- Email: [email protected]
FasL and Bax on brain and infiltrating T cells in the central nervous system is closely associated with apoptotic cell death during autoimmune encephalomyelitis
Journal of Neuroimmunology 106 (2000) 165–171 www.elsevier.com / locate / jneuroin
Coexpression of Fas / FasL and Bax on brain and infiltrating T cells in the central nervous system is closely associated with apoptotic cell death during autoimmune encephalomyelitis Toshihiko Kohji, Yoh Matsumoto* Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2 -6 Fuchu, Tokyo 183 -8526, Japan Received 15 December 1999; received in revised form 28 January 2000; accepted 31 January 2000
Abstract Recent studies have suggested that autoimmune inflammation elicited in the central nervous system (CNS) is subsided by apoptotic cell death of inflammatory cells. To elucidate the molecular mechanism of apoptosis of infiltrating T and other cells occurring in the CNS during autoimmune encephalomyelitis, we determined the type of apoptotic cells and the localization of apoptosis-related molecules (Fas, FasL, Bax, Bcl-2 and active caspase 3) by immunohistochemistry. Double labeling with the TUNEL method and cell-type markers showed that infiltrating T cells and microglia / macrophages underwent apoptosis, while astrocytes and neurons did not. Staining for apoptosis-related molecules revealed that infiltrating T cells and microglia / macrophages, but not astrocytes and neurons, expressed both Fas–FasL and Bax. The distribution and cell type of active caspase 3-positive cells were essentially the same as those of TUNEL-positive cells. These findings suggest that coexpression of Fas / FasL and Bax is closely associated with apoptotic cell death of infiltrating T cells and microglia in the CNS. Furthermore, astrocytes which express Fas and FasL, but not Bax, may play an important role in regulating inflammation in the CNS by inducing apoptotic cell death of infiltrating T cells and microglia, both of which have an inflammationpromoting nature. 2000 Elsevier Science B.V. All rights reserved. Keywords: Experimental autoimmune encephalomyelitis; Apoptosis; Fas; Bax
1. Introduction Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease that is inducible in susceptible strains of animals by immunization with brainspecific antigens such as myelin basic protein (MBP) (Lassmann, 1983). Rats usually develop acute and monophasic EAE after immunization with MBP and do not show relapse of the clinical signs. Recent studies have indicated that infiltrating T cells are eliminated from the central nervous system (CNS) by apoptotic cell death at the peak and recovery stages of EAE (Pender et al., 1992; Schmied et al., 1993). In a previous study, we demonstrated by double staining with the terminal deoxynu-
Abbreviations: EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; MBP, myelin basic protein *Corresponding author. Tel.: 181-423-25-3881, extn. 4719; fax: 181423-21-8678. E-mail address: [email protected] (Y. Matsumoto)
cleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and immunocytochemistry for astrocytes and microglia that astrocytes are more closely associated with apoptotic cells than microglia / macrophages, suggesting that astrocytes, rather than microglia, induce programmed cell death of infiltrating inflammatory cells (Kohji et al., 1998). In addition, we noticed that some microglia / macrophages also undergo apoptosis. However, the molecular mechanisms of apoptosis of these inflammatory and brain cells remain poorly understood. In the present study, we determined by immunohistochemistry the type of apoptotic cells and the localization of apoptosis-related molecules in the CNS during EAE to elucidate the relationship between the presence or absence of the molecules and apoptotic cell death. For this purpose, we selected Fas, FasL, caspase 3, Bax and Bcl-2 from a large number of apoptosis-related molecules because these molecules play a key role in induction or prevention of Fas-mediated apoptosis (Adams and Cory, 1998; Green and Reed, 1998). As suggested previously and confirmed here, only infiltrating T cells and microglia / macrophages,
0165-5728 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00238-1
166
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
but not astrocytes and neurons, showed apoptosis. The distribution and cell type of active caspase 3-positive cells were essentially the same as those of TUNEL-positive cells. Furthermore, we found that T cells and microglia / macrophages expressed all the apoptosis-related molecules examined (Fas, FasL, Bax and Bcl-2), whereas astrocytes did not express Bax. Neurons showed only Bcl-2 immunoreactivities. These findings suggest that coexpression of Fas / FasL and Bax is closely associated with apoptotic cell death of brain and infiltrating T cells.
2. Materials and methods
2.1. Animals and EAE induction Lewis rats were purchased from Seiwa (Fukuoka, Japan) and bred in our animal facility. Rats at age 8–12 weeks were used throughout the experiments. Acute EAE was induced as described previously (Ohmori et al., 1992). Briefly, each rat was injected in the hind footpads bilaterally with an emulsion containing 100 mg of MBP in CFA (Mycobacterium tuberculosis H37Ra, 5 mg / ml). Immunized rats were observed daily for clinical signs of EAE. The clinical stage of EAE was divided into four (Grade 1, floppy tail; Grade 2, mild paraparesis; Grade 3, severe paraparesis; Grade 4, tetraparesis or moribund condition). In this study, tissue sampling was performed at the early (days 10–12 post-immunization, Grade 1), peak (days 13–15 post-immunization, Grades 3 and 4) and recovery (days 21–23 post-immunization, Grade 0) stages of EAE. At each stage, the spinal cord tissues of three rats were examined and the findings were compared with those of normal unimmunized rats.
2.2. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ( TUNEL) DNA fragmentation was detected by in situ nick end labeling as described previously (Kohji et al., 1998). Both frozen and paraffin-embedded sections were used in this study. Frozen sections were air dried and fixed with 1% paraformaldehyde for 30 min at room temperature and treated with a solution of 0.1% Triton X-100 and 0.1% sodium citrate for 2 min at 48C. Paraffin-embedded sections were deparaffinized and rehydrated, and endogenous peroxidase was blocked by incubation in 0.3% H 2 O 2 in methanol. Subsequently, slides were incubated in a terminal deoxynucleotidyl transferase (TdT) buffer solution (140 mM sodium cacodylate, 1 mM cobalt chloride, 30 mM Tris–HCl, pH 7.2) containing 0.15 U / ml TdT (Gibco BRL, Tokyo) and 0.004 nmol / ml digoxigenindUTP (Boehringer-Mannheim, Tokyo) for 60 min at 378C, and then in the TB buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min. They were allowed to react with alkaline phosphatase (AP)-labeled anti-digox-
igenin antibody (Boehringer-Mannheim) for 60 min. Positive cells were finally visualized by incubating sections with the Sigma FAST E BCIP/ NBT Buffered Substrate Tablet solution (Sigma) containing levamisole (0.24 mg / ml) which blocks endogenous AP.
2.3. Immunohistochemical staining After incubation with normal sheep or goat serum, sections were allowed to react with the first antibody for 60 min. The first antibodies used in the present study were R73 (anti-TCR ab, 1:800), OX42 (anti-microglia / macrophage, 1:800), anti-glial fibrillary acidic protein (GFAP), anti-Fas (1:1600, Wako, Tokyo), anti-FasL (1:800, Wako), anti-Bax (P-19) (1:2400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bcl-2 (N-19) (1:200, Santa Cruz Biotechnology) and anti-activated caspase 3 (1:500, Pharmigen). For the detection of microglia, biotinylated tomato lectin was also used (Acarin et al., 1994; Kohji et al., 1998). Then, sections were incubated with biotinylated anti-mouse or rabbit IgG (Vector, Burlingame, CA, USA) followed by horseradish peroxidase (HRP)-labeled VECTASTAINÆ Elite ABC Kit (Vector). HRP binding sites were detected with the Sigma FAST E DAB Peroxidase Substrate Tablet Set solution (Sigma). For GFAP immunostaining, the peroxidase anti-peroxidase method was applied using Histogen PAP rabbit Universal Kit ´ CA, USA). Control sections for (BioGenex, San Ramon, the staining were prepared by omitting the first antibodies or replacing them with LN2 (IgG1), which is against human B cells (Seikagaku Corp.), or with W6 / 32 (IgG2a), which is against human MHC class I molecules (Serotec). These negative controls gave no specific staining (data not shown).
2.4. Double staining for apoptotic cells and brain cells Double staining for TUNEL-positive cells and cell-type markers such as T cells, microglia and astrocytes was performed using frozen sections. In the first step, apoptotic cells were detected by the TUNEL method and AP developed in the BCIP/ NBT solution as a blue color. After washing, slides were stained for T cells, microglia or astrocytes using HRP as an enzyme. HRP developed in the DAB solution as a brown color.
3. Results
3.1. Identification of the type of cells undergoing apoptosis in the CNS We first determined the type of cells undergoing apoptosis in the CNS under normal and pathological conditions. As reported previously (Kohji et al., 1998) and confirmed here, there were no TUNEL-positive cells in the normal
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
CNS (data not shown). During EAE, a large number of TUNEL-positive cells were found in the CNS parenchyma mainly at the peak stage. Double staining revealed that the majority of apoptotic cells were also stained positively with T cell marker (Fig. 1A) and that some were microglia / macrophages (Fig. 1B). However, astrocytes were negative for TUNEL throughout the course of EAE (Fig. 1C). In addition, there were no TUNEL-positive cells with the morphological features of neurons (not shown).
3.2. Localization of apoptosis-related molecules As apoptosis-related molecules, we examined the presence or absence of Fas, FasL, Bax, Bcl-2 and active caspase 3 by immunohistochemistry. Under normal conditions, no apoptosis-related molecules except Bcl-2 were detectable on cells in the CNS (Table 1). Bcl-2 was expressed weakly on some neurons in the spinal cord (data not shown). During EAE, apoptosis-related molecules were expressed on a variety of cells in the CNS (Fig. 2A,C,E,G,I, early stage; Fig. 2B,D,F,H,J, peak stage). As shown in Fig. 2A, a few mononuclear and small stellate cells stained positively for Fas at the early stage of EAE (arrows). At the peak stage of the disease, Fas-positive cells increased in number and many Fas-positive small stellate cells were detectable in the parenchyma (Fig. 2B). The staining pattern for FasL was similar to that for Fas (Fig. 2C,D). Bax immunoreactivities were weakly detectable at the early stage (Fig. 2E) and increased at the peak
167
Table 1 Expression of apoptosis-related molecules on brain and inflammatory cells in the CNS Fas
FasL
Bax
Bcl-2
Active caspase 3
TUNEL
Normal T cells Microglia / MF Astrocytes Neurons
n.d.a 2b 2 2
n.d. 2 2 2
n.d. 2 2 2
n.d. 2 2 1
n.d. 2 2 2
n.d. 2 2 2
EAE T cells Microglia / MF Astrocytes Neurons
11 11 1 2
11 11 11 2
11 11 2 2
11 11 11 1
11 1 2 2
11 1 2 2
a
Not detectable. T cells are not detectable in the CNS parenchyma of normal rats. b According to the number of positive cells in each staining, histological findings are graded into three categories: 2, no positive cells; 1, a few positive cells; 11, many positive cells.
stage (Fig. 2F). At both stages, inflammatory cells and some small stellate cells were positive for Bax. Bcl-2 immunoreactivities were mainly detectable on cells with long processes at the early stage (Fig. 2G), but all the components in the CNS were stained more or less positively at the peak stage (Fig. 2H). In active caspase staining, the majority of positive cells were round mononuclear cells (Fig. 2I,J). Some small stellate cells were also stained positively (data not shown).
Fig. 1. Identification of the type of cells undergoing apoptosis. TUNEL-positive (blue) TCRab-positive (brown) T cells are frequently seen in the parenchyma (arrows in A). Some microglia (brown) are positive for TUNEL (blue) (arrows in B). Astrocytes are negative for TUNEL (C). (A) Double staining with anti-TCRab (R73) and TUNEL. (B) Double staining with tomato lectin for microglia and TUNEL. (C) Double staining with anti-GFAP antibody and TUNEL. Original magnification 3600.
168
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
Fig. 2. Expression of Fas (A, B), FasL (C, D), Bax (E, F), Bcl-2 (G, H) and active caspase 3 (I, J) in the spinal cord at the early (A, C, E, G, I) and peak (B, D, F, H, J) stages of acute EAE. Some positive cells in (A), (I) and (J) are indicated by arrows. Original magnification: (A, C, E, G, I), 3350; (B, D, F, H, J), 3280.
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
3.3. Relationship between apoptotic cells and cells expressing apoptosis-related molecules As shown in Table 1, cells expressing Fas / FasL and Bax underwent apoptotic cell death, whereas cells lacking at least one of these molecules did not show apoptosis. To determine the type of cells expressing Fas and Bax in more detail, sets of serial sections, 10 mm thick, were stained for these molecules and markers for microglia and astrocytes (Fig. 3). As shown in Fig. 3A, Fas immunoreactivities were detected on small stellate cells (arrows) and long fibers running radially from the spinal cord surface (arrowheads). Comparison of this staining pattern with that of
169
microglia (Fig. 3B) and astrocytes (Fig. 3C) revealed that the former and latter staining pattern overlapped with microglial and astroglial staining, respectively. These findings indicate that the Fas molecules are expressed on some, but not all, microglia and astrocytes. On the other hand, Bax immunoreactivities were present only on small stellate cells (Fig. 3D) which were also positive by microglial staining (Fig. 3E). However, astrocytes did not show Bax immunoreactivities (Fig. 3D,F). Table 1 summarizes all the findings obtained by the present examination. During EAE, T cells and microglia / macrophages showed apoptosis. Fas and FasL were expressed on T cells, microglia / macrophages and astrocytes, but not on neurons.
Fig. 3. Identification of Fas-expressing and Bax-expressing cells using serial sections. Fas immunoreactivities are present on small stellate cells (arrows in A) and long fibers (arrowheads in A). Fas-positive stellate cells and fibers are also positive for microglia marker (arrows in B) and astrocyte marker (arrowhead in C), respectively. On the other hand, Bax immunoreactivities (D) are present only on microglia (E), but not on astrocytes (F). Two sets of serial sections were stained for Fas, microglia and astrocytes (A, B and C) and for Bax (D), microglia (E) and astrocytes (F). Original magnification 3200.
170
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
Bax was detectable on T cells and microglia / macrophages, whereas Bcl-2 expression was observed on all the above cells (Table 1).
4. Discussion EAE is a T cell-mediated autoimmune disease characterized by the presence of inflammatory cells mainly comprising T cells and macrophages in the CNS. In acute EAE, autoimmune inflammation in the CNS begins around day 10 post-immunization, reaches a maximal level on day 12 and subsides by day 21 (Matsumoto et al., 1993). In earlier studies, we showed that the majority of T cells do not proliferate vigorously in the CNS (Ohmori et al., 1992) and others later demonstrated that this phenomenon is attributable to apoptotic cell death of T cells in the CNS (Pender et al., 1991, 1992; Schmied et al., 1993). Based on these findings, we recently reported two important findings (Kohji et al., 1998). First, the majority of apoptotic infiltrating T cells are found in the CNS parenchyma. Second, astrocytes are more frequently associated with apoptotic cells than microglia. These findings suggest that astrocytes play an important role in the induction of apoptosis. In the present study, we found that infiltrating T cells and microglia / macrophages undergo apoptosis in the CNS during EAE and that coexpression of Fas / FasL and Bax on these cells is closely associated with apoptosis. White et al. (1998) performed similar experiments using inflammatory cells isolated from the spinal cord of rats with EAE and reported that expression of Fas / FasL, but not Bax, on these cells influences the susceptibility to apoptosis. However, this finding does not deny the role of Bax in apoptotic processes in the CNS. As clearly shown here, astrocytes which express Fas and FasL, but not Bax, did not show apoptosis throughout the course of the disease, strongly suggesting that expression of Fas and FasL is not sufficient for apoptotic cell death. Recently, it was demonstrated that there are two main signal transduction pathways for mediating Fas–FasL-induced apoptosis (Scaffidi et al., 1998). One is the FADD (Fas-associated death domain)-mediated pathway in which FADD recruited after ligand binding activates caspase 8. The other is the mitochondrial pathway in which cytochrome c released from mitochondria activates caspases. The molecular mechanism of the latter pathway has been elucidated very recently. Caspase 8 activated after crosslinking of Fas with FasL cleaves full-length Bid into truncated Bid (tBid) (Li et al., 1998). tBid then translocates from the cytosol to mitochondria and is associated with Bax. This complex stimulates the release of cytochrome c from mitochondria which induces apoptosis (Desagher et al., 1999). Bcl-2, on the other hand, suppresses the apoptosis-inducing function of Bax by forming Bax–Bcl-2 heterodimers (Oltvai et al., 1993). Taken together, Bax and Bcl-2 are key molecules representing
pro- and anti-apoptosis function, respectively, in the mitochondrial pathway. The findings obtained in the present study suggest that the mitochondrial pathway mainly operates in apoptotic cell death in the CNS. Consistent with our findings, Spanaus et al. (1998) recently demonstrated that cultured activated microglia undergo Fas-mediated apoptosis which is induced by down-regulation of Bcl-2 but not Bax. Since microglia present MBP to MBP-reactive T cells as APC and induce T cell proliferation (Matsumoto et al., 1992), apoptotic cell death of this cell type seems to be essential for the regulation of autoimmune inflammation in the CNS. With regard to oligodendrocytes, we did not succeed in staining this cell type with anti-cyclic nucleotide phosphodiesterase (CNPase), probably because the antibody we used is not appropriate for immunohistochemistry. However, previous studies demonstrated that oligodendroglia which express Fas and Bcl-2, but not FasL and Bax, do not show apoptosis (Bonetti et al., 1997). Neurons express only Bcl-2 among the molecules examined and do not undergo apoptosis under normal and pathological conditions. It is interesting to note the role of astrocytes in apoptotic cell death in the CNS. As demonstrated previously (Matsumoto et al., 1992, 1993), astrocytes do not present antigen to T cells and rather suppress antigen-driven T cell proliferation. Moreover, astrocytes are closely associated with apoptotic cells during autoimmune inflammation in the spinal cord (Kohji et al., 1998). These findings strongly suggest that astrocytes may induce apoptosis of infiltrating T cells and microglia. Recently, it has been demonstrated that cultured astrocytes induce apoptosis of T line cells via the Fas–FasL pathway (Choi et al., 1999). Since astrocytes express Fas and FasL, but not Bax, and do not undergo apoptosis by themselves, this type of cell may play a critical role in regulation of inflammation in the CNS by inducing apoptosis of infiltrating T cells and cells supporting inflammation, i.e. microglia (Matsumoto et al., 1992), during the recovery from inflammation in the CNS. In the present study, we have shown that coexpression of Fas / FasL and Bax plays an important role in apoptotic cell death of brain and infiltrating T cells in the CNS. By examining apoptosis-associated molecules in more detail, the precise mechanisms of formation and subsidization of autoimmune inflammation in the CNS will be elucidated.
Acknowledgements We thank Y. Kawazoe, K. Kohyama and K. Nomura for technical assistance. This study was supported, in part, by Grants-in-Aid (10357005, 09480216 and 09670682) from the Ministry of Education, Japan.
References ´ Acarin, L., Vela, J.M., Gonzalez, B., Castellano, B., 1994. Demonstration of poly-N-acetyl lactosamine residues in ameboid and ramified
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171 microglial cells in rat brain by tomato lectin binding. J. Histochem. Cytochem. 42, 1003–1041. Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Bonetti, B., Pohl, J., Gao, Y., Raine, C.S., 1997. Cell death during autoimmune demyelination: effector but not target cells are eliminated by apoptosis. J. Immunol. 159, 5733–5741. Choi, C., Park, J., Lee, J., Lim, J., Shin, E., Ahn, Y., Kim, C., Kim, S., Kim, J., Choi, I., Choi, I., 1999. Fas ligand and Fas are expressed constitutively in human astrocytes and the expression increases with IL-1, IL-6, TNF-a, or IFN-g. J. Immunol. 162, 1889–1895. Desagher, K., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., Martinou, J., 1999. Bidinduced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144, 891– 901. Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312. Kohji, T., Tanuma, N., Aikawa, Y., Kojima, T., Matsumoto, Y., 1998. Interaction between apoptotic cells and reactive brain cells in the central nervous system of rats with autoimmune encephalomyelitis. J. Neuroimmunol. 82, 168–174. Lassmann, H. (Ed.), 1983. Comparative Neuropathology of Chronic Experimental Allergic Encephalomyelitis and Multiple Sclerosis, Springer, Berlin, pp. 9–94. Li, H., Zhu, H., Xu, C., Yuan, J., 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491–501. Matsumoto, Y., Ohmori, K., Fujiwara, M., 1992. Immune regulation by brain cells in the central nervous system: microglia but not astrocytes present myelin basic protein to encephalitogenic T cells under in vivo-mimicking conditions. Immunology 76, 209–216. Matsumoto, Y., Hanawa, H., Tsuchida, M., Abo, T., 1993. In situ inactivation of infiltrating T cells in the central nervous system with
171
autoimmune encephalomyelitis. The role of astrocytes. Immunology 79, 381–390. Ohmori, K., Hong, Y., Fujiwara, M., Matsumoto, Y., 1992. In situ demonstration of proliferating cells in the rat central nervous system during experimental autoimmune encephalomyelitis. Evidence suggesting that most infiltrating T cells do not proliferate in the target organ. Lab. Invest. 66, 54–62. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619. Pender, M.P., Nguyen, K.B., McCombe, P.A., Kerr, J.F.R., 1991. Apoptosis in the nervous system in experimental allergic encephalomyelitis. J. Neurol. Sci. 104, 81–87. Pender, M.P., McCombe, A., Yoog, G., Nguyen, K.B., 1992. Apoptosis of ab T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: its possible implication for recovery and acquired tolerance. J. Autoimmun. 5, 401–410. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K.J., Debatin, K., Krammer, P.H., Peter, M.E., 1998. Two CD95 (APO-1 / Fas) signaling pathways. EMBO J. 17, 1675–1687. Schmied, M., Breitschopf, H., Gold, R., Zischler, H., Rothe, G., Wekerle, H., Lassmann, H., 1993. Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell death as a mechanism to control inflammation in the brain. Am. J. Pathol. 143, 446–452. Spanaus, K.S., Schlapbach, R., Fontana, A., 1998. TNF-a and IFN-g render microglia sensitive to Fas ligand-induced apoptosis by induction of Fas expression and down-regulation of Bcl-2 and Bcl-xL. Eur. J. Immunol. 28, 4398–4408. White, C.A., McCombe, P.A., Pender, M.P., 1998. The role of Fas, Fas ligand and Bcl-2 in T cell apoptosis in the central nervous system in experimental autoimmune encephalomyelitis. J. Neuroimmunol. 82, 47–55.
Coexpression of Fas / FasL and Bax on brain and infiltrating T cells in the central nervous system is closely associated with apoptotic cell death during autoimmune encephalomyelitis Toshihiko Kohji, Yoh Matsumoto* Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2 -6 Fuchu, Tokyo 183 -8526, Japan Received 15 December 1999; received in revised form 28 January 2000; accepted 31 January 2000
Abstract Recent studies have suggested that autoimmune inflammation elicited in the central nervous system (CNS) is subsided by apoptotic cell death of inflammatory cells. To elucidate the molecular mechanism of apoptosis of infiltrating T and other cells occurring in the CNS during autoimmune encephalomyelitis, we determined the type of apoptotic cells and the localization of apoptosis-related molecules (Fas, FasL, Bax, Bcl-2 and active caspase 3) by immunohistochemistry. Double labeling with the TUNEL method and cell-type markers showed that infiltrating T cells and microglia / macrophages underwent apoptosis, while astrocytes and neurons did not. Staining for apoptosis-related molecules revealed that infiltrating T cells and microglia / macrophages, but not astrocytes and neurons, expressed both Fas–FasL and Bax. The distribution and cell type of active caspase 3-positive cells were essentially the same as those of TUNEL-positive cells. These findings suggest that coexpression of Fas / FasL and Bax is closely associated with apoptotic cell death of infiltrating T cells and microglia in the CNS. Furthermore, astrocytes which express Fas and FasL, but not Bax, may play an important role in regulating inflammation in the CNS by inducing apoptotic cell death of infiltrating T cells and microglia, both of which have an inflammationpromoting nature. 2000 Elsevier Science B.V. All rights reserved. Keywords: Experimental autoimmune encephalomyelitis; Apoptosis; Fas; Bax
1. Introduction Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease that is inducible in susceptible strains of animals by immunization with brainspecific antigens such as myelin basic protein (MBP) (Lassmann, 1983). Rats usually develop acute and monophasic EAE after immunization with MBP and do not show relapse of the clinical signs. Recent studies have indicated that infiltrating T cells are eliminated from the central nervous system (CNS) by apoptotic cell death at the peak and recovery stages of EAE (Pender et al., 1992; Schmied et al., 1993). In a previous study, we demonstrated by double staining with the terminal deoxynu-
Abbreviations: EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; MBP, myelin basic protein *Corresponding author. Tel.: 181-423-25-3881, extn. 4719; fax: 181423-21-8678. E-mail address: [email protected] (Y. Matsumoto)
cleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and immunocytochemistry for astrocytes and microglia that astrocytes are more closely associated with apoptotic cells than microglia / macrophages, suggesting that astrocytes, rather than microglia, induce programmed cell death of infiltrating inflammatory cells (Kohji et al., 1998). In addition, we noticed that some microglia / macrophages also undergo apoptosis. However, the molecular mechanisms of apoptosis of these inflammatory and brain cells remain poorly understood. In the present study, we determined by immunohistochemistry the type of apoptotic cells and the localization of apoptosis-related molecules in the CNS during EAE to elucidate the relationship between the presence or absence of the molecules and apoptotic cell death. For this purpose, we selected Fas, FasL, caspase 3, Bax and Bcl-2 from a large number of apoptosis-related molecules because these molecules play a key role in induction or prevention of Fas-mediated apoptosis (Adams and Cory, 1998; Green and Reed, 1998). As suggested previously and confirmed here, only infiltrating T cells and microglia / macrophages,
0165-5728 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00238-1
166
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
but not astrocytes and neurons, showed apoptosis. The distribution and cell type of active caspase 3-positive cells were essentially the same as those of TUNEL-positive cells. Furthermore, we found that T cells and microglia / macrophages expressed all the apoptosis-related molecules examined (Fas, FasL, Bax and Bcl-2), whereas astrocytes did not express Bax. Neurons showed only Bcl-2 immunoreactivities. These findings suggest that coexpression of Fas / FasL and Bax is closely associated with apoptotic cell death of brain and infiltrating T cells.
2. Materials and methods
2.1. Animals and EAE induction Lewis rats were purchased from Seiwa (Fukuoka, Japan) and bred in our animal facility. Rats at age 8–12 weeks were used throughout the experiments. Acute EAE was induced as described previously (Ohmori et al., 1992). Briefly, each rat was injected in the hind footpads bilaterally with an emulsion containing 100 mg of MBP in CFA (Mycobacterium tuberculosis H37Ra, 5 mg / ml). Immunized rats were observed daily for clinical signs of EAE. The clinical stage of EAE was divided into four (Grade 1, floppy tail; Grade 2, mild paraparesis; Grade 3, severe paraparesis; Grade 4, tetraparesis or moribund condition). In this study, tissue sampling was performed at the early (days 10–12 post-immunization, Grade 1), peak (days 13–15 post-immunization, Grades 3 and 4) and recovery (days 21–23 post-immunization, Grade 0) stages of EAE. At each stage, the spinal cord tissues of three rats were examined and the findings were compared with those of normal unimmunized rats.
2.2. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ( TUNEL) DNA fragmentation was detected by in situ nick end labeling as described previously (Kohji et al., 1998). Both frozen and paraffin-embedded sections were used in this study. Frozen sections were air dried and fixed with 1% paraformaldehyde for 30 min at room temperature and treated with a solution of 0.1% Triton X-100 and 0.1% sodium citrate for 2 min at 48C. Paraffin-embedded sections were deparaffinized and rehydrated, and endogenous peroxidase was blocked by incubation in 0.3% H 2 O 2 in methanol. Subsequently, slides were incubated in a terminal deoxynucleotidyl transferase (TdT) buffer solution (140 mM sodium cacodylate, 1 mM cobalt chloride, 30 mM Tris–HCl, pH 7.2) containing 0.15 U / ml TdT (Gibco BRL, Tokyo) and 0.004 nmol / ml digoxigenindUTP (Boehringer-Mannheim, Tokyo) for 60 min at 378C, and then in the TB buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min. They were allowed to react with alkaline phosphatase (AP)-labeled anti-digox-
igenin antibody (Boehringer-Mannheim) for 60 min. Positive cells were finally visualized by incubating sections with the Sigma FAST E BCIP/ NBT Buffered Substrate Tablet solution (Sigma) containing levamisole (0.24 mg / ml) which blocks endogenous AP.
2.3. Immunohistochemical staining After incubation with normal sheep or goat serum, sections were allowed to react with the first antibody for 60 min. The first antibodies used in the present study were R73 (anti-TCR ab, 1:800), OX42 (anti-microglia / macrophage, 1:800), anti-glial fibrillary acidic protein (GFAP), anti-Fas (1:1600, Wako, Tokyo), anti-FasL (1:800, Wako), anti-Bax (P-19) (1:2400, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bcl-2 (N-19) (1:200, Santa Cruz Biotechnology) and anti-activated caspase 3 (1:500, Pharmigen). For the detection of microglia, biotinylated tomato lectin was also used (Acarin et al., 1994; Kohji et al., 1998). Then, sections were incubated with biotinylated anti-mouse or rabbit IgG (Vector, Burlingame, CA, USA) followed by horseradish peroxidase (HRP)-labeled VECTASTAINÆ Elite ABC Kit (Vector). HRP binding sites were detected with the Sigma FAST E DAB Peroxidase Substrate Tablet Set solution (Sigma). For GFAP immunostaining, the peroxidase anti-peroxidase method was applied using Histogen PAP rabbit Universal Kit ´ CA, USA). Control sections for (BioGenex, San Ramon, the staining were prepared by omitting the first antibodies or replacing them with LN2 (IgG1), which is against human B cells (Seikagaku Corp.), or with W6 / 32 (IgG2a), which is against human MHC class I molecules (Serotec). These negative controls gave no specific staining (data not shown).
2.4. Double staining for apoptotic cells and brain cells Double staining for TUNEL-positive cells and cell-type markers such as T cells, microglia and astrocytes was performed using frozen sections. In the first step, apoptotic cells were detected by the TUNEL method and AP developed in the BCIP/ NBT solution as a blue color. After washing, slides were stained for T cells, microglia or astrocytes using HRP as an enzyme. HRP developed in the DAB solution as a brown color.
3. Results
3.1. Identification of the type of cells undergoing apoptosis in the CNS We first determined the type of cells undergoing apoptosis in the CNS under normal and pathological conditions. As reported previously (Kohji et al., 1998) and confirmed here, there were no TUNEL-positive cells in the normal
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
CNS (data not shown). During EAE, a large number of TUNEL-positive cells were found in the CNS parenchyma mainly at the peak stage. Double staining revealed that the majority of apoptotic cells were also stained positively with T cell marker (Fig. 1A) and that some were microglia / macrophages (Fig. 1B). However, astrocytes were negative for TUNEL throughout the course of EAE (Fig. 1C). In addition, there were no TUNEL-positive cells with the morphological features of neurons (not shown).
3.2. Localization of apoptosis-related molecules As apoptosis-related molecules, we examined the presence or absence of Fas, FasL, Bax, Bcl-2 and active caspase 3 by immunohistochemistry. Under normal conditions, no apoptosis-related molecules except Bcl-2 were detectable on cells in the CNS (Table 1). Bcl-2 was expressed weakly on some neurons in the spinal cord (data not shown). During EAE, apoptosis-related molecules were expressed on a variety of cells in the CNS (Fig. 2A,C,E,G,I, early stage; Fig. 2B,D,F,H,J, peak stage). As shown in Fig. 2A, a few mononuclear and small stellate cells stained positively for Fas at the early stage of EAE (arrows). At the peak stage of the disease, Fas-positive cells increased in number and many Fas-positive small stellate cells were detectable in the parenchyma (Fig. 2B). The staining pattern for FasL was similar to that for Fas (Fig. 2C,D). Bax immunoreactivities were weakly detectable at the early stage (Fig. 2E) and increased at the peak
167
Table 1 Expression of apoptosis-related molecules on brain and inflammatory cells in the CNS Fas
FasL
Bax
Bcl-2
Active caspase 3
TUNEL
Normal T cells Microglia / MF Astrocytes Neurons
n.d.a 2b 2 2
n.d. 2 2 2
n.d. 2 2 2
n.d. 2 2 1
n.d. 2 2 2
n.d. 2 2 2
EAE T cells Microglia / MF Astrocytes Neurons
11 11 1 2
11 11 11 2
11 11 2 2
11 11 11 1
11 1 2 2
11 1 2 2
a
Not detectable. T cells are not detectable in the CNS parenchyma of normal rats. b According to the number of positive cells in each staining, histological findings are graded into three categories: 2, no positive cells; 1, a few positive cells; 11, many positive cells.
stage (Fig. 2F). At both stages, inflammatory cells and some small stellate cells were positive for Bax. Bcl-2 immunoreactivities were mainly detectable on cells with long processes at the early stage (Fig. 2G), but all the components in the CNS were stained more or less positively at the peak stage (Fig. 2H). In active caspase staining, the majority of positive cells were round mononuclear cells (Fig. 2I,J). Some small stellate cells were also stained positively (data not shown).
Fig. 1. Identification of the type of cells undergoing apoptosis. TUNEL-positive (blue) TCRab-positive (brown) T cells are frequently seen in the parenchyma (arrows in A). Some microglia (brown) are positive for TUNEL (blue) (arrows in B). Astrocytes are negative for TUNEL (C). (A) Double staining with anti-TCRab (R73) and TUNEL. (B) Double staining with tomato lectin for microglia and TUNEL. (C) Double staining with anti-GFAP antibody and TUNEL. Original magnification 3600.
168
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
Fig. 2. Expression of Fas (A, B), FasL (C, D), Bax (E, F), Bcl-2 (G, H) and active caspase 3 (I, J) in the spinal cord at the early (A, C, E, G, I) and peak (B, D, F, H, J) stages of acute EAE. Some positive cells in (A), (I) and (J) are indicated by arrows. Original magnification: (A, C, E, G, I), 3350; (B, D, F, H, J), 3280.
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
3.3. Relationship between apoptotic cells and cells expressing apoptosis-related molecules As shown in Table 1, cells expressing Fas / FasL and Bax underwent apoptotic cell death, whereas cells lacking at least one of these molecules did not show apoptosis. To determine the type of cells expressing Fas and Bax in more detail, sets of serial sections, 10 mm thick, were stained for these molecules and markers for microglia and astrocytes (Fig. 3). As shown in Fig. 3A, Fas immunoreactivities were detected on small stellate cells (arrows) and long fibers running radially from the spinal cord surface (arrowheads). Comparison of this staining pattern with that of
169
microglia (Fig. 3B) and astrocytes (Fig. 3C) revealed that the former and latter staining pattern overlapped with microglial and astroglial staining, respectively. These findings indicate that the Fas molecules are expressed on some, but not all, microglia and astrocytes. On the other hand, Bax immunoreactivities were present only on small stellate cells (Fig. 3D) which were also positive by microglial staining (Fig. 3E). However, astrocytes did not show Bax immunoreactivities (Fig. 3D,F). Table 1 summarizes all the findings obtained by the present examination. During EAE, T cells and microglia / macrophages showed apoptosis. Fas and FasL were expressed on T cells, microglia / macrophages and astrocytes, but not on neurons.
Fig. 3. Identification of Fas-expressing and Bax-expressing cells using serial sections. Fas immunoreactivities are present on small stellate cells (arrows in A) and long fibers (arrowheads in A). Fas-positive stellate cells and fibers are also positive for microglia marker (arrows in B) and astrocyte marker (arrowhead in C), respectively. On the other hand, Bax immunoreactivities (D) are present only on microglia (E), but not on astrocytes (F). Two sets of serial sections were stained for Fas, microglia and astrocytes (A, B and C) and for Bax (D), microglia (E) and astrocytes (F). Original magnification 3200.
170
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171
Bax was detectable on T cells and microglia / macrophages, whereas Bcl-2 expression was observed on all the above cells (Table 1).
4. Discussion EAE is a T cell-mediated autoimmune disease characterized by the presence of inflammatory cells mainly comprising T cells and macrophages in the CNS. In acute EAE, autoimmune inflammation in the CNS begins around day 10 post-immunization, reaches a maximal level on day 12 and subsides by day 21 (Matsumoto et al., 1993). In earlier studies, we showed that the majority of T cells do not proliferate vigorously in the CNS (Ohmori et al., 1992) and others later demonstrated that this phenomenon is attributable to apoptotic cell death of T cells in the CNS (Pender et al., 1991, 1992; Schmied et al., 1993). Based on these findings, we recently reported two important findings (Kohji et al., 1998). First, the majority of apoptotic infiltrating T cells are found in the CNS parenchyma. Second, astrocytes are more frequently associated with apoptotic cells than microglia. These findings suggest that astrocytes play an important role in the induction of apoptosis. In the present study, we found that infiltrating T cells and microglia / macrophages undergo apoptosis in the CNS during EAE and that coexpression of Fas / FasL and Bax on these cells is closely associated with apoptosis. White et al. (1998) performed similar experiments using inflammatory cells isolated from the spinal cord of rats with EAE and reported that expression of Fas / FasL, but not Bax, on these cells influences the susceptibility to apoptosis. However, this finding does not deny the role of Bax in apoptotic processes in the CNS. As clearly shown here, astrocytes which express Fas and FasL, but not Bax, did not show apoptosis throughout the course of the disease, strongly suggesting that expression of Fas and FasL is not sufficient for apoptotic cell death. Recently, it was demonstrated that there are two main signal transduction pathways for mediating Fas–FasL-induced apoptosis (Scaffidi et al., 1998). One is the FADD (Fas-associated death domain)-mediated pathway in which FADD recruited after ligand binding activates caspase 8. The other is the mitochondrial pathway in which cytochrome c released from mitochondria activates caspases. The molecular mechanism of the latter pathway has been elucidated very recently. Caspase 8 activated after crosslinking of Fas with FasL cleaves full-length Bid into truncated Bid (tBid) (Li et al., 1998). tBid then translocates from the cytosol to mitochondria and is associated with Bax. This complex stimulates the release of cytochrome c from mitochondria which induces apoptosis (Desagher et al., 1999). Bcl-2, on the other hand, suppresses the apoptosis-inducing function of Bax by forming Bax–Bcl-2 heterodimers (Oltvai et al., 1993). Taken together, Bax and Bcl-2 are key molecules representing
pro- and anti-apoptosis function, respectively, in the mitochondrial pathway. The findings obtained in the present study suggest that the mitochondrial pathway mainly operates in apoptotic cell death in the CNS. Consistent with our findings, Spanaus et al. (1998) recently demonstrated that cultured activated microglia undergo Fas-mediated apoptosis which is induced by down-regulation of Bcl-2 but not Bax. Since microglia present MBP to MBP-reactive T cells as APC and induce T cell proliferation (Matsumoto et al., 1992), apoptotic cell death of this cell type seems to be essential for the regulation of autoimmune inflammation in the CNS. With regard to oligodendrocytes, we did not succeed in staining this cell type with anti-cyclic nucleotide phosphodiesterase (CNPase), probably because the antibody we used is not appropriate for immunohistochemistry. However, previous studies demonstrated that oligodendroglia which express Fas and Bcl-2, but not FasL and Bax, do not show apoptosis (Bonetti et al., 1997). Neurons express only Bcl-2 among the molecules examined and do not undergo apoptosis under normal and pathological conditions. It is interesting to note the role of astrocytes in apoptotic cell death in the CNS. As demonstrated previously (Matsumoto et al., 1992, 1993), astrocytes do not present antigen to T cells and rather suppress antigen-driven T cell proliferation. Moreover, astrocytes are closely associated with apoptotic cells during autoimmune inflammation in the spinal cord (Kohji et al., 1998). These findings strongly suggest that astrocytes may induce apoptosis of infiltrating T cells and microglia. Recently, it has been demonstrated that cultured astrocytes induce apoptosis of T line cells via the Fas–FasL pathway (Choi et al., 1999). Since astrocytes express Fas and FasL, but not Bax, and do not undergo apoptosis by themselves, this type of cell may play a critical role in regulation of inflammation in the CNS by inducing apoptosis of infiltrating T cells and cells supporting inflammation, i.e. microglia (Matsumoto et al., 1992), during the recovery from inflammation in the CNS. In the present study, we have shown that coexpression of Fas / FasL and Bax plays an important role in apoptotic cell death of brain and infiltrating T cells in the CNS. By examining apoptosis-associated molecules in more detail, the precise mechanisms of formation and subsidization of autoimmune inflammation in the CNS will be elucidated.
Acknowledgements We thank Y. Kawazoe, K. Kohyama and K. Nomura for technical assistance. This study was supported, in part, by Grants-in-Aid (10357005, 09480216 and 09670682) from the Ministry of Education, Japan.
References ´ Acarin, L., Vela, J.M., Gonzalez, B., Castellano, B., 1994. Demonstration of poly-N-acetyl lactosamine residues in ameboid and ramified
T. Kohji, Y. Matsumoto / Journal of Neuroimmunology 106 (2000) 165 – 171 microglial cells in rat brain by tomato lectin binding. J. Histochem. Cytochem. 42, 1003–1041. Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Bonetti, B., Pohl, J., Gao, Y., Raine, C.S., 1997. Cell death during autoimmune demyelination: effector but not target cells are eliminated by apoptosis. J. Immunol. 159, 5733–5741. Choi, C., Park, J., Lee, J., Lim, J., Shin, E., Ahn, Y., Kim, C., Kim, S., Kim, J., Choi, I., Choi, I., 1999. Fas ligand and Fas are expressed constitutively in human astrocytes and the expression increases with IL-1, IL-6, TNF-a, or IFN-g. J. Immunol. 162, 1889–1895. Desagher, K., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., Martinou, J., 1999. Bidinduced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144, 891– 901. Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312. Kohji, T., Tanuma, N., Aikawa, Y., Kojima, T., Matsumoto, Y., 1998. Interaction between apoptotic cells and reactive brain cells in the central nervous system of rats with autoimmune encephalomyelitis. J. Neuroimmunol. 82, 168–174. Lassmann, H. (Ed.), 1983. Comparative Neuropathology of Chronic Experimental Allergic Encephalomyelitis and Multiple Sclerosis, Springer, Berlin, pp. 9–94. Li, H., Zhu, H., Xu, C., Yuan, J., 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491–501. Matsumoto, Y., Ohmori, K., Fujiwara, M., 1992. Immune regulation by brain cells in the central nervous system: microglia but not astrocytes present myelin basic protein to encephalitogenic T cells under in vivo-mimicking conditions. Immunology 76, 209–216. Matsumoto, Y., Hanawa, H., Tsuchida, M., Abo, T., 1993. In situ inactivation of infiltrating T cells in the central nervous system with
171
autoimmune encephalomyelitis. The role of astrocytes. Immunology 79, 381–390. Ohmori, K., Hong, Y., Fujiwara, M., Matsumoto, Y., 1992. In situ demonstration of proliferating cells in the rat central nervous system during experimental autoimmune encephalomyelitis. Evidence suggesting that most infiltrating T cells do not proliferate in the target organ. Lab. Invest. 66, 54–62. Oltvai, Z.N., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619. Pender, M.P., Nguyen, K.B., McCombe, P.A., Kerr, J.F.R., 1991. Apoptosis in the nervous system in experimental allergic encephalomyelitis. J. Neurol. Sci. 104, 81–87. Pender, M.P., McCombe, A., Yoog, G., Nguyen, K.B., 1992. Apoptosis of ab T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: its possible implication for recovery and acquired tolerance. J. Autoimmun. 5, 401–410. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K.J., Debatin, K., Krammer, P.H., Peter, M.E., 1998. Two CD95 (APO-1 / Fas) signaling pathways. EMBO J. 17, 1675–1687. Schmied, M., Breitschopf, H., Gold, R., Zischler, H., Rothe, G., Wekerle, H., Lassmann, H., 1993. Apoptosis of T lymphocytes in experimental autoimmune encephalomyelitis. Evidence for programmed cell death as a mechanism to control inflammation in the brain. Am. J. Pathol. 143, 446–452. Spanaus, K.S., Schlapbach, R., Fontana, A., 1998. TNF-a and IFN-g render microglia sensitive to Fas ligand-induced apoptosis by induction of Fas expression and down-regulation of Bcl-2 and Bcl-xL. Eur. J. Immunol. 28, 4398–4408. White, C.A., McCombe, P.A., Pender, M.P., 1998. The role of Fas, Fas ligand and Bcl-2 in T cell apoptosis in the central nervous system in experimental autoimmune encephalomyelitis. J. Neuroimmunol. 82, 47–55.