Divergent Roles for p55 and p75 Tumor Necrosis Factor Receptors in the Pathogenesis of MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Cellular Immunology 205, 24 –33 (2000) doi:10.1006/cimm.2000.1706, available online at http://www.idealibrary.com on

Divergent Roles for p55 and p75 Tumor Necrosis Factor Receptors in the Pathogenesis of MOG 35-55-Induced Experimental Autoimmune Encephalomyelitis Graig C. Suvannavejh,* ,1 Hae-Ock Lee,† ,1 Josette Padilla,* Mauro C. Dal Canto,‡ Terrance A. Barrett,† and Stephen D. Miller* ,2 *Department of Microbiology-Immunology, †Department of Medicine, and ‡Department of Pathology and the Interdepartmental Immunobiology Center, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, Illinois 60611 Received June 12, 2000; accepted August 29, 2000

sensitization with myelin proteins or their immunogenic epitopes (1– 4). The disease is characterized by a mononuclear-rich infiltrate in the perivascular spaces of the spinal cord and brain and demyelination resulting from bystander activation of local and/or recruited macrophages (1). Based on both the histopathology and the clinical course of affected animals, EAE has been widely used as an animal model of the human demyelinating disease multiple sclerosis (MS). Though it is widely believed that Th1 cytokines are the primary mediators of the initial inflammation in EAE, the exact role of tumor necrosis factor (TNF) remains controversial. Much of the literature has supported a necessary role for TNF in the initiation of clinical disease. Indeed, TNF mRNA as measured by polymerase chain reaction (PCR) and in situ hybridization has been shown to correlate well with the onset of acute clinical disease and relapses (5– 8). Transgenic mice expressing TNF under control of a CNS-specific promoter display spontaneous T cell infiltration and demyelination (9, 10). In addition, the administration of neutralizing antibodies to TNF, depending on the timing of administration, has been effective in not only delaying the onset of disease, but also in completely ameliorating the appearance of clinical symptoms (11, 12). Similar results have been found using soluble TNF–IgG fusion proteins (13, 14). The exact pathogenic role for TNF in EAE, however, has not clearly been established, as some recent studies using TNF knockout mice have proved conflicting. Indeed, some reports have demonstrated TNF knockout mice to be protected from clinical disease (15, 16). However, other studies have shown TNF-␣ knockout mice to be either equally as or more susceptible than controls (17, 18). This ambiguity regarding the role of TNF also extends to other spontaneous and experimental models of autoimmunity, such as the nonobese diabetic mouse and collagen-induced arthritis (19 –22).

To clarify the role of tumor necrosis factor (TNF) in the inflammatory aspects of autoimmunity vs its potential role in the apoptotic elimination of autoreactive effector cells, we assessed the roles of the p55 (TNFR1/Tnfrsf1a/CD120a) and p75 (TNFR2/ Tnfrsf1b/CD120b) TNF receptors in the pathogenesis of MOG 35-55 -induced experimental autoimmune encephalomyelitis (EAE). TNFR p55/p75 ⴚ/ⴚ double knockout mice were completely resistant to clinical disease. TNFR p55 ⴚ/ⴚ single knockout mice were also totally resistant to EAE, exhibiting reduced MOG 35-55specific proliferative responses and Th1 cytokine production, despite displaying equivalent DTH responses. Importantly, IL-5 was significantly increased in p55 ⴚ/ⴚ mice. In contrast, p75 ⴚ/ⴚ knockout mice exhibited exacerbated EAE, enhanced Th1 cytokine production, and enhanced CD4 ⴙ and F4/80 ⴙ CNS infiltration. Thus, p55/TNFR1 is required for the initiation of pathologic disease, whereas p75/ TNFR2 may be important in regulating the immune response. These results have important implications for therapies targeting p55 and p75 receptors for treating autoimmune diseases. © 2000 Academic Press Key Words: TNF; TNF receptor; p55; p75; EAE; MS; CNS; autoimmunity; inflammation; regulation.

INTRODUCTION Experimental autoimmune encephalomyelitis (EAE) is a CD4 ⫹ Th1 cell-mediated, inflammatory, demyelinating disease of the central nervous system (CNS) that can be induced in susceptible strains of mice by 1

G. C. Suvannavejh and H.-O. Lee are co-first authors of this paper. 2 To whom correspondence and reprint requests should be addressed at Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Avenue, W213, Chicago, IL 60611. Fax: 312-503-1154. E-mail: [email protected]. 0008-8749/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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DIVERGENT ROLE OF p55 AND p75 TNF RECEPTORS IN EAE

TNF exerts pleiotropic effects, and its importance as a biological mediator is highlighted by its production by a wide variety of cell types. Although TNF is involved in numerous physiologic cellular activities and processes, the two most significant roles of TNF-␣ are in inflammation and apoptosis/activation-induced cell death. TNF exerts its physiologic actions by binding to one of two cognate receptors that are distinguished by their molecular weight and cellular distribution, p55 (TNFR1/Tnfrsf1a) and p75 (TNFR2/Tnfrsf1b) (23–26). Although the role of TNF in EAE has been mainly attributed to its inflammatory properties, both p55 and p75 receptors have been implicated in apoptotic mechanisms, which lends support to a possible role in the elimination of activated, and potentially autoreactive, cells (27, 28). Since TNF has been reported to be involved in both inflammatory and apoptotic processes, we sought to establish exact physiologic roles for each receptor during the pathogenesis and resolution of EAE. Using mice with targeted disruption of the p55 and/or p75 TNF receptors, we demonstrate that p55 is critical for the initiation of inflammation and subsequent demyelination. In contrast, p75 has a potential role in TNF-mediated elimination of autoreactive effector cells and regulation of autoimmunity. MATERIALS AND METHODS Mice and Reagents Male and female p55 (TNFR1/Tnfrsf1a/CD120a) and p75 (TNFR2/Tnfrsf1b/CD120b) singly deficient mice, as well as p55/p75 doubly deficient mice, were originally obtained as a kind gift from Immunex Corporation (Seattle, WA). Colonies were maintained and housed in the Center for Experimental Animal Research at Northwestern University. The p75 single knockout and p55/p75 double knockout mice were originally made on the 129 background and then backcrossed onto the B6 background for at least four generations. The p55 single knockout mice were made directly on the B6 background. Additional C57BL/6 wild-type mice, B6.MRL-Faslpr mice, as well as C57BL/6-Tnfrsf1btm1lmx (p75 knockout) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were usually 6 –10 weeks of age at the time of priming. Myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP) peptides, MOG35-55 and PLP 139-151, were purchased from Peptides International (Cleveland, OH).

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toxin (List Biological Laboratories, Campbell, CA) was administered iv in 200 ␮l of saline immediately after immunization, as well as 48 h later. Mice were monitored and graded daily for symptoms of EAE using a clinical scoring system as follows: 0, absence of clinical symptoms; 1, limp tail or hind limb weakness, such that legs fall through the cage top; 2, limp tail and hind limb weakness; 3, partial hind limb paralysis, such that one leg drags; 4, full hind limb paralysis; 5, death. In Vitro Proliferation and Cytokine Assays At 8 –10 days postimmunization, draining lymph node (DLN) cells and splenocytes were harvested. For T cell proliferation, 5 ⫻ 10 5 cells were cultured in 200 ␮l of DMEM-10 (Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 1 mM glutamine, 1% Pen-Strep, 1 mM nonessential amino acids, and 5 ⫻ 10 ⫺5 M 2-mercaptoethanol) in triplicate with varying concentrations (1, 10, and 100 ␮M) of MOG 35-55, PLP 139-151, PLP 178-191, and TMEV VP2 viral peptide (VP2 70-86) in 96-well microtiter plates. The cells were pulsed at 72 h with 1 ␮Ci [ 3H]thymidine and harvested at 96 h. The results are expressed as stimulation index (SI) ⫽ mean CPM of antigen containing wells/mean CPM of control wells. [3H]Thymidine incorporation was determined using a TopCount NXT (Packard Instruments, Meriden, CT). For cytokine assays, 5 ⫻ 10 6 cells were activated in the presence or the absence of MOG35-55 in vitro in 24-well plates. Supernatants were collected after 48 h. The concentrations of IL-2, IL-4, IL-5, and IFN-␥ present in these supernatants were quantitated with indirect ELISA assays (Endogen Inc., Woburn, MA). Delayed-Type Hypersensitivity (DTH) Assay DTH responses were quantitated using a 24-h ear swelling assay. Prechallenge ear thickness was determined using a Mitutoyo Model 7326 engineer’s micrometer (Schlesinger’s Tools, Brooklyn, NY). DTH responses were elicited by injecting 10 ␮g of peptide (in 10 ␮l of saline) into the dorsal surface of the ear using a Hamilton syringe fitted with a 30-gauge needle. Twenty-four hours after ear challenge, the increase in ear thickness over prechallenge measurements was determined. Results are expressed in units 10⫺4 in. ⫾SEM. Ear swelling responses were the result of mononuclear cell infiltration and typical DTH kinetics (i.e., minimal swelling at 4 h and maximal swelling at 24 h).

Disease Induction and Clinical Assessment of EAE

Histologic Evaluation of Demyelination

Mice were immunized with 200 ␮g MOG 35-55 emulsified in complete Freund’s adjuvant (CFA) supplemented with 4 mg/ml H37Ra Mycobacterium tuberculosis (Difco, Detroit, MI). The emulsion was injected subcutaneously on the dorsal aspect over three spots in a total volume of 100 ␮l. In addition, 200 ng pertussis

Mice were anaesthetized and sacrificed by total body perfusion through the left ventricle using chilled 3% glutaraldehyde in phosphate-buffered saline (PBS), pH 7.3. Spinal cords were dissected out, cut into 1-mmthick segments, and postfixed in OsO 4, dehydrated, and embedded in Epon (Electron Microscopy Services,

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Fort Washington, PA). Toluidine blue-stained sections from 10 segments per mouse were read and scored blinded as follows: ⫾, mild inflammation without demyelination; 1 ⫹, inflammation with focal demyelination; 2 ⫹, inflammation with multiple foci of demyelination; 3 ⫹, marked inflammation with bilateral converging areas of demyelination; 4 ⫹, extensive bilateral areas of demyelination and remyelination. Preparation, Storage, and Sectioning of Tissues Mice were anesthetized and perfused with 1⫻ PBS. Spinal cords were removed by dissection, and 2- to 3-mm spinal cord blocks were immediately frozen in OCT (Miles Laboratories; Elkhart, IN) in liquid nitrogen. The blocks were stored at ⫺80°C in plastic bags to prevent dehydration. Sections 5– 6 ␮m thick from the lumbar region (approximately L2–L3) were cut on a Reichert-Jung Cryocut 1800 cryotome (Leica Instruments; Deerfield, IL), mounted on Superfrost Plus electrostatically charged slides (Fisher), air-dried, and stored at ⫺80°C. Immunohistochemical Analysis Slides were stained using a Tyramide Signal Amplification (TSA) Direct Kit (NEN Life Science Products; Boston, MA) according to the manufacturer’s instructions. Lumbar sections from each group were thawed, air-dried, fixed in acetone at room temperature, and rehydrated in 1⫻ PBS. Nonspecific staining was blocked using anti-CD16/CD32 (Fc␥ III/II receptor, 2.4G2; Pharmingen; San Diego, CA), and an avidin/ biotin blocking kit (Vector Laboratories; Burlingame, CA) in addition to the blocking reagent provided by the TSA kit. Slides were stained with the following biotinconjugated antibodies: anti-macrophage (F4/80) (Caltag; Burlingame, CA); anti-CD4 (H129.19; Pharmingen), anti-I-A b (AF6-120.1; Pharmingen). Sections were counterstained with DAPI (Sigma) and then coverslipped with Vectashield mounting medium (Vector). Slides were examined by epifluorescence using a chroma triple-band filter (Chroma Technology Corp., Brattleboro, VT). Four to eight serial lumbar sections from each sample per group were analyzed at 100⫻ and 400⫻ magnification throughout the entire spinal cord section, which included gray and white matter and dorsal, ventral, and lateral regions. Statistical Analyses Comparison of the percentage of animals showing clinical disease between any two groups of mice was done by ␹ 2 using Fisher’s exact probability. Comparisons of the mean day of onset of relapse and mean peak disease severity between any two groups of mice were analyzed by the Student’s t test. P values ⬍0.05 were considered significant.

RESULTS p55/p75 ⫺/⫺ and p55 ⫺/⫺ Mice Are Resistant to MOGInduced EAE, but p75 ⫺/⫺ Mice Exhibit Exacerbated Clinical Disease To determine whether TNF receptors may be involved in the initiation of and/or resolution from EAE, we initially compared EAE development in p55/p75 ⫺/⫺ and wild-type control mice after priming with MOG35-55. Wild-type B6 mice developed initial clinical disease starting at approximately day 9, whereas mice deficient for both p55 and p75 were completely resistant to EAE induction (Fig. 1A). In an attempt to determine the individual roles of the two receptors, disease was induced in p55 ⫺/⫺ and p75 ⫺/⫺ mice. Similar to the p55/ p75 double knockouts, p55 ⫺/⫺ knockout mice were resistant to disease induction (Fig. 1B). Interestingly, p75 ⫺/⫺ mice were fully susceptible to disease and actually exhibited a more severe disease state than did wild-type controls (Fig. 1B, Table 1). We also compared the clinical disease in mice deficient for another TNFR family member, CD95/Fas/APO-1 (data not shown). Like p55 and p75, Fas can deliver a death signal via binding of death domains that allow for the docking of adapter proteins, such as Fas-associated death domain adapter protein (FADD). FADD can bind to FLICE/ caspases-8 to initiate the death cascade. Fas-deficient B6 lpr mice showed a significantly reduced disease incidence [21/34 vs 21/23 in wild-type controls (P 0.029)], but the mean peak disease severity in animals displaying clinical signs was similar to that of controls [2.1 ⫾ 0.3 vs 2.4 ⫾ 0.5 in wild-type controls (P 0.219)], consistent with previously reported data (29, 30). Differences in clinical disease between wild-type and TNFR-deficient mice became clearer when clinical data from four separate experiments were compiled. As shown in Table 1, no disease was observed in a large number of p55/75 ⫺/⫺ or p55 ⫺/⫺ mice (0/16 and 0/26, respectively, vs 21/23 in wild-type controls). In contrast, p75 single knockout mice displayed a disease incidence equivalent to that of wild-type control mice (91.30 and 88.89%, respectively), but interestingly showed a slight, but significant, delay in the mean day of onset (MDO) (13.3 vs 10.6 days, P 0.004). More importantly, p75-deficient mice exhibited a significantly enhanced mean peak disease score when compared to controls (3.0 ⫾ 0.2 vs 2.4 ⫾ 0.2, P 0.033). Absence of CNS Infiltration and Demyelination in p55 ⫺/⫺ Mice, but Severe Histopathology in p75 ⫺/⫺ Mice To investigate the degree of mononuclear infiltration and demyelination in the CNS, representative mice from each strain were sacrificed and spinal cords analyzed for histopathology. Naı¨ve, unimmunized wildtype B6 mice (Fig. 2A) and the TNFR1/2 knockouts

DIVERGENT ROLE OF p55 AND p75 TNF RECEPTORS IN EAE

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both TNFR1 and TNFR2 mice was paralleled by the pattern of CNS histopathology. Lack of CNS Mononuclear Cell Infiltrates and MHC Class II Expression in the Absence of TNFR1, but not TNFR2 To characterize the nature of the CNS infiltrate in the different strains, we performed immunohistochemistry on CNS tissue (Fig. 3). The CNS of wild-type B6 mice immunized with MOG 35-55 displayed moderate infiltration by CD4 ⫹ and F4/80 ⫹ cells, as well as modest MHC class II expression. In contrast, the CNS of both p55 ⫺/⫺ and p55/p75 ⫺/⫺ mice was completely free of CD4 ⫹ and F4/80 ⫹ infiltrates, as well as MHC class II staining. Single p75-deficient mice, which exhibited severe disease, showed significantly more extensive CD4 ⫹, F4/80 ⫹, and MHC class II staining than wildtype controls. p55 Knockout Mice Display Decreased Proliferation, but Normal MOG-specific DTH Responses

FIG. 1. Comparison of MOG 35-55-induced EAE clinical disease courses between wild-type and TNFR-deficient C57BL/6 mice. (A) Wild-type and p55/p75 ⫺/⫺ B6 mice were immunized with 200 ␮g of MOG 35-55/CFA on day 0. (B) Wild-type, p55 ⫺/⫺, and p75 ⫺/⫺ B6 mice were immunized with 200 ␮g of MOG 35-55/CFA on day 0. All mice were scored for clinical signs of EAE as described under Materials and Methods. Results are plotted as the mean clinical score for all of the animals in each treatment group vs day postimmunization. Numbers in parentheses indicate incidence of disease. Results are representative of four separate experiments (see Table 1 for compilation of the data).

(Fig. 2D) showed no signs of infiltration or demyelination, as all of the myelin sheaths within the section were intact. MOG 35-55-immunized wild-type B6 mice showed moderate signs of inflammation and demyelination, noticeably in the superficial zone of the spinal cord white matter (Fig. 2B). p55 ⫺/⫺ mice showed only minimal inflammation limited to the leptomeninges and no parenchymal involvement was observed (Fig. 2C). In contrast, p75 ⫺/⫺ mice displayed severe inflammation and demyelination (Fig. 2E). Although not shown, demyelination in p75-deficient mice extended in a confluent pattern throughout anterior and lateral columns of the spinal cord. Thus, clinical disease in

To determine whether the absence of p55 or p75 caused qualitative changes in antigen-specific recall responses, draining lymph node cells (DLNC) and splenocytes (SPL) were isolated and T cell proliferation responses tested in vitro. As shown in Fig. 4, DLNC isolated from p55 ⫺/⫺ mice displayed significant defects in their ability to proliferate in response to the immunizing MOG 35-55 peptide when compared to control B6 mice (SI, 2.7 vs 14.9). Interestingly, splenic T cells proliferated comparably to the controls. DLNC proliferative responses of p75-deficient mice were also somewhat reduced compared to wild-type controls (SI, 5.7 vs 14.9), whereas splenic proliferative responses were comparable to those of controls. Proliferative responses to the immunizing MOG 35-55 peptide were dose-dependent and the T cells failed to respond to PLP 139-151, a non-cross-reactive control peptide (data not shown). DTH responses were assessed to determine the ability of TNFR-deficient mice to mount a peripheral Th1 response. As shown in Fig. 5, wild-type B6 controls and both p55 ⫺/⫺ and p75 ⫺/⫺ mice exhibited significant DTH upon challenge with MOG 35-55, but not upon challenge with PLP 139-151. MOG Peptide-Specific Cytokine Responses in p55 ⫺/⫺ and p75 ⫺/⫺ Mice To determine whether the Th1 and Th2 cytokine profile might explain the disease resistance in p55 ⫺/⫺ mice and/or disease exacerbation in p75 ⫺/⫺ mice, we performed cytokine ELISAs on culture supernatants from DLN and splenic T cells harvested 8 days postimmunization that were cultured in vitro with MOG 35-55. Both DLNC and splenic T cells from wild-type control mice showed robust Th1 cytokine production (Figs. 6A

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TABLE 1 Summary of EAE Disease Parameters in Wild-Type, p55/p75 ⴚ/ⴚ, p55 ⴚ/ⴚ, and p75 ⴚ/ⴚ C57Bl/6 Mice Genotype Disease parameter a

Wild-type B6

p55 ⫺/⫺p75 ⫺/⫺

P55 ⫺/⫺

p75 ⫺/⫺

Incidence Mean day of onset Mean peak clinical score Histology

21/23 (91%) 10.6 ⫾ 0.5 2.4 ⫾ 0.2 Inflammation; demyelination

0/16 (0%)* na na None

0/26 (0%)* na na None

32/36 (89%) 13.3 ⫾ 0.6** 3.0 ⫾ 0.2*** Severe inflammation and demyelination

⫹ ⫹ ⫹

— — —

— — —

⫹⫹⫹ ⫹⫹⫹ ⫹⫹

CNS IHC b CD4 F4/80 MHC class II a

Mean day of onset and mean peak clinical score values were calculated as described in Materials and Methods. The data provided reflect the cumulative total of four separate experiments. b Values assigned for CNS immunohistochemical staining are semiquantitative and are based on a comparison with background levels found in naı¨ve control CNS sections. * Disease incidence was significantly reduced compared to that of the wild-type B6 controls, P ⬍ 0.001. ** Mean day of onset was significantly delayed compared to that of the wild-type B6 controls, P ⬍ 0.05. *** Mean peak clinical score was significantly more severe than that of the wild-type B6 controls, P ⬍ 0.03.

and 6B), but weak Th2 cytokine production (Figs. 6C and 6D). In contrast, splenic T cells from p55 ⫺/⫺ mice produced substantially fewer Th1 cytokines (IFN-␥ and IL-2), but produced significant levels of IL-5 in both spleen and DLN compartments (Fig. 6D). Similar cytokine patterns were observed with T cells cultured at the peak of acute disease (day 15–16). Thus, p55 ⫺/⫺ mice showed a preferential skewing toward a Th2 phenotype. Although a Th2 cytokine profile was observed in p55 ⫺/⫺ mice, p75 ⫺/⫺ mice showed equivalent or greater Th1 cytokine production, particularly in DLN T cells (Figs. 6A and 6B), but failed to produce detectable Th2 cytokines (Figs. 6C and 6B).

required. In addition, a knowledge of receptor functions is important because of the multiplicity of the TNF family members and the receptors that bind

DISCUSSION TNF and its receptors play a primary pathogenic role in inflammatory, autoimmune conditions. Thus, many therapeutic approaches to treating autoimmune diseases are now aimed at neutralizing the biological functions of TNF. Neutralizing antibodies to TNF and soluble TNFR fusion proteins have been recently demonstrated to be efficacious in treating collagen-induced arthritis, rheumatoid arthritis, and Crohn’s disease, illustrating the effectiveness of this experimental approach (31–33). TNF/TNFR interactions mediate and/or participate in many functions, including cellular proliferation, upregulation of adhesion molecules and MHC molecules, nitric oxide production, cellular cytotoxicity, and apoptosis/activation-induced cell death (34). It remains to be firmly established whether p55 and p75 mediate distinct events or are redundant in function. Thus, a greater knowledge of the individual signal transduction pathways and the ultimate effector functions mediated by TNFR1 and TNFR2 is

FIG. 2. Histological analysis of spinal cord sections from wild-type, p55 ⫺/⫺, p75 ⫺/⫺, and p55/p75⫺/⫺ C57BL/6 mice. Spinal cords from one to two representative mice were examined for histological signs of mononuclear cell infiltration and demyelination at 18 days postimmunization. (A) Naı¨ve, nonimmunized wild-type control C57/BL6 mouse; (B) MOG 35-55-immunized wild-type control C57/BL6 mouse; (C) MOG35-55immunized p55⫺/⫺ mouse; (D) MOG35-55-immunized p55/p75⫺/⫺ mouse; (E) MOG 35-55-immunized p75⫺/⫺ mouse. All sections are 1-␮m-thick, Epon-embedded sections stained with toluidine blue; ⫻220.

DIVERGENT ROLE OF p55 AND p75 TNF RECEPTORS IN EAE

FIG. 3. Immunohistochemical analysis of CD4 ⫹, F4/80 ⫹, and MHC class II ⫹ cells in the CNS of wild-type and TNFR knockout mice during chronic EAE. Spinal cord sections were isolated from MOG 35-55-primed mice at 30 days postimmunization and prepared as described under Materials and Methods. A, C, E, and G were stained for CD4 (green) and F4/80 (red). B, D, F, and H were stained for I-A b (red). (A and B) MOG 35-55-immunized wild-type control C57/BL6 mouse; (C and D) MOG 35-55-immunized p55/p75 ⫺/⫺ mouse; (E and F) MOG 35-55-immunized p55 ⫺/⫺ mouse; and (G and H) MOG 35-55-immunized p75 ⫺/⫺ mouse. ⫻100.

them. TNF exists as both TNF-␣ and TNF-␤ (LT-␣), which can both bind to either TNFR1 or TNFR2 (35). In addition, LT␤, which exists as a membrane-bound form in association with LT-␣, binds it own receptor, LT␤R (36). The data in the current report demonstrate functional differences between the two receptors in the context of the pathogenesis of EAE, a CD4 ⫹ T-cellmediated inflammatory demyelinating disease. Mice deficient for both TNFR1 and TNFR2 were completely protected from development of MOG 35-55-induced EAE. Taking advantage of single TNFR knockout mice to delineate the specific biologic contribution of each receptor, we observed an absolute requirement for p55 in the induction of clinical EAE, as p55 ⫺/⫺ mice were completely resistant to disease. In contrast, p75-deficient mice exhibited a significantly enhanced disease severity. Moreover, p75 ⫺/⫺ mice displayed significant increases in MOG-specific proinflammatory cytokine

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production in DLNC, the number of CNS infiltrating CD4 ⫹ and F4/80 ⫹ cells, and the degree of CNS inflammation and demyelination. However, p55-deficient mice exhibited relatively poor proliferative responses, decreased Th1, but increased Th2 cytokine production, and a lack of CNS cellular infiltration and demyelination. These findings are consistent with divergent roles for the individual TNF receptors in CNS autoimmunity with p55 (TNFR1) important for T cell priming, trafficking, and/or mediation of CNS inflammation and demyelination, whereas p75 (TNFR2) apparently acts to limit pathology, perhaps by elimination of autoreactive CD4 ⫹ and F4/80 ⫹ cells and/or by playing a role in remyelination. Due to the pleiotropic effects of TNF in T-cell-mediated inflammatory processes, the failure of p55-deficient mice to develop MOG 35-55-specific clinical EAE in these studies may be due to multiple effects. First, p55 signaling may play a critical role in the effector phases of inflammatory demyelination by causing direct apoptosis/cytotoxicity of oligodendrocytes. In agreement with our findings, Eugster et al. found that p75 knockout mice showed enhanced disease compared to wildtype controls, whereas p55 and p55/p75 double knockout mice showed significantly reduced disease and demyelination (though not absent, as reported here) (37). We speculate that the differences may relate to the cleanliness of our animal facility. A role for TNFR1 in effector tissue damage is also supported by findings in other T-cell-dependent autoimmune models, including

FIG. 4. MOG 35-55-specific T cell proliferative responses in lymph node and spleen of wild-type and TNFR-deficient C57BL/6 mice. Draining lymph node and splenic cells isolated from MOG 35-55/CFAprimed mice 8 days postimmunization were cultured with PLP 56-70. Data represent the mean thymidine uptake of triplicate cultures stimulated for a total of 96 h with 100 ␮M peptide and are plotted as ⌬ CPM ⫾SEM (background counts subtracted). T cells from all groups failed to respond to OVA 323-339 as an irrelevant peptide control (data not shown). Results are representative of three separate experiments. Proliferative responses in the DLN of p55 ⫺/⫺ and p75 ⫺/⫺ mice were significantly less than those of wild-type controls. Numbers in parentheses represent MOG 35-55-specific stimulation indices (SI). SI values greater than 2.0 are considered significant.

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FIG. 5. Peripheral MOG35-55-specific DTH responses are normal in TNFR1- and TNFR2-deficient C57BL/6 mice. DTH responses to MOG 35-55 were assessed in groups of four to five mice per group on day 35 postimmunization. The results are expressed as ⌬ 24-h ear swelling (background swelling subtracted) in units of 10⫺4 in. ⫾ SEM in response to ear challenge with 10 ␮g of MOG 35-55. Background ear swelling responses upon challenge with PLP139-151 as an irrelevant peptide control were negligible. These results are representative of two separate experiments.

experimental autoimmune myocarditis, diabetes in the NOD mouse, and collagen-induced arthritis, all of which showed that mice lacking functional p55 exhibit

reduced inflammatory infiltrates and clinical disease (21, 38, 39). In addition, recent studies in which exogenous production of p55 TNFR fusion proteins was induced in the target organ have demonstrated a dramatic amelioration of several inflammatory autoimmune diseases (21, 22). A critical role for p55 in CNS pathogenesis is also supported by the findings of Akassoglou et al., who reported that mice expressing transgenic TNF-␣ in the CNS which were deficient in p55 expression, but not in p75 expression, were protected from CNS inflammation and demyelination (40). The failure of p55 ⫺/⫺ mice to develop EAE could also result from a requirement for TNF/p55 interactions in trafficking of encephalitogenic T cells and F4/80 effector macrophages to the CNS (40). The absence of infiltrating CD4 ⫹ and F4/80 ⫹ cells in the spinal cords of MOG-primed p55-deficient mice is consistent with this possibility. Inefficient homing of inflammatory cells could relate to the demonstrated role of TNF/p55 signaling in synergizing with other cytokines, such as IFN-␥, in upregulating expression of MHC class II on CNS-resident APCs (38). More importantly, lack of p55/TNF interactions may result in a failure to upregulate adhesion molecules (41, 42), such as VLA-4, VCAM, and ICAM, which are critical in the migration

FIG. 6. MOG 35-55-specific Th1 and Th2 cytokine production from lymph node T cells of wild-type and TNFR-deficient C57BL/6 mice. Draining lymph node and splenic cells isolated from MOG35-55/CFA-primed mice 8 days postimmunization were cultured with MOG35-55. Culture supernatants were collected after 48 h and assayed for IFN-␥ (A), IL-2 (B), IL-4 (C), and IL-5 (D) levels by ELISA. Data are plotted as mean cytokine levels from triplicate cultures in pg/ml and are representative of two separate experiments. No antigen controls were below the range of detection.

DIVERGENT ROLE OF p55 AND p75 TNF RECEPTORS IN EAE

and trafficking of peripheral immune cells into the CNS in EAE (43– 45). A recent study showed that p55 and p75 mRNAs are upregulated in high endothelial venules (HEV) in the lymph nodes and CNS before the onset of clinical EAE (46). Importantly, Selmaj et al. showed that administration of a p55 binding protein correlated with a downregulation of VCAM and VLA-4, inhibition of inflammation, and prevention of clinical EAE (47). However, the role of p55 in the trafficking of inflammatory cells may differ among species. For example, a recent study showed that administration of p55 TNFR-IgG fusion protein prevented clinical EAE in Lewis rats, despite the fact that effector T lymphocytes were fully capable of trafficking into the CNS (48). Disease inhibition may relate to the failure of p55deficient mice to develop robust Th1 cytokine responses, indicating a possible role for p55 in the priming of encephalitogenic T cells. Interestingly, we were able to detect a substantial increase in MOG 35-55-specific IL-5 cytokine production in p55 ⫺/⫺ mice. The physiological consequence of the enhanced IL-5 production has yet to be determined, but it may represent a Th1 to Th2 cytokine “switch” that could promote an anti-inflammatory environment that may suppress or regulate proinflammatory Th1 cell activation. Last, it is also possible that defective B cell responses may play some role in the lack of inflammatory disease, as p55deficient mice display pronounced abnormalities in the numbers of splenic B220 ⫹ cells (49). As anti-MOG humoral responses have been shown to be an important (50, 51), though not necessary (52, 53), component in the pathogenesis of MOG peptide-induced EAE, B cell defects could affect peptide presentation in the periphery or the CNS. The possible role of p75 in the regulation of autoimmunity is an interesting, but not a surprising, finding. Consistent with the observed exacerbated clinical EAE disease course, our data showed that MOG 35-55-specific draining lymph node T cells from p75-deficient mice produced significantly higher levels of the Th1 cytokines IFN-␥ and IL-2 than did wild-type B6 control mice. Apoptotic cell death of encephalitogenic T cells mediated by Fas/FasL interactions contributes to disease recovery in the Lewis rat (54) and disease remission the SJL mouse EAE (55) models. In addition, a number of other studies have indicated an important role for p75 in the apoptotic elimination of activated T cells. Activated mature CD8 ⫹ cells apoptose primarily via TNF/p75 interactions (28). In addition p75-deficient mice, but not p55-deficient or p55/p75 doubledeficient mice, exhibited exacerbated responses in a model of pulmonary inflammation (56). Bachmann et al. (57) showed that apoptosis of CD3 ⫹ T cells is significantly reduced in p75 knockout mice. A critical role for TNFR in T cell apoptosis in MS may explain the results of a recent clinical trial in which treatment with lener-

31

cept, a recombinant soluble p55 immunoglobulin fusion protein previously reported to inhibit induction of EAE in Lewis rats (58), was found to exacerbate the number and severity of MS clinical disease relapses (59). Collectively, these studies suggest that p75/TNF interactions play a critical role in the immunoregulation of inflammatory responses. It should be mentioned that Croxford et al. have reported that neutralization of TNF with soluble dimeric p75 TNFR was effective in ameliorating EAE using both a DNA– cationic liposome complex and retroviral vectors (60, 61). In summary, our findings show that there are divergent roles and effector functions for the p55 and p75 signaling in the pathogenesis of EAE. p55 (TNFR1) apparently plays a predominant proinflammatory role, whereas p75 (TNFR2) may play a critical role in disease regulation. The current evidence indicates that blocking TNF/p55 interactions is effective in ameliorating clinical signs of EAE, while promoting TNF/p75 interactions may prove beneficial in modulating chronic and relapsing forms of autoimmune disease. We are currently performing adoptive transfer studies utilizing wild-type and TNFR1/2-deficient mice which should allow us to more clearly determine which receptor is critical for the CNS infiltration of inflammatory cells and which may be important in regulation of the autoimmune response. Thus, despite overall similarities between the p55 and p75 receptors, differences in effector function and tissue-specific receptor expression may confound and compromise attempts to effectively neutralize the desired actions of TNF in therapeutic applications. Further investigation of the exact cellular and temporal expression of TNF and its receptors in the CNS is required to clarify these issues. Overall, the current data provide important insights into the design of effective TNFR-directed therapeutic strategies in treating inflammatory autoimmune diseases, such as multiple sclerosis. ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service/National Institutes of Health Grants NS26543, NS30871, and NS13011 and by National Multiple Sclerosis Society Grant RG2893.

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