Receptor 6-Deficient Mice Autoimmune Encephalomyelitis by Death ...

The Journal of Immunology

Resistance to Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis by Death Receptor 6-Deficient Mice Clint S. Schmidt,1,2* Jingyong Zhao,1,* Jana Chain,* Deena Hepburn,† Bruce Gitter,† George Sandusky,‡ Subba Chintalacharuvu,* Andrew Glasebrook,* and Songqing Na3* Genetic disruption of death receptor 6 (DR6) results in enhanced CD4ⴙ T cell expansion, Th2 differentiation, and humoral responses after stimulation. However, the in vivo consequences of DR6 targeting (DR6ⴚ/ⴚ) during the initiation and progression of inflammatory autoimmune disease are unclear. Using a myelin oligodendrocyte glycoprotein (MOG35–55)induced model of experimental autoimmune encephalomyelitis, DR6ⴚ/ⴚ mice were found to be highly resistant to both the onset and the progression of CNS disease compared with wild-type (WT) littermates. DR6ⴚ/ⴚ mice exhibited fewer inflammatory foci along with minimal demyelination and perivascular cuffing of inflammatory cells. Consistent with these observations, mononuclear cell infiltration, including CD4ⴙ T cells and macrophages, in the spinal cord of DR6ⴚ/ⴚ mice was dramatically reduced. Furthermore, CD4ⴙ T cells from DR6ⴚ/ⴚ mice exhibited profoundly reduced cell surface expression of VLA-4 before and after stimulation. Compared with WT mice, DR6ⴚ/ⴚ mice exhibited significantly increased autoantigeninduced T cell proliferative responses along with greater numbers of IL-4-producing and similar or slightly higher numbers of IFN-␥-producing CD4ⴙ T cells. DR6ⴚ/ⴚ CD4ⴙ T cells secreted higher levels of the Th2 cytokine, IL-4, and similar levels of the Th1 cytokine, IFN-␥, compared with WT cells. Taken together, our data demonstrate that DR6 plays an important role in regulating leukocyte infiltration and function in the induction and progression of experimental autoimmune encephalomyelitis. The Journal of Immunology, 2005, 175: 2286 –2292.

M

ultiple sclerosis (MS)4 is an autoimmune demyelinating disease characterized by inappropriate host immune responses to CNS Ags (1). Although the etiology of MS is unclear, the use of murine experimental autoimmune encephalomyelitis (EAE) models has emphasized the role of inflammatory mediators and CD4⫹ Th1 cells during both the induction and the progression of the disease (2, 3). In addition, there is increasing awareness that certain members of the TNF/TNFR superfamily play a critical role(s) in autoimmune disease by regulating inflammation and lymphoid tissue homeostasis (4, 5). Specific TNF members, such as lymphotoxin-␣, or pairs of TNF/ TNFR family members, CD40/CD154 and CD134 (OX40)/ CD134L, have been shown to be critical for the development of EAE (6 –11). Despite these previous studies, the complete identities of factors required for autoimmune disease induction and progression remain to be elucidated. Death receptor 6 (DR6) is a member of the TNFR family that is expressed in lymphoid tissues and contains a cytoplasmic death *Stress and Immune Response, †Neuroscience Research, and ‡BioTechnology Discovery Research, Lilly Research Laboratories, Eli Lilly, Indianapolis, IN 46285 Received for publication October 27, 2004. Accepted for publication May 9, 2005. 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

C.S.S. and J.Z. contributed equally to this work.

2

Current address: Dendreon Corp., Seattle, WA 98121.

domain (12). Efforts to decipher the immunological role(s) of DR6 have focused on DR6-deficient (DR6⫺/⫺) mice, which have been shown to exhibit enhanced CD4⫹ T cell proliferation, Th2 cytokine production, and humoral responses after stimulation (13–15). Based on the immunological phenotype of DR6⫺/⫺ mice, we wanted to determine whether the absence of this receptor could influence the outcome of strongly Th1-based EAE development. To address these issues, a myelin oligodendrocyte glycoprotein (MOG35–55)-induced model of EAE was performed on DR6⫺/⫺ and wild-type (WT) littermates (6, 16, 17). We found that DR6⫺/⫺ mice respond to the initial immunogen, although they are highly resistant to the development of CNS pathology. DR6⫺/⫺ mice exhibited reduced mononuclear cell infiltration, including CD4⫹ T cells, inflammation, and demyelination (DM) in the brain and spinal cord. DR6⫺/⫺ splenocytes respond robustly to ex vivo restimulation and show a significantly greater number of IL-4-producing cells that secrete higher amounts of IL-4, with little or no difference in IFN-␥ compared with WT cells. Lastly, MOG35–55-activated DR6⫺/⫺ CD4⫹ T cells were incapable of transferring disease to naive WT recipients. Conversely, activated WT CD4⫹ T cells were capable of transferring disease to naive DR6⫺/⫺ mice, suggesting that the resistance to EAE by DR6⫺/⫺ mice is due to impaired cell infiltration of CD4⫹ T cells into the CNS, consistent with a reduced expression of VLA-4 observed on DR6⫺/⫺ CD4⫹ T cells. Based on these observations, DR6 appears to be another member of the TNFR superfamily that is required for the establishment of inflammatory CNS autoimmune disease.

3

Address correspondence to Dr. Songqing Na, Stress and Immune Response, Lilly Corporate Center, Eli Lilly, Indianapolis, IN 46285. E-mail address: na_songqing@ lilly.com 4

Abbreviations used in this paper: MS, multiple sclerosis; DM, demyelination; DR6, death receptor 6; EAE, experimental autoimmune encephalomyelitis; FI, focal inflammation; MFI, multifocal inflammation; MOG, myelin oligodendrocyte glycoprotein; PVC, perivascular cuffing with inflammatory cell; WT, wild type. Copyright © 2005 by The American Association of Immunologists, Inc.

Materials and Methods Mice The generation and maintenance of DR6⫺/⫺ mice along with WT littermates were previously described (13). All animals were kept in American Association for Accreditation of Laboratory Animal Care-accredited, 0022-1767/05/$02.00

The Journal of Immunology pathogen-free facilities and provided standard laboratory diet and water ad libitum.

Induction of EAE Six- to 10-wk-old female DR6⫺/⫺ and WT mice were immunized by s.c. administration at two sites on the back of each mouse with 300 ␮g of MOG35–55 (MEVGWYRSPFSRVVHLYRNGK; Peptides International) emulsified in a total of 200 ␮l of CFA (Difco) containing 500 ␮g of Mycobacterium tuberculosis H37 Ra (Difco) on days 0 and 7, supplemented with i.p. injections of 500 ng of pertussis toxin (Calbiochem) on days 0 and 2 (6). Randomized mice were observed daily for clinical symptoms of EAE and were scored independently by two investigators in a blinded manner for severity using the following scale: 0 ⫽ no symptoms, 0.5 ⫽ distal weak or spastic tail, 1 ⫽ completely limp tail, 1.5 ⫽ limp tail and hind limb weakness (feet slip through cage grill), 2.0 ⫽ unilateral partial hindlimb paralysis, 2.5 ⫽ bilateral partial hindlimb paralysis, 3.0 ⫽ complete bilateral hindlimb paralysis, 3.5 ⫽ complete hindlimb and unilateral partial forelimb paralysis, and 4.0 ⫽ moribund or death.

Histology, immunohistochemistry, and tissue pathology MOG35–55-immunized DR6⫺/⫺ and WT mice were killed on days 22–28, and brains and spinal cords were removed, fixed overnight in immunohistochemical staining zinc fixative (BD Pharmingen) solution, and then transferred to 70% ethanol before processing through paraffin. Five- to 7-␮m sections were generated by a microtome, and sections were placed on positively charged slides. Slides were baked overnight at 60°C, deparaffinized in xylene, and rehydrated through graded alcohols to water (18). Rehydrated tissue sections were immersed in Luxol Fast Blue 0.1% alcoholic (Poly Scientific R&D) solution at 56°C for 24 h, followed by washing with distilled water and differentiation with 0.05% lithium carbonate, and then were counterstained with Cresyl Echt Violet 0.1% aqueous and dehydrated through graded alcohols before permanent mounting and light microscope examination (19). H&E (Sigma-Aldrich) staining was performed according to a standard protocol (18). For immunohistochemical staining, sections were deparaffinized, rehydrated, and quenched for endogenous peroxidase activity with 1% H2O2/ 0.1% Triton X-100 and then blocked with 15% normal rabbit serum. Endogenous biotin was blocked with an avidin/biotin blocking kit (Vector Laboratories). Sections were incubated overnight at 4°C with primary Ab at an optimal concentration prepared in Ab diluent (DakoCytomation). Slides were then incubated with biotinylated rabbit anti-rat IgG (Vector Laboratories). For CD45 (Serotec) and I-A/I-E (BD Pharmingen) staining, sections were additionally processed with the ABC Elite-HRP complex (Vector Laboratories). For CD4 (Serotec) and CD11b (Serotec) staining, sections were incubated with streptavidin-HRP and amplified with thymic shared Ag, followed by incubation with streptavidin-HRP. Color development was performed using the 3,3⬘-diaminobenzidine substrate kit for peroxidase (Vector Laboratories), according to the kit protocol. All wash steps between incubations were performed with TBS/0.5% Tween 20 (TBS-T). Slides were counterstained with Nuclear Fast Red (Vector Laboratories), dehydrated, and mounted with Clearium (Surgipath). For the negative control, equivalent concentrations of rat isotype control IgG2b (Serotec) were used to replace the primary Ab and were processed according to the above protocol. Randomized samples of brain and spinal cord from DR6⫺/⫺ and WT mice were prepared as described above and analyzed under light microscopy for focal inflammation (FI), multifocal inflammation (MFI), DM, and perivascular cuffing with inflammatory cells (PVC). The severity of the above parameters was graded as follows: 0 ⫽ normal, 1 ⫽ minimal, 2 ⫽ moderate, and 3 ⫽ severe.

Flow cytometric analysis Cell subset analysis was performed by preparing cell suspensions from RBC-lysed spleen and lymph nodes. Cells were suspended (1–2 ⫻ 106 cells/sample) in PBS containing 0.1% BSA (fraction V; Invitrogen Life Technologies) and initially blocked with Fc Block (BD Pharmingen) at 4°C for 30 min. CD4⫹ T cells were purified by negative selection using autoMACS (Miltenyi Biotec). For VLA-4 and ICAM-1 expression analysis, cells were first activated in 96-well tissue culture plates coated with antiCD3 Ab (2C11; 1 ␮g/ml; BD Pharmingen) for 72 h. The cell surface expression of integrins was examined by flow cytometry. For analysis of spinal cord mononuclear cell infiltrates, mononuclear cells from the spinal cords of the mice were isolated by Percoll gradient centrifugation with modifications (20, 21). Briefly, pooled spinal cords were dissociated by glass homogenization in ice-cold GKN buffer (8.00 g/l NaCl, 0.40 g/l KCl, 3.56 g/l Na2HPO4䡠12H2O, 0.78 g/l NaH2PO4䡠2H2O, and 2 g/l D-(⫹)-glucose (pH 7.4)) with 0.02% BSA. Dissociated tissues were washed once in

2287 GKN/BSA and enzymatically digested for 30 min at 37°C with 250 ␮g/ml type II collagenase (Sigma-Aldrich) (21). The digested spinal cord preparation was passed through a 40-␮m pore size cell strainer, washed once with GKN/BSA, and resuspended in 30% isotonic Percoll (Pharmacia Biotech). The suspension was fractionated with a 30:37:70% Percoll gradient by centrifugation at 500 ⫻ g for 30 min at 25°C. Cells were collected from the 37:70% interface and washed with PBS containing 2% FCS and 0.1% BSA. All flow cytometric data were collected and analyzed with a Cytomics FC500 (Beckman Coulter) using RXP software (Beckman Coulter). The following staining Abs were purchased from BD Pharmingen: antiCD45-CyChrome (20-F1), anti-CD4-PE (L3T4), anti-CD8a-FITC (53-6.7), anti-CD54-FITC (3E2), and isotype control Abs (rat anti-mouse IgG-FITC and rat anti-mouse IgG-PE). Anti-CD11b-PE (M1/70) and antiCD49d-PE (PS2) were purchased from Biolegend. F4/80-FITC (MCA497F) was purchased from Serotec.

Splenocyte cell culture and proliferation assay Splenocyte cell suspensions were isolated from MOG35–55-immunized WT and DR6⫺/⫺ mice on day 28, along with naive WT mice, by homogenizing spleens between frosted glass slides (Fisher Scientific) and removing RBC with ACK lysing buffer (BioWhittaker). Pooled splenocytes from six individual mice from the same group were plated in triplicate in 96-well, round-bottom plates at 2 ⫻ 105 cells/well in 200 ␮l of complete RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 2 mM L-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 5.5 ⫻ 10⫺5 M 2-ME, and 5% FCS (all supplements from Invitrogen Life Technologies) containing either 0 –50 ␮g/ml MOG35–55 (Peptides International) or control OVA323–339 peptide (ISQAVHAAHAEINEAGR; Research Genetics) and cultured at 37°C in 5% CO2. Proliferation was measured by incorporation of [methyl-3H]thymidine (1 ␮Ci/well; ICN Radiochemicals) during the last 8 h of culture using a filtermate harvester (Packard Instruments) and a 1450 Microbeta liquid scintillation counter (Pharmacia Biotech).

ELISPOT and cytokine analysis ELISPOT was performed using mouse IL-4 (catalog no. EL404) and mouse IFN-␥ (catalog no. EL485) ELISPOT kits according to the manufacturer’s instructions (R&D Systems). Basically, the freshly isolated splenocyte cells from immunized WT and DR6⫺/⫺ mice were plated in HL-1/1% glutamine-supplemented medium at 1 ⫻ 106/well, with or without MOG35–55 at 0, 5, and 50 ␮g/ml. The plates were incubated at 37°C in 5% CO2 for 24 h (IFN-␥) and 48 h (IL-4). The resulting spots were counted with an ImmunoSpot Analyzer (Cellular Technology) specifically designed for the ELISPOT assay. Cytokine levels produced by cultured splenocytes from MOG35–55-immunized DR6⫺/⫺ and WT mice along with naive WT spleens were analyzed by removing 100 ␮l of cell culture supernatant/well after 60 h of culture as described above. Supernatants were filtered using Millipore plates (Mabvnob) and stored at ⫺80°C. Cytokines were analyzed with a R&D Systems Multianalyte profiling kit (catalog no. LUM000) with the following beads: IL-4 (catalog no. LUM413) and IFN-␥ (catalog no. LUM485).

Passive EAE transfer Age-matched female WT and DR6⫺/⫺ mice were immunized with 300 ␮g of MOG35–55 in CFA. On days 15–20, mice were killed, and spleens and lymph nodes were removed. CD4⫹ T cells were enriched by positive selection using magnetic anti-CD4 microbeads and an autoMACS magnetic cell sorter (Miltenyi Biotec). The purity of isolated CD4⫹ T cells was 93–94%, determined by flow cytometry. Purified T cells were cultured in 1 ml (5 ⫻ 105 cells/well) of a Costar 24-well tissue culture plate (Corning Glass) in complete RPMI 1640 medium at 37°C in 5% CO2 with 5 ⫻ 105 irradiated (3000 rad from a 137Cs source at 250 rad/min) WT T cell-depleted splenocytes that had been pulsed with 10 ␮g/ml MOG35–55. After 48 h, 100 U/ml rhIL-2 (R&D Systems) was added to each well, followed by incubation for another 48 h. After MOG35–55 stimulation, CD4⫹ T cells were harvested and washed twice with Ca2⫹- and Mg2⫹-free PBS, and 1 ⫻ 107 cells were injected i.v. into either WT or DR6⫺/⫺ naive recipient mice. Recipient mice were given 500 ng of pertussis toxin i.p. on days 0 and 2 of adoptive transfer and were monitored daily for symptoms of EAE as described above.

Statistical analysis Disease scores were analyzed using repeated measures ANOVA (SAS version 8.1; SAS Institute). A statistically significant difference in disease progression between two groups corresponds to p ⬍ 0.05 for a two-sided

2288 significance test. For cell proliferation and cytokine analyses, log values were analyzed by JMP using Tukey’s method.

Results

DR6⫺/⫺ mice are resistant to MOG35–55-induced EAE Because previous studies suggest that DR6 plays a regulatory role in CD4⫹ Th2 cell and humoral immune responses, we investigated whether targeted disruption of this gene would also influence the outcome of Th1-mediated autoimmune diseases such as EAE (3, 13–15). A MOG35–55-induced EAE was chosen for experiments in DR6⫺/⫺ mice as a human MS model in which CD4⫹ T cells play critical roles in the induction and progression of the disease (16, 17, 22, 23). As depicted in Fig. 1, MOG35–55-immunized DR6⫺/⫺ mice failed to develop any severe EAE symptoms throughout the course of experiments up to 28 days after immunization. The resistance to EAE induction in DR6⫺/⫺ mice was also observed up to 45 days after immunization (data not shown). This contrasts with identically treated WT mice, which exhibited severe paralytic symptoms that peaked between days 20 and 25. Mice, either WT or DR6⫺/⫺, immunized with CFA alone displayed no EAE symptoms (data not shown). To visualize the histopathological features of these phenomena, brains and spinal cords from representative mice in each group were removed and analyzed for cellular infiltration and tissue destruction. WT mice exhibited massive infiltration into myelinated areas throughout the brain from Bregma 2.34 to Bregma ⫺5.68 (Fig. 2A and data not shown) and perivascular regions of the spinal cord with profound DM, whereas tissue samples from DR6⫺/⫺ mice were similar to those from mice that were either not immunized or had just received CFA alone (Fig. 2A and data not shown). Consistent with these observations, flow cytometric analysis of spinal cord mononuclear cell infiltrates showed that DR6⫺/⫺ mice had a ⬎12-fold reduction of CD4⫹ T cells compared with WT mice (0.2 vs 2.5%; Fig. 2B, top panels). Furthermore, the number of infiltrated macrophages (CD45highF4/80⫹) was ⬎30 times higher in WT spinal cord compared with DR6⫺/⫺ (1.33 vs 0.04%; Fig. 2B, lower panels). These data suggest that lack of DR6 resulted in decreased cell infiltration during the course of the disease.

DR6 IN CNS AUTOIMMUNITY To further quantify the histopathological features depicted in Fig. 2A, brain and spinal cord tissue from cohorts (n ⫽ 6) of representative mice from each group were subjected to blinded pathology analysis. Fig. 2C depicts the number of mice in each group that had a particular severity score for the following parameters: FI, MFI, DM, and PVC. Samples from DR6⫺/⫺ mice exhibited few of these pathological parameters, with only two of six mice showing the minimal severity score of 1 with respect to FI in the brain and PVC in the spinal cord. In contrast, all samples from WT mice showed some level of severity in all parameters scored. To further analyze the cell population infiltrating the CNS of WT and DR6⫺/⫺ mice, spinal cords from both groups were subjected to immunochemistry staining. Fig. 2D illustrates a profound lack of infiltrating CD4⫹, CD45⫹, CD11b⫹, and MHC II⫹ mononuclear cells in DR6⫺/⫺ spinal cord compared with that in WT controls. These results suggest that the resistance to EAE disease by DR6⫺/⫺ mice was primarily due to defective mononuclear cell infiltration into the CNS during the progression of EAE. Specificity and magnitude of DR6⫺/⫺ responses to MOG35–55 immunization To determine whether the lack of cell infiltration into the CNS of DR6⫺/⫺ mice is due to a defective T cell response to the initial immunizations, splenocytes from MOG35–55-immunized DR6⫺/⫺ and WT mice along with age-matched naive WT mice were assayed on day 21 after immunization for their ex vivo proliferative responses to either MOG35–55 or control OVA323–339 peptides. As shown in Fig. 3A, DR6⫺/⫺ splenocytes proliferated in response to MOG35–55 restimulation to a greater level than their WT counterparts, whereas naive WT samples exhibited only background proliferation. No MOG35–55-specific responses were detected from WT and DR6⫺/⫺ mice immunized with CFA alone (data not shown). These data suggest that DR6⫺/⫺ mice had even greater responses to the initial MOG35–55 immunization and are consistent with previous studies of CD4⫹ T cell hyperproliferation from these animals (13, 14). Additionally, this enhanced recall response was peptide specific because proliferative responses to the control OVA323–339 peptide was at background levels for all cultures tested (data not shown). These results indicate that although DR6⫺/⫺ responded specifically and robustly to initial immunizations, the resulting immune response did not cause CNS inflammatory disease. Increased IL-4-producing cell frequency and augmented IL-4 production in DR6⫺/⫺ splenocyte cultures

FIGURE 1. DR6⫺/⫺ mice exhibit reduced clinical scores for MOG35– -induced EAE. Groups of DR6-deficient (DR6⫺/⫺; n ⫽ 18; F) and WT (n ⫽ 14; E) mice were immunized on days 0 and 7 with MOG35–55, as described in Materials and Methods, and were scored daily for clinical symptoms of EAE. The values shown represent the mean, and error bars represent the SEM. ⴱ, p ⬍ 0.01. The data shown are representative of three independent experiments.

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Previous studies of DR6⫺/⫺ CD4⫹ T cells have shown a pronounced enhancement of Th2 cytokine production in these mice upon stimulation (13, 14). To determine whether any differences in Th1 vs Th2 cytokine profiles may have contributed to the EAE resistance in DR6⫺/⫺ mice, we examined both IL-4- and IFN-␥producing cells by ELISPOT and their respective cytokine production by the supernatants of MOG35–55 peptide-stimulated splenocyte cultures. A significant increase in IL-4-producing cell frequency was observed on both days 14 and 21 after immunization (Fig. 3, B and C, left panel) in DR6⫺/⫺ splenocytes. In contrast, there was no statistical difference in IFN-␥-producing cell frequency, even though an increase in cell frequency was observed on postimmunization day 21 at a high concentration of MOG35–55, but did not reach statistical significance (Fig. 3, B and C, right panel). Consistent with the ELISPOT results, a marked increase in IL-4 secretion was observed in DR6⫺/⫺ cultures compared with WT cells in an Ag dose-dependent fashion, especially on day 21 (Fig. 3, D and E, left panel) without any difference in IFN-␥

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FIGURE 2. Mononuclear cell infiltration in brain and spinal cord from DR6⫺/⫺ and WT mice. A, Fixed 7-␮m sections from brain (top; magnification, ⫻20; day 22) and spinal cord (bottom; magnification, ⫻20; day 22) from both WT (left) and DR6-deficient (DR6⫺/⫺; right) mice, depicting mononuclear cell infiltration and DM. The brain sections (stained with Luxol Fast Blue and counterstained with H&E) shown are from Bregma 0.14 mm and depict the myelin of the anterior commissure, posterior. Longitudinal spinal cord sections were stained with Luxol Fast Blue, and DMs are indicated by arrows. The images shown are representative of six mice from both WT and DR6⫺/⫺ MOG35–55-immunized groups. B, Mononuclear cells were isolated from five pooled mouse spinal cords, stained with the indicated Abs, and examined by flow cytometry. The percentage of each individual cell population is indicated. Macrophages are represented as CD45highF4/80⫹ cells. The results shown are representative of three independent experiments with similar results. C, Groups (n ⫽ 6) of 6- to 10-wk-old DR6⫺/⫺ and WT littermate control mice were immunized on days 0 and 7 with MOG35–55 and were subsequently killed on day 28 for collection of brains and spinal cords as described in Materials and Methods. Randomized, blinded samples underwent subjective pathological scoring for FI, MFI, DM, and PVC. The severity of the above parameters was graded as follows: 0 ⫽ normal, 1 ⫽ minimal, 2 ⫽ moderate, and 3 ⫽ severe. Values shown with bars represent the number of mice with a particular pathology score, as indicated on the x-axis (total of six mice per graph). D, Immunohistology of spinal cord. Spinal cord sections were processed and stained with the primary Ab and subsequently with biotinylated rabbit anti-rat IgG and streptavidin-HRP. Positively stained cells are indicated by arrows.

secretion (Fig. 3, D and E, right panel). There were undetectable levels of cytokines in the naive WT cultures or when control OVA323–339 peptide was used for restimulation (data not shown).

These data suggest that DR6⫺/⫺ modulates Th2 cytokine production, which is important in regulating disease induction and progression (24, 25).

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DR6 IN CNS AUTOIMMUNITY

FIGURE 3. Peptide-specific recall responses from MOG35–55-immunized DR6⫺/⫺ and WT mice. A, Splenocytes from groups (n ⫽ 6) of MOG35–55-immunized DR6-deficient (DR6⫺/⫺) and WT mice along with unimmunized WT mice (WT Naive) were harvested on day 21 and analyzed ex vivo for proliferative responses to MOG35–55 at the concentrations indicated. Cells were cultured in triplicate in 96-well plates for 60 h, and proliferation was measured by [3H]thymidine incorporation, expressed as counts per minute (cpm), during the final 8 h of culture. The values shown represent the mean of triplicate determinations for each peptide concentration, and error bars represent the SEM. ⴱ, p ⬍ 0.05. The data shown are representative of three independent experiments. B and C, Splenocytes from groups (n ⫽ 6) of MOG35–55-immunized, DR6-deficient (DR6⫺/⫺) and WT mice were harvested on day 14 (B) and day 21 (C) and were analyzed ex vivo for ELISPOT in the presence of MOG35–55 at the concentrations indicated. Each point was determined in triplicate, and the value shown at each concentration of MOG35–55 represents the mean frequency of IL-4- and IFN-␥-producing cells; the error bars represent the SEM. ⴱ, p ⬍ 0.05. D and E, IL-4 and IFN-␥ production analysis of splenocyte culture. Splenocytes from groups (n ⫽ 6) of MOG35–55-immunized, DR6-deficient (DR6⫺/⫺) and WT mice were harvested on day 14 (D) and day 21 (E) and were analyzed ex vivo for IL-4 and IFN-␥ production in response to MOG35–55 restimulation at the indicated concentrations. Cells were cultured in triplicate in 96-well plates for 60 h, and culture supernatant levels for IL-4 and IFN-␥ were determined as described in Materials and Methods. Values shown represent the mean of triplicate determinations, and error bars represent the SEM. ⴱ, p ⬍ 0.05.

DR6⫺/⫺ CD4⫹ T cells express lower levels of cell surface VLA-4 An important event in the pathogenesis of MS is the recruitment of pathogenic lymphocytes and inflammatory macrophages to the CNS. Previous studies have shown that integrins on lymphocyte, such as VLA-4 and ICAM-1, are involved in EAE induction and progression (26, 27). To test whether the resistance to EAE by DR6⫺/⫺ mice was due to the effect of T cell integrin expression, we examined the cell surface level of VLA-4 and ICAM-1 on CD4⫹ T cells by flow cytometry before and after in vitro stimulation. As shown in Fig. 4, cell surface expression of VLA-4 (CD49d) was profoundly reduced on DR6⫺/⫺ cells compared with WT cells. On day 0 before stimulation, 53.3% of WT CD4⫹ T cells were VLA-4⫹, compared with only 37.4% of DR6⫺/⫺ CD4⫹ T cells. After 72 h of stimulation in vitro, the percentage of WT CD4⫹ T cells that were VLA-4⫹ was increased to 83.5%, whereas DR6⫺/⫺ CD4⫹ T cells were profoundly lower than WT (increased only to 58.2%), even though both WT and DR6⫺/⫺ CD4⫹ T cells increased VLA-4 expression after activation (30 and 20.8%, respectively; Fig. 4, left panels). Compared with VLA-4 expression,

there was no difference in cell surface expression of ICAM-1 (CD54) between WT and DR6⫺/⫺ T cells after anti-CD3 stimulation, although DR6⫺/⫺ T cells exhibited slightly higher levels before stimulation by anti-CD3 (Fig. 4, right panels). In contrast to the decreased expression of VLA-4 on DR6⫺/⫺ T cells, CD44 and CD25 expressions were increased on DR6⫺/⫺ CD4⫹ T cells after stimulation (13) (data not shown). These data suggest that DR6 may regulate VLA-4 expression on activated CD4⫹ T cells and, consequently, could partially explain the reduction of T cell infiltration into the CNS of DR6⫺/⫺ mice (Fig. 2, B and D). MOG35–55-activated DR6⫺/⫺ CD4⫹ T cells fail to passively transfer EAE disease After T cell activation and differentiation, mechanisms of EAE disease development include the migration of these T cells and macrophages into the CNS (28). The data presented above, however, clearly show that DR6⫺/⫺ mice responded to the MOG35–55 immunogen without the development of EAE. Therefore, it was possible that in this particular knockout mouse, some as yet

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FIGURE 4. Reduced VLA-4 expression on CD4⫹ T cells in DR6⫺/⫺ mice after stimulation. Purified CD4⫹ T cells (4 ⫻ 105) from both WT and DR6⫺/⫺ mouse splenocytes were cultured for 72 h in 96-well tissue culture plates that were precoated with anti-CD3 Ab. Before and after stimulation, cells were washed and stained with anti-CD49d-PE (VLA-4) (A), CD54-FITC (ICAM-1) (B), and control isotype Ab (PE or FITC conjugated). The stained cells were analyzed with flow cytometry (Cytomics FC500; Beckman Coulter). The data shown are representative of three independent experiments.

unidentified anatomical factor(s) prevents trafficking of these activated cells across the blood-brain barrier into the CNS. To test this hypothesis, MOG35–55-activated CD4⫹ T cells from DR6⫺/⫺ and WT littermates were adoptively transferred into naive DR6⫺/⫺ and WT mice and monitored for clinical EAE symptoms. As shown in Fig. 5, MOG35–55-activated CD4⫹ T cells from DR6⫺/⫺ mice were incapable of inducing strong EAE symptoms whether they were transferred to naive DR6⫺/⫺ or WT mice. In contrast, MOG35–55-activated CD4⫹ T cells from WT mice caused observable paralytic symptoms regardless of whether naive recipient mice were DR6⫺/⫺ or WT. These data suggest that there was an intrinsic defect of encephalitogenic CD4⫹ T cells from DR6⫺/⫺ mice to infiltrate into and accumulate in the CNS.

Discussion Previously it has been demonstrated that several TNF/TNFR family members, such as CD40/CD40L, CD134 (OX40)/CD134L, and lymphotoxin-␣, are critical for EAE development (6 –11). DR6 is

FIGURE 5. MOG35–55-activated CD4⫹ T cells from DR6⫺/⫺ mice fail to passively transfer EAE to naive WT recipients. Cohorts (n ⫽ 6) of DR6-deficient (DR6⫺/⫺) and WT mice were immunized with MOG35–55 on day 0, and on day 15, splenic CD4⫹ T cells from each group were stimulated ex vivo with MOG35–55 for adoptive transfer to naive recipients, as described in Materials and Methods. CD4⫹ T cells (1 ⫻ 107/mouse) were injected i.v. on day 0, and EAE scoring was continued until day 33. DR6⫺/⫺ or WT CD4⫹ T cells were transferred into age-matched naive DR6⫺/⫺ or WT recipients. The values shown represent the mean values of three mice from each transferred group, and error bars represent the SD. The data shown are representative of two independent experiments.

a recently identified member of the TNFR family, and the absence of this receptor is known to cause enhanced CD4⫹ proliferation, Th2 cytokine production, and humoral responses (12–15). These previous studies, however, did not address whether the unique immune phenotype of DR6⫺/⫺ mice is capable of influencing the outcome of system-wide, Th1-based autoimmune disease. In the present study we have used DR6⫺/⫺ mice to ascertain a possible role(s) of this DR during MOG35–55-induced EAE. It is generally accepted that encephalitogenic CD4⫹ Th1 responses are necessary for EAE disease progression (29), although the effects of Th2 differentiation and effector function in this model are more ambiguous. The traditional Th1-Th2 paradigm dictates that Th2 responses are protective (30). Our data indicate that CD4⫹ T cells from DR6⫺/⫺ mice are capable of producing high levels of Th2 cytokines (13), and the mice are highly resistant to MOG35–55-induced EAE induction (Fig. 1). Interestingly, cytokine ELISPOT analysis showed that DR6⫺/⫺ splenocyte cultures from MOG35–55-immunized animals exhibited 2- to 3-fold as many IL4-producing cells on both days 14 and 21 after immunization (Fig. 3, B and C), whereas no difference in IFN-␥-producing cells was observed, except for the slight increase at high concentrations of MOG35–55 on day 21, but this did not reach significance (Fig. 3, B and C). As a result, higher levels of IL-4 were produced in DR6⫺/⫺ splenocyte cultures in an Ag-dose dependent fashion compared with WT culture, with little difference in IFN-␥ production (Fig. 3, D and E). IL-4 is a determining factor for Th2 differentiation. The early production of IL-4 during T cell activation can autoregulate its own expression and drive Th2 differentiation (24). In previous studies using herpes simplex virus-based vectors expressing Th2 cytokine IL-4, Broberg et al. (25) showed that overexpression of IL-4 can significantly abolish EAE disease without down-regulation of Th1 cytokines, suggesting that IL-4 plays an important role in directing Th differentiation and modulating EAE disease. Treatment with myelin basic protein, glatiramer acetate, or altered peptide ligand has led to increased Th2 responses in vivo and has inhibited and reduced EAE incidence and severity in animals (31, 32). Current MS therapeutic treatments, including type 1 IFNs and glatiramer acetate, have demonstrated the promotion of Th2 responses as a common effect (31, 32). Therefore, modulation of the DR6 signaling pathway and the potential Th1Th2 equilibrium may be able to control the extent of inflammatory autoimmune responses and potentially provide therapeutic benefits. EAE is characterized by acute onset of paralysis and perivascular and parenchymal infiltration of the CNS by encephalitogenic CD4⫹ T cells and macrophages. Interaction of VLA-4 on T cells

2292 with endothelial VCAM-1 has been demonstrated to be required for CD4⫹ T cell infiltration into the CNS, and the loss of encephalogenic potential correlates with a decreased level of cell surface VLA-4 and a corresponding loss of the ability of T cells to enter the CNS (26). Furthermore, anti-VLA-4 Ab treatment reduces cellular infiltration, inhibits the development of autoimmune encephalomyelitis, and even reverses the existing symptoms by preventing inflammatory cells from crossing the blood-brain barrier (27). Most importantly, a clinical trial of Natalizumab, a humanized mAb against ␣4 integrin (VLA-4), in MS patients led to significantly fewer new inflammatory CNS lesions compared with the placebo group and reduced clinical relapses by ⬃50% (33). In our current study the absence of DR6 correlated with down-regulated VLA-4 expression on CD4⫹ T cells (Fig. 4), which may partially contribute to the inability of T cells to infiltrate the CNS and to the resistance of EAE induction by DR6⫺/⫺ mice. The mechanism of DR6 regulating VLA-4 expression is not clear at the present time and will be the subject of future studies.

Acknowledgments We thank Dr. Marcel Bergeron for instrumental discussion; Drs. Jonathon Sedgwick, Jia En Chin, and Chandrasekar Venkataraman for critical review of the manuscript; Jian Wang for technical help; and Dr. Stephen J. Iturria for statistical analysis.

Disclosures The authors have no financial conflict of interest.

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