelin mimic peptide HI574–586, or a control nonself, nonmimic peptide, OVA323–339, and observed for the development of clinical EAE. B, Separate groups of SJL mice (n = 5–7) were immunized on day 0 with 100 µg of PLP139–151 or on both days 0 and 7 with 100 µg of HI574–586. Additional groups of mice were primed on days 0 and 7 with the indicated combinations of a suboptimal dose (20 µg) of PLP139–151 (sPLP139–151), 100 µg of HI574–586, or 100 µg of the control OVA323–339 peptide. All mice were observed for clinical signs of EAE for 38 days. Only mice primed on day 0 with the optimal dose of PLP139–151 developed clinical disease. C, Separate groups of SJL mice (n = 10) were infected i.c. with 3 x 107 PFU of PLP139-BeAn, HI-BeAn, WT-TMEV, or OVA-BeAn, and observed for 70 days for clinical signs of demyelinating disease. *, Disease severity was significantly more severe in PLP-BeAn-infected mice compared with HI-BeAn-infected mice; p < 0.05. **, Disease onset was significantly earlier in PLP-BeAn- and HI-BeAn-infected mice compared with OVA-BeAn- and WT-TMEV-infected mice; p < 0.05. These results in each panel are representative of at least three separate experiments.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3. HI574–586 immunization of SJL mice induces early myelin-specific CD4+ T cell proliferative responses, but not Th1 differentiation. SJL mice were immunized s.c. with 100 µg of PLP139–151 or HI574–586 peptides in CFA. A, Splenic CD4+ T cell proliferative responses were assessed 16 days thereafter in response to restimulation with PLP139–151, HI574–586, or the immunodominant TMEV capsid peptide, VP270–86. T cell proliferation was determined at 96 h, and results are expressed as counts per minute (x10–3) ± SEM from triplicate cultures. Restimulation with PLP139–151 and HI574–586 peptides induced significant T cell proliferation in both PLP139–151- and HI574–586-immunized groups. Neither group responded to challenge with the control VP270–86 peptide. B, In vivo differentiation of CD4+ Th1 cells was assessed by DTH ear swelling assays on day 14 postpriming. Results are expressed as mean {Delta}24-h ear swelling (responses of naive SJL mice subtracted) ± SEM in groups of four to five mice. C, IFN-{gamma} levels (nanograms per milliliter) from the supernatants of the proliferative cultures (A) were determined by ELISA as described in Materials and Methods. *, Values significantly above control levels; p < 0.05. These results in each panel are representative of at least three separate experiments.

 
Rechallenge with either HI574–586 or PLP139–151 peptide in mice immunized against PLP139–151, induced significant DTH responses (in vivo peptide rechallenge), increased T cell proliferation, and significant IFN-{gamma} secretion (in vitro peptide rechallenge) when compared with naive mice (*, p < 0.05) (Fig. 3). Mice immunized with HI574–586 exhibited significant levels of T cell proliferation when rechallenged with either HI574–586 or PLP139–151 compared with naive mice (Fig. 3A). However, mice immunized to HI574–586, in contrast to PLP139–151-immunized mice, responded only to DTH rechallenge with HI574–586 (*, p < 0.05), but not PLP139–151 (Fig. 3B). In addition, HI574–586-immunized mice only secreted significant quantities of IFN-{gamma} in response to HI574–586 (*, p < 0.05), but not PLP139–151 (Fig. 3C). Rechallenge with VP270–86 did not induce either T cell proliferation or IFN-{gamma} secretion in either HI574–586- or PLP139–151-immunized groups, and was comparable with PBS and naive control groups (Fig. 3).

By day 14 post-PLP139–151 immunization, mice exhibit clinical signs of disease, and the presence of inflammatory infiltrate can be identified in the CNS of these mice (24). Immunohistochemistry of the spinal cord and brain, taken from mice 14 days following immunization against either HI574–586 or OVA323–339, were analyzed for the presence of CD4+ and CD8+ T cells, B220+ B cells, or F4/80+ monocytes/macrophages (Fig. 4). Although mice primed with HI574–586 exhibited cross-reactive T cells to PLP139–151 as measured by T cell proliferation, no signs of clinical disease were observed (Fig. 2A), and there was minimal presence of any inflammatory infiltrate in the spinal cord of these mice, or in mice immunized against OVA323–339 in comparison with PLP139–151-immunized mice (Fig. 4A). However, there was sparse staining for CD4+ T cells and F4/80+ monocytes/macrophages, compared with OVA323–339-immunized mice, in the white matter of the cerebellum of HI574–586-immunized mice (Fig. 4B). CD8+ T cells and B220+ B cells were not present at this early time point in either of the groups (data not shown).



View larger version (73K):
[in this window]
[in a new window]
  		FIGURE 4. CD4+ T cells infiltrate the cerebellum of mice immunized with the HI574–586 mimic peptide. Groups of SJL mice (n = 8) were immunized s.c. with 100 µg of PLP139–151, HI574–586, or OVA323–339 peptide in CFA on day 0. At day 14 postimmunization, mice were anesthetized and perfused, and sections of spinal cord (A) or cerebellum (B) were removed. Immunohistochemistry of 5-µm-thick slices demonstrated the presence of CD4+ T cells and F4/80+ monocytes/macrophages present in both the brain and cerebellum of PLP139–151/CFA-immunized mice, but only in the cerebellum, but not spinal cord, of HI574–586/CFA-immunized mice. No infiltrating cells were observed in OVA323–339-immunized mice. Quantification of immunohistochemistry was determined on representative sections using ImageJ software. The percentages indicated in the lower left corner of each panel indicate the percentage of positive pixels per area of the photomicrograph.

 
Double priming with HI574–586 or prepriming with a suboptimal dose of PLP139–151 does not induce clinical disease

To determine whether multiple immunizations with the mimic peptide could induce clinical disease, mice were immunized with HI574–586 (100 µg) in CFA on days 0 and +7 (Fig. 2B). To test whether preimmunization with PLP139–151 could lower the threshold for HI574–586-induced EAE, mice were first immunized with a suboptimal dose of PLP139–151 (sPLP139–151; 20 µg), and then immunized on day +7 with HI574–586 (100 µg) or vice versa (Fig. 2B). Negative controls consisted of mice immunized with HI574–586 (100 µg) or a suboptimal dose of PLP139–151 (20 µg), which received a secondary immunization against OVA323–339 (100 µg), or two immunizations (days 0 and +7) with the suboptimal dose of PLP139–151 (20 µg) (Fig. 2B). Positive controls consisted of mice immunized with an optimal dose of PLP139–151 (100 µg) on day 0. Only mice primed with the optimal dose of PLP139–151 (100 µg) exhibited clinical disease (Fig. 2B).

Infection of the CNS with the HI-BeAn mimic-expressing virus induces early-onset, nonprogressive clinical disease associated with induction of Th1 autoreactivity to PLP139–151

To determine whether exposure to the mimic peptide in the context of an active virus infection could induce clinical disease, mice were infected i.c. with 3 x 107 PFU of TMEV expressing the PLP139–151 mimic peptide, HI574–586 (HI-BeAn) (Fig. 2C). Positive controls consisted of mice infected i.c. with 3 x 107 PFU of the virus expressing the immunodominant PLP139–151 peptide (PLP139-BeAn) or WT-TMEV. Negative controls consisted of mice infected with 3 x 107 PFU of the virus expressing the nonencephalitogenic peptide OVA323–339 (OVA-BeAn) (Fig. 2C). Mice infected with PLP139-BeAn or HI-BeAn exhibited clinical disease with a significantly earlier onset (days 5–14 p.i.; **, p < 0.05) in comparison to mice infected with either OVA-BeAn or WT-TMEV (onset, days 38–54 p.i.) (Fig. 2C). However, mice infected with HI-BeAn experienced a nonprogressive clinical disease, with a significantly ameliorated clinical score (*, p < 0.05 between days 32 and 68 p.i.) compared with PLP139-BeAn-infected mice (Fig. 2C). The clinical disease exhibited by the HI-BeAn-infected mice was atypical of either the usual WT-TMEV or EAE-type diseases reported previously (20). In these mice, disease consisted of ruffled fur, mild waddling gait, and an arched back, present for the duration of the experiment, but with no worsening over time (Fig. 2C). Both WT-TMEV- and OVA-BeAn-infected mice exhibited a progressive clinical disease over the duration of the experiment (70 days) compared with the HI-BeAn-infected mice (Fig. 2C). Mice infected with the {Delta}Cla-BeAn parental virus did not exhibit disease signs (data not shown) (21).

To determine whether infection with HI-BeAn induced the cross-activation of self-reactive PLP139–151-specific T cells, HI-BeAn-infected mice were analyzed for T cell proliferation, DTH, and IFN-{gamma} responses to PLP139–151. Mice infected with either HI-BeAn or PLP139-BeAn responded similarly via T cell proliferation upon in vitro challenge with HI574–586 or PLP139–151, as well as the immunodominant virus peptide VP270–86 (Fig. 5A). In addition, mice infected with either PLP139-BeAn or HI-BeAn responded significantly to DTH rechallenge with either the self PLP139–151 peptide or the HI574–586 mimic peptide (*, p < 0.05) (Fig. 5B). This is in contrast to responses seen in SJL mice primed with HI574–586/CFA (Fig. 3B). T cells from HI-BeAn-infected mice also secreted significant amounts of IFN-{gamma} in response to VP270–86, HI574–586, and PLP139–151 (*, p < 0.05) rechallenge compared with naive mice (Fig. 5C). In contrast, T cells from mice infected with PLP139-BeAn only responded to rechallenge with VP270–86 and PLP139–151 but not to HI574–586 (Fig. 5C). These results demonstrate that exposure of mice to the HI574–586 mimic peptide in the context of an active, persistent, replicating virus infection, but not in the context of CFA immunization, leads to clinical disease, concomitant with induction of a potent Th1 response cross-reactive with the self PLP139–151 myelin epitope. IL-4 was not produced by restimulation of T cells from HI-BeAn-infected mice with either PLP139–151 or HI574–586, indicating lack of activation of Th2 cells (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 5. HI-BeAn infection of SJL mice induces robust differentiation of myelin-specific CD4+ Th1 cells. Separate groups of SJL mice (n = 10) were infected i.c. with 3 x 107 PFU of PLP139-BeAn or HI-BeAn. A, Splenic CD4+ T cell proliferative responses were assessed 16 days thereafter in response to restimulation with PLP139–151, HI574–586, or the immunodominant TMEV capsid peptide, VP270–86. T cell proliferation was determined at 96 h and results expressed as counts per minute (x10–3) ± SEM from triplicate cultures. Restimulation with PLP139–151, HI574–586, and VP270–86 peptides induced significant T cell proliferation in both the PLP139-BeAn- and HI-BeAn-infected mice. B, In vivo differentiation of CD4+ Th1 cells was assessed by DTH ear swelling assays on day 14 postpriming. Results are expressed as mean {Delta}24-h ear swelling (responses of naive SJL mice subtracted) ± SEM in groups of four to five mice. C, IFN-{gamma} levels (nanograms per milliliter) from the supernatants of the proliferative cultures (A) were determined by ELISA as described in Materials and Methods. *, Values significantly above control levels; p < 0.05. These results in each panel are representative of at least three separate experiments.

 
Persistent preinfection of the CNS by HI-BeAn is required for severe clinical disease following the cross-reactive activation of PLP139–151 T cells by HI574–586 immunization

The difference in route of Ag exposure between the HI574–586 mimic peptide-immunized and -infected groups may account for the difference in disease incidence observed. The activation of resident CNS cells following HI-BeAn infection may increase disease incidence. However, mice cannot be immunized with CFA in the CNS, and TMEV administered by peripheral routes rapidly crosses the blood-brain barrier into the CNS (J. K. Olson and S. D. Miller, unpublished observations). Therefore, to determine whether activation of CNS-resident cells by persistent virus infection is necessary for HI-BeAn-induced autoimmunity, mice were preinfected with either HI-BeAn or OVA-BeAn, and then immunized with HI574–586, OVA323–339, or PBS emulsified in CFA day +14 p.i. Additionally, we tested whether a transient infection of the CNS by {Delta}Cla-BeAn, a nonpathogenic variant of WT-TMEV that fails to persist, was sufficient to provide the necessary stimuli to allow induction of disease following HI574–586 immunization. Mice preinfected with HI-BeAn and then immunized against HI574–586 developed a significantly more severe disease (*, p < 0.01) compared with mice infected with HI-BeAn and subsequently immunized with either OVA323–339 or PBS in CFA (Fig. 6A). All HI-BeAn-infected groups developed the typical early-onset, nonprogressive disease. However, only the HI574–586/CFA-immunized group began to develop more severe clinical disease by day 30 p.i. By day 50 p.i., mice had reached a average clinical score of 2.3, which did not increase in severity, as late as day 110 p.i. (Fig. 6A). In contrast, preinfection of the CNS of mice with the nonpersistent {Delta}Cla-BeAn variant, or OVA-BeAn, did not predispose mice to a more severe disease following HI574–586 or OVA323–339 immunization (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 6. Preinfection with HI-BeAn before HI574–586 immunization induces a severe clinical disease. Separate groups of SJL mice (n = 10) were infected i.c. with 3 x 107 PFU HI-BeAn and then treated s.c. with PBS or immunized with 100 µg of either HI574–586/CFA or OVA323–339/CFA at day +14 p.i. ({uparrow}). A, Mice were observed for clinical signs of demyelinating disease for 114 days. Mice preinfected with HI-BeAn and then immunized against HI574–586 developed clinical disease that was significantly more severe than that observed in HI-BeAn-infected mice treated with PBS or immunized against the control OVA323–339 peptide. B and C, T cell-proliferative responses were determined at day 35 post-initial infection in response to restimulation with either HI574–586 or PLP139–151. T cell proliferation was determined at 96 h, and results are expressed as counts per minute (x10–3) ± SEM from triplicate cultures. D, IFN-{gamma} levels (nanograms per milliliter) from the supernatants of the proliferative cultures (B and C) were determined by ELISA as described in Materials and Methods. *, Values significantly above control levels; p < 0.05. These results in each panel are representative of at least three separate experiments.

 
HI-BeAn-infected mice subsequently immunized against with HI574–586/CFA developed significantly greater T cell proliferative responses upon in vitro rechallenge with HI574–586 compared with the control groups (Fig. 6B). HI-BeAn-infected mice immunized with either OVA323–339 or PBS/CFA had similar T cell proliferative responses, suggesting that priming with an irrelevant Ag had no nonspecific or bystander effect upon HI-specific T cell proliferation (Fig. 6B). Following rechallenge with PLP139–151 T cell responses were greatest in the HI-immunized group (Fig. 6C). However, there was not as great an effect between the groups as seen with HI rechallenge (Fig. 6B). In addition, proliferative counts were substantially lower in the PLP rechallenge group compared with the HI group (12 x 103 vs 40 x 103 cpm, respectively; Fig. 6, B and C). Splenocytes from mice infected with HI-BeAn and immunized against HI574–586 secreted significant greater amounts of IFN-{gamma} (*, p < 0.05) in vitro, compared with controls, when rechallenged with either HI574–586 or PLP139–151 (Fig. 6D).

Severe clinical disease is associated with lesions in the cerebellum and brainstem and the spread of inflammatory infiltrate from the thoracic to lumbar region of spinal cord

Spinal cord and brain removed at day 50 p.i. was cryosectioned for immunohistochemical analysis of CD4+ and CD8+ T cells, B220+ B cells, or F4/80+ monocyte/macrophage infiltration (Fig. 7). Mice infected with HI-BeAn and later immunized with PBS/CFA contained significant CD4+ T cell infiltrates in white matter perivascular lesions in the thoracic, but not lumbar regions of the spinal cord (Fig. 7A). In addition, diffuse F4/80+ staining was also observed in the white matter of the thoracic spinal cord (Fig. 7A). HI-BeAn-infected mice, subsequently immunized with HI574–586/CFA, showed diffuse CD4+ and F4/80+ staining in the thoracic region of the spinal cord similar to that seen in PLP-BeAn mice (Fig. 7A). In contrast to the other groups, diffuse CD4+ and F4/80+ staining was also observed in the lumbar white matter of HI-BeAn plus HI574–586/CFA mice and PLP-BeAn mice, suggesting the trafficking of inflammatory infiltrate along the spinal cord (Fig. 7A). The significantly enhanced clinical disease scores (Fig. 6A) and infiltration of CD4+ T cells and F4/80+ macrophages in HI-BeAn plus HI574–586/CFA mice as reflected by quantitative image analyses (Fig. 7) also correlated with increased myelin loss in these areas as determined by anti-PLP-FITC staining (data not shown). In contrast, HI-BeAn-infected mice that were immunized against OVA323–339 had markedly less CD4+ and F4/80+ staining present (Fig. 7A).



View larger version (65K):
[in this window]
[in a new window]
  		FIGURE 7. Severe clinical disease in HI-BeAn-infected plus HI574–586/CFA-immunized mice is associated with increased inflammatory infiltrates in the lumbar region of the spinal cord, cerebellum, and brainstem. Groups of SJL mice (n = 5–8) were infected i.c. with 3 x 107 PFU of HI-BeAn and then immunized s.c. with either PBS or 100 µg of either HI574–586/CFA or OVA323–339/CFA at day +14 p.i. At day 50 p.i., mice were anesthetized and perfused, and sections of spinal cord were dissected into the thoracic and lumbar regions (A), and the brainstem and cerebellum (B) were removed. Immunohistochemistry of 5-µm-thick tissue slices was performed, by staining for the presence of CD4+ T cells and F4/80+ monocytes/macrophages. Immunohistochemistry of the brain and spinal cord from PLP-BeAn-infected mice (infected i.c. with 3 x 107 PFU) was used as a positive control. Quantification of immunohistochemistry was determined on representative sections using ImageJ software. The percentages indicated in the lower left corner of each panel indicate the percentage of positive pixels per area of the photomicrograph.

 
In brain sections, CD4+ T cells were observed only in the white matter of the brainstem adjacent to the cerebellum and in the main region of the brainstem of mice infected with HI-BeAn and subsequently immunized with HI574–586/CFA similar to PLP-BeAn-infected mice (Fig. 7B). F4/80+ cells were observed in large quantities in the white matter of the cerebellum and brainstem of the HI-BeAn plus HI574–586/CFA group, although the HI-BeAn plus PBS/CFA and HI-BeAn plus OVA323–339/CFA groups also contained minimal F4/80+ staining in the cerebellar white matter (Fig. 7B). In all groups, both CD8+ and B220+ cells were present at low numbers in both the spinal cord white matter and in the cerebellar white matter and brainstem (data not shown). Naive mice were negative for CD4+ and CD8+ T cells, B220+ B cells, and F4/80+ monocytes/macrophages in both the spinal cord and brain (Fig. 7).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study clearly demonstrates that the viral delivery of a myelin PLP HI mimic peptide can induce cross-activation of autoreactive PLP139–151-specific CD4+ T cells and CNS autoimmune disease, via molecular mimicry. In addition, the current results highlight the importance of studying molecular mimicry in the context of pathogen-induced innate inflammatory immune signals.

The primary TCR and MHC class II binding sites of PLP139–151, a dominant encephalitogenic myelin peptide in SJL mice, have been defined by multiple amino acid substitutions (19, 25, 26). Analysis of these sites determined epitopes from infectious pathogens, which shared sequence homology at the primary TCR and MHC residues. A peptide from serine protease IV (HI574–586) secreted by HI bacteria, a natural mouse pathogen, contains structural similarities to the native PLP139–151 peptide (19). Although this sequence has a limited sequence homology, it shares the primary TCR contact site at position 144 and the primary and secondary I-As contact residues at positions 145 and 148. The HI574–586 peptide has been shown to bind I-As and to activate T cell clones derived from PLP139–151-primed SJL mice (19).

In this study, we report that i.c. infection of mice with the HI-BeAn variant of TMEV induces a rapid onset, nonprogressive form of clinical disease, wherein the early cross-reactive induction of IFN-{gamma}-producing PLP139–151-specific Th1 cells temporally correlates with disease onset (10–14 days p.i.) (Figs. 2 and 5). In comparison, WT-TMEV- or OVA-BeAn-infected groups develop late-onset disease and PLP139–151-specific T cell responses that are first evident 40–55 days p.i. arising via epitope spreading rather than molecular mimicry (14, 21). Mice infected with the PLP139-BeAn virus, a model of molecular identity, also exhibited an early-onset clinical disease, but with more severe clinical symptoms and a progressive clinical course (Fig. 2C) (21). Interestingly, as previously reported (21), mice infected with OVA-BeAn develop a similar late-onset disease profile as seen with WT-TMEV infection. Recent tolerance experiments indicate that disease severity is significantly inhibited in HI-BeAn-infected mice pretolerized to PLP139–151, demonstrating that PLP139–151-specific T cells activated by cross-reactivity to HI574–586, are critical for the early-onset HI-BeAn-induced disease.4 Furthermore, disease induction does not correlate with enhanced responses to TMEV epitopes (Fig. 5) or to any observed differences in virus replication in vivo or in vitro (data not shown).

Following i.c. infection with HI-BeAn, CD4+ T cells respond equally in T cell proliferation assays following rechallenge with the mimic HI574–586 or self PLP139–151 peptides (Fig. 5). This is an important finding, which suggests that the host can process the HI574–586 mimic sequence from the leader sequence of HI-BeAn and present it in an inflammatory context to PLP139–151-specific T cells. Furthermore, in vivo presentation of the HI574–586 mimic sequence can induce differentiation of PLP139–151-reactive Th1 cells, as measured by IFN-{gamma} ELISA and in vivo DTH responses to PLP139–151. Therefore, the early disease and associated enhanced CNS inflammation (Fig. 7) are likely the result of an immune response to virus-infected cells expressing the mimic epitope in the CNS, and/or due to a response to other endogenous CNS APCs presenting the self PLP139–151 epitope that are not virus infected. However, despite the presence of IFN-{gamma}-producing PLP139–151-reactive T cells, HI-BeAn-infected mice develop only a mild, nonprogressive form of clinical disease, compared with PLP139-BeAn-infected mice. The I-As molecule has a lower affinity for the mimic HI574–586 peptide compared with that of PLP139–151 (IC50, 385 and 87 nM, respectively) (19), and therefore, potential cross-reactive T cells may not be fully reactivated when they encounter the self-peptide in vivo. Alternatively, the nonprogressive disease observed may be due to the relative proportion of HI574–586-specific and PLP139–151-specific T cells activated following HI-BeAn infection. The HI574–586 population of CD4+ T cells may therefore be a subpopulation of the total PLP139–151 population or may be separate to the PLP139–151 population, with only minor overlap, as we have previously proposed (10). The identity of the cross-reactive repertoire is currently under investigation in our laboratory.

Another possible explanation could be that, whereas the cross-activated PLP139–151 Th1 cells were encephalitogenic, the HI574–586-activated population could differentiate to a Th2 phenotype and may be protective against disease induction. However, rechallenge of CD4+ T cells isolated from either OVA-BeAn- or HI-BeAn-infected mice, with either PLP139–151 or HI574–586, did not induce the Th2-type cytokines IL-4 or IL-5, as measured by ELISPOT and ELISA (data not shown). Although significant numbers of CD4+ T cells were observed by immunohistochemistry in the white matter of the thoracic region of the spinal cord in HI-BeAn-infected mice, the majority of CD4+ infiltrate appeared to be perivascular in nature (Fig. 7A). These data may support the idea that T cells activated by HI-BeAn may not be fully activated upon encountering PLP139–151 epitopes in the CNS, and therefore do not efficiently traffic into the spinal cord parenchyma. Further evidence for deficient trafficking is seen by the relative lack of inflammatory infiltrate in the lumbar region of the spinal cord, where lesions are normally seen during EAE and TMEV disease (24). However, a number of CD4+ T cells do traffic through the thoracic spinal cord, and these may represent the proportion of T cells that have a high affinity for PLP139–151. We have previously shown that mice infected with PLP139-BeAn develop severe clinical disease associated with widespread inflammation of the spinal cord (21, 23). In this instance, it is likely that the majority of CD4+ T cells are PLP139–151 specific with a high affinity for this epitope in vivo.

Initial studies on molecular mimicry demonstrated that immunization of rabbits against a hepatitis B viral epitope, that shared 6 aa with an encephalitogenic MBP peptide, could induce T cell reactivity to MPB (9). Despite this autoreactive T cell component, and inflammatory infiltrate in the cerebral cortex, clinical disease did not develop (9). Other studies have suggested the likelihood of molecular mimicry playing a role in the induction of autoimmunity, where immunization with viral sequences mimicking self-peptides can activate autoreactive T cells (17, 27, 28). Studies in other infectious autoimmune models, such as myocarditis and herpes stromal keratitis, also suggest the likelihood of molecular mimicry playing a role in their pathogenesis (29, 30, 31). Immunization with the HI mimic peptide can activate PLP139–151-specific CD4+ T cells, but does not induce clinical disease (Fig. 2) (19). We demonstrate that the mechanism behind the inability of HI574–586 immunization to induce clinical disease is due to the lack of Th1 differentiation of the cross-activated CD4+ T cells. This is in marked contrast to the potent Th1 IFN-{gamma} response recalled by PLP139–151 in mice infected with the HI-BeAn virus (Fig. 5C) and further supports the importance of using infectious pathogens for studies of molecular mimicry. Although CD4+ T cells from HI574–586-immunized mice proliferate significantly in response to PLP139–151 rechallenge in vitro, they do not produce significant quantities of IFN-{gamma} (Fig. 5C), a Th1 cytokine thought to be important in the induction of EAE, TMEV, and MS. In addition, HI574–586-immunized mice demonstrate minimal in vivo DTH responses to PLP139–151 rechallenge compared with rechallenge with the cognate Ag (Fig. 3B).

Interestingly, in mice immunized with PLP139–151, which induces potent clinical disease, in vitro rechallenge with HI574–586 not only induces CD4+ T cell proliferation, but also significant CD4+ Th1 differentiation as measured by IFN-{gamma} secretion and DTH responses. Furthermore, double immunization with HI574–586 also failed to induce clinical disease (Fig. 2). Therefore, it appears that PLP139–151 is a stronger immunogen than HI574–586 with regard to in vivo CD4+ Th1 differentiation. Again, this may reflect the lower affinity of the HI574–586 peptide for I-As compared with that of PLP139–151, or the subpopulation hypothesis discussed above (10). Therefore, rechallenge with PLP139–151 in HI574–586-immunized mice would target all HI574–586-specific CD4+ T cells. In contrast, rechallenge with HI574–586 in PLP139–151-immunized mice would target only a small percentage of the total PLP139–151-specific CD4+ T cells.

During the disease course of EAE and TMEV-induced demyelinating disease, inflammatory infiltrates consisting mainly of CD4+ T cells and F4/80+ monocytes can be observed in the white matter of the spinal cord, which have been shown to correlate with clinical disease severity (24). Therefore, we studied the inflammatory infiltrate present in the spinal cord and brain of mice immunized with HI574–586/CFA. Throughout the spinal cord, there was little presence of inflammatory infiltrates, consistent with the lack of clinical disease. However, CD4+ T cells and F4/80+ monocytes/macrophages were present in the cerebellum of HI574–586-, but not OVA323–339-immunized mice. This suggests that the cerebellum may be the primary CNS entry point for activated cells from the periphery. However, HI574–586-specific T cells may lack sufficient affinity to PLP139–151 in vivo to allow further activation in the CNS. Therefore, in the absence of activated resident CNS APC, such as microglia, myelin epitopes may be presented in a noninflammatory context, i.e., with minimal costimulatory molecule expression. This may have a negative impact on the cascade of progressive events leading to severe clinical disease, such as chemokine secretion, adhesion molecule expression, and secondary recruitment of inflammatory cells. This is in contrast to the situation of potent induction of PLP139–151-directed autoimmunity in HI-BeAn-infected mice, where the cross-activated CD4+ T cells secrete high levels of IFN-{gamma}. In addition, they also encounter the CNS in an inflammatory state due to the presence of numerous innate immune molecules and up-regulation of MHC and costimulatory molecules directly induced by TMEV infection of CNS-resident microglia and astrocytes (32).

Activation of innate immune responses by TMEV appears to be a critical determinant in explaining the difference in disease susceptibility between the mimic peptide immunization vs infection paradigms. It is likely that the type of virus encoding the mimic epitope, and its cell tropism, are important factors in setting the threshold for disease susceptibility upon infection with pathogens encoding molecular mimics. Previous reports have demonstrated that a vaccinia virus encoding the whole PLP construct failed to induce clinical disease in SJL/J mice, although mice were more susceptible to later induction of PLP139–151-induced EAE (33). In contrast, infection of mice with vaccinia virus engineered to express an encephalitogenic epitope of MPB protected mice from EAE induction by subsequent MPB immunization (34). Vaccinia virus was administered either by i.p. injection or by tail scarification (34). It is likely that different cell types will be exposed to the virus by these routes, which may influence the ability of the pathogen to induce clinical disease. Vaccinia virus can infect many cell types, which may lack APC function and costimulatory molecule expression, as opposed to TMEV, which appears to have tropism for APCs. We have recently shown that TMEV infection of microglia, which normally do not express MHC or costimulatory molecules, induces these cells to up-regulate type I IFNs, IL-12, IL-18, MHC class I and II, and costimulatory molecules and to acquire Ag presentation function (32).

From these studies, it is possible to draw parallels to benign MS, where patients experience a single clinical episode with little or no further exacerbation. Therefore, infection of the target organ with a mimic-expressing virus during childhood may activate a population of autoreactive T cells and induce no clinical signs or only mild symptoms. Various risk factors have been described, which are thought to be involved in inducing or exacerbating relapses in MS patients, such as stress, trauma, elevated temperatures, infections, and vaccination (35). Therefore, it is tempting to speculate that a normally innocuous stimulus later in life may reactivate those autoreactive T cells to induce severe clinical disease (10). To test this hypothesis, mice were preinfected with HI-BeAn, and immunized 14 days later with either HI574–586 or OVA323–339. Mice preinfected with HI-BeAn and immunized with HI574–586 developed a more severe disease than mice infected with HI-BeAn alone, or those later immunized with OVA323–339. This suggests that induction of severe disease requires a secondary stimulus containing the original mimic. In addition, preinfection with a persistent virus that expresses a nonself, nonmimic epitope (OVA-BeAn) or a transient infection with {Delta}Cla-BeAn, which fails to persist in the CNS, did not predispose mice to a more severe disease upon HI574–586 immunization (data not shown). The exacerbated disease was associated with an increase in HI574–586-specific T cell proliferation and IFN-{gamma} secretion, and increased numbers of CD4+ T cells in both the thoracic and lumbar regions of spinal cord. In addition, significant numbers of CD4+ T cells and F4/80+ monocytes/macrophages were observed in the white matter of the brainstem adjacent to the cerebellum and in the main region of the brainstem of these mice, in contrast to mice infected with HI-BeAn and immunized against OVA323–339. However, disease in mice infected with HI-BeAn and subsequently immunized against HI574–586 was slightly less severe than in mice infected with PLP-BeAn alone. This may be due to the differential effects of the HI peptide on the immune system as described above.

This study demonstrates the cross-reactive potential of a myelin mimic sequence from an infectious pathogen, delivered by a recombinant neurotropic virus, and suggests that molecular mimicry is a potential mechanism for the induction of a T cell-mediated CNS autoimmune disease. This initial response can be further exacerbated following reactivation of the autoreactive T cells. In contrast, immunization with the identical mimic peptide multiple times in CFA failed to induce clinical disease highlighting the importance of studying molecular mimics in the context of innate immune and other stimuli present during ongoing infection. Collectively, these data suggest that molecular mimicry could be an important factor in the pathogenesis of some forms of MS by expanding a population of memory T cells that cross-react with myelin epitopes. The autoreactive cells may then be further expanded later in life by a number of differing stimuli, which could include reinfection with the same mimic-expressing virus. Alternatively, other unrelated stimuli, which normally may be nonpathogenic, may cause the restimulation of the autoreactive T cells, either specifically or by a bystander mechanism. Therefore, we are currently studying the effect of repeated viral infections of the CNS and/or the periphery either by viruses containing mimic or irrelevant Ags. In addition, the need for the mimic-expressing virus to persist in the CNS is also being addressed.


   Acknowledgments
 
We thank Dr. Kaori Sakuishi (National Center of Neurology and Psychiatry, Tokyo, Japan) for consultation on and interpretation of the immunohistochemical data.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by U.S. Public Health Service Grants NS-40460 and NS-23349. J.L.C. is a fellow of the National Multiple Sclerosis Society (Postdoctoral Research Fellowship Award FG-1456-A-1). Back

2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: s-d-miller{at}northwestern.edu Back

3 Abbreviations used in this paper: MS, multiple sclerosis; MBP, myelin basic protein; PLP, proteolipid protein; HHV-6, human herpesvirus-6; TMEV, Theiler’s murine encephalomyelitis virus; EAE, experimental autoimmune encephalomyelitis; WT, wild type; HI, Haemophilus influenzae; i.c., intracerebral; DTH, delayed-type hypersensitivity. Back

4 J. L. Croxford, J. K. Olson, H. A. Anger, and S. D. Miller. Initiaiton and exacerbation of CNS autoimmune demyelination via virus-induced molecular mimicry: implicaitons for MS pathogenesis. Submitted for publication. Back

Received for publication March 16, 2004. Accepted for publication November 8, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kurtzke, J. F.. 1993. Epidemiologic evidence for multiple sclerosis as an infection. Clin. Microbiol. Rev. 6:382.[Abstract/Free Full Text]
  2. ter Meulen, V., M. Katz. 1997. The proposed viral etiology of multiple sclerosis and related demyelinating disease. C. S. Raine, and H. F. McFarland, and W. W. Tourtellotte, eds. Multiple Sclerosis: Clinical and Pathogenetic Basis 287. Chapman and Hall, London.
  3. Padgett, B. L., D. L. Walker. 1976. New human papovaviruses. Prog. Med. Virol. 22:1.[Medline]
  4. Osame, M., K. Usuku, S. Izumo, N. Ijichi, H. Amitani, A. Igata, M. Matsumoto, M. Tara. 1986. HTLV-I associated myelopathy, a new clinical entity. Lancet 1:1031.[Medline]
  5. Gessain, A., F. Barin, J. C. Vernant, O. Gout, L. Maurs, A. Calender, G. de The. 1985. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet 2:407.[Medline]
  6. Olsson, T., J. Sun, J. Hillert, B. Hojeberg, H. P. Ekre, G. Andersson, O. Olerup, H. Link. 1992. Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis. Eur. J. Immunol. 22:1083.[Medline]
  7. Scholz, C., K. T. Patton, D. E. Anderson, G. J. Freeman, D. A. Hafler. 1998. Expansion of autoreactive T cells in multiple sclerosis is independent of exogenous B7 costimulation. J. Immunol. 160:1532.[Abstract/Free Full Text]
  8. de Rosbo, N. K., M. Hoffman, I. Mendel, I. Yust, J. Kaye, R. Bakimer, S. Flechter, O. Abramsky, R. Milo, A. Karni, A. Ben-Nun. 1997. Predominance of the autoimmune response to myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis: reactivity to the extracellular domain of MOG is directed against three main regions. Eur. J. Immunol. 27:3059.[Medline]
  9. Fujinami, R. S., M. B. Oldstone. 1985. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science 230:1043.[Abstract/Free Full Text]
  10. Croxford, J. L., J. K. Olson, S. D. Miller. 2002. Epitope spreading and molecular mimicry as triggers of autoimmunity in the Theiler’s virus induced demyelinating disease model of multiple sclerosis. Autoimmun. Rev. 1:251.[Medline]
  11. Tejada-Simon, M. V., Y. C. Zang, J. Hong, V. M. Rivera, J. Z. Zhang. 2003. Cross-reactivity with myelin basic protein and human herpesvirus-6 in multiple sclerosis. Ann. Neurol. 53:189.[Medline]
  12. Lipton, H. L.. 1975. Theiler’s virus infection in mice: an unusual biphasic disease process leading to demyelination. Infect. Immun. 11:1147.[Abstract/Free Full Text]
  13. Miller, S. D., S. J. Gerety. 1990. Immunologic aspects of Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease. Semin. Virol. 1:263.
  14. Miller, S. D., C. L. Vanderlugt, W. S. Begolka, W. Pao, R. L. Yauch, K. L. Neville, Y. Katz-Levy, A. Carrizosa, B. S. Kim. 1997. Persistent infection with Theiler’s virus leads to CNS autoimmunity via epitope spreading. Nat. Med. 3:1133.[Medline]
  15. Karpus, W. J., J. G. Pope, J. D. Peterson, M. C. Dal Canto, S. D. Miller. 1995. Inhibition of Theiler’s virus-mediated demyelination by peripheral immune tolerance induction. J. Immunol. 155:947.[Abstract]
  16. Vanderlugt, C. L., S. D. Miller. 2002. Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat. Rev. Immunol. 2:85.[Medline]
  17. Wucherpfennig, K. W., J. L. Strominger. 1995. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80:695.[Medline]
  18. Ufret-Vincenty, R. L., L. Quigley, N. Tresser, S. H. Pak, A. Gado, S. Hausmann, K. W. Wucherpfennig, S. Brocke. 1998. In vivo survival of viral antigen-specific T cells that induce experimental autoimmune encephalomyelitis. J. Exp. Med. 188:1725.[Abstract/Free Full Text]
  19. Carrizosa, A. M., L. B. Nicholson, M. Farzan, S. Southwood, A. Sette, R. A. Sobel, V. K. Kuchroo. 1998. Expansion by self antigen is necessary for the induction of experimental autoimmune encephalomyelitis by T cells primed with a cross-reactive environmental antigen. J. Immunol. 161:3307.[Abstract/Free Full Text]
  20. Gautam, A. M., R. Liblau, G. Chelvanayagam, L. Steinman, T. Boston. 1998. Viral peptide with limited homology to a self peptide can induce clinical signs of experimental autoimmune encephalomyelitis. J. Immunol. 161:60.[Abstract/Free Full Text]
  21. Olson, J. K., J. L. Croxford, M. Calenoff, M. C. Dal Canto, S. D. Miller. 2001. A virus-induced molecular mimicry model of multiple sclerosis. J. Clin. Invest. 108:311.[Medline]
  22. Lipton, H. L., J. Kratochvil, P. Sethi, M. C. Dal Canto. 1984. Theiler’s virus antigen detected in mouse spinal cord 21/2 years after infection. Neurology 34:1117.[Abstract/Free Full Text]
  23. Olson, J. K., T. N. Eagar, S. D. Miller. 2002. Functional activation of myelin-specific T cells by virus-induced molecular mimicry. J. Immunol. 169:2719.[Abstract/Free Full Text]
  24. Begolka, W. S., C. L. Vanderlugt, S. M. Rahbe, S. D. Miller. 1998. Differential expression of inflammatory cytokines parallels progression of central nervous system pathology in two clinically distinct models of multiple sclerosis. J. Immunol. 161:4437.[Abstract/Free Full Text]
  25. McRae, B. L., S. D. Miller. 1994. Fine specificity of CD4+ T cell responses to the dominant encephalitogenic PLP 139–151 peptide in SJL/J mice. Neurochem. Res. 19:997.[Medline]
  26. Kuchroo, V. K., J. M. Greer, D. Kaul, G. Ishioka, A. Franco, A. Sette, R. A. Sobel, M. B. Lees. 1994. A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T cell repertoire. J. Immunol. 153:3326.[Abstract]
  27. Hemmer, B., B. T. Fleckenstein, M. Vergelli, G. Jung, H. McFarland, R. Martin, K. H. Wiesmuller. 1997. Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone. J. Exp. Med. 185:1651.[Abstract/Free Full Text]
  28. Gran, B., B. Hemmer, M. Vergelli, H. F. McFarland, R. Martin. 1999. Molecular mimicry and multiple sclerosis: degenerate T-cell recognition and the induction of autoimmunity. Ann. Neurol. 45:559.[Medline]
  29. Zhao, Z. S., F. Granucci, L. Yeh, P. A. Schaffer, H. Cantor. 1998. Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection. Science 279:1344.[Abstract/Free Full Text]
  30. Panoutsakopoulou, V., M. E. Sanchirico, K. M. Huster, M. Jansson, F. Granucci, D. J. Shim, K. W. Wucherpfennig, H. Cantor. 2001. Analysis of the relationship between viral infection and autoimmune disease. Immunity 15:137.[Medline]
  31. Bachmaier, K., N. Neu, L. M. de la Maza, S. Pal, A. Hessel, J. M. Penninger. 1999. Chlamydia infections and heart disease linked through antigenic mimicry. Science 283:1335.[Abstract/Free Full Text]
  32. Olson, J. K., A. M. Girvin, S. D. Miller. 2001. Direct activation of innate and antigen presenting functions of microglia following infection with Theiler’s virus. J. Virol. 75:9780.[Abstract/Free Full Text]
  33. Barnett, L. A., J. L. Whitton, Y. Wada, R. S. Fujinami. 1993. Enhancement of autoimmune disease using recombinant vaccinia virus encoding myelin proteolipid protein. J. Neuroimmunol. 44:15.[Medline]
  34. Barnett, L. A., J. L. Whitton, L. Y. Wang, R. S. Fujinami. 1996. Virus encoding an encephalitogenic peptide protects mice from experimental allergic encephalomyelitis. J. Neuroimmunol. 64:163.[Medline]
  35. Whitaker, J. N., G. W. Mitchell. 1997. Clinical features of multiple sclerosis. C. S. Raine, and H. F. McFarland, and W. W. Tourtellotte, eds. Multiple Sclerosis: Clinical and Pathogenetic Basis 3. Chapman and Hall, London.



This article has been cited by other articles:


Home page
 J. Exp. Med.Home page
M. J. Turner, E. R. Jellison, E. G. Lingenheld, L. Puddington, and L. Lefrancois
Avidity maturation of memory CD8 T cells is limited by self-antigen expression
J. Exp. Med., July 14, 2008; (2008) jem.20072390.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Sanchez-Ruiz, L. Wilden, W. Muller, W. Stenzel, A. Brunn, H. Miletic, D. Schluter, and M. Deckert
Molecular Mimicry between Neurons and an Intracerebral Pathogen Induces a CD8 T Cell-Mediated Autoimmune Disease
J. Immunol., June 15, 2008; 180(12): 8421 - 8433.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
M. Holdener, E. Hintermann, M. Bayer, A. Rhode, E. Rodrigo, G. Hintereder, E. F. Johnson, F. J. Gonzalez, J. Pfeilschifter, M. P. Manns, et al.
Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection
J. Exp. Med., June 9, 2008; 205(6): 1409 - 1422.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. M. Ercolini and S. D. Miller
Molecular Mimics Can Induce Novel Self Peptide-Reactive CD4+ T Cell Clonotypes in Autoimmune Disease
J. Immunol., November 15, 2007; 179(10): 6604 - 6612.
[Abstract] [Full Text] [PDF]