Roles of TNF-Related -Inducing Ligand in Experimental Autoimmune Encephalomyelitis

This information is current as Brendan Hilliard, Andreas Wilmen, Cheryl Seidel, of October 1, 2021. Tzu-Shang T. Liu, Rüdiger Göke and Youhai Chen J Immunol 2001; 166:1314-1319; ; doi: 10.4049/jimmunol.166.2.1314 http://www.jimmunol.org/content/166/2/1314 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Roles of TNF-Related Apoptosis-Inducing Ligand in Experimental Autoimmune Encephalomyelitis1

Brendan Hilliard, Andreas Wilmen, Cheryl Seidel, Tzu-Shang T. Liu, Ru¨diger Go¨ke, and Youhai Chen2

TRAIL, the TNF-related apoptosis-inducing ligand, induces apoptosis of tumor cells, but not normal cells; the roles of TRAIL in nontransformed tissues are unknown. Using a soluble TRAIL , we examined the consequences of TRAIL blockade in an animal model of multiple sclerosis. We found that chronic TRAIL blockade in mice exacerbated experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. The exacerbation was evidenced primarily by increases in disease score and degree of inflammation in the CNS. Interestingly, the degree of apoptosis of inflammatory cells in the CNS was not affected by TRAIL blockade, suggesting that TRAIL may not regulate apoptosis of inflammatory cells in experimental autoimmune encephalomyelitis. By contrast, myelin oligodendrocyte glycoprotein-specific Th1 and Th2 cell responses were sig- Downloaded from nificantly enhanced in animals treated with the soluble TRAIL receptor. Based on these observations, we conclude that unlike TNF, which promotes autoimmune inflammation, TRAIL inhibits autoimmune encephalomyelitis and prevents activation of autoreactive T cells. The Journal of Immunology, 2001, 166: 1314–1319.

he TNF-related apoptosis-inducing ligand, TRAIL,3 is a mal cells, leaving the host organ systems unharmed (18, 19). To newly identified member of the TNF superfamily (1). Un- explore the roles of TRAIL in vivo, we have recently studied the T like other members of the TNF superfamily that interact consequences of chronic TRAIL blockade in an animal model of http://www.jimmunol.org/ with one or two specific receptors, TRAIL can potentially interact rheumatoid arthritis (20). We found that chronic TRAIL blockade with five different receptors. These include (DR4, exacerbated autoimmune arthritis, whereas intraarticular TRAIL TRAIL-R1), (DR5, TRAIL-R2), gene transfer diminished it. Additionally, we found that unlike Fas (DcR1, TRAIL-R3, TRID), (DcR2, TRAIL-R4, ligand or TNF, TRAIL did not mediate activation-induced cell TRUNDD) (2–8), and a soluble receptor called death of T lymphocytes. Instead, TRAIL prevented cell cycle pro- (9). TRAIL and its receptors are constitutively expressed in a va- gression of T cells and inhibited their differentiation into effector riety of cell types, including lymphocytes, NK cells, and neural cells (20). In this study, we examine the roles of TRAIL in an

cells (1, 2, 4, 10–15). The levels of TRAIL expression in lympho- animal model of multiple sclerosis. We propose that unlike many by guest on October 1, 2021 cytes can be markedly up-regulated following cell activation (5, other members of the TNF family that promote autoimmune en- 16, 17). cephalomyelitis, TRAIL inhibits experimental autoimmune en- While the presence of multiple TRAIL receptors strongly sug- cephalomyelitis (EAE) and prevents activation of encephalitogenic gests that TRAIL be involved in multiple processes, the precise T cells. roles of TRAIL in health and disease are unknown. In vitro studies suggest that TRAIL induces apoptosis of some, but not all, tumor Materials and Methods cell lines (2, 6). This may be mediated through DR4 and DR5, Mice which are capable of activating the caspase cascade. The presence of DcR1 and DcR2, which do not contain functional death do- Female C57BL/6 mice, 4–6 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in the University of mains, blocks TRAIL-induced apoptosis (2, 6). TRAIL may not Pennsylvania (Philadelphia, PA) animal care facilities and were acclimated induce apoptosis of most nontransformed cells (2, 6). In vivo ad- for 5–7 days before being used for experiments. ministration of rTRAIL selectively kills tumor cells, but not nor- Induction and clinical evaluation of EAE ␮ Department of Molecular and Cellular Engineering, Institute for Human Gene Ther- Mice were immunized by s.c. injection with 400 g of myelin oligoden- apy, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 drocyte glycoprotein (MOG) 38–50 peptide emulsified in CFA containing 500 ␮gofMycobacterium tuberculosis H37 RA (Difco, Detroit, MI). Per- Received for publication April 5, 2000. Accepted for publication October 16, 2000. tussis toxin, 250 ng per mouse, was injected i.v. on the day of immuniza- The costs of publication of this article were defrayed in part by the payment of page tion and 48 h later. Mice were evaluated daily and scored for EAE, as charges. This article must therefore be hereby marked advertisement in accordance follows (21): 0, no disease; 1, tail paralysis; 2, hind limb weakness; 3, hind with 18 U.S.C. Section 1734 solely to indicate this fact. limb paralysis; 4, hind limb paralysis plus forelimb paralysis; 5, moribund 1 This work was supported by grants from the National Institutes of Health (NS40188, or dead. NS36581, NS40447, and AR44914). R.G. is a fellow of the Heisenberg program of the Deutsche Forschungsgemeinschaft. Production of soluble rTRAIL receptor 2 Address correspondence and reprint requests to Dr. Youhai Chen, 511 BRB-II/III, Department of Molecular and Cellular Engineering, University of Pennsylvania Soluble DR5 (sDR5) was produced using the Pichia pastoris system, as School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: follows (20). The cDNA that contains the full-length extracellular domain [email protected] of the human DR5 (a gift from Dr. W. El-Deiry (University of Pennsyl- 3 Abbreviations used in this paper: TRAIL, TNF-related apoptosis-inducing ligand; vania, Philadelphia, PA)) (22) was cloned into pGAPZa (Invitrogen, Carls- DcR, decoy receptor; DR, death receptor; EAE, experimental autoimmune encepha- bad, CA), which contains a PAOX1 promoter and a six-histidine tag as well lomyelitis; HSA, human serum albumin; L, ligand; MOG, myelin oligodendrocyte as a zeocin resistance gene. Several recombinant P. pastoris clones with glycoprotein; sDR, soluble DR. zeocin resistance were generated, which secreted up to 25 mg sDR5 per

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 1315

lected 40 h later, and cytokine concentrations were determined by ELISA. For proliferation assays, 0.5 ϫ 106 cells/well were used. [3H]Thymidine was added to the culture at 48 h, and cells were harvested 16 h later. Radioactivity was determined using a flatbed beta counter (Wallac, Gaithersburg, MD). MTT assay L929 cells were first cultured in flat-bottom 96-well plate, at 2 ϫ 104 cells/well, in 100 ␮l AIM-V medium (Life Technologies). Sixteen hours later, actinomycin D was added to the culture at 1 ␮g/well and cells were cultured for another 2 h. rTRAIL was then added, and culture was contin- ued for an additional 5 h. MTT was added only for the last hour of the culture. At the end of the culture, medium was removed and DMSO (100 ␮l/well) was added, and absorbance was determined at 595 nm. The per- centage of dead cells was calculated using untreated cells as control (25). FIGURE 1. sDR5 blocks TRAIL-induced apoptosis of mouse L929 cells. L929 cells were treated with different concentrations of rTRAIL (Bi- Results omol Research Laboratory, Plymouth Meeting, PA) with or without 5 Generation of a soluble rTRAIL receptor that blocks TRAIL ␮g/ml of sDR5. The percentage of dead cells was determined by MTT function assay, as described in Materials and Methods. The rTRAIL receptor used in this study was the sDR5 produced in the yeast P. pastoris system (26). To assess the biological activities of

liter of yeast culture. The recombinant was purified by Ni ion col- sDR5, we studied TRAIL-induced apoptosis of mouse L929 cells. As Downloaded from umn chromatography and treated with polymyxin B agarose (Sigma, St. shown in Fig. 1, TRAIL induces apoptosis of L929 cells in a dose- Louis, MO). The purity of the sDR5 was confirmed by PAGE and Coo- dependent manner. This was completely blocked by addition of massie blue staining; sDR5, which was 26 kDa, was the only protein band sDR5. In parallel experiments, we also studied TRAIL-induced apo- present. The purified sDR5 contains 1–2 ng of LPS per mg of protein, as determined by Limulus amebocyte lysate assay. This was comparable with ptosis of other tumor cell lines, including human Jurkat cells and the BSA and human serum albumin (HSA) purchased from Sigma, which K562 cells. The sDR5 selectively blocked TRAIL-induced apoptosis contain 1–4 ng LPS per mg of protein. In previous experiments, we have determined that this level of LPS had no effect on the development of EAE http://www.jimmunol.org/ or MOG-specific immune responses using LPS-free PBS as a control. In vitro, purified sDR5 does not induce proliferation of lymphocytes regard- less of the amount of the sDR5 in the culture (1–100 ␮g/ml) (unpublished data). Histopathological studies Mice were perfused with PBS and 10% Formalin phosphate. Spinal cords were first embedded in paraffin, cut into five pieces, and then sectioned at 5 ␮m and stained with luxol fast blue and cresyl violet (23). The total area of tissue section and the area of inflammation were measured using the by guest on October 1, 2021 Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) in a blinded manner. The percentage of the spinal cord with inflammation was calculated as follows: (area of the spinal cord that is infiltrated by inflam- matory cells/total area of the spinal cord sections measured) ϫ 100. A total of 10 tissue sections from cervical, thoracic, lumbar, and sacral spinal cord was analyzed for each animal. Apoptosis studies TUNEL staining was performed on paraffin-embedded Formalin-fixed spinal cord sections, as described (24). Briefly, sections were dewaxed in xylene, hydrated in water/ethanol, and washed in PBS. Endogenous peroxidase activ-

ity was inactivated by incubating the tissue in 3% H2O2. Fragmented DNA in apoptotic cells was labeled with digoxigenin-conjugated dUTP (Roche Mo- lecular Biochemicals, Indianopolis, IN) using TdT enzyme (Clontech, Palo Alto, CA). The labeled DNA was then detected by peroxidase-conjugated anti-digoxigenin Ab (Roche) using diaminobenzidine as substrate. Counter- stain was performed with methyl green. The number of apoptotic cells in the lesions was determined by microscopy. Reagents and ELISA

Mouse MOG38–50 peptide was synthesized using Fmoc solid-phase meth- ods and purified through HPLC by Research Genetics (Huntsville, AL). FIGURE 2. Exacerbation of EAE by TRAIL blockade. C57BL/6 mice, Pertussis toxin was purchased from List Biological Laboratories (Camp- six to eight mice per group, were immunized with MOG peptide to induce bell, CA). The following reagents were purchased from PharMingen (San Diego, CA): rat anti-mouse IL-2 (clone JES6-1A12), IL-4 (BVD4-1D11), EAE, as described in Materials and Methods. sDR5 or a control protein ␮ and IFN-␥ (R4-6A2) mAb; biotin-labeled rat anti-mouse IL-2 (clone JES6- (HSA), 200 g/mouse, was injected i.p. once every other day. Data pre- 5H6), IL-4 (BVD6-24G2), and IFN-␥ (XMG112D) mAb; recombinant sented are representative of three experiments. A, sDR5 injections were mouse IL-2, IL-4, IL-10, and IFN-␥. Quantitative ELISA for IL-2, IL-4, performed 7 days after disease onset until the end of the experiment. B, and IFN-␥ was performed as per manufacturer’s recommendations. sDR5 injections were performed from the day of immunization to the day of disease onset (17 days after immunization). The incidence of the disease Cell culture was more than 90% for all the experiments. Only mice that developed EAE For cytokine assays, splenocytes were cultured at 1.5 ϫ 106 cells/well in 0.2 were included for calculating mean clinical scores. The differences be- ml of DMEM (Life Technologies, Grand Island, NY) containing 10% FBS tween the two groups are statistically significant, as determined by Mann- Ͻ and various amounts of MOG38–50 peptide. Culture supernatants were col- Whitney test (p 0.01) for A, but not for B. 1316 TRAIL IN AUTOIMMUNE ENCEPHALOMYELITIS

FIGURE 3. Inflammation and apoptosis in the spinal cord. Mice were treated as in Fig. 2A and sacrificed 42 days after immunization. Spinal cord was treated and examined for histology and apoptosis, as described in Materials and Methods. A and B, Luxol fast blue stain- ing of spinal cords from sDR5 (A)- and HSA (B)-treated mice (original magnification ϫ100). C and D, TUNEL staining of spinal cords from sDR5 (C)- and HSA (D)- treated mice (original magnification ϫ200). Arrows in- dicates apoptotic nuclei, which are shown in brown. Downloaded from

of these cells (20) (data not shown). Thus, sDR5 is biologically active of TRAIL blockade during the inductive phase of the disease.

and can be used to block TRAIL function in vitro. Thus, MOG-immunized mice were treated with sDR5 or a control http://www.jimmunol.org/ protein from the day of immunization until the day of disease onset TRAIL blockade exacerbates MOG-induced autoimmune (when 1 mouse of 12 developed signs of EAE). As shown in Fig. encephalomyelitis 2B, no significant differences between control and sDR5-treated To investigate the roles of TRAIL in vivo, we examined the conse- groups were observed with respect to disease onset or severity. quences of TRAIL blockade in an animal model of multiple sclerosis. This suggests that TRAIL blockade during the inductive phase of

C57BL/6 mice were immunized with MOG38–50 peptide to induce EAE alone may not be sufficient to affect the disease course. EAE. Eight days after disease onset (when ϳ90% of the mice had developed signs of EAE), mice were injected i.p. with either 200 ␮g TRAIL blockade enhances the formation of inflammatory lesions of sDR5 or a control protein once every other day for a total of 17 in the CNS by guest on October 1, 2021 days. As shown in Fig. 2A, C57BL/6 mice developed typical EAE To investigate the effect of TRAIL blockade on the formation of starting ϳ18 days after immunization. Injection of sDR5 significantly inflammatory lesions in the CNS, we performed quantitative his- exacerbated the disease. The mean maximal disease score in the con- topathological studies of spinal cords. As shown in Fig. 3, A and B, trol group was 2 Ϯ 0.5. This was increased to 3.3 Ϯ 0.4 in the sDR5- treated group. One of six mice died from EAE in the sDR5-treated group, whereas none died in the control group. To determine whether the effect of TRAIL blockade is limited to the effector phase of EAE, we also investigated the consequences

FIGURE 5. Quantitative analysis of the degree of apoptosis in the CNS. FIGURE 4. Quantitative analysis of the degree of inflammation in the C57BL/6 mice, four mice per group, were treated as in Fig. 2A and sac- CNS. C57BL/6 mice, six mice per group, were treated as in Fig. 2A and rificed 42 days after immunization. Spinal cord was treated and examined sacrificed 42 days after immunization. Spinal cord was treated and exam- for apoptosis, as described in Materials and Methods. The numbers of ined for histology, as described in Materials and Methods. Each data point apoptotic nuclei/mm2 of inflamed tissue were counted and plotted against represents a percentage of spinal cord section that is inflamed. The hori- the percentages of the corresponding spinal cord sections that were in- zontal bars represent the means of respective groups. The differences be- flamed. The differences between the two groups are not statistically sig- tween the two groups are statistically significant, as determined by Student nificant, as determined by Mann-Whitney test (p Ͼ 0.05). f, Mice treated t test (p Ͻ 0.05). f, Mice treated with HSA. E, Mice treated with sDR5. with HSA. E, Mice treated with sDR5. The Journal of Immunology 1317

FIGURE 6. MOG-specific proliferation and cytokine pro- duction in vitro. C57BL/6 mice, six mice per group, were immunized with MOG peptide to induce EAE. sDR5 or HSA, 200 ␮g/mouse, was injected i.p. once every other day for a total of 16 days as in Fig. 2B. Mice were sacrificed 10 days Downloaded from after the last injection of sDR5, and splenocytes were tested for anti-MOG proliferative and cy- tokine responses, as described in Materials and Methods. Data presented are representa- http://www.jimmunol.org/ tive of two experiments. by guest on October 1, 2021

inflammatory lesions were readily detectable in both control and The vast majority of apoptotic cells were localized within the inflam- sDR5-treated mice. The inflammatory lesions consisted mostly of matory lesions. In sections that did not contain inflammatory lesions, lymphocytes and macrophages as well as granulocytes and micro- few or no apoptotic cells were detected. To compare the degree of glial cells. To quantify the degree of inflammation, the area of apoptosis between control and sDR5-treated groups, we first calcu- spinal cord sections that showed signs of infiltration as well as the lated the number of apoptotic cells/mm2 of spinal cord that showed total area of each spinal cord sections were measured using the signs of inflammation. The number of apoptotic cells was then plotted Image-Pro Plus software. The degree of inflammation was then against the degree of inflammation in the same section. As shown in evaluated based on the percentage of the spinal cord areas that Fig. 5, no statistically significant differences in the degree of apoptosis show signs of inflammation. As shown in Fig. 4, mice treated with were observed between control and sDR5-treated groups. We then sDR5 had significantly more severe inflammation than control examined whether neural cell death was affected by TRAIL blockade mice. The extent of demyelination correlated well with the degree in EAE. Apoptotic neural cells were identified based on their mor- of inflammation and clinical score. Mice with similar disease phology and anatomic location in addition to TUNEL staining. We scores exhibited similar degrees of demyelination and inflamma- found that the number of apoptotic neural cells per cross section of tion regardless of treatment groups (data not shown). spinal cord for control and sDR5-treated animals were 3.6 Ϯ 2.9 and 4.3 Ϯ 1.4, respectively ( p Ͼ 0.05, as determined by ANOVA). In TRAIL blockade does not affect apoptosis of inflammatory cells mice that did not develop EAE, few or no apoptotic neural cells were in the CNS detected. Taken together, these results strongly suggest that TRAIL Some members of the TNF family are capable of inducing apoptosis may not regulate apoptosis in the CNS during EAE. of normal and/or tumor cells. TRAIL has been shown to induce ap- optosis of some, but not all, tumor cell lines (2–8). The effect of sDR5 TRAIL blockade enhances anti-MOG T cell responses on EAE can be explained by its blockade of TRAIL-induced apopto- EAE is a T cell-mediated autoimmune disease. To determine sis of inflammatory cells. To test this theory, we examined the effect whether TRAIL blockade affected the functions of encephalito- of TRAIL blockade on apoptosis of inflammatory cells and neural genic T cells, we studied anti-MOG T cell responses in mice fol- cells in the CNS. As shown in Fig. 3C/D, apoptotic cells were readily lowing TRAIL blockade. Mice were immunized with MOG pep- detectable in the spinal cords of both control and sDR5-treated mice. tide to induce EAE and treated with either sDR5 or BSA for a total 1318 TRAIL IN AUTOIMMUNE ENCEPHALOMYELITIS of 16 days. Anti-MOG T cell responses were determined ex vivo degrees of apoptosis in the CNS and arthritic joints (20) were not 10 days after the last injection of sDR5. As shown in Fig. 6, affected by TRAIL blockade suggests that TRAIL may not regu- splenocytes of BSA-treated mice produced primarily Th1-type cy- late apoptosis of inflammatory cells in these systems. Experiments tokines (i.e., IL-2 and IFN-␥) in response to MOG peptide. This are underway to investigate whether different types of inflamma- was significantly increased in mice treated with sDR5. A small but tory cells respond differently to TRAIL signals in inflammation. significant amount of IL-4 was also detected in sDR5-treated Thus, by in vivo TRAIL blockade, we have established that, group. Interestingly, lymphocyte proliferative responses were unlike TNF, TRAIL inhibits autoimmune encephalomyelitis and comparable between BSA- and sDR5-treated groups. These results prevents activation of autoreactive T cells. Because EAE is an suggest that TRAIL blockade enhances functions of both Th1- and animal model for human multiple sclerosis and because TRAIL Th2-type cells in vivo. and its receptors are also expressed by human cells, results re- ported in this study may be important not only for our understand- Discussion ing of the pathogenesis of EAE, but also for designing therapeutic The TNF family of play crucial roles in a number of strategies for the treatment of autoimmune diseases such as mul- biological processes, including apoptosis, immunity, inflamma- tiple sclerosis. tion, and development. TRAIL is a newly identified member of the TNF family, which, unlike TNF, induces apoptosis of tumor cells, Acknowledgments but not normal cells. However, both TRAIL and TRAIL receptors We thank Dr. W. El-Deiry for the human DR5 cDNA. are expressed in normal nontransformed tissues; the roles of TRAIL in these tissues are unknown and are the subjects of this

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